RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

RhoGTPase is involved in PDGF-BB-mediated VSMC phenotypic modulation. RhoGDIs are key factors in regulating RhoGTPase activation. In the present study, we investigated the regulatory effect of RhoGDI1 on the activation of RhoGTPase in VSMC transformation and neointima formation. Western blot and co-immunoprecipitation assays showed that the PDGF receptor inhibition by crenolanib promoted RhoGDI1 polyubiquitination and degradation. Inhibition of RhoGDI1 degradation via MG132 reversed the decrease in VSMC phenotypic transformation. In addition, RhoGDI1 knockdown significantly inhibited VSMC phenotypic transformation and neointima formation in vitro and in vivo. These results suggest that PDGF-BB promotes RhoGDI1 stability via the PDGF receptor and induces the VSMC synthetic phenotype. The co-immunoprecipitation assay showed that PDGF-BB enhanced the interaction of RhoGDI1 with Cdc42 and promoted the activation of Cdc42; these enhancements were blocked by crenolanib and RhoGDI1 knockdown. Moreover, RhoGDI1 knockdown and crenolanib pretreatment prevented the localization of Cdc42 to the plasma membrane (PM) to activate and improve the accumulation of Cdc42 on endoplasmic reticulum (ER). Furthermore, Cdc42 inhibition or suppression significantly reduced VSMC phenotypic transformation and neointima formation in vitro and in vivo. This study revealed the novel mechanism by which RhoGDI1 stability promotes the RhoGDI1-Cdc42 interaction and Cdc42 activation, thereby affecting VSMC phenotypic transformation and neointima formation.

Diverse roles of microRNA-145 in regulating smooth muscle (dys)function in health and disease

MicroRNAs are short, non-coding RNAs that target messenger RNAs for degradation. miR-145 is a vascular-enriched microRNA that is important for smooth muscle cell (SMC) differentiation. Under healthy circumstances, SMC exist in a contractile, differentiated phenotype promoted by miR-145. In cases of disease or injury, SMC can undergo reversible dedifferentiation into a synthetic phenotype, accompanied by inhibition of miR-145 expression. Vascular disorders such as atherosclerosis and neointimal hyperplasia are characterised by aberrant phenotypic switching in SMC. This review will summarise the physiological roles of miR-145 in vascular SMC, including the molecular regulation of differentiation, proliferation and migration. Furthermore, it will discuss the different ways in which miR-145 can be dysregulated and the downstream impact this has on the progression of vascular pathologies. Finally, it will discuss whether miR-145 may be suitable for use as a biomarker of vascular disease.

Kahweol, a Diterpenoid Molecule, Inhibits CTGF-Dependent Synthetic Phenotype Switching and Migration in Vascular Smooth Muscle Cells

Vascular smooth muscle cell (VSMC) phenotype switching from contractile to synthetic is essential for proliferation and migration in vascular pathophysiology. Connective tissue growth factor (CTGF) is a matricellular protein involved in cell adhesion, migration, and proliferation. Kahweol, a diterpene molecule in arabica coffee beans, has been reported to have anti-inflammatory, antiproliferative, and apoptotic effects in many cells. However, in VSMCs, the effects of kahweol on CTGF activities have not been investigated. Thus, in this study, the effects and associated mechanisms of kahweol in CTGF-dependent phenotype switching and migration in VSMCs were examined. Experiments were performed on primary rat aortic smooth muscle cells and a rat VSMC line, A7r5. Western blot analysis was used to determine the protein levels. The mRNA levels of synthetic markers were measured by qRT-PCR. Migration of VSMCs was evaluated by wound healing and transwell assays. Kahweol reduced the angiotensin II (Ang II)-induced CTGF expression. Further, kahweol inhibited expressions of synthetic phenotype markers of VSMC. The kahweol-reduced synthetic marker protein levels were reversed by the administration of rCTGF. However, expressions of contractile phenotype markers of VSMC were not affected. Kahweol suppressed Ang II-stimulated VSMC migration. Moreover, kahweol downregulated Ang II-induced p-FAK, p-Erk, and Yes-associated protein (YAP) protein expressions. Taken together, in Ang II-stimulated VSMCs, kahweol inhibited CTGF-dependent synthetic phenotype switching and migration, with focal adhesion kinase (FAK), Erk, and YAP involved in the underlying mechanisms of the kahweol effects. These results suggest that kahweol has a potential as a therapeutic agent to inhibit CTGF, which is a molecular target in sclerogenic vascular disease.

