Platelet-derived microvesicles regulate vascular smooth muscle cell energy metabolism via PRKAA after intimal injury.

Vascular intimal injury initiates various cardiovascular disease processes. Exposure to subendothelial collagen can cause platelet activation, leading to collagen-activated platelet-derived microvesicles (aPMVs) secretion. In addition, vascular smooth muscle cells (VSMCs) exposed to large amounts of aPMVs undergo abnormal energy metabolism; they proliferate excessively and migrate after the loss of endothelium, eventually contributing to neointimal hyperplasia. However, the roles of aPMVs in VSMC energy metabolism are still unknown. Our carotid artery intimal injury model indicated that platelets adhered to injured blood vessels. In vitro, phosphorylated Pka (cAMP-dependent protein kinase) content was increased in aPMVs. We also found that aPMVs significantly reduced VSMC glycolysis and increased oxidative phosphorylation, and promoted VSMC migration and proliferation by upregulating phosphorylated PRKAA (α catalytic subunit of AMP-activated protein kinase) and phosphorylated FoxO1. Compound C, an inhibitor of PRKAA, effectively reversed the enhancement of cellular function and energy metabolism triggered by aPMVs in vitro and neointimal formation in vivo. We show that aPMVs can affect VSMC energy metabolism through the Pka-PRKAA-FoxO1 signaling pathway and this ultimately affects VSMC function, indicating that the shift in VSMC metabolic phenotype by aPMVs can be considered a potential target for the inhibition of hyperplasia. This provides a new perspective for regulating the abnormal activity of VSMCs after injury.

Defective autophagy triggered by arterial cyclic stretch promotes neointimal hyperplasia in vein grafts via the p62/nrf2/slc7a11 signaling pathway.

Autophagy is an adaptation mechanism to keep cellular homeostasis, and its deregulation is implicated in various cardiovascular diseases. After vein grafting, hemodynamic factors play crucial roles in neointimal hyperplasia, but the mechanisms are poorly understood. Here, we investigated the impacts of arterial cyclic stretch on autophagy of venous smooth muscle cells (SMCs) and its role in neointima formation after vein grafting. Rat jugular vein graft were generated via the 'cuff' technique. Autophagic flux in venous SMCs is impaired in 3-day, 1-week and 2-week grafted veins. 10%-1.25 Hz cyclic stretch (arterial stretch) loaded with FX5000 stretch system on venous SMCs blocks cellular autophagic flux in vitro and shows no significant impact on activity of mTORC1 and AMPK. Microtubule depolymerization but not lysosome dysfunction nor autophagosome/amphisome-lysosomal membrane fusion blockade is involved in the impairment of autophagic flux. Microtubule stabilization, induced by paclitaxel treatment and external stents intervention respectively, restores venous SMC autophagy and ameliorates neointimal hyperplasia in vivo. Moreover, autophagy impairment causes accumulation of the cargo receptor p62, which sequesters keap1 to p62 aggregates and results in the stabilization and nuclear translocation of nrf2 to modulate its target antioxidative gene SLC7A11. p62 silencing abrogates the increases of nrf2 and slc7a11 protein expression, glutathione level and venous SMC proliferation triggered by arterial cyclic stretch in vitro, and further hinders nrf2 nuclear translocation, reduces neointimal thickness after vein grafting in vivo. p62 (T349A) mutation also inhibited venous SMC proliferation and alleviated neointimal formation in vivo. These findings suggest that stabilization of microtubules to rescue autophagic flux or direct silencing of p62 are potential therapeutic strategies for neointimal hyperplasia.

Extracellular matrix stiffness controls VEGF165 secretion and neuroblastoma angiogenesis via the YAP/RUNX2/SRSF1 axis.

Aberrant variations in angiogenesis have been observed in tumor tissues with abnormal stiffness of extracellular matrix (ECM). However, it remains largely unclear how ECM stiffness influences tumor angiogenesis. Numerous studies have reported that vascular endothelial growth factor-A (VEGF-A) released from tumor cells plays crucial roles in angiogenesis. Hence, we demonstrated the role of ECM stiffness in VEGF-A release from neuroblastoma (NB) cells and the underlying mechanisms. Based on 17 NB clinical samples, a negative correlation was observed between the length of blood vessels and stiffness of NB tissues. In vitro, an ECM stiffness of 30 kPa repressed the secretion of VEGF165 from NB cells which subsequently inhibited the tube formation of human umbilical vein endothelial cells (HUVECs). Knocked down VEGF165 in NB cells or blocked VEGF165 with neutralizing antibodies both repressed the tube formation of HUVECs. Specifically, 30 kPa ECM stiffness repressed the expression and nuclear accumulation of Yes-associated protein (YAP) to regulate the expression of Serine/Arginine Splicing Factor 1 (SRSF1) via Runt-related transcription factor 2 (RUNX2), which may then subsequently induce the expression and secretion of VEGF165 in NB tumor cells. Through implantation of 3D col-Tgels with different stiffness into nude mice, the inhibitory effect of 30 kPa on NB angiogenesis was confirmed in vivo. Furthermore, we found that the inhibitory effect of 30 kPa stiffness on NB angiogenesis was reversed by YAP overexpression, suggesting the important role of YAP in NB angiogenesis regulated by ECM stiffness. Overall, our work not only showed a regulatory effect of ECM stiffness on NB angiogenesis, but also revealed a new signaling axis, YAP-RUNX2-SRSF1, that mediates angiogenesis by regulating the expression and secretion of VEGF165 from NB cells. ECM stiffness and the potential molecules revealed in the present study may be new therapeutic targets for NB angiogenesis.

Endothelial microparticles induced by cyclic stretch activate Src and modulate cell apoptosis.

Endothelial microparticles (EMPs) are involved in various cardiovascular pathologies and play remarkable roles in communication between endothelial cells (ECs), which are constantly exposed to mechanical cyclic stretch (CS) following blood pressure. However, the roles of EMPs induced by CS in EC homeostasis are still unclear. Both fluorescence resonance energy transfer (FRET) and western blotting revealed the activation of Src in ECs was significantly increased by 5% CS-induced EMPs. Furthermore, proteomic analysis revealed that the contents were obvious different in the EMPs between 5%- and 15%-group. Based on the bioinformatic analysis, CD151 on EMPs was predicted to activate Src, which was further confirmed by both FRET and western blotting. Moreover, the expression of CD151 on EMPs was significantly increased by 5% CS and involved in the binding of EMPs to ECs. EC apoptosis, which was significantly decreased by 5% CS-derived EMPs, showed obvious increase after pretreatment with Src inhibitor in target ECs. Our present research suggests that mechanical stretch changes the components of EMPs, which in turn modulates EC apoptosis by Src activation. CD151 expressed on CS-induced EMPs may play important roles in EC communication and homeostasis.

Endothelial microvesicles induced by physiological cyclic stretch inhibit ICAM1-Dependent leukocyte adhesion.

Physiological cyclic stretch (CS), caused by artery deformation following blood pressure, plays important roles in the homeostasis of endothelial cells (ECs). Here, we detected the effect of physiological CS on endothelial microvesicles (EMVs) and their roles in leukocyte recruitment to ECs, which is a crucial event in EC inflammation. The results showed compared with the static treatment, pretreatment of 5%-CS-derived EMVs with ECs significantly decreased the adherence level of leukocytes. Comparative proteomic analysis revealed 373 proteins differentially expressed between static-derived and 5%-CS-derived EMVs, in which 314 proteins were uniquely identified in static-derived EMVs, 34 proteins uniquely in 5%-CS-derived EMVs, and 25 proteins showed obvious differences. Based on the proteomic data, Ingenuity Pathways Analysis predicted intercellular adhesion molecule 1 (ICAM1) in EMVs might be the potential molecule involved in EC-leukocyte adhesion. Western blot and flow cytometry analyses confirmed the significant decrease of ICAM1 in 5%-CS-derived EMVs, which subsequently inhibited the phosphorylation of VE-cadherin at Tyr731 in target ECs. Moreover, leukocyte adhesion was obviously decreased after pretreatment with ICAM1 neutralizing antibody. Our present research suggested that physiological stretch changes the components of EMVs, which in turn inhibits leukocyte adhesion. ICAM1 expressed on CS-induced EMVs may play an important role in maintaining EC homeostasis.

Circular RNA circTET3 mediates migration of rat vascular smooth muscle cells by targeting miR-351-5p.

Abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are the pathological basis of hyperplasia during vein graft disease. It remains unknown if circular RNAs (circRNAs) are involved in vein graft disease. In the present study, a rat vein graft model was constructed by the "cuff" technique, and whole transcriptome deep sequencing was applied to identify differential circRNAs in the grafted vein compared to the control. We identified a novel circRNA, named circTET3, whose structure was verified by Sanger sequencing and RNase R digestion. CircTET3 was increased in the grafted vein and stably located in the cytoplasm as detected by fluorescence in situ hybridization. Knockdown of circTET3 suppressed VSMC migration by acting as an endogenous miR-351-5p sponge detected by RNA pull-down and dual-luciferase reporter assays. PTPN1 was the targeted gene due to the competitive binding of circTET3 to miR-351-5p. This regulatory pathway may serve as a potential therapeutic avenue against intimal hyperplasia in vein graft disease.

Single-cell analyses reveal functional classification of dendritic cells and their potential roles in inflammatory disease.

Dendritic cells (DCs) have crucial roles in immune-related diseases. However, it is difficult to explore DCs because of their rareness and heterogeneity. Although previous studies had been performed to detect the phenotypic characteristics of DC populations, the functional diversity has been ignored. Using a combination of flow cytometry, single-cell quantitative PCR, and bioinformatic analysis, we depicted the DC panorama with not only phenotypic but also functional markers. Functional classification of DCs in mouse lymphoid tissue (spleen) and nonlymphoid tissue (liver) was performed. The results revealed that expression of macrophage scavenger receptor 1 ( MSR1) and C-C motif chemokine receptors ( CCR) 1, CCR2, and CCR4 were elevated in liver DCs, suggesting increased lipid uptake and migration abilities. The enriched expression of costimulatory molecule CD80, TLR9, and TLR adaptor MYD88 in spleen DCs indicated a more-mature phenotype, enhanced pathogen recognition, and T-cell stimulation abilities. Furthermore, we compared DCs in the atherosclerotic mouse models with healthy controls. In addition to the quantitative increase in DCs in the liver and spleen of the apolipoprotein E-knockout ( ApoE-/-) mice, the functional expression patterns of the DCs also changed at the single-cell level. These results promote our understanding of the participation of DCs in inflammatory diseases and have potential applications in DC clinical assessment.-Shi, Q., Zhuang, F., Liu, J.-T., Li, N., Chen, Y.-X., Su, X.-B., Yao, A.-H., Yao, Q.-P., Han, Y., Li, S.-S., Qi, Y.-X., Jiang, Z.-L. Single-cell analyses reveal functional classification of dendritic cells and their potential roles in inflammatory disease.

CTGF regulates cyclic stretch-induced vascular smooth muscle cell proliferation via microRNA-19b-3p.

Cyclic stretch regulates proliferation of vascular smooth muscle cells (VSMCs) during hypertension-induced vascular remodeling, but the underlying mechanisms remain to be studied. Connective tissue growth factor (CTGF) has been reported associated with several cellular function such as proliferation，migration and adhesion. Herein, the role of CTGF in VSMCs was investigated in response to mechanical cyclic stretch. Here we show that CTGF is up-regulated both in vivo and in vitro during hypertension. Overexpression of CTGF markedly promoted VSMC proliferation, whereas CTGF knockdown attenuated cyclic stretch-induced proliferation. Furthermore, 3'UTR reporter assays revealed that microRNA-19b-3p (miR-19b-3p) directly regulates CTGF expression. Under pathological condition (e.g. 15% cyclic stretch), miR-19b-3p expression was significantly down-regulated; conversely miR-19b-3p overexpression blocked VSMC proliferation. Taken together, these findings indicate that pathological cyclic stretch induces vascular remodeling by promoting VSMC proliferation via miR-19b-3p/CTGF pathway, and point to CTGF as a potential therapeutic target for hypertension.

MicroRNA-29a Involvement in Phenotypic Transformation of Venous Smooth Muscle Cells Via Ten-Eleven Translocation Methylcytosinedioxygenase 1 in Response to Mechanical Cyclic Stretch.

Mechanical stimuli play an important role in vein graft restenosis and the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are pathological processes contributing to this disorder. Here, based on previous high-throughput sequencing data from vein grafts, miR-29a-3p and its target, the role of Ten-eleven translocation methylcytosinedioxygenase 1 (TET1) in phenotypic transformation of VSMCs induced by mechanical stretch was investigated. Vein grafts were generated by using the "cuff" technique in rats. Deep transcriptome sequencing revealed that the expression of TET1 was significantly decreased, a process confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. MicroRNA-seq showed that miR-29a-3p was significantly up-regulated, targeting TET1 as predicted by Targetscan. Bioinformatics analysis indicated that the co-expressed genes with TET1 might modulate VSMC contraction. Venous VSMCs exposed to 10%-1.25 Hz cyclic stretch by using the Flexcell system were used to simulate arterial mechanical conditions in vitro. RT-qPCR revealed that mechanical stretch increased the expression of miR-29a-3p at 3 h. Western blot analysis showed that TET1 was significantly decreased, switching contractile VSMCs to cells with a synthetic phenotype. miR-29a-3p mimics (MI) and inhibitor (IN) transfection confirmed the negative impact of miR-29a-3p on TET1. Taken together, results from this investigation demonstrate that mechanical stretch modulates venous VSMC phenotypic transformation via the mediation of the miR-29a-3p/TET1 signaling pathway. miR-29a-3p may have potential clinical implications in the pathogenesis of remodeling of vein graft restenosis.

MicroRNA-129-1-3p regulates cyclic stretch-induced endothelial progenitor cell differentiation by targeting Runx2.

Endothelial progenitor cells (EPCs) are vital to the recovery of endothelial function and maintenance of vascular homeostasis. EPCs mobilize to sites of vessel injury and differentiate into mature endothelial cells (ECs). Locally mobilized EPCs are exposed to cyclic stretch caused by blood flow, which is important for EPC differentiation. MicroRNAs (miRNAs) have emerged as key regulators of several cellular processes. However, the role of miRNAs in cyclic stretch-induced EPC differentiation remains unclear. Here, we investigate the effects of microRNA-129-1-3p (miR-129-1-3p) and its novel target Runt-related transcription factor 2 (Runx2) on EPC differentiation induced by cyclic stretch. Bone marrow-derived EPCs were exposed to cyclic stretch with a magnitude of 5% (which mimics physiological mechanical stress) at a constant frequency of 1.25 Hz for 24 hours. The results from a miRNA array revealed that cyclic stretch significantly decreased miR-129-1-3p expression. Furthermore, we found that downregulation of miR-129-1-3p during cyclic stretch-induced EPC differentiation toward ECs. Meanwhile, expression of Runx2, a putative target gene of miR-129-1-3p, was increased as a result of cyclic stretch. A 3'UTR reporter assay validated Runx2 as a direct target of miR-129-1-3p. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Runx2 inhibited EPC differentiation into ECs and attenuated EPC tube formation via modulation of vascular endothelial growth factor (VEGF) secretion from EPCs in vitro. Our findings demonstrated that cyclic stretch suppresses miR-129-1-3p expression, which in turn activates Runx2 and VEGF to promote endothelial differentiation of EPCs and angiogenesis. Therefore, targeting miR-129-1-3p and Runx2 may be a potential therapeutic strategy for treating vessel injury.

Platelet-derived microparticles promote endothelial cell proliferation in hypertension via miR-142-3p.

Endothelial cells (ECs) are located at the interface between flowing blood and the vessel wall, and abnormal EC proliferation induced by pathologic environments plays an important role in vascular remodeling in hypertensive conditions. Exchanges of information between blood components and ECs are important for EC function. Hence, the present study sought to determine how platelets induce EC dysfunction under hypertensive conditions. EC proliferation was increased in renal hypertensive rats established by abdominal aortic coarctation compared with control rats and that elevated thrombin in plasma promoted platelet activation, which may induce the release of platelet-derived microparticles (PMPs). MicroRNA (MiR) array and qPCR revealed a higher level of miR-142-3p in platelets and PMPs. In vitro, PMPs delivered miR-142-3p into ECs and enhanced their proliferation via Bcl-2-associated transcription factor (BCLAF)1 and its downstream genes. These results indicate that PMPs deliver miR-142-3p from activated platelets into ECs and that miR-142-3p may play important roles in EC dysfunction in hypertensive conditions and may be a novel therapeutic target for maintaining EC homeostasis in hypertension.-Bao, H., Chen, Y.-X., Huang, K., Zhuang, F., Bao, M., Han, Y., Chen, X.-H., Shi, Q., Yao, Q.-P., Qi, Y.-X. Platelet-derived microparticles promote endothelial cell proliferation in hypertension via miR-142-3p.

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.

Cyclic stretch induces VEGFA alternative splicing via Serine/Arginine-Rich Splicing Factor 1.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by high cyclic stretch is crucial in the vascular remodeling during hypertension. Vascular endothelial growth factor A (VEGFA) alternative splicing plays important roles in the pathological process of vascular diseases and remodeling. However, the roles of VEGFA isoforms in modulating VSMC functions in response to cyclic stretch remain unclear. We hypothesize that high cyclic stretch may induce VEGFA alternative splicing via Serine/arginine-rich splicing factor 1 (SRSF1) which subsequently induce VSMC proliferation. In the present research, hypertensive rat model was established using the abdominal aortic constriction method. In comparison with sham-operated group, immunohistology staining showed translocation of SRSF1 into nuclei in hypertensive rat thoracic aorta, and RT-PCR detected a shift of VEGFA expression pattern, including the increased expression of VEGFA120 and VEGFA164, but not VEGFA188.Then VSMCs were subjected to cyclic stretch in vitro using a Flexercell strain unit. VEGFA ELISA assay showed 15% cyclic stretch increased the secretion of VEGFA which significantly increased proliferation of VSMCs. Western blot and immunofluorescence detected accumulation of SRSF1 in nuclei after 15% cyclic stretch application. Furthermore, SRSF1-specific siRNA transfection reversed the VEGFA secretion induced by pathological high cyclic stretch. Our present results suggested that pathologically high cyclic stretch induces the shuttling of SRSF1 which results in the secretive pattern splicing of VEGFA and finally contributes to the proliferation of VSMCs.

Mechanobiology and Vascular Remodeling: From Membrane to Nucleus.

Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to hemodynamic forces in vivo, including flow shear stress and cyclic stretch caused by the blood flow. Numerous researches revealed that during various cardiovascular diseases such as atherosclerosis, hypertension, and vein graft, abnormal (pathological) mechanical forces play crucial roles in the dysfunction of ECs and VSMCs, which is the fundamental process during both vascular homeostasis and remodeling. Hemodynamic forces trigger several membrane molecules and structures, such as integrin, ion channel, primary cilia, etc., and induce the cascade reaction processes through complicated cellular signaling networks. Recent researches suggest that nuclear envelope proteins act as the functional homology of molecules on the membrane, are important mechanosensitive molecules which modulate chromatin location and gene transcription, and subsequently regulate cellular functions. However, the studies on the roles of nucleus in the mechanotransduction process are still at the beginning. Here, based on the recent researches, we focused on the nuclear envelope proteins and discussed the roles of pathological hemodynamic forces in vascular remodeling. It may provide new insight into understanding the molecular mechanism of vascular physiological homeostasis and pathophysiological remodeling and may help to develop hemodynamic-based strategies for the prevention and management of vascular diseases.

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.

Activatable and Cell-Penetrable Multiplex FRET Nanosensor for Profiling MT1-MMP Activity in Single Cancer Cells.

We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.

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.

MicroRNA-34a targets Forkhead box j2 to modulate differentiation of endothelial progenitor cells in response to shear stress.

Flow shear stress plays important roles in modulating differentiation of endothelial progenitor cells (EPCs). MicroRNAs are crucial for diverse cellular processes, but the expressions and functions of microRNAs in EPCs responding to mechanical stimuli remain unclear. We sought to determine the effects of microRNA-34a (miR-34a) and a novel target Forkhead box j2 (Foxj2) on shear stress-induced EPC differentiation. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15dyn/cm(2) with parallel plate flow chamber system. Real time RT-PCR showed that shear stress significantly increased miR-34a expression, which was accompanied by the endothelial differentiation of EPCs. Whereas Foxj2, a putative target of miR-34a predicted by multiple algorithms, was suppressed in this process. Dual luciferase reporter assays, as well as miR-34a mimics and inhibitor treatment were used to confirm the interplay between miR-34a and Foxj2. Our results revealed an inverse correlation of miR-34a and Foxj2 expressions implicated in the endothelial differentiation of EPCs. MiR-34a contributed to this process by up-regulating the expressions of endothelial cell markers, and down-regulating smooth muscular cell markers. In addition, Foxj2 overexpression attenuated endothelial differentiation of EPCs, while Foxj2 siRNA had the opposite effect. These data suggested a unique mechanism that shear stress induces the expression of miR-34a, which targets to Foxj2 and promotes endothelial differentiation of EPCs. The results provide new insights into miR-34a/Foxj2 on shear stress-induced EPC differentiation.

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.

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/cm(2) 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.