Mesenchymal stem cells (MSCs) of placental origin hold great promise in tissue engineering and regenerative medicine for diseases affecting cartilage and bone. However, their utility has been limited by their tendency to undergo premature senescence and phenotypic drift into adipocytes. This study aimed to explore the potential involvement of a specific subset of aging and antiaging genes by measuring their expression prior to and following in vitro-induced differentiation of placental MSCs into chondrocytes and osteoblasts as opposed to adipocytes. The targeted genes of interest included the various LMNA/C transcript variants (lamin A, lamin C, and lamin A∆10), sirtuin 7 (SIRT7), and SM22α, along with the classic aging markers plasminogen activator inhibitor 1 (PAI-1), p53, and p16INK4a. MSCs were isolated from the decidua basalis of human term placentas, expanded, and then analyzed for phenotypic properties by flow cytometry and evaluated for colony-forming efficiency. The cells were then induced to differentiate in vitro into chondrocytes, osteocytes, and adipocytes following established protocols. The mRNA expression of the targeted genes was measured by RT-qPCR in the undifferentiated cells and those fully differentiated into the three cellular lineages. Compared to undifferentiated cells, the differentiated chondrocytes demonstrated decreased expression of SIRT7, along with decreased PAI-1, lamin A, and SM22α expression, but the expression of p16INK4a and p53 increased, suggesting their tendency to undergo premature senescence. Interestingly, the cells maintained the expression of lamin C, which indicates that it is the primary lamin variant influencing the mechanoelastic properties of the differentiated cells. Notably, the expression of all targeted genes did not differ from the undifferentiated cells following osteogenic differentiation. On the other hand, the differentiation of the cells into adipocytes was associated with decreased expression of lamin A and PAI-1. The distinct patterns of expression of aging and antiaging genes following in vitro-induced differentiation of MSCs into chondrocytes, osteocytes, and adipocytes potentially reflect specific roles for these genes during and following differentiation in the fully functional cells. Understanding these roles and the network of signaling molecules involved can open opportunities to improve the handling and utility of MSCs as cellular precursors for the treatment of cartilage and bone diseases.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

Bone stromal cells are critical for bone homeostasis and regeneration. Growing evidence suggests that non-stem bone niche cells support bone homeostasis and regeneration via paracrine mechanisms, which remain to be elucidated. Here, we show that physiologically quiescent SM22α-lineage stromal cells expand after bone injury to regulate diverse processes of intramembranous bone regeneration. The majority of SM22α-lineage cells neither act as stem cells in vivo nor show their expression patterns. Dysfunction of SM22α-lineage niche cells induced by loss of platelet-derived growth factor receptor β (PDGFRβ) impairs bone repair. We further show that PDGFRβ-triggered hydrogen sulfide (H2S) generation in SM22α-lineage niche cells facilitates osteogenesis and angiogenesis and suppresses overactive osteoclastogenesis. Collectively, these data demonstrate that non-stem SM22α-lineage niche cells support the niche for bone regeneration with a PDGFRβ/H2S-dependent regulatory mechanism. Our findings provide further insight into non-stem bone stromal niche cell populations and niche-regulation strategy for bone repair.

The placental labyrinth is important for the exchange of nutrients and gases between the mother and the embryo in mice. This interface contains cells of both trophoblast and allantoic mesodermal origin that together produce maternal blood sinuses and placental blood vessels. However, the molecular mechanisms that take place during process of placental labyrinth development, especially concerning fetal capillaries, are not well understood. SREBP cleavage-activating protein (SCAP), a membrane protein, is required for the synthesis of fatty acids and cholesterol. Recently, when we crossed the offspring of the cross between smooth muscle 22 alpha (SM22α)- Cre recombinase (Cre) mice and SCAPloxp/loxp mice to research the function of SCAP in vascular smooth muscle cells (VSMCs) during certain pathological processes, we found that there were no resultant SM22α-Cre-specific SCAP knockout (KO) pups (SM22α-Cre+SCAPflox/flox; hereafter referred to as SCAP KO). Through anatomic studies of these embryos and placentas, we found that SCAP KO resulted in defective placental vessels and abnormal fetal morphology. Further immunohistochemical and immunocytochemical analyses suggested that SCAP is knocked out in the pericytes of the placental labyrinth. Compared to wildtype mice, SCAP KO placentas had abnormal vasculature in the labyrinth and lower levels of angiogenesis. By using RNA-seq and western blotting, we found that the expression of some genes and proteins in SCAP KO placentas was changed, including those related to pericyte/endothelial interactions genes and angiogenesis. Our results suggest that the proper organizational structure of the placental labyrinth depends on SCAP expression in pericytes.

