Qing-Ping Yao

Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis

AIMS Low shear stress (LSS) plays a significant role in vascular remodelling during atherogenesis, which involves migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). The aim of the present study is to elucidate the molecular mechanisms by which LSS induces vascular remodelling. METHODS AND RESULTS Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague-Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm(2), were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDIalpha) in the LSS vessels. Rho-GDIalpha signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDIalpha in the LSS-induced vascular remodelling. A decrease in Rho-GDIalpha expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDIalpha expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDIalpha siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis. CONCLUSION The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDIalpha. The effect of Rho-GDIalpha on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.

Association of SIRT1 expression with shear stress induced endothelial progenitor cell differentiation.

Shear stress imposed by blood flow is crucial for differentiation of endothelial progenitor cells (EPCs). Histone deacetylase SIRT1 has been shown to play a pivotal role in many physiological processes. However, association of SIRT1 expression with shear stress‐induced EPC differentiation remains to be elucidated. The present study was designed to determine the effect of SIRT1 on EPC differentiation induced by shear stress, and to seek the underlying mechanisms. Human umbilical cord blood‐derived EPCs were exposed to laminar shear stress of 15 dyn/cm2 by parallel plate flow chamber system. Shear stress enhanced EPC differentiation toward endothelial cells (ECs) while inhibited to smooth muscle cells (SMCs). The expressions of phospho‐Akt, SIRT1 and histone H3 acetylation (Ac‐H3) in EPCs were detected after exposure to shear stress for 2, 6, 12, and 24 h, respectively. Shear stress significantly activated Akt phosphorylation, augmented SIRT1 expression and downregulated Ac‐H3. SIRT1 siRNA in EPCs diminished the expression of EC markers, but increased the expression of SMC markers, and resulted in upregulation of Ac‐H3. Whereas, resveratrol, an activator of SIRT1, had the opposite effects on both EPC differentiation and histone H3 acetylation. Wortmannin, an inhibitor of PI3‐kinase, suppressed endothelial differentiation of EPCs, decreased SIRT1, and upregulated Ac‐H3 expression. In addition, SIRT1 promoted tube formation of EPCs in matrix gels. These results provided a mechanobiological basis of shear stress‐induced EPC differentiation into ECs and suggest that PI3k/Akt‐SIRT1‐Ac‐H3 pathway is crucial in such a process. J. Cell. Biochem. 113: 3663–3671, 2012.

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.

Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

Significance The proliferation of vascular smooth muscle cells (VSMCs) in response to excessive cyclic stretch is crucial in vascular remodeling in hypertension. To elucidate the molecular mechanism, we studied the mechanobiological roles of emerin and lamin A/C, two important components of nuclear envelope proteins localized beneath the inner nuclear membrane. We found that emerin and lamin A/C play significant roles in the mechanical modulation of VSMC proliferation. The repressed expression of emerin and lamin A/C mediates the stretch-induced VSMC proliferation, which is important in vascular remodeling during hypertension. Emerin and lamin A/C bind to the respective sequencing-specific motifs of transcription factors to mediate the molecular mechanisms underlying the hyperstretch-induced VSMC dysfunction. Cyclic stretch is an important inducer of vascular smooth muscle cell (VSMC) proliferation, which is crucial in vascular remodeling during hypertension. However, the molecular mechanism remains unclear. We studied the effects of emerin and lamin A/C, two important nuclear envelope proteins, on VSMC proliferation in hypertension and the underlying mechano-mechanisms. In common carotid artery of hypertensive rats in vivo and in cultured cells subjected to high (15%) cyclic stretch in vitro, VSMC proliferation was increased significantly, and the expression of emerin and lamin A/C was repressed compared with normotensive or normal (5%) cyclic stretch controls. Using targeted siRNA to mimic the repressed expression of emerin or lamin A/C induced by 15% stretch, we found that VSMC proliferation was enhanced under static and 5%-stretch conditions. Overexpression of emerin or lamin A/C reversed VSMC proliferation induced by 15% stretch. Hence, emerin and lamin A/C play critical roles in suppressing VSMC hyperproliferation induced by hyperstretch. ChIP-on-chip and MOTIF analyses showed that the DNAs binding with emerin contain three transcription factor motifs: CCNGGA, CCMGCC, and ABTTCCG; DNAs binding with lamin A/C contain the motifs CVGGAA, GCCGCYGC, and DAAGAAA. Protein/DNA array proved that altered emerin or lamin A/C expression modulated the activation of various transcription factors. Furthermore, accelerating local expression of emerin or lamin A/C reversed cell proliferation in the carotid artery of hypertensive rats in vivo. Our findings establish the pathogenetic role of emerin and lamin A/C repression in stretch-induced VSMC proliferation and suggest mechanobiological mechanism underlying this process that involves the sequence-specific binding of emerin and lamin A/C to specific transcription factor motifs.

