WO2013185214A1 - Pla2g7/lp-pla2 as biomarker and therapeutic target in the prevention and treatment of calcific aortic valve disease - Google Patents

Abstract

The present invention relates to the treatment and/or prevention of calcific aortic vascular disease (CAVD) and valve calcification. Particularly, the invention provides a target for intervention in the treatment or prevention of CAVD through the administration of an of inhibitor PLA2G7 expression or Lp-PLA2 enzymatic activity. Also, the invention provides 5 means to treat hypertension related to decreased arterial compliance and vascular calcification. The invention also relates to the use of inhibitors of Lp-PLA2 to prevent structural valve degeneration (SVD) as the major cause of bioprosthetic valve failure leading to bioprostheses (BPs) stenosis or regurgitation.

Description

PLA2G7/LP-PLA2 AS BIOMARKER AND THERAPEUTIC TARGET IN THE PREVENTION AND TREATMENT OF CALCIFIC AORTIC VALVE DISEASE

Cross-reference

[0001] This application claims priority from US provisional application 60/657,969 filed on June 11 , 2012, which is herein incorporated by reference in its entirety.

Field of the invention

[0002] The present invention relates to the prevention and/or treatment of calcific aortic valve disease disease (CAVD) also known as calcific aortic valve stenosis that stems from tissue calcification. Particularly, the invention provides for the quantification of expression of the PLA2G7 gene or the activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) as a biomarker for the diagnosis or prognosis of CAVD. More particularly, the invention provides a target for intervention in the treatment or prevention of CAVD through the administration of an inhibitor of the expression of PLA2G7 or an inhibitor of the enzymatic activity of Lp-PLA2. Also, the invention provides means to treat: (1 ) hypertension related to decreased arterial compliance due to vascular tissue calcification and/or (2) degradation of bioprosthetic valves.

Background of the invention

[0003] There is so far no medical treatment for calcific aortic valve disease (CAVD) that could prevent the progression of the pathological process once established. CAVD is the most prevalent heart valve disorder in humans. Incipient disorder, also called aortic sclerosis, is characterized by the calcification and thickening of aortic valve tissues.

Progressive and relentless calcification of the aortic valve leads to the development of a significant stenosis in a matter of years. Risk factors for CAVD includes: age,

hypertension, diabetes, male gender and dyslipidemia. It is estimated that CAVD is present in 2-4% of the population over 65 years of age. Once the process is severe and accompanied by symptoms, the only possible cure is an aortic valve replacement (AVR). In North America alone, 100 000 AVR are performed annually. It is worth emphasizing that this figure represents just the tip of the iceberg, since the vast majority of patients have a mild-to-moderate AS, which is prone to progression in a matter of years. So far, there is no pharmacological treatment that could prevent further disease progression when detected as a mild or moderate-to-severe aortic stenosis. Hence, it is possible to envision that if a medical treatment was available it could be possible to stop the disease process and prevent AVR for a large number of afflicted patients.

[0004] Progressive calcification of the aortic valve has been ascribed to an active process which relies on cellular transformation upon different signals. The development of CAVD relies on different mechanisms, which include lipid infiltration, inflammation and calcification. Calcification of the aortic valve is the predominant feature of the

pathobiological process. Also, although CAVD shares some risk factors with

atherosclerosis, it is an independent and distinct pathobiological process. (1 ) To that effect, the anatomy as well as the mechanical stress and biology of the aortic valve are dissimilar and unrelated to the vascular wall.

[0005] On this score, patients with a bicuspid aortic valve (BAV) have high rate of CAVD occurring at an early age (usually in the 5^th decade). BAV have a higher mechanical stress, which may exacerbate a predisposition to calcify valve tissue. Several mechanisms may be at play during pathological calcification of the aortic valve, but the major determinants remain to be elucidated. To this effect, recent studies indicate that the fate of valve interstitial cells (VICs), the main cellular component of the aortic valve, largely determines the occurrence of pathological calcification. Studies have highlighted that during calcification, VICs are progressively modified toward osteob!astic-like cells which promote calcification. [0006] Lipoprotein- Associated Phospholipase A2 (Lp-PLA₂), also previously known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase) is a member of the super family of phospholipase A2 enzymes that are involved in hydrolysis of lipoprotein lipids or phospholipids. It is secreted by several cells that play a major role in the systemic inflammatory response to injury, including lymphocytes, monocytes, macrophage, T lymphocytes and mast cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2; EC 3.1.1.47; NP_001 161829.1 ) or PLA2G7 (official gene symbol;

NM_001168357.1 ) or (LDL-PLA2, PAFAD, PAFAH, PAF acetylhydrolase) is an enzyme that uses oxidized lipids derived from low-density lipoproteins (ox-LDL) and produce lysophosphatidylcholine (LPC), a down-product of enzymatic activity, which may increase inflammation.(2) The aortic valve is infiltrated by ox-LDL.(3, 4) PLA2G7 is expressed by macrophages, which are present within CAVD tissues. Also, Lp-PLA2 is bound in circulation to LDL.(5) Hence the presence of Lp-PI_A2 within the aortic valve could come from macrophages and/or circulating LDL infiltrating the aortic valve.

[0007] During the conversion of LDL to its oxidised form, Lp-PLA2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso- phosphatidylcholine and an oxidatively modified fatty acid. Lp-PLA2 hydrolyzes the sn2 position of a truncated phospholipid associated with oxidized LDL. As a result, there is a generation of two inflammatory cell homing mediators (non-esterified fatty acids (NEFA) and LYSO PC) Both NEFA and LYSO PCs are chemoattractants for circulating monocytes, play a role in the activation of macrophages and increase oxidative stress as well as affecting the functional and the immediate responses of T lymphocytes. Lp-PLA2 is bound in humans and pigs to the LDL molecule via lipoprotein B, and once in the arterial wall the oxidized LDL is susceptible to hydrolysis by LpPLA2.

[0008] Both products of Lp-PLA₂ action are potent chemoattractants for circulating monocytes. As such, this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic 'fatty streak' associated with the early stages of atherosclerosis, and inhibition of the Lp-PLA2 enzyme may be useful in preventing the build up of this fatty streak (by inhibition of the formation of lysophosphatidylcholine), and therefore is thought to be useful in the treatment of atherosclerosis. [0009] In addition, it is proposed that Lp-PLA2 plays a direct role in LDL oxidation. This is due to the polyunsaturated fatty acid-derived lipid peroxide products of Lp-PLA2 action contributing to and enhancing the overall oxidative process. In keeping with this idea, Lp- PLA2 inhibitors inhibit LDL oxidation. Lp-PLA2 inhibitors may therefore have a general application in any disorder that involves lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, myocardial infarction and reperfusion injury.

[0010] Although Lp-PLA2 has been implicated in the pathobiological process of atherosclerosis, calcific aortic valve disease (CAVD) is not atherosclerosis.(l ) Albeit there is an overlap in clinical risk factors, the pathobiology of CAVD is different. For instance, the genetics and processes related to clinical events as well as disease progression are not related. To this effect, cellular components as well as hemodynamics on the aortic valve are different. Patients with a bicuspid aortic valve are at high risk of developing CAVD, which indicate that genetic risk factors as well as particular hemodynamic and biology of the aortic valve participate to the development/progression of CAVD. Also, the mechanisms related to clinical events in CAVD involve increased leaflet stiffness and calcification, not plaque progression and rupture such as found in atherosclerosis. Hence, CAVD is a distinct pathological entity and drugs that are efficient for the treatment of one disorder are not necessarily of efficacy in the other. For instance, statins reduce the number of events in patients with coronary artery disease but have no efficacy to prevent events related to CAVD as well as clinical hemodynamic progression of aortic stenosis. [0011] A trial with Darapladib in 330 patients showed that when compared to the placebo, Darapladib did not reduce atherosclerotic plaque deformability nor total plaque volume. However, it reduced significantly atherosclerotic plaque necrotic core. (6) Studies are undergoing to demonstrate if Darapladib could reduce the number of coronary artery disease events in a large Phase III trial. Hence, so far, the efficacy of PLA2G7/LP-PLA2 inhibitors to prevent the clinical events related to coronary artery disease is not established. There is no indication or data on PLA2G7/LP-PLA2 inhibitors regarding CAVD.

[0012] US2009/0081703 and WO2011/137419 disclose an association between Lp-PLA2 activity and thrombosis (such as deep vein thrombosis). WO2008/140449 teach a correlation between Lp-PLA2 activity and disease ad disorders with abnormal blood brain barrier function and that this enzyme can be used as a target for the treatment of neurodegenerative diseases such as Alzheimer's, Huntington's, Parkinson's diseases or vascular dementia. No mention is made of calcific aortic valve disease.

Bioprostheses (BP) structural valve degeneration (SVD)

[0013] Replacement of aortic valves with bioprostheses (BPs) has the main advantage that it may obviate the need of chronic anticoagulation. However, one major drawback of BPs is that structural valve degeneration (SVD) contributes to the failure of these valve substitutes. (7) To this effect, freedom of reoperation for SVD is between 40-75% at 15 years after an aortic valve replacement (AVR). However, it should be underlined that incidence of SVD based solely on reintervention underestimates the true incidence of BP dysfunction, since operation maybe denied by the either the patients and/or surgeon particularly in elderly patients with important co-morbidities. Hence, when based on echocardiographic criteria the incidence of BP dysfunction is between 20-30% 5 to 10 years after AV .(8, 9)

[0014] Despite that pre-implant treatment with anti-calcifying agents has evolved over the last 20 years, the incidence of SVD has remained high. Studies have indicated that several risk factors such as age at implantation, dyslipidemia, diabetes, renal failure, metabolic syndrome (MetS) and increased ApoB/ApoA-l ratio are associated with SVD. (9) Analyses of explanted BPs for SVD have revealed that a high percentage of prostheses are infiltrated by inflammatory cells and oxidized-low-density lipoproteins (ox-LDL). (10) These findings suggest that SVD may be modulated by metabolic risk factors of the recipient. Moreover, the presence of lipid-derived factors and inflammatory cells in explanted BPs suggest that an active process related to lipid metabolism may be implicated in SVD. Lipoprotein-associated phospholipase A2 (Lp-PLA2) uses ox-LDL as a substrate and produces free fatty acids and lysophosphatidylcholine (LPC), which has proinflammatory activity.(11 ) [0015] In this work we have identified that PLA2G7 is expressed within the aortic valve and/or blood plasma of patients suffering from CAVD. We show that PLA2G7 promotes aortic valve inflammation and calcification. Hence, targeting PLA2G7 expression or Lp- PLA2 activity represents a potential marker and its inhibition may represent a new form of medical treatment for CAVD as well as for hypertension caused by vascular calcification. [0016] We have also found that Lp-PLA2 may be associated with SVD of BPs and therefore posit that inhibition of Lp-PLA2 may represent a new form of prevention or treatment of structural valve degradation of bioprosthesis.

Summary of the invention

[0017] In the present work we present evidence that inhibitors of PLA2G7 expression and/or Lp-PLA2 activity may prevent calcific disease (CD) (such as calcific aortic valve disease and/or aortic valve stenosis and/or aortic valve calcification and/or vascular calcification and/or hypertension related to decreased arterial compliance due to vascular tissue calcification).

[0018] We also present evidence that inhibitors of PLA2G7 expression and/or Lp-PLA2 activity may prevent structural valve degeneration of bioprosthesis. [0019] Particularly, the invention provides, for the first time, that the expression of the PLA2G7 gene and/or activity of the Lp-PLA2 enzyme is a key regulator of the calcifying process.

[0020] Particularly, we present convincing evidence that inhibitors of PLA2G7 expression or Lp-PLA2 activity may be considered as potential pharmaceutical agents for the treatment of CAVD.

[0021] Particularly, the invention provides means to prevent or treat CAVD by the administration of an inhibitor of PLA2G7/Lp-PLA2 in a subject.

[0022] The invention further provides a method for preventing or treating CAVD in a subject, including human, suffering therefrom, comprising the step of administering a pharmaceutically effective amount of a PLA2G7/Lp-PLA2 inhibitor.

[0023] Alternatively, the invention is directed to the use of an inhibitor of PLA2G7/Lp-PLA2 for the manufacture of a medicament for the prevention or treatment of CAVD in a subject.

[0024] The invention further provides an inhibitor of PLA2G7/Lp-PLA2 for use to prevent or treat CAVD in a subject.

[0025] The invention further provides the use of an inhibitor of PLA2G7/Lp-PLA2 for preventing or treating CAVD in a subject.

[0026] The present invention further provides a method for identifying an inhibitor of CAVD, comprising the steps of: a) contacting PLA2G7/Lp-PLA2 with a potential inhibitor thereof; and b) measuring PLA2G7/Lp-PLA2 expression or activity; whereby inhibition of PLA2G7/Lp-PLA2 expression or activity is an indication that said compound is a potential inhibitor of CAVD.

[0027] In an embodiment, there is provided a method of preventing and/or treating calcific disease in a subject, the method comprising: identifying a subject with, or at risk of developing tissue calcification; and administering to the subject in need thereof a pharmaceutical composition comprising an agent that inhibits expression of PLA2G7 gene and/or activity of Lp-PLA2 protein. [0028] In an embodiment, there is provided a method of preventing and/or treating a subject with or at risk of calcific disease comprising: identifying a subject with, or at risk of developing tissue calcification; and administering to a subject in need thereof a pharmaceutical composition comprising an agent which inhibits expression of PLA2G7 gene and/or activity of Lp-PLA₂ protein, wherein inhibition of the PLA2G7 gene or Lp-PLA2 protein reduces or stops a symptom of CAVD.

[0029] In an embodiment, there is provided a method of preventing and/or treating CAVD in a subject in need thereof, comprising: (i) screening the subject for likelihood of having or developing tissue calcification; and (ii) administering to the subject a pharmaceutical composition comprising an agent which inhibits expression of PLA2G7 gene and/or activity of Lp-PLA2 protein, wherein inhibition of PLA2G7 gene or Lp-PLA2 protein reduces or slows tissue calcification, wherein the subject is identified to have an increased risk or likelihood of developing calcific aortic valve disease determined by step (i).

[0030] In an embodiment, there is provided a method for preventing calcification of a prosthetic aortic valve in a subject, comprising: (i) screening the subject for likelihood of having or developing valve calcification; and (ii) administering to the subject a

pharmaceutical composition comprising an agent which inhibits expression of PLA2G7 gene and/or activity of Lp-PLA2 protein, wherein inhibition of PLA2G7 gene or Lp-PLA2 protein reduces or slows valvular calcification. [0031] In an embodiment, there is provided an inhibitor of PLA2G7 expression or Lp-PLA2 activity for use to prevent or treat calcific disease, or bioprosthetic valve calcification in a subject.

