The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks' follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio

Functional interactions between the Forkhead transcription factor FOXK1 and the MADS-box protein SRF

The combinatorial control of gene expression by the association of members of different families of transcription factors is a common theme in eukaryotic transcriptional control. The MADS-box transcription factors SRF and Mcm1 represent paradigms for such regulation through their interaction with numerous partner proteins. For example, in Saccharomyces cerevisiae, Mcm1 interacts with the forkhead transcription factor Fkh2. Here, we identify a novel interaction between SRF and the Forkhead transcription factor FOXK1 in human cells. The importance of this interaction is shown for the regulation of the SRF target genes SM α-actin and PPGB. The binding of FOXK1 to the SM α-actin and PPGB promoters requires the presence of SRF on the promoter. FOXK1 acts as a transcriptional repressor and it represses SM α-actin and PPGB expression. Thus FOXK1 represents an additional member of the growing repertoire of transcription factors that can interact with SRF and modulate the transcriptional output from SRF-regulated promoters

Bindarit inhibits human coronary artery smooth muscle cell proliferation, migration and phenotypic switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100-300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation

Vitamin D treatment prevents uremia-induced reductions in aortic microRNA-145 attenuating osteogenic differentiation despite hyperphosphatemia

In chronic kidney disease, systemic inflammation and high serum phosphate (P) promote the de-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells, increasing the propensity for medial calcification and cardiovascular mortality. Vascular microRNA-145 (miR-145) content is essential to maintain VSMC contractile phenotype. Because vitamin D induces aortic miR-145, uremia and high serum P reduce it and miR-145 directly targets osteogenic osterix in osteoblasts, this study evaluated a potential causal link between vascular miR-145 reductions and osterix-driven osteogenic differentiation and its counter-regulation by vitamin D. Studies in aortic rings from normal rats and in the rat aortic VSMC line A7r5 exposed to calcifying conditions corroborated that miR-145 reductions were associated with decreases in contractile markers and increases in osteogenic differentiation and calcium (Ca) deposition. Furthermore, miR-145 silencing enhanced Ca deposition in A7r5 cells exposed to calcifying conditions, while miR-145 overexpression attenuated it, partly through increasing α-actin levels and reducing osterix-driven osteogenic differentiation. In mice, 14 weeks after the induction of renal mass reduction, both aortic miR-145 and α-actin mRNA decreased by 80% without significant elevations in osterix or Ca deposition. Vitamin D treatment from week 8 to 14 fully prevented the reductions in aortic miR-145 and attenuated by 50% the decreases in α-actin, despite uremia-induced hyperphosphatemia. In conclusion, vitamin D was able to prevent the reductions in aortic miR-145 and α-actin content induced by uremia, reducing the alterations in vascular contractility and osteogenic differentiation despite hyperphosphatemia

Blood-borne origin of neointimal smooth muscle cells in transplant arteriosclerosis

Transplant arteriosclerosis (TA) is a major complication after solid organ transplantation. TA is characterized by persistent perivascular inflammation and concentric intimal thickening consisting of α-actin-positive vascular smooth muscle (VSM) cells. The current view on TA is that donor-derived medial VSM cells of affected arteries migrate and proliferate into the subendothelial space, resulting in luminal narrowing. Following this concept, the VSM cells present in the arteriosclerotic lesions are of donor origin. In this study, the authors analyzed the origin (donor vs recipient) of endothelium (EC) and neointimal α-actin-positive VSM cells in 2 different experimental transplant models. Aortic and cardiac allografting was performed in the PVG (RT-1c) to AO (RT-1u) rat strain combination. Aorta recipients were not immunosuppressed, whereas cardiac allograft recipients were intrathymically immune modulated to prevent acute rejection. Transplants were performed from female donor to male recipient rats. The α-actin-positive VSM cells present in arteriosclerotic lesions, in aortic as well as cardiac allografts, were of recipient, rather than donor, origin. Following aortic allografts, the ECs are completely replaced by host-derived ECs, whereas in cardiac allografts the ECs are still of donor origin.</p

Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

OBJECTIVE: Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. APPROACH AND RESULTS: In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1β, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CONCLUSIONS: CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies

A mouse bone marrow stromal cell line, TBR-B, shows inducible expression of smooth muscle-specific genes

AbstractWe established an in vitro culture system which mimicked the differentiation pathway of smooth muscle cell, using TBR-B, a bone marrow stromal cell line derived from transgenic mice harboring temperature-sensitive SV40 large T-antigen gene. TBR-B cells have the potential to express smooth muscle-specific genes including h1-calponin, h-caldesmon, SM22α and α-actin, only after cultured in the differentiation medium for 2 weeks. The differentiation state of TBR-B was well controlled by using different culture medium. Using this cell line, we also found that ascorbic acid is a potent factor inducing the expression of h1-calponin and α-actin. TBR-B cells will serve as a useful tool for elucidating the regulatory mechanisms of smooth muscle-specific gene expression, and for identifying compounds that regulate the differentiation state of vascular smooth muscle cells

Endocardial-mesenchymal transition underlies fusion of the conotruncal ridges during embryonic cardiac outflow tract septation

The embryonic cardiac outflow tract (conotruncus) is a single tubular chamber that connects the right ventricle with the aortic arch arteries. It contains two opposite, long and helical mesenchymal cushions covered by endocardial cells (conotruncal ridges). Conotruncal division (septation) gives rise to the adult right and left outflows together with the aortic and pulmonary valves. It takes place by fusion of the two opposite ridges and formation of the conotruncal septum. Although the participation of neural crest cells in septation is well established, the mechanism of fusion of the conotruncal ridges remains unknown. Defects in fusion have been shown to produce bicuspid aortic valve, the most prevalent human congenital cardiac malformation, in a hamster model. Three fusion mechanisms have been proposed to operate during embryonic development: epithelial adhesion, epithelial apoptosis and epithelial-mesenchymal transition (EMT). The first mechanism entails the expression of adhesion molecules and the maintenance of the identity of cells in contact, whereas in the other two, epithelial cells covering the fusing structures disappear by apoptosis or by transforming into mesenchymal cells. The objective of this study is to elucidate the mechanism involved in the fusion of the conotruncal ridges. Immunofluorecence techniques were used in ED 11-12 hamster embryos. The results indicate that the mechanism of EMT, but not epithelial adhesion or apoptosis, is involved in the process of fusion of the conotruncal ridges. The EMT mechanism associated with conotruncal septation seems to be uncoupled from the process of formation of the endocardial cushions, which takes place at early stages. With these results, we can raise the hypothesis that defects in the EMT process may lead to different morphological types of bicuspid aortic valve.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This study was supported by P10-CTS-6068 (Junta de Andalucía), CGL2014-52356-P and CGL2017-85090-P (Ministerio de Economía y Competitividad), contract UMAJI75 (Junta de Andalucía, European Social Fund), and Universidad de Málaga