Loss of Transforming Growth Factor Beta Signaling in Aortic Smooth Muscle Cells Causes Endothelial Dysfunction and Aortic Hypercontractility

Objective: Humans and mice with loss-of-function variants of genes in the TGF-β (transforming growth factor beta) signaling pathway develop aortic aneurysms. These aneurysms could be caused by decreased aortic smooth muscle cell (SMC) contractile-protein levels and impaired aortic SMC contractile-unit function. Accordingly, we investigated whether loss of SMC TGF-β signaling in mice alters aortic contractile-protein levels and aortic contractility. Approach and Results: We used immunoblotting, wire myography, histological analyses, and measurements of aortic nitric oxide and superoxide levels to assess aortic contractile-protein levels and vasomotor function in mice with SMC-specific deletion of the type 2 TGF-β receptor (TBR2 SMΔ mice). Aortic contractile-protein levels were not altered in TBR2 SMΔ mice. Surprisingly, TBR2 SMΔ mice had increased aortic contractility and severe endothelial dysfunction. Endothelial dysfunction was manifested as decreased relaxation to acetylcholine (Emax 37% versus 97%; P <0.0001), decreased aortic nitric oxide (50%; P =0.005), decreased endothelial nitric oxide synthase activation (31%; P =0.002), and lower aortic levels of phosphorylated vasodilator-stimulated phosphoprotein (an indicator of nitric oxide bioavailability: 65%; P <0.0001). Aortic hypercontractility was reduced by mechanical denudation of endothelium and was eliminated by pretreatment of TBR2 SMΔ and control aortas with a nitric oxide synthase inhibitor, revealing a significant positive interaction between aortic hypercontractility and absence of endothelium-derived nitric oxide ( P <0.05 for both denudation and nitric oxide inhibition). Conclusions: Aortic aneurysms that develop in TBR2 SMΔ mice are not caused by decreased SMC contractility. Loss of physiological SMC TGF-β signaling causes endothelial dysfunction leading to aortic hypercontractility. Endothelial dysfunction may contribute to vascular pathologies associated with abnormal TGF-β signaling.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling

Objective: The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. Conclusions: SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

Ubiquitin Pathway Is Associated with Worsening Left Ventricle Function after Mitral Valve Repair: A Global Gene Expression Study

The molecular mechanism for worsening left ventricular (LV) function after mitral valve (MV) repair for chronic mitral regurgitation remains unknown. We wished to assess the LV transcriptome and identify determinants associated with worsening LV function post-MV repair. A total of 13 patients who underwent MV repair for chronic primary mitral regurgitation were divided into two groups, preserved LV function (N = 8) and worsening LV function (N = 5), for the study. Specimens of LV from the patients taken during surgery were used for the gene microarray study. Cardiomyocyte cell line HL-1 cells were transfected with gene-containing plasmids and further evaluated for mRNA and protein expression, apoptosis, and contractile protein degradation. Of 67,258 expressed sequence tags, microarrays identified 718 genes to be differentially expressed between preserved-LVF and worsening-LVF, including genes related to the protein ubiquitination pathway, bone morphogenetic protein (BMP) receptors, and regulation of eIF4 and p70S6K signaling. In addition, worsening-LVF was associated with altered expressions of genes pathologically relevant to heart failure, such asdownregulated apelin receptors and upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). HL-1 cardiomyocyte cells transfected with ubiquitination-related genes demonstrated activation of the protein ubiquitination pathwaywith an increase in the ubiquitin activating enzyme E1 (UAE-E1). It also led to increased apoptosis, downregulated and ubiquitinated X-linked inhibitor of apoptosis protein (XIAP), and reduced cell viability. Overexpression of ubiquitination-related genes also resulted in degradation and increased ubiquitination of α-smooth muscle actin (SMA). In conclusion, worsening-LVF presented differential gene expression profiles from preserved-LVF after MV repair. Upregulation of protein ubiquitination-related genes associated with worsening-LVF after MV repair may exert adverse effects on LV through increased apoptosis and contractile protein degradation.

Drug-eluting stent specifically designed to target vascular smooth muscle cell phenotypic modulation attenuated restenosis through the YAP pathway

Vascular smooth muscle cell (SMC) phenotypic modulation contributes to the development of restenosis. A sorafenib-eluting stent was specifically designed to target SMC phenotypic modulation to inhibit in-stent restenosis in the present study. SMC contractile protein from the freshly isolated rat aorta was expressed at a high level, but its expression was dramatically reduced after SMCs were cultured in 10% FBS for 1 wk. After sorafenib treatment, SMC contractile protein expression was markedly upregulated. We further observed that Yes-associated protein (YAP) expression was attenuated after sorafenib treatment in a dose-dependent manner. Overexpression of YAP by lentivirus reversed the expression of sorafenib-induced SMC contractile protein and increased the expression of cyclin D. Mechanistically, sorafenib regulated the serum response factor-myocardin (SRF-Myocd) complex through competitive binding of YAP to Myocd and increased SRF binding to CArG-containing regions of SMC-specific contractile genes within intact chromatin, thereby controlling the activity of smooth muscle-specific gene transcription. In a rabbit carotid model, the sorafenib-eluting stent (SFES) dramatically inhibited in-stent restenosis and upregulated SMC contractile protein expression. Overexpression of YAP blocked the antirestenosis effect of SFES and repressed contractile smooth muscle-specific genes in vivo, indicating that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. We demonstrated that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. Targeting SMC phenotypic modulation by drug-eluting stent represents an attractive therapeutic approach for the treatment of occlusive vascular diseases. NEW & NOTEWORTHY In the present study, we demonstrated that sorafenib regulates smooth muscle cell (SMC) phenotypic modulation from a proliferative to a contractile state. Sorafenib induced a myocardin-serum response factor interaction and increased SMC contractile gene transcription through the Yes-associated protein pathway. Moreover, local delivery of sorafenib regulating SMC phenotypic modulation represents a promising strategy in the design of drug-eluting stents.

Combined effect of chronic partial occlusion and orthostatic load on the saphenous vein network: A varicosity model in the rat

Objectives We tested the combined effects of chronic flow obstacle and gravitation on the saphenous vein network of rats. Methods A narrowing clip (500 µm, partial occlusion) was administered on the saphenous vein main branch for 4, 8 and 12 weeks, either separately or in combination with chronic orthostatic load (tilted tube-cages for four weeks). Resulting network changes were studied on plastic casts, by video-microscopy, histochemistry–immunohistochemistry and image analysis. Results A rich collateral venous network developed containing newly formed masses of retrograde conducting small veins. Their walls had less dense elastica, less contractile protein, increased cell division activity and macrophage invasion, and were more sensitive to chronic gravitational load. Conclusions Hemodynamic disturbance induces remodeling of the saphenous vein network. Walls of veins being in the process of flow-induced morphological remodeling are weak and more sensitive to gravitational load. Reticular vein conglomerates, veins with local dilations, and convoluted courses were observed.