[0032] In an embodiment, there is provided use of an inhibitor of PLA2G7 expression or Lp-PLA2 activity for preventing or treating calcific disease or bioprosthetic valve calcification in a subject.

[0033] In an embodiment, there is provided use of an agent that inhibits expression of PLA2G7 gene or activity of Lp-PLA2 enzyme for the preparation of a medicament for treatment and/or prevention of calcific disease in a subject.

[0034] In accordance with a particular embodiment, the invention provides a method, an inhibitor, or a use, wherein the calcific disease is selected from a group consisting of: aortic valve stenosis (AS), calcific aortic valve disease (CAVD) and hypertension related to increased vascular calcification.

[0035] In accordance with a particular embodiment, the invention provides a method, an inhibitor, or a use, wherein the calcific disease is structural valve degradation of bioprosthesis.

[0036] In an embodiment, there is provided a method for diagnosing or prognosing a vascular or valvular calcification disorder in a subject, comprising the steps of: quantifying Lp-PLA2 activity (or PLA2G7 expression) in plasma of said subject; and determining if said activity/expression is above a threshold or control level; wherein if said

activity/expression is above said threshold or control level, said subject is at risk of developing calcific disease.

[0037] In an embodiment, there is provided a method for predicting prosthetic valve calcification (degradation) in a subject having undergone prosthetic valve replacement, comprising the steps of: identifying a subject having undergone prosthetic valve replacement following a diagnosis of aortic valve disorder; obtaining a plasma sample from said subject; quantifying Lp-PLA2 activity (or PLA2G7 expression) in said sample; and determining if said activity/expression is above a predetermined threshold or control level; wherein if said activity/expression is above said threshold or control level, said subject is at risk of calcifying the prosthetic valve. Detailed description of the invention

Abbreviations

[0038] For convenience, certain abbreviations employed herein are presented here.

[0039] ALP: alkaline phosphatase; AS: aortic stenosis or aortic valve stenosis; ; cAMP: 3'- 5'-cyclic adenosine monophosphate; CAVD: calcific aortic valve disease; CD: calcific disease; ENPP1 : ectonucleotide pyrophosphatase/phosphodisterase-1 ; Lp-PLA2:

lipoprotein-associated phospholipase A2 (encoded by PLA2G7 gene); LPC:

lysophosphatidylcholine; ox-LDL: oxidized low-density lipoprotein; PKA: protein kinase A; OPN: osteopontin; P2Y₂: G-protein-coupled receptor-2; Pit1 : Sodium-dependent phosphate cotransporter 1 (encoded by SLC20A1 gene); VIC: valve interstitial cells ; VSMC: vascular smooth muscle cells; and ΔΨιη: mitochondrial membrane potential. Definitions

[0040] For convenience, certain terms employed herein are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] The term "about" as used herein refers to a margin of + or - 5% of the number indicated. For sake of precision, the term about when used in conjunction with, for example, 90%, means 90% +/- 4.5% i.e. from 86.5% to 94.5%.

[0043] As used herein, the terms "administering," and "introducing" are used interchangeably and refer to the placement of the agents that inhibit Lp-PLA₂ as disclosed herein into a subject by a method or route which results in at least partial localization of the agents at a desired site. The compounds of the present invention can be administered by any appropriate route which results in an effective treatment in the subject.

[0044] The term "agent" refers to any entity which is normally not present or not present at the levels being administered in the cell. Agent can be selected from a group comprising: chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof. A nucleic acid sequence can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc- PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide or fragment thereof can be any protein of interest, for example, but are not limited to: mutated proteins; therapeutic proteins and truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins can also be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. Alternatively, the agent can be intracellular within the cell as a result of introduction of a nucleic acid sequence into the cell and its transcription resulting in the production of the nucleic acid and/or protein inhibitor OfLp-PLA₂ within the cell. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

[0045] The term "calcific disease" (CD) as used herein means an increase in aortic valve stiffness and/or diminished vascular compliance that is caused by aorta calcification. Amongst others, calcification of the aortic valve. Symptoms of CD include, but are not limited to: dyspnea, angor, and syncope. Different forms of CD can be embodied as AS, CAVD, and hypertension.

[0046] The term "disease" or "disorder" is used interchangeably herein, and refers to any alteration in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also relate to a ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition or affectation.

[0047] The term "effective amount" as used herein refers to the amount of therapeutic agent of pharmaceutical composition to reduce or stop vascular or valve calcification. For example, an effective amount using the methods as disclosed herein would be considered as the amount sufficient to reduce or stabilize a symptom of the disease or disorder, for example when assessed by Doppler echocardiography or computed tomographic imaging (CTI). An effective amount as used herein would also include an amount sufficient to prevent or delay calcification, alter the course of progression of calcification (for example but not limited to, slow the progression of calcification), or reverse calcification. [0048] The term "inhibiting" as used herein means that the expression of PLA2G7 gene or activity of Lp-PLA₂ protein or variants or homologues thereof is reduced to an extent, and/or for a time, sufficient to produce the desired effect. The reduction in activity can be due to affecting one or more characteristics of Lp-PLA₂ including decreasing its catalytic activity or by inhibiting a co-factor of Lp-PLA₂ or by binding to Lp-PLA2 with a degree of avidity that is such that the outcome is that of treating or preventing vascular or valvular calcification. In particular, inhibition of Lp-PLA₂ can be determined using an assay for Lp- PLA₂ inhibition, well known in the art, for example, but not limited to, by using the bioassay for Lp-PLA₂ protein as disclosed in WO2008/140449, US7531316 and US2010/0256919. [0049] The term "gene" used herein can be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3'- untranslated sequences and regulatory sequences). The coding region of a gene can be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene can also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and miRNA) optionally comprising 5'- or 3' untranslated sequences linked thereto. A gene can also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'- untranslated sequences linked thereto. [0050] As used herein, "gene silencing" or "gene silenced" in reference to an activity of n RNAi molecule, for example a siRNA or miRNA refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about interference molecule. In one preferred embodiment, the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[0051] As used herein, the term "Lp-PLA2" refers to the protein target to be inhibited by the methods as disclosed herein. Lp-PLA2 is used interchangeably with Lp-PLA2 and lipoprotein associated phospholipase A2, also previously known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase). Human Lp-PLA2 is encoded by nucleic acid corresponding to accession No: U20157 or Ref Seq ID: NM_005084 or and the human Lp-PLA₂ corresponds to protein sequence corresponding to accession No: NP_005075, which are disclosed in US 5,981 ,252.

[0052] The term "prevent" or " prevention" as used herein means the prevention of the disease or progression of the disease, as well as prevention of aggravation of symptoms. [0053] As used herein, the term "RNAi" refers to any type of interfering RNA, including but are not limited to, siRNAi, shRNAi, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of downstream processing of the RNA.

[0054] As used herein an "siRNA" refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene, for example Lp-PLA₂. The double stranded RNA siRNA can be formed by the complementary strands. In one embodiment, a siRNA refers to a nucleic acid that can form a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0055] As used herein "shRNA" or "small hairpin RNA" (also called stem loop) is a type of siRNA. In one embodiment, these shRNAs are composed of a short, e.g. about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.

[0056] The terms "microRNA" or "miRNA" are used interchangeably herein are endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. Endogenous microRNA are small RNAs naturally present in the genome which are capable of modulating the productive utilization of mRNA. The term artificial microRNA includes any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the productive utilization of mRNA. MicroRNA sequences have been described in publications such as Lim, et al., Genes & Development, 17, p. 991-1008 (2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294, 862 (2001 ), Lau et al., Science 294, 858-861 (2001 ), Lagos-Quintana et al, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science 294, 853-857 (2001 ), and Lagos-Quintana et al, RNA, 9, 175-179 (2003), which are incorporated by reference. Multiple microRNAs can also be incorporated into a precursor molecule. Furthermore, miRN A-like stem-loops can be expressed in cells as a vehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs) for the purpose of modulating the expression of endogenous genes through the miRNA and or RNAi pathways. [0057] As used herein, "double stranded RNA" or "dsRNA" refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of a single RNA molecule that doubles back on itself to form a two-stranded structure. For example, the stem loop structure of the progenitor molecules from which the single- stranded miRNA is derived, called the pre-miRNA (Bartel et al. 2004. Cell 116:281 -297), comprises a dsRNA molecule.

[0058] The terms "subject" and "individual" are used interchangeably herein, and refer to a mammal, particularly a human, to whom treatment including prophylactic treatment is provided. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. [0059] As used herein, the term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with tissue calcification. As used herein, the term treating is used to refer to the stabilization or reduction of a symptom and/or a biochemical marker of calcific disease.

Description of the figures

[0060] Figure 1 : Expression of Lp-PLA2 in CAVD. A) Gene expression profiles of the PLA2/PLA2G family of genes in control, non-calcified aortic valves (VAHN21 , VAHN24, VAHN47) and stenotic aortic valves (VAHC79, VAHC8, VAHC220, VAHC147, VAHC15). Each row represents a different probe set tagging a specific enzyme indicated at the right. The fold-change comparing the expression of CAVD versus control, non-calcified aortic valves is indicated in parentheses. The asterisks represent probe sets that are claim significant based on the whole microarray experiment. B and C) In a larger group of patients both the number of Lp-PLA2(PLA2G7) transcript and enzyme activity were increased in CAVD tissues when compared to control valves. D) Lp-PLA2 activity was increased in the blood plasma of subjects with CAVD. E) A thin layer chromatography showed the presence of lysophosphatidylcholine (LPC) in CAVD valves (VAHC1-3). [0061] Figure 2: Immunodetection of Lp-PLA2 and ox-LDL. A-C)

Immunohistochemistry studies showed that in control non-mineralized aortic valves (A) Lp-PLA2 was not expressed, whereas there was a strong immunostaining in CAVD, which appears as cellular marking (arrows) (B) or as a diffuse staining (arrows) (C). D) In areas of diffuse staining, Lp-PLA2 co-localized with ox-LDL (arrows). [0062] Figure 3: Lp-pLA2 and indices of CAVD severity. A) The number of Lp-PLA2 transcript in CAVD valves increased significantly with the severity of the remodeling score of the aortic valve (to stabilize variances the number of Lp-PLA2 has been log

transformed). B-C) The number of Lp-PLA2 transcript in CAVD valves correlated significantly with the weight of the aortic valve (B), and the peak transaortic gradient (C) (Spearman's correlation). D) Within CAVD valves, enzyme activity of Lp-PLA2 correlated significantly with the amount of calcium (Spearman's correlation). ^*p<0.05 compared to remodelling score 2; # p<0.05 compared to remodelling score 3.

[0063] Figure 4: LPC induces mineralization of VICs. A) In isolated valve interstitial cells (VICs), lysophosphatidylcholine (LPC) exacerbated the mineralization induced by phosphate (P0₄). B) In isolated VICs medium oxidized-LDL (ox-LDL) exacerbated mineralization induced by P0₄. C) In isolated VICs, exposition to LPC (1 nM) increased the expression of ENPP1 , ALP, Pit-1 , and osteopontin (OPN). D-E) In isolated VICs, LPC (1 nM) increased enzyme activity of both ALP (D) and ENPP (E). F) In isolated VICs, mineralization induced by both P0₄ and LPC was abrogated by a treatment with

ARL67156 (ARL), a competitive inhibitor of ectonucleotidase enzymes. ^*p<0.05 compared to control (Ctn); # p<0.05 compared to mineralizing medium (P0₄); * p<0.05 compared to P0 + lysophosphatidylcholine (LPC).

[0064] Figure 5: LPC mediates apoptosis of VICs. A) In isolated valve interstitial cells (VICs), a treatment with lysophosphatidylcholine (LPC) increased the activation of caspase 3/7. B-C) In isolated VICs, LPC decreased the mitochondrial membrane potential (ΔΨι^η). D) In isolated VICs, LPC promoted the release of cytochrome c in the cytosol.

[0065] Figure 6: LPC promotes apoptosis-mediated mineralization of VICs through a PKA pathway. A-B) Mineralization of isolated VICs induced by P0₄ and LPC was prevented by ZVAD-fmk, a caspase inhibitor (A), a PKA inhibitor (PKAi) (B). C) In isolated VICs LPC promoted PKA activity. D) Mineralization of isolated VICs induced by P0₄ and LPC was amplified by a treatment with forskolin. ^*p<0.05 compared to control (Ctn); # p<0.05 compared to LPC in panel (C) and to mineralizing medium (P0₄) in panel (A-B and D); I p<0.05 compared to P0₄ + lysophosphatidylcholine (LPC) t p<0.05 compared to P0₄ + forskolin.

[0066] Figure 7: Proportion of examined bioprosthetic heart valves that expressed Lp- PLA2, oxidized-LDL (ox-LDL) and macrophages (CD68+).

[0067] Figure 8: Immunohistochemistry studies in explanted bioprostheses (BPs). A) Lp- PLA2 was expressed around calcific nodules (empty space following cut sectioning). B and C) A cellular pattern of expression of Lp-PLA2 was observed (B), which co-localized with macrophages (CD68+ cells) (C). D and E) Lp-PLA2 appears as diffuse staining (D), which co-localized with oxidized-LDL (ox-LDL) (E).

[0068] Figure 9: Relationships between markers measured in explanted bioprotheses (BPs) with quantitative morphometric analyses. A and B) Correlations between Lp-PLA2 and oxidized-LDL (ox-LDL) (A) and with the density of macrophages (B). C) Correlation between the density of macrophages and the content in ox-LDL.

Detailed description of particular embodiments

[0069] The present invention relates to methods for treatment and/or prevention of vascular and valvular calcification in a mammal by inhibition of Lp-PLA2, for example inhibition of expression of PLA2G7 gene and/or activity of Lp-PLA2 protein. In particular, inhibition is carried out by the administration of an effective amount of an agent that inhibits Lp-PLA2 activity or PLA2G7 expression.

[0070] In some embodiments, the agent that inhibits Lp-PLA₂ can inhibit the expression of the PLA2G7 gene, for example inhibit the translation of PLA2G7 RNA to produce the Lp- PLA2 protein. In alternative embodiments, the agent that inhibits Lp-PLA₂ can inhibit Lp- PLA2 enzymatic activity. Any agent for use in the methods and uses herein disclosed is encompassed.

[0071] More particularly, agents selected from Darapladib (aka NCT00799903, CAS 356057-34-6; lUPAC name: N-(2-diethylaminoethyl)-2-[2-[(4-fluorophenyl)methylsulfanyl]- 4-0X0-6, 7-dihydro-5H-cyclopenta[d]pyrimidin-1-yl]-N-[[4-[4-(trifluoromethyl)

phenyl]phenyl]methyl]acetamide) from GlaxoSmithKline, and corresponding to structure I:

is encompassed as a potential agent to treat calcific disease as disclosed herein.

[0072] In some embodiments, the agent can be a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof.