To investigate the effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells. The highly expression plasmids of 27nt-miRNA and anti-27nt-miRNA, and negative control plasmids were constructed, packaged with lentivirus and transfected into human umbilical cord mesenchymal stem cells (hUCMSCs). Collagen IV was added to induce hUCMSCs differentiation into blood vessel smooth muscle cells (VSMCs). The cell viability was measured by MTT assay. The expression of SMA, SM22α at mRNA and protein levels was determined by RT-PCR, immunocytochemical staining and Western blotting. Compared with the negative control group, the viability of the 27nt-miRNA overexpression group was decreased by 20.48% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was significantly increased (P<0.05); the viability of Anti-27nt-miRNA group was increased 18.07% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was decreased (P<0.05). In summary, 27nt-miRNA promotes mesenchymal stem cells differentiation into vascular smooth muscle cells and inhibits cells viability.
为探讨27nt-miRNA 对间充质干细胞向血管平滑肌细胞分化影响，构建27nt-miRNA 过表达、反义序列Anti-27nt-miRNA 以及阴性对照的表达质粒，慢病毒包装后分别转染人脐带间充质干细胞 (hUCMSC)，加入Ⅳ型胶原诱导hUCMSC 定向分化为血管平滑肌细胞。四唑盐 (MTT) 比色法检测分化后细胞活力，免疫细胞化学染色法检测分化后细胞SM22α (兔抗平滑肌22α，smooth muscle 22α) 的表达，Western 印迹法和RT-PCR 检测分化后细胞内的SMA (兔抗平滑肌肌动蛋白，smooth muscle actin) mRNA、SM 22α mRNA 及其蛋白质表达情况。经检测，27nt-miRNA 过表达分化组与阴性对照组相比，细胞活力下降20.48% (P<0.05)，SMA mRNA、SM22α mRNA及其蛋白质表达量明显升高 (P<0.05)；而Anti-27nt-miRNA 分化组细胞活力上升了18.07% (P<0.05)，SMA mRNA、SM22α mRNA 及其蛋白质表达量下降 (P<0.05)。综上所述，27nt-miRNA 能够促进间充质干细胞向血管平滑肌细胞分化，并且抑制分化后的细胞活力。.

Thoracic aorta perivascular adipose tissue (T-PVAT) has critical roles in regulating vascular homeostasis. However, the developmental characteristics and cellular lineage of adipocyte in the T-PVAT remain unclear. We show that T-PVAT contains three long strip-shaped fat depots, anterior T-PVAT (A-T-PVAT), left lateral T-PVAT (LL-T-PVAT), and right lateral T-PVAT (RL-T-PVAT). A-T-PVAT displays a distinct transcriptional profile and developmental origin compared to the two lateral T-PVATs (L-T-PVAT). Lineage tracing studies indicate that A-T-PVAT adipocytes are primarily derived from SM22α+ progenitors, whereas L-T-PVAT contains both SM22α+ and Myf5+ cells. We also show that L-T-PVAT contains more UCP1+ brown adipocytes than A-T-PVAT, and L-T-PVAT exerts a greater relaxing effect on aorta than A-T-PVAT. Angiotensin II-infused hypertensive mice display greater macrophage infiltration into A-T-PVAT than L-T-PVAT. These combined results indicate that L-T-PVAT has a distinct development from A-T-PVAT with different cellular lineage, and suggest that L-T-PVAT and A-T-PVAT have different physiological and pathological functions.

Transgelin-1 (SM22α) has been recognized as a smooth muscle marker and a tumor suppressor, but many details of the working mechanisms remain unclear. Transgelin-1 belongs to the calponin family of actin-binding proteins with an N-terminal calponin homology domain (CH-domain) and a C-terminal calponin-like module (CLIK23). Here, we demonstrate that transgelin-1 interacts with actin stress fibers and podosomes in smooth muscle cells via its type-3 CH-domain, while CLIK23 is dispensable for the binding to the actin structures. We further suggest that the EF-hand motif in transgelin-1 contributes to proper folding of the CH-domain and in turn to the interaction with the actin structures. These results are in contrast to the ones reported in in vitro studies that demonstrated CLIK23 was necessary for the transgelin-1-actin binding, while the CH-domain was not. Besides, within cells, transgelin-1 phosphorylation at Ser181 in CLIK23 did not affect its colocalization with the actin structures, while the same phosphorylation was reported in in vitro studies to negatively regulate actin binding. Thus, our results suggest the molecular basis of intracellular interaction between transgelin-1 and actin, distinct from that in vitro. The actin binding capability intrinsic to CLIK23 may not appear within cells probably because of the weaker competition for actin binding compared to other actin binding molecules.

UNASSIGNED: Sirtuin 1 (SIRT1) inhibits nuclear factor kappa B (NF-κB) activity in response to the inflammatory cytokine tumour necrosis factor alpha (TNF-α). Smooