WO1995026745A1 - TRANSFORMING GROWTH FACTOR-β INHIBITS INDUCIBLE NITRIC OXYDE SYNTHASE GENE TRANSCRIPTION - Google Patents

Abstract

Methods of treating hypotension and septic shock by introducing into a mammal suspected of needing such treatment an effective amount of transforming growth factor-β or an active fragment thereof to inhibit transcription of a gene encoding inducible nitric oxide synthase but not a gene encoding constitutive nitric oxide synthase.

Description

TRANSFORMING GROWTH FACTOR-θ INHIBITS INDUCIBLE NITRIC OXIDE SYNTHASE GENE TRANSCRIPTION

Background of the Invention

The field of invention is treatment for hypotension. Septic shock, the most common cause of death in intensive care units, is characterized by severe and often irreversible hypotension (Parrillo, J. E. , 1993, N. Engl . J. tied. 328:1471-1477). The hemodynamic changes associated with septic shock are mediated in part by nitric oxide (NO) generated after the induction of inducible nitric oxide synthase (iNOS) by cytokines such as interleukin 1-3 (IL1-_/5) and tumor necrosis factor-α (TNF-α) (Moncada, S. , Palmer, R. M. , and Higgs, E. A.,

1991, Pharmacol Rev. 43:109-42). NO is a short-lived, free radical gas that accounts for the biologic properties of endothelium-derived relaxing factor. NO has a variety of functions including vasodilation and neurotransmission, and tumoricidal as well as microbicidal activities. NO is synthesized from L-arginine by nitric oxide synthase (NOS) . Two classes of NOS have been identified: a constitutive, calcium- dependent isozyme (cNOS) that is present in tissues under basal conditions, and an inducible, calcium-independent isozyme (iNOS) that is negligibly present under basal conditions and requires cytokine or endotoxin activation for expression. Glucocorticoids, which prevent the induction of iNOS activity (Radomski, M. W. , Palmer, R. M. , and Moncada, S.,1990, Proc . Natl . Acad . Sci . U S A. 87:10043-7), have been extensively evaluated as a potential therapy for septic shock. However, glucocorticoids do not improve survival in patients with septic shock (Bone, R. C, Fisher, C. F. , Jr., Clemmer, T. P., Slotman, G. J. , Metz, C. A., and Balk, R. A., 1987, N. Engl . J. tied . 317:653-658).

Summary of the Invention The invention provides a method of treatment of hypotension, including that associated with septic shock, which is effective both before and after induction of iNOS gene transcription. The methods of the invention are based on the discovery that transforming growth factor β (TGF3) inhibits iNOS gene transcription and NO production in smooth muscle cells both before and after the induction of transcription by the cytokine IL1-_/9.

In one aspect, the invention features a method of treating hypotension by introducing into a mammal suspected of needing treatment for hypotension an effective amount of TGFβ or an active fragment of TGF_/9. By the term "active" is meant having the ability to inhibit transcription of the iNOS gene. Active fragments of TGFβ can be generated by methods known to those skilled in the art, e.g., proteolytic cleavage or expression of recombinant peptides. As used herein, the term "fragment", as applied to TGFβ , is a polypeptide having an amino acid sequence corresponding to a portion of TGF3 which is least 10 contiguous amino acids in length, but is less than full length mature TGFβ . Fragments are typically at least 20 contiguous amino acids, more typically at least 30 contiguous amino acids, usually at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids, more preferably at least 60 contiguous amino acids, and most preferably at least 70 to 80 or more contiguous amino acids in length.

A mammal, such as a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, or human, suspected of needing treatment for hypotension is one that has severe inflammation, e.g., toxic shock syndrome, or is in septic shock. Severe inflammation, for example, may be the result of an infection with a viral or bacterial pathogen.

A second aspect of the invention features a method of inhibiting transcription of a gene encoding iNOS in an e dothelial cell of a mammal, by identifying a mammal having an endothelial cell in need of inhibition of iNOS gene transcription and contacting the endothelial cells of such a mammal with an effective amount of TGFβ or an active fragment of TGF>5. A mammal in need of inhibition of iNOS gene transcription may be one afflicted with hypotension, e.g., that associated with severe inflammation or septic shock. TGF? used in the claimed method may be TGFβ-1 , TGFβ-2, or a mixture of TGFβ-1 and TGFβ-2. A mixture of TGFβ-1 and TGFβ-2 may be a combination of both intact proteins or a combination of fragments of each protein. A recombinant fusion protein which contains portions of each protein, e.g. , the amino- terminal domain of TGF/3-1 fused to the carboxy-terminal domain of TGF_/9-2 or the amino-terminal domain of TGF3-2 fused to the carboxy-terminal domain of TGF_/9-1 can also be used in the claimed method. Preferably, the endothelial cell is a smooth muscle cell and more preferably, a smooth muscle cell of vascular origin. Transcription of iNOS may have been induced by a variety of factors associated with inflammation such as lipopolysaccharide (LPS) (a component of gram negative bacterial cell walls) or a cytokine, e.g., interleukin 1- β (ILl->9) or tumor necrosis factor-α (TNF-α) . Preferably, TGFβ inhibits iNOS transcription at a dose which does not inhibit transcription of a gene encoding cNOS. TGF3 may be administered with intravenous fluids as well as in combination with other anti-inflammatory agents, e.g., antibiotics; glucocorticoids, such as dexamethasone (Dex) ; and/or pressors, such as epinephrine or norepinephrine.

Brief Description of the Drawings Fig. IA is a photograph of a Northern blot showing dose-dependent rat smooth muscle cell (RASMC) iNOS gene transcription induced by IL1-3. RASMC were treated for 24 hours with the indicated concentrations of ILl-,3. Total RNA was extracted at the end of the incubation period. Northern blot analyses were performed in each of the experiments using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to a ³²P-labeled RASMC iNOS probe. The filters were also hybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to assess loading differences.

Fig. IB is a photograph of a Northern blot showing a time course of RASMC iNOS gene transcription induced by IL1-/3. RASMC were exposed to IL1-3 (10 ng/ml) and total RNA was extracted from the cells at the indicated times. RNA was also extracted from control cells, which received no IL1-/3, at the indicated times. Northern blot analyses were performed in each of the experiments using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to a ³²P-labeled RASMC iNOS probe. The filters were also hybridized with a GAPDH probe to assess loading differences.

Fig. 2 is a photograph of a Northern blot analysis showing the effect of cyclohexi ide (CHX) on RASMC iNOS gene transcription. RASMC were exposed to no stimulus (control), IL1-_/5 (10 ng/ml), CHX (10 μg/ml) , or a combination of CHX (10 μg/ l) and IL1-/3 (10 ng/ml) at the indicated times. CHX was applied to the cells 1 hour before IL1-3 was applied. Total RNA was extracted from the cells, and Northern blot analysis was performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²P-labeled RASMC iNOS and GAPDH probes. Fig. 3A is a photograph of a Northern blot and a corresponding bar graph showing a dose-dependent effect of dexamethasone (Dex) on RASMC iNOS gene transcription induced by IL1-_/5. RASMC were exposed to no stimulus (control), IL1-3 (10 ng/ml) alone, IL1-3 (10 ng/ml) plus pretreatment (30 minutes) with increasing doses of Dex as indicated, or Dex alone. Total RNA was extracted from the cells 24 hours after exposure to ILl-,3. Northern blot analyses were performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²p- labeled RASMC iNOS and GAPDH probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that hybridized to the GAPDH probe. The corrected density was then plotted as a percentage of IL1-/3 stimulation.

Fig. 3B is a photograph of a Northern blot and a corresponding bar graph showing a time course of Dex treatment on IL1-3-induced RASMC iNOS gene transcription. Northern blot analysis was performed as described in Fig. 3A. RASMC were exposed to no stimulus (control) , IL1-/9 (10 ng/ml) alone, IL1-9 (10 ng/ml) plus pretreatment (30 minutes) or posttreatment (2 and 6 hours) with Dex (10"⁶M) , or Dex alone (10^~6 M) .

Fig. 4A is a photograph of a Northern blot and a corresponding bar graph showing the dose-dependent effect of TGF_/9-1 on ILl-/3-induced or TNFα-induced RASMC iNOS gene transcription. RASMC were exposed to no stimulus (control), IL1-3 (10 ng/ml) alone, ILl-ø (10 ng/ml) plus pretreatment (30 minutes) with increasing doses of TGFβ-1 as indicated, or TGF3-1 alone. RASMC were also exposed to TNF-α (100 ng/ml) alone or TNF-α (100 ng/ml) plus pretreatment (30 minutes) with increasing doses of TGF3- 1. Total RNA was extracted from the cells 24 hours after exposure to ILl-,9. Northern blot analyses were performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²P-labeled RASMC iNOS and GAPDH probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that hybridized to the GAPDH probe. The corrected density was then plotted as a percentage of IL1-_/3 stimulation.

Fig. 4B is a photograph of a Northern blot and a corresponding bar graph showing a time course of TGFJ-1 treatment on IL-l3-induced RASMC iNOS gene transcription. RASMC were exposed to no stimulus (control) , IL1-_/9 (10 ng/ml) alone, IL1-J (10 ng/ml) plus pretreatment (30 minutes) or posttreatment (2 and 6 hours) with TGFβ-1 (10 ng/ml) , or TGFβ-1 alone (10 ng/ml) . Total RNA was extracted from the cells 24 hours after exposure to IL1- β . Northern blot analyses were performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²P-labeled RASMC iNOS and GAPDH probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that hybridized to the GAPDH probe. The corrected density was then plotted as a percentage of IL1-3 stimulation. Fig. 5 is a bar graph showing a time course of

TGF3-1 and Dex treatment on iNOS gene transcription after prolonged IL1-3 stimulation. RASMC were exposed to IL1-3 (10 ng/ml) for 24 hours (black bar) , then either TGF?-l (10 ng/ml, gray bars) or Dex (10^~6 M, white bars) were added to the cells. Total RNA was extracted 4, 8, 16, and 24 hours after the initial 24 hours of IL1-0 stimulation. Northern blot analyses were performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²P-labeled RASMC iNOS and GAPDH probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that hybridized to the GAPDH probe. The corrected density was then plotted as a percentage of ILl-β stimulation.

Fig. 6A is a line graph showing the effect of TGF_/9-1 and Dex on iNOS mRNA stability and transcriptional rate in RASMC. RASMC were stimulated with IL1-5 (10 ng/ml) for 24 hours. Then RASMC were co-incubated with vehicle, TGF_/3-1 (10 ng/ml), or Dex (10^~6 M) and IL1-3 for 3 hours. After this coincubation period, actinomycin D (ACD, 10 μg/ml) was administered to the RASMC. Total RNA was extracted from the RASMC at the indicated times after administration of ACD. Northern blot analysis was performed using 10 μg of total RNA per lane. After electrophoresis the RNA was transferred to nitrocellulose filters, which were hybridized to ³²P-labeled SMC iNOS and GAPDH probes. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that hybridized to the GAPDH probe. The corrected density was then plotted as a percentage of the 0-hour value against time (in log scale) .

Fig. 6B is a photograph of a Northern blot. Confluent RASMC were either not stimulated (control) or stimulated with ILl-,5 for 24 hours followed by 6 hours of coincubation with vehicle (IL1-/3) , TGFβ-1 (ILl-,5 + TGFβ- 1) , or Dex (IL1-_/S + Dex) . Nuclei were isolated, and in vitro transcription was allowed to resume in the presence of [α-³²P] UTP. Equal amounts of ³²P-labelled, in vitro transcribed RNA probes from each group were hybridized to 1 μg of denatured iNOS and β-actin cDNA that had been immobilized on nitrocellulose filters.

Fig. 7 is a bar graph showing the effects of TGFβ- 1 and Dex on ILl-,9 stimulated extracellular nitrite accumulation in RASMC. RASMC were exposed to no stimulus (ILl->9 control) , IL1-3 alone (10 ng/ml) , TGFβ-1 alone (10 ng/ml, TGF3-1 control) , IL1-_/9 (10 ng/ml) plus pretreatment (30 minutes) or posttreatment (2 and 6 hours) with TGFβ-1 (10 ng/ml) , Dex alone (10^-6 M, Dex control) , or IL1-/3 plus pretreatment (30 minutes) or posttreatment (2 and 6 hours) with Dex (10^-6 M) . Extracellular nitrite accumulation was assayed at 72 hours. NO production was expressed in μM units. The values represent the mean ± SEM (n=6) . The numbers above the bars signify the fold increase in nitrite accumulation from the appropriate control. IL1-9 stimulation and its appropriate control (black bars) , TGF/3-1 treatment of ILl-,9 stimulated RASMC and their appropriate control (gray bars) , and Dex treatment of ILl-,9 stimulated RASMC and their appropriate controls (white bars) .

Fig. 8 is a line graph showing blood pressure over time of control rats.

Fig. 9 is a line graph showing blood pressure over time of a rat treated with TGF3.

Fig. 10 is a line graph showing blood pressure over time of rats treated with LPS.

Figs. 11-14 are line graphs showing blood pressure over time of rats treated with LPS followed by TGFβ . Fig. 15 is a DNA sequence encoding TGFβ-1 (SEQ ID N0:3) .

Fig. 16 is a DNA sequence encoding TGFJ-2 (SEQ ID NO:4) .

Fig. 17 is an amino acid sequence of TGFβ-1 (SEQ ID N0:5) . Fig. 18 is an amino acid sequence of TGFβ-2 (SEQ ID NO:6) .

Detailed Description Described below are experiments which established that TGFfl inhibits transcription of the iNOS gene before as well as after its induction by mediators of inflammation such as iLl-β and LPS.

Cell culture

RASMC were harvested from male Sprague-Dawley rats (200-250 grams) by enzymatic dissociation using methods known in the art. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, JRH Biosciences, Lenexa, KS) and supplemented with 10% fetal calf serum, penicillin (100 U/ml) , streptomycin (100 μg/ml) , and 25 M Hepes (pH 7.4) (Sigma Chemical, St. Louis, MO). The cultured cells were stained with an antibody to smooth muscle cell specific α-actin to assess the purity of the cultures. This study demonstrated that greater than 99% of the cells were of smooth muscle cell origin. RASMC were passaged every 4 to 7 days, and experiments were performed on cells 4 to 6 passages from primary culture. After the cells had grown to confluence, they were placed in 2% fetal calf serum 16 hours before the experiments. Recombinant human IL1-0 (Collaborative Biomedical, Bedford, MA) and murine TNF-α (Genzyme, Cambridge, MA) were dissolved in Dulbecco's phosphate buffered saline (PBS, JRH Biosciences, Lenexa, KS) and stored at -80°C until use. Human TGFfl-1 and anti-human TGFβ-1 IgG (Collaborative Biomedical, Bedford, MA) were also stored at -80°C and diluted in medium prior to use. Dex (Sigma Chemical, St. Louis, MO) was dissolved in ethanol and stored at 4°C. Dex was further diluted in medium prior to use. In the Dex experiments, ethanol was added to the control cells in the same concentration to exclude the effect of the vehicle.

Amplification of RASMC iNOS cDNA fragment An iNOS cDNA fragment from ILl-β-stimulated RASMC RNA was amplified by reverse transcription polymerase chain reaction (PCR) using degenerative primers based on the known DNA sequence of macrophage iNOS. The sequences of the forward (5' GAYGGNAARCAYGAYTT 3') (SEQ ID NO: 1) and reverse (5' ACNTCCTCCAGGATNTTGTA 3') (SEQ ID NO: 2) primers were used to amplify a 432-base pair fragment. The PCR fragment was then subcloned and sequenced by the dideoxy chain termination method. The sequence of the PCR fragment was found to be identical to the recently published RASMC iNOS sequence (Nunokawa et al., 1993, Biochem . Biophys . Res . Commun . 191:89-94) . The fragment was then labeled as described below and used as a probe in Northern blot experiments.

RNA blot hybridization Total RNA was obtained from cultured cells by guanidinium isothiocyanate extraction and centrifuged through cesium chloride. The RNA was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters. The filters were hybridized with a random-primed, ³²P-labeled iNOS cDNA probe. The hybridized filters were then washed in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate solution at 55°C and autoradiographed with Kodak XAR film at -80°C for 12 to 24 hours or stored on phosphor screens for 6 to 8 hours. To correct for differences in RNA loading, the filters were washed in a 50% formamide solution at 80°C and rehybridized with a rat GAPDH probe. The filters were scanned and radioactivity was measured on a Phosphorlmager running - li ¬ the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) .

Nuclear run-on analysis

Confluent RASMC were either not stimulated (control) or stimulated with ILl-β for 24 hours followed by 6 hours of coincubation with vehicle (ILl-β) , TGFβ-1 (ILl-β + TGFfl-1) , or Dex (ILl-β + Dex) . The cells were subsequently lysed and nuclei isolated. The nuclear suspension (200 μl) was incubated with 0.5 mM each of CTP, ATP, and GTP and with 125 μCi of ³²P-labeled UTP (3,000 Ci/mmol, DuPont - New England Nuclear). The samples were extracted with phenol/chloroform, precipitated, and resuspended at equal counts/min/ml in hybridization buffer (6.5 x 10⁶ cpm/ml) . Denatured probes (1 μg) slot-blotted on nitrocellulose filters were hybridized at 40°C for four days in the presence of formamide. cDNAs for the iNOS and β-actin genes were used as probes. The filters were scanned and radioactivity was measured on a Phosphorlmager running the ImageQuant software. The amount of sample hybridizing to the iNOS probe was divided by that hybridizing to the β-actin probe and the corrected density was reported as a percentage decrease from ILl-β stimulation.

Nitrite assay

To determine the amount of NO produced by RASMC, a stable product of NO oxidation, N0₂" (nitrite) , was measured, using standard methodology. An equal number of cells was used in each of the experiments, and the cells were cultured in DMEM without phenol red for the experiments assessing nitrite concentrations. An aliquot of cell supernatant was mixed with an equal volume of Griess reagent (one part 0.1% napthylethylenediamine dihydrochloride added to one part 1% sulfanilamide in 5% phosphoric acid) and allowed to stand at room temperature for 10 minutes. Nitrite levels in the cell supernatants were subsequently measured with a microplate reader at an absorbance wavelength of 560 nm, and converted to micromolar units (μM) .

Animal studies

LPS was administered intraperitoneally at a dose of 6 mg/kg to male Sprague-Dawley rats (200-250 grams) to cause severe hypotension. Control animals were given vehicle alone. The vehicle used for the delivery of LPS was phosphate buffered saline (PBS) , and the vehicle used for TGFβ-1 administration was 1% bovine serum albumin (BSA) . To evaluate the effect of TGFβ-1 on LPS-induced hypotension in vivo, TGFβ-1 in vehicle was administered at various doses intraperitoneally or intravenously (as a bolus or as a continuous infusion) as indicated in Example 2 and Figs. 8-14. Blood pressure of each animal was recorded prior to any treatment and then at 30 minute intervals following LPS administration and TGFβ-1 treatment.

Generation of active fragments of TGFβ

Fragments of TGFβ can be generated by enzymatic digestion, e.g., trypsin digestion, of the purified TGFβ protein which is commercially available (Collaborative Biomedical, Bedford, MA) . Peptide fragments can also be generated de novo using a peptide synthesizer.

Alternatively, reco binant fragments of any length can be produced by appropriate genetic manipulations of the cloned TGFβ cDNA, incorporating the resulting cDNA fragment into an appropriate vector and expressing the recombinant protein fragment in a bacterial or eucaryotic cell. Such standard molecular biology methods are known in the art and are described in Ausubel et al., 1993, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York. The cDNA encoding TGFβ-l is shown in Fig. 15 (SEQ ID NO:3), and the cDNA encoding TGFβ-2 is shown in Fig. 16 (SEQ ID NO:4) .

The amino acid sequence of TGFβ-l is shown in Fig. 17 (SEQ ID NO:5), and the amino acid sequence of TGFβ-2 is shown in Fig. 18 (SEQ ID NO:6). The carboxy-terminal 112 amino acids of each sequence represent the mature active TGFβ-l molecule and TGFfl-2 molecule, respectively. Fragments of TGFβ-l or TGFθ-2 derived from this region of the molecule can be made by expressing the appropriate cDNA or by enzymatic digestion. Such fragments will generally be greater than 10 amino acids in length but less than 112 amino acids in length and can be readily tested for iNOS gene transcription inhibitory activity in vitro using RASMC as described above (see RNA blot hybridization. Nuclear run-on analysis and Nitrite assay) or in vivo (see Animal Studies) .

