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 modulate proliferation of vascular smooth muscle cells during cyclic stretch application.

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.

SIRT1 and FOXO Mediate Contractile Differentiation of Vascular Smooth Muscle Cells under Cyclic Stretch.

BACKGROUND/AIMS: Physiological mechanical stretch in vivo helps to maintain the quiescent contractile differentiation of vascular smooth muscle cells (VSMCs), but the underlying mechanisms are still unclear. Here, we investigated the effects of SIRT1 in VSMC differentiation in response to mechanical cyclic stretch. METHODS AND RESULTS: Rat VSMCs were subjected to 10%-1.25Hz-cyclic stretch in vitro using a FX-4000T system. The data indicated that the expression of contractile markers, including α-actin, calponin and SM22α, was significantly enhanced in VSMCs that were subjected to cyclic stretch compared to the static controls. The expression of SIRT1 and FOXO3a was increased by the stretch, but the expression of FOXO4 was decreased. Decreasing SIRT1 by siRNA transfection attenuated the stretch-induced expression of contractile VSMC markers and FOXO3a. Furthermore, increasing SIRT1 by either treatment with activator resveratrol or transfection with a plasmid to induce overexpression increased the expression of FOXO3a and contractile markers, and decreased the expression of FOXO4 in VSMCs. Similar trends were observed in VSMCs of SIRT1 (+/-) knockout mice. The overexpression of FOXO3a promoted the expression of contractile markers in VSMCs, while the overexpression of FOXO4 demonstrated the opposite effect. CONCLUSION: Our results indicated that physiological cyclic stretch promotes the contractile differentiation of VSMCs via the SIRT1/FOXO pathways and thus contributes to maintaining vascular homeostasis.

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.

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.

Involvement of BK channel in differentiation of vascular smooth muscle cells induced by mechanical stretch.

The differentiation of vascular smooth muscle cells (VSMCs), which are exposed to mechanical stretch in vivo, plays an important role in vascular remodeling during hypertension. Here, we demonstrated the mechanobiological roles of large conductance calcium and voltage-activated potassium (BK) channels in this process. In comparison with 5% stretch (physiological), 15% stretch (pathological) induced the de-differentiation of VSMCs, resulting in significantly decreased expressions of VSMC markers, i.e., α-actin, calponin and SM22. The activity of BK channels, assessed by patch clamp recording, was significantly increased by 15% stretch and was accompanied by an increased alternative splicing of BK channel α-subunit at the stress axis-regulated exons (STREX). Furthermore, transfection of whole BK or STREX-deleted BK plasmids revealed that STREX was important for BK channels to sense mechanical stretch. Using thapsigargin (TG) which induces endoplasmic reticulum (ER) stress, and xbp1-targeted siRNA transfection which blocks ER stress, the results revealed that ER stress was contribute to stretch-induced alternative splicing of STREX. Our results suggested that during hypertension, pathological stretch may induce the ER stress in VSMCs, which affects the alternative splicing and activity of BK channels, and subsequently modulates VSMC differentiation.

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-ß1 (TGF-ß1)-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-ß1 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-ß1 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.

Endothelial insulin-like growth factor-1 modulates proliferation and phenotype of smooth muscle cells induced by low shear stress.

Endothelial cells (ECs) are directly exposed to shear stress and modulate the neighboring vascular smooth muscle cells (VSMCs), which plays important roles in vascular remodeling during atherosclerosis. Our previous research revealed that insulin-like growth factors (IGFs) might participate in low shear stress (LowSS) induced vascular remodeling, which remains to be elucidated. Using EC/VSMC co-cultured parallel-plate flow chamber, LowSS (5 dyn/cm(2)) was applied and normal shear stress (NSS, 15 dyn/cm(2)) was used as control. LowSS induced IGF-1 secretion from ECs, which subsequently phosphorylated IGF-1 receptor (IGF-1R) on co-cultured VSMCs, then increased Akt phosphorylation and Sirt2 expression. Decreasing IGF-1 in ECs by RNA interference (RNAi) reversed these effects on VSMCs. Exogenous IGF-1 increased IGF-1R and Akt phosphorylation, Sirt2 expression, and proliferation of VSMCs, and induced VSMCs towards synthetic phenotype. PI3 K/Akt specific inhibitor wortmannin decreased Sirt2 expression, proliferation, and synthetic phenotype transformation of VSMCs, but had no effect on IGF-1R. Sirt2 RNAi repressed VSMC proliferation and phenotypic transformation, but had no effect on IGF-1R and Akt. Taken together, LowSS induces the secretion of IGF-1 from ECs, which subsequently paracrine influences the co-cultured VSMCs via IGF-1R and Akt phosphorylation, and Sirt2 expression, then results in the proliferation and synthetic phenotype transformation.

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.

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.

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/cm(2) 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.

