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 β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-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 β-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.

Cyclic tensile strain facilitates proliferation and migration of human aortic smooth muscle cells and reduces their apoptosis via miRNA-187-3p

“…For example, CTS was able to induce expression of thrombin on the surface of rat thoracic aortic smooth muscle cells via an integrin-related signaling pathway, and ultimately caused a phenotypic shift in smooth muscle cells [ 42 ]. CTS induces VSMCs to secrete connective tissue growth factor and promotes endothelial progenitor cell differentiation and angiogenesis [ 43 ]. CTS stimulates VSMCs alignment through redox-dependent activation of Notch3 [ 44 ].…”

Mechanical regulation of signal transduction in angiogenesis

“…BAEC (Sato et al, 1987;Sumpio et al, 1987;Letsou et al, 1990;Malek and Izumo, 1992;Awolesi et al, 1995;Jacobs et al, 1995;Hishikawa and Lüscher, 1997;Kataoka et al, 1998;Kohn et al, 2015;Mahmoud et al, 2021) HUVEC (Illi et al, 2003;Zheng et al, 2004;Yu et al, 2009;Dickinson et al, 2012;Jalali et al, 2015;Zhao et al, 2018;Tsaryk et al, 2022) HMEC-1 (Ades et al, 1992;Wilson et al, 2009;Groothuis et al, 2010;Xie et al, 2020;Katari et al, 2021) Material Collagen (Krishnan et al, 2008;Lee et al, 2013;Mason et al, 2013;Wilkins et al, 2015) Matrigel (Ades et al, 1992;Mammoto et al, 2009;Wilson et al, 2009;Yan et al, 2020) Fibrin (Groothuis et al, 2010;Morin and Tranquillo, 2013;Wilkins et al, 2015) PDMS (Dickinson et al, 2012;Rosenfeld et al, 2016;Sivarapatna et al, 2017;Zhao et al, 2018;Zeinali et al, 2021) Polyacrylamide gel (Deroanne et al, 2001;Saunders and Hammer, 2010;Jalali et al, 2015;Santos et al, 2015;…”

“…To study the effect of ligand availability and curvature, endothelial cells are seeded on micro-patterns to form islands, lines, half-channels, or channels so that the phenotypes and the behaviors across various geometries can be compared, and the key geometric characteristic contributing to endothelial sprouting can be identified ( Figure 2 ) ( Dike et al, 1999 ; Mandrycky et al, 2020 ). To interrogate how different degrees of stretch affects angiogenesis, cells are first seeded on an elastic film, often with a compatible refractive index to facilitate imaging-based observations ( Yu et al, 2009 ; Yan et al, 2020 ; Lien and Wang, 2021 ). A prescribed strain will then be applied to the elastic film ( Figure 3 ), through a motorized stepper, a vacuum, or other stretching mechanisms.…”

CCN2-induced lymphangiogenesis is mediated by the integrin αvβ5–ERK pathway and regulated by DUSP6

“…CCN2 directly binds to integrin αvβ3 and promotes endothelial cell adhesion, migration, and survival 27 . In vivo, it has been shown to promote angiogenesis in CAM assays, collagen pellet implantation, and Matrigel plug assays 28 , 29 . CCN2 is highly expressed in endothelial cells during development, and Ccn2 knockout mice have impaired angiogenesis in the growth plate, resulting in skeletal abnormalities and perinatal lethality 30 .…”