The six-transmembrane protein Stamp2 ameliorates pulmonary vascular remodeling and pulmonary hypertension in mice.

Six-transmembrane protein of prostate (Stamp2) protects from diabetes and atherosclerosis in mice via anti-inflammatory mechanisms. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PAH), we investigated the role of Stamp2. Stamp2 expression was substantially reduced in the lung of humans with idiopathic PAH, as well as in experimental PAH. In Stamp2-deficient mice, hypoxia modestly aggravated pulmonary vascular remodeling and right ventricular pressure compared to WT. As endothelial cell (EC) and pulmonary arterial smooth muscle cell (PASMC) phenotypes drive remodeling in PAH, we explored the role of Stamp2. Knock-down of Stamp2 in human EC neither affected apoptosis, viability, nor release of IL-6. Moreover, Stamp2 deficiency in primary PASMC did not alter mitogenic or migratory properties. As Stamp2 deficiency augmented expression of inflammatory cytokines and numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages may drive vascular remodeling. Thus, PASMC responses were assessed following treatment with conditioned media of primary Stamp2-/- or WT macrophages. Stamp2-/- supernatants induced PASMC proliferation and migration stronger compared to WT. A cytokine array revealed CXCL12, MCP-1 and IL-6 as most relevant candidates. Experiments with neutralizing antibodies confirmed the role of these cytokines in driving Stamp2's responses. In conclusion, Stamp2 deficiency aggravates pulmonary vascular remodeling via cross-talk between macrophages and PASMC. Despite a substantial pro-inflammatory response, the hemodynamic effect of Stamp2 deficiency is modest suggesting that additional mechanisms apart from inflammation are necessary to induce severe PAH.

Genetic Ablation of PDGF-Dependent Signaling Pathways Abolishes Vascular Remodeling and Experimental Pulmonary Hypertension.

OBJECTIVE: Despite modern therapies, pulmonary arterial hypertension (PAH) harbors a high mortality. Vascular remodeling is a hallmark of the disease. Recent clinical studies revealed that antiremodeling approaches with tyrosine-kinase inhibitors such as imatinib are effective, but its applicability is limited by significant side effects. Although imatinib has multiple targets, expression analyses support a role for platelet-derived growth factor (PDGF) in the pathobiology of the disease. However, its precise role and downstream signaling events have not been established. APPROACH AND RESULTS: Patients with PAH exhibit enhanced expression and phosphorylation of ß PDGF receptor (ßPDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLCγ at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of ßPDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated ßPDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLCγ (ßPDGFR(F3/F3)). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in ßPDGFR(F3/F3) pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in ßPDGFR(F3/F3) mice compared with wild-type littermates. These alterations led to protection from hypoxia-induced PAH and right ventricular hypertrophy. CONCLUSIONS: By means of a genetic approach, our data provide definite evidence that the activated ßPDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLCγ activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.

Class IA Phosphatidylinositol 3-Kinase Isoform p110α Mediates Vascular Remodeling.

OBJECTIVE: Neointima formation after vascular injury remains a significant problem in clinical cardiology, and current preventive strategies are suboptimal. Phosphatidylinositol 3'-kinase is a central downstream mediator of growth factor signaling, but the role of phosphatidylinositol 3'-kinase isoforms in vascular remodeling remains elusive. We sought to systematically characterize the precise role of catalytic class IA phosphatidylinositol 3'-kinase isoforms (p110α, p110ß, p110δ), which signal downstream of receptor tyrosine kinases, for vascular remodeling in vivo. APPROACH AND RESULTS: Western blot analyses revealed that all 3 isoforms are abundantly expressed in smooth muscle cells. To analyze their significance for receptor tyrosine kinases-dependent cellular responses, we used targeted gene knockdown and isoform-specific small molecule inhibitors of p110α (PIK-75), p110ß (TGX-221), and p110δ (IC-87114), respectively. We identified p110α to be crucial for receptor tyrosine kinases signaling, thus affecting proliferation, migration, and survival of rat, murine, and human smooth muscle cells, whereas p110ß and p110δ activities were dispensable. Surprisingly, p110δ exerted noncatalytic functions in smooth muscle cell proliferation, but had no effect on migration. Based on these results, we generated a mouse model of smooth muscle cell-specific p110α deficiency (sm-p110α(-/-)). Targeted deletion of p110α in sm-p110α(-/-) mice blunted growth factor-induced cellular responses and abolished neointima formation after balloon injury of the carotid artery in mice. In contrast, p110δ deficiency did not affect vascular remodeling in vivo. CONCLUSIONS: Receptor tyrosine kinases-induced phosphatidylinositol 3'-kinase signaling via the p110α isoform plays a central role for vascular remodeling in vivo. Thus, p110α represents a selective target for the prevention of neointima formation after vascular injury, whereas p110ß and p110δ expression and activity do not play a significant role.