PDGF-BB and TGF-{beta}1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress.

Shear stress, especially low shear stress (LowSS), plays an important role in vascular remodeling during atherosclerosis. Endothelial cells (ECs), which are directly exposed to shear stress, convert mechanical stimuli into intracellular signals and interact with the underlying vascular smooth muscle cells (VSMCs). The interactions between ECs and VSMCs modulate the LowSS-induced vascular remodeling. With the use of proteomic analysis, the protein profiles of rat aorta cultured under LowSS (5 dyn/cm(2)) and normal shear stress (15 dyn/cm(2)) were compared. By using Ingenuity Pathway Analysis to identify protein-protein association, a network was disclosed that involves two secretary molecules, PDGF-BB and TGF-ß1, and three other linked proteins, lamin A, lysyl oxidase, and ERK 1/2. The roles of this network in cellular communication, migration, and proliferation were further studied in vitro by a cocultured parallel-plate flow chamber system. LowSS up-regulated migration and proliferation of ECs and VSMCs, increased productions of PDGF-BB and TGF-ß1, enhanced expressions of lysyl oxidase and phospho-ERK1/2, and decreased Lamin A in ECs and VSMCs. These changes induced by LowSS were confirmed by using PDGF-BB recombinant protein, siRNA, and neutralizing antibody. TGF-ß1 had similar influences on ECs as PDGF-BB, but not on VSMCs. Our results suggest that ECs convert the LowSS stimuli into up-regulations of PDGF-BB and TGF-ß1, but these two factors play different roles in LowSS-induced vascular remodeling. PDGF-BB is involved in the paracrine control of VSMCs by ECs, whereas TGF-ß1 participates in the feedback control from VSMCs to ECs.

Normal shear stress and vascular smooth muscle cells modulate migration of endothelial cells through histone deacetylase 6 activation and tubulin acetylation.

Endothelial cells (ECs) line the innermost of the blood vessel wall and are constantly subjected to shear stress imposed by blood flow. ECs were also influenced by the neighboring vascular smooth muscle cells (VSMCs). The bidirectional communication between ECs and VSMCs modulates vascular homeostasis. In this study, the involvement of histone deacetylase 6 (HDAC6) in modulating migration of ECs co-cultured with VSMCs by the normal level of laminar shear stress (NSS) was investigated. ECs was either cultured alone or co-cultured with VSMCs under static conditions or subjected to NSS of 15 dyne/cm2 by using a parallel-plate co-culture flow chamber system. It was demonstrated that both NSS and VSMCs could increase EC migration. The migration level of ECs co-cultured with VSMCs under NSS was not higher than that under the static condition. The process of EC migration regulated by VSMCs and NSS was associated with the increased expression of HDAC6 and low level of acetylated tubulin. The increase in HDAC6 expression was accompanied by a time-dependent decrease in the acetylation of tubulin in ECs co-cultured with VSMCs. Inhibition of the HDAC6 by siRNA or tributyrin, an inhibitor of HDACs, induced a parallel alteration in the migration and the acetylated tubulin of ECs co-cultured with VSMCs. It was observed by immunofluorescence staining that the acetylated tubulin was distributed mostly around the cell nucleus in ECs co-cultured with VSMCs. The results suggest that the NSS may display a protective function on the vascular homeostasis by modulating EC migration to a normal level in a VSMC-dependent manner. This modulation process involves the down-regulation of acetylated tubulin which results from increased HDAC6 activity in ECs.

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho-GDIalpha, 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-GDIalpha) 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-GDIalpha "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-GDIalpha expression. When siRNAs of Rho-GDIalpha and Rac1 were co-transfected to VSMCs, the expressions of Rho-GDIalpha and phospho-Rac1 were both decreased, and the effects of Rho-GDIalpha "knock-down" were blocked. Rho-GDIalpha "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-GDIalpha, which subsequently verified phosphorylations of Rac1 and p38.

Vascular smooth muscle cells promote endothelial cell adhesion via microtubule dynamics and activation of paxillin and the extracellular signal-regulated kinase (ERK) pathway in a co-culture system.

Interaction between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in vascular biology. Cell adhesion to the extracellular matrix provides critical environmental information necessary for cell migration, proliferation, differentiation and survival. In this study, the role of VSMCs in EC adhesion was demonstrated by using a co-culture system. It was shown that the co-cultured VSMCs significantly increased the number of adherent ECs, and induced an increase of total focal adhesion area in ECs. These changes were associated with a low microtubule-to-tubulin ratio, and activation of extracellular signal-regulated kinase (ERK) and paxillin. Both the EC adhesion state and activation of the ERK/paxillin pathway by the co-cultured VSMCs could be inhibited by trichostatin A (TSA). As an inhibitor of histone deacetylase, TSA acts by modulating microtubule polymerization state. Taken together, these data suggest that the co-cultured VSMCs promote EC adhesion by modulating the microtubule cytoskeleton polymerization state, which in turn activates the ERK pathway and up-regulates phosphorylated paxillin expression to accelerate focal adhesion formation.