[0073] In some embodiments, the agent is a nucleic acid agent, for example but are not limited to an NAi agent, for example but are not limited to siRNA, shRNA, miRNA, dsRNA or ribozyme or a variant thereof.

[0074] In some embodiments, the agent that inhibits the protein activity of Lp-PLA2 is a small molecule, for example, but not limited to, a small molecule reversible or irreversible inhibitor of Lp-PLA2 protein. In some embodiments, such a small molecule is a pyrimidione-based compound.

[0075] In some embodiments, a small molecule inhibitor of Lp-PLA2 is, for example, but not limited to, 1 -(N-(2-(diethyiamino)ethyl)-/V-(4-(4-trifluoromethylphenyl)benzyl)- aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one (which is also known as SB480848) or a salt thereof. In some embodiments, a small molecule inhibitor of Lp-PLA2 is, for example, but not limited to, /V-(2-diethylaminoethyl)-2-[2-(2-(2,3- difluorophenyl)ethyl)-4-oxo-4,5,6J-tetrahydro-cyclopentapyrimidin-l-yl]-/V-4'- trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof. In some embodiments, a small molecule inhibitor of Lp-PLA2 is, for example but not limited to, Λ/-(1-(2-

Methoxyethyl)piperidin-4-yl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-1-yl]-/V-(4'- trifluoromethylbiphenyl-4-ylmethyl)acetamide or a salt thereof. In some embodiments, a small molecule inhibitor of Lp-PLA2 is, for example but not limited to, methyl 2-[4-({[2-[2- (2,3-difluorophenyl)ethyl]-4-oxopyrido[2,3-d]pyrimidin-1(4H)-yl]-acetyl} {[4'- (trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidinyl]-2-methylpropanoate or a salt thereof.

[0076] In some embodiments where a subject is administered a pharmaceutical composition comprising an inhibitor of Lp-PLA2, the methods can further comprise administering to the subject additional therapeutic agents, for example but not limited to a therapeutic agent used in the treatment of CD.

[0077] For example, the additional therapeutic agent is another agent that prevents or slows down tissue calcification such as, for example, inhibitors of ENPP-1 or agonists of the P2Y₂ receptor.

[0078] In some embodiments, the methods as disclosed herein for the treatment and/or prevention of CD are applicable to subjects, for example mammalian subjects. Particularly, with respect to the methods, use and compounds of the present invention, the test compound is particularly an inhibitor of PLA2G7 expression or Lp-PLA2 activity, more particularly the inhibitor is Darapladib (aka NCT00799903) from GlaxoSmithKline.

[0079] Particularly, with respect to the different embodiments of the present invention such as methods and uses, the mammalian subject is a human.

LP-PLA2

[0080] Lp-PLA2 is also referred to in the art as aliases Lp-PLA2, LDL-PLA2, lipoprotein associated phospholipase A2, PLA2G7, phospholipase A2 (group VII), or Platelet

Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase or PAFAH). Human Lp-PLA2 is encoded by nucleic acid corresponding to GenBank Accession No: U20157 or Ref Seq ID: NM_005084 and the human Lp-PLA2 corresponds to protein sequence corresponding to GenBank Accession No: NP_005075, which are disclosed in US5,981 ,252.

[0081] Phospholipase A2 enzyme Lipoprotein Associated Phospholipase A2 (Lp-PLA2), the sequence, isolation and purification thereof, isolated nucleic acids encoding the enzyme, and recombinant host cells transformed with DNA encoding the enzyme are disclosed in WO95/00649 (SmithKline Beecham pic). A subsequent publication from the same group further describes this enzyme ((WO 95/09921 ) wherein it is referred to as LDL-PLA2 and later referred to the enzyme PAF-AH.

[0082] It has been shown that Lp-PLA2 is responsible for the conversion of

phosphatidylcholine to lysophosphatidylcholine, during the conversion of low density lipoprotein (LDL) to its oxidised form. The enzyme is known to hydrolyse the sn-2 ester of the oxidised phosphatidylcholine to give lysophosphatidylcholine and an oxidatively modified fatty acid. Both products of Lp-PLA2 action are biologically active with lysophosphatidylcholine, in particular having several pro-atherogenic activities ascribed to it including monocyte chemotaxis and induction of endothelial dysfunction, both of which facilitate monocyte-derived macrophage accumulation within the artery wall.

[0083] The inventor has found that PLA2G7 is highly expressed within aortic valve tissues and blood plasma of patients with CAVD. Furthermore, the number of PLA2G7 transcripts was related to several indices of aortic valve disease, including transaortic gradient. In addition, it was found that, the down-product derived from PLA2G7 activity, LPC, induced calcification of VICs, the main cellular component of the aortic valve.

Lp-PLA2 as a therapeutic target for screening and identifying potential inhibitors of CD

[0084] In a particular embodiment, the present invention relates to an assay for identifying an inhibitor of calcific disease (CD), comprising the steps of: a) contacting PLA2G7 with a potential inhibitor thereof; and b) measuring PLA2G7 expression; whereby inhibition of PLA2G7 expression is an indication that said compound is a potential inhibitor of CD.

[0085] In an alternative embodiment, the present invention relates to an assay for identifying an inhibitor of calcific disease (CD), comprising the steps of: a) contacting Lp- PLA2 with a potential inhibitor thereof; and b) measuring Lp-PLA2 activity; whereby inhibition of Lp-PLA2 activity is an indication that said compound is a potential inhibitor of CD. Lp-PLA2 activity can be measured with assays that are well known in the art, for example, such as the ones disclosed in US7531316 (and references therein) and WO2010/0256919.

Agents that inhibit Lp-PLA2

[0086] In some embodiments, the present invention relates to the inhibition of Lp-PLA2. In some embodiments, inhibition is inhibition of nucleic acid transcripts encoding Lp-PLA2, for example inhibition of messenger RNA (mRNA). In alternative embodiments, inhibition of Lp-PLA2 is inhibition of the PLA2G7 gene expression and/or inhibition of activity of the gene product of PLA2G7, for example the polypeptide or protein of Lp-PLA2, or isoforms thereof. As used herein, the term "gene product" refers to RNA transcribed from a gene, or a polypeptide encoded by a gene or translated from RNA.

[0087] In some embodiments, inhibition of Lp-PLA2 is done by an agent. One can use any agent, for example but are not limited to nucleic acids, nucleic acid analogues, peptides, phage, phagemids, polypeptides, peptidomimetics, ribosomes, aptamers, antibodies, small or large organic or inorganic molecules, or any combination thereof. In some embodiments, agents useful in methods of the present invention include agents that function as inhibitors of Lp-PLA expression, for example inhibitors of mRNA encoding Lp- PLA2.

[0088] Agents useful in the methods as disclosed herein can also inhibit gene expression (i.e. suppress and/or repress the expression of the gene). Such agents are referred to in the art as "gene silencers" and are commonly known to those of ordinary skill in the art. Examples include, but are not limited to a nucleic acid sequence, for an RNA, DNA or nucleic acid analogue, and can be single or double stranded, and can be selected from a group comprising nucleic acid encoding a protein of interest, oligonucleotides, nucleic acids, nucleic acid analogues, for example but are not limited to peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acids (LNA) and derivatives thereof etc. Nucleic acid agents also include, for example, but are not limited to nucleic acid sequences encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (miRNA), antisense oligonucleotides.etc. [0089] As used herein, agents useful in the method as inhibitors of PLA2G7 expression and/or inhibition of Lp-PLA2 protein function can be any type of entity, for example but are not limited to chemicals, nucleic acid sequences, nucleic acid analogues, proteins, peptides or fragments thereof. In some embodiments, the agent is any chemical, entity or moiety, including without limitation, synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety. For example, in some embodiments, the chemical moiety is a pyrimidinone-based compound as disclosed in the art. [0090] In alternative embodiments, agents useful in the methods as disclosed herein are proteins and/or peptides or fragment thereof, which inhibit the gene expression of PLA2G7 or the function of the Lp-PLA2 protein. Such agents include, for example but are not limited to protein variants, mutated proteins, therapeutic proteins, truncated proteins and protein fragments. Protein agents can also be selected from a group comprising mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. [0091] Alternatively, agents useful in the methods as disclosed herein as inhibitors of Lp- PLA2 can be a chemical, small molecule, large molecule or entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having the chemical moieties as disclosed in the art. [0092] In accordance with a particular embodiment of the invention, the agent is a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof. Particularly, the nucleic acid agent is an RNAi agent. More particularly, the RNAi agent is a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof. [0093] In accordance with a particular embodiment of the invention further comprises the administration to the subject of an additional therapeutic agent against valvular or vascular calcification. Particularly, this additional therapeutic agent is an inhibitor of ENPP-1 or an agonist of P2Y₂ receptor.

Small molecules [0094] In some embodiments, agents that inhibit Lp-PLA2 are small molecules.

Irreversible or reversible inhibitors of Lp-PLA2 can be used in the methods and uses of the present invention.

[0095] Irreversible inhibitors of Lp-PLA2 are disclosed in patent applications WO

96/13484, W096/19451 , WO 97/02242, W097/217675, W097/217676, WO 97/41098, and WO97/41099 (SmithKline Beecham pic) which are specifically incorporated in their entirety herein by reference and disclose inter alia various series of 4- thionyl/sulfinyl/sulfonyl azetidinone compounds which are inhibitors of the enzyme Lp- PLA2. These are irreversible acylating inhibitors.

[0096] Lp-PLA2 inhibitors effective in humans are commonly known by persons of ordinary skill and include those undergoing evaluation, for example undergoing pre-clinical and clinical assessment including Phase II and Phase III clinical trials. A number of applications have been filed and published by SmithKlineBeecham (now

GlaxoSmithKline). A list of relevant published applications assigned to same is:

WO01/60805, WO02/30904, WO03/016287, WO00/66567, WO03/042218,

WO03/042206, WO03/042179, WO03/041712, WO03/086400, WO03/087088,

WO02/3091 1 , WO99/24420, WO00/66566, WO00/68208, WO00/10980, and

WO2005/021002, which are specifically incorporated in their entirety herein by reference.

[0097] Other Lp-PLA2 inhibitors useful in the methods as disclosed herein are described in published patent applications, for example WO2006/063791 , WO2006/06381 1 , WO2006/063812, WO2006/063813, and US2006/106017. Lp-PLA2 inhibitors also include known agents, for example but not limited to include the use of statins with Niacin (see www.genengnews.com/news/bnitem. aspx?name=6724568) and fenofibrate (see

www.genengnews.com/news/bnitem.aspx?name=14817756&taxid==19).

[0098] All of the applications set out in the above paragraphs are incorporated herein by reference. It is believed that any or all of the compounds disclosed in these documents are useful for prophylaxis or treatment of tissue calcification and disorders stemming therefrom, including, for example, but not limited to aortic stenosis, calcific valve aortic disease or hypertension related to decreased arterial compliance due to vascular calcification and/or structural valve degradation of bioprosthesis. [0099] In a particular embodiment, Lp-PLA2 inhibitors as disclosed in U.S. 6,649,619 and 7,153,861 (WO 01/60805) and U.S. 7,169,924 (WO 02/3091 1 ), are useful in the methods disclosed herein for the prophylaxis or for the treatment of vascular or valvular

calcification. In some embodiments, the Lp-PLA2 inhibitors as disclosed in U.S.

2005/0033052A1 and WO02/30904, WO03/042218, WO03/042206, WO03/042179 , WO03/041712, WO03/086400, and WO03/87088 and WO2008/140449 are reversible Lp- PLA2 inhibitors. [00100] In some embodiments, the small molecule is 1-(N-(2-(diethylamino)ethyl)-N- (4-(4-trittuoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one or SB480848 or a salt thereof. In a further embodiment, the small molecule is N-(2-diethylaminoethyl)-2-[2-(2-( 2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7- tetrahydro-cyclopentapyrimidin-1 -yl]-N-(4'-trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof. Alternatively, the small molecule is N-(1-(2-Methoxyethyl)piperidin-4-yl)-2- [2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-1-yl]-N-(4'-trifluoromethylbiphenyl-4- ylmethyl)acetamide; or a salt thereof. Still, particularly, the small molecule is methyl 2-[4- ({[2-[2-(2,3-difluorophenyl)ethyl]-4-oxopyrido[2,3-d]pyrimidin-1 (4H)-yl]acetyl}{[4'- (trifluoromethyl)-4-biphenylyl]methyl}amino)-1-piperidinyl]-2-methylpropanoate or a salt thereof. Most particularly, the small molecule is Darapladib (GlaxoSmithkline).

LP-PLA2 as a diagnostic or prognostic marker of CD

[00101] Measure of the PLA2G7 gene expression or of the Lp-PLA2 enzyme can be a useful markers for the diagnostic of disease or for assessing disease progression before surgery, or before and after start of therapy. Lp-PLA2 activity can be measured in a plurality of samples from mammals with assays that are well known in the art, for example, such as the ones disclosed in US7531316 (and references therein) and

WO2010/0256919.

[00102] In particularly, the invention provides that, when the subject is determined to be at risk of developing or progressing to the disease stage, he/she is prescribed an agent that inhibits Lp-PLA2 activity as defined herein.

Subjects

[00103] In accordance with a particular embodiment of the invention, the subject is a mammal. In particular, the subject is a human. Control levels

[00104] In order to determine whether the level of Lp-PLA2 referred to herein is greater than normal, the normal level (i.e. control) of the relevant control population or individual needs to be determined. The relevant control population or individual may be defined based on, for example, ethnic background, family history or risk background or any other characteristic that may affect normal levels of the marker. The relevant population or individual for establishing the normal level of Lp-PLA2 is preferably selected on the basis of absence of calcific heart valve disease.

[00105] Particularly, the control population or individual is selected from the group consisting of: an individual in a normal population devoid of CVD symptoms or a biological sample from the same subject but taken prior to developing symptoms of CVD or a biological sample from the same subject having developed CVD symptoms but taken earlier in time when the disease was less developed (i.e. less severe).

[00106] Once the normal level is determined (for a population or an individual), the measured level can be compared and the significance of the difference determined using standard statistical methods. If there is a statistically significant difference between the measured level and the normal level, then there is a significant risk that the individual from whom the levels have been measured will develop or has developed CAVD.

[00107] Particularly, there is a significant difference when the sample level is increased by at least about 10% compared to the control level, particularly at least about 15%, more particularly at least about 20%.

[00108] Of course, the present invention teaches that a certain level when higher than normal levels is indicative of calcific heart valve condition. It can be seen that the level of sensitivity and specificity can be altered by altering the control level. In some situations, e.g. when screening large numbers of subjects at low risk of CVD, it is important to have high specificity. In other situations, it may be important to have a balance between high sensitivity and specificity, e.g. when considering individual subjects at high risk of CVD, a balance between high sensitivity and specificity is needed.

