Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm.

BACKGROUND: The fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood. METHODS: In this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRß, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib. RESULTS: Single-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation. CONCLUSION: Our findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Administration of H2S improves erectile dysfunction by inhibiting phenotypic modulation of corpus cavernosum smooth muscle in bilateral cavernous nerve injury rats.

Phenotypic modulation of Corpus Cavernosum Smooth Muscle Cells (CCSMCs) is an important step in the development and progression of bilateral cavernous nerve injury induced erectile dysfunction (BCNI-ED). To investigate the effect of exogenous hydrogen sulfide (H2S) on the phenotypic modulation of CCSMCs in BCNI-ED rats, a total of 18 male Sprague-Dawley rats were equally divided into 3 groups, including sham-operated (Sham) group, BCNI group and BCNI treated with NaHS (BCNI + NaHS) group. The treated group received intraperitoneal injection of NaHS (100 µmol kg-1day-1) for 4 weeks starting day 1 postoperatively. Erectile function was measured by the ratio of intracavernous pressure (ICP)/mean arterial pressure (MAP), and relevant tissues were harvested for Immunohistochemistry, Hematoxylin and eosin (H&E), Masson's trichrome staining, H2S fluorescent probe WSP-1 and Western blot. The primary CCSMCs were isolated and pretreatment with NaHS before exposed to PDGF-BB (platelet-derived growth factor). Relative expression mRNA and protein of phenotypic biomarkers, RhoA, ROCK-1 and cell cycle proteins were detected. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST) and H2S levels in penile tissue was significantly decreased in the BCNI group compared with the Sham group. Compared with the BCNI group, administration of NaHS significantly increased the ratio of ICP/MAP, ratio of smooth muscle to collagen, expressions of a-SMA, calponin and decreased the expression of OPN, collagen-I, RhoA, ROCK1 in the penile tissue. PDGF-BB-treated CCSMCs exhibited higher expression of OPN, RhoA, ROCK1, and lower α-SMA, calponin, which were attenuated by NaHS pretreatment. NaHS suppressed RhoA/ROCK activity and decreased the expression of CDK2, Cyclin E1, while increased the expression of P27kip1 induced by PDGF-BB in CCSMCs. Taken together, this study indicated that exogenous H2S inhibited the phenotypic modulation of CCSMCs by suppressing RhoA/ROCK1 signaling and affecting its downstream factor, CDK2, Cyclin E1, P27kip1, thereby improved BCNI rat erectile function.

Metformin inhibits intracranial aneurysm formation and progression by regulating vascular smooth muscle cell phenotype switching via the AMPK/ACC pathway.

BACKGROUND: The regulation of vascular smooth muscle cell (VSMC) phenotype plays an important role in intracranial aneurysm (IA) formation and progression. However, the underlying mechanism remains unclear. Metformin is a 5' AMP-activated protein kinase (AMPK) agonist that has a protective effect on vasculature. The present study investigated whether metformin modulates VSMC phenotype switching via the AMPK/acetyl-CoA carboxylase (ACC) pathway during IA pathogenesis. METHODS: Adult male Sprague-Dawley rats (n = 80) were used to establish an elastase-induced IA model. The effects of metformin on AMPK activation and VSMC phenotype modulation were examined. We also established a platelet-derived growth factor (PDGF)-BB-induced VSMC model and analyzed changes in phenotype including proliferation, migration, and apoptosis as well as AMPK/ACC axis activation under different doses of metformin, AMPK antagonist, ACC antagonist, and their combinations. RESULTS: Metformin decreased the incidence and rupture rate of IA in the rat model and induced a switch in VSMC phenotype from contractile to synthetic through activation of the AMPK/ACC pathway, as evidenced by upregulation of VSMC-specific genes and decreased levels of pro-inflammatory cytokines. AMPK/ACC axis activation inhibited the proliferation, migration, and apoptosis of VSMCs, in which phenotypic switching was induced by PDGF-BB. CONCLUSIONS: Metformin protects against IA formation and rupture by inhibiting VSMC phenotype switching and proliferation, migration, and apoptosis. Thus, metformin has therapeutic potential for the prevention of IA.

Mechanotransduction and Stiffness-Sensing: Mechanisms and Opportunities to Control Multiple Molecular Aspects of Cell Phenotype as a Design Cornerstone of Cell-Instructive Biomaterials for Articular Cartilage Repair.

Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-ß1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-ß1- and interleukin 1 beta (IL-1ß)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.