Induction of Thoracic Aortic Remodeling by Endothelial-Specific Deletion of MicroRNA-21 in Mice

MicroRNAs (miRs) are known to have an important role in modulating vascular biology. MiR21 was found to be involved in the pathogenesis of proliferative vascular disease. The role of miR21 in endothelial cells (ECs) has well studied in vitro, but the study in vivo remains to be elucidated. In this study, miR21 endothelial-specific knockout mice were generated by Cre/LoxP system. Compared with wild-type mice, the miR21 deletion in ECs resulted in structural and functional remodeling of aorta significantly, such as diastolic pressure dropping, maximal tension depression, endothelium-dependent relaxation impairment, an increase of opening angles and wall-thickness/inner diameter ratio, and compliance decrease, in the miR21 endothelial-specific knockout mice. Furthermore, the miR21 deletion in ECs induced down-regulation of collagen I, collagen III and elastin mRNA and proteins, as well as up-regulation of Smad7 and down-regulation of Smad2/5 in the aorta of miR21 endothelial-specific knockout mice. CTGF and downstream MMP/TIMP changes were also identified to mediate vascular remodeling. The results showed that miR21 is identified as a critical molecule to modulate vascular remodeling, which will help to understand the role of miR21 in vascular biology and the pathogenesis of vascular diseases.

Neuropeptide Y Stimulates Proliferation and Migration of Vascular Smooth Muscle Cells from Pregnancy Hypertensive Rats via Y1 and Y5 Receptors

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in pathophysiological remodeling of arteries during hypertension in pregnancy. However, the mechanisms involved in this process remain unclear. We hypothesized that Neuropeptide Y (NPY), which is a potent mitogenic peptide, participates in modulating proliferation and migration of VSMCs during hypertension in pregnancy. Using pregnant hypertensive rats, induced by intraperitoneal injection of L-nitro-arginine methylester (L-NAME), the plasma concentration of NPY was detected. Open angle, which reflects the non-uniform remodeling with high sensitivity, was used to detect the pathophysiological vascular remodeling in vivo. The results revealed that NPY concentration and artery open angle were both significantly increased in rats with hypertension in pregnant. The underlying mechanism of elevated NPY on vascular remodeling were further analyzed by using cultured VSMCs in vitro. In cultured VSMCs, NPY most effectively stimulated the migration and proliferation of VSMCs at 10-6 mol/L, similar to the plasma concentration in L-NAME hypertension in pregnant rats. NPY up-regulated the expressions of both Y1 and Y5 receptors, increased the phosphorylations of STAT3 on Tyr705 and Ser727 residues, and induced the expression of c-Fos. The NPY-induced VSMCs proliferation was reduced by Y5 receptor antagonist, and fully blocked by combinations with other antagonist, such as Y2+Y5, Y1+Y5, and Y1+Y2+Y5. In contrast, the NPY-induced VSMC migration was blocked by either Y receptor antagonist or any combination of Y receptor antagonists. These results suggest that the elevated plasma concentration of NPY during hypertension in pregnancy may induce VSMC proliferation mainly via Y5 receptor, which subsequently modulate STAT3 and c-Fos signaling pathways to result in the vascular remodeling. These results also suggest that NPY mainly acts on VSMCs in vitro via Y1, Y5 receptors and in vascular tissues in vivo via Y5 receptor.

