Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor, Elafin, to Maintain Graft Perfusion During Acute Rejection.

The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.

Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.

Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

Insulin resistance in pulmonary arterial hypertension.

Although obesity, dyslipidemia and insulin resistance (IR) are well known risk factors for systemic cardiovascular disease, their impact on pulmonary arterial hypertension (PAH) is unknown. The present authors' previous studies indicate that IR may be a risk factor for PAH. The current study has investigated the prevalence of IR in PAH and explored its relationship with disease severity. Clinical data and fasting blood samples were evaluated in 81 nondiabetic PAH females. In total, 967 National Health and Nutrition Examination Surveys (NHANES) females served as controls. The fasting triglyceride to high-density lipoprotein cholesterol ratio was used as a surrogate of insulin sensitivity. While body mass index was similar in NHANES versus PAH females (28.6 versus 28.7 kg.m(-2)), PAH females were more likely to have IR (45.7 versus 21.5%) and less likely to be insulin sensitive (IS; 43.2 versus 57.8%). PAH females mostly (82.7%) had New York Heart Association (NYHA) class II and III symptoms. Aetiology, NYHA class, 6-min walk-distance and haemodynamics did not differ between IR and IS PAH groups. However, the presence of IR and a higher NYHA class was associated with poorer 6-months event-free survival (58 versus 79%). Insulin resistance appears to be more common in pulmonary arterial hypertension females than in the general population, and may be a novel risk factor or disease modifier that might impact on survival.

Leishmania (L.) amazonensis: fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages.

[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a.

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Pathobiology of pulmonary hypertension. Extracellular matrix.

Changes in the extracellular matrix underlie the structural and functional abnormalities in the vessel wall that lead to progressive pulmonary vascular disease. Studies are reviewed aimed at addressing the cellular and molecular programs that regulate the production of the extracellular matrix describing new ways to arrest proliferation and migration of smooth muscle cells and to induce apoptosis. The latter can lead to the reversal of pathology at least in experimental animal models.

Arterial elastase activity after balloon angioplasty and effects of elafin, an elastase inhibitor.

Increased proteolytic activity may be a factor in intimal hyperplasia after balloon angioplasty (BA). The objectives of this study were to assess elastase activity after BA in a rabbit arterial double-injury model and the effects of elastase inhibition. Elastase activity increased immediately after BA, reached an 8-fold peak at 1 week, and declined to baseline levels by 4 weeks. Elastin zymography showed that the elastase activity was associated predominantly with a molecular mass of 25 kDa. Elastase activity was significantly inhibited in vitro by elafin and phenylmethylsulfonyl fluoride, selective inhibitors of serine elastases. A second group of animals was transfected after BA with a plasmid containing the cDNA for either elafin or a control (chloramphenicol acetyltransferase, CAT) construct by using a hemagglutinating virus of Japan-liposome transfection technique. Arterial segments were obtained at 48 hours, 1 week, and 4 weeks to assess transgene expression, arterial wall elastase activity, and intimal cross-sectional area, respectively. Elafin transgene expression was evident at 48 hours and resulted in a significant (80%) inhibition of elastase activity compared with chloramphenicol acetyltransferase-transfected arteries. There was a 43% reduction in intimal cross-sectional area in elafin-transfected arteries (0.28+/-0.22 versus 0.16+/-0.07 mm(2) for CAT-transfected versus elafin-transfected arteries, respectively; P<0.05). These data suggest that an early increase in serine elastase activity after BA contributes to intimal hyperplasia. Serine elastase inhibition may be a potential therapeutic approach to inhibit intimal hyperplasia.

Tumor necrosis factor-alpha induces fibronectin synthesis in coronary artery smooth muscle cells by a nitric oxide-dependent posttranscriptional mechanism.

Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (P<0.05). This correlated with increased NO production (P<0.05) and fibronectin synthesis (P<0.05). TNF-alpha induction of fibronectin synthesis was abrogated by the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (P<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (P<0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P<0.05). Northern blotting showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. TNF-alpha increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.

Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice.

We cloned a rat vascular chymase (RVCH) from smooth muscle cells (SMCs) that converts angiotensin I to II and is up-regulated in SMC from spontaneously hypertensive vs. normotensive rats. To determine whether increased activity of RVCH is sufficient to cause hypertension, transgenic mice were generated with targeted conditional expression of RVCH to SMC, with the use of the tetracycline-controlled transactivator (tTA). We confirmed conditional expression of RVCH by mRNA, protein, and chymase activity in the absence, but not in the presence, of dietary doxycycline. The systolic blood pressure (mmHg), measured by carotid artery cannulation at 10-12 weeks of age, was higher in tTA+/RVCH+ mice than in nonbinary transgenic littermates (136 +/- 4 vs. 109 +/- 3) (P < 0.05), as were the diastolic and mean pressures. Hypertension was completely reversed by doxycycline, suggesting a causal link with chymase expression. Medial thickening of mesenteric arteries from tTA+/RVCH+ mice vs. littermates (0.82 +/- 0.1 vs. 0.42 +/- 0.02) (P < 0.05) was associated with increased SMC proliferation, as judged by positive immunoreactivity, with the use of an antibody to the proliferating cell nuclear antigen. These structural changes were prevented by doxycycline. Perfusion myography of mesenteric arteries from tTA+/RVCH+ mice also revealed increased vasoconstriction in response to phenylephrine and impaired metacholine-induced vasodilatation when compared with littermate controls or with the doxycyline-treated group. Our studies suggest that up-regulation of this vascular chymase is sufficient to cause a hypertensive arteriopathy, and that RVCH may be a candidate gene and a therapeutic target in patients with high blood pressure.

A novel vascular smooth muscle chymase is upregulated in hypertensive rats.

While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell-expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.

Hemodynamic unloading leads to regression of pulmonary vascular disease in rats.

OBJECTIVE: Treatment options for patients with advanced pulmonary vascular disease caused by a congenital heart defect are still mainly limited to heart-lung transplantation or lung transplantation with repair of the cardiac lesion. Because we have previously shown that the structural changes associated with pulmonary hypertension can be reversed by stress unloading in an organ culture model, we now investigate whether hemodynamic unloading will lead to regression of pulmonary vascular disease in the intact animal. METHODS: Right middle and lower lobectomy and monocrotaline injection were performed in Lewis rats (n = 22) to cause pulmonary vascular disease from a combined hemodynamic and toxic injury. Twenty-eight days later the left lungs were examined (n = 10) or exposed to normal pulmonary artery pressure for an additional 14 (n = 5) or 28 (n = 7) days by transplantation into healthy recipients. Pulmonary artery pressure, ventricular weight, and pulmonary artery morphology were evaluated in each group. RESULTS: Pulmonary hypertension (50 vs 16 mm Hg; P <.001) and right ventricular hypertrophy (right ventricular/left ventricular weight 0.69 vs 0.32; P <.001) associated with pulmonary artery medial hypertrophy (28.2% vs 7.2% wall thickness; P <.001) and muscularization of small pulmonary arteries (92.3% vs 19.4%; P <.001) developed by day 28 (compared with untreated controls). However, transplantation into healthy recipients effectively unloaded the lungs (mean pulmonary artery pressure 17 and 24 mm Hg at 14 and 28 days after transplantation) and resulted in progressive normalization of medial hypertrophy (15.6% and 12.1% at 14 and 28 days) and muscularization (65.1% and 42.2% at 14 and 28 days) relative to nontransplanted controls (P <.005 in each case). CONCLUSIONS: Hemodynamic unloading of lungs with pulmonary vascular disease results in progressive normalization of pulmonary artery structure. These results are the first to provide a rationale for attempting to induce regression of pulmonary vascular disease by pressure unloading of the pulmonary circulation. Methods to mechanically unload the pulmonary circulation should be critically evaluated as a strategy for staged surgical repair of congenital heart defects despite presumed irreversible pulmonary hypertension.

Infection of Vero cells with Coxiella burnetii phase II: relative intracellular bacterial load and distribution estimated by confocal laser scanning microscopy and morphometry.

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is an intracellular pathogen not yet grown axenically. Confocal laser fluorescence microscopy and morphometry were used to measure relative C. burnetii phase II loads and their intracellular distribution in aldehyde fixed and DAPI stained Vero cell monolayers. The fluorescence of single horizontal optical sections provided useful information on relative loads of bacteria in cells and vacuoles. The relative density of the bacteria in the vacuoles was inferred from ratios of fluorescence to vacuolar section areas. Relative bacterial loads, bacterial densities and section areas of large vacuoles increased exponentially between days 2 and 4 of the infection of gamma-irradiated host cells, stabilized between days 4 and 6, and decreased thereafter. Estimated minimum doubling times were higher for the overall complement of the intracellular organisms (about 12 h) than for bacteria that were confined to larger vacuoles (about 10 h).

Gene transfer of the serine elastase inhibitor elafin protects against vein graft degeneration.

BACKGROUND: Leukocyte infiltration and serine elastase activity lead to smooth muscle cell proliferation in association with posttransplant coronary arteriopathy and may also be involved in vein graft neointimal formation. A number of therapies have targeted cellular proliferation, but the inhibition of serine elastase-mediated extracellular matrix remodeling has not been investigated as a potential strategy to prevent neointimal formation and subsequent atherosclerotic degeneration in vein grafts. METHODS AND RESULTS: We studied jugular vein grafts 48 hours after interposition into the carotid arteries of rabbits and demonstrated inflammatory cell infiltration and elevated serine elastase activity, a stimulus for matrix remodeling and deposition of elastin. Therefore, elastolytic activity in vein grafts was targeted through transient expression of the selective serine elastase inhibitor elafin with hemagglutinating virus of Japan liposome-mediated gene transfer. Elafin transfection reduced inflammation by 60% at 48 hours and neointimal formation by approximately 50% at 4 weeks after implantation. At 3 months, a 74% decrease in neointimal elastin deposition correlated with protection against cholesterol-induced macrophage infiltration and lipid accumulation, which were both reduced by approximately 50% in elafin-transfected grafts relative to controls. CONCLUSIONS: Gene transfer of the selective serine elastase inhibitor elafin in vein grafts is effective in reducing the early inflammatory response. Although transient expression of elafin delays neointimal formation, it is also sufficient to cause an alteration in elastin content of the extracellular matrix, making it relatively resistant to atherosclerotic degeneration.

Synthesis of extracellular matrix and adhesion through beta(1) integrins are critical for fetal ventricular myocyte proliferation.

Extracellular matrix (ECM) regulates vascular smooth muscle cell proliferation. The role of ECM in myocardial growth is unexplored. We sought to determine whether human fetal ventricular myocytes (HFVMs) produce ECM and whether synthesis and attachment to ECM are necessary for their epidermal growth factor (EGF)-dependent and -independent proliferation. Cultured HFVMs proliferate in the presence but not absence of serum and EGF, as determined by increase in cell number and [(3)H]thymidine and [(14)C]leucine incorporation (measures of DNA and protein synthesis, respectively). Using a cyanogen bromide digestion technique to measure collagen and elastin and using affinity chromatography for fibronectin, we found that HFVMs synthesized collagen and fibronectin but not elastin. HFVMs grown on exogenous ECM (including fibronectin and type I collagen and laminin) demonstrated no change in proliferation or DNA and protein synthesis with or without EGF. However, inhibition of collagen synthesis using cis-4-hydroxyproline resulted in a decrease in EGF-related HFVM proliferation and DNA and protein synthesis, which was reversed by exposure to L-proline but not by growth on type I collagen. Use of beta(1) but not beta(3) integrin antibody to inhibit cell interaction with ECM resulted in a decrease in HFVM proliferation and DNA and protein synthesis in response to EGF. Furthermore, EGF-dependent proliferation was enhanced by alpha(1)beta(1) and alpha(5)beta(1) antibodies that act as functional ligands, but not alpha(3)beta(1), the only beta(1) subtype expressed in adult myocytes. In conclusion, proliferating HFVMs synthesize collagen and fibronectin. The proliferative response of HFVMs to EGF requires the synthesis of collagen as well as attachment to specific alpha/beta(1) integrin heterodimers.

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own.

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.

Isolated ductus arteriosus aneurysm in the fetus and infant: a multi-institutional experience.

OBJECTIVES: The purpose of this study was to describe the clinical characteristics and outcome and to elucidate the pathogenesis of ductus arteriosus aneurysm (DAA). BACKGROUND: Ductus arteriosus aneurysm is a rare lesion that can be associated with severe complications including thromboembolism, rupture and death. METHOD: We reviewed the clinical records, diagnostic imaging studies and available histology of 24 cases of DAA, diagnosed postnatally (PD) in 15 and antenatally (AD) in 9 encountered in five institutions. RESULTS: Of PD cases, 13 presented at <2 months, and all AD cases were detected incidentally after 33 weeks of gestation during a late trimester fetal ultrasound study. Of the 24, only 4 had DAA-related symptoms and 6 had associated syndromes: Marfan, Smith-Lemli-Opitz, trisomies 21 and 13 and one possible Ehlers-Danlos. Three had complications related to the DAA: thrombus extension into the pulmonary artery, spontaneous rupture, and asymptomatic cerebral infarction. Six underwent uncomplicated DAA resection for ductal patency, DAA size or extension of thrombus. In the four examined, there was histologic evidence of reduced intimal cushions in two and abnormal elastin expression in two. Five of the 24 died, with only one death due to DAA. Of 19 survivors, all but one remain clinically asymptomatic at a median follow-up of 35 months; however, two have developed other cardiac lesions that suggest Marfan syndrome. A review of 200 consecutive third trimester fetal ultrasounds suggests an incidence of DAA of 1.5%. CONCLUSIONS: Ductus arteriosus aneurysm likely develops in the third trimester perhaps due to abnormal intimal cushion formation or elastin expression. Although it can be associated with syndromes and severe complications, many affected infants have a benign course. Given the potential for development of other cardiac lesions associated with connective tissue disease, follow-up is warranted.

Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor.

Progression of pulmonary hypertension is associated with increased serine elastase activity and the proteinase-dependent deposition of the extracellular matrix smooth muscle cell survival factor tenascin-C (refs. 1,2). Tenascin-C amplifies the response of smooth muscle cells to growth factors, which are also liberated through matrix proteolysis. Recent organ culture studies using hypertrophied rat pulmonary arteries have shown that elastase inhibitors suppress tenascin-C and induce smooth muscle cell apoptosis. This initiates complete regression of the hypertrophied vessel wall by a coordinated loss of cellularity and extracellular matrix. We now report that elastase inhibitors can reverse advanced pulmonary vascular disease produced in rats by injecting monocrotaline, an endothelial toxin. We began oral administration of the peptidyl trifluoromethylketone serine elastase inhibitors M249314 or ZD0892 21 days after injection of monocrotaline. A 1-week treatment resulted in 92% survival, compared with 39% survival in untreated or vehicle-treated rats. Pulmonary artery pressure and muscularization were reduced by myocyte apoptosis and loss of extracellular matrix, specifically elastin and tenascin-C. After 2 weeks, pulmonary artery pressure and structure normalized, and survival was 86%, compared with 0% in untreated or vehicle-treated rats. Although concomitant treatment with various agents can reduce pulmonary hypertension, we have documented complete regression after establishment of malignant monocrotaline-induced disease.