Modulation of the airway smooth muscle phenotype in a murine asthma model and effects of nuclear factor-κB inhibition.

Objective: Phenotype modulation of airway smooth muscle (ASM) is a unique characteristic of asthma and is considered to regulate airway remodeling, airway hyperresponsiveness (AHR) and inflammation. The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in phenotypic modulation. Thus, models of acute and chronic asthma were established and pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor was delivered by intraperitoneal injection. Methods: The Penh value was measured using the BUXCO WBP system. Lung tissues were subjected to histologic analysis. Phenotypic markers of ASM and COL1A1 mRNA levels were measured by RT-PCR. Expression levels of phosphorylated p65 (pP65) and α-SMA were detected by Western blot. Serum cytokine levels were quantified by RayBiotech ELISA array. Results: PDTC intervention decreased the Penh values in both the acute and chronic models. The ASM area and the airway collagen area were decreased in the PDTC intervention group. A decrease in phenotypic markers were detected in both the acute and chronic models in time-dependent manner, and PDTC intervention partially reversed the phenotypic modulation. The effect of PDTC intervention on systemic inflammation was also verified. Conclusion: These results revealed the existence of a dynamic ASM phenotype modulation procedure in asthma development and that targeting NF-κB by PDTC was effective to mitigate ASM phenotype modulation and major asthmatic pathological features.

Irisin reverses platelet derived growth factor-BB-induced vascular smooth muscle cells phenotype modulation through STAT3 signaling pathway.

Vascular smooth muscle cells (VSMCs) phenotype modulation toward a synthetic phenotype is the main cause of cardiovascular disease. As a newly discovered myokine, Irisin is thought to be a promising candidate for the treatment of metabolic disturbances, as well as cardiovascular disease. However, no evidence has been shown for the direct effect of Irisin on VSMCs phenotype modulation and its underling mechanisms. The aim of this study was to explore the effect of Irisin on VSMCs phenotype modulation and the mechanisms involved. In the present study, it was found that Irisin restored the PDGF-BB-induced VSMCs phenotype modulation which exhibited down-regulation of smooth muscle cells (SMC) expression and up-regulation of matrix synthesis related marker expression, as well as proliferative phenotype. Moreover, our research demonstrated that Irisin further activated STAT3 signaling pathways. Finally, by applying an STAT3 inhibitor, WP1066, we revealed the roles of STAT3 in the PDGF-BB-induced VSMCs phenotype modulation when they were treated with Irisin. Taken together, these results demonstrated that Irisin may play a crucial role in regulating VSMCs phenotype modulation via the STAT3 signaling pathway.

Reversing the Epithelial-Mesenchymal Transition in Metastatic Cancer Cells Using CD146-Targeted Black Phosphorus Nanosheets and a Mild Photothermal Treatment.

Cancer metastasis leads to most deaths in cancer patients, and the epithelial-mesenchymal transition (EMT) is the key mechanism that endows the cancer cells with strong migratory and invasive abilities. Here, we present a nanomaterial-based approach to reverse the EMT in cancer cells by targeting an EMT inducer, CD146, using engineered black phosphorus nanosheets (BPNSs) and a mild photothermal treatment. We demonstrate this approach can convert highly metastatic, mesenchymal-type breast cancer cells to an epithelial phenotype (i.e., reversing EMT), leading to a complete stoppage of cancer cell migration. By using advanced nanomechanical and super-resolution imaging, complemented by immunoblotting, we validate the phenotypic switch in the cancer cells, as evidenced by the altered actin organization and cell morphology, downregulation of mesenchymal protein markers, and upregulation of epithelial protein markers. We also elucidate the molecular mechanism behind the reversal of EMT. Our results reveal that CD146-targeted BPNSs and a mild photothermal treatment synergistically contribute to EMT reversal by downregulating membrane CD146 and perturbing its downstream EMT-related signaling pathways. Considering CD146 overexpression has been confirmed on the surface of a variety of metastatic, mesenchymal-like cancer cells, this approach could be applicable for treating various cancer metastasis via modulating the phenotype switch in cancer cells.

SET knockdown attenuated phenotype modulation and calcium channel associated markers of airway smooth muscle cells in asthmatic mice.

BACKGROUND: Dysfunctional phenotype modulation and calcium channels in airway smooth muscle cells (ASMCs) are important characteristics of airway remodeling in chronic asthma. However, the mechanisms underlying these pathological processes remain unclear. SET (I2PP2A, inhibitor-2 of protein phosphatase 2A) has many significant functions and is involved in various physiological and pathological processes. This study aimed to determine the function of SET in chronic asthma. METHODS: BALB/c mice were sensitized by ovalbumin injection and repeated inhalation of ovalbumin. The Penh value was measured using the Buxco whole body plethysmography system. A short hairpin RNA of the SET gene was designed and transfected into ASMCs derived from asthmatic mice. Flow cytometry of Annexin-V/propidium iodide staining was used for evaluating cell apoptosis. Western blot was adopted to measure the expression levels of ASMCs phenotype modulation markers and calcium channel-associated proteins. RESULTS: The results showed that shRNA targeting SET significantly decreased the expression of SET, and enhanced the apoptosis of ASMCs. SET knockdown promoted the expression of contractile phenotype markers such as α-SMA (alpha smooth muscle Actin), SM-MHC (smooth muscle Myosin heavy chain), and calponin, and inhibited the expression of synthetic phenotype markers including vimentin and CD44. The expression of the calcium channel-related proteins STIM1 (Stromal interaction molecule 1) and Orai1 were also inhibited after SET knockdown. CONCLUSIONS: These data demonstrated that SET participated in the development of airway dysfunction in asthma, suggesting that the silencing of SET may be a new therapeutic target for the treatment of asthma patients.

The Potential Role of hsa_circ_0005505 in the Rupture of Human Intracranial Aneurysm.

Objective: Recently, abundant number of studies have revealed many functions of circular RNAs in multiple diseases, however, the role of circular RNA in the rupture of human intracranial aneurysm is still unknown. This study aims to explore the potential functions of circular RNA in the rupture of human intracranial aneurysms. Methods: The differentially expressed circular RNAs between un-ruptured intracranial aneurysms (n = 5) and ruptured intracranial aneurysms (n = 5) were analyzed with the Arraystar human circRNAs microarray. Quantitative real-time PCR (qPCR) was used to verify the results of the circRNA microarray. The role of circular RNA in intracranial aneurysm rupture was assessed in vitro. MTT assay, CCK-8 assay, Caspase3/7 assay, assay of cell apoptosis and Celigo wound healing was conducted to evaluate the relationship between circular RNA and the rupture of human intracranial aneurysms. Results: A total of 13,175 circRNA genes were detected. Among them 63 circRNAs upregulated and 54 circRNAs downregulated significantly in ruptured intracranial aneurysms compared with un-ruptured intracranial aneurysms (p < 0.05 Fold Change > 1.5). Five upregulated circRNAs were selected for further study (hsa_circ_0001947, hsa_circ_0043001, hsa_circ_0064557, hsa_circ_0058514, hsa_circ_0005505). The results of qPCR showed only hsa_circ_0005505 significantly upregulated (p < 0.05). The expression of hsa_circ_0005505 was higher in ruptured intracranial aneurysm tissues. And our in vitro data showed that hsa_circRNA_005505 promotes the proliferation, migration and suppresses the apoptosis of vascular smooth muscle cell. Conclusion: This study revealed an important role of hsa_circ_0005505 in the proliferation, migration and apoptosis of vascular smooth muscle cell, and indicated that hsa_circ_0005505 may associate with the pathological process of intracranial aneurysms.

[CRISPR/Cas9-mediated TEAD1 knockout induces phenotypic modulation of corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction].

OBJECTIVE: To construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED. OBJECTIVE: Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively. OBJECTIVE: The primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED. OBJECTIVE: We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.

Bacillus Responses to Plant-Associated Fungal and Bacterial Communities.

Some members of root-associated Bacillus species have been developed as biocontrol agents due to their contribution to plant protection by directly interfering with the growth of pathogens or by stimulating systemic resistance in their host. As rhizosphere-dwelling bacteria, these bacilli are surrounded and constantly interacting with other microbes via different types of communications. With this review, we provide an updated vision of the molecular and phenotypic responses of Bacillus upon sensing other rhizosphere microorganisms and/or their metabolites. We illustrate how Bacillus spp. may react by modulating the production of secondary metabolites, such as cyclic lipopeptides or polyketides. On the other hand, some developmental processes, such as biofilm formation, motility, and sporulation may also be modified upon interaction, reflecting the adaptation of Bacillus multicellular communities to microbial competitors for preserving their ecological persistence. This review also points out the limited data available and a global lack of knowledge indicating that more research is needed in order to, not only better understand the ecology of bacilli in their natural soil niche, but also to better assess and improve their promising biocontrol potential.