Hematopoietic PI3Kδ deficiency aggravates murine atherosclerosis through impairment of regulatory T cells.

Chronic activation of the adaptive immune system is a hallmark of atherosclerosis. As PI3Kδ is a key regulator of T and B-cell differentiation and function, we hypothesized that alleviation of adaptive immunity by PI3Kδ inactivation may represent an attractive strategy counteracting atherogenesis. As expected, lack of hematopoietic PI3Kδ in atherosclerosis-prone Ldlr-/- mice resulted in hindered T- and B-cell numbers, CD4+ effector T cells, Th1 response, and immunoglobulin levels. However, despite markedly impaired peripheral proinflammatory Th1 cells and atheromatous CD4+ T cells, the unexpected net effect of hematopoietic PI3Kδ deficiency was aggravated vascular inflammation and atherosclerosis. Further analyses revealed that PI3Kδ deficiency impaired numbers, immunosuppressive functions, and stability of regulatory CD4+ T cells (Tregs), whereas macrophage biology remained largely unaffected. Adoptive transfer of wild-type Tregs fully restrained the atherosclerotic plaque burden in Ldlr-/- mice lacking hematopoietic PI3Kδ, whereas PI3Kδ deficient Tregs failed to mitigate disease. Numbers of atheroprotective B-1 and proatherogenic B-2 cells as well serum immunoglobulin levels remained unaffected by adoptively transferred wild-type Tregs. In conclusion, we demonstrate that hematopoietic PI3Kδ ablation promotes atherosclerosis. Mechanistically, we identified PI3Kδ signaling as a powerful driver of atheroprotective Treg responses, which outweigh PI3Kδ driven proatherogenic effects of adaptive immune cells like Th1 cells.

Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

RATIONALE: Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia. OBJECTIVES: To investigate whether hypoxia alters PDGF ß receptor (ßPDGFR) signaling in the pulmonary vasculature. METHODS: The impact of chronic hypoxia on signal transduction by the ßPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo. MEASUREMENTS AND MAIN RESULTS: Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect ßPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated ßPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced ßPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. ßPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH. CONCLUSIONS: Hypoxia reduces expression and activity of ßPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the ßPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.

Coagulation-independent effects of thrombin and Factor Xa: role of protease-activated receptors in pulmonary hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is a devastating disease with limited therapeutic options. Vascular remodelling of pulmonary arteries, characterized by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a hallmark of PAH. Here, we aimed to systematically characterize coagulation-independent effects of key coagulation proteases thrombin and Factor Xa (FXa) and their designated receptors, protease-activated receptor (PAR)-1 and -2, on PASMCs in vitro and experimental PAH in vivo. METHODS AND RESULTS: In human and murine PASMCs, both thrombin and FXa were identified as potent mitogens, and chemoattractants. FXa mediated its responses via PAR-1 and PAR-2, whereas thrombin signalled through PAR-1. Extracellular-signal regulated kinases 1/2, protein kinase B (AKT), and sphingosine kinase 1 were identified as downstream mediators of PAR-1 and PAR-2. Inhibition of FXa or thrombin blunted cellular responses in vitro, but unexpectedly failed to protect against hypoxia-induced PAH in vivo. However, pharmacological inhibition as well as genetic deficiency of both PAR-1 and PAR-2 significantly reduced vascular muscularization of small pulmonary arteries, diminished right ventricular systolic pressure, and right ventricular hypertrophy upon chronic hypoxia compared to wild-type controls. CONCLUSION: Our findings indicate a coagulation-independent pathogenic potential of thrombin and FXa for pulmonary vascular remodelling via acting through PAR-1 and PAR-2, respectively. While inhibition of single coagulation proteases was ineffective in preventing experimental PAH, our results propose a crucial role for PAR-1 and PAR-2 in its pathobiology, thus identifying PARs but not their dedicated activators FXa and thrombin as suitable targets for the treatment of PAH.

