WO1996008268A1

WO1996008268A1 - Inhibition of endothelin-1 to reduce inflammatory processes

Info

Publication number: WO1996008268A1
Application number: PCT/US1995/011937
Authority: WO
Inventors: Mary E. Russell; Mohamed Sayegh; Bruno Watschinger
Original assignee: President And Fellows Of Harvard College; Brigham & Women's Hospital
Priority date: 1994-09-14
Filing date: 1995-09-12
Publication date: 1996-03-21
Also published as: EP0784478A1; JPH10508830A; CA2199939A1

Abstract

The vasoactive and mitogenic peptide, endothelin-1 (ET-1) is upregulated within grafts, and methods of diagnosing and treating graft rejection, arteriosclerosis, and other cell-mediated inflammatory conditions are provided. These methods involve identifying and reducing expression of ET-1.

Description

INHIBITION OF ENDOTHELIN-1 TO REDUCE INFLAMMATORY

PROCESSES

Background of the Invention The field of the invention is inflammation and tissue and organ transplant rejection.

Tissue, cardiac, and other solid-organ transplants are often compromised by acute or chronic rejection, typically characterized by the development of transplant- associated arteriosclerosis. This limits the long-term survival of grafts in humans, since the progressive thickening and occlusion of the blood vessel eventually results in ischemic injury to the organ or tissue. This stenosis occurs only in the grafted tissue and not in host arteries, and indicates that the pathological inflammatory processes occur only in the donor tissue. Histologically, lesions related to graft rejection differ from common chronic arteriosclerosis, in that transplant arteriosclerosis involves the artery in a concentric rather than eccentric fashion; lipid accumulation is less common in the early development of the transplant- associated lesion, and the development of the disease is faster. Monocytes and macrophages accumulate in the early stages of graft rejection, followed by macrophage and smooth muscle cell accumulation, and in the later, more obliterative stage, smooth muscle cells predominate. It is likely that an immune response stimulates expression of a variety of growth factors and cytokines which are involved in the pathogenesis of graft rejection.

Endothelin-1 (ET-1) , a 21 a ino acid peptide, was first isolated from the supernatant of cultured endothelial cells. Besides having potent vasoconstrictive effects, ET-1 has been shown to exert a mitogenic effect on endothelial and smooth muscle cells in vitro and in the neointima after injury from balloon angioplasty in vivo.

Plasma ET-1 levels are elevated at various time points after uncomplicated transplantation of hearts, other solid organs, and in patients with severe symptomatic atherosclerosis. In atherosclerotic lesions, ET-1 protein and mRNA are increased compared with normal vessels.

ET-1 binds to two distinct G-protein coupled receptors, ET_A and ET_B. These receptors are expressed in endothelial cells and smooth muscle cells, but in the course of normal chronic atherosclerosis, receptor expression decreases.

Summary of the Invention We have discovered that endothelin-1 mRNA and protein are upregulated in grafts undergoing acute or chronic rejection, and are expressed by infiltrating mononuclear cells.

Accordingly, in one aspect, the invention features a method to reduce graft rejection, graft-induced arteriosclerosis, or other graft-induced chronic cell- mediated inflammatory processes in a mammal. The method(s) involve inhibiting the expression of ET-1 or ET-1 activity on the transplanted or inflamed tissue by administering a therapeutic compound to the graft either before or after implantation to the host mammal. A therapeutic compound is a drug, nucleic acid, chemical, or protein compounded in a suitable excipient which will have a beneficial effect, such as inhibiting graft rejection or arteriosclerosis. The terms •^■grafted" and "transplanted" are used interchangeably herein, and indicate a graft of any tissue transplanted between genetically nonidentical individuals or species. Allografts are between genetically nonidentical individuals of the same species, xenografts are between genetically nonidentical members of different species. The methods of the invention can be used for both types of grafts. These terms are distinguishable from an isograft, meaning a tissue or organ transplanted between genetically identical individuals or the same individual. "ET-l activity" means the biological functions of ET-1, vasoconstriction and mitogenic properties which are exerted on, e.g., smooth muscle cells and endothelial cells. Preferably, an alloimmune-induced arteriosclerosis aspect of the graft rejection is treated by this method. By "alloimmune" is meant an immunologic response induced by the engrafted tissue, regardless of its source (e.g., allografts, xenografts). In preferred embodiments, the graft being spared from rejection is a solid organ, more preferably a heart. The source and host animal for the graft or inflammation is a mammal, preferably a rodent, and more preferably a human patient. Preferably, transcription or translation of ET-1 is reduced or inhibited in mononuclear cells which have infiltrated the graft and/or endothelial cells. These cells are preferably host mononuclear cells but may also be donor mononuclear cells. By "mononuclear cells" is meant macrophages, monocytes, lymphocytes, and any other cell types which are typically referred to as mononuclear in the art.

In other embodiments, the inhibition of the effects of ET-1 is brought about by inhibiting transcription or translation of ET-l mRNA. In preferred embodiments, this regulation of expression occurs in mononuclear cells and/or endothelial cells, more preferably mononuclear cells which have infiltrated the graft or area of inflammation. Alternatively, the inhibition of ET-l effects is achieved by means of inhibiting translation of ET-l mRNA into ET-l polypeptide, preferably by introducing antisense DNA into ET-l expressing cells (e.g., mononuclear cells). By antisense DNA is meant any DNA of 15 nucleotides or longer which is complementary to a region of the ET-l nucleotide sequence (SEQ ID NO: ), and can be identified and generated using methods well known in the art.

The diagnostic assays and therapeutics of the invention encompass the use of isolated DNA containing part or all of the sequence of a mammalian ET-l. In preferred embodiments, the DNA encodes human ET-l, however, any mammalian DNA encoding ET-l is included for use in the invention as long as it has preferably 50% sequence identity with the human ET-l sequence, more preferably 70% and most preferably over 90% sequence identity with the human sequence. Also included are vectors containing the isolated DNA; cells, which can be prokaryotic or eukaryotic, containing the isolated DNA; and the use of methods of manufacturing recombinant ET-l known in the art, such as methods of culturing the cells containing isolated ET-l DNA under conditions permitting expression of the DNA. An "isolated DNA", as used herein, refers to a DNA sequence which may be single stranded or double stranded, sense or antisense and which has been removed from the sequences which flank it in a naturally occurring state, e.g., the sequences adjacent to the DNA sequence in a genome in which it naturally occurs. The term includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Other embodiments include inhibiting ET-l activity by use of a blocking agent (e.g., physically), inhibiting agent (an agent that restrains or retards physiologic, chemical, or enzymatic action; depression or arrest of function) , or antagonizing agent (an agent which opposes or resists the action of another; agents that tend to neutralize or impede the action or effect of others; e.g., something which binds or interacts with the ET-l receptor and doesn't have the biological activity of ET-

1). There are two types of G-protein linked ET-l receptors, ET_A and ET_B; either or both of these receptor types are subject to blocking, inhibiting or antagonizing in preferred embodiments of the invention. In preferred embodiments, these receptor's function is inhibited by interfering with any portion of the G-protein cascade, including subsequent cellular effects (e.g., calcium influx into the receptor-bearing cell) . Agents working on the receptor may be peptide (at least 5 amino acids in length) or non-peptide. ET-l expression or activity preferably may be inhibited by agents which directly interact with a translated ET-l product, e.g., an endothelin converting enzyme inhibitor to prevent posttranslational modifications which may be essential for ET-l activity. In preferred embodiments, whichever inhibitor of ET-l activity or expression is used results in a reduction of smooth muscle cell migration or proliferation in the intimal layer of arteries of the graft. The intima is defined as the region between the lumen and the internal elastic lamina of the artery; the media is defined as the region between the internal and external elastic laminae; and the adventitia is defined as the region outside the external elastic lamina of the artery. The graft may be treated prior to transplantation; preferably by suffusing, soaking, or perfusing it with a therapeutic composition of the invention.

The invention also features a method of detecting graft rejection, graft-induced arteriosclerosis, or any other graft pathophysiology in a transplant recipient or patient. The method involves analyzing a sample from the patient for increased expression of ET-l in the graft, which, if present, indicates ongoing pathophysiology. This method can be performed and ET-l expression detected very soon following the transplant, within 1 day, preferably within 3 days, more preferably within 5 days. It can be performed throughout the life of the graft. In preferred embodiments, the increased expression is detected in graft-infiltrating mononuclear cells. Another featured method to detect graft rejection, graft-induced arteriosclerosis, or any other graft pathophysiology in a transplant recipient, is an in vivo method which involves detection of a labelled molecule which is targeted specifically for cells producing ET-l or ET-l itself. In a preferred embodiment, a detectable label is linked to an ET-l specific antibody or antibody fragment (Fab, Fab₂) , administered to a graft recipient, and the graft imaged. This label is preferably radioactive, more preferably has a short half-life, and most preferably is ^1:llIn. Use of this label has been reported in Yasuda et al . , 1987, Circulation 76:306-311; Frist et al . , 1987, Circulation 76(Suppl V):V81-V85; both hereby incorporated by reference. However, the label can be a fluorescent label for in vitro use, or a spin label, or any other suitable label known to those skilled in the art. All methods are suitable for use in vitro, e.g., by in situ hybridization, and can be detected by any standard method, e.g., x-ray techniques, epifluorescence microscopy, NMR, etc. An elevated level of label in the graft is indicative of graft pathophysiology. Tissue can be obtained from the graft or from a blood sample which is then enriched using standard methods for mononuclear cells. Mononuclear cell activation, or an upregulation of ET-l expression, can be assayed using standard techniques such as PCR, Northern, Southern, and dot blotting; in situ hybridization, antibody assays such as ELISAs, Western blots, etc., and such activation in peripheral blood samples will be indicative of graft rejection, graft-arteriosclerosis, or other graft pathophysiology.

Another method featured in the invention is delivery of gene therapy to a grafted tissue, either prior to engraftment or after engraftment. The gene therapy may involve delivering a DNA encoding ET-l polypeptide, polypeptide analog, or a fragment thereof; or encoding an antisense DNA, which is complementary to at least 15 nucleotides of the sequence of ET-l, preferably SEQ ID NO:4. Delivery is effected by administering the nucleic acid in a suitable vector for expressing therapeutic amounts of gene transcript to the graft and/or infiltrating mononuclear cells.

Additionally featured is a method of screening test compounds for potential utility as therapeutic compounds for use in the methods of the invention relating to inhibiting ET-l activity and expression. The method includes administering a test compound to a graft either prior to or following transplant into a mammal, allowing sufficient time for graft rejection to progress, if present, removing a sample of the graft and a sample of normal host tissue or tissue from a control animal, and comparing the expression of ET-l in the samples. Samples will be normalized to produce a value of ET- 1/cell. If the expression of ET-l in the graft is less than 3-4 fold greater than the baseline expression in the control tissue ("differential expression") , it will indicate that the test compound has potential utility to inhibit expression or activity of ET-l and may further be useful to inhibit graft rejection, arteriosclerosis, and pathophysiology. The term "differentially expressed" refers to the ET-l gene transcript in a graft which is substantially greater or less than the amount of the same transcript found in the surrounding host tissue or in normal controls. By the term "gene transcript" is meant a mRNA or cDNA. Peptides derived from the sequence of ET-l (SEQ ID NO:3 (amino acid) or SEQ ID NO:4 (nucleotide) ) may be used to generate antibodies or directly for therapeutic purposes (e.g., competition with endogenous ET-l polypeptide for receptor sites) . Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering, e.g., cloning the gene or a portion of the gene encoding ET-l into an expression vector as described above. Also included in therapies of the invention are the use of analogs of the above peptides. Analogs can differ from the native peptide by conservative amino acid replacements which alter the sequence but do not adversely affect the functioning of the resulting polypeptide, or by modifications which do not affect the sequence, or by both. Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivitization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Other ET-l-related polypeptides which can be used in therapeutic compositions of the invention include polypeptide analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases. Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as a therapeutic. Such mimetics, and methods of incorporating them into polypeptides, are well known in the art. Similarly, the replacement of an L-amino acid residue with a D-amino acid residue is a standard way of rendering the polypeptide less sensitive to proteolysis. Also useful are amino-terminal blocking groups such as t- butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,- dinitrophenyl.

This invention offers improvements over the prior art in terms of more directed therapeutics, diagnostics, and screening for new therapeutics. The graft-rejecting screening tests can be performed very soon following transplantation, allowing a much sooner therapeutic intervention (e.g., immunosuppression, compositions of this invention) ; this will enhance graft survival and make possible grafts between individuals with larger histocompatibility differences than has been possible previously.

It was previously unknown that ET-l is upregulated in grafts, and that infiltrating host mononuclear cells are the primary source of the ET-l in rejecting or arteriosclerosing grafts; knowledge of this expression pattern in cardiac and other transplantation will allow direct targeting by therapeutics of this detrimental cell-mediated inflammatory mechanism. Other features and advantages of the invention will be apparent from the following detailed description and other embodiments of the invention, and from the claims.

Brief Description of the Drawings Fig. 1A is a graph showing the time course of ET-l mRNA expression in the acute cardiac graft rejection model. Corrected levels of expression were derived by normalizing ET-l reverse transcriptase PCR values against those for a control gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) , and are shown in relative units. There was a significant increase in graft mRNA at day 5 when compared with the paired host heart and allografts at day 3 and 7 (p<0.002 and p<0.01, respectively) (n=3/group/time point) .

Fig. IB is a comparison of ET-l mRNA expression in allografts, isografts, and paired spleens in the acute (day 5 after transplantation) and the chronic allograft rejection (day 75 after engraftment) models. ET-l mRNA expression is significantly upregulated in rejecting allografts (p <0.02 in acute rejection and p<0.0007 in chronic rejection (n=2-3/group/time point) .

Fig. 1C is a graph showing the time course of ET-l mRNA expression in the chronic cardiac allograft rejection model. Corrected levels of expression were derived as above, and are shown in relative units. There was a significant increase in allograft mRNA at days 7, 28 and 75 when compared with the paired host heart (p=0.02, 0.003 and 0.001, respectively) (n=2/group/time point) .

Fig. 2 is the amino acid sequence of human ET-l. Fig. 3 is the cDNA sequence of human mRNA for ET- 1.

Detailed Description Acute and chronic rejection are major complications which develop following cardiac transplantation. Levels of endothelin-1 (ET-l) are elevated in plasma from patients with grafts and those with symptomatic vascular atherosclerosis but little is known about its role in these processes. To determine the role of ET-l in graft rejection, we examined expression of ET-l during acute cardiac graft rejection and chronic rejection in which graft arteriosclerosis is a characteristic feature (Adams et al., Transplantation , 53(5) :1115-19 (1992)). The heterotopic cardiac transplant model allowed us to study the graft and the recipient's host heart (exposed to the same circulation but without development of vascular lesions) , which was not removed during transplantation.

Our study using these acute and chronic transplant rejection models is the first in vivo documentation to show that mononuclear cells as well as endothelial cells can be induced to produce ET-l in response to alloimmune stimuli. This suggests a pivotal role for these inflammatory cells and the cytokines they produce in stimulating local upregulation of ET-l in vivo, and in contributing to graft rejection and arteriosclerosis.

EXPERIMENTAL METHODS:

Heterotopic cardiac transplantation:

Adult male Lewis, WF and F-344 rats (8-10 weeks old) were obtained from Harlan Sprague-Dawley (Inc.) and Charles River Laboratories (Kingston, NY) . Abdominal heterotopic rat cardiac transplantation was performed as described below, and in (Sayegh et al., Transplantation, 51:296-99 (1991)), hereby incorporated by reference. In the acute rejection model, Lewis rats received WF cardiac grafts which reject usually between day 6 and 8 after engraftment, as defined by the cessation of palpable heart beat (Sayegh et al., 1991, supra) . In these animals, organs were harvested at day 3, 5 and 7 after engraftment (n=3/group/time point) . In the chronic rejection studies (detailed below by way of example) , Lewis grafts were transplanted into F-344 rats (Adams et al., 1992, supra , and harvested at day 7, 28, and 75, representing various stages of graft arteriosclerosis after transplantation (n=2/group/time point) . Isografts harvested at day 5 (WF -> WF; n=3) and day 75 (LEW -> LEW, n=3) served as controls for the acute and chronic allograft rejection, respectively. Transplant function was followed by daily palpation of the heartbeat. Midventricular sections of the grafts, the paired host heart and in some cases the spleens were processed for mRNA extraction and histological evaluation, as described in Russell et al., Proc . Natl . Acad. Sci . USA, 90:6086-90 (1993); Russell et al., J . Clin . Invest . , 94: (1994), hereby incorporated by reference. The animals received no immunosuppression, in order to study the influence of unmodified immunologic injury on ET-l expression. Specific details of the procedure are provided below to illustrate one of the allograft models.

Lewis to F344 rat cardiac transplantation

The combination of Lewis rat donors and F344 rat recipients results in long-term graft survival and a time-dependent development of arteriosclerotic lesions that resemble those in human transplant vessels and organs upon histological examination (Cramer et al., Transplantation 47:414-419, 1993; Adams et al., 1992, supra) , and thus this is a suitable animal model for allograft rejection and arteriosclerosis, as well as other inflammatory diseases. Immunohistochemical studies using antibodies against monocytes, T-cells, and smooth muscle cells have shown that arteriosclerotic lesions develop in 3 distinct stages (Cramer et al., 1992, J. Heart Lung Transplant , 11:458-466; Adams et al.. Transplantation , 56:794-799 (1993)). In the first 30 days, the neointimal lesions are composed of infiltrating inflammatory cells (rather than smooth muscle cells) , which are predominantly macrophages with fewer lymphocytes. Between 45 and 90 days, the infiltrating inflammatory cell population in the neointima decreases as intimal smooth muscle cells appear. In the last phase (beyond 90 days) , the neointima is maximally expanded, often obliterative, and composed predominantly of smooth muscle cells with fewer infiltrating mononuclear cells. The early and persistent presence of monocytes/macrophages in the first stage of arteriosclerosis suggests a prominent role for the macrophage in the initial phase of chronic rejection. To date there are few studies examining specific molecular mechanisms that may regulate the infiltration or function of macrophages in chronically rejecting hearts.

Heterotopic abdominal cardiac transplantation was performed using Lewis donor hearts as described (Adams et al., 1992, supra) in an allogeneic combination involving F344 recipients for the chronic rejection model, and WF donor hearts into Lewis hosts for the acute rejection model. At the time of harvest, both the host (recipient) and the transplanted hearts were collected for histologic analysis and RNA extraction. The host heart served as a reference that had been exposed to the same circulation but was normal on histologic examination. ET-l Gene Transcript Analysis:

ET-l oligonucleotides spanning exon/intron borders within the coding region were chosen using the MAC VECTOR* program (Int. Biotechnologies Inc, New Haven, CT) . The 5'primer (AT GGA TTA TTT TCC CGT GAT C; SEQ ID NO:l) and 3'primer (C TGT AGT CAA TGT GCT CGG; SEQ ID NO:2) generated a 614 bp fragment. PCR studies were carried out on a GeneAmp system 9600 (total vol, 25μl) as described in (Russell et al., supra (1993); Russell et al., supra (1994)). Reaction conditions included 1.25μl cDNA, lμM (each) 5'and 3'primer, lOmM TrisHCL/50mM KCl/1.5mM MgCl2/0.001% (wt/vol) gelatin/800/iM dNTPs/0.625 unit of A pliTaq DNA polymerase. The amplified product was cloned directly into the PCRII vector (Invitrogen, San Diego, CA) . Sequence analysis (Sequenase 2.0 Kit, United States Biochemical) by the dideoxynucleotide chain-termination method confirmed that the amplified PCR fragment, had a 100% identity with the previously submitted rat ET-l sequence (GenBank accession number m647ll; Sakurai et al. Biochem. Biophys . Res . Commun . , 175:44-47 (1991)).

.ET-l Gene Transcript Assay

In an effort to conserve RNA we measured gene transcript levels using a published reverse transcription PCR assay (Russell et al., 1994, J. Clin . Invest . , supra) . To identify the optimum PCR conditions for accurate measurement of gene transcript levels, we established the logarithmic assay range with respect to cycle number and starting template concentration against different dilutions of cDNA. Subsequent measurements of ET-l transcript levels were then completed at 28 Cycles using 1.25 μl cDNA in a 25_/xl reaction (denaturation at 94°C for 15 sec, annealing at 50°C for 20 sec, and extension at 72°C for 60 sec with a final extension of 7 min at the end of all cycles. Glyceraldehyde-3- phosphate dehydrogenase (G3PDH; amplification for 22 cycles at 94°C, 58°C and 72°C) , which represents an ubiquitously expressed mRNA, was used as an internal reference to reflect total cellular RNA, as previously described (Russell et al., 1993, Proc . Natl . Acad . Sci . USA. , supra , Russell et al., 1994, J. Clin . Invest , supra .

For semiquantitative PCR analysis 150000 cpm of ³²P dCTP (800Ci/mMol; 1 Ci=37GBq; DuPont/NEN) was included per reaction. Negative control experiments were performed by substituting water for cDNA or omitting reverse transcriptase in cDNA synthesis. Agarose gels (1%) containing the amplified products were dried and exposed to storage phosphor plates for 20-24 hrs.

Incorporated ³²P in the amplified band was measured on a PhosphorImager system (Imagequant software. Molecular Dynamics, Sunnyvale, CA) . Corrected values were derived by dividing the measured ³²P value for ET-l by the mean G3PDH value (obtained from quadruplicates) for the sample. Relative transcript levels were then determined from cDNA sets that included negative control samples (for which reverse transcriptase had been omitted during cDNA synthesis or water used instead of cDNA) performed in the same evaluation. The results represent the mean corrected levels obtained by pooling the levels obtained from 2 or 3 animals per group that had been analyzed in quadruplicate. Differences in the corrected ET-l mRNA expression were examined in three different studies: (A) - Time course in the acute rejection model, (B) -

Comparison of allografts, paired spleens and isografts for the acute (day 5) and the chronic (day 75) model and (C) - Time course in the chronic rejection model. Results from each experimental set were subjected to multivariate analysis of variance (MANOVA) without replication. Individual comparisons were made and the level of significance was corrected by the Bonferroni method.

Immunohi s tochemi s try Cardiac grafts, paired host hearts and spleens and isografts were embedded in OCT (Optimal Cutting Temperature compound) and stored at -70°C until sectioning on a cryostat. ET-l staining was performed using a polyclonal rabbit anti-ET-1 antibody raised against human ET-l and kindly provided by Biomedica,

Vienna, Austria. This antibody has previously been used to identify ET-l in human renal endothelial cells (Watschinger et al., Clin . Nephrol . , 41(2):86-93 (1994)). Negative controls included omission of the primary antibody and use of an irrelevant primary antibody (von Willebrand factor) .

The examples below are intended to illustrate the invention, and are not limiting.

EXAMPLE 1: ET-l mRNA expression ET-l mRNA expression increases in acute cardiac allograft rejection (WF into LEW) model :

ET-l gene transcript levels in acutely rejected allografts were significantly higher than in the paired host hearts at day 5, but not on day 3 and day 7 (n=3/group; p<0.002). (Figure 1A) . ET-l levels obtained from the allografts on day 7 were significantly lower than those on day 5 (p<0.02). Transcripts from the paired host hearts were low and comparable at all time points (day 3,5,7). As shown in Figure 1C, ET-l transcripts levels in allografted hearts at day 5 were significantly higher (p<0.05), than in day 5 control isografts, suggesting that the rejection process rather than the surgical procedure is responsible for the ET-l increase. ET-l mRNA expression in matched recipient spleens during the rejection process was significantly less than in the graft (p<0.02) and comparable to host heart and isograft transcript levels indicating that the microenvironment of the rejecting graft is crucial for the induction of ET-l synthesis in mononuclear cells.

Sustained increase in ET-l mRNA levels in chronic cardiac allograft rejection (LEW into F344) model :

ET-l transcript levels were significantly higher in cardiac allografts compared with paired host hearts at the time points examined (day 7, 28, 75) . These points were chosen to examine three stages of arteriosclerosis (Day 7 - when the adhesion of scattered mononuclear cells to the lumen is seen; day 28 - when mild degrees of concentric intimal thickening are first apparent, and day 75 when neointimal thickening and smooth muscle cell proliferation is a typical feature (Johnson et al., J. Heart Transplant , 8:349-59 (1989)). The upregulation of ET-l was seen early (day 7) and was sustained over time (p<0.003) (Figure 1C) . ET-l transcript levels in the paired host hearts were low throughout the observation period and comparable to those seen in the acute rejection model (Figure IB) . The spleens of the allograft-rejecting animals expressed significantly less ET-l mRNA (p<0.0007) than allografts at day 75, comparable to matched host heart and day 75 isografts (Figure 1C) , again indicating that there is intragraft upregulation of ET-l by stimuli in the allograft.

EXAMPLE 2: Immunohistochemical studies Expression of ET-l gene products as identified by a rabbit anti ET-l polyclonal antibody Host hearts

Endothelial cells of arteries, venules and a few capillaries stained positive for ET-l. Acute cardiac allograft rejection (WF into LEW) In contrast to normal hearts where only endothelial cells stained positive for ET-l, allografts undergoing acute rejection showed focal, positive staining for ET-l in mononuclear cells as well. ET-l immunopositive cells were seen as early as day 3, with a subsequent increase in the extent of labelling. At the later time point (day 7), the intensity of labelling per cell was reduced. Chronic cardiac alloσraft rejection (LEW into F344 )

In the chronically rejecting allograft, by day 7 endothelial cells and many of the mononuclear cells (>75%) showed a dense labelling with the anti-ET-1 antibody. After 28 days, the labelling of the endothelial cells was reduced in intensity and primarily restricted to endothelial cells adjacent to ET-l positive mononuclear cells and interstitial dendritic cells. Occasionally ET-l positive cells could be seen within the thickened intima. In contrast, on day 75 a marked increase in the intensity of discrete mononuclear cells labelling plus focal vascular intimal cell staining and medial smooth muscle cell staining was seen.

We have demonstrated that the allogeneic stimulus of a rejecting allograft leads to local upregulation of ET-1-mRNA and protein expression within the transplanted organ. Two distinct patterns of mRNA ET-l regulation were seen in our studies. In the acute allograft rejection model ET-l mRNA expression shows a distinct peak on day 5 after engraftment with a consecutive decrease on day 7. This reduction may reflect the decreasing viability of cells capable of RNA or protein synthesis, as the ongoing immunological process leads to necrosis of the allografted tissue (Sayegh et al., 1991, supra . In chronic rejection a significant increase in ET-l expression occurs by day 7 and persists throughout day 75. The upregulation of ET-l mRNA is restricted to allografts and does not occur in the paired host spleens or hearts, nor in isografts, and indicates that the allogeneic stimulus plays a crucial role in the local upregulation of ET-l expression.

IFN-gamma, TNF-alpha and IL-1, have been shown to induce ET-l production in cells maintained in culture (Lee et al., J. Biol . Chem . , 266:16188-192 (1991), Ohta et al., Biochem . Biophys . Res . Commun . , 169:578-84

(1990), Marsden et al.. Am . J. Physiol . , 262:C854-C861 (1992)). It has been recently established that graft infiltrating cells are the main source of these and other inflammatory cytokines during graft rejection (reviewed in (Halloran et al.. Transplant Proc , 21(l):26-30 (1989); Russell et al.. Transplantation , in press (1994)). Finally, in our study we used seriate sections to demonstrate that TNF-alpha and ET-l localized to the same set of mononuclear cells in transplant vessels. These cells are macrophages, monocytes, and lymphocytes. Macrophages and monocytes have been shown to express ET-l in vitro following stimulation (e.g., with phorbol esters) , but lymphocytes have not been shown to express ET-l (Ehrenreich et al . , 1990, supra) . Our findings suggest that lymphocytes may also play a role in the expression of ET-l in grafts. Taken together, these findings suggest that cytokines associated with the allogeneic milieu may contribute to increased ET-l production within the rejecting graft. Arteriosclerotic changes associated with chronic rejection develop in stages (Adams et al., 1992, supra) . Early in the course (day 7 to 14) mononuclear cells adhere to the vessel wall. At day 28 when intimal thickening due to inflammatory cells occurs, marked ET-l immunostaining was observed in monocytes and macrophages in the neointima in addition to the strong staining of vascular endothelial cells. At day 75, when the neointima is typically composed of both mononuclear and smooth muscle cells, ET-l staining showed marked increases in both neointimal cell types and some staining in medial cells.

Activated macrophages have been shown to play an important role in the development of graft arteriosclerosis (Russell et al., 1993, Proc . Natl . Acad. Sci. USA, supra , Russell et al., 1994, Transplantation, supra . It is likely that these macrophages might be supplying the necessary cytokines to stimulate endothelial cells to produce ET-l, or alternatively they could be contributing to the mononuclear cell production of ET-l.

The presence of ET-l immunoreactivity within the arteriosclerotic lesions of transplant vessels suggests a common role for ET-l, as a vasoactive substance and as a potential mitogen, in the development of various forms of arteriosclerosis.

EXAMPLE 3: Assays to detect rejection of a graft

Methods to detect rejection or arteriosclerotic changes of a graft involve detection of expression of the ET-l gene either at the level of transcription, e.g, by PCR, Northern blot, differential mRNA display, or in situ hybridization, or at the level of translation/protein production, e.g., by FACS, Western blot, or in situ immunostaining. Such detection methods provide a means for early detection of events which lead to graft rejection, and thus facilitate early intervention to prevent or inhibit rejection of the transplanted organ (e.g., by immunosuppression) . For example, ET-l transcript or protein levels in transplanted heart samples obtained by endomyocardial biopsy could serve as clinical markers of mononuclear cell infiltration. These levels might provide prognostic information about the degree of acute or chronic rejection or the rate at which arteriosclerosis is progressing. One advantage of such a diagnostic approach is that the diagnostic methods of the invention can be performed on a very small amount of tissue which may be obtained using standard biopsy techniques known in the art. Specimens of the transplanted tissue can be obtained by biopsy using common medical practice and prepared for analysis using standard histological or nucleic acid methodology. For example, tissues can be set into OCT or paraffin, or frozen prior to cutting into thin sections for histological evaluation, or the RNA and protein extracted and analyzed (e.g., by PCR, in situ hybridization, Western, Northern, or Southern blotting, etc.) .

Also included are diagnostic methods involving imaging of the graft in vivo or in vitro . One method involves linking a detectable label to a molecule which will target ET-l producing cell or ET-l itself. For example, antibodies or fragmented antibodies (Fab, Fab₂) specific for ET-l can be linked to a label (e.g., a radiolabel, a fluorescent label, a spin label, etc.) and administered to the human or animal which has received a tissue graft (or to a sample retrieved from the patient) . Following a suitable circulation/incubation time, appropriate means are used to image or detect the label (e.g., epifluorescence, x-ray detection, NMR) . If the assay is being done in vivo, the grafted tissue will be imaged, and the presence of detectable label in the graft as compared to control tissues (e.g., normal host tissue) will be indicative of upregulation of ET-l in the graft, and initiation of rejection or graft-induced arteriosclerosis. Jn vitro, standard in situ hybridization techniques can be used, or ELISA or Western blotting assays to detect an increase in expression of ET-l in the grafted tissue. Fab fragments can have two morphologies: a single binding site (univalent Fab) or two linked binding sites (bivalent Fab₂) . These can be produced and labelled using methods well known in the art (e.g. , papain digestion) , and the term Fab is used to include either form.

EXAMPLE 4: Therapeutic applications of ET-l Activity or Expression Inhibitors

Nucleic Acid Therapeutics

As described above, an increase in the amount of

ET-l gene transcript in a graft indicates graft rejection. Thus, graft rejection in patients may be decreased or inhibited using gene therapy in which the antisense strand of the upregulated ET-l gene is introduced into the cells in which the gene is transcribed. The antisense strand (either RNA or DNA) may be directly introduced into the cells in a form that is capable of binding to the transcripts, or a vector containing sequence which, once within the target cells, is transcribed into the appropriate antisense mRNA, may be the therapeutic administered to the patient's cells. Antisense nucleic acid which hybridizes to the complementary coding strand of DNA can decrease or inhibit production of the polypeptide product encoded by the upregulated ET-l gene, by associating with the normally single-stranded mRNA transcript, and thereby interfere with translation. Other gene therapies may involve the use of recombinant mutants (e.g., to compete with endogenous peptide for binding on ET-l receptors, or interfere with ET-l effects at the level of the gene promoter or enhancers) . The isolated DNA may be introduced into target cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others.

A therapeutic composition is provided which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid which is capable of inhibiting expression of ET-l gene in grafts and other inflammatory conditions, either directly or by encoding a transcript which inhibits expression of the gene. The therapeutic composition may also include a gene delivery system as described above.

Other Therapeutics Other therapeutic compositions are provided which are capable of inhibiting or reducing the effects of ET-l (e.g., the ET-l receptor antagonist SB 209670; Douglas et al . , 1994, Circ . Res . 75:190) on allografts, xenografts, or in other inflammatory conditions, such as autoimmune disorders, myocarditis, endocarditis, Lyme disease, and other chronic cell-mediated inflammatory conditions. The compounds may be peptide or non-peptide, and have actions on the expression of ET-l (e.g., transcription or translation of the gene) ; act on the ET-l receptors ET_A and ET_B at any level (e.g., binding or antagonism of the binding site, interference with the G-protein/kinase cascade which is activated following receptor binding) ; or act as posttranslational inhibitors on ET-l (e.g., Endothelin converting enzyme inhibitors) . It is likely that ET-l receptors may also be upregulated in inflamed or allografted tissues (e.g., on migrating or proliferating smooth muscle cells and endothelial cells) , so drugs and therapies directed to ET-l receptors are likely to be particularly useful in reducing the effects of ET-l in these conditions.

Administration of Therapeutics

Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A therapeutically effective amount is an amount of the DNA of the invention which is capable of producing a medically desirable result in a treated animal, e.g., downregulation of the differentially expressed allograft gene.

The compositions of the invention can be formulated for pharmaceutical, veterinary, and organ culture use (e.g., treatment of tissue or organ prior to transplantation) , optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form. In using the compounds of the invention, conventional pharmaceutical, veterinary, or culture practice may be employed to provide suitable formulations or compositions. Thus, for human or animal use, the formulations of this invention can be administered by parenteral administration, for example, intravenous, subcutaneous, intramuscular, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal, vaginal, or topical administration.

The formulations of this invention may also be administered by the use of surgical implants which release the compounds of the invention. These devices could be readily implanted into the graft prior to transplantation, and could be mechanical or passive. Mechanical devices, such as pumps, are well known in the art, as are passive devices (e.g., consisting of a polymer matrix which contains therapeutic formulations; these polymers may slowly dissolve or degrade to release the compound, or may be porous and allow release via pores) . Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations can be found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

The concentration of the compound in the formulations of the invention will vary depending upon a number of factors, including the dosage to be administered, and the route of administration. In general terms, the non-nucleotide therapeutics of the invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. General dose ranges are from about 0.01 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered is likely to depend upon the extent of progression of rejection, the overall health of the patient, and the route of administration.

Peptides may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Such methods are well known to those of ordinary skill in the art. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, intraperitoneal, and inhalation.

Dosages for the nucleic acid therapies of the invention will vary, but a preferred dosage for intravenous administration of nucleic acid therapies is from approximately 10⁶ to 10²² copies of the nucleic acid molecule.

EXAMPLE 5: Screening Assay for ET-l Inhibitors/Antagonists Screening for ET-l inhibitors and ET-l receptor antagonists can also be accomplished in vivo using the rat heterotopic cardiac transplant model described herein and in Sayegh et al., 1991, supra , and Adams et al., 1992, supra . For example, the organ to be grafted can be perfused or soaked in a solution containing a candidate compound prior to transplantation. The organ can then transplanted and monitored for indications of rejection. Transplant rejection can be monitored using conventional methods, e.g., sacrifice of the animal followed by gross examination of the tissue and histological studies, as well as the diagnostic assays of the invention, e.g., evaluating a tissue biopsy for the differential expression of ET-l in host and graft tissue. A decrease in gene expression over non-treated graft controls or a reduction in the physical characteristics of transplant rejection would indicate that the candidate compound inhibits graft rejection and could be useful in inhibiting symptoms of other chronic inflammatory conditions (e.g., autoimmune disorders, carditis, etc.). Test compounds may be any sort of drug, chemical, nucleic acid, polypeptide. A potential source of non- nucleic acid test compounds are those related to known immunosuppressive compounds (e.g., cyclosporin) and those related to known ET-l inhibitors, antagonist, and blockers (e.g., SB 209670), although any compound may be chosen for use in this screening assay. Nucleic acid test compounds are likely to be composed of nucleotides which are complementary (antisense DNA) to some portion of the ET-l expression machinery (e.g., the coding region of the ET-l gene, signal sequences, promoters, etc.) or encode a polypeptide which may mimic ET-l polypeptide but is biologically inactive (e.g., to compete for receptor binding with endogenous ET-l) ; the encoded polypeptide may have a tertiary structure which physically blocks the receptor or binds the ET-l polypeptide or gene. Potential protein test compounds may have these features as well, although any nucleic acid or protein compound or molecule may be used in the screening assay.

Additional Uses/Advantages

The methods of the invention should enhance long term graft survival due to early diagnosis and effective counter-measures to acute and chronic rejection by inhibition of ET-l activity and expression. Our findings show that rodent cardiac allograft rejection models may be used to elucidate the precise in-vivo role of ET-l as a vasoactive and/or mitogenic factor after cardiac transplantation. The availability of specific ET- receptor antagonists will provide potential therapeutic strategies to prevent or ameliorate graft arteriosclerosis.

Measurements of ET-l gene transcript or polypeptide product levels may serve as clinical or diagnostic indicators of mononuclear cell infiltration, chronic inflammation, transplant rejection, and arteriosclerosis. ET-l may be used to determine that mononuclear cells are activated, given that the results described herein show that ET-l transcripts are expressed in mononuclear cells in the cardiac allograft, but not in host tissue. All or part of the human or any mammalian ET-l DNA sequence which has at least 50% sequence identity, preferably 70%, more preferably 90% sequence identity with the human ET-l sequence can be used as a hybridization probe to identify ET-l upregulation for the purpose of diagnosing transplant rejection. Portions of the DNA can also be used as PCR primers to amplify ET-l sequences to identify expression of these genes in grafts for the purpose of diagnosing rejection, using e.g., differential display technology (Russell et al . , 1993, supra) .

All or part of the ET-l DNA can be cloned into an expression vector and used to produce polypeptides of ET- 1 for the purpose of immunizing animals to generate polyclonal or monoclonal antibodies. Such antibodies can then be used for therapeutic applications as described above or for diagnostic applications such as identification of ET-l polypeptides in grafts indicating ongoing transplant rejection. Fusion proteins of ET-l containing components known to block specific inflammatory factors may also serve as a way of modulating the inflammatory response. DNA containing a sequence that encodes part or all of the amino acid sequence of ET-l can be subcloned into an expression vector, using a variety of methods known in the art. For example, a recombinant polypeptide can be expressed as a fusion protein with maltose binding protein produced in E. coli . Using the maltose binding protein fusion and purification system (New England Biolabs) , the cloned human cDNA sequence can be inserted downstream and in frame of the gene encoding maltose binding protein (malE) , and the alE fusion protein can then be overexpressed. In the absence of convenient restriction sites in the human cDNA sequence, PCR can be used to introduce restriction sites compatible with the vector at the 5' and 3' end of the cDNA fragment to facilitate insertion of the cDNA fragment into the vector. Following expression of the fusion protein, it can be purified by affinity chromatography. For example, the fusion protein can be purified by virtue of the ability of the maltose binding protein portion of the fusion protein to bind to a ylose immobilized on a column.

To facilitate protein purification, the pMalE plasmid contains a factor Xa cleavage site upstream of the site into which the cDNA is inserted into the vector. Thus, the fusion protein purified as described above can then be cleaved with factor Xa to separate the maltose binding protein from recombinant human cDNA gene product. The cleavage products can be subjected to further chromatography to purify recombinant polypeptide from the maltose binding protein.

The purified recombinant gene product can then be used to raise polyclonal or monoclonal antibodies against the ET-l using well-known methods (see Coligan et al., eds., Current Protocols in Immunology, 1992, Greene Publishing Associates and Wiley-Interscience) . To generate monoclonal antibodies, a mouse can be immunized with the recombinant protein, and antibody-secreting B cells isolated and immortalized with a non-secretory myeloma cell fusion partner. Hybridomas are then screened for production of ET-1-specific antibody and cloned to obtain a homogenous cell population which produces a monoclonal antibody. Downregulation of ET-l expression in grafts will enhance the longevity of transplant survival and survival of the transplant recipient. Regulation of ET-l expression in inflammatory conditions such as autoimmune disorders, cardiac inflammation, Lyme disease, or other chronic cell-mediated inflammatory process will lead to better management of these conditions, resulting in less tissue scarring and degradation, and improvement in quality and length of life.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: President and Fellows of Harvard

College (ii) TITLE OF INVENTION: INHIBITION OF ENDOTHELIN-1

TO REDUCE INFLAMMATORY PROCESSES

(iii) NUMBER OF SEQUENCES: 4 (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

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version

#1.30B

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US95/ (B) FILING DATE: 12-SEP-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/307,354

(B) FILING DATE: 14-SEP-1994

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Clark, Paul T.

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 05433/013WO1

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGGATTATT TTCCCGTGAT C 21

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTGTAGTCAA TGTGCTCGG 19

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY : linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15

Leu Asp lie lie Trp 20

( 2 ) INFORMATION FOR SEQ ID NO : 4 :

( i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 1167 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

5 (ii) MOLECULE TYPE: cDNA

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

CTGCGCCAGG CGAACGGGTC CTGCGCCTCC TGCAGTCCCA GCTCTCCACC GCCGCGTGCG 60

CCTGCAGACG CTCCGCTCGC TGCCTTCTCT CCTGGCAGGC GCTGCCTTTT CTCCCCGTTA 120

AAGGGCACTT GGGCTGAAGG ATCGCTTTGA GATCTGAGGA ACCCGCAGCG CTTTGAGGGA 180 lOCTGAAGCTG TTTTTCTTCG TTTTCCTTTG GGTTCAGTTT GAACGGGAGG TTTTTGATCC 240

CTTTTTTTCA GAATGGATTA TTTGCTCATG ATTTTCTCTC TGCTGTTTGT GGCTTGCCAA 300

GGAGCTCCAG AAACAGCAGT CTTAGGCGCT GAGCTCAGCG CGGTGGGTGA GAACGGCGGG 360

GAGAAACCCA CTCCCAGTCC ACCCTGGCGG CTCCGCCGGT CCAAGCGCTG CTCCTGCTCG 420

TCCCTGATGG ATAAAGAGTG TGTCTACTTC TGCCACCTGG ACATCATTTG GGTCAACACT 480 lβCCGAGCACG TTGTTCCGTA TGGACTTGGA AGCCCTAGGT CCAAGAGAGC CTTGGAGAAT 540

TTACTTCCCA CAAAGGCAAC AGACCGTGAG AATAGATGCC AATGTGCTAG CCAAAAAGAC 600

AAGAAGTGCT GGAATTTTTG CCAAGCAGGA AAAGAACTCA GGGCTGAAGA CATTATGGAG 660

AAAGACTGGA ATAATCATAA GAAAGGAAAA GACTGTTCCA AGCTTGGGAA AAAGTGTATT 720

TATCAGCAGT TAGTGAGAGG AAGAAAAATC AGAAGAAGTT CAGAGGAACA CCTAAGACAA 780

2SCCAGGTCGG AGACCATGAG AAACAGCGTC AAATCATCTT TTCATGATCC CAAGCTGAAA 840

GGCAAGCCCT CCAGAGAGCG TTATGTGACC CACAACCGAG CACATTGGTG ACAGACTTCG 900

GGGCCTGTCT GAAGCCATAG CCTCCACGGA GAGCCCTGTG GCCGACTCTG CACTCTCCAC 960

CCTGGCTGGG ATCAGAGCAG GAGCATCCTC TGCTGGTTCC TGACTGGCAA AGGACCAGCG 1020

TCCTCGTTCA AAACATTCCA AGAAAGGTTA AGGAGTTCCC CCAACCATCT TCACTGGCTT 1080

2βCATCAGTGG TAACTGCTTT GGTCTCTTCT TTCATCTGGG GATGACAATG GACCTCTCAG 1140

CAGAAACACA CAGTCACATT CGAATTC 1167

What is claimed is:

Claims

1. A method of reducing graft rejection in a mammal, said method comprising administering to said mammal, or contacting said graft with, a compound which inhibits endothelin-1 activity in host mononuclear cells associated with said graft.

2. The method of claim l wherein said rejection is associated with alloimmune-induced arteriosclerosis.

3. A method of reducing graft arteriosclerosis in a mammal, said method comprising administering to said mammal a compound which inhibits expression of endothelin-1 in arteries of said graft.

4. The method of claim 1 or 3 wherein said graft is a solid organ.

5. The method of claim 4 wherein said organ is a heart.

6. The method of claim 1 or claim 3 wherein said mononuclear cells infiltrate said graft.

7. The method of claim 1 or claim 3 wherein said compound reduces transcription of endothelin-1 mRNA in said host mononuclear cells.

8. The method of claim 1 or claim 3 wherein said compound reduces translation of endothelin-1 mRNA in said host mononuclear cells.

9. The method of claim 8 wherein said reduction of translation is achieved by contacting said endothelin-

1 mRNA with antisense DNA complementary to said endothelin-1 mRNA.

10. The method of claim 8 wherein said reduction of translation is achieved by contacting said endothelin- 1 mRNA with antisense DNA complementary to a region of SEQ ID NO:4.

11. The method of claim 1 or 3 wherein said compound is an antagonist of an endothelin-1 receptor.

12. The method of claim 11 wherein said antagonist is a polypeptide at least 5 amino acids long.

13. The method of claim 11 wherein said antagonist is an antibody specific for said receptor.

14. The method of claim 11 wherein said antagonized receptor is ET_A.

15. The method of claim 11 wherein said antagonized receptor is ET_B.

16. The method of claim 1 or 3 wherein said compound is an inhibitor of the G-protein cascade linked to ET_A or ET_B.

17. The method of claim 1 or 3 wherein said therapeutic composition comprises an inhibitor of endothelin-1 posttranslational modification.

18. The method of claim 2 or 3 wherein said inhibition reduces smooth muscle cell migration or proliferation in the intimal layer of said arteries.

19. The method of claim 1 or claim 3 wherein said graft is suffused with said compound prior to transplantation.

20. A method of detecting graft rejection, graft- induced arteriosclerosis, or other graft pathophysiology in a transplant patient, said method comprising analyzing a sample from said patient for increased expression of endothelin-1 in said graft said increased expression being indicative of said pathophysiology.

21. The method of claim 20 wherein said increased expression is detected in graft-infiltrating mononuclear cells.

22. An in vivo method of detecting graft rejection, graft-induced arteriosclerosis, or other graft pathophysiology in a transplant patient, said method comprising administering to said patient an endothelin-1 specific antibody or Fab linked to a detectable label and imaging said graft, an elevated level of said label in said graft being indicative of said pathophysiology.

23. A method of modulating the expression of endothelin-1 in grafted tissue, said method comprising delivering a DNA encoding endothelin-1 polypeptide, polypeptide analog, or a fragment thereof; or encoding an antisense DNA, complementary to at least 15 nucleotides of the sequence of endothelin-1, to said tissue in a vector suitable to express a therapeutic quantity of gene transcript.

24. A method of determining whether a test compound inhibits the expression or activity of endothelin-1, said method comprising the steps of a) administering said test compound to a graft heterotopically implanted in a mammal, b) allowing sufficient time for graft rejection to progress, if present, - 37 - c) removing a sample of said graft and a sample of analogous host tissue, and d) comparing expression of endothelin-1 per cell in said samples, wherein differential expression of endothelin-1 less than four fold between said samples is indicative of said test compound's efficacy to inhibit said expression or activity of endothelin-1.