Heterogeneity of Microglia Phenotypes: Developmental, Functional and Some Therapeutic Considerations.

BACKGROUND: Microglia play a pivotal role in maintaining homeostasis in complex brain environment. They first exist as amoeboid microglial cells (AMCs) in the developing brain, but with brain maturation, they transform into ramified microglial cells (RMCs). In pathological conditions, microglia are activated and have been classified into M1 and M2 phenotypes. The roles of AMCs, RMCs and M1/M2 microglia phenotypes especially in pathological conditions have been the focus of many recent studies. METHODS: Here, we review the early development of the AMCs and RMCs and discuss their specific functions with reference to their anatomic locations, immunochemical coding etc. M1 and M2 microglia phenotypes in different neuropathological conditions are also reviewed. RESULTS: Activated microglia are engaged in phagocytosis, production of proinflammatory mediators, trophic factors and synaptogenesis etc. Prolonged microglia activation, however, can cause damage to neurons and oligodendrocytes. The M1 and M2 phenotypes featured prominently in pathological conditions are discussed in depth. Experimental evidence suggests that microglia phenotype is being modulated by multiple factors including external and internal stimuli, local demands, epigenetic regulation, and herbal compounds. CONCLUSION: Prevailing views converge that M2 polarization is neuroprotective. Thus, proper therapeutic designs including the use of anti-inflammatory drugs, herbal agents may be beneficial in suppression of microglial activation, especially M1 phenotype, for amelioration of neuroinflammation in different neuropathological conditions. Finally, recent development of radioligands targeting 18 kDa translocator protein (TSPO) in activated microglia may hold great promises clinically for early detection of brain lesion with the positron emission tomography.

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells.

Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

Intermedin reduces neointima formation by regulating vascular smooth muscle cell phenotype via cAMP/PKA pathway.

BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) dedifferentiation contributes to neointima formation, which results in various vascular disorders. Intermedin (IMD), a cardiovascular paracrine/autocrine polypeptide, is involved in maintaining circulatory homeostasis. However, whether IMD protects against neointima formation remains largely unknown. The purpose of this study is to investigate the role of IMD in neointima formation and the possible mechanism. METHODS: IMD1-53 (100ng/kg/h) or saline water was used on rat carotid-artery balloon-injury model. The mouse left common carotid-artery ligation-injury model was established using IMD-transgenic and C57BL/6J mice. Immunohistochemistry and immunofluorescence staining was used to detect the protein expression in rat carotid arteries. Radioimmunoassay was used to determine the serum IMD level. The hematoxylin andeosin staining was used for carotid arteries morphological testing. In vitro, for rat primary cultured VSMC phenotype transition, proliferation and migration assays, platelet-derived growth factor-BB (PDGF-BB) reagent and IMD1-53 peptide were added to the culture media at the final concentration of 20 ng/mL and 10-7mol/L respectively. Quantification of VSMC proliferation involved MTT and BrdU assay and migration was detected by wound-healing assay. Western blot and realtime PCR were used to detect the protein and mRNA levels of tissues or cells. RESULTS: With the rat carotid-artery balloon-injury model, IMD was significantly downregulated in injured arteries and plasma. Exogenous IMD1-53 greatly inhibited neointima formation and prevented VSMC from switching to a synthetic phenotype. With the left common carotid-artery ligation-injury model, IMD-transgenic mice showed less neointima formation than C57BL/6J mice. PDGF-BB reduced IMD mRNA expression in rat primary cultured VSMCs but increased that of its receptors, calcitonin receptor-like receptor or receptor activity-modifying proteins. Furthermore, PDGF-BB promoted VSMC proliferation and migration and transformed VSMCs to the synthetic phenotype, which was reversed with IMD1-53 treatment. Mechanistically, IMD1-53 maintained the contractile VSMC phenotype via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. CONCLUSIONS: IMD attenuated neointima formation both in the rat model of carotid-artery balloon injury and mouse model of common carotid-artery ligation injury. IMD protection may be mediated by maintaining a VSMC contractile phenotype via the cAMP/PKA pathway.

miRNA-181a/b Regulates Phenotypes of Vessel Smooth Muscle Cells Through Serum Response Factor.