[00109] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees

Centigrade, and pressure is at or near atmospheric. Examples

Example 1 - Elevated expression of Lp-PLA2 in calcific aortic valve disease:

implication for valve mineralization

Material and methods Patients

We examined 40 aortic valves that were explanted from patients at the time of aortic valve replacement for CAVD. Control non-calcified aortic valves (n=20) with normal

echocardiographic analyses were obtained during heart transplant procedures. Patients with a history of rheumatic disease, endocarditis, and inflammatory diseases were excluded. Valves with moderate to severe aortic valve regurgitation (grade >2) were excluded. Patients with reduced left ventricular ejection fraction (LVEF) (<40%) were also excluded in order to eliminate potential patients with low-flow, low-gradient AS in whom correlations with the gradient would not be reliable. All patients underwent a

comprehensive Doppler echocardiographic examination preoperatively. Doppler echocardiographic measurements were performed including the left ventricular stroke volume and transvalvular gradients using the modified Bernoulli equation. The protocol was approved by the local ethical committee and informed consent was obtained from the subjects.

Procurement of tissues for analyses and cell culture

[00110] CAVD valves and control, non-calcified valves were obtained respectively from aortic valve replacements and heart transplantation procedures. VICs were isolated from control, non-calcified aortic valves. In each patient, overnight fasting plasma was collected for laboratory measurements of lipids and glucose. For all patients included in this study, a transthoracic Doppler-echocardiography was performed preoperatively. The hemodynamic severity of the stenosis was assessed by the measurements of peak transvalvular gradients. The protocol was approved by the local ethical committee and informed consent has been obtained from the subjects.

Valve interstitial cells isolation and in vitro analyses of calcification

[00111] Human valve interstitial cells (VICs) were isolated by collagenase digestion. To provoke calcification, cells were incubated for 7 days with a pro-calcifying medium containing: DMEM + 5% FBS, 10^"7 M insulin, 50 pg /ml ascorbic acid and NaH₂P0₄ at 2 mM. In some experiments lysophosphatidylcholine (LPC) (1 nM) was added to the medium.

Microarray

[00112] A total of five CAVD valves and five normal aortic valves were selected for microarray analysis. Expression studies were performed using the human U133 plus 2.0 Affymetrix GeneChip microarrays (Affymetrix, Santa Clara, CA, USA). Arrays were processed using a standard Affymetrix double amplification protocol using 80 ng of RNA. Expression values were extracted using the Robust Multichip Average (RMA) method. Quality control assessment was performed with the FlexArray software version RC3 and the affy package that is part of the Bioconductor project (www.bioconductor.org/). Two microarrays interrogating control valves failed quality control and were discarded from the analysis. The Significant Analysis of Microarrays (SAM) method was used to claim significant regulation. The minimum fold change and the delta values was set to 2 and 0.76, respectively, in order to reach a false discovery rate below 5% (4.85%). [00113] The microarray dataset can be found in the National Center for

Biotechnology Information's Gene Expression Omnibus (GEO) repository

(www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE12644.

Real-time PCR

[00114] RNA was extracted from valves explanted from patients. RNA was also extracted from cells during in vitro experiments. Total RNA was isolated with RNeasy micro kit from Qiagen (Qiagen, Mississauga, ON, Canada). The RNA extraction protocol was performed according to manufacturer's instructions using 100 mg of tissue. The quality of total RNA was monitored by capillary electrophoresis (Experion, Biorad, Mississauga, ON, Canada). Four (4) μg of RNA was reverse transcribed using the

Quantitec Reverse Transcription Kit from Qiagen. Quantitative real-time PCR (q-PCR) was performed with Quantitec SYBR Green PCR kit from Qiagen on the Rotor-Gene 6000 system (Corbett Robotics Inc, San Francisco, CA, USA). Primers for the following transcripts were obtained from Invitrogen (Burlington, ON, Canada): PLA2G7, Runx2, osteopontin, ALP, BMP2. The expression of the cyclophilinA (CycloA) gene (Invitrogen, Burlington, ON, Canada) was used as a reference gene to normalize the results. Determination of calcium concentrations

[00115] A segment of valve tissue was kept in liquid nitrogen until determination of the calcium content. Cusps were cut in small pieces and treated with HCI 6N at 90°C during 24 hours. Treated tissues were then centrifuged at 4400 RPM during 30 min and supernatants were collected. Calcium content was determined by the Arsenazo III method (Synermed, Monterey Park, CA, USA), which relies on the specific reaction of Arsenazo III with calcium to produce a blue complex. Results are measured at 650 nm on the the Modular P800 Elecsys of Roche Diagnostics apparatus (Roche Diagnostics, Laval, Que, Canada. This reaction is specific for calcium. Magnesium is prevented from forming a complex with the reactive. Results were expressed as mg of calcium per wet weight of tissue (Ca mg/g ww). In isolated cells calcium was also measured by the Arsenazo III method and results reported as percent changes.

Determination of alkaline phosphatase activity in isolated cells

[00116] VICs were washed with PBS, transferred in 200 μΙ of 0.2% NP-40

0.2%,1 mM MgCI₂ and then sonicated. Alkaline phosphatase (ALP) activity was assayed using p-nitrophenyl phosphate as substrate (PNPP) (Sigma, Oakville, Ont., Canada). Samples were incubated in presence of substrate for 30 min at 37°C. The alkaline phosphatase activity was then measured by absorbance reading at 410nm. The assay was carried out in triplicate. Results were normalized to protein content. Enzyme activity of PLA2G 7/LP-PLA2

[00117] Enzyme activity of PLA2G7/LP-PLA2 was measured in aortic valves and blood plasma with the PAF acetylhydrolase assay kit following instructions of the manufacturer (Cayman Chemical, Burlington, Ont., Canada).

Statistical analyses

[00118] For the comparisons of groups with regards to q-PCR analyses, enzyme activity, and the amount of valvular calcium, the results were expressed as means ±SEM. For continuous data, values were compared between groups with Student t-test or ANOVA when two or more than two groups were compared, respectively. Post hoc Tukey analyses were done when the p value of the ANOVA was <0.05. Correlations between variables were determined using Spearman's coefficients. A p value <0.05 was considered as statistically significant. Statistical analysis was performed with a commercially available software package JMP IN 8.1.

Results

Transcriptomic of PLA2 genes Transcriptomic of phospholipase A2 genes in calcific aortic valves

[00119] A tissue-based microarray experiment was conducted to explore the gene expression pattern of phospholipase A2 (PLA2/PLA2G) family of genes. Two control valves failed quality control and were discarded from subsequent analyses. Figure 1A shows a heat map of normalized expression values for PLA2/PLA2G genes for each sample. A total of 15 probe sets were available to study the expression of PLA2G3, PLA2G4A, PLA2G5, PLA2G6, PLA2G7, PLA2G10, PLA2G12A, and PLA2G12B genes. The expression of PLA2G4A and PLA2G5 were significantly down-regulated in stenotic valves. In contrast, the expression of PLA2G7 (Lp-PLA2) was increased by 4.2-folds in stenotic aortic valves (Figure 1 A). PLA2G4A and PLA2G5 are respectively cytosolic and secreted enzymes that hydrolyze membrane phospholipids. On the other hand, Lp-PLA2 (PLA2G7) is a cellular enzyme that is also secreted, which can metabolize ox-LDL into lysophosphatidylcholine (LPC), a powerful inflammatory metabolite. The result of the microarray experiment was then confirmed with q-PC analyses in a larger group of patients (Table 1). Compared to control non-calcified aortic valves the amount of Lp-PLA2 transcript was increased in stenotic aortic valves by 2.3-folds (Figure 1 B). Considering the age differences between patients with CAVD and controls we have repeated the analyses after matching 20 patients for age (±1 year) (Table 2). In this analysis, we found in age- matched groups that patients with CAVD had a 3.5-folds increase in mRNA encoding for Lp-PLA2 when compared to control subjects (control: 15.3±3.9 copies/CycloA vs. CAVD: 53.6±17.0; p=0.03). Similarly, the activity of the Lp-PLA2 enzyme was increased by 1.9- folds in stenotic aortic valve tissues compared to control non-mineralized valves (Figure 1 C). Within the blood plasma, the activity of Lp-PLA2 was significantly elevated in CAVD patients when compared to control (Figure 1 D). In the age-matched subjects the plasma Lp-PLA2 activity was also significantly elevated in patients with CAVD (control: 25.7±1.6 nmol/min/ml vs. CAVD 33.7±1.6 nmol/min/ml; p=0.001). However, enzyme activity in aortic valve and blood plasma did not correlate significantly (r=-0.10; p=0.75). We then documented by thin layer chromatography that LPC, the end-product of Lp-PLA2 activity, is present in stenotic valves (mean concentration: 177±23 ng/g of tissue) (Figure 1 E).

Table 1. Clinical characteristics of patients

Control valves CAVD p-value

Age 51 ± 3 70 ± 1 < 0.0001

Male (%) 78 56 NS

Smoking (%) 11 7 NS

Hypertension (%) 33 67 0.04

Diabetes (%) 11 26 NS

Coronary heart disease (%) 64 49 0.01

Bicuspid aortic valves (%) 0 30 < 0.0001

BMI (kg/m²) 27.9 ± 1.5 27.6 1 0.5 NS

Statins (%) 73 61 NS

Ang II receptor blockers (%) 11 16 NS

Aortic valve area (cm ) 0.77+0.03

Aortic mean gradient(mmHg) 43 1 2 -

Triglycerides (mmol/L) 1.39 ± 0.15 1.40 1 0.08 NS

LDL (mmol/L) 2.42 + 0.28 2.26 1 0.12 NS

HDL (mmol/L) 1.66 ± 0.37 1.33 1 0.04 NS

Creatinin (mmol/L) 92.2 1 9.5 92.2 ± 3.42 NS

Creatinin clearance(ml/min) 88.7 ± 9.05 63.1 1 2.8 0.01

BMI: body mass index; LDL: Low-density lipoprotein; HDL: High-density lipoprotein

Table 2. Clinical characteristics of age-matched patients

Control valves CAVD p-value

Age 52 + 2 53 1 3 NS

Male (%) 70 70 NS

Smoking (%) 10 20 NS

Hypertension (%) 30 40 NS

Diabetes (%) 10 0 NS

Coronary heart disease (%) 70 20 0.02

Bicuspid aortic valves (%) 0 70 < 0.0001 B I (kg/m ) 27.2 ± 1.5 25.8 + 0.9 NS

Statins (%) 80 30 0.07

Ang II receptor blockers (%) 10 20 NS

2

Aortic valve area (cm ) -- 0.80 ± 0.10 -

Aortic mean gradient(mmHg) - 46 ± 3

Triglycerides (mmol/L) 1.32 ± 0.16 1.60 + 0.27 NS

LDL (mmol/L) 2.36 ± 0.25 2.42 ± 0.29 NS

HDL (mmol/L) 1.55 + 0.34 1.31 ± 0.13 NS

Creatinin (mmol/L) 90.3 ± 8.7 84.4 ± 7.6 NS

Creatinin clearance(ml/min) 88.8 ± 8.0 87.5 ± 8.4 NS

BMI: body mass index; LDL: Low-density lipoprotein; HDL: High-density lipoprotein

Relationships between the expression of Lp-PLA2 and oxidized-LDL

[00120] Immunohistochemistry studies confirmed a higher expression of Lp-PLA2 within stenotic valves (Figures 2A and B). We found that immunostaining of Lp-PLA2 in control non-mineralized aortic valve was faint or non-detectable (Figure 2A), whereas there was a strong staining in stenotic aortic valves (Figures 2B and C). Two distinct patterns of expression were observed for Lp-PLA2 in stenotic valves. First, Lp-PLA2 was expressed in cellular-rich inflammatory infiltrates, which are abundant in areas of tissue remodelling and mineralization (Figure 2B). Second, Lp-PLA2 expression appeared as a diffuse immunostaining (Figure 2C), which co-localized with ox-LDL (Figure 2D). Ox-LDL is a powerful promoter of Lp-PLA2 expression as well as a substrate for the enzyme. We then measured the blood profile of patients and correlated the results with the amount of Lp-PLA2 transcript within the aortic valve. Among the different blood lipid variables, the number of Lp-PLA2 transcripts within stenotic aortic valves significantly correlated with the blood plasma level of ox-LDL (r=0.33; p=0.01 ) and LDL levels (r=0.29;p=0.02) (Table 3).

Table 3. Correlation matrix between the number of Lp-PLA2 transcripts and clinical data

Variables R P

Age -0.22 0.09

Cholesterol 0.26 0.04

LDL 0.29 0.02

HDL 0.03 0.79 Triglycerides 0.04 0.74

ox-LDL 0.33 0.01

LDL: low-density lipoprotein; HDL: high-density lipoprotein

Expression of Lp-PLA2 within the aortic valve is related to tissue remodelling and the hemodynamic severity of aortic stenosis

[00121] We next documented the relationships between Lp-PLA2 and indices of CAVD disease activity and severity. The number of Lp-PLA2 transcripts increased with the severity of the remodelling score of stenotic aortic valves (pANOVA=0.002) (Figure 3A). Also, the number of Lp-PLA2 transcript correlated with the weight of the aortic valve, a marker of active remodelling that is also related to the severity of aortic stenosis (r=0.56; p=0.0009) (Figure 3B). In addition, the level of Lp-PLA2 transcript also correlated with the peak transaortic gradient (r=0.56; p=0.0004) (Figure 3C). In the same line, the level of mRNA transcript encoding Lp-PLA2 was significantly correlated with the amount of calcium measured within CAVD valves (r=0.27; p=0.03). However, the enzymatic activity of Lp-PLA2 in CAVD tissues had a better correlation with the concentration of calcium (r=0.56, p=0.01 ) (Figure 3D). Lysophosphatidylcholine induces mineralization of the aortic valve trough a PKA pathway

[00122] Lysophosphatidylcholine (LPC) is one of the main products derived from ox- LDL following the action of Lp-PLA2 and present within CAVD tissues. We thus next tested the effect of LPC on the mineralization of VIC cultures. We found that small amount of LPC (1 nM) increased mineralization of VIC cultures treated with the mineralizing medium (P0₄) (Figure 4A). LPC is normally bound to lipoproteins and its content is elevated in ox-LDL. (7) Furthermore, it has been shown that the high content of LPC in ox- LDL is largely dependent on Lp-PLA2 activity.(8) We thus treated VICs with ox-LDL. In this experiment, similarly to LPC, ox-LDL increased mineralization of VICs treated with the mineralizing medium by 2.6-folds (Figure 4B). In isolated VICs, LPC elevated the expression of ENPP1 , ALP, the phosphate transporter Pit-1 (SLC20A1) and osteopontin (OPN) (Figure 4C). The enzyme activity of ALP and ENPP1 were also increased following a treatment of VIC cultures with LPC (1nM) (Figures 4D and E). We have previously shown that a high expression of the ectonucleotidase enzymes contribute to the mineralization of VIC cultures by elevating phosphate levels, whereby apoptosis-mediated mineralization is promoted. We then treated VIC cultures with ARL67156, an

ectonucleotidase inhibitor. It is worth to highlight that ARL67156 is an inhibitor of ENPP1 , ENTPD1 and ENTPD3 and that only ENPP1 is expressed significantly by human VICs.(9) In this experiment, ARL67156 prevented the mineralization induced by the mineralizing medium and LPC (Figure 4F), suggesting that LPC-mediated mineralization relies on the expression of ENPP1 , which, in turn, promotes mineralization. We then hypothesized that apoptosis might be implicated in LPC-induced mineralization. First, by using the detection of activated caspase 3/7 assay we found that LPC induced the activation of the effector caspases (Figure 5A). Also, on exposure to LPC, there was also a loss of the

mitochondrial membrane potential (ΔΨηι) in VIC culture, indicating that mitochondrial- dependent pathway is involved in LPC-mediated apoptosis (Figures 5B and C).