Hybrid fusion proteins can be generated by cloning a cDNA encoding a portion or all of TGFβ-l in frame with a cDNA encoding a portion or all of TGFfl-2. For example, fusion proteins can be made in which the amino-terminal domain of TGFfl-1 is fused to the carboxy-terminal domain of TGFB-2 or the amino-terminal domain of TGFβ-2 is fused to the carboxy-terminal domain of TGFfl-1 by cloning the appropriate genetic construct into an expression vector. Host cells (procaryotic or eucaryotic) are then transformed with the expression vector and allowed to produce the recombinant fusion protein. The fusion protein can then be purified using methods known in art.

Treatment of hypotension and septic shock The experiments described below test the effects of Dex and TGFβ-l on iNOS gene transcription after its induction by ILl-β. The results of these experiments indicate that Dex prevented the induction of iNOS gene transcription only when given before the addition of IL1- β. In contrast, TGF3-1 inhibited iNOS gene transcription and NO production in RASMC both before and after the addition of ILl-β. TGFβ-l inhibited iNOS gene transcription even after 24 hours of ILl-fl stimulation. The discovery that TGFβ-l mediates inhibition of SMC iNOS gene transcription after its induction by cytokines forms the basis of the claimed methods of treating septic shock.

EXAMPLE 1: TGFβ-mediated inhibition of iNOS in vitro Primary smooth muscle cells were extracted from Sprague-Dawley mice as described above. RASMC were exposed to cytokines to induce NO production and iNOS gene expression and the effect of TGF/3 on these processes was evaluated.

Prolonged induction of an iNOS gene transcription by IL1- β in RASMC

Northern blot analysis was performed with RNA from RASMC, and the blots were hybridized to a rat SMC cDNA probe. The hybridization pattern of iNOS mRNA demonstrated a characteristic three distinct band pattern, with the lowest and most prominent band at approximately 4.4 kilobases (kb) . ILl-β caused a dose- dependent increase in level of iNOS mRNA in RASMC (Fig. IA) . Maximal induction of iNOS gene transcription occurred at an ILl-β dose of 10 ng/ml, although as little as 1 ng/ml promoted an increase in mRNA levels. Increasing the dose of ILl-β beyond 10 ng/ml did not further increase iNOS mRNA levels. In response to 10 ng/ml of ILl-β, iNOS message was induced at 6 hours (Fig. IB) . Induction could be detected as early as 2 hours, and this single dose of ILl-β promoted a dramatic and prolonged induction of iNOS gene transcription that peaked at 48 hours (Fig. IB) . At 72 hours of ILl-β stimulation, the iNOS mRNA level remained significantly elevated above base line. This prolonged induction of SMC iNOS does not appear to be ILl-β specific, as TNF-α produced an analogous induction pattern.

Induction of RASMC iNOS gene bv ILl-β did not require protein synthesis

To determine whether induction of iNOS gene transcription required protein synthesis de novo, RASMC were treated with the protein synthesis inhibitor, cycloheximide (10 μg/ml) (Sigma Chemical, St. Louis, MO) , for 1 hour before adding ILl-β (10 ng/ml) . This dose of cycloheximide completely inhibited leucine uptake in RASMC. Cycloheximide did not prevent ILl-β induction of iNOS gene transcription at 6 and 12 hours (Fig. 2) indicating that ILl-β-mediated induction of RASMC iNOS gene transcription does not require new protein synthesis. In contrast, macrophages stimulated by LPS and IFN-γ (Lorsbach, R.B, Murphy, W.J., Lowenstein, C.J., and Russel, S.W., 1993, J. Biol . Chem . 268:1908-1913), hepatocytes stimulated by multiple cytokines (Geller, D.A. , Nussler, A.K. , DiSilvio, M. , Lowenstein, C.J., Shapiro, R.A. , Wang, S.C., Simmons, R.L., and Billiar, T.R., 1993, Proc . Natl . Acad. Sci . USA 90"533-526) , and insulin-producing cells stimulated by ILl-β (Eizirik, D.L., Bjorklund, A., and Welsh, N. , 1993, FEBS Lett . 317:62-66) require new protein synthesis for induction of iNOS gene transcription. Dex failed to inhibit iNOS gene transcription when administered after ILl-β

To assess the effect of glucocorticoids on the induction of iNOS gene transcription by ILl-β, RASMC were treated with Dex 30 minutes before ILl-fl (10 ng/ml) , and total RNA was extracted after 24 hours for Northern analysis. Dex inhibited ILl-fl-induced iNOS gene transcription in a dose-dependent manner (Fig. 3A) . To determine whether Dex inhibited iNOS gene transcription after its induction by ILl-β, RASMC were treated with 10~⁶ M of Dex before (30 minutes) or after (2 and 6 hours) the addition of ILl-fl. Dex decreased iNOS gene transcription by 58% when given before ILl-fl; however, it failed to inhibit iNOS gene transcription when administered after ILl-fl (Fig. 3B and 5) . This response also occurred at the level of NO production. The failure of Dex to inhibit iNOS gene transcription and NO production after their induction by cytokines may help explain the ineffectiveness of glucocorticoids in the treatment of septic shock.

TGFfl-1 inhibits iNOS gene transcription after its induction by cytokines

To determine whether TGFfl-l inhibited cytokine- induced iNOS gene transcription, RASMC were treated with TGFfl-l for 30 minutes before the addition of ILl-fl_^ or

TNF-α. TGFfl-l inhibited iNOS induction by ILl-fl and TNF- α in a dose-dependent fashion (Fig. 4A) . At 10 ng/ml (4 x 10^"7 M) , TGFfl-l markedly inhibited transcription of the iNOS gene induced by ILl-β by 92%. Similarly, TGFfl-l inhibited transcription of the iNOS gene induced by TNF-α by 98%. To demonstrate that the inhibition of iNOS gene transcription by TGFfl-l was specific, a TGFfl-l antibody was administered to the RASMC along with TGFfl-l for 30 minutes before ILl-fl. The TGFfl-l antibody completely blocked the inhibitory effect of TGFβ-l on ILl-fl-induced iNOS gene transcription.

To determine if TGFfl-l would have this dramatic inhibitory effect on iNOS gene transcription even after induction by ILlfl-1, TGFfl-l (10 ng/ml) was administered either before or after the addition of ILl-fl. TGFfl-l completely inhibited iNOS induction when given 30 minutes before ILl-fl (Fig. 4B) . In contrast to the effect of Dex, TGFfl-l markedly inhibited iNOS gene transcription when given 2 hours (98%) and 6 hours (90%) after the addition of ILl-β. To determine if the effect of TGFfl-l was related to preventing a further increase in iNOS, or if TGFfl-l actually reduced iNOS transcription, TGFfl-l or Dex was administered to RASMC after 24 hours of ILl-fl stimulation, and iNOS mRNA levels were assessed serially over the next 24 hours. Even after this prolonged ILl-fl stimulation, TGFfl-l (10 ng/ml) was able to reduce the amount of iNOS mRNA detected in the cells (Fig. 5) . Dex (10~⁶ M) , however, was unable to reverse the ILlfl-mediated induction of iNOS gene transcription. In fact, iNOS mRNA levels continued to increase after the administration of Dex (Fig. 5) .

TGFfl-l decreased iNOS gene transcription rate after its induction by ILl-fl To understand the mechanism by which TGFfl-l, but not Dex, downregulates iNOS gene transcription after its induction by ILl-fl, experiments were performed to assess stability and transcriptional rate. The half-life of the iNOS mRNA was determined by measuring mRNA levels in the presence of the transcription inhibitor actinomycin D (ACD). RASMC were stimulated with ILl-fl (10 ng/ml) for 24 hours. Then the cells were incubated with vehicle, TGFfl-l (10 ng/ml), or Dex (10^~6 M) in the presence of ILl- fl for 3 hours. Thereafter, ACD (10 μg/ml) was administered to the RASMC and iNOS message was measured at 1, 2, 4, and 8 hours. The half-life of iNOS mRNA stimulated by ILl-fl alone was approximately 1.4 hours (Fig. 6A) . TGFfl-l did not alter iNOS mRNA stability; however, Dex lengthened the iNOS mRNA half-life to approximately 4 hours. Nuclear run-on experiments were performed to assess the transcriptional rate. ILl-fl markedly increased the transcriptional rate of the SMC iNOS gene (Fig. 6B) . TGFfl-l decreased the transcriptional rate of the SMC iNOS gene by 65% compared to ILl-fl stimulation alone. Dex also decreased the transcriptional rate, but only modestly (31%) . Thus, the suppressive effect of TGFfl-l on the level of SMC iNOS mRNA was mediated mainly at the transcriptional level. A modest decrease in transcriptional rate in conjunction with a greater than doubling of mRNA half-life would explain the ineffectiveness of Dex to decrease the level of iNOS in cells after its induction by ILl-fl.

TGFfl-l. but not Dex. inhibited NO production induced by ILl-fl

To determine the effect of Dex and TGFβ-l on NO production, a stable product of NO oxidation, N0₂- (nitrite) , was measured by a standard assay 72 hours after ILl-fl stimulation (10 ng/ml) . ILl-fl promoted a marked increase in nitrite levels (Fig. 7) , which was completely blocked by coadministration of 10~³ M N^-monomethyl-L-arginine (LNMMA, Calbiochem, La Jolla, CA) , a competitive inhibitor of NO production. Dex (10~⁶ M) administered before (30 minutes) or after (2 and 6 hours) ILl-fl stimulation appeared to partially suppress NO production. However, when Dex plus ILl-fl and ILl-fl alone were compared with their respective controls, the fold increase in nitrite levels did not differ. In contrast, a single administration of TGFfl-l (10 ng/ml) totally inhibited NO production. This inhibitory effect of TGFfl-l occurred even after the induction of iNOS was initiated by ILl-fl.

EXAMPLE 2: Treatment of LPS-induced hypotension with TGFfl-l in vivo

The effect of TGFfl-l on septic shock-associated hypotension was evaluated in vivo using Sprague-Dawley rats. The results of this study are shown in Figs. 8-14.

To induce septic shock in the rats, bacterial endotoxin, LPS, was administered to the eight animals at a dose of 6 mg/kg of body weight. Four rats received no further treatment and four rats were treated with TGFβ. As a control, 3 rats received vehicle alone (PBS) , an one rat received TGFfl alone (20 μg/kg iv bolus + 20 ng/kg/min infusion) . The four rats which received LPS in the absence of further treatment developed severe hypotension (Fig. 10). Three out of these four rats that.received LPS alone died within a four hour period. As shown in Fig. 8 and 9, the blood pressure of the four control rats remained unchanged. Each of the four rats treated with both LPS and TGFβ-l received TGFfl-l 90 minutes after LPS administration using one of the following protocols: Fig. 11 shows the effect on blood pressure of 20 μg/kg TGFfl-l delivered intravenously in a bolus; Fig. 12 shows the effect of 40 μg/kg delivered intravenously in a bolus; Fig. 13 shows data from an animal which received an intravenous bolus of 20 μg/kg TGFfl-l followed by 20 μg/kg TGFfl-l intraperitoneally; and Fig. 14 shows results from a rat treated with a 20 μg/kg intravenous bolus of TGFfl-l followed by a continuous infusion of 200 ng/kg/min of TGFfl-l. None of the rats which received TGFfl-l treatment for LPS-induced hypotension (see Figs. 11-14) died. These results indicate that TGFfl prevented or reversed the hypotension caused by LPS. Clinical applications and advantages

Septic shock is the most common cause of death in intensive care units, and overall the 13th most common cause of death in the United States. Despite an extensive effort, attempts to treat septic shock have not been very successful. It has now been found that TGFfl prevents septic shock-associated hypotension and death in animals.

TGFfl or an active fragment thereof can be administered to an animal characterized by the aberrant upregulation of iNOS transcription, e.g., a patient with severe hypotension associated with septic shock. TGFfl or active fragments thereof may be administered in any manner which is medically acceptable, e.g., combined with any non-toxic, pharmaceutically-acceptable carrier substance suitable for administration to animals or humans. Methods of delivery may include parenteral routes such as intravascular, intravenous, intra- arterial, subcutaneous, intramuscular, or intraperitoneal injections or transdermal or transmucosal methods.

Sustained release administration may also be used, e.g., depot injections or erodible implants.

In some cases, it may be advantageous to administer TGFfl in combination with drugs that affect other points of a pathway leading to severe hypotension or septic shock. For example, TGFfl may be administered either simultaneously or sequentially with another therapeutic agent, e.g., an antibiotic or anti- inflammatory drug, to treat the underlying infection. Co-administration of TGFfl with a glucocorticoid drug may increase the vascular response to therapeutic vasoconstrictors, e.g., pressors such as epinephrine or norepinephrine. Combination therapy may also allow administration of a lower dose of each drug, thus lessening potential side effects of therapy associated with high doses of either drug alone, e.g., fibrosis associated with administration of high doses of TGFfl.

Dosages of TGFfl will vary, depending on factors such as route of administration, the condition of the patient, and whether or not other drugs are being administered. It may be desirable to administer an initial bolus of TGFfl followed by a continuous infusion to maintain the optimal concentration to prevent or treat septic shock. For example, an initial dose can be in the range of 0.2-200 μg/kg followed by a continuous infusion of 2 ng-2 μg/kg/min to provide an adequate concentration of TGFfl in the blood. This protocol may be adjusted to provide for a lower concentration of TGFfl for a longer period, or a higher concentration for a shorter period of treatment.

The methods of the invention provide two important advantages over existing methods of treating septic shock: (1) TGFfl is effective even after induction of iNOS gene transcription, and (2) TGFfl inhibits iNOS but not cNOS.

Because of the importance of NO in the pathogenesis of septic shock, therapeutic options for regulating the NO pathway have been extensively explored. Glucocorticoids have been evaluated as a therapy for septic shock, but their use has not improved the survival rate of patients with septic shock. This failure is probably related to the inability of glucocorticoids to inhibit iNOS after its induction.

Efforts have also focused on directly inhibiting NO production as a way of treating septic shock. For example, L-arginine analogs that competitively inhibit NO production are currently being evaluated in animal models of septic shock and humans. Unfortunately, these analogs block both the iNOS and cNOS pathways, and they have been observed to cause detrimental effects during experimental production are currently being evaluated in animal models of septic shock and humans. Unfortunately, these analogs block both the iNOS and cNOS pathways, and they have been observed to cause detrimental effects during experimental septic shock. At higher doses, such NOS inhibitors, e.g., LNMMA, may abolish all NO-dependent vasodilation. In the presence of circulating vasoconstrictors during septic shock, the removal of all NO-dependent vasodilation may lead to a further decrease in tissue perfusion and an increase in fatal organ damage. In contrast to these L-arginine analogs, TGFfl-l does not inhibit cNOS in vascular endothelial cells. Thus, selective inhibition of iNOS and the ability of TGFβ-l to inhibit iNOS expression after its induction provide important advantages in the treatment of septic shock. Furthermore, TGF,9-1 not only inhibited IL1_/9- induced iNOS gene transcription, it actually appeared to reduce the level of iNOS mRNA in cells that had been subjected to a prolonged ILl-fl induction period. This effect is in sharp contrast to that of the glucocorticoid, Dex, which allowed a continued induction of iNOS gene transcription in the presence of ILl-,9. The fact that TGFfl-l is able to inhibit iNOS gene transcription after it has been induced is important because iNOS expression would almost certainly be initiated by the time septic shock is diagnosed.

The claimed methods provide a novel, more effective, and markedly safer approach to the treatment of hypotension and septic shock. SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Mu-En Lee

Mark A. Perrella

(ii) TITLE OP INVENTION: TRANSFORMING GROWTH

FACTOR-^ INHIBITS INDUCIBLE NITRIC OXIDE SYNTHASE GENE TRANSCRIPTION

(iϋ) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: MS-DOS (Version 5.0)

(D) SOFTWARE: WordPerfect (Version 5.1)

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 5 April 1994

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Janis K. Fraser

(B) REGISTRATION NUMBER: Reg. No. 34,819

(C) REFERENCE/DOCKET NUMBER: 05433/007001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAYGGNAARC AYGAYTT 17

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ACNTCCTCCA GGATNTTGTA 20

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2745

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ACCTCCCTCC GCGGAGCAGC CAGACAGCGA GGGCCCCGGC CGGGGGCAGG GGGGACGCCC 60

CGTCCGGGGC ACCCCCCCCG GCTCTGAGCC GCCCGCGGGG CCGGCCTCGG CCCGGAGCGG 120

AGGAAGGAGT CGCCGAGGAG CAGCCTGAGG CCCCAGAGTC TGAGACGAGC CGCCGCCGCC 180

CCCGCCACTG CGGGGAGGAG GGGGAGGAGG AGCGGGAGGA GGGACGAGCT GGTCGGGAGA 240

AGAGGAAAAA AACTTTTGAG ACTTTTCCGT TGCCGCTGGG AGCCGGAGGC GCGGGGACCT 300

CTTGGCGCGA CGCTGCCCCG CGAGGAGGCA GGACTTGGGG ACCCCAGACC GCCTCCC TT 360

GCCGCCGGGG ACGCTTGCTC CCTCCCTGCC CCCTACACGG CGTCCCTCAG GCGCCCCCAT 420

TCCGGACCAG CCCTCGGGAG TCGCCGACCC GGCCTCCCGC AAAGACTTTT CCCCAGACCT 480

CGGGCGCACC CCCTGCACGC CGCCTTCATC CCCGGCCTGT CTCCTGAGCC CCCGCGCATC 540

CTAGACCCTT TCTCCTCCAG GAGACGGATC TCTCTCCGAC CTGCCACAGA TCCCCTATTC 600

AAGACCACCC ACCTTCTGGT ACCAGATCGC GCCCATCTAG GTTATTTCCG TGGGATACTG 660

AGACACCCCC GGTCCAAGCC TCCCCTCCAC CACTGCGCCC TTCTCCCTGA GGAGCCTCAG 720

CTTTCCCTCG AGGCCCTCCT ACCTTTTGCC GGGAGACCCC CAGCCCCTGC AGGGGCGGGG 780

CCTCCCCACC ACACCAGCCC TGTTCGCGCT CTCGGCAGTG CCGGGGGGCG CCGCCTCCCC 840

CATGCCGCCC TCCGGGCTGC GGCTGCTGCC GCTGCTGCTA CCGCTGCTGT GGCTACTGGT 900

GCTGACGCCT GGCCCGCCGG CCGCGGGACT ATCCACCTGC AAGACTATCG ACATGGAGCT 960

GGTGAAGCGG AAGCGCATCG AGGCCATCCG CGGCCAGATC CTGTCCAAGC TGCGGCTCGC 1020

CAGCCCCCCG AGCCAGGGGG AGGTGCCGCC CGGCCCGCTG CCCGAGGCCG TGCTCGCCCT 1080

GTACAACAGC ACCCGCGACC GGGTGGCCGG GGAGAGTGCA GAACCGGAGC CCGAGCCTGA 1140

GGCCGACTAC TACGCCAAGG AGGTCACCCG CGTGCTAATG GTGGAAACCC ACAACGAAAT 1200

CTATGACAAG TTCAAGCAGA GTACACACAG CATATATATG TTCTTCAACA CATCAGAGCT 1560

CCGAGAAGCG GTACCTGAAC CCGTGTTGCT CTCCCGGGCA GAGCTGCGTC TGCTGAGGAG 1320

GCTCAAGTTA AAAGTGGAGC AGCACGTGGA GCTGTACCAG AAATACAGCA ACAATTCCTG 1380

GCGATACCTC AGCAACCGGC TGCTGGCACC CAGCGACTCG CCAGAGTGGT TATCTTTTGA 1440

TGTCACCGGA GTTGTGCGGC AGTGGTTGAG CCGTGGAGGG GAAATTGAGG GCTTTCGCCT 1500

TAGCGCCCAC TGCTCCTGTG ACAGCAGGGA TAACACACTG CAAGTGGACA TCAACGGGTT 1560

CACTACCGGC CGCCGAGGTG ACCTGGCCAC CATTCATGGC ATGAACCGGC CTTTCCTGCT 1620

TCTCATGGCC ACCCCGCTGG AGAGGGCCCA GCATCTGCAA AGCTCCCGGC ACCGCCGAGC 1680

CCTGGACACC AACTATTGCT TCAGCTCCAC GGAGAAGAAC TGCTGCGTGC GGCAGCTGTA 1740

CATTGACTTC CGCAAGGACC TCGGCTGGAA GTGGATCCAC GAGCCCAAGG GCTACCATGC 1800

CAACTTCTGC CTCGGGCCCT GCCCCTACAT TTGGAGCCTG GACACGCAGT ACAGCAAGGT 1860 CCTGGCCCTG TACAACCAGC ATAACCCGGG CGCCTCGGCG GCGCCGTGCT GCGTGCCGCA . 1920

GGCGCTGGAG CCGCTGCCCA TCGTGTACTA CGTGGGCCGC AAGCCCAAGG TGGAGCAGCT 1980

GTCCAACATG ATCGTGCGCT CCTGCAAGTG CAGCTGAGGT CCCGCCCCGC CCCGCCCCGC 2040

CCCGGCAGGC CCGGCCCCAC CCCGCCCCGC CCCCGCTGCC TTGCCCATGG GGGCTGTATT 2100

TAAGGACACC GTGCCCCAAG CCCACCTGGG GCCCCATTAA AGATGGAGAG AGGACTGCGG 2160

ATCTCTGTGT CATTGGGCGC CTGCCTGGGG TCTCCATCCC TGACGTTCCC CCACTCCCAC 2220

TCCCTCTCTC TCCCTCTCTG CCTCCTCCTG CCTGTCTGCA CTATTCCTTT GCCCGGCATC 2280

AAGGCACAGG GGACCAGTGG GGAACACTAC TGTAGTTAGA TCTATTTATT GAGCACCTTG 2340

GGCACTGTTG AAGTGCCTTA CATTAATGAA CTCATTCAGT CACCATAGCA ACACTCTGAG 2400

ATGGCAGGGA CTCTGATAAC ACCCATTTTA AAGGTTGAGG AAACAAGCCC AGAGAGGTTA 2460

AGGGAGGAGT TCCTGCCCAC CAGGAACCTG CTTTAGTGGG GGATAGTGAA GAAGACAATA 2520

AAAGATAGTA GTTCAGGCCA GGCGGGGTGC TCACGCCTGT AATCCTAGCA CTTTTGGGAG 2580

GCAGAGATGG GAGGATACTT GAATCCAGGC ATTTGAGACC AGCCTGGGTA ACATAGTGAG 2640

ACCCTATCTC TACAAAACAC TTTTAAAAAA TGTACACCTG TGGTCCCAGC TACTCTGGAG 2700

GCTAAGGTGG GAGGATCACT TGATCCTGGG AGGTCAAGGC TGCAG 2745

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1695

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CAAGCAGGAT ACGTTTTTCT GTTGGGCATT GACTAGATTG TTTGCAAAAG TTTCGCATCA 60

AAAACAAACA ACAACAACAA AAAACCAAAC AACTCTCCTT GATCTATACT TTGAGAATTG 120

TTGATTTCTT TTTTTTTATT CTGACTTTTA AAAACAACTT TTTTTTCCAC TTTTTTAAAA 180

AATGCACTAC TGTGTGCTGA GCGCTTTTCT GATCCTGCAT CTGGTCACGG TCGCGCTCAG 240

CCTGTCTACC TGCAGCACAC TCGATATGGA CCAGTTCATG CGCAAGAGGA TCGAGGCGAT 300

CCGCGGGCAG ATCCTGAGCA AGCTGAAGCT CACCAGTCCC CCAGAAGACT ATCCTGAGCC 360

CGAGGAAGTC CCCCCGGAGG TGATTTCCAT CTACAACAGC ACCAGGGACT TGCTCCAGGA 420

GAAGGCGAGC CGGAGGGCGG CCGCCTGCGA GCGCGAGAGG AGCGACGAAG AGTACTACGC 480 CAAGGAGGTT TACAAAATAG ACATGCCGCC CTTCTTCCCC TCCGAAAATG CCATCCCGCC 540

CACTTTCTAC AGACCCTACT TCAGAATTGT TCGATTTGAC GTCTCAGCAA TGGAGAAGAA 600

TGCTTCCAAT TTGGTGAAAG CAGAGTTCAG AGTCTTTCGT TTGCAGAACC CAAAAGCCAG 660

AGTGCCTGAA CAACGGATTG AGCTATATCA GATTCTCAAG TCCAAAGATT TAACATCTCC 720

AACCCAGCGC TACATCGACA GCAAAGTTGT GAAAACAAGA GCAGAAGGCG AATGGCTCTC 780

CTTCGATGTA ACTGATGCTG TTCATGAATG GCTTCACCAT AAAGACAGGA ACCTGGGATT 840

TAAAATAAGC TTACACTGTC CCTGCTGCAC TTTTGTACCA TCTAATAATT ACATCATCCC 900

AAATAAAAGT GAAGAACTAG AAGCAAGATT TGCAGGTATT GATGGCACCT CCACATATAC 960

CAGTGGTGAT CAGAAAACTA TAAAGTCCAC TAGGAAAAAA AACAGTGGGA AGACCCCACA 1020

TCTCCTGCTA ATGTTATTGC CCTCCTACAG ACTTGAGTCA CAACAGACCA ACCGGCGGAA 1080

GAAGCGTGCT TTGGATGCGG CCTATTGCTT TAGAAATGTG CAGGATAATT GCTGCCTACG 1140

TCCACTTTAC ATTGATTTCA AGAGGGATCT AGGGTGGAAA TGGATACACG AACCCAAAGG 1200

GTACAATGCC AACTTCTGTG CTGGAGCATG CCCGTATTTA TGGAGTTCAG ACACTCAGCA 1260

CAGCAGGGTC CTGAGCTTAT ATAATACCAT AAATCCAGAA GCATCTGCTT CTCCTTGCTG 1320

CGTGTCCCAA GATTTAGAAC CTCTAACCAT TCTCTACTAC ATTGGCAAAA CACCCAAGAT 1380

TGAACAGCTT TCTAATATGA TTGTAAAGTC TTGCAAATGC AGCTAAAATT CTTGGAAAAG 1440

TGGCAAGACC AAAATGACAA TGATGATGAT AATGATGATG ACGACGACAA CGATGATGCT 1500

TGTAACAAGA AAACATAAGA GAGCCTTGGT TCATCAGTGT TAAAAAATTT TTGAAAAGGC 1560

GGTACTAGTT CAGACACTTT GGAAGTTTGT GTTCTGTTTG TTAAAACTGG CATCTGACAC 1620

AAAAAAAGTT GAAGGCCTTA TTCTACATTT CACCTACTTT GTAAGTGAGA GAGACAAGAA 1680

GCAAATTTTT TTAAA 1695

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 394

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu 1 5 10 15

Trp Leu Leu Val Leu Thr Pro Gly Pro Pro Ala Ala Gly Leu Ser T r 20 25 30 Cys Lys Thr lie Asp Met Glu Leu Val Lys Arg LyB Arg lie Glu Ala 35 40 45 lie Arg Gly Gin lie Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 50 55 60

Gin Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 65 70 75 80

Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90 95

Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 100 105 110

Met Val Glu Thr His Asn Glu lie Tyr Asp Lys Phe Lys Gin Ser Thr 115 120 125

His Ser lie Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140

Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Arg 145 150 155 160

Leu Lys Leu Lys Val Glu Gin His Val Glu Leu Tyr Gin Lys Tyr Ser 165 170 175

Asn Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp 180 185 190

Ser Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gin Trp 195 200 205

Leu Ser Arg Gly Gly Glu He Glu Gly Phe Arg Leu Ser Ala His Cys 210 215 220

Ser Cys Asp Ser Arg Asp Asn Thr Leu Gin Val Asp He Asn Gly Phe 225 230 235 240

Thr Thr Gly Arg Arg Gly Asp Leu Ala Thr He His Gly Met Asn Arg 245 250 255

Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gin His Leu 260 265 270

Gin Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser 275 280 285

Ser Thr Glu Lys Asn Cys Cys Val Arg Gin Leu Tyr He Asp Phe Arg ^~ 290 295 300

Lys Asp Leu Gly Trp Lys Trp He His Glu Pro Lys Gly Tyr His Ala 305 310 315 320

Asn Phe Cys Leu Gly Pro Cys Pro Tyr He Trp Ser Leu Asp Thr Gin 325 330 335

Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gin His Asn Pro Gly Ala Ser 340 345 350 Ala Ala Pro Cys Cys Val Pro Gin Ala Leu Glu Pro Leu Pro He Val 355 360 365

Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gin Leu Ser Asn Met He

370 375 380

Val Arg Ser Cys Lys Cys Ser Cys Asp Ser 385 390

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 414

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met His Tyr Cys Val Leu Ser Ala Phe Leu He Leu His Leu Val Thr 1 5 10 15

Val Ala Leu Ser Leu Ser Thr Cys Ser Thr Leu Asp Met Asp Gin Phe 20 25 30

Met Arg Lys Arg He Glu Ala He Arg Gly Gin He Leu Ser Lys Leu 35 40 45

Lys Leu Thr Ser Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60

Pro Glu Val He Ser He Tyr Asn Ser Thr Arg Asp Leu Leu Gin Glu 65 70 75 80

Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu 85 90 95

Glu Tyr Tyr Ala Lys Glu Val Tyr Lys He Asp Met Pro Pro Phe Phe 100 105 110

Pro Ser Glu Asn Ala He Pro Pro Thr Phe Tyr Arg Pro Tyr Phe Arg 115 120 125

He Val Arg Phe Asp Val Ser Ala Met Glu Lys Asn Ala Ser Asn Leu 130 135 140

Val Lys Ala Glu Phe Arg Val Phe Arg Leu Gin Asn Pro Lys Ala Arg 145 150 155 160

Val Pro Glu Gin Arg He Glu Leu Tyr Gin He Leu Lys Ser Lys Asp 165 170 175

Leu Thr Ser Pro Thr Gin Arg Tyr lie Asp Ser Lys Val Val Lys Thr 180 185 190

Arg Ala Glu Gly Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His 195 200 205 Glu Trp Leu His His Lys Asp Arg Asn Leu Gly Phe Lys He Ser Leu 210 215 220

His Cys Pro Cys Cys Thr Phe Val Pro Ser Asn Asn Tyr He He Pro 225 230 235 240

Asn Lys Ser Glu Glu Leu Glu Ala Arg Phe Ala Gly He Asp Gly Thr 245 250 255

Ser Thr Tyr Thr Ser Gly Asp Gin Lys Thr He Lys Ser Thr Arg Lys 260 265 270

Lys Asn Ser Gly Lys Thr Pro His Leu Leu Leu Met Leu Leu Pro Ser 275 280 285

Tyr Arg Leu Glu Ser Gin Gin Thr Asn Arg Arg Lys Lys Arg Ala Leu 290 295 300

Asp Ala Ala Tyr Cys Phe Arg Asn Val Gin Asp Asn Cys Cys Leu Arg 305 310 315 320

Pro Leu Tyr He Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp He His 325 330 335

Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr 340 345 350

Leu Trp Ser Ser Asp Thr Gin His Ser Arg Val Leu Ser Leu Tyr Asn 355 360 365

Thr He Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser Gin Asp 370 375 380

Leu Glu Pro Leu Thr He Leu Tyr Tyr He Gly Lys Thr Pro Lys He 385 390 395 400

Glu Gin Leu Ser Asn Met He Val Lys Ser Cys Lys Cys Ser 405 410

What is claimed is:

Claims

1. A method of treating hypotension comprising introducing into a mammal suspected of needing treatment for hypotension an effective amount of transforming growth factor fl (TGFfl) or an active fragment thereof.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said mammal is a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog or cat.

4. The method of claim 1, wherein said mammal is characterized by having severe inflammation.

5. The method of claim l , wherein said mammal is in septic shock.

6. The method of claim 1, further comprising introducing into said mammal an anti-inflammatory agent.

7. The method of claim l, wherein said anti- inflammatory agent is a glucocorticoid.

8. A method of inhibiting transcription of a gene encoding inducible nitric oxide synthase (iNOS) in an endothelial cell of a mammal, comprising identifying a mammal having an endothelial cell in need of inhibition of iNOS gene transcription and contacting said cell with an effective amount of TGFfl or an active fragment thereof.

9. The method of claim 8, wherein said mammal is a human.

10. The method of claim 8, wherein said mammal is a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog or cat.

11. The method of claim 8, wherein said mammal is characterized as having severe inflammation.

12. The method of claim 8, wherein said mammal is in septic shock.

13. The method of claim 8, wherein said TGFfl is TGFfl-l.

14. The method of claim 8, wherein said TGFfl is TGFfl-2.

15. The method of claim 8, wherein said TGFfl is a mixture of TGFfl-l and TGFfl-2.

16. The method of claim 8, wherein said endothelial cell is a smooth muscle cell.

17. The method of claim 16, wherein said smooth muscle cell is a vascular smooth muscle cell.

18. The method of claim 8, wherein said amount of TGFfl or active fragment thereof is effective to inhibit said iNOS gene transcription but does not inhibit transcription of a gene encoding constitutive nitric oxide synthase (cNOS) in said cell.

19. The method of claim 8, further comprising introducing into said mammal an anti-inflammatory agent.

20. The method of claim 19, wherein said anti- inflammatory agent is a glucocorticoid.