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Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis.

ABSTRACT: Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. In vivo study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague-Dawley (SD) rat carotid artery injury model. In vitro, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for 12 h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and ?-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/?-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. Wnt signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an in vivo Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and ?-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.

SUBMITTER: Yan J

PROVIDER: S-EPMC7755638 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis.

Yan Jing J Wang Wen-Bin WB Fan Yang-Jing YJ Bao Han H Li Na N Yao Qing-Ping QP Huo Yun-Long YL Jiang Zong-Lai ZL Qi Ying-Xin YX Han Yue Y

Frontiers in cell and developmental biology 20201209

Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. <i>In vivo</i> study r ...[more]

PMID: 33363166

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Project description:Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Project description:Objective: This investigation examined the effect of velvet antler (VA) on endothelial progenitor cells (EPCs) and the associated effects to promote angiogenesis and repair vascular endothelial injury in rats with myocardial infarction (MI). Methods: VA was analyzed by liquid chromatography-mass spectrometry. Male Sprague Dawley rats were randomly divided into four groups: sham, MI, VA, and VA + DAPT (gamma-secretase inhibitor IX, a specific blocker of the Notch signaling pathway) group. The rats underwent ligation of the left anterior descending coronary artery for the establishment of MI. Sham-operated rats were used as controls. Blood was taken from the orbital plexus on the first and third days after the operation, and all rats were euthanized on the 7th day after surgery. The blood samples were used to detect the contents of circulating endothelial progenitor cells (CEPCs) and vascular endothelial growth factor (VEGF). Echocardiography was used to test the cardiac function. Cardiac tissue was used for immunohistochemistry and electron microscope, and the marginal zone of the MI tissue was used for western blot and reverse transcription-quantitative polymerase chain reaction. Results: The number of basically qualitative metabolites is 445. Among them, there are 74 substances with relative content greater than 0.05%. VA increased the concentration of CEPCs and VEGF in serum, CD133 content and microvessel density (MVD), and protected the morphology of microvascular endothelial cells in the marginal area of MI at 7 days post-MI surgery. CEPCs and MVD in the VA +DAPT group were lower than those of VA group. VA increased the protein expressions of Jagged-1, Notch1, NICD and HES1, and the mRNA expressions of Hes1 and Hey2, while some of the effects could be suppressed by DAPT. Conclusion: These results suggest that VA promotes the mobilization of EPCs to promote angiogenesis and repair vascular endothelial cell damage in post-MI rats, and these effects may be due to activation of the Notch signal pathway.

Project description:Dickkopf-1 (DKK1) was recently shown to play an important role in cardiovascular disease. The aim of this work was to assess the role of DKK1 in the regulation of smooth muscle cell function by mechanical stretch and the mechanisms underlying this process. Methods: Wild-type C57BL/6J mice were subjected to sham or abdominal aortic constriction (AAC) surgery. The expression level of DKK1 was examined by immunohistochemical staining and Western blotting. Analyses of DKK1 function in vascular smooth muscle cell (VSMC) proliferation and migration were performed. Transcriptome sequencing analysis was performed to identify the differentially expressed genes and pathways regulated by DKK1. Smooth muscle-specific Dkk1 knockout mice were used to confirm the function of DKK1 in vivo. Chromatin immunoprecipitation (ChIP) was used to confirm DNA-protein interactions. Promoter luciferase analysis was used to detect transcription factor activity. Results: We found that AAC significantly increased DKK1 protein levels in the thoracic aorta and coronary artery in vivo. In vitro, high-level stretch (18%) induced the expression of DKK1 in VSMCs. Knocking down DKK1 inhibited VSMC proliferation and migration under high-level stretch (18%). We identified ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as a target gene of DKK1. Knockdown of UHRF1 with small interfering RNAs partially reversed the regulatory effect of recombinant DKK1 on VSMCs. Specific deletion of DKK1 in VSMCs was sufficient to attenuate the AAC-induced upregulation of UHRF1, thickening of arterial media and increase in VSMC proliferation. Furthermore, we found that DKK1 regulated UHRF1 expression through the YAP-TEAD pathway. TEAD1 and TEAD4 bound directly to the promoter of UHRF1, and blocking the YAP-TEAD interaction inhibited UHRF1 upregulation due to DKK1. Conclusions: This study reveals that DKK1 mediates the mechanical stretch regulation of smooth muscle cell function by modulating UHRF1 expression through the YAP-TEAD pathway.

Project description:Large, elastic arteries buffer the pressure wave originating in the left ventricle and are constantly exposed to higher amplitudes of cyclic stretch (10%) than muscular arteries (2%). As a crucial factor for endothelial and smooth muscle cell function, cyclic stretch has, however, never been studied in ex vivo aortic segments of mice. To investigate the effects of cyclic stretch on vaso-reactivity of mouse aortic segments, we used the Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). The aortic segments were clamped at frequencies of 6-600 bpm between two variable preloads, thereby mimicking dilation as upon left ventricular systole and recoiling as during diastole. The preloads corresponding to different transmural pressures were chosen to correspond to a low, normal or high amplitude of cyclic stretch. At different time intervals, cyclic stretch was interrupted, the segments were afterloaded and isometric contractions by ?1-adrenergic stimulation with 2 ?M phenylephrine in the absence and presence of 300 ?M L-NAME (eNOS inhibitor) and/or 35 ?M diltiazem (blocker of voltage-gated Ca2+ channels) were measured. As compared with static or cyclic stretch at low amplitude (<10 mN) or low frequency (0.1 Hz), cyclic stretch at physiological amplitude (>10 mN) and frequency (1-10 Hz) caused better ex vivo conservation of basal NO release with time after mounting. The relaxation of PE-precontracted segments by addition of ACh to stimulate NO release was unaffected by cyclic stretch. In the absence of basal NO release (hence, presence of L-NAME), physiological in comparison with aberrant cyclic stretch decreased the baseline tension, attenuated the phasic contraction by phenylephrine in the absence of extracellular Ca2+ and shifted the smaller tonic contraction more from a voltage-gated Ca2+ channel-mediated to a non-selective cation channel-mediated. Data highlight the need of sufficient mechanical activation of endothelial and vascular smooth muscle cells to maintain basal NO release and low intracellular Ca2+ in the smooth muscle cells in large arteries. Both phenomena may play a vital role in maintaining the high compliance of large arteries.

Project description:BackgroundVascular dementia (VaD) mainly results from cerebral vascular lesions and tissue changes, which contribute to neurodegenerative processes. Effective therapeutic approaches to targeting angiogenesis may reduce mortality of VaD. Endothelial progenitor cells (EPCs) play a key role in postnatal angiogenesis. Many exosomal microRNAs (exo-miRNAs) have been reported to involve in the development of dementia. The present study was designed to investigate whether the expression profile of the exo-miRNAs is significantly altered in patients with VaD and to reveal the function of differentially expressed miRNAs and the relevant mechanisms in EPC-mediated angiogenesis in VaD rat model.ResultsExosomes isolated from serum of patients with VaD (n = 7) and age-matched control subjects (n = 7), and miRNA sequencing and bioinformatics analysis found that circulating exosome miRNA-155-5p, miRNA-154-5p, miR-132-5p, and miR-1294 were upregulated in patients with VaD. The expression of miRNA-154-5p was further verified to be upregulated in clinical samples (n = 23) and 2-vessel occlusion-induced VaD rat model by reverse transcription quantitative PCR (RT-qPCR). Notably, miRNA-154-5p inhibition in bone marrow-EPCs (BM-EPCs) from VaD rats improved EPC functions, including tube formation, migration, and adhesion, and elevated concentrations of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α). The mRNA levels of ICAM-1, VCAM-1, and MCP-1 were reduced in miRNA-154-5p-inhibited EPCs. In addition, miRNA-154-5p inhibition increased the level of superoxide dismutase (SOD), and decreased reactive oxygen species (ROS) in EPCs. PRKAA2 was chosen as a promising target gene of miR-154-5p, and miRNA-154-5p inhibition upregulated the protein expression of AMPKα2. Furthermore, upregulation of miR-154-5p markedly diminished EPC functions and inhibited angiogenesis following EPC transplantation in VaD rats.ConclusionCirculating exo-miR-154-5p was upregulated in patients with VaD, and miR-154-5p upregulation was associated with impaired EPC functions and angiogenesis in VaD rat model. Therefore, miR-154-5p is a promising biomarker and therapeutic strategy for VaD.

Dataset Information

Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis.

Publications

Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis.

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