Histone deacetylases modulate vascular smooth muscle cell migration induced by cyclic mechanical strain.

The migration of vascular smooth muscle cells (VSMCs) is found to participate in vascular remodeling which is pivotal in the pathogenesis of vascular diseases, for instance atherosclerosis and restenosis. However, the underlying mechanisms of how mechanical strain influence VSMC migration remain to be elucidated. Histone deacetylases (HDACs) are involved in chromatin remodeling and modification of both histone and nonhistone transcription regulatory proteins, thus HDACs modulate genes important for complex biological processes. But whether HDACs take part in modulating migration of VSMCs induced by mechanical strain is poorly understood. Here, we showed that cyclic strain of 1 Hz at 10% elongation for 48 h significantly inhibited the migration of cultured VSMCs compared to the static one. The cyclic strain upregulated the levels of acetylased histone H3 and HDAC7 while downregulated the level of HDAC3/4 in VSMCs. Furthermore, the mechanically induced VSMC migration was diminished by treatment with tributyrin, a HDAC inhibitor. We also observed hyperacetylation of histone H3 and reduced expression of HDAC7 upon tributyrin treatment. These results provide convincing evidence that HDACs are involved in the migration of VSMCs induced by mechanical strain through chromatin remodeling. Thus, inhibition of HDAC may be beneficial in preventing the migration of VSMCs in treating proliferative vascular diseases.

A proteomic analysis of aorta from spontaneously hypertensive rat: RhoGDI alpha upregulation by angiotensin II via AT(1) receptor.

Arteries undergo remodeling as a consequence of increased wall stress during hypertension. However, the molecular mechanisms of the vascular remodeling are largely unknown. Proteomics is a powerful tool to screen for differentially expressed proteins, but little effort was made on vascular disease research, especially on hypertension. In the present study, the differentially expressed proteins in aortas from 18-week-old spontaneously hypertensive rats (SHR) and their normotensive counterpart, Wistar Kyoto rats (WKY), were examined by two-dimensional electrophoresis (2-DE). We found 50 proteins to be differentially expressed, among which 27 were highly or only expressed in SHR and 23 in WKY. Using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) and online data search, nine proteins, including Rho GDP dissociation inhibitor alpha (RhoGDIalpha), were identified with high confidence. Further, the upregulation of RhoGDIalpha was verified at both mRNA and protein level in SHR. In addition, when cultured vascular smooth muscle cells (VSMCs) from aortas of SHR and WKY were treated with angiotensin II (Ang II) and antagonist of angiotensin II type I (AT(1)) receptor, L158809, respectively, RhoGDIalpha was upregulated by Ang II and downregulated by L158809 in VSMCs of SHR. These results demonstrate that vascular remodeling results in significant alterations in the protein expression profile of the aorta during hypertension and suggest that the upregulation of RhoGDIalpha in hypertension is induced by Ang II via AT(1) receptor.

Role of cyclic strain frequency in regulating the alignment of vascular smooth muscle cells in vitro.

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-beta1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-beta1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-beta1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.

Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain.

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway.

Coculture with endothelial cells enhances vascular smooth muscle cell adhesion and spreading via activation of beta1-integrin and phosphatidylinositol 3-kinase/Akt.

The interactions between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) play significant roles in the homeostasis of the blood vessel during vascular remodeling. Cell adhesion and spreading are an essential process for VSMC migration, survival and proliferation in the events of vascular physiology and pathophysiology. However, effects of ECs on adhesion and spreading of VSMCs have not been characterized yet. Here, the interaction of ECs and VSMCs on adhesion and spreading of VSMCs were investigated by using a coculture system. The results showed that VSMCs cocultured with ECs exhibited a significant increase in the number of adherent and spreading cells, and much more mRNA (twofold, P<0.01) and protein (threefold, P<0.05) expression of beta(1)-integrin comparing to the control, i.e., VSMCs cultured alone. Furthermore, the enhanced functional activity of beta(1)-integrin expression was confirmed by FACS. A beta(1)-integrin blocking antibody (P5D2) could inhibit the EC-induced VSMC adhesion and spreading. It was demonstrated that in correspondence with enhanced cell adhesion, ECs also prompted focal adhesion complex assembly and stress fiber formation of VSMCs. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway was more pronouncedly activated in response to VSMC attachment. Our results for the first time show that coculture with ECs enhances VSMC adhesion and spreading by up-regulating beta(1)-integrin expression and activating the PI3K/Akt pathway, suggesting that the interaction between ECs and VSMCs serves an important role in vascular homeostasis and remodeling.