CN114404592A

CN114404592A - Application of TET2 as target in treating ischemic vascular diseases

Info

Publication number: CN114404592A
Application number: CN202210121023.2A
Authority: CN
Inventors: 孙爱军; 葛均波; 洪文轩
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-02-09
Filing date: 2022-02-09
Publication date: 2022-04-29

Abstract

The invention discloses application of TET2 as a target in treating ischemic vascular diseases. The invention verifies the role of TET2 in endothelial cell tubulation and invasion through tubulation and Transwell experiments at the cellular level. The endothelial cell specific TET2 knockout mouse is used for verifying the effect of TET2 in blood flow recovery at an animal level, and the tissue specific TET2 knockout mouse can obviously improve the blood flow recovery in a lower limb ischemia model. The invention finds a new therapeutic target for clinically treating ischemic vascular diseases, particularly peripheral arterial diseases, and the targeted endothelial cell deletion TET2 is expected to realize accurate treatment of the ischemic vascular diseases, particularly the peripheral arterial diseases.

Description

Application of TET2 as target in treating ischemic vascular diseases

Technical Field

The invention relates to application of TET2 as a target in treating ischemic vascular diseases, belonging to the technical field of biological medicines.

Background

Angiogenesis is critical to tissue regeneration or repair in the context of wound healing, muscle and bone repair, hypoxia, and chronic ischemia, among others. Limb ischemia is one of the most common peripheral arterial diseases, and is associated with the occurrence of cardiovascular events including stroke and myocardial infarction. At the end of the 90 s of the 20 th century, humans first tried gene therapy to treat severe ischemic limb disease. To date, there have been few other treatments to restore blood flow to ischemic tissue, other than intravascular treatment. Therefore, the key regulator of angiogenesis is searched as a therapeutic target point, and the important significance is achieved on promoting the blood flow recovery of living bodies.

TET (Ten-eleven translocation) proteins are a newly discovered class of DNA demethylases, the catalytic activity of which depends on alpha-ketoglutaric acid (alpha-KG) and Fe²⁺. The TET family includes three members of TET1, TET2, and TET 3. TET proteins can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine and thereby initiate the process of DNA demethylation. The TET2 protein was first discovered in leukemia patients and early findings focused mainly on the relationship between TET2 and hematologic malignancies because it plays an important role in the hematopoiesis. With the progress of the research, researchers found that TET2 also has an irreplaceable role in the development of other diseases. In the aspect of cardiovascular, Martin, K.A team finds that DNA demethylase TET2 participates in the regulation of vascular injury processes such as atherosclerosis and the like by maintaining the DNA hypomethylation level of a promoter region of a smooth muscle specific gene. The Sano S team found that TET2 was involved in the regulation of acute myocardial ischemic injury by affecting the differentiation of the M1 and M2 phenotypes, which are myeloid mononuclear macrophages. Therefore, TET2 is suggested to play an important role in the occurrence and development of cardiovascular diseases. However, no studies have been made to date to reveal whether TET2 in endothelial cells plays a regulatory role in angiogenesis in ischemic vascular diseases and its specific mechanism.

Angiogenesis occurs in various physiological and pathological processes of the body and becomes one of the markers of the pathological processes. In the process, a large number of resident cells and mobilized cells actively participate, wherein endothelial cells play an important role. Endothelial cells in the capillary wall can obtain invasion and mobilization ability in a budding mode, and the endothelial cells are continuously migrated and proliferated under the guidance of VEGF, so that continuous elongation of buds is promoted. In this manner, endothelial cells gradually fuse to form a continuous lumen, which in turn is angiogenesis. In ischemic vascular diseases, endothelial cells are targeted, the function of the endothelial cells is enhanced, and the target is expected to become an effective target for clinical treatment.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to promote the blood flow recovery of a living body through a key regulator of angiogenesis so as to realize the aim of treating ischemic vascular diseases, particularly peripheral arterial diseases.

In order to solve the technical problems, the invention provides application of an agent for inhibiting expression and/or activity of TET2 in preparing a medicament for treating ischemic vascular diseases.

Preferably, said agent that inhibits expression and/or activity of TET2 comprises an siRNA specific for TET 2.

Preferably, the sequence of the specific siRNA of TET2 is: GGGUAAGCCAAGAAAGAAATT or AGAAAGAAAUCCAGGUGAATT.