The phenotypic modulation of vessel smooth muscle cells (VSMCs) plays a crucial role in the physiological and pathological conditions of vasculature in response to local environmental changes. The phenotypic transition of VSMCs is largely modulated by the serum response factor (SRF). miR-181a and miR-181b are members of the well-studied miR-181 family and both have complementary sequence in the 3' untranslated region (UTR) of SRF gene. In this article, evidence insinuates that miR-181a/b was involved in VSMCs differentiation through upregulating synthetic marker genes and downregulating contractile ones, respectively. We also confirmed the roles of the miR-181a/b in promoting SMC proliferation, migration, and synthetic phenotype transformation. In addition, miR-181a/b was indicated as directly targeting at 3' UTR of SRF by dual-luciferase assay and Western blot assay. In a word, miR-181a/b is one of the factors involved in VSMC differentiation toward a synthetic phenotype through targeting at SRF. These findings may provide a potential therapeutic approach that miR-181a/b took part in regulating the vessel disorders.

T-cadherin promotes vascular smooth muscle cell dedifferentiation via a GSK3ß-inactivation dependent mechanism.

Participation of the cadherin superfamily of adhesion molecules in smooth muscle cell (SMC) phenotype modulation is poorly understood. Immunohistochemical analyses of arterial lesions indirectly suggest upregulated expression of atypical glycosylphosphatidylinositol-anchored T-cadherin on vascular SMCs as a molecular indicator of the dedifferentiated/proliferative phenotype. This study investigated the role of T-cadherin in SMC phenotypic modulation. Morphological, molecular and functional SMC-signature characteristics of rat, porcine and human arterial SMCs stably transduced with respect to T-cadherin upregulation (Tcad+) or T-cadherin-deficiency (shTcad) were compared with their respective control transductants (E-SMCs or shC-SMCs). Tcad+-SMCs displayed several characteristics of the dedifferentiated phenotype including loss of spindle morphology, reduced/disorganized stress fiber formation, decay of SMC-differentiation markers (smooth muscle α-actin, smooth muscle myosin heavy chain, h-caldesmon), gain of SMC-dedifferentiation marker calmodulin, reduced levels of myocardin, nuclear-to-cytoplasmic redistribution of the myocardin related transcription factors MRTFA/B and increased proliferative and migratory capacities. T-cadherin depletion enforced features of the differentiated SMC phenotype. PI3K/Akt is a major signal pathway utilized by T-cadherin in SMCs and we investigated mTORC1/S6K1 and GSK3ß axes as mediators of T-cadherin-induced dedifferentiation. Inhibition of mTORC1/S6K1 signalling by rapamycin suppressed proliferation in both E-SMCs and Tcad+-SMCs but failed to restore expression of contractile protein markers in Tcad+-SMCs. Ectopic adenoviral-mediated co-expression of constitutively active GSK3ß mutant S9A in Tcad+-SMCs restored the morphological and molecular marker characteristics of differentiated SMCs and normalized rate of proliferation to that in control SMCs. In conclusion our study demonstrates that T-cadherin promotes acquisition of the dedifferentiated phenotype via a mechanism that is dependent on GSK3ß inactivation.

Insoluble elastin reduces collagen scaffold stiffness, improves viscoelastic properties, and induces a contractile phenotype in smooth muscle cells.

Biomaterials with the capacity to innately guide cell behaviour while also displaying suitable mechanical properties remain a challenge in tissue engineering. Our approach to this has been to utilise insoluble elastin in combination with collagen as the basis of a biomimetic scaffold for cardiovascular tissue engineering. Elastin was found to markedly alter the mechanical and biological response of these collagen-based scaffolds. Specifically, during extensive mechanical assessment elastin was found to reduce the specific tensile and compressive moduli of the scaffolds in a concentration dependant manner while having minimal effect on scaffold microarchitecture with both scaffold porosity and pore size still within the ideal ranges for tissue engineering applications. However, the viscoelastic properties were significantly improved with elastin addition with a 3.5-fold decrease in induced creep strain, a 6-fold increase in cyclical strain recovery, and with a four-parameter viscoelastic model confirming the ability of elastin to confer resistance to long term deformation/creep. Furthermore, elastin was found to result in the modulation of SMC phenotype towards a contractile state which was determined via reduced proliferation and significantly enhanced expression of early (α-SMA), mid (calponin), and late stage (SM-MHC) contractile proteins. This allows the ability to utilise extracellular matrix proteins alone to modulate SMC phenotype without any exogenous factors added. Taken together, the ability of elastin to alter the mechanical and biological response of collagen scaffolds has led to the development of a biomimetic biomaterial highly suitable for cardiovascular tissue engineering.