Suppressed nuclear envelope proteins activate autophagy of vascular smooth muscle cells during cyclic stretch application

Dysfunctions of vascular smooth muscle cells (VSMCs) play crucial roles in vascular remodeling in hypertension, which correlates with pathologically elevated cyclic stretch due to increased arterial pressure. Recent researches reported that autophagy, a life-sustaining process, was increased in hypertension. However, the mechanobiological mechanism of VSMC autophagy and its potential roles in vascular remodeling are still unclear. Using renal hypertensive rats in vivo and FX5000 stretch application Unit in vitro, the autophagy of VSMCs was detected. The results showed that LC3II remarkably enhanced in hypertensive rats and 15% cyclic stretch (mimic the pathologically increased mechanical stretch in hypertension), and the activity of mammalian target of rapamycin (mTOR) was suppressed in 15% cyclic stretch. Administration of autophagy inhibitors, bafilomycin A1 and chloroquine, repressed VSMC proliferation efficiently, but did not affect the degradation of two important nuclear envelope (NE) proteins, lamin A/C and emerin. Using RNA interference to decline the expression of lamin A/C and emerin, respectively, we discovered that autophagy was upregulated under both static and 5% cyclic stretch conditions, accompanying with the decreased mTOR activity. During 15% cyclic stretch application, mTOR inhibition was responsible for autophagy elevation. Chloroquine administration in vivo inhibited the expression of PCNA (marker of proliferation) of abdominal aorta in hypertensive rats. Altogether, these results demonstrated that pathological cyclic stretch suppresses the expression of lamin A/C and emerin which subsequently represses mTOR pathway so as to induce autophagy activation. Blocking autophagic flux may be a practicable way to relieve the pathological vascular remodeling in hypertensive.     

Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia.     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Background

The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.

Results

Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [³H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.

Conclusions

Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.     

Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.     

Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator–activated receptor-γ coactivator-1α

In varicose veins, vascular smooth muscle cells (VSMCs) often show abnormal proliferative and migratory rates and phenotypic transition. This study aimed to investigate whether microRNA (miR)-202 and its potential target, peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), were involved in VSMC phenotypic transition. miR-202 expression was analyzed in varicose veins and in VSMCs conditioned with platelet-derived growth factor. The effect of miR-202 on cell proliferation and migration was assessed. Furthermore, contractile marker SM-22α, synthetic markers vimentin and collagen I, and PGC-1α were analyzed by Western blot analysis. The modulation of PGC-1α expression by miR-202 was also evaluated. In varicose veins and proliferative VSMCs, miR-202 expression was upregulated, with decreased SM-22α expression and increased vimentin and collagen I expression. Transfection with a miR-202 mimic induced VSMC proliferation and migration, whereas a miR-202 inhibitor reduced cell proliferation and migration. miR-202 mimic constrained luciferase activity in HEK293 cells that were cotransfected with the PGC-1α 3′-untranslated region (3′-UTR) but not those with mutated 3′-UTR. miR-202 suppressed PGC-1α protein expression, with no influence on its messenger RNA expression. PGC-1α mediated VSMC phenotypic transition and was correlated with reactive oxygen species production. In conclusion, miR-202 affects VSMC phenotypic transition by targeting PGC-1α expression, providing a novel target for varicose vein therapy.     

Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/β-catenin axis

Characterized by abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), neointima hyperplasia is a hallmark of vascular restenosis after percutaneous vascular interventions. Vaccinia-related kinase 1 (VRK1) is a stress adaption-associated ser/thr protein kinase that can induce the proliferation of various types of cells. However, the role of VRK1 in the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury remains unknown. We observed increased expression of VRK1 in VSMCs subjected to platelet-derived growth factor (PDGF)-BB by western blotting. Silencing VRK1 by shVrk1 reduced the number of Ki-67-positive VSMCs and attenuated the migration of VSMCs. Mechanistically, we found that relative expression levels of β-catenin and effectors of mTOR complex 1 (mTORC1) such as phospho (p)-mammalian target of rapamycin (mTOR), p-S6, and p-4EBP1 were decreased after silencing VRK1. Restoration of β-catenin expression by SKL2001 and re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) both abolished shVrk1-mediated inhibitory effect on VSMC proliferation and migration. siTsc1 also rescued the reduced expression of β-catenin caused by VRK1 inhibition. Furthermore, mTORC1 re-activation failed to recover the attenuated proliferation and migration of VSMC resulting from shVrk1 after silencing β-catenin. We also found that the vascular expression of VRK1 was increased after injury. VRK1 inactivation in vivo inhibited vascular injury-induced neointima hyperplasia in a β-catenindependent manner. These results demonstrate that inhibition of VRK1 can suppress the proliferation and migration of VSMC and neointima hyperplasia after vascular injury via mTORC1/β-catenin pathway.     

