Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

.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.<0.001 vs. no atherosclerosis or control group).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

Transforming Growth Factor β1 Oppositely Regulates the Hypertrophic and Contractile Response to β-Adrenergic Stimulation in the Heart

BACKGROUND: Neuroendocrine activation and local mediators such as transforming growth factor-β₁ (TGF-β₁) 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-β₁, the renin-angiotensin system, and the β-adrenergic system in the heart. METHODS: Transgenic mice overexpressing TGF-β₁ (TGF-β₁-Tg) were treated with a β-blocker, an AT₁-receptor antagonist, or a TGF-β-antagonist (sTGFβR-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-β₁-Tg mice displayed an increased heart-to-body-weight ratio involving both fibrosis and myocyte hypertrophy. TGF-β₁ overexpression increased the hypertrophic responsiveness to β-adrenergic stimulation. In contrast, the inotropic response to β-adrenergic stimulation was diminished in TGF-β₁-Tg mice, albeit unchanged basal contractility. Treatment with sTGF-βR-Fc completely prevented the cardiac phenotype in transgenic mice. Chronic β-blocker treatment also prevented hypertrophy and ANF induction by isoprenaline, and restored the inotropic response to β-adrenergic stimulation without affecting TGF-β₁ levels, whereas AT₁-receptor blockade had no effect. The impaired contractile reserve in TGF-β₁-Tg mice was accompanied by an upregulation of mitochondrial uncoupling proteins (UCPs) which was reversed by β-adrenoceptor blockade. UCP-inhibition restored the contractile response to β-adrenoceptor stimulation in vitro and in vivo. Finally, cardiac TGF-β₁ and UCP expression were elevated in heart failure in humans, and UCP--but not TGF-β₁--was downregulated by β-blocker treatment. CONCLUSIONS: Our data support the concept that TGF-β₁ acts downstream of angiotensin II in cardiomyocytes, and furthermore, highlight the critical role of the β-adrenergic system in TGF-β₁-induced cardiac phenotype. Our data indicate for the first time, that TGF-β₁ directly influences mitochondrial energy metabolism by regulating UCP3 expression. β-blockers may act beneficially by normalizing regulatory mechanisms of cellular hypertrophy and energy metabolism

Proteinkinase C-abhängige Phosphorylierung zyklisch-Nukleotid-gesteuerter Ionenkanäle aus den Sehzellen des Rindes

In retinal rod and cone photoreceptor cells cyclic nucleotide-gated (CNG) channels mediatethe electrical response to stimulation by light. CNG channels are composed of differentsubunits, designated CNC$\alpha$ or CNC$\beta$ subunits. CNG channels are gated by the internalmessenger molecule cGMP and are modulated by small Ca$^{2+}$-binding proteins. This PhD-thesisshows that the CNG channels from rod and cone photoreceptor cells are phosphorylatedby protein kinase C (PKC). PKC-dependent phosphorylation of homomeric heterologouslyexpressed cone CNG channels composed of CNC$\alpha$2 subunits was investigatedelectrophysiologically in inside-out patches. Phosphorylation of two serine residues (serine577 and serine 579) in the cGMP-binding pocket of the CNC$\alpha$2 subunit decreases the ligandsensitivity of cone CNG channels by a factor of 2 - 3. The decrease in ligand sensitivity iscompletely abolished in mutant channels in which the two serine residues were replaced byalanine.The auxiliary CNC$\beta$1 a subunit of the rod CNG channel is phosphorylated by PKC too. The $\textit{invitro}$ phosphorylation of different recombinant fusion proteins shows that the phosphorylationsites are localized in the vicinity of the N-terminal calmodulin (CaM)-binding domain.Binding of CaM to the CNC$\beta$1a subunit reduces the degree of phosphorylation, whereasphosphorylation did not prevent CaM from binding.GARP2 is an N-terminal splice form of the CNC$\beta$1a subunit. In contrast to the CNC$\alpha$2 andCNC$\beta$1a subunit, GARP2 is not phosphorylated by PKC. Gel filtration experiments showedthat GARP2 binds the light-activated cGMP-hydrolyzing enzyme phosphodiesterase. In thisway GARP2 may organize an "adaptational" signaling complex at the disk rim of the rodphotoreceptor cell that controls cGMP turnover during daylight

Ligand sensitivity of the α2 subunit from the bovine cone cGMP-gated channel is modulated by protein kinase C but not by calmodulin

Homomeric cyclic nucleotide-gated (CNG) channels composed of α2 subunits from bovine cone photoreceptors were heterologously expressed in the human embryonic kidney (HEK) 293 cell line. Modulation of cGMP sensitivity by protein kinase C (PKC)-mediated phosphorylation and by binding of calmodulin (CaM) was investigated in inside-out patches.A peptide encompassing the putative CaM-binding site within the N-terminus of the channel protein binds Ca2+-CaM with high affinity, yet the ligand sensitivity of α2 channels is not modulated by CaM.PKC-mediated phosphorylation increased the activation constant (K1/2) for cGMP from 19 to 56 μm and decreased the Hill coefficient (from 2.5 to 1.5). The change in ligand sensitivity involves phosphorylation of the serine residues S577 and S579 in the cGMP-binding domain. The increase in K1/2 was completely abolished in mutant channels in which the two serine residues were replaced by alanine.An antibody specific for the δ isoform of PKC strongly labels the cone outer segments.Modulation of cGMP affinity of bovine α2 CNG channels by phosphorylation could play a role in the regulation of photoreceptor sensitivity

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

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

Class IA Phosphatidylinositol 3-Kinase Isoform p110 alpha 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 (p110 alpha, p110 beta, p110 delta), 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 p110 alpha (PIK-75), p110 beta (TGX-221), and p110 delta (IC-87114), respectively. We identified p110 alpha to be crucial for receptor tyrosine kinases signaling, thus affecting proliferation, migration, and survival of rat, murine, and human smooth muscle cells, whereas p110 beta and p110 delta activities were dispensable. Surprisingly, p110 delta 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 p110 alpha deficiency (sm-p110 alpha(-/-)). Targeted deletion of p110 alpha in sm-p110 alpha(-/-) mice blunted growth factor-induced cellular responses and abolished neointima formation after balloon injury of the carotid artery in mice. In contrast, p110 delta deficiency did not affect vascular remodeling in vivo. Conclusions-Receptor tyrosine kinases-induced phosphatidylinositol 3'-kinase signaling via the p110 alpha isoform plays a central role for vascular remodeling in vivo. Thus, p110 alpha represents a selective target for the prevention of neointima formation after vascular injury, whereas p110 beta and p110 delta expression and activity do not play a significant role