BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype.
OBJECTIVE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).
METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.
RESULTS: Notably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.
CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Thoracic aortic dissection (TAD) is a highly dangerous cardiovascular disorder caused by weakening of the aortic wall, resulting in a sudden tear of the internal face. Progressive loss of the contractile apparatus in vascular smooth muscle cells (VSMCs) is a major event in TAD. Exploring the endogenous regulators essential for the contractile phenotype of VSMCs may aid the development of strategies to prevent TAD. Krüppel-like factor 15 (KLF15) overexpression was reported to inhibit TAD formation; however, the mechanisms by which KLF15 prevents TAD formation and whether KLF15 regulates the contractile phenotype of VSMCs in TAD are not well understood. Therefore, we investigated these unknown aspects of KLF15 function. We found that KLF15 expression was reduced in human TAD samples and β-aminopropionitrile monofumarate-induced TAD mouse model. Klf15KO mice are susceptible to both β-aminopropionitrile monofumarate- and angiotensin II-induced TAD. KLF15 deficiency results in reduced VSMC contractility and exacerbated vascular inflammation and extracellular matrix degradation. Mechanistically, KLF15 interacts with myocardin-related transcription factor B (MRTFB), a potent serum response factor coactivator that drives contractile gene expression. KLF15 silencing represses the MRTFB-induced activation of contractile genes in VSMCs. Thus, KLF15 cooperates with MRTFB to promote the expression of contractile genes in VSMCs, and its dysfunction may exacerbate TAD. These findings indicate that KLF15 may be a novel therapeutic target for the treatment of TAD.

Alterations in the vascular smooth muscle cells (VSMC) phenotype play a critical role in the pathogenesis of several cardiovascular diseases, including hypertension, atherosclerosis, and restenosis after angioplasty. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs (approximately 19-25 nucleotides in length) that function as regulators in various physiological and pathophysiological events. Recent studies have suggested that aberrant miRNAs\' expression might underlie VSMC phenotypic transformation, appearing to regulate the phenotypic transformations of VSMCs by targeting specific genes that either participate in the maintenance of the contractile phenotype or contribute to the transformation to alternate phenotypes, and affecting atherosclerosis, hypertension, and coronary artery disease by altering VSMC proliferation, migration, differentiation, inflammation, calcification, oxidative stress, and apoptosis, suggesting an important regulatory role in vascular remodeling for maintaining vascular homeostasis. This review outlines recent progress in the discovery of miRNAs and elucidation of their mechanisms of action and functions in VSMC phenotypic regulation. Importantly, as the literature supports roles for miRNAs in modulating vascular remodeling and for maintaining vascular homeostasis, this area of research will likely provide new insights into clinical diagnosis and prognosis and ultimately facilitate the identification of novel therapeutic targets.

Background Abnormal regulation of vascular smooth muscle cells is regarded as the iconic pathological change of aortic dissection (AD). Herein, we aim to identify circ_0022920 as a crucial regulator in AD. Methods and Results Microarray analysis of circular RNAs, messenger RNAs, and micro RNAs in patients with AD was performed, and we identified that circ_0022920 was significantly downregulated in these patients. The Pearson correlation analysis uncovered the negative correlation between miR-650 and circ_0022920 or TGFβR1 (transforming growth factor beta receptor 1). Angiotensin II was used to treat human aortic vascular smooth muscle cells (HASMCs) and mice as models for AD. Hematoxylin and eosin and Masson\'s trichrome staining were used to analyze AD histopathology. Cell proliferation was analyzed with Cell Counting Kit-8 assay and EdU incorporation. Cell migration was assessed with transwell and wound healing assays. Enhanced circ_0022920 expression dramatically inhibited HASMC proliferation and migration and maintained contractile marker expression induced by angiotensin II, whereas miR-650 exerted opposite effects. MiR-650 was a target of circ_0022920. MiR-650 targeted IRF1 (interferon regulatory factor 1) and thus negatively regulated TGFβR1 expression to promote HASMC proliferation and migration and inhibit contractile marker expression. Circ_0022920 suppressed the progression of AD in vivo. Conclusions Circ_0022920 modulates the contractile phenotype of HASMCs via regulating the miR-650-IRF1-TGFβR1 axis in angiotensin II-induced models for AD, which provides potential therapeutic targets for AD.

OBJECTIVE: Vascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear.
METHODS: We used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development.
RESULTS: Here, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation.
CONCLUSIONS: Bves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

The in-stent restenosis (IRS) after the percutaneous coronary intervention contributes to the major treatment failure of stent implantation. MicroRNAs have been revealed as powerful gene medicine to regulate endothelial cells (EC) and smooth muscle cells (SMC) in response to vascular injury, providing a promising therapeutic candidate to inhibit IRS. However, the controllable loading and eluting of hydrophilic bioactive microRNAs pose a challenge to current lipophilic stent coatings. Here, we developed a microRNA eluting cardiovascular stent via the self-healing encapsulation process based on an amphipathic poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) triblock copolymer spongy network. The miR-22 was used as a model microRNA to regulate SMC. The dynamic porous coating realized the uniform and controllable loading of miR-22, reaching the highest dosage of 133 pmol cm-2. We demonstrated that the sustained release of miR-22 dramatically enhanced the contractile phenotype of SMC without interfering with the proliferation of EC, thus leading to the EC dominating growth at an EC/SMC ratio of 5.4. More importantly, the PCEC@miR-22 coated stents showed reduced inflammation, low switching of SMC phenotype, and low secretion of extracellular matrix, which significantly inhibited IRS. This work provides a simple and robust coating platform for the delivery of microRNAs on cardiovascular stent, which may extend to other combination medical devices, and facilitate practical application of bioactive agents in clinics.