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

Aims

This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms.

Methods

Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression.

Results

Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis.

Conclusions

Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.     

Mechanical Stretch Suppresses microRNA-145 Expression by Activating Extracellular Signal-Regulated Kinase 1/2 and Upregulating Angiotensin-Converting Enzyme to Alter Vascular Smooth Muscle Cell Phenotype

Phenotype modulation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of various vascular diseases, including hypertension and atherosclerosis. Several microRNAs (miRNAs) were found involved in regulating the VSMC phenotype with platelet-derived growth factor (PDGF) treatment, but the role of miRNAs in the mechanical stretch-altered VSMC phenotype is not clear. Here, we identified miR-145 as a major miRNA contributing to stretch-altered VSMC phenotype by miRNA array, quantitative RT-PCR and gain- and loss-of-function methods. Our data demonstrated that 16% stretch suppressed miR-145 expression, with reduced expression of contractile markers of VSMCs cultured on collagenI; overexpression of miR-145 could partially recover the expression in stretched cells. Serum response factor (SRF), myocardin, and Kruppel-like factor 4 (KLF4) are major regulators of the VSMC phenotype. The effect of stretch on myocardin and KLF4 protein expression was altered by miR-145 mimics, but SRF expression was not affected. In addition, stretch-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and up-regulated angiotensin-converting enzyme (ACE) were confirmed to be responsible for the inhibition of miR-145 expression. Mechanical stretch inhibits miR-145 expression by activating the ERK1/2 signaling pathway and promoting ACE expression, thus modulating the VSMC phenotype.     

Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells

The pulsatile nature of blood pressure and flow creates hemodynamic stimuli in the forms of cyclic stretch and shear stress, which exert continuous influences on the constituents of the blood vessel wall. Vascular smooth muscle cells (VSMCs) use multiple sensing mechanisms to detect the mechanical stimulus resulting from pulsatile stretch and transduce it into intracellular signals that lead to modulations of gene expression and cellular functions, e.g., proliferation, apoptosis, migration, and remodeling. The cytoskeleton provides a structural framework for the VSMC to transmit mechanical forces between its luminal, abluminal, and junctional surfaces, as well as its interior, including the focal adhesion sites, the cytoplasm, and the nucleus. VSMCs also respond differently to the surrounding structural environment, e.g., two-dimensional versus three-dimensional matrix. In vitro studies have been conducted on cultured VSMCs on deformable substrates to elucidate the molecular mechanisms by which the cells convert mechanical inputs into biochemical events, eventually leading to functional responses. The knowledge gained from research on mechanotransduction in vitro, in conjunction with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes involved in vascular remodeling and adaptation in health and disease.     

培养的血管平滑肌细胞ERα和ERβ的表达与细胞表型转变及增殖时相有关

目的探讨雌激素对血管平滑肌细胞（VSMC）增殖的双重效应机制。方法采用Westernblot、电镜形态定量及细胞计数的方法,动态检测原代培养大鼠VSMC在有或无10^-8mol/L17β-雌二醇（E2）存在下,雌激素受体（ER）α和β表达变化与细胞表型转变及增殖时相的关系。结果无E2存在时,VSMC在从收缩型向合成型转变（原代培养第0到5天）及活跃增殖（第5到12天）过程中,ERβ表达无明显变化,但ERα表达明显上升,导致ERα/ERβ比值升高。这种变化并不随VSMC表型的恢复及增殖停止而逆转。有E2存在时,ERα/ERβ比值在第5天时低于对照组,而第9天后各时点均高于对照组;这种影响与E2对不同状态VSMC的不同作用基本对应,即延长原代收缩型SMC的增殖潜伏期,但促进已发生表型转变的VSMC增殖。结论雌激素对不同表型VSMC的双重效应与表型转变前后ERα/ERβ比值变化有关。     

Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.