Stamp2 Protects From Maladaptive Structural Remodeling and Systolic Dysfunction in Post-Ischemic Hearts by Attenuating Neutrophil Activation.

The six-transmembrane protein of prostate 2 (Stamp2) acts as an anti-inflammatory protein in macrophages by protecting from overt inflammatory signaling and Stamp2 deficiency accelerates atherosclerosis in mice. Herein, we describe an unexpected role of Stamp2 in polymorphonuclear neutrophils (PMN) and characterize Stamp2's protective effects in myocardial ischemic injury. In a murine model of ischemia and reperfusion (I/R), echocardiography and histological analyses revealed a pronounced impairment of cardiac function in hearts of Stamp2-deficient- (Stamp2-/- ) mice as compared to wild-type (WT) animals. This difference was driven by aggravated cardiac fibrosis, as augmented fibroblast-to-myofibroblast transdifferentiation was observed which was mediated by activation of the redox-sensitive p38 mitogen-activated protein kinase (p38 MAPK). Furthermore, we observed increased production of reactive oxygen species (ROS) in Stamp2-/- hearts after I/R, which is the likely cause for p38 MAPK activation. Although myocardial macrophage numbers were not affected by Stamp2 deficiency after I/R, augmented myocardial infiltration by polymorphonuclear neutrophils (PMN) was observed, which coincided with enhanced myeloperoxidase (MPO) plasma levels. Primary PMN isolated from Stamp2-/- animals exhibited a proinflammatory phenotype characterized by enhanced nuclear factor (NF)-κB activity and MPO secretion. To prove the critical role of PMN for the observed phenotype after I/R, antibody-mediated PMN depletion was performed in Stamp2-/- mice which reduced deterioration of LV function and adverse structural remodeling to WT levels. These data indicate a novel role of Stamp2 as an anti-inflammatory regulator of PMN and fibroblast-to-myofibroblast transdifferentiation in myocardial I/R injury.

Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents.

Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3'-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell-specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

PDGF-BB protects cardiomyocytes from apoptosis and improves contractile function of engineered heart tissue.

Platelet-derived-growth-factor-BB (PDGF-BB) can protect various cell types from apoptotic cell death, and induce hypertrophic growth and proliferation, but little is known about its direct or indirect effects on cardiomyocytes. Cardiac muscle engineering is compromised by a particularly high rate of cardiomyocyte death. Here we hypothesized that PDGF-BB stimulation can (1) protect cardiomyocytes from apoptosis, (2) enhance myocyte content in and (3) consequently optimize contractile performance of engineered heart tissue (EHT). We investigated the effects of PDGF-receptor activation in neonatal rat heart monolayer- and EHT-cultures by isometric contraction experiments, cytomorphometry, (3)H-thymidine and (3)H-phenylalanine incorporation assays, quantitative PCR (calsequestrin 2, alpha-cardiac and skeletal actin, atrial natriuretic factor, alpha- and beta-myosin heavy chain), immunoblotting (activated caspase 3, Akt-phosphorylation), and ELISA (cell death detection). PDGF-BB did not induce hypertrophy or proliferation in cardiomyocytes, but enhanced contractile performance of EHT. This effect was concentration-dependent (E(max) 10 ng/ml) and maximal only after transient PDGF-BB stimulation (culture days 0-7; total culture duration: 12 days). Improvement of contractile function was associated with higher cardiomyocyte content, as a consequence of PDGF-BB mediated protection from apoptosis (lower caspase-3 activity particularly in cardiomyocytes in PDGF-BB treated vs. untreated EHTs). We confirmed the anti-apoptotic effect of PDGF-BB in monolayer cultures and observed that PI3-kinase inhibition with LY294002 attenuated PDGF-BB-mediated cardiomyocyte protection. We conclude that PDGF-BB does not induce hypertrophy or proliferation, but confers an anti-apoptotic effect on cardiomyocytes. Our findings suggest a further exploitation of PDGF-BB in cardiomyocyte protection in vivo and in vitro.

Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation.

OBJECTIVE: Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229+/-9% and 362+/-54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 microM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2+/-0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2+/-0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 microM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 microM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner. CONCLUSIONS: VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which-in contrast to Akt and Erk-appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.

PI3-kinase/Akt-dependent antiapoptotic signaling by the PDGF alpha receptor is negatively regulated by Src family kinases.

Regulation of growth factor dependent cell survival is crucial for development and disease progression. Here, we report a novel function of Src kinases as a negative regulator of platelet-derived growth factor (PDGF) dependent cell survival. We characterized a series of PDGF alpha receptor (PDGFRA) mutants, which lack the binding sites for Src, phosphatidylinositol 3'-kinase (PI3K), SHP-2 or phospholipase C-gamma. We found that PDGFRA-dependent cell survival was mainly mediated through activation of PI3K, and was negatively regulated by Src. Characterization of the downstream signaling events revealed that PI3K activates the protein kinase Akt, which in turn phosphorylates and thus inactivates proapoptotic Forkhead transcription factors. Src phosphorylates the ubiquitin-ligase c-Cbl, which is required for degradation of the activated receptor. Consequently, overexpression of c-Cbl prevented PDGFRA-mediated cell survival, whereas it did not affect this response, when Src was unable to associate with the receptor. This novel function of Src in antiapoptotic signaling introduces Src kinases as an interesting therapeutic target in apoptosis related diseases.

Imatinib mesylate for the treatment of pulmonary arterial hypertension.

INTRODUCTION: Despite recent advances, pulmonary arterial hypertension (PAH) remains a devastating disease which harbors a poor prognosis. Novel therapeutic approaches directly targeting pulmonary vascular remodeling are warranted. AREAS COVERED: This review delineates the current limitations in the management of PAH and focuses on a novel, anti-proliferative therapeutic concept. It will help readers understand the mechanisms of receptor tyrosine kinase signaling, with a special focus on platelet-derived growth factor (PDGF) receptors and their role in the pathobiology of PAH. Furthermore, it provides a comprehensive summary regarding the rationale, efficacy and safety of the tyrosine kinase inhibitor imatinib mesylate , which potently inhibits the PDGF receptor, as an additional treatment option in PAH. EXPERT OPINION: PDGF is a potent mitogen for pulmonary vascular smooth muscle cells and represents an important mediator of pulmonary vascular remodeling. Imatinib mesylate, a compound that inhibits the Bcr-Abl kinase and was developed for the treatment of chronic myeloid leukemia, also targets PDGF receptors. Both experimental and clinical data indicate that it reverses the vascular remodeling process even when it is fully established. Results from Phase II and III clinical trials suggest potent and prolonged efficacy in patients with severe PAH (i.e., pulmonary vascular resistance > 800 dynes*s*cm(-5)). Future studies should evaluate the long-term clinical efficacy and safety of imatinib, including patients with less impaired hemodynamics. Based on the current knowledge, this compound is likely to become an additional treatment option for patients with PAH and has the potential to at least partially correct the pathology of the disease.

Disruption of platelet-derived growth factor-dependent phosphatidylinositol 3-kinase and phospholipase Cγ 1 activity abolishes vascular smooth muscle cell proliferation and migration and attenuates neointima formation in vivo.

