miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Apoptosis repressor with caspase recruitment domain is dramatically reduced in cardiac, skeletal, and vascular smooth muscle during hypertension

Apoptosis repressor with caspase recruitment domain (ARC) is a unique anti-apoptotic protein with a distinct tissue distribution. In addition, unlike most anti-apoptotic proteins which act on one pathway, ARC can inhibit apoptosis mediated by both the death-receptor and mitochondrial signaling pathways. In this study, we confirm previous reports showing high levels of ARC protein in rat heart and skeletal muscle, but demonstrate for the first time that ARC is also expressed in rat aorta. Immunoblot analysis on endothelium-denuded aorta as well as immunohistochemical analysis on intact aorta demonstrated that ARC was highly expressed in smooth muscle. Immunoblot analysis also found that ARC protein was severely downregulated in skeletal muscle (−82%; P < 0.001), heart (−80%; P < 0.001), and aorta (−71%; P < 0.001) of spontaneously hypertensive rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats. Decreased ARC levels were also confirmed in tissues of hypertensive animals by immunohistochemical analysis. Collectively, this data suggests that ARC protein is expressed in vascular smooth muscle and is significantly reduced in several target tissues during hypertension.     

Comparison of patient and staff temple dose during fluoroscopically guided coronary angiography,implantable cardiac devices,and electrophysiology procedures

There is a paucity of literature comparing patient and staff dose during coronary angiography (CA), implantable cardiac devices, permanent pacemakers (PPM) and electrophysiology (EP) procedures and little noting dose to staff other than cardiologists. This study sought to compare patient and occupational dose during a range of fluoroscopically guided cardiac procedures. Radiation dose levels for the patients (n = 1651), cardiologists (n = 24), scrub (n = 32) and scout nurses (n = 35) were measured in a prospective single-centre study between February 2017 and August 2019. A comparison of dose during CA, device implantation, PPM insertion and EP studies was performed. Three angiographic units were used, with dosimeters worn on the temple of staff. Results indicated that occupational dose during PPM was significantly higher than other procedures. The cardiologist had the highest mean dose during biventricular implantable cardioverter-defibrillators; levels were approximately five times that of ‘normal’ pacemaker insertions. Transcatheter aortic valve implantations (TAVI) were associated with relatively high mean doses for both staff and patients and had a statistically significant higher (>2 times) mean patient dose area product than all other categories. TAVI workups were also related to higher mean cardiologist and scrub nurse dose. It was observed that the mean scrub nurse dose can exceed that of the cardiologist. The highest mean dose for Scout nurses were recorded during EP studies. Given the significantly higher temple dose associated with PPM insertion, cardiologists should consider utilizing ceiling mounted lead shields, lead glasses and/or skull caps where possible. Efforts should also be made to minimize the use of DSA during TAVI and TAVI workups to reduce cardiologist, nurse and patient dose.     

The role of SIRT6 in the differentiation of vascular smooth muscle cells in response to cyclic strain

Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-β1 (TGF-β1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-β1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule-1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-β1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain.     

血流阻断的缺血预处理技术在肝癌切除术中的临床应用价值

目的:探讨血流阻断的缺血预处理技术在肝癌切除术中的临床应用价值。方法:选取2010年4月至2013年7月我院收治的96名原发性肝癌并采用肝脏部分切除术进行治疗的患者,将患者随机分为观察组和对照组,每组各48例,观察组在肝脏部分切除术阻断肝门血流前先分别给予1个5 min缺血和再灌注的处理。对照组不采取任何干预措施。术前、术后1天、3天、7天时分别进行生化检查,并于术前及术后1h对Fas-mRNA表达、Caspasc-3活性及AI进行测定,观察记录患者术后的并发症情况、手术时间、术中出血量以及住院时间。结果:术后1天、3天、7天时两组间的AST、ALT、TBIL等生化指标的含量情况相比,观察组均显著优于对照组(P0.05);术后l d,两组患者ALB均有不同程度的降低,对照组低于观察组(P0.05);术后住院时间观察组为13.28±3.85天,对照组为19.48±4.92天,观察组明显低于对照组(P0.05);术后1h,两组患者的Fas-mRNA表达、Caspasc-3活性相比于阻断前均显著提高,但观察组提高幅度明显低于对照组,差异有统计学意义(P0.05);两组阻断前均未见肝细胞凋亡,术后l h时,两组组均可见肝细胞凋亡,且对照组明显高于观察组组(P0.05)。结论:血流阻断的缺血预处理技术具有操作简便、副作用小的重要特点,应用于肝癌切除术中在保护肝功能方面具有显著的优势。     

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.     

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.     

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na⁺-K⁺ pump’s β₁-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na⁺-K⁺ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na⁺-K⁺ pump activity is an important determinant of vascular tone through effects on [Ca²⁺]_i, we have examined the role of oxidative regulation of the Na⁺-K⁺ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β₁-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na⁺-K⁺ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K⁺-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na⁺-K⁺ ATPase and decrease in K⁺-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β₁-subunit. Knock-out of FXYD1 dramatically decreased K⁺-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β₁-subunit glutathionylation, and enhanced K⁺-induced vasorelaxation. Ang II inhibits the Na⁺-K⁺ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β₁-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na⁺-K⁺ pump and may have an important protective role in the vasculature under conditions of oxidative stress.     

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.