Correspondingly, we found that LPC promoted cytochrome c release within the cytosol of VICs (Figure 5D). To further document the role of apoptosis in LPC-mediated

mineralization of VICs, we have used the general caspase inhibitor ZVAD-fmk. ZVAD-fmk abolished the mineralization of VIC cultures induced by LPC and the calcifying medium (Figure 6A). Expression of ectonucleotidases and mineralization of vascular smooth muscle cells has been previously shown to rely on the activation of PKA pathway.(10) Furthermore, studies have highlighted the role of PKA in apoptosis. (11 ) We thus hypothesized that PKA could mediate LPC-induced mineralization of VIC cultures. In this regard, inhibition of PKA with PKA inhibitor fragment (6-22) amide prevented

mineralization of VIC cultures induced by the mineralizing medium and LPC (Figure 6B), suggesting that cAMP-dependent activation of PKA is involved in LPC-mediated mineralization. LPC is one potent agonist of the G protein-coupled receptor G2A, which may signal through PKA.(12) We have thus measured PKA activity in VICs and found that LPC increased PKA activity by 21 %. Furthermore, this effect with LPC was synergistic with the mineralizing medium increasing PKA activity by 40% (Figure 6C). To further prove the implication of the PKA pathway in the mineralization of VIC cultures, we have used forskolin, an agent that increase adenylate cyclase activity and augment cell content in cAMP. To this effect, stimulation of cAMP production with forskolin treatment increased mineralization of VIC cultures induced by mineralizing medium and LPC (Figure 6D). We next measured the level of cAMP in control and stenotic aortic valves to document the activation of the cAMP pathway in vivo. In control non-mineralized aortic valve the amount of cAMP was very low and beyond detection level in 5 samples, whereas it was detected in 4 stenotic aortic valves from 5 patients (mean value: 16.38±8.75 pmol/mg protein), indicating activation of the cAMP/PKA pathway in stenotic aortic valve tissues. Discussion

[00123] A recent report has documented that the mass of Lp-PLA2 within the blood plasma is elevated in patients with CAVD. (13) In the present study we identified for the first time that Lp-PLA2 is highly expressed within stenotic aortic valve tissues and that it contributes to valve mineralization by the production of LPC. We next showed that LPC- mediated mineralization of human VICs relies on ectonucleotidase enzyme and apoptosis through a P A pathway.

Origin of Lp-PLA2 in CAVD

[00124] Lp-PLA2 is significantly expressed by platelets and macrophages. A significant fraction of Lp-PLA2 is bound to lipoproteins in the bloodstream. (14) In this work we documented that transcripts encoding for Lp-PLA2 were highly expressed in CAVD valves, indicating that the enzyme is locally synthesized. This is in line with the immunohistochemistry studies, which showed the expression of Lp-PLA2 by inflammatory cells. However, it should be pointed out that immunohistochemistry studies also showed a diffuse staining of Lp-PLA2 in CAVD tissues, suggesting that the enzyme is locally secreted and released by macrophages and/or transported by the lipoproteins within stenotic aortic valves.

[00125] We previously showed that plasma level of ox-LDL is associated with the remodelling of stenotic aortic valves. (15) In addition, we also documented that the proportion of circulating small, dense, LDL is related to the amount of ox-LDL within CAVD tissues. (16) Of interest, studies found that a higher proportion of Lp-PLA2 is associated with small, dense LDL.(17) Hence, it is possible that small, dense LDLs, which have a higher oxidation rate, promote the accumulation ox-LDL, which in turn promotes the expression of Lp-PLA2 within the aortic valve during CAVD. (18) Also, it is worth to underline that oxidized phospholipids are transported and sequestered in the blood plasma by Lp(a).(14) To this effect, a recent work has highlighted that gene polymorphism of Lp(a) is significantly associated with CAVD at the genome-wide level. (19)

Lp-PLA2-derived lysophosphatidylcholine and mineralization of the aortic valve

[00126] In this study, the transcripts levels and enzyme activity of Lp-PLA2 correlated with several indices of disease activity. More specifically, the number transcripts were significantly correlated with the remodelling score and the weight of the aortic valve. It is worth noting that the weight of the aortic valve, a simple and convenient

measurement, is an independent predictor of the hemodynamic severity of CAVD.(20, 21 ) Mineralization of the aortic valve is certainly an important culprit, which is related with the severity of CAVD.(22) To this effect we identified that both the number of transcript encoding for Lp-PLA2 and enzyme specific activity correlated with the amount of calcium within CAVD. It is worth noting that Lp-PLA2 activity better correlated (r=0.56) with the valvular concentration of calcium than the number of transcript (r=0.27). This suggest that tissue enzyme activity, which may take into account several factors, such as the total amount of Lp-PLA2 and differences in enzyme activity related to the genotype, is a better correlate of pathological mineralization of the aortic valve.

[00127] The hydrolysis of oxidized phospholipids generates LPC, a powerful proinflammatory and atherogenic compound. (23) A recent study using VSMCs found that LPC induce the expression of ALP and mineralization of cell cultures. (24) In the present study, we documented that LPC-induced mineralization relied on the expression of phosphate-related genes including ENPP1 , ALP, Pit-1 and osteopontin. Whereas ENPP1 and ALP uses nucleotides to generate PPi and Pi, Pit-1 is a cell membrane pi-transporter that plays an important role in the mineralization of the aortic valve. (25). Expression of ectonucleotidase genes, Pit-1 and osteopontin is highly regulated by the concentration of extra-cellular phosphate. (26) In the present study we found that ARL67156, which inhibits ENPP1 , abrogated LPC-induced mineralization. It is worth to highlight that a complete knockdown of ENPP1 is associated with ectopic mineralization by lowering the mineralization inhibitor PPi to exceptional low levels. (27) For instance, the tiptoe walking mice (ttw) with invalidation of the ENPP1 gene develop extensive mineralization of ligament. (28) On the other hand, overexpression of ENPP1 , such as during pathologic mineralization, is promoting calcification by several mechanisms. (29) In this regard, when highly expressed ENPP1 contributes to elevate the Pi/PPi ratio whereby mineralization is promoted. (9) In addition, it should be pointed out that ALP activity was increased following LPC treatment. ALP is potent enzyme that transforms PPi into Pi. (30) Hence, it is possible that ENPP1 and ALP works in tandem and generate important amount of Pi and, in doing so, promote LPC-mediated mineralization.

[00128] Phosphate is a strong promoter of mineralization through programmed-cell death. (31) It is worth noting that LPC induced mitochondrial-dependent apoptosis of VIC cultures. Hence, the present findings suggest that LPC induced the expression of phosphate-related genes, such as ENPP1 , ALP and Pit-1 , which, in turn, promoted apoptosis-mediated mineralization of VIC cultures. Phosphate-induced expression of ENPP1 and ALP as well as mineralization of VSMCs has been shown to be dependent on the PKA pathway. (10) In the present work, we documented that LPC-induced

mineralization was abrogated by PKA inhibition, whereas a treatment with forskolin, an activator of adenylate cyclase, exacerbated the process. Furthermore, LPC promoted a strong increase in PKA activity in VICs. These facts suggest that LPC-induced the expression of phosphate-generating enzymes and promoted mineralization through a PKA dependent pathway. Clinical implications

[00129] There is actually no medical treatment to prevent the progression of CAVD. Statins have been used in randomized trials and have been shown to be inefficient to prevent the progression of aortic stenosis.(32) Although statins reduce the level of circulating Lp-PLA2 by decreasing LDL concentration they do not prevent the de novo synthesis and secretion by macrophages. (33) Hence, it is likely that statins although reducing blood plasma level of LDL do not impact local secretion of Lp-PLA2 within the aortic valve. Accordingly, we documented in this study that Lp-PLA2 activity was elevated in stenotic aortic valves and that tissue activity did not correlate with blood plasma enzyme activity. These findings are in accordance with a previous study, which shows that enzyme activity of Lp-PLA2 in carotid endarterectomy specimen was not associated with blood plasma activity.(34) More recently, a human study showed that Lp-PLA2 mass in the blood plasma is not increased following an inflammatory stimulus. Instead Lp-PLA2 secretion is increased by several-folds in macrophages exposed to a lipid load. Taken together these findings suggest that Lp-PLA2 is preferentially secreted by tissue macrophages rather than by circulating leukocytes. (35) Also, it should be pointed out that the pro-inflammatory effect of Lp-PLA2 is controversial. To this effect, by using platelet- activating factor (PAF) and ox-LDL as substrates Lp-PLA2 may lower oxidative stress. (36) But on the other hand, it generates lysophospholipids, which have pro-inflammatory activity and, as shown in the present study, promote mineralization of VICs. [00130] Lp-PLA2 is considered as a potential target in the treatment of

atherosclerosis. Darapladib is a potent inhibitor of Lp-PLA2 under investigation in patients with coronary artery disease and has been shown in a swine model to decrease atherosclerotic plaque volume and to decrease the lipid core content.(37) It is worth to mention that although CAVD shares some features with atherosclerosis, such as lipid deposition and inflammation, it is a distinct pathobiological process. Hence findings in atherosclerosis cannot be transposed directly to CAVD before proper investigations have been performed.

[00131] We found a high level of PLA2G7 within the aortic valve as well as in blood plasma of patients with CAVD, suggesting that, in vivo, expression and enzymatic activity of PLA2G7 play an important role in CAVD. Also, considering that PLA2G7 activity was elevated in the blood plasma of patients with CAVD, it could represent an interesting biomarker. Hence, PLA2G7 mass and activity within the blood plasma could help to detect patients with CAVD as well as their disease activity.

[00132] From the above discussion it thus appears that PLA2G7 must be considered as a novel potential target in the treatment of CAVD. We should point out that inhibitors of PLA2G7, that are already in use and/or that could be produced in the future, could potentially be used in the treatment of CAVD. In this regard, inhibitors of PLA2G7 such as Darapladib (GlaxoSmithkline) is a potential candidate in the treatment of CAVD. CAVD thus could represent a novel indication for inhibitors of PLA2G7, including

Darapladib (GlaxoSmithkline).

Limitations

[00133] Sofar, we have examined CAVD with advanced pathological mineralization. Nevertheless, the present study may also possibly be transposed to nascent disease process since it documented several points suggesting that Lp-PLA2 is involved during the development of CAVD.

Conclusion [00134] Lp-PLA2 is highly expressed in human CAVD. Several lines of evidence documented in this work suggest that Lp-PLA2 activity may contribute to the pathological mineralization of the aortic valve. CAVD is a highly prevalent condition and there is, so far, no medical treatment to prevent its progression. The present study gives impetus to realize further study in order to support Lp-PLA2 as a novel therapeutic target for CAVD. [00135] In conclusion, PLA2G7/Lp-PLA2 should be considered as a novel pharmacological target and biomarker of CAVD. Hence, pharmacological inhibition of PLA2G7/Lp-PLA2 in CAVD represents a potential novel indication for this class of medication. Example 2 - Elevated plasma activity of Lp-PLA2 is associated with structural valve degeneration of bioprostheses

Methods Study patients

[00136] From June 2008 to June 2010, 203 consecutive patients with a BP in the aortic position were prospectively recruited for this study that included a Doppler- echocardiography, and a blood sample. The protocol was approved by the local ethical committee and informed consent was obtained from the subjects. The baseline characteristics of this cohort were previously reported in (9). For this study, 6 patients were not having enough blood plasma for the measurement of Lp-PLA2 mass and activity and therefore were excluded leaving 197 patients for the analyses.

[00137] All patients underwent isolated aortic valve replacement procedure at the Quebec Heart & Lung Institute at least 3 years ago with a complete Doppler

echocardiographic examination available at 12±6 months postoperatively. Exclusion criteria were as follows: (1 ) Presence of > mild paravalvular regurgitation; (2) Significant concomitant mitral valve disease, defined by > mild mitral regurgitation or mitral valve effective orifice area (EOA) < 1.5 cm²; (3) Subvalvular flow acceleration precluding measurement of BP valve EOA; (4) Left ventricular (LV) systolic dysfunction defined by a LV ejection fraction < 50%; (5) Congestive heart failure with New York Heart Association Class III or IV. All patients recruited in the study had a clinical examination, a complete plasma glycemic and lipid profile, and a complete Doppler echocardiographic study.

Echocardiographic measurements

[00138] All Doppler echocardiographic studies were reviewed by the same cardiologist (H.M.). Operators were blinded to results of clinical and laboratory data. Peak transprosthetic flow velocity was determined by continuous-wave Doppler. Mean transprosthetic gradient was calculated using the modified Bernoulli equation.

Bioprosthetic valve effective orifice area (EOA) was calculated using the standard continuity equation. The absolute and annualized changes in mean gradient and EOA were calculated as follows:

Absolute change = (value at last follow-up echo - value at 1-year postop echo)

Annualized change = (value at last follow-up echo - value at 1-year postop echo) / time between 1 year and last follow-up echocardiographic exams

[00139] Prosthetic regurgitation was detected by color Doppler echocardiography and the origin of the jet was visualized in several views to differentiate periprosthetic from transprosthetic regurgitation. Transprosthetic regurgitation severity was assessed as recommended by the American Society of Echocardiography and classified as mild, moderate or severe. (38) Worsening of valve regurgitation was defined as an increase of at least 1/3 class in the severity of regurgitation during follow-up according to the following scheme: from none or mild to moderate or from moderate to severe.

SVD was defined as an increase in transprosthetic mean gradient≥ 10 mmHg and/or worsening of transprosthetic regurgitation ≥1/3 class between 1-year and last follow-up echocardiograms.

Clinical and Operative Data

[00140] Previous and current medical history included history of smoking, documented diagnoses of hypertension (patients receiving antihypertensive medications or having known but untreated hypertension [blood pressure > 140/90 mmHg]), diabetes (fasting glucose≥ 7 mmol/l), hypercholesterolemia (patients receiving cholesterol-lowering medication or, in the absence of such medication, having a total plasma cholesterol level > 240 mg/l) coronary heart disease (history of myocardial infarction or coronary artery stenosis on coronary angiography), renal insufficiency (estimated glomerular filtration rate < 60 ml/min/1.73 m²), and detailed information on current medication were collected. Body weight, height and waist circumference were measured following standardized

procedures. Blood pressure, heart rate and NYHA class were also assessed. The clinical identification of patients with the features of the metabolic syndrome was based on the modified criteria proposed by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III). Operative data including model and size of bioprosthetic valves were also recorded. Laboratory Data

[00141] Overnight fasting plasma was collected and immediately processed by the laboratory for the measurement of glucose, total cholesterol, low density-cholesterol (LDL), high density-cholesterol (HDL), and triglyceride (TG) levels. Plasma ox-LDL was measured by sandwich ELISA with the monoclonal antibody 4E6 (Mercodia, Uppsala, Sweden) directed against the modified apoB-100 of ox-LDL. The test was conducted according to the manufacturer instructions and optical density was read at 450 nm.

Results were expressed as units per liter (U/l). Blood plasma Lp-PLA2 activity was measured by a colorimetric activity method (Cayman). The level of Lp-PLA2 activity in nmol/min/ml was calculated from the absorption curve (410 nm). The assay was carried out in duplicate. Plasma Lp-PLA2 mass was determined by ELISA kit R&D systems according to manufacturer instruction. (Minneapolis, MN, USA).

Immunostaining and histologic analyses

[00142] Bioprosthetic valves (n=39) were obtained from patients undergoing reoperation for SVD. Samples were taken at the time of surgery. Cusps were embedded in optimum cutting temperature (OCT) compound (TissueTek, Miles Laboratories,

Elkhart, Ind, USA) and frozen in liquid nitrogen for immunohistological analysis.

Immunostaining analyses were performed with the following antibodies: Lp-PLA2 (Abgent, San Diego, CA, USA), ox-LDL (Accurate chemical, Hornby, ON, Canada) and CD68 (macrophages) (Cedarlane, Hornby, Ont, Can). Slides were then incubated with EnVision Dual Link System-HRP, followed by AEC substrate (Dako, Carpinteria, CA, USA). Non- immunized mouse serum was used as a negative control in all immunohistology experiments. Using a microscope at 10X magnification, the total area of each valve was determined with Image Pro Plus Version 6.1 image analysis software. Using a 40X magnification, amount of stainings, represented by red pixels, was measured on the entire valve section. Then, the percentage of pixels on the total area of the valves was calculated.

Statistical Analyses

[00143] Results are expressed as mean ± SEM and compared using unpaired Student's test. Categorical data were expressed as a percentage and compared with the χ ² test. Correlations between variables were determined using linear regressions or Spearman's coefficients. A multiple logistic regression analysis was used to identify the factors independently associated with bioprosthetic valve SVD. Multiple linear regression analyses were used to identify the factors that are independently associated with Lp-PLA2 mass. Variables with p values < 0.1 on univariate analysis were entered into the multivariate models. Age at implantation was forced into the models. A p value < 0.05 was considered as statistically significant.

Results

Clinical data and characteristics of patients with SVD

[00144] In this study, 197 patients were enrolled and Lp-PLA2 mass and activity were measured. Table 4 presents the clinical data of patients with and without SVD. Forty one patients (21%) had echocardiographic evidence of SVD. Patients with SVD had a longer follow-up time (9.1 ±0.7 vs. 7.6±0.2 yrs; p=0.01 ). When compared to patients without SVD, those with SVD had a similar proportion of risk factors and medication. However, in patients with SVD there was a tendency for having a lower BMI (p=0.1 ) and lower proportion of patients with diabetes (p=0.1 ). The use of statins (p=0.1 ), angiotensin converting enzyme inhibitors (ACEi) (p=0.4) and angiotensin receptor blockers (ARBs) (p=0.7) were similar among the two groups.

Table 4. Clinical characteristics of patients used for SVD

Variables All patients No SVD SVD p-value*

(n=197) (n=156) <n=41)

Age at implantation, yrs 67 ± 1 68 ± 1 66 ± 1 0.3

Male 138 (70) 110 (70) 28 (68) 0.7

BMI, kg/m² 28 ± 0.4 28 ± 0.4 27 ± 0.7 0.1

Follow-up time, yrs 7.9 + 0.2 7.6 + 0.2 9.1 + 0.7 0.01

Risk factors

Hypertension 139 (70) 113 (72) 26 (63) 0.2

Diabetes 42 (21 ) 37 (23) 5 (12) 0.1

Obesity 52 (26) 42 (27) 10 (24) 0.7

History of hypercholesterolemia 153 (77) 124 (79) 29 (70) 0.2

Metabolic syndrome 85 (43) 64 (41 ) 21 (51 ) 0.2

History of smoking 121 (61 ) 97 (62) 24 (58) 0.6

Coronary artery disease 94 (48) 73 (47) 21 (51 ) 0.6

Medications Statin 153 (77) 125 (80) 28 (68) 0.1

ACEi 63 (33) 52 (34) 11 (28) 0.4

ARB 53 (27) 41 (27) 12 (30) 0.7

Operative data

Stented bioprosthesis 141 (71 ) 11 1 (71 ) 30 (73) 0.7

Porcine bioprosthesis 118 (60) 92 (59) 26 (63) 0.6

Bioprosthesis size, mm 23.9 + 0.16 24.0 ± 0.2 23.7 ± 0.3 0.5

PPM 20 (10) 16 (10) 4 (10) 0.9

CABG 78 (39) 57 (36) 21 (51 ) 0.08

Mean gradient

Early post-operative, mm Hg 11.8 1 0.4 11.9 ± 0.4 11.6 ± 0.9 0.7

Last follow-up, mm Hg 14.9 + 0.5 12.5 ± 0.4 24 ± 1.1 <0.0001

Progression, mm Hg 3.1 ± 0.5 0.7 ± 0.3 12.5 ± 0.9 <0.0001

Progression rate, mm Hg/yr 0.47 ± 0.08 0.09 ± 0.05 1.9 + 0.25 <0.0001

LVEF, %

Early post-operative 63 ± 0.6 63 ± 0.7 63 ± 1.5 0.9

Last follow-up 63 ± 0.6 63 ± 0.7 64 + 1.4 0.8

SVD: structural valve degeneration; ACEi: angiotensin-converting enzyme inhibitors; ARB: angiotensin receptor blockers; PPM: patient-prosthesis mismatch; CABG: coronary artery bypass graft; LVEF: left ventricular ejection fraction; ^*p value refers to the comparison between SVD and No SVD groups; Values are expressed as means ± SEM or n (%).

[00145] Doppler echocardiographic analyses indicate that early postoperative transprosthetic mean gradient were similar among patients with or without SVD (p=0.7) (Table 4). During the follow-up patients with SVD increased significantly the mean transprosthetic gradient (Table 4). Aortic regurgitation (AR) was absent or mild in every patient after discharge following AVR with a BP. At the follow-up, intraprosthetic AR was documented as a cause of failure in 8 (20 %) patients with SVD. The development of SVD was not associated with global LV dysfunction; mean LVEF were similar (p=0.8) in patients with and without SVD.

Blood lipid profile and predictors of SVD

[00146] On univariate analysis, we documented that SVD was associated with LDL cholesterol (p=0.02), ox-LDL (p=0.02), apoB/ApoA-l (p=0.007), Lp-PLA2 mass (p=0.03) and activity (p=0.005) (Table 5). In a first multivariable model including both Lp-PLA2 mass and activity we found that only Lp-PLA2 activity (OR: 1.10, 95%CI: 1.01 -1.22; p=0.04 was significantly associated with SVD. In a second model after adjustment for age at implantation, foliow-up time, LDL, ox-LDL and ApoB/ApoA-l ratio entered as quartiles, Lp-PLA2 activity (OR: 1.09, 95%CI: 1.01-1.18; p=0.03) remained associated with SVD. Also, when statin was forced into the multivariate model, it did not reach statistical significance (OR: 1 .01 , 95%CI: 0.36-2.66; p=0.93), whereas blood plasma Lp-PLA2 activity (OR1.08, 95%CI: 1.01 -1.18; p=0.02) remained significantly associated with SVD.

Table 5. Laboratory data.

variables All patients No SVD SVD p-value*

(n=197) (n=156) (n=41)

Glycemia, mmol/l 5.8 ± 0.1 5.7 ± 0.1 6 ± 0.2 0.1

Triglycerides, mmol/l 1.43 ± 0.05 1.4 ± 0.06 1.5 ± 0.12 0.2

HDL cholesterol, mmol/l 1.35 ± 0.02 1.35 ± 0.02 1.34 ± 0.07 0.5

LDL cholesterol, mmol/l 2.27 ± 0.06 2.19 ± 0.06 2.55 ± 0.16 0.02

ApoB/ApoA-l Ratio 0.43 ± 0.01 0.41 ± 0.01 0.49 ± 0.03 0.007

Creatinin ( mol/l) 96.2 ± 2.6 96.2 ± 3.0 96.1 ± 5.2 0.5 ox-LDL (U/l) 35.3 ± 0.6 34.5 ± 0.6 38.5 ± 1.7 0.02

Lp-PLA2 mass (ng/ml) 137.0 + 3.3 133.2 ± 3.4 151.8 ± 9.2 0.03

Lp-PLA2 activity 25.6 ± 0.4 25.0 ± 0.4 27.6 ± 0.9 0.005

(nmol/min/ml)

SVD: structural valve degeneration; ^*p value refers to the comparison between SVD and No SVD groups; Values are expressed as means ±SEM.

Correlates of blood plasma Lp-PLA2 activity

[00147] We next assess the relationships between Lp-PLA2 activity with the clinical variables and blood plasma analyses. First, patients with the metabolic syndrome (MetS) had elevated Lp-PLA2 activity (26.5±0.5 nmol/min/ml vs. 24.8+0.5 nmol/min/ml; p=0.01 ) as well as those with history of active smocking (26.1 ±0.5 nmol/min/ml vs. 24.7+0.6 nmol/min/ml; p=0.03). Second, the Lp-PLA2 activity was lower in patients with diabetes (24.2±0.8 nmol/min/ml vs. 25.9±0.4 nmol/min/ml; p=0.03), hypertension (25.1 ±0.4 nmol/min/ml vs. 26.6±0.7 nmol/min/ml; p=0.03) those under ARBs treatment (24.4+0.6 nmol/min/ml vs. 25.9±0.4 nmol/min/ml; p=0.02) and under statins (25.1 ±0.4 nmol/min/ml vs. 27.3±0.8 nmol/min/ml; p=0.008). In addition, there was a tendency for Lp-PLA2 activity to inversely correlate with age (p=0.06) (Table 6). Among the blood lipid variables Lp- PLA2 correlated positively and significantly with the LDL blood level (r=0.24, p=0.0007), ox-LDL plasma level (r=0.27, p<0.0001 ), apoB/apoA-l ratio (r=0.44, p<0.0001 ) and Lp- PLA2 mass (r=0.61 , p<0.0001 ). In addition, blood plasma Lp-PLA2 activity also correlated inversely with the HDL blood level (r— 0.33, p<0.0001 ). In a multivariate logistic regression analysis after correction for hypertension, diabetes, MetS, smocking, statins, ARBs, LDL and ox-LDL we found that age (β= -0.08, SE= 0.03; p=0.02) was independently and inversely related to Lp-PLA2 activity, whereas Lp-PLA2 mass (β=0.06, SE 0.007;

p<0.0001 ) was positively associated with the activity of Lp-PLA2 (r² adjusted=0.43;

p<0.0001 ).

Table 6. Correlation matrix between the Lp-PLA2 activity and clinical data

variables r P

Age -0.13 0.06

Triglycerides (mmol/l) 0.10 0.1

HDL (mmol/l) -0.33 <0.0001

LDL (mmol/l) 0.24 0.0007

ApoB/ApoA-l Ratio 0.44 <0.0001 ox-LDL (U/l) 0.27 <0.0001

Lp-PLA2 mass (nmol/min/ml) 0.61 <0.0001

Expression of Lp-PLA2 in explanted bioprostheses for S VD

[00148] The expression pattern of Lp-PLA2 was next evaluated in group of 39 patients in whom BPs were explanted for SVD. The clinical characteristics of these patients are presented in Table 7. Expression of Lp-PLA2 was documented by immunohistochemistry and found in 30 BPs (77%), whereas expression of ox-LDL and macrophages (CD68) was documented in 95% (37/39) and 74% (29/39) of patients respectively (Figure 7). It is worth to highlight that expression of Lp-PLA2 in bioprosthetic tissues appears as both a diffuse (Figures 8A and D) and a cellular pattern (Figure 8B ). In this regard, we found that Lp-PLA2 co-localized with macrophages (CD68+) (Figure 8B and C) and ox-LDL (Figure 8D and E). We next quantified the amount Lp-PLA2, ox-LDL and CD68 in histological sections with morphometric analyses. Of interest, we found that the level Lp-PLA2 staining in BPs correlated significantly with the amount of ox-LDL (r=0.71 , p<0.0001 ) (Figure 9A) and macrophages content (r=0.50, p=0.008) (Figure 9B ) within explanted tissues. In addition, the density of macrophages positively correlated with the level of ox-LDL (r=0.45, p=0.01 ) within bioprosthetic tissues (Figure 9C). Table 7: Clinical characteristics of patients used for IHC quantitative analysis

Variables Patients (n=39)

Age 67 ± 2

Male (%) 71.8

Smoking (%) 12.8

Hypertension (%) 69.2

Diabetes (%) 15.4

BMI (kg/rr 26.74 + 0.9

Triglycerides (mmol/l) 1.41 ± 0.10

LDL (mmol/l) 2.29 ± 0.19

HDL (mmol/l) 1.19 ± 0.06

Creatinin (μmol/l) 96.7 ± 5.2

Creatinin clearance (ml/min) 67.4 ± 4.4

Mean gradient (mm Hg) 21.1 ± 3.2

Stented bioprosthesis (%) 22 (56)

Porcine bioprosthesis (%) 22 (56)

IHC: immunohistochemistry; BMI: body mass index;

Discussion

[00149] In this work we identified, to our knowledge for the first time, that blood plasma Lp-PLA2 activity was independently related to the SVD of BPs. In addition, we identified by using immunohistochemistry studies that a high proportion (77%) of explanted BPs for SVD had the expression of Lp-PLA2. Quantitative morphometric analyses of bioprosthetic tissues revealed that Lp-PLa2 positively correlated with the density of macrophages and the amount of ox-LDL within BPs. Taken together these findings suggest that Lp-PLA2 and lipid metabolism could play a role in the development of SVD following the implantation of a BP.

Cardiometabolic risk factors and SVD

[00150] Although pre-implant treatment of BPs has evolved over the last several years there is still a relatively high proportion of SVD. To this effect, it is estimated that 20- 30% of BPs have echocardiographic signs of SVD 5-10 years after implantation, whereas 30-50% have SVD at 15 years. (7) In this work we relied on echocardiographic definition of SVD, which has the advantage of being unbiased when compared to a definition based solely only on reintervention. In this regard, surgery for SVD may be denied by the surgeon or the patient in function of the risk factors particularly in an elderly population. In the present study we found that 21% of patients had echocardiographic evidence of BP dysfunction after a mean follow-up of 8 years, which is in accordance with previous studies. (8, 9) Age at implantation has been consistently shown to predict SVD. However, studies performed in the last decade have also emphasized that cardiometabolic risk factors are associated with SVD. In this regard, MetS and diabetes have been shown to be predictors of SVD. (8) Also, patients with low adiponectin blood levels and a high percentage of circulating small, dense LDL particles are at a higher risk to develop SVD.(39) More recently, the ApoB/ApoA-l ratio was associated with SVD of BPs.(9) Taken together these findings suggest that a dysmetabolic state characterized by an atherogenic dyslipidemia is possibly involved in the SVD following implantation of BPs. In the present work the finding that blood plasma Lp-PLA2 activity was associated with SVD gives support to the hypothesis that lipid-derived factor(s) may participate to SVD. Moreover, we also documented that Lp-PLA2 was present in a majority of explanted BPs.

Lp-PLA2 and SVD

[00151] Lp-PLA2 is transported in the blood plasma by circulating lipoproteins. More than two-thirds is transported in circulation by LDL, whereas the remaining fraction is carried by HDL. (40) Lp-PLA2 is also secreted by macrophages following exposition to oxidized phospholipids. (41 ) In the present study, we documented that both the mass and activity of blood plasma Lp-PLA2 were elevated in patients with SVD. However, after correction for covariates only the activity of Lp-PLA2 remained significantly associated with SVD. As previously demonstrated the mass of Lp-PLA2 is a correlate of enzyme activity, but not all the variation in activity is explained by the mass.(42) Hence, it is possible that the activity, which takes into the account both the mass and the genotype, gives a better assessment of the role of Lp-PLA2 and function. Besides, we also found in explanted tissues that Lp-PLA2 was present in 77% of explanted BPs and that it correlated positively with the density of macrophages. Furthermore, immunohistochemistry studies showed that Lp-PLA2 was expressed by macrophages and also appeared as a diffuse staining. Thus, it is possible that Lp-PLA2 is either secreted in the BPs and/or is transported by blood plasma lipoproteins within bioprosthetic tissues.

[00152] A human study has recently shown that in response to an inflammatory stimulus the circulating level of Lp-PLA2 is not modified. However, following a challenge with a lipid load the macrophages release a great quantity of Lp-PLA2.(43) These results indicate that leukocytes in circulation do not appreciably contribute to the amount of circulating Lp-PLA2, whereas peripheral secretion by tissue macrophages is likely an important source of Lp-PLA2 found within the bloodstream. Hence, it is possible that elevated Lp-PLA2 levels in patients with SVD may reflect the secretion of the enzyme by peripheral tissues including the bioprosthetic valves.

[00153] Failure of BPs is intimately linked to the mineralization of the prosthetic leaflets and degradation of the extracellular matrix. Shetty et al. identified in explanted BPs that lipid, glycosaminoglycans and matrix metalloproteinases (MMPs) were expressed within the leaflets.(10) Hence, it is possible that lipid infiltration and retention are involved and that, in turn, the accumulation of ox-LDL in BPs promotes the recruitment and activation of macrophages. It then follows that expression of Lp-PLA2 by macrophages locally in the BP tissue contributes to produce LPC, which, in turn, may increase inflammation in a vicious circle. A recent investigation has documented that LPC promotes the mineralization of vascular cells.(44) Hence, it is possible that local production of Lp- PLA2 in BP tissue contributes to exacerbate the inflammatory process and the

degradation of bioprosthetic leaflets.

Clinical implications

[00154] SVD remains a major hurdle to a widespread utilization of SVD. To this effect, SVD is an important cause of morbidity in patients with BPs. This study along with previous reports suggest that lipid-derived factors may contribute to promote SVD. We previously reported in the native aortic valve that the small, dense LDL phenotype was associated with a greater accumulation of ox-LDL and inflammation in the aortic leaflets. (45) Of interest, the small, dense LDLs carry a greater load of Lp-PLA2.(46) Hence, it is possible that different factors contribute to exacerbate lipid infiltration of the BPs, which entrains inflammation and tissue degradation. Considering that there is a novel pharmaceutical inhibitor of Lp-PLA2, Darapladib, the present study may have some clinical implications. (47) In this regard, in light of the present findings, which shows that both circulating and local secretion of Lp-PLA2 are involved in the SVD process, it is possible that targeting this enzyme may help to prevent or delay tissue degradation.

However, before any conclusion can be made it is necessary to conduct clinical trials evaluating this hypothesis. Nonetheless, it should be pointed out that the independent association between blood plasma enzyme activity of Lp-PLA2 and SVD found in this study suggest that it may, at least, be used as a useful clinical marker.

[00155] The present cross-sectional study gives a snapshot view of the SVD process and does not provide prospective information. However, based on these results, it is possible to hypothesize that temporal changes in Lp-PLA2 activity may predict future events. Nevertheless, this study identified that during the follow-up period after implantation of a BP the enzyme activity of Lp-PLA2 is an independent predictor of SVD. Furthermore, the study of explanted BPs has buttresses our findings and suggests that local expression of the enzyme may contribute to bioprosthetic tissue degradation.

Conclusion [00156] The present study has identified Lp-PLA2 as an independent predictor of SVD. Analyses of explanted BPs also revealed that the enzyme is present locally and its amount correlates with bioprosthetic tissue levels of oxidized lipids and inflammation. Hence, taken together these findings suggest that Lp-PLA2 potentially plays a role in the SVD process. [00157] The present invention has been described in terms of particular

embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

[00158] All articles, references, weblinks, database reference numbers, patent applications and patents referred to herein are specifically incorporated herein by reference in their entirety. Reference List

(1 ) Otto CM. Calcific aortic stenosis--time to look more closely at the valve. N Engl J Med 2008 September 25;359(13):1395-8.

(2) Tselepis AF, Rizzo M, Goudevenos IA. Therapeutic modulation of lipoprotein-associated phospholipase A2 (Lp-PLA2). Curr Pharm Des 201 1 November;17(33):3656-61.

(3) Mohty D, Pibarot P, Despres JP et al. Association between plasma LDL particle size, valve accumulation of oxidized LDL, and inflammation in patients with aortic stenosis. Arterioscler Thromb Vase Biol 2008 January;28(1 ): 187-93.

(4) Cote C, Pibarot P, Despres JP et al. Association between circulating oxidised low-density lipoprotein and fibrocalcific remodelling of the aortic valve in aortic stenosis. Heart 2008 September;94(9):1175-80.

(5) Pentikainen MO, Oksjoki R, Oorni K, Kovanen PT. Lipoprotein lipase in the arterial wall: linking LDL to the arterial extracellular matrix and much more. Arterioscler Thromb Vase Biol 2002 February 1 ;22(2):211-7.

(6) Serruys PW et al. Circulation 2008;1 18:1172-1182 and McConnell JP, Clin Chem 2009;55:21-23.

(7) Stengel D, Antonucci M, Gaoua W, et al. Inhibition of LPL expression in human monocyte- derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine. Arterioscler Thromb Vase Biol 1998; 18:1172-80.

(8) Steinbrecher UP, Pritchard PH. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase. J Lipid Res 1989; 30:305-15. 9.

Cote N, El HD, Pepin A, et al. ATP acts as a survival signal and prevents the mineralization of aortic valve. J Mol Cell Cardiol 2012; 52:1191-202.

(10) Huang MS, Sage AP, Lu J, Demer LL, Tintut Y. Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification. Biochem Biophys Res Commun 2008; 374:553-8.

(1 1 ) Ferretti AC, Mattaloni SM, Ochoa JE, Larocca MC, Favre C. Protein kinase A signals apoptotic activation in glucose-deprived hepatocytes: participation of reactive oxygen species. Apoptosis 2012; 17:475-91.

(7a) Ruel M, Kulik A, Rubens FD, Bedard P, Masters RG, Pipe AL, Mesana TG. Late incidence and determinants of reoperation in patients with prosthetic heart valves. Eur J Cardiothorac Surg 2004;25(3):364-370.

(8a) Briand M, Pibarot P, Despres JP, Voisine P, Dumesnil JG, Dagenais F, Mathieu P.

Metabolic syndrome is associated with faster degeneration of bioprosthetic valves. Circulation 2006;114(1 Suppl):l512-I517.

(9a) Mahjoub H, Mathieu P, Senechal M, Larose E, Dumesnil J, Despres JP, Pibarot P.

ApoB/ApoA-l ratio is associated with increased risk of bioprosthetic valve degeneration. J Am Coll Cardiol 20 3;61 (7):752-761.

(10a) Shetty R, Pibarot P, Audet A, Janvier R, Dagenais F, Perron J, Couture C, Voisine P, Despres JP, Mathieu P. Lipid-mediated inflammation and degeneration of bioprosthetic heart valves. Eur J Clin Invest 2009;39(6):471-480. (11 a) acphee CH, Nelson JJ, Zalewski A. Lipoprotein-associated phospholipase A2 as a target of therapy. Curr Opin Lipidol 2005;16(4):442-446.

(12) Lin P, Ye RD. The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis. J Biol Chem 2003; 278:14379-86.

(13) Kolasa-Trela R, Fil K, Bazanek , et al. Lipoprotein-associated phospholipase A2 is elevated in patients with severe aortic valve stenosis without clinically overt atherosclerosis. Clin Chem Lab Med 2012; 50:1825-31.

(14) Karabina SA, Gora S, Atout R, Ninio E. Extracellular phospholipases in atherosclerosis.

Biochimie 2010; 92:594-600.

(15) Cote C, Pibarot P, Despres JP, et al. Association between circulating oxidised low-density lipoprotein and fibrocalcific remodelling of the aortic valve in aortic stenosis. Heart 2008; 94:1175-80.

(16) Mohty D, Pibarot P, Despres JP, et al. Association between plasma LDL particle size, valvular accumulation of oxidized LDL, and inflammation in patients with aortic stenosis. Arterioscler Thromb Vase Biol 2008; 28:187-93.

(17) Tselepis AD, Dentan C, Karabina SA, Chapman MJ, Ninio E. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb Vase Biol 1995; 15:1764-73.

(18) Wang WY, Li J, Yang D, Xu W, Zha RP, Wang YP. OxLDL stimulates lipoprotein- associated phospholipase A2 expression in THP-1 monocytes via PI3K and p38 MAPK pathways. Cardiovasc Res 2010; 85:845-52.

(19) Thanassoulis G, Campbell CY, Owens DS, et al. Genetic associations with valvular calcification and aortic stenosis. N Engl J Med 2013; 368:503-12.

(20) Cote N, Couture C, Pibarot P, Despres JP, Mathieu P. Angiotensin receptor blockers are associated with a lower remodelling score of stenotic aortic valves. Eur J Clin Invest 2011 ; 41 :1172-9.

(21 ) Roberts WC, Ko JM. Relation of weights of operatively excised stenotic aortic valves to preoperative transvalvular peak systolic pressure gradients and to calculated aortic valve areas. J Am Coll Cardiol 2004; 44:1847-55.

(22) Messika-Zeitoun D, Aubry MC, Detaint D, et al. Evaluation and clinical implications of aortic valve calcification measured by electron-beam computed tomography. Circulation 2004; 110:356-62.

Macphee CH, Moores KE, Boyd HF, et al. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor. Biochem J 1999; 338 ( Pt 2):479-87.

(24) Vickers KC, Castro-Chavez F, Morrisett JD. Lyso-phosphatidylcholine induces osteogenic gene expression and phenotype in vascular smooth muscle cells. Atherosclerosis 2010; 211 :122-9. (25) El Husseini D, Boulanger MC, Fournier D, et al. High Expression of the Pi-Transporter SLC20A1/Pit1 in Calcific Aortic Valve Disease Promotes Mineralization through Regulation of Akt-1. PLoS One 2013; 8:e53393.

(26) Beck GR, Jr., Zerler B, Moran E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci U S A 2000; 97:8352-7.

(27) Rutsch F, Vaingankar S, Johnson K, et al. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol 2001 ; 158:543-54.

(28) Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet 1998; 19:271-3.

(29) Johnson K, Hashimoto S, Lotz M, Pritzker K, Goding J, Terkeltaub R. Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification. Arthritis Rheum 2001 ; 44:1071-81.

(30) Towler DA. Inorganic pyrophosphate: a paracrine regulator of vascular calcification and smooth muscle phenotype. Arterioscler Thromb Vase Biol 2005; 25:651-4.

(31 ) Mune S, Shibata M, Hatamura I, et al. Mechanism of phosphate-induced calcification in rat aortic tissue culture: possible involvement of Pit-1 and apoptosis. Clin Exp Nephrol 2009; 13:571-7.

(32) Chan KL, Teo K, Dumesnil JG, Ni A, Tarn J. Effect of Lipid lowering with rosuvastatin on progression of aortic stenosis: results of the aortic stenosis progression observation: measuring effects of rosuvastatin (ASTRONOMER) trial. Circulation 2010; 121 :306-14.

(33) Zalewski A, Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. Arterioscler Thromb Vase Biol 2005; 25:923-31.

(34) Vickers KC, Maguire CT, Wolfert R, et al. Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis. J Lipid Res 2009; 50:1735-43.

(35) Ferguson JF, Hinkle CC, Mehta NN, et al. Translational studies of lipoprotein-associated phospholipase A(2) in inflammation and atherosclerosis. J Am Coll Cardiol 2012; 59:764- 72.

(36) Silva IT, Mello AP, Damasceno NR. Antioxidant and inflammatory aspects of lipoprotein- associated phospholipase A(2) (Lp-PLA(2)): a review. Lipids Health Dis 2011 ; 10:170.

(37) Wilensky RL, Shi Y, Mohler ER, III, et al. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development. Nat Med 2008; 14:1059-66.

(38) Zoghbi WA, Chambers JB, Dumesnil JG, Foster E, Gottdiener JS, Grayburn PA, Khandheria BK, Levine RA, Marx GR, Miller FA, Jr., Nakatani S, Quinones MA, Rakowski H, Rodriguez LL, Swaminathan M, Waggoner AD, Weissman NJ, Zabalgoitia M. Recommendations for evaluation of prosthetic valves with echocardiography and doppler ultrasound: a report From the American Society of Echocardiography's Guidelines and Standards Committee and the Task Force on Prosthetic Valves, developed in conjunction with the American College of Cardiology Cardiovascular Imaging Committee, Cardiac Imaging Committee of the American Heart Association, the European Association of Echocardiography, a registered branch of the European Society of Cardiology, the Japanese Society of Echocardiography and the Canadian Society of Echocardiography, endorsed by the American College of Cardiology Foundation, American Heart Association, European Association of Echocardiography, a registered branch of the European Society of Cardiology, the Japanese Society of Echocardiography, and Canadian Society of Echocardiography. J Am Soc Echocardiogr 2009;22(9):975-1014.

(39) Shetty R, Girerd N, Cote N, Arsenault B, Despres JP, Pibarot P, Mathieu P. Elevated proportion of small, dense low-density lipoprotein particles and lower adiponectin blood levels predict early structural valve degeneration of bioprostheses. Cardiology 2012;121(1 ):20-26.

(40) Karabina SA, Gora S, Atout R, Ninio E. Extracellular phospholipases in atherosclerosis.

Biochimie 2010;92(6):594-600.

(41 ) Wang WY, Li J, Yang D, Xu W, Zha RP, Wang YP. OxLDL stimulates lipoprotein- associated phospholipase A2 expression in THP-1 monocytes via PI3K and p38 MAPK pathways. Cardiovasc Res 2010;85(4):845-852.

(42) Suchindran S, Rivedal D, Guyton JR, Milledge T, Gao X, Benjamin A, Rowell J, Ginsburg GS, McCarthy JJ. Genome-wide association study of Lp-PLA(2) activity and mass in the

Framingham Heart Study. PLoS Genet 2010;6(4):e1000928.

(43) Ferguson JF, Hinkle CC, Mehta NN, Bagheri R, Derohannessian SL, Shah R, Mucksavage Ml, Bradfield JP, Hakonarson H, Wang X, Master SR, Rader DJ, Li M, Reilly MP. Translational studies of lipoprotein-associated phospholipase A(2) in inflammation and atherosclerosis. J Am Coll Cardiol 2012;59(8):764-772.

(44) Vickers KC, Castro-Chavez F, Morrisett JD. Lyso-phosphatidylcholine induces osteogenic gene expression and phenotype in vascular smooth muscle cells. Atherosclerosis 2010;211(1 ):122-129.

(45) Mohty D, Pibarot P, Despres JP, Cote C, Arsenault B, Cartier A, Cosnay P, Couture C, Mathieu P. Association between plasma LDL particle size, valvular accumulation of oxidized LDL, and inflammation in patients with aortic stenosis. Arterioscler Thromb Vase Biol 2008;28(1 ): 187-193.

(46) Tselepis AD, Dentan C, Karabina SA, Chapman MJ, Ninio E. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb

Vase Biol 995; 15( 0): 1764- 773.

(47) Wilensky RL, Shi Y, Mohler ER, III, Hamamdzic D, Burgert ME, Li J, Postle A, Fenning RS, Bollinger JG, Hoffman BE, Pelchovitz DJ, Yang J, Mirabile RC, Webb CL, Zhang L, Zhang P, Gelb MH, Walker MC, Zalewski A, Macphee CH. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development. Nat Med

2008;14(10):1059-1066.

Claims

1. Use of an inhibitor of PLA2G7 expression or Lp-PLA2 activity for preventing or treating calcific disease or prosthetic valve calcification in a subject.

2. The use of claim 1 , wherein the calcific disease is selected from a group consisting of: aortic valve stenosis (AS), calcific aortic valve disease (CAVD), hypertension related to decreased vascular calcification.

3. The use of claim 1 or 2, wherein the subject is a mammal.

4. The use of claim 3, wherein the mammal is a human.

5. The use of claim 1 or 2 wherein the inhibitor is a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof.

6. The use of claim 5, wherein the nucleic acid agent is an RNAi agent.

7. The use of claim 6 wherein the RNAi agent is a siRNA, shRNA, mi^'RNA, dsRNA or ribozyme or variants thereof.

8. The use of claim 5, wherein the small molecule is 1-(N-(2-(diethylamino)ethyl)-N- (4-(4-trittuoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one or SB480848 or a salt thereof.

9. The use of claim 5, wherein the small molecule is /V-(2-diethylaminoethyl)-2-[2-(2-( 2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-cyclopentapyrimidin-1-yl]-A/-(4'- trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof.

10. The use of claim 5, wherein the small molecule is A/-(1-(2-Methoxyethyl)piperidin-4- yl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4/-/-quinolin-1-yl]-/V-(4'-trifluoromethylbiphenyl-4- ylmethyl)acetamide; or a salt thereof

11. The use of claim 5, wherein the small molecule is methyl 2-[4-({[2-[2-(2,3- difluorophenyl)ethyl]-4-oxopyrido[2,3-c]pyrimidin-1 (4/-/)-yl]acetyl}{[4'-(trifluoromethyl)-4- biphenylyl]methyl}amino)-1 -piperidinyl]-2-methylpropanoate or a salt thereof.

12. The use of claim 5, wherein the small molecule is Darapladib.

13. The use of claim 1 or 2, further comprising an additional therapeutic agent against valvular or vascular calcification.

14. The use of claim 13, wherein said additional therapeutic agent is an inhibitor of ENPP-1 or an agonist of P2Y₂ receptor.

15. Use of an agent that inhibits expression of PLA2G7 gene or activity of Lp-PLA₂ protein for the preparation of a medicament for treatment and/or prevention of calcific disease in a subject.

16. The use of claim 15, wherein the calcific disease is selected from a group consisting of: aortic valve stenosis (AS), calcific aortic valve disease (CAVD), hypertension related to decreased vascular calcification.

17. The use of claim 15 or 16, wherein the subject is a mammal.

18. The use of claim 17, wherein the mammal is a human.

19. The use of claim 15 or 16 wherein the inhibitor is a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof.

20. The use of claim 19, wherein the nucleic acid agent is an RNAi agent.

21. The use of claim 20 wherein the RNAi agent is a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.

22. The use of claim 19, wherein the small molecule is 1-(N-(2-(diethylamino)ethyl)-N- (4-(4-trittuoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one or SB480848 or a salt thereof.

23. The use of claim 19, wherein the small molecule is /\/-(2-diethylaminoethyl)-2-[2-(2- ( 2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-cyclopentapyrimidin-1-yl]-/V-(4'- trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof.

24. The use of claim 19, wherein the small molecule is /V-(1-(2-Methoxyethyl)piperidin- 4-yl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-^

ylmethyl)acetamide; or a salt thereof

25. The use of claim 19, wherein the small molecule is methyl 2-[4-({[2-[2-(2,3- difluorophenyl)ethyl]-4-oxopyrido[2,3-o pyrimidin-1 (4H)-yl]acetyl}{[4'-(trifluoromethyl)-4- biphenylyl]methyl}amino)-1 -piperidinyl]-2-methylpropanoate or a salt thereof.

26. The use of claim 19, wherein the small molecule is Darapladib.

27. The use of claim 15 or 16, further comprising an additional therapeutic agent against valvular or vascular calcification.

28. The use of claim 27, wherein said additional therapeutic agent is an inhibitor of ENPP-1 or an agonist of P2Y₂ receptor.

29. A method for treating calcific disease in a subject suffering therefrom, comprising administering to the subject an effective amount of an inhibitor of PLA2G7 gene expression and/or of Lp-PLA₂ enzymatic activity.

30. A method for preventing calcific disease (CD) in a subject at risk of developing CD, comprising administering to the subject an effective amount of an inhibitor of PLA2G7 gene expression and/or of Lp-PLA₂ enzymatic activity, wherein inhibition of the PLA2G7 gene or Lp-PLA₂ protein reduces or stops a symptom of calcific disease.

31. The method of claim 30, wherein the calcific disease is selected from a group consisting of: aortic valve stenosis (AS), calcific aortic valve disease (CAVD), hypertension related to decreased vascular calcification.

32. The method of claim 30 or 31 , wherein the subject is a mammal.

33. The method of claim 32, wherein the mammal is a human.

34. The method of claim 30 or 31 wherein the inhibitor is a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof.

35. The method of claim 34, wherein the nucleic acid agent is an RNAi agent.

36. The method of claim 35 wherein the RNAi agent is a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.

37. The method of claim 34, wherein the small molecule is 1 -(N-(2- (diethylamino)ethyl)-N-(4-(4-trittuoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-

5 fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one or SB480848 or a salt thereof.

38. The method of claim 34, wherein the small molecule is A/-(2-diethylaminoethyl)-2- [2-(2-( 2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-cyclopentapyrimidin-1 -yl]-A/-(4'- trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof.

39. The method of claim 34, wherein the small molecule is Λ/-(1-(2-

10 Methoxyethyl)piperidin-4-yl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4H-quinolin-1 -yl]-A/-(4'- trifluoromethylbiphenyl-4-ylmethyl)acetamide; or a salt thereof

40. The method of claim 34, wherein the small molecule is methyl 2-[4-({[2-[2-(2,3- difluorophenyl)ethyl]-4-oxopyrido[2,3-<¾pyrimidin-1 (4H)-yl]acetyl}{[4'-(trifluoromethyl)-4- biphenylyl]methyl}amino)-1 -piperidinyl]-2-methylpropanoate or a salt thereof.

15 41. The method of claim 34, wherein the small molecule is Darapladib.

42. The method of claim 30 or 31 , further comprising the administration of an additional therapeutic agent against valvular or vascular calcification.

43. The method of claim 42, wherein said additional therapeutic agent is an inhibitor of ENPP-1 or an agonist of P2Y₂ receptor. 0

44. An inhibitor of PLA2G7 expression or Lp-PLA2 activity for use to prevent or treat calcific disease, or prosthetic valve calcification in a subject.

45. The inhibitor of claim 44, wherein the calcific disease is selected from a group consisting of: aortic valve stenosis (AS), calcific aortic valve disease (CAVD), hypertension related to decreased vascular calcification. 5

46. The inhibitor of claim 43 or 44, wherein the subject is a mammal.

47. The inhibitor of claim 46, wherein the mammal is a human.

48. The inhibitor of claim 43 or 44 selected from the group consisting of: a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or a fragment thereof.

49. The inhibitor of claim 48, wherein the nucleic acid agent is an RNAi agent.

50. The inhibitor of claim 49 wherein the RNAi agent is a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.

51. The inhibitor of claim 48, wherein the small molecule is 1-(N-(2- (diethylamino)ethyl)-N-(4-(4-trittuoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4- fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one or SB480848 or a salt thereof.

52. The inhibitor of claim 48, wherein the small molecule is A/-(2-diethylaminoethyl)-2- [2-(2-( 2,3-difluorophenyl)ethyl)-4-oxo-4,5,6,7-tetrahydro-cyclopentapyrimidin-1-yl]-A7-(4'- trifluoromethyl-biphenyl-4-ylmethyl)acetamide or a salt thereof.

53. The inhibitor of claim 48, wherein the small molecule is Λ/-(1-(2- Methoxyethyl)piperidin-4-yl)-2-[2-(2,3-difluorobenzylthio)-4-oxo-4/- -quinolin-1-yl]-/\/-(4'- trifluoromethylbiphenyl-4-ylmethyl)acetamide; or a salt thereof

54. The inhibitor of claim 48, wherein the small molecule is methyl 2-[4-({[2-[2-(2,3- difluorophenyl)ethyl]-4-oxopyrido[2,3-c]pyrimidin-1 (4H)-yl]acetyl}{[4'-(trifluoromethyl)-4- biphenylyl]methyl}amino)-1 -piperidinyl]-2-methylpropanoate or a salt thereof.

55. The inhibitor of claim 48, wherein the small molecule is Darapladib.

56. A composition for the prevention or treatment of calcific disease, or prosthetic valve calcification in a subjectof comprising the inhibitor according to any one of claims 44 to 55 in admixture with a pharmaceutically acceptable excipient.

57. The composition of claim 56, further comprising an additional therapeutic agent against valvular or vascular calcification.

58. The composition of claim 57, wherein said additional therapeutic agent is an inhibitor of ENPP-1 or an agonist of P2Y₂ receptor.

59. A method for diagnosing or prognosing a vascular or valvular calcification disorder in a subject, comprising the steps of:

- quantifying Lp-PLA2 activity or PLA2G7 expression in plasma of said subject; and

- determining if said activity/expression is above a predetermined threshold level; wherein if said activity/expression is above said predetermined threshold level, said subject is at risk of developing calcific disease.

60. A method for predicting prosthetic valve calcification in a subject having undergone prosthetic valve replacement, comprising the steps of:

- identifying a subject having undergone prosthetic valve replacement following a diagnosis of aortic stenosis;

- obtaining a plasma sample from said subject;

- quantifying Lp-PLA2 activity or PLA2G7 expression in said sample; and

- determining if said activity/expression is above a predetermined threshold level; wherein if said activity/expression is above said predetermined threshold level, said subject is at risk of calcifying the prosthetic valve.

61. The method of claim 59 or 60, wherein said subject at risk is prescribed an agent that inhibits Lp-PLA2 activity.

62. An assay for identifying an inhibitor of calcific disease (CD), comprising the steps of:

a) contacting PLA2G7 with a potential inhibitor thereof; and

b) measuring PLA2G7 expression;

whereby inhibition of PLA2G7 expression is an indication that said compound is a potential inhibitor of CD.

63. An assay for identifying an inhibitor of calcific disease (CD), comprising the steps of:

a) contacting Lp-PLA2 with a potential inhibitor thereof; and

b) measuring Lp-PLA2 activity;

whereby inhibition of Lp-PLA2 activity is an indication that said compound is a potential inhibitor of CD.

64. A method of preventing and/or treating calcific disease in a subject in need thereof, comprising:

(i) screening the subject for likelihood of having or developing tissue calcification; and

(ii ) administering to the subject a pharmaceutical composition comprising an agent which inhibits expression of PLA2G7 gene and/or activity of Lp-PLA₂ protein, wherein inhibition of PLA2G7 gene or Lp-PLA₂ protein reduces or slows tissue calcification.

wherein the subject is identified to have an increased risk or likelihood of developing calcific disease determined by step (i).

65. A method for preventing calcification of a prosthetic aortic valve in a subject, comprising:

(i) screening the subject for likelihood of having or developing valve calcification; and

(ii ) administering to the subject a pharmaceutical composition comprising an agent which inhibits expression of PLA2G7 gene and/or activity of Lp-PLA₂ protein, wherein inhibition of PLA2G7 gene or Lp-PLA₂ protein reduces or slows valvular calcification.