MicroRNA-33 protects against neointimal hyperplasia induced by arterial mechanical stretch in the grafted vein

Aims Mechanical factors play significant roles in neointimal hyperplasia after vein grafting, but the mechanisms are not fully understood. Here, we investigated the roles of microRNA-33 (miR-33) in neointimal hyperplasia induced by arterial mechanical stretch after vein grafting. Methods and results Grafted veins were generated by the ‘cuff’ technique. Neointimal hyperplasia and cell proliferation was significantly increased, and miR-33 expression was decreased after 1-, 2-, and 4-week grafts. In contrast, the expression of bone morphogenetic protein 3 (BMP3), which is a putative target of miR-33, and the phosphorylation of smad2 and smad5, which are potential downstream targets of BMP3, were increased in the grafted veins. miR-33 mimics/inhibitor and dual luciferase reporter assay confirmed the interaction of miR-33 and BMP3. miR-33 mimics attenuated, while miR-33 inhibitor accelerated, proliferation of venous smooth muscle cells (SMCs). Moreover, recombinant BMP3 increased SMC proliferation and P-smad2 and P-smad5 levels, whereas BMP3-directed siRNAs had the opposite effect. Then, venous SMCs were exposed to a 10%-1.25 Hz cyclic stretch (arterial stretch) by using the FX4000 cyclic stretch loading system in vitro to mimic arterial mechanical conditions. The arterial stretch increased venous SMC proliferation and repressed miR-33 expression, but enhanced BMP3 expression and smad2 and smad5 phosphorylation. Furthermore, perivascular multi-point injection in vivo demonstrated that agomiR-33 not only attenuates BMP3 expression and smad2 and smad5 phosphorylation, but also slows neointimal formation and cell proliferation in grafted veins. These effects of agomiR-33 on grafted veins could be reversed by local injection of BMP3 lentivirus. Conclusion The miR-33-BMP3-smad signalling pathway protects against venous SMC proliferation in response to the arterial stretch. miR-33 is a target that attenuates neointimal hyperplasia in grafted vessels and may have potential clinical applications.

Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress.

The dysfunction of vascular endothelial cells (ECs) influenced by flow shear stress is crucial for vascular remodeling. However, the roles of nuclear envelope (NE) proteins in shear stress-induced EC dysfunction are still unknown. Our results indicated that, compared with normal shear stress (NSS), low shear stress (LowSS) suppressed the expression of two types of NE proteins, Nesprin2 and LaminA, and increased the proliferation and apoptosis of ECs. Targeted small interfering RNA (siRNA) and gene overexpression plasmid transfection revealed that Nesprin2 and LaminA participate in the regulation of EC proliferation and apoptosis. A protein/DNA array was further used to detect the activation of transcription factors in ECs following transfection with target siRNAs and overexpression plasmids. The regulation of AP-2 and TFIID mediated by Nesprin2 and the activation of Stat-1, Stat-3, Stat-5 and Stat-6 by LaminA were verified under shear stress. Furthermore, using Ingenuity Pathway Analysis software and real-time RT-PCR, the effects of Nesprin2 or LaminA on the downstream target genes of AP-2, TFIID, and Stat-1, Stat-3, Stat-5 and Stat-6, respectively, were investigated under LowSS. Our study has revealed that NE proteins are novel mechano-sensitive molecules in ECs. LowSS suppresses the expression of Nesprin2 and LaminA, which may subsequently modulate the activation of important transcription factors and eventually lead to EC dysfunction.

SIRT1 and Connexin40 Mediate the Normal Shear Stress-Induced Inhibition of the Proliferation of Endothelial Cells Co-Cultured with Vascular Smooth Muscle Cells

Background: Shear stress imposed by blood flow directly impacts endothelial cells (ECs), which are simultaneously influenced by neighboring vascular smooth muscle cells (VSMCs). However, the mechanisms by which shear stress and VSMCs modulate EC proliferation remain to be elucidated. Methods: ECs, cultured alone or co-cultured with VSMCs, were subjected to a normal level of laminar shear stress (NSS) of 15 dyne/cm2 or kept under static conditions by using a parallel-plate flow chamber system, respectively. Results: BrdU incorporation assay and flow cytometry revealed that NSS inhibited EC proliferation with or without VSMCs. Western blot analysis demonstrated that NSS down-regulated the expression of Connexin40 (Cx40) in both ECs cultured alone and ECs co-cultured with VSMCs, accompanied by the increased expression of SIRT1. Moreover, salermide, an inhibitor of SIRT1, as well as SIRT1-specifc siRNA transfection inhibited the effect of NSS on EC proliferation and Cx40 expression. In contrast, resveratrol, a SIRT1 activator, induced an alteration in ECs similar to the application of NSS. Conclusion: NSS inhibits the proliferation of ECs via SIRT1 and Cx40 in the presence or absence of VSMCs. The data suggest that NSS plays a protective role in vascular homeostasis by maintaining EC proliferation at a normal level.

Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells

Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.