The invention also provides a medicament or a pharmaceutical composition for treating ischemic vascular diseases, which comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients comprise an agent for inhibiting the expression and/or activity of TET 2.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention verifies the effect of TET2 in endothelial cell tubulation and invasion at the cellular level through tubulation and Transwell experiments, verifies the effect of TET2 in blood flow recovery by using an endothelial cell specificity TET2 knockout mouse at the animal level, finds a new treatment target for treating ischemic vascular diseases, particularly peripheral arterial diseases, and has good clinical application prospect;

2. the invention provides an application of TET2 in preparation of a medicine for treating ischemic vascular diseases, which can remarkably improve blood flow recovery in a lower limb ischemia model by knocking TET2 out through tissue specificity, target endothelial cell deletion TET2 and hopefully realize accurate treatment of ischemic vascular diseases, particularly peripheral arterial diseases.

Drawings

FIG. 1 is a graph of RT-qPCR to measure the mRNA expression level of TET2 after different times of hypoxic treatment;

FIG. 2 shows the Western blotting method for detecting the expression level of TET2 protein after different times of hypoxia treatment;

FIG. 3 is a graph showing RT-qPCR detection of the mRNA expression level of TET2 24h after HUVEC cells were transfected with siScramble (NC) and si-TET2(siTET2-1, siTET2-2 and siTET2-3), respectively;

FIG. 4 shows the level of TET2 protein expression 24h after Western blotting of HUVEC cells transfected with siScramble (NC) and siTET2(siTET2-1, siTET2-2 and siTET2-3), respectively;

FIG. 5 shows the results of a Matrigel tube formation experiment for detecting the tube forming ability of endothelial cells;

FIG. 6 shows the results of the endothelial cell invasion ability measured by the Transwell assay;

FIG. 7 shows Doppler scan imaging results and quantitative statistics results; wherein WT represents a wild mouse, CDH5-TET2 represents a TET2 knockout mouse;

FIG. 8 shows the results of measuring the density of capillaries by immunofluorescence staining; wherein WT represents a wild mouse, CDH5-TET2 represents a TET2 knockout mouse;

FIG. 9 shows the real-time fluorescent quantitative PCR detection of VEGF expression level in the tissue of ischemic lower limb mice of each experimental group; wherein WT represents a wild mouse, CDH5-TET2 represents a TET2 knockout mouse;

FIG. 10 shows the ELISA detection of the total VEGF protein level in the tissues of the lower limb ischemic mice of each experimental group; wherein WT represents a wild mouse, CDH5-TET2 represents a TET2 knockout mouse.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1 hypoxia was detected in association with TET2 in endothelial cells

To verify whether hypoxia affects the expression level of TET2 in endothelial cells, transcription and expression levels of TET2 were examined at different times of hypoxia treatment of Human Umbilical Vein Endothelial Cells (HUVECs) by RT-qPCR method and western blotting method, respectively, and as a result, the transcription and protein expression levels of TET2 were down-regulated with hypoxia time dependence as shown in fig. 1 and 2.

Example 2 inhibition of TET2 enhances endothelial cell tubulogenesis and invasiveness

(1) Knockout of HUVEC cells TET 2:

to further investigate whether TET2 was involved in regulating angiogenesis, at the cellular level, this example used Lipofectamine 3000(Invitrogen) in HUVEC cells, according to the instructions, to transfect siScramble (NC group, negative control group), siTET2 (three pairs of interfering sequences were used in the experiment, siTET 2-1/siRNA-1: ACAAGAAAGUAGAGGGUAUTT, siTET 2-2/siRNA-2: GGGUAAGCCAAGAAAGAAATT, siTET 2-3/siRNA-3: AGAAAGAAAUCCAGGUGAATT, respectively), and after 24h of transfection, the transcription and protein expression levels of Human Umbilical Vein Endothelial Cell (HUVEC) TET2 were examined by RT-qPCR and Western blotting, respectively, as shown in FIGS. 3 and 4, indicating that TET2 was successfully knocked out.

(2) Matrigel tube formation assay for endothelial cell tube formation ability:

the day before the experiment Matrigel was placed in an ice box and placed in a 4 degree freezer to allow the gel to melt slowly overnight. A24-well plate was layered with 200. mu.L of a basement membrane substrate (BD biosciences, san Diego, Calif.) and placed in a 37-degree incubator for 30 minutes. Next, HUVEC cells that successfully knocked out TET2 and HUVEC cells of the negative control group were collected, counted and plated (1X 10)⁵Cells/well) were incubated for 6 hours under hypoxic conditions, images were collected, the capillary network length was quantified using ImageJ software (NIH), and changes in endothelial cell function were detected, with the results shown in fig. 5, which indicate that TET2 deletion in endothelial cells significantly promotes endothelial cell tube-forming ability.