Neutralization of S100A4 induces stabilization of atherosclerotic plaques: role of smooth muscle cells

Aims During atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they switch from a contractile to a synthetic phenotype. From porcine coronary artery, we isolated spindle-shaped (S) SMCs exhibiting features of the contractile phenotype and rhomboid (R) SMCs typical of the synthetic phenotype. S100A4 was identified as a marker of R-SMCs in vitro and intimal SMCs, in pig and man. S100A4 exhibits intra- and extracellular functions. In this study, we investigated the role of extracellular S100A4 in SMC phenotypic transition. Methods and Results S-SMCs were treated with oligomeric recombinant S100A4 (oS100A4), which induced nuclear factor (NF)-κB activation. Treatment of S-SMCs with oS100A4 in combination with platelet-derived growth factor (PDGF)-BB induced a complete SMC transition toward a pro-inflammatory R-phenotype associated with NF-κB activation, through toll-like receptor-4. RNA sequencing of cells treated with oS100A4/PDGF-BB revealed a strong upregulation of pro-inflammatory genes and enrichment of transcription factor binding sites essential for SMC phenotypic transition. In a mouse model of established atherosclerosis, neutralization of extracellular S100A4 decreased area of atherosclerotic lesions, necrotic core, and CD68 expression and increased α-smooth muscle actin and smooth muscle myosin heavy chain expression. Conclusion We suggest that the neutralization of extracellular S100A4 promotes the stabilization of atherosclerotic plaques. Extracellular S100A4 could be a new target to influence the evolution of atherosclerotic plaques. Translational perspective Our studies indicate that extracellular S100A4 is causally related to atherosclerotic plaque progression putting it forward as a prospective therapeutic target for plaque stabilization and/or regression.

ESCRT-III and ER–PM contacts maintain lipid homeostasis

Saturating transposon mutagenesis screen identified the ESCRTs as synthetic genetic interactors in ER–PM contact mutant. The synthetic phenotype is caused by defects in lipid synthesis. Other ESCRT complexes, and VPS4 do not have a synthetic growth phenotype, indicating that only ESCRT-III proteins function in this lipid regulation pathway.

P0888FIBROBLAST GROWTH FACTOR 23 PRODUCES ARTERIAL STIFFNESS THROUGH CHANGES IN VASCULAR SMOOTH MUSCLE CELL PHENOTYPE

Background and Aims In patients with chronic kidney disease (CKD), high levels of c-terminal fibroblast growth factor 23 (FGF23) are associated with cardiovascular disease and mortality. Vascular smooth muscle cells (VSMC) may present two clearly differentiated functional phenotypes, contractile and synthetic. The abundance of synthetic phenotype is associated with vascular dysfunction and it is unknown whether in FGF23 may promote transition from a contractile to a synthetic phenotype in VSMC causing vascular stiffness. The present study was conducted to evaluate whether FGF23 affects VSMC phenotype and arterial stiffness. Method and Results High levels of FGF23 promoted VSMC transition from a contractile to a synthetic phenotype. These effects were mediated through FGFR1 and Ras/MAPK signaling activation. Inhibition of both pathways enhanced contractile phenotype of VSMC. The pro-contractile microRNAs, miR-221 and miR-222 were reduced by FGF23 and miR-221 transfection recovered the contractile phenotype of VSMC decreased by FGF23. In experimental rats, exogenous infusion of FGF23 produced an increase in vascular wall thickness with VSMC exhibiting synthetic phenotype and reduction of plasma levels of miR-221. Functional studies performed on aortic arterial rings revealed that passive and active forces were altered in rats treated with FGF23. In a group of CKD stage 2-3 patients with rather high levels of FGF23 it was observed an increased in pulse pressure reflecting vascular stiffness together with low plasma levels of miR-221 and miR-222. Conclusion FGF23 favors the transition of VSMC from contractile to synthetic phenotype causing vascular dysfunction and arterial stiffness; this may be a mechanism by which FGF23 contribute directly to the development of vascular disease in CKD patients.