|Home | About | Journals | Submit | Contact Us | Français|
Proliferation of pulmonary arterial smooth muscle cells, endothelial-dysfunction, oxidative stress and inflammation promote the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exerts anti-oxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteries remain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonary hypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment (25 mg/kg/day, p.o., from day 1 post-monocrotaline) attenuated right ventricular systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (TNFα, IL-1β, IL-6, PDGFα/β) and limited leukocyte infiltration in the lung. Resveratrol also inhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol a) increased expression of eNOS, b) decreased oxidative stress and c) improved endothelial function in small pulmonary arteries. Pulmonary hypertension was associated with an up-regulation of NAD(P)H oxidase in small pulmonary arteries, which was significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory, anti-oxidant and anti-proliferative effects in the pulmonary arteries, which may contribute to prevention of pulmonary hypertension.
Pulmonary hypertension is a syndrome that encompasses several diseases, all of which have in common increased pulmonary artery pressures. Idiopathic (“primary”) pulmonary hypertension is a rare disease caused by genetic defects in the bone morphogenetic protein signaling pathways. Common causes of “secondary” forms of pulmonary hypertension include (i) pulmonary hypertension associated with chronic obstructive pulmonary disease, (ii) pulmonary embolism, and (iii) pressure/volume overload-related pulmonary hypertension. In addition, pulmonary hypertension often develops in patients with autoimmune diseases or as a severe side effect of anorectic drug treatment. Despite the diverse etiological differences, many similarities in the pathological alterations in pulmonary arteries occur among the various forms of pulmonary hypertension, i.e. vascular remodeling, including cellular proliferation in both the intima and media; endothelial dysfunction/increased vasoconstriction; and activation of inflammatory processes (e.g. inflammatory cytokine expression, monocyte infiltration).
Current therapies for chronic pulmonary hypertension are designed to reduce pulmonary arterial resistance by inducing vasodilation (e.g. NO inhalation, stimulation of cGMP production by phosphodiesterase inhibitors, endothelin receptor antagonists, prostacyclin analogs). These therapeutic approaches mainly provide symptomatic relief. Novel treatments are required to prevent progression of pulmonary hypertension by interfering with the pathomechanisms of the disease at multiple levels. For example, in a pre-clinical setting experimental therapeutics that exert anti-mitogenic effects on proliferation of pulmonary arterial smooth muscle cell (PASMC)1–3 in addition to promoting vasodilation show promise in enhancing overall prognosis.
Resveratrol (3,5,4'-trihydroxystilbene) is a dietary polyphenolic compound, which exerts significant anti-oxidant, anti-inflammatory and endothelial protective effects in the systemic circulation4–7. Importantly, our previous study shows that resveratrol inhibits proliferation of cultured aortic smooth muscle cells8. Thus, we hypothesize that resveratrol exerts anti-proliferative, anti-oxidant, anti-inflammatory and endothelial protective effects in the pulmonary circulation and prevents the progression of pulmonary hypertension.
To test this hypothesis we investigated the chronic efficacy of oral resveratrol treatment in monocrotaline-treated rats. Rats develop severe pulmonary hypertension after a single injection of monocrotaline (MCT)9 and this model mimics several key aspects of both primary and secondary forms of human pulmonary hypertension, including vascular remodeling, proliferation of pulmonary arterial smooth muscle cells, oxidative stress, endothelial dysfunction and up-regulation of inflammatory cytokines and leukocyte infiltration. We particularly addressed the question of whether resveratrol exerts beneficial effects on proliferation of pulmonary arterial smooth muscle cells, pulmonary arterial endothelial function and inflammation.
A detailed Methods section can be found in the Online Supplement (please see http://hyper.ahajournals.org).
Animal use protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College, Valhalla, NY. To assess preventive effects of resveratrol on MCT-induced pulmonary hypertension, adult male Sprague-Dawley rats (300 g) were randomized to the following groups (n=6 animals in each group) to receive either vehicle of resveratrol in the drinking water: control animals for 14 and 21 days; MCT (60 mg/kg s.c.) -treated animals for 14 and 21 days; MCT-treated animals receiving resveratrol (25 mg/kg/day, p.o., in the drinking water) from day 1 to day 14 or day 21 after MCT injection.
Three or 5 weeks after MCT administration, rats were anesthetized with ketamine (60 mg/kg i.m.) and xylazine (3 mg/kg i.m.). A microtip Millar pressure catheter was introduced into the right ventricle through the jugular vein to measure right ventricular pressure. After hemodynamic measurements the thorax was opened and pieces of the left lung and isolated segments of small intrapulmonary arteries were processed for histological evaluation or frozen in liquid nitrogen for subsequent biochemical measurements.
The heart was dissected, and the ratio of the right ventricular free wall weight divided by the length of the tibia was calculated as an index of right ventricular hypertrophy (which is unaffected by changes in body weight).
Immunofluorescent labeling for α-smooth muscle actin was performed to visualize the medial layer of the vessels. Index of smooth muscle hypertrophy of small pulmonary arteries (d: 50–150 μm) was calculated. Cell proliferation was also assessed in the walls of distal pulmonary vessels by use of a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The percentage of PCNA-positive cells was calculated in 10 randomly chosen fields. To quantify the rate of endothelial cell apoptosis, TUNEL assay was performed as described7.
To assess the anti-proliferative effects of resveratrol in vitro, primary human pulmonary arterial smooth muscle cells (PASMC) were stimulated in vitro with PDGF (10 ng/mL) in the presence or absence of resveratrol. Cultured cells were stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole and cell number was determined using a hemocytometer. The inhibitory effect of resveratrol on cell proliferation was also tested using a colony formation assay. In other experiments PASMC grown in a 96 well plate were stimulated in vitro with TGFβ (10 ng/mL), IL-1β (10 ng/mL) or IL-6 (10 ng/mL) in the presence or absence of resveratrol. Cell proliferation was assessed by measuring the fluorescence of the DNA-binding CyQUANT dye (Invitrogen) in cell lysates with a fluorescence microplate reader. The effect of resveratrol on apoptotic cell death (annexin V and TUNEL staining) in PASMC and pulmonary arterial endothelial cells (PAEC) were quantified by flow cytometry (Guava Easycyte). Resveratrol-induced inhibition of the cell cycle in PASMC was assessed by the Guava Cell Cycle Assay by flow cytometry.
Acetylcholine-induced relaxation was assessed in segments of small intrapulmonary arteries pre-contracted by phenylephrine. In separate experiments the vasorelaxant effect of in vitro administration of resveratrol (10−7 to 3×10−4 mol/L) was also assessed.
Production of O2.- in isolated intrapulmonary arteries was assessed using the dihydroethidine staining method, as described10. The cell-permeant oxidative fluorescent indicator dye C-H2DCFDA was used to assess peroxide levels in isolated intrapulmonary arteries, as we reported5. Pressure overload-induced oxidative stress in the right ventricle was assessed by dihydroethidine staining and lucigenin chemiluminscence assay, as reported11.
Western blotting was performed to assess protein expression of Nox-1, gp91phox and eNOS in small intrapulmonary arteries.
Real time RT-PCR technique was used to analyze mRNA expression, as reported11.
Immunolabeling on lung sections was performed for the rat monocyte/macrophage marker ED1. The number of ED1-positive cells was determined in 10 randomly chosen fields.
Data were normalized to the respective control mean values and are expressed as means ± S.D. or S.E.M. Statistical analyses of data were performed by Student's t-test or by two-way ANOVA followed by a Tukey's post hoc test, as appropriate. p<0.05 was considered statistically significant.
Rats challenged with monocrotaline (MCT) consistently developed significant pulmonary hypertension within 14 days, which progressively increased until day 21. Consequently, right ventricular systolic pressure (RVSP) was increased significantly as compared with the saline-challenged group (Fig. 1A). Resveratrol treatment from day 1 normalized RVSP in MCT-injected rats at both the 2- and 3- week period (Fig. 1A). Mean systemic arterial pressure was comparable in each group (data not shown). In the MCT groups, a significant RV hypertrophy (Fig. 1B) associated with RV oxidative stress (Fig. S1; please see http://hyper.ahajournals.org) developed as a consequence of increased pulmonary pressures. Resveratrol treatment prevented MCT-induced RV hypertrophy (Fig. 1B) and oxidative stress.
Smooth muscle cell mass in the small pulmonary arteries was markedly increased in the MCT groups, both at day 14 and day 21 (Fig. 1C,D), as compared with control animals. Resveratrol treatment normalized medial wall thickness preventing the increase in PASMC mass in vessels of MCT-treated rats (Fig. 1C,D). Medial hypertrophy of pulmonary resistance vessels was associated with an increased number of PCNA-positive proliferating vascular cells in MCT-induced pulmonary hypertension (Fig. 1E and Fig. S2; please see http://hyper.ahajournals.org). In parallel to normalization of vessel morphology, the number of PCNA-positive cells was considerably reduced in animals treated with resveratrol (Fig. 1E and Fig. S2; please see http://hyper.ahajournals.org). Neither MCT- pulmonary hypertension nor resveratrol treatment elicited apoptotic cell death in small pulmonary arteries (Fig. 1F and Fig. S2; please see http://hyper.ahajournals.org).
PDGF (10 ng/ml for 48 h) stimulated proliferation in cultured PASMCs, which was prevented by resveratrol (Fig. 2A). The inhibitory effect of resveratrol on PASMC proliferation was also tested using a colony formation assay (Fig. 2B). We confirmed that resveratrol potently inhibited PASMC colony formation and proliferation in a physiologically relevant concentration range (Fig. 2B–C). Resveratrol also inhibited proliferation of PASMCs stimulated by IL-1β, TGFβ or IL-6 (Fig. 2D). In the concentration range, in which the anti-proliferative effects were evident, resveratrol did not increase significantly apoptotic cell death either in PASMC or PAEC (Fig. 2E–F). Using flow cytometry, we demonstrated that the inhibitory effect of resveratrol on cell proliferation could be attributed to the cell cycle arrest in S phase (Fig. 2G–I). Resveratrol significantly inhibited cytokine-induced NF-κB activation in PASMCs (Fig. S3; please see http://hyper.ahajournals.org).
Development of pulmonary hypertension was associated with dysregulation of the expression of components of BMP-4 signaling pathway. Resveratrol treatment normalized alterations in BMP receptors (ACVR1, ACVRL1), BMP antagonists (chordin) and SMAD signaling molecules (SMAD1/4) in the lung (Fig S4; please see http://hyper.ahajournals.org).
Development of pulmonary hypertension was associated with up-regulation of mRNA expression of IL-6, IL-1, TNFα, PDGFα, PDGFβ, TGFβ, MCP-1, iNOS and ICAM-1 in the lung of MCT-treated rats (Fig. 3). Resveratrol treatment significantly attenuated expression of each inflammatory markers (Fig. 3).
In the lungs of MCT-treated rats the number of ED-1 positive leukocytes was significantly increased (Fig 4.). Resveratrol treatment prevented infiltration of ED-1 positive cells (Fig. 4.). Thus, signaling for leukocyte invasion into lung tissue is markedly attenuated by resveratrol treatment.
Acetylcholine-induced relaxation was impaired in small pulmonary arteries of MCT-treated rats and relaxation was restored following resveratrol treatment (Fig. 5A). Vascular relaxations to the NO donor SNAP were unaffected in MCT rats (not shown). In small pulmonary arteries of MCT-treated rats, ROS generation was elevated, whereas resveratrol treatment significantly attenuated oxidative stress (detected with dihydroethidine [Fig. 5B] and DCF fluorescence [data not shown] methods). Resveratrol in vitro did not elicit significant vasorelaxation in the physiological relevant concentration range (up to 10 μmol/L; Fig. 5C). In small pulmonary arteries of MCT-treated rats expression of NAD(P)H oxidase subunits was up-regulated (Fig. 5D,E and Fig. S4, please see http://hyper.ahajournals.org). Attenuation of vascular oxidative stress by resveratrol (Fig. 5B) was associated with down-regulation of NOX-1 and gp91phox, and improved eNOS expression (5D–F and Fig. S5, please see http://hyper.ahajournals.org).
Resveratrol belongs to a new class of drugs12 that exhibits cytoprotective, anti-oxidative and anti-inflammatory vasoprotective properties in the systemic circulation4. Our studies show that resveratrol in the pulmonary arteries of MCT-treated rats improved endothelial function, attenuated oxidative stress and inhibited inflammation. Resveratrol treatment also inhibited vascular remodeling thus preventing development of pulmonary hypertension. In addition, treatment of cultured PASMCs with resveratrol inhibited cell proliferation recapitulating in vitro the effects observed in vivo on MCT-treated animals.
Previous studies focused on the effects of resveratrol on the systemic circulation but provided little information on its pulmonary effects. In our study, we found a prominent prevention of progression of pulmonary hypertension in response to resveratrol therapy (Fig. 1A). Accordingly, reducing right heart load also prevented adaptive hypertrophy (Fig. 1B) and strain-induced oxidative stress (Fig. S1; please see http://hyper.ahajournals.org.) in the right ventricle of MCT-treated rats. Further studies are needed to determine whether resveratrol treatment can reverse/delay the progression of the disease once pulmonary hypertension is established, which is the ultimate clinical relevance of a new therapeutic paradigm.
Structural changes observed in MCT-induced pulmonary hypertension resemble characteristics of human pulmonary hypertension in terms of marked medial wall thickening (Fig. 1C–D) resulting in a dramatic increase in pulmonary arterial resistance13. The current study is in agreement with previous reports2 in that smooth muscle cell proliferation markedly increases in pulmonary resistance vessels of MCT-challenged animals (Fig. 1C–E). Resveratrol treatment resulted in near normal vessel morphology and reduced pulmonary arterial smooth muscle proliferation (Fig. 1C–E) without increasing rate of apoptosis (Fig. 1F). The lack of increased apoptotic cell death may explain the lack of adverse effects and good tolerability of resveratrol treatment in clinical studies.
It is likely that inhibition of pulmonary arterial smooth muscle proliferation and vascular remodeling is predominantly a direct effect of resveratrol, because resveratrol also effectively inhibited proliferation of cultured PASMCs, arresting the cell cycle in S phase (Fig. 2). These findings are in agreement with our previous studies that demonstrate that the antiproliferative properties of resveratrol are attributed to induction of p53 and heat shock protein HSP27 in human aortic smooth muscle cells8, 14.
Current literature supports contributory effects of inflammation to the development and progression of pulmonary hypertension15. In animal models of pulmonary hypertension, an increased presence and activity of inflammatory cells (including macrophages, polymorphonuclear neutrophils, lymphocytes and mast cells) are routinely observed15. Indeed, our study documents a substantial increase in ED-1 positive cells in the lungs of MCT-treated rats (Fig. 4), which is accompanied by a significant up-regulation of inflammatory cytokines16, 17 and growth factors (TNFα. IL-1β, IL-6, PDGFα, PDGFβ, TGFβ) and adhesion molecules (Fig. 3). There is increasing appreciation of inflammation in various forms of clinical pulmonary hypertension based on evidence ranging from increased plasma levels of inflammatory cytokines to pulmonary infiltration of inflammatory cells15. In humans a variety of diverse inflammatory conditions (e.g. viral infections, autoimmune diseases) culminate in severe pulmonary hypertension. It has been generally accepted that inflammatory cytokines and growth factors can cause pulmonary vasoconstriction and promote proliferation of vascular cells. Recent studies suggest that disruption of PDGF signaling pathways prevents development and progression of MCT-induced pulmonary hypertension in rats2. Importantly, we found that resveratrol treatment in MCT-treated rats normalized expression of inflammatory cytokines and growth factors (including PDGFα/β; Fig. 3) and significantly decreased the number of infiltrating ED-1 positive cells in the lung (Fig. 4). The mechanisms by which resveratrol interferes with inflammatory processes in the lung are not well understood. Recently we showed that resveratrol effectively blocks NF-κB activation in endothelial cells, thus limiting expression of inflammatory cytokines and adhesion molecules and attenuating monocyte adhesiveness6. Resveratrol also effectively inhibits cytokine-induced NF-κB activation in PASMCs (Fig. S3; please see http://hyper.ahajournals.org). NF-κB activation is known to regulate PASMC proliferation in vitro 18, 19. Because development of pulmonary hypertension in MCT-treated rats is associated with NF-κB activation20, and administration of an inhibitor of NF-κB prevents pulmonary hypertension in MCT-treated rodents21, we speculate that inhibition of NF-κB by resveratrol may contribute to its therapeutic potential in pulmonary hypertension.
Mutations in BMP receptors have been identified in patients with PAH, implicating BMP signaling in pulmonary hypertension. Importantly, recent studies have demonstrated that the expression and function of the BMP/Smad signaling axis are perturbed in MCT-treated animals as well22. Because BMP signals antagonize PASMC proliferation induced by inflammatory cytokines, altered BMP/Smad signaling may play a role in the pathogenesis of pulmonary hypertension. Resveratrol normalized the expression of many components of the BMP/Smad signaling pathway (Fig. S4; please see http://hyper.ahajournals.org). Further studies are needed to elucidate whether these effects contribute to the therapeutic benefits of resveratrol treatment.
There is solid evidence in MCT-treated rats, that oxidative injury to the pulmonary vascular endothelium precedes PASMC proliferation and medial hypertrophy in the distal pulmonary vascular bed and the rise in pulmonary artery pressure. We found that pulmonary hypertension in small pulmonary arteries is associated with endothelial dysfunction (Fig. 5A), oxidative stress (Fig. 5B) and up-regulation of NAD(P)H oxidases (Fig. 5D,E). By contrast, resveratrol treatment significantly improved pulmonary arterial endothelial function (Fig. 5A) and attenuated oxidative stress and NADPH oxidase expression (Fig. 5D–E, Fig. S5 please see http://hyper.ahajournals.org.). These findings are significant because recent studies on p91phox knockout mice suggest that NAD(P)H oxidases contribute to the development of pulmonary hypertension23. The activity and expression of vascular NAD(P)H oxidases are known to be regulated by inflammatory cytokines (e.g. TNFα, TGFβ24), the expression of which is up-regulated in lungs of rats with pulmonary hypertension (Fig. 2). Thus, we suggest that the observed resveratrol-induced down-regulation of NAD(P)H oxidase (Fig. 5) is due to the resveratrol-induced attenuation of inflammatory cytokine production (Fig. 3).
We confirmed that eNOS is down-regulated (Fig. 5E) in rats with pulmonary hypertension16,25. Importantly, this attenuation was corrected by resveratrol (Fig. 5E). Previous studies showed that resveratrol directly regulates eNOS expression at the level of transcription in cultured endothelial cells5. Since some studies suggest that treatment with NO donors26 or overexpression of eNOS itself may attenuate pulmonary hypertension27, it is possible that up-regulation of eNOS contributes to the therapeutic action of resveratrol. We cannot exclude the possibility that resveratrol, in addition to up-regulating eNOS, may also affect BH4 levels and/or caveolin25 and thus directly influence eNOS activity. Previously, resveratrol was shown to upregulate the expression of Nrf2-regulated antioxidant gene battery, including heme oxygenase-1 (HO-1) in systemic arteries5. In the present study, resveratrol treatment also induced HO-1 in the pulmonary arteries of MCT-treated rats (Fig. S6, please see http://hyper.ahajournals.org.), which is known to confer antiproliferative and vasoprotective effects28.
We demonstrate that treatment with resveratrol prevents the development of pulmonary hypertension in a recognized animal model of the disease. The demonstrated anti-proliferative action of resveratrol on PASMCs may be contributory to its beneficial effects. Furthermore, we provide evidence for anti-inflammatory, anti-oxidative and endothelial protective effects in the beneficial actions of resveratrol in pulmonary arteries. Since phase I clinical trials are currently underway for anti-cancer efficacy of oral resveratrol treatment in humans12, use of resveratrol as a new therapy for pulmonary hypertension might therefore be plausible. Future studies should determine i) whether resveratrol treatment can reverse or delay the progression of established pulmonary hypertension, ii) whether resveratrol may potentiate the effects of current therapies, and iii) whether resveratrol can exert direct cardioprotective effects preventing right ventricular failure in patients with pulmonary hypertension.
Sources of funding This work was supported by grants from the American Federation for Aging Research (to AC), the American Diabetes Association (to ZU), the NIH (HL077256 and HL43023, HL31069), and the Hungarian Scientific Research Fund (OTKA-K68758).