Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats.

OBJECTIVE: Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity). METHODS AND RESULTS: PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 µM PFT. CONCLUSIONS: Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis.

Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension.

Mutations in the gene encoding bone morphogenetic protein (BMP) receptor type 2 (BMPR-2) have been reported in pulmonary arterial hypertension (PAH), but their functional relevance remains incompletely understood. BMP receptor expression was evaluated in human lungs and in cultured pulmonary artery smooth muscle cells (PASMCs) isolated from 19 idiopathic PAH patients and nine heritable PAH patients with demonstrated BMPR-2 mutations. BMP4-treated PASMCs were assessed for Smad and p38 mitogen-activated protein kinase (MAPK) signalling associated with mitosis and apoptosis. Lung tissue and PASMCs from heritable PAH patients presented with decreased BMPR-2 expression and variable increases in BMPR-1A and BMPR-1B expression, while a less important decreased BMPR-2 expression was observed in PASMCs from idiopathic PAH patients. Heritable PAH PASMCs showed no increased phosphorylation of Smad1/5/8 in the presence of BMP4, which actually activated the p38MAPK pathway. Individual responses varied from one mutation to another. PASMCs from PAH patients presented with an in vitro proliferative pattern, which could be inhibited by BMP4 in idiopathic PAH but not in heritable PAH. PASMCs from idiopathic PAH and more so from heritable PAH presented an inhibition of BMP4-induced apoptosis. Most heterogeneous BMPR-2 mutations are associated with defective Smad signalling compensated for by an activation of p38MAPK signalling, accounting for PASMC proliferation and deficient apoptosis.

Cirrhosis ameliorates monocrotaline-induced pulmonary hypertension in rats.

Common bile duct ligation (CBDL) induces biliary cirrhosis and pulmonary vasodilatation. We tested whether CBDL ameliorates monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. Five groups of rats were studied: controls; rats dosed with MCT (60 mg.kg(-1) subcutaneously); CBDL; rats dosed with MCT followed by CBDL on day 7; and rats dosed with MCT followed by CBDL (day 7) and L-NAME therapy between days 24 and 28. 28-day survival was 26% in the MCT group and 72% in the MCT+CBDL group. Pulmonary vascular resistance measured on days 21 and 28 increased in the MCT and MCT+CBDL+L-NAME groups, but returned to normal in the MCT+CBDL group on day 28. Pulmonary artery (PA) medial hypertrophy persisted in MCT+CBDL rats. PA inflammation increased in MCT+CBDL rats, with accumulation of both intra- and perivascular macrophages. Exhaled nitric oxide (NO) levels decreased in the MCT group and increased in the MCT+CBDL group, which showed upregulation of inducible NO synthase and normal endothelial NO synthase. Blood endothelin (ET)-1 increased in CBDL, MCT, and MCT+CBDL rats. Levels of ET(B) receptors increased and ET(A) receptors decreased in the MCT+CBDL group, whereas the opposite changes occurred in the MCT group. Biliary cirrhosis induces pulmonary vasodilation that ameliorates MCT-induced PH and improves survival. Upregulation of inducible NO synthase and ET(B) receptor and downregulation of ET(A) receptor may be involved.

Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice.

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.

Lessons from oncology to understand and treat pulmonary hypertension.

Pulmonary artery hypertension (PAH) is now considered to be a proliferative disorder characterised by unexplained proliferation of pulmonary artery smooth muscle cells (PA-SMCs) and pulmonary artery endothelial cells (PA-ECs). An abnormal phenotype of PA-SMCs and PA-ECs has been described in PAH and some analogies now appear between pulmonary vascular cells from patients with PAH and cancer cells. Such analogies are discussed here with respect to essential hallmarks of cancer cells and with the hope that new treatments targeted at one or more of these cancer cell abnormalities may be appropriate for PAH.

Fractalkine-induced smooth muscle cell proliferation in pulmonary hypertension.

Pulmonary hypertension is characterised by a progressive increase in pulmonary arterial resistance due to endothelial and smooth muscle cell proliferation resulting in chronic obstruction of small pulmonary arteries. There is evidence that inflammatory mechanisms may contribute to the pathogenesis of human and experimental pulmonary hypertension. The aim of the study was to address the role of fractalkine (CX3CL1) in the inflammatory responses and pulmonary vascular remodelling of a monocrotaline-induced pulmonary hypertension model. The expression of CX3CL1 and its receptor CX3CR1 was studied in monocrotaline-induced pulmonary hypertension by means of immunohistochemistry and quantitative reverse-transcription PCR on laser-captured microdissected pulmonary arteries. It was demonstrated that CX3CL1 was expressed by inflammatory cells surrounding pulmonary arterial lesions and that smooth muscle cells from these vessels had increased CX3CR1 expression. It was then shown that cultured rat pulmonary artery smooth muscle cells expressed CX3CR1 and that CX3CL1 induced proliferation but not migration of these cells. In conclusion, the current authors proposed that fractalkine may act as a growth factor for pulmonary artery smooth muscle cells. Chemokines may thus play a role in pulmonary artery remodelling.

The serotonin pathway in pulmonary hypertension.

The nature of the primary defect responsible for triggering and maintaining pulmonary artery smooth muscle (PA-SMC) proliferation in pulmonary artery hypertension (PH) is poorly understood but may be either an inherent characteristic of PA-SMCs or a secondary response to an external abnormality, such as up-regulation of growth factors. In previous studies, we found that cultured PA-SMCs from patients with idiopathic PH (iPH) had an abnormally strong proliferative response to serotonin or serum (which contains high levels of serotonin). This abnormal response is due to overexpression of the serotonin transporter (5-HTT) which mediates the mitogenic action of serotonin. That 5-HTT plays a key role in pulmonary vascular remodeling is supported by experimental studies showing that transgenic animals overexpressing 5-HTT in smooth muscle (at a level close to that seen in PH) spontaneously develop pulmonary vascular remodeling and PH. Conversely, mice with targeted S-HTT gene disruption are protected against hypoxic PH, and selective 5-HTT inhibitors reverse or prevent experimental PH. In patients with chronic lung disease, a close association has been found between a 5-HTT gene polymorphism and the severity of pulmonary hypertension. Agents capable of selectively inhibiting 5-HTT-mediated PA-SMC proliferation deserve to be investigated as potential treatments for pulmonary hypertension.

[Serotonin and pulmonary arterial hypertension]. / Sérotonine et hypertension artérielle pulmonaire.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is related to hypertrophic remodelling of the pulmonary vessels. Proliferation of pulmonary vascular smooth muscle cells (PA-SMCs) plays a key role in these changes. BACKGROUND: The PA-SMCs of patients suffering from idiopathic or secondary PAH are characterized by abnormally increased in vitro proliferation in response to serotonin or serum. Serotonin transporter (5-HTT), the mediator of the mitogenic activity of serotonin, and the expression of which is increased in the course of idiopathic or secondary PAH, is the basis of these effects. The overexpression of 5-HTT, selectively induced in vascular smooth muscle by transgenesis in the mouse, leads to the development of PAH. Treatment with selective 5-HTT inhibitors prevents or leads to complete reversal of experimental hypoxic or inflammatory PAH. The presence of polymorphism of the gene causing over expression of the protein and proliferation of PA-SMCs seems to determine the severity of certain types of human PAH, notably that complicating COPD. VIEWPOINT: There is a potential therapeutic application of 5-HTT inhibitors in human PAH. A clinical study is currently taking place in France. CONCLUSION: The exploration of the role of 5-HTT and the mechanisms leading to its overexpression in PAH, as well as the interactions between 5-HTT and the BMPR2 gene, should lead to a significant increase in the understanding of the pathophysiology of the disease.

Deficiency of the 5-hydroxytryptamine transporter gene leads to cardiac fibrosis and valvulopathy in mice.

BACKGROUND: Serotonin (5-hydroxytryptamine; 5-HT) overproduction is responsible for cardiac valvular disease in patients with carcinoid tumors. Reduced 5-HT inactivation is one proposed mechanism of the valvulopathy observed in individuals treated with the appetite suppressants fenfluramine and phentermine. One key protein limiting systemic availability of 5-HT is the 5-HT transporter (5-HTT) expressed by platelets and pulmonary vascular cells; 5-HTT is responsible for 5-HT uptake and subsequent inactivation of the amine passing through the lung. Here we investigated whether 5-HTT-deficient (5-HTT-KO) mice developed structural and/or functional cardiac abnormalities and valvulopathy. METHODS AND RESULTS: Cardiac endothelial cells expressed large amounts of 5-HTT in wild-type mice. 5-HTT deficiency appeared to be associated with marked interstitial, perivascular, and valvular fibrosis as evidenced by staining of cardiac collagen in 5-HTT-KO mice. Histological analysis provided evidence for valvulopathy characterized by valvular hyperplasia and prominent fibrosis at the attachment site and base of the leaflets. Echocardiography revealed an increase in left ventricular lumen diameter and a decrease in left ventricular diameter fractional shortening. Although 5-HT1B receptors mediated the 5-HT-induced collagen secretion by human cardiac myofibroblasts, the contribution of this receptor type to valvulopathy was ruled out because double-KO mice deficient in both 5-HTT and 5-HT1B receptors showed the same cardiac alterations as 5-HTT-KO mice. CONCLUSIONS: The present results establish a link between 5-HTT and the development of cardiac fibrosis and valvulopathy in vivo. 5-HTT-KO mice represent an especially relevant model for studying the mechanisms by which 5-HT induces valvulopathy.

Smooth muscle cell matrix metalloproteinases in idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) results from persistent vasoconstriction, smooth muscle growth and extracellular matrix (ECM) remodelling of pulmonary arteries (PAs). Matrix metalloproteinases (MMPs) are matrix-degrading enzymes involved in ECM turnover, and in smooth muscle cell (SMC) and endothelial cell migration and proliferation. MMP expression and activity are increased in experimental PAH. Therefore, this study investigated whether similar changes occur in idiopathic PAH (IPAH; formerly known as primary pulmonary hypertension). Both in situ and in vitro studies were performed on PAs from patients undergoing lung transplantation for IPAH and from patients treated by lobectomy for localised lung cancer, who served as controls. In IPAH, MMP-tissue inhibitor of metalloproteinase (TIMP) imbalance was found in cultured PA-SMCs, with increased TIMP-1 and decreased MMP-3. MMP-2 activity was markedly elevated as a result of increases in both total MMP-2 and proportion of active MMP-2. In situ zymography and immunolocalisation showed that MMP-2 was associated with SMCs and elastic fibres, and also confirmed the MMP-3-TIMP-1 imbalance. In conclusion, the findings of this study were consistent with a role for the matrix metalloproteinase-tissue inhibitor of metalloproteinase system in pulmonary vascular remodelling in idiopathic pulmonary arterial hypertension. The matrix metalloproteinase-tissue inhibitor of metalloproteinase imbalance may lead to matrix accumulation, and increased matrix metalloproteinase-2 activity may contribute to smooth muscle cell migration and proliferation. Whether these abnormalities are potential therapeutic targets deserves further investigation.

Pathobiology of pulmonary arterial hypertension.

Recent years have witnessed important advances in the understanding of the pathophysiology of primary pulmonary hypertension (PPH). Both genetic and mechanistic studies have succeeded in identifying new molecular pathways relevant to the process of pulmonary vascular remodelling, which underlies PPH. Mutations in the type II bone morphogenetic protein (BMP) receptor (BMPR)-II are now considered to be the genetic basis for familial PPH and approximately 30% of cases of sporadic PPH. The identification of the relevance of the BMP pathway to the aetiology of PPH now raises many questions about the link between the BMPR-II mutant genotype and the PPH phenotype. As PPH does not develop in all subjects with BMPR-II mutations, environmental or associated genetic factors may play a crucial role. Among these, the finding of an association between PPH and the L-allelic variant of the serotonin transporter (5-HTT) gene indicates that 5-HTT, which controls smooth muscle hyperplasia, probably contributes to susceptibility to PPH or is an important modifier of the PPH phenotype. Recognition of these molecular pathways should provide insight into the pathogenesis not only of primary pulmonary hypertension, but also of secondary forms of pulmonary hypertension. This should soon lead to the development of new and more selective therapeutic approaches to pulmonary hypertension.

Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension.

Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is a hallmark pathological feature of primary pulmonary hypertension (PPH). Here we found that PA-SMCs from patients with PPH grow faster than PA-SMCs from controls when stimulated by serotonin or serum and that these effects are due to increased expression of the serotonin transporter (5-HTT), which mediates internalization of indoleamine. In the presence of 5-HTT inhibitors, the growth stimulatory effects of serum and serotonin were markedly reduced and the difference between growth of PA-SMCs from patients and controls was no longer observed. As compared with controls, the expression of 5-HTT was increased in cultured PA-SMCs as well as in platelets and lungs from patients with PPH where it predominated in the media of thickened pulmonary arteries and in onion-bulb lesions. The L-allelic variant of the 5HTT gene promoter, which is associated with 5-HTT overexpression and increased PA-SMC growth, was present in homozygous form in 65% of patients but in only 27% of controls. We conclude that 5-HTT activity plays a key role in the pathogenesis of PA-SMC proliferation in PPH and that a 5HTT polymorphism confers susceptibility to PPH.

Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats.

The appetite suppressant dexfenfluramine, which inhibits neuronal 5-HT uptake and elevates plasma 5-HT levels, has been associated with an increase in the relative risk of developing primary pulmonary hypertension. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PA-SMCs), an effect that depends upon activity of the 5-HT transporter (5-HTT). To investigate the relationship between dexfenfluramine and pulmonary hypertension, we examined 1) the effect of dexfenfluramine on 5-HT uptake by PA-SMCs and the mitogenic response of these cells to 5-HT, and 2) 5-HTT mRNA in lung tissue from normoxic and chronically hypoxic rats during and at discontinuation of a 4-week dexfenfluramine treatment (2 mg/kg/day). In cultured PA-SMCs, dexfenfluramine (10(-6) M) markedly reduced [3H]5-HT uptake and [3H]thymidine incorporation in response to 5-HT (10(-6) M). In lungs from rats exposed to 4-week hypoxia (10% O(2)), 5-HTT mRNA levels were higher than in normoxic rats (233.5 +/- 22.5 versus 121.8 +/- 4.8 amol/mg of RNA, P < 0.05), but were not affected by concomitant treatment with dexfenfluramine. One week after discontinuation of dexfenfluramine, 5-HTT mRNA levels increased substantially, this effect being additive with that of hypoxia (364.0 +/- 13.1 in hypoxic versus 164.2 +/- 10 amol/mg of RNA in normoxic rats). When exposure to 2 weeks of hypoxia followed discontinuation of a 4-week treatment, right ventricular hypertrophy was more severe and muscularization of distal pulmonary arteries more marked (P < 0.01) than in rats pretreated with the vehicle. These data show that, in rats, the increased 5-HTT expression that follows dexfenfluramine discontinuation promotes the development of hypoxic pulmonary hypertension.

Adenovirus-mediated atrial natriuretic protein expression in the lung protects rats from hypoxia-induced pulmonary hypertension.

Endogenous as well as exogenous atrial natriuretic peptide (ANP) attenuates the development of chronic hypoxic pulmonary hypertension (CHPH) in rats. We built a recombinant adenovirus type 5 containing ANP cDNA under the control of the Rous sarcoma virus long terminal repeat (Ad.ANP). The efficiency of this vector in delivering the ANP gene was first examined in rat primary cultures of pulmonary vessel smooth muscle cells (SMCs) in comparison with Ad.beta GAL. Conditioned medium collected from Ad.ANP-infected cells (1000 TCID(50)/cell) contained 5 x 10(9) M immunoreactive ANP and elicited relaxation of isolated rat pulmonary arteries preconstricted with phenylepinephrine. To examine the effects of adenovirus-mediated ANP expression in the CHPH rat lung, Ad.ANP or Ad.beta GAL was administered via the tracheal route. Immunoreactive ANP was detected in bronchoalveolar fluid as early as 4 days and until 10-17 days after Ad.ANP administration (5 x 10(8) TCID(50)). Lung ANP immunostaining was mainly localized in bronchial and alveolar epithelial cells. As compared with Ad.beta GAL-treated controls, rats given Ad.ANP (5 x 10(8) TCID(50)) on the day before a 2-week exposure to hypoxia (10% O(2)) had lower values for pulmonary artery pressure (32.1 +/- 1.93 vs. 35.5 +/- 2 mmHg, p < 0.01) and Fulton's index (0.52 +/- 0.089 vs. 0.67 +/- 0.12, p < 0.001) and less severe right ventricular hypertrophy and distal vessel muscularization. These results suggest that induction of ANP expression in the lung may hold promise in the treatment of pulmonary hypertension.

Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension.

Structural remodelling of pulmonary vessels is an important feature of pulmonary hypertension (PH), which reflects distal artery muscularization and matrix remodelling. The matrix metalloproteinases (MMPs) are involved in extracellular matrix turnover and hence, in smooth muscle cell migration and endothelial cell migration and proliferation. Among the MMPs, gelatinases (MMP-2 and MMP-9) can degrade basement membrane components and promote cell proliferation and migration. This study evaluated gelatinases in pulmonary vessels during progressive PH in two rat models: exposure to hypoxia or monocrotaline. Zymography of tissue homogenates revealed an association of progression of hypoxic PH with a time-dependent increase in gelatinase MMP-2 activity, specific to pulmonary vessels. Increased MMP-2 activity was also found 30 days postmonocrotaline. Reverse transcription polymerase chain reaction demonstrated upregulation of MMP-2 messenger ribonucleic acid. Immunolocalization showed MMP-2 throughout the pulmonary vasculature, from the trunk to the distal vessels, with strong staining of the intima, media and adventitia. MMP-2 was found in its active form and gelatinolytic activity was correlated to PH severity. Activity localization by in situ zymography corroborated with the immunolocalization findings. In conclusion, the authors demonstrated that matrix metalloproteinase-2 activity is increased in pulmonary vessels during progression of pulmonary hypertension, probably as a result of involvement in the matrix turnover associated with vascular remodelling during pulmonary hypertension.

Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats.

Chronic hypoxic pulmonary hypertension (PH) is associated with vasoconstriction and structural remodeling of pulmonary vessels including narrowing of the arterial lumen and loss of distal functional arteries. To test whether lung overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) is beneficial in hypoxic PH, recombinant adenovirus encoding the human VEGF 165 gene under the control of a cytomegalovirus promoter (Ad. VEGF) or control vector containing no gene in the expression cassette (Ad.Null) was administered intratracheally to rats. With Ad. VEGF (10(8) plaque-forming units [pfu]), VEGF protein was present in bronchoalveolar lavage fluid as early as 2 d and until 17 d after gene transfer, but was not detected in serum. Only small patchy areas of mononuclear cells without cell damage, edema, or hemorrhage were observed on lung histology with no significant change in lung permeability. In rats pretreated with Ad.VEGF (10(8) pfu) 2 d before a 2-wk exposure to hypoxia (10% O(2)), lower values versus Ad. Null-pretreated controls were found for pulmonary artery pressure (25 +/- 1 versus 30 +/- 2 mm Hg, P < 0.05), right ventricular over left ventricular-plus-septum weight (0.37 +/- 0.01 versus 0.47 +/- 0. 02, P < 0.001), normalized wall thickness of 50- to 200-microm vessels (P < 0.001), and muscularization of distal vessels (P < 0. 001). Pretreatment with Ad.VEGF (10(8) pfu) increased endothelial nitric oxide synthase activity in lung tissue and partially restored endothelium-dependent vasodilation in isolated lungs from chronically hypoxic rats, as assessed by improvement of ionophore A23187-induced vasodilation and attenuation of endothelin-1 (300 pmol)-induced vasoconstriction, an effect abolished in the presence of nitro-L-arginine methylester. We conclude that adenoviral-mediated VEGF overexpression in the lungs attenuates development of hypoxic PH, in part by protecting endothelium-dependent function.

Imbalance between platelet vascular endothelial growth factor and platelet-derived growth factor in pulmonary hypertension. Effect of prostacyclin therapy.

Focal vascular injury and impaired endothelial function are features of pulmonary hypertension (PH) that lead to enhanced platelet endothelial cell interactions. Vascular endothelial growth factor (VEGF) is contained in platelets and released at sites of vascular injury to promote endothelial repair and wound healing in combination with platelet-derived nonspecific mitogens such as platelet-derived growth factor (PDGF). The overall balance between platelet VEGF and PDGF was investigated in 21 patients with primary PH, 8 with secondary PH, and 27 with chronic hypoxemic lung disease (CHLD), as well as in 29 control subjects. Platelet VEGF content was increased in patients with primary and secondary PH as compared with control subjects (518 +/- 89, 675 +/- 156, and 166 +/- 29 fg/10(5) platelets, respectively; p < 0.01), whereas platelet PDGF content was similar in the three groups (31 +/- 2, 36 +/- 4, and 33 +/- 3 pg/10(5) platelets, respectively; NS). Patients treated with a continuous prostacyclin infusion had a higher platelet VEGF but a similar platelet PDGF content as compared with untreated patients. Moderate increases in platelet VEGF and PDGF contents were observed in the CHLD patients. We conclude that patients with primary or secondary PH have an increase in platelet VEGF content, but not in platelet PDGF content, and that their platelet VEGF content increases further in response to prostacyclin infusion. We suggest that imbalance between platelet VEGF and PDGF is beneficial to patients with PH.

Inhibition of matrix metalloproteinases by lung TIMP-1 gene transfer or doxycycline aggravates pulmonary hypertension in rats.

Chronic hypoxic pulmonary hypertension (PH) results from persistent vasoconstriction, excess muscularization, and extracellular matrix remodeling of pulmonary arteries. The matrix metalloproteinases (MMPs) are a family of proteinases implicated in extracellular matrix turnover and hence in smooth muscle and endothelial cell migration and proliferation. Because MMP expression and activity are increased in PH, we designed the present study to investigate whether inhibition of lung MMPs in rats subjected to chronic hypoxia (CH) contributes to or protects against vascular remodeling and PH. To achieve lung MMP inhibition, rats exposed to 10% O(2) for 15 days were treated with either doxycycline (20 mg/kg per day by gavage starting 2 days before and continuing throughout the CH period) or a single dose of recombinant adenovirus (Ad) for the human tissue inhibitors of metalloproteinases-1 (hTIMP-1) gene (Ad.hTIMP-1, 10(8) plaque-forming units given intratracheally 2 days before CH initiation). Control groups either received no treatment or were treated with an adenovirus containing no gene in the expression cassette (Ad.Null). Efficacy of hTIMP-1 gene transfer was assessed both by ELISA on bronchoalveolar lavages and by hTIMP-1 immunofluorescence on lung sections. MMP inhibition in lungs was evaluated by in situ zymography and gelatinolytic activity assessment using [(3)H]gelatin. Rats treated with either doxycycline or Ad.hTIMP-1 had higher pulmonary artery pressure and right heart ventricular hypertrophy more severe than their respective controls. Worsening of PH was associated with increased muscularization and periadventitial collagen accumulation in distal arteries. In conclusion, our study provides compelling evidence that MMPs play a pivotal role in protecting against pulmonary artery remodeling.