Abdominal aortic aneurysm (AAA) is life-threatening, for which efficient nonsurgical treatment strategy has not been available so far. Several previous studies investigating the therapeutic effect of mesenchymal stem cells (MSCs) in AAA indicated that MSCs could inhibit aneurysmal inflammatory responses and extracellular matrix destruction, and suppress aneurysm occurrence and expansion. Vascular smooth muscle cell (VSMC) phenotypic plasticity is reported to be predisposed in AAA initiation and progression. However, little is known about the effect of MSCs on VSMC phenotypic modulation in AAA. In this study, we investigate the therapeutic efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) in elastase-induced AAA model and evaluate the effect of UC-MSC on VSMC phenotypic regulation. We demonstrate that the intravenous injection of UC-MSC attenuates elastase-induced aneurysmal expansion, reduces elastin degradation and fragmentation, inhibits MMPs and TNF-α expression, and preserves and/or restores VSMC contractile phenotype in AAA. Taken together, these results highlight the therapeutic and VSMC phenotypic modulation effects of UC-MSC in AAA progression, which further indicates the potential of applying UC-MSC as an alternative treatment candidate for AAA.

Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif-containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif-containing 28 (TRIM28) and tripartite motif-containing 27 (TRIM27) in VSMCs. We observed that siRNA-mediated knockdown of TRIM28 and TRIM27 inhibited platelet-derived growth factor (PDGF)-induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor-β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N-terminal RING-B boxes-Coiled-Coil domain. Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs.

Electrospun uniaxially aligned ultrafine fibers show great promise in constructing vascular grafts mimicking the anisotropic architecture of native blood vessels. However, understanding how the stiffness of aligned fibers would impose influences on the functionality of vascular cells has yet to be explored. The present study aimed to explore the stiffness effects of electrospun aligned fibrous substrates (AFSs) on phenotypic modulation in vascular smooth muscle cells (SMCs). A stable jet coaxial electrospinning (SJCES) method was employed to generate highly aligned ultrafine fibers of poly(l-lactide- co-caprolactone)/poly(l-lactic acid) (PLCL/PLLA) in shell-core configuration with a remarkably varying stiffness region from 0.09 to 13.18 N/mm. We found that increasing AFS stiffness had no significant influence on the cellular shape and orientation along the fiber direction with the cultured human umbilical artery SMCs (huaSMCs) but inhibited the cell adhesion rate, promoted cell proliferation and migration, and especially enhanced the F-actin fiber assembly in the huaSMCs. Notably, higher fiber stiffness resulted in significant downregulation of contractile markers like alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain, calponin, and desmin, whereas upregulated the gene expression of pathosis-associated osteopontin ( OPN) in the huaSMCs. These results allude to the phenotype of huaSMCs on stiffer AFSs being miserably modulated into a proliferative and pathological state. Consequently, it adversely affected the proliferation and migration behavior of human umbilical vein endothelial cells as well. Moreover, stiffer AFSs also revealed to incur significant upregulation of inflammatory gene expression, such as interleukin-6 ( IL-6), monocyte chemoattractant protein-1 ( MCP-1), and intercellular adhesion molecule-1 ( ICAM-1), in the huaSMCs. This study stresses that although electrospun aligned fibers are capable of modulating native-like oriented cell morphology and even desired phenotype realization or transition, they might not always direct cells into correct functionality. The integrated fiber stiffness underlying is thereby a critical parameter to consider in engineering structurally anisotropic tissue-engineered vascular grafts to ultimately achieve long-term patency.

The tubular porous poly(ɛ-caprolactone) (PCL) scaffold was fabricated by electrospinning. After then, the scaffold\'s surface was firstly eroded by hexyldiamine to endow amine group, and heparin was covalently grafted to the surface to get surface heparin modified scaffold (ShPCL scaffold). It was found that ShPCL scaffold can induce smooth muscle cells (SMCs) to penetrate the scaffold surface, while the SMCs cannot penetrate the surface of PCL scaffold. Subsequently, the rabbit SMCs were seeded on the ShPCL scaffold and cultured for 14 days. It was found the expression of α-smooth muscle actin in ShPCL scaffold maintained much higher level than that in culture plate, which implied the SMC differentiation in ShPCL scaffold. Furthermore, the immunefluorescence staining of the cross-sections of ShPCL scaffold exhibited the expression of calponin in ShPCL scaffold can be detected after 7 and 14 days, whereas the expression of smooth muscle myosin heavy chain can also be detected at 14 days. These results proved that penetrated SMCs preferably differentiated in to contractile phenotype. The successful SMC penetration and the contractile phenotype expression implied ShPCL scaffold is a suitable candidate for regenerating smooth muscle layer in vascular tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2806-2815, 2017.