(3) Endothelial cell invasion capacity was detected by Transwell migration invasion assay:

HUVEC cells successfully knocking out TET2 and negative control group HUVEC cells (1X 10)⁴Cells/well) were plated in the upper chamber of a Transwell chamber and the lower chamber was replaced with serum-free medium and incubated for an additional 24 hours in the absence of oxygen. The next day, the cells in the chamber were fixed with 4% paraformaldehyde solution and then usedStaining with a 1% crystal violet solution, finally removing cells in the upper chamber with a cotton swab, collecting images, counting the number of the cells with ImageJ software, and detecting the invasion capacity of endothelial cells, wherein the result is shown in FIG. 6, and the invasion capacity of endothelial cells knocked out by surface TET2 is remarkably enhanced.

Example 3 mouse endothelial cell knockout of TET2 accelerates blood flow recovery in ischemic tissues

(1) Construction of endothelial cell TET2 conditional knockout mice

TET2-Flox mice are constructed by CRE-Floxp technology, and are hybridized with CDH5-ERT2-CRE mice to successfully obtain endothelial cell TET2 specific conditional knockout mice.

(2) Construction of a lower limb ischemia model

All experimental mice WT and TET2KO mice were prepped one day prior to femoral artery ligation and were depilated on the abdomen and both lower limbs with depilatory cream. Pentobarbital sodium 8mg/mL was anesthetized by intraperitoneal injection at 10 mL/kg. The mice were supine, secured with double lower limb tape, and left exposed to the right lower limb surgical field. A2.5-3 cm longitudinal incision is cut along the direction of the blood vessel, and the blood vessel and the nerve are exposed by careful blunt separation with a micro-forceps. The common femoral artery has two upward branches, and the two branches are used as positioning to separate the femoral artery. The superficial femoral artery at the lower surface was also isolated. The thread passes through the lower part of the femoral artery, is knotted and is sewn on the skin to form a lower limb ischemia model. Laser Doppler perfusion blood flow observation shows whether the operation is successful, and Doppler scanning results show that the lower limb blood flow recovery of the skin TET2 knockout mouse is obviously enhanced compared with that of a wild control group, as shown in figure 7. The results of immunofluorescence staining for detecting the capillary density show that the capillary density of the muscle tissue of the lower limb of the TET2 knockout mouse is obviously increased compared with that of a wild control group, as shown in FIG. 8.

(3) Detection of VEGF expression in ischemic lower limb tissues of model mice

Because the expression peak period of VEGF in the ischemic tissues of the lower limbs is 3d or 7d after operation, mRNA and ELISA detection of VEGF are carried out on the muscle tissues of the lower limbs after 3d and 7d of modeling. The real-time fluorescent quantitative PCR detection result of the expression level of VEGF in the tissue shows that the VEGF transcription level of the muscle tissue of the lower limb of a TET2 knockout mouse is obviously increased compared with that of a wild control group, as shown in FIG. 9. ELISA detection results show that the total VEGF protein level of the muscle tissue of the lower limbs of TET2 knockout mice is obviously increased compared with that of a wild control group, as shown in FIG. 10.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.

Claims

1. Use of an agent that inhibits the expression and/or activity of TET2 in the manufacture of a medicament for the treatment of an ischemic vascular disease.

2. The use of claim 1, wherein said agent that inhibits the expression and/or activity of TET2 comprises an siRNA specific for TET 2.

3. The use of claim 2, wherein said siRNA specific for TET2 has the sequence:

GGGUAAGCCAAGAAAGAAATT；

or AGAAAGAAAUCCAGGUGAATT.

4. A medicament or pharmaceutical composition for the treatment of ischemic vascular diseases comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient comprising an agent that inhibits the expression and/or activity of TET 2.

5. The drug or pharmaceutical composition for the treatment of ischemic vascular disease of claim 4, wherein said agent that inhibits the expression and/or activity of TET2 comprises siRNA specific for TET 2.

6. The drug or pharmaceutical composition for treating ischemic vascular disease of claim 5, wherein said siRNA specific to TET2 has the sequence:

GGGUAAGCCAAGAAAGAAATT；

or AGAAAGAAAUCCAGGUGAATT.