OBJECTIVES: We tested the hypothesis whether selective blunting of platelet-derived growth factor (PDGF)-dependent vascular smooth muscle cell (VSMC) proliferation and migration is sufficient to prevent neointima formation after vascular injury. BACKGROUND: To prevent neointima formation and stent thrombosis after coronary interventions, it is essential to inhibit VSMC proliferation and migration without harming endothelial cell function. The role of PDGF-a potent mitogen and chemoattractant for VSMC that does not affect endothelial cells-for neointima formation remains controversial. METHODS: To decipher the signaling pathways that control PDGF beta receptor (ßPDGFR)-driven VSMC proliferation and migration, we characterized 2 panels of chimeric CSF1R/ßPDGFR mutants in which the binding sites for ßPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase [PI3K], GTPase activating protein of ras, SHP-2, phospholipase Cγ 1 [PLCγ]) were individually mutated. Based on in vitro results, the importance of PDGF-initiated signals for neointima formation was investigated in genetically modified mice. RESULTS: Our results indicate that the chemotactic response to PDGF requires the activation of Src, PI3K, and PLCγ, whereas PDGF-dependent cell cycle progression is exclusively mediated by PI3K and PLCγ. These 2 signaling molecules contribute to signal relay of the ßPDGFR by differentially regulating cyclin D1 and p27(kip1). Blunting of ßPDGFR-induced PI3K and PLCγ signaling by a combination mutant (F3) completely abolished the mitogenic and chemotactic response to PDGF. Disruption of PDGF-dependent PI3K and PLCγ signaling in mice expressing the F3 receptor led to a profound reduction of neointima formation after balloon injury. CONCLUSIONS: Signaling by the activated ßPDGFR, particularly through PI3K and PLCγ, is crucial for neointima formation after vascular injury. Disruption of these specific signaling pathways is sufficient to attenuate pathogenic processes such as vascular remodeling in vivo.

Transforming growth factor ß1 oppositely regulates the hypertrophic and contractile response to ß-adrenergic stimulation in the heart.

BACKGROUND: Neuroendocrine activation and local mediators such as transforming growth factor-ß1 (TGF-ß1) contribute to the pathobiology of cardiac hypertrophy and failure, but the underlying mechanisms are incompletely understood. We aimed to characterize the functional network involving TGF-ß1, the renin-angiotensin system, and the ß-adrenergic system in the heart. METHODS: Transgenic mice overexpressing TGF-ß1 (TGF-ß1-Tg) were treated with a ß-blocker, an AT1-receptor antagonist, or a TGF-ß-antagonist (sTGFßR-Fc), were morphologically characterized. Contractile function was assessed by dobutamine stress echocardiography in vivo and isolated myocytes in vitro. Functional alterations were related to regulators of cardiac energy metabolism. RESULTS: Compared to wild-type controls, TGF-ß1-Tg mice displayed an increased heart-to-body-weight ratio involving both fibrosis and myocyte hypertrophy. TGF-ß1 overexpression increased the hypertrophic responsiveness to ß-adrenergic stimulation. In contrast, the inotropic response to ß-adrenergic stimulation was diminished in TGF-ß1-Tg mice, albeit unchanged basal contractility. Treatment with sTGF-ßR-Fc completely prevented the cardiac phenotype in transgenic mice. Chronic ß-blocker treatment also prevented hypertrophy and ANF induction by isoprenaline, and restored the inotropic response to ß-adrenergic stimulation without affecting TGF-ß1 levels, whereas AT1-receptor blockade had no effect. The impaired contractile reserve in TGF-ß1-Tg mice was accompanied by an upregulation of mitochondrial uncoupling proteins (UCPs) which was reversed by ß-adrenoceptor blockade. UCP-inhibition restored the contractile response to ß-adrenoceptor stimulation in vitro and in vivo. Finally, cardiac TGF-ß1 and UCP expression were elevated in heart failure in humans, and UCP--but not TGF-ß1--was downregulated by ß-blocker treatment. CONCLUSIONS: Our data support the concept that TGF-ß1 acts downstream of angiotensin II in cardiomyocytes, and furthermore, highlight the critical role of the ß-adrenergic system in TGF-ß1-induced cardiac phenotype. Our data indicate for the first time, that TGF-ß1 directly influences mitochondrial energy metabolism by regulating UCP3 expression. ß-blockers may act beneficially by normalizing regulatory mechanisms of cellular hypertrophy and energy metabolism.

Profilin-1 is expressed in human atherosclerotic plaques and induces atherogenic effects on vascular smooth muscle cells.

BACKGROUND: Profilin-1 is an ubiquitous actin binding protein. Under pathological conditions such as diabetes, profilin-1 levels are increased in the vascular endothelium. We recently demonstrated that profilin-1 overexpression triggers indicators of endothelial dysfunction downstream of LDL signaling, and that attenuated expression of profilin-1 confers protection from atherosclerosis in vivo. METHODOLOGY: Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans. PRINCIPAL FINDINGS: In coronary arteries from patients with coronary heart disease, we found markedly enhanced profilin expression in atherosclerotic plaques compared to the normal vessel wall. Stimulation of rat aortic and human coronary VSMCs with recombinant profilin-1 (10(-6) M) in vitro led to activation of intracellular signaling cascades such as phosphorylation of Erk1/2, p70(S6) kinase and PI3K/Akt within 10 minutes. Furthermore, profilin-1 concentration-dependently induced DNA-synthesis and migration of both rat and human VSMCs, respectively. Inhibition of PI3K (Wortmannin, LY294002) or Src-family kinases (SU6656, PP2), but not PLCγ (U73122), completely abolished profilin-induced cell cycle progression, whereas PI3K inhibition partially reduced the chemotactic response. Finally, we found that profilin-1 serum levels were significantly elevated in patients with severe atherosclerosis in humans (p<0.001 vs. no atherosclerosis or control group). CONCLUSIONS: Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target.

Influence of cell treatment with PDGF-BB and reperfusion on cardiac persistence of mononuclear and mesenchymal bone marrow cells after transplantation into acute myocardial infarction in rats.

Bone marrow cells are used for cell therapy after myocardial infarction (MI) with promising results. However, cardiac persistence of transplanted cells is rather low. Here, we investigated strategies to increase the survival and cardiac persistence of mononuclear (MNC) and mesenchymal (MSC) bone marrow cells transplanted into infarcted rat hearts. MNC and MSC (male Fischer 344 rats) were treated with different doses of PDGF-BB prior to intramyocardial injection into border zone of MI (syngeneic females, permanent LAD ligation) and hearts were harvested after 5 days and 3 weeks. In additional experiments, untreated MNC and MSC were injected immediately after permanent or temporary LAD ligation and hearts were harvested after 48 h, 5 days, 3 weeks, and 6 weeks. DNA of the hearts was isolated and the number of donor cells was determined by quantitative real-time PCR with Y chromosome-specific primers. There was a remarkable though not statistically significant (p = 0.08) cell loss of approximately 46% between 5 days and 3 weeks in the control group, which was completely inhibited by treatment with high dose of PDGF-BB. Forty-eight hours after reperfusion only 10% of injected MSC or 1% for MNC were found in the heart, decreasing to 1% for MSC and 0.5% for MNC after 6 weeks. These numbers were lower than after permanent LAD ligation for both MNC and MSC at all time points studied. Treatment with PDGF-BB seems to prevent loss of transplanted bone marrow cells at later times presumably by inhibition of apoptosis, while reperfusion of the occluded artery enhances cell loss at early times putatively due to enhanced early wash-out. Further investigations are needed to substantially improve the persistence and survival of grafted bone marrow cells in infarcted rat hearts, in order to fully explore the therapeutic potential of this novel treatment modality for myocardial repair.

Differential effects of red and white wines on inhibition of the platelet-derived growth factor receptor: impact of the mash fermentation.

AIMS: Moderate wine consumption is associated with a significant reduction of cardiovascular mortality. The molecular basis of this phenomenon remains unknown. Platelet-derived growth factor (PDGF) is an important contributor to atherogenesis. We investigated the effects of selected red and white wines on PDGF receptor (PDGFR) signalling in rat and human vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: All red wines concentration dependently inhibited the ligand-induced tyrosine phosphorylation of the PDGFR, downstream signalling events such as mitogen activated protein (MAP) kinase activation (Erk 1/2) and induction of immediate early genes (Egr-1, c-fos), and PDGF-induced cellular responses, whereas all white wines had no effect. At concentrations achieved after wine consumption in humans, all red wines completely abolished PDGF-dependent VSMC proliferation and migration. Red wines also inhibited PDGFR phosphorylation in vascular tissue, and in human coronary smooth muscle cells. Quantitative analyses of all tested wines and of samples collected at various time points (Days 0-16) of the 'mash fermentation', which is only performed for red wine, revealed that flavonoids of the catechin family, which potently inhibit PDGFR signalling, are extracted from grape seeds and skins during this process and therefore accumulate specifically in red wine. The accumulation of flavonoids correlated with the inhibitory potency of red wines on PDGFR signalling. Furthermore, this procedure could be imitated by incubation of wines with shredded grape seeds, and flavonoid-enriched white wine inhibited the PDGFR as potently as red wines. CONCLUSION: Only red wines abrogate a critical pathogenic mechanism during atherogenesis, PDGFR signalling, in VSMCs. This effect is mediated by non-alcoholic constituents, which accumulate during the mash fermentation. Our findings offer a molecular explanation for the vasoprotective effects particularly of red wine. Therefore, future epidemiological studies should consider differential protective effects of red and white wine in vivo.

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species.

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

Systematic evaluation of anti-apoptotic growth factor signaling in vascular smooth muscle cells. Only phosphatidylinositol 3'-kinase is important.

Peptide growth factors contribute to the pathogenesis of cardiovascular diseases by inducing a variety of cellular responses including anti-apoptotic effects. Several of the signaling molecules that are activated by growth factor receptors such as Src family kinases (Src), phosphatidylinositol 3'-kinase (PI3K), phospholipase Cgamma (PLCgamma), Ras, and SHP-2 were shown to mediate survival signals. We systematically investigated the relative contribution of each signaling molecule for growth factor-dependent cell survival in vascular smooth muscle cells (VSMC). Our approach was the use of mutated plateletderived growth factor (PDGF) beta-receptors (betaPDGFR) in which the tyrosine residues required for binding of each signaling molecule were individually mutated to phenylalanine. To bypass endogenous PDGFR in VSMC we used chimeric receptors (ChiRs), containing the extracellular domain of the macrophage colony-stimulating factor (M-CSF) receptor and the cytoplasmic domain of the wild type (WT) or mutated betaPDGFR. Selective activation of the ChiR-WT with M-CSF significantly reduced apoptosis to the same extent as PDGF-BB in non-transfected cells. Deletion of the binding site for PI3K, but not for Src, RasGAP, SHP-2, or PLCgamma, completely abolished the anti-apoptotic effect. Consistently, a ChiR mutant that only binds PI3K was fully able to mediate cell survival as efficiently as the ChiR-WT. Furthermore, the PDGF-dependent anti-apoptotic effect in non-transfected cells was completely abolished by the PI3K inhibitor wortmannin, whereas inhibitors of Src, PLCgamma, ERK, or p38 MAP kinase had no effect. The exploration of downstream signaling events revealed that PDGF-BB activates the anti-apoptotic Akt signaling pathway in a PI3K-dependent manner. Moreover, Akt phosphorylates and thus inactivates the pro-apoptotic proteins BAD and Forkhead transcription factors (FKHR, FKHRL1). We conclude that growth factor-dependent cell survival in VSMC is mediated only by activation of the PI3K/Akt pathway, whereas all other receptor-associated signaling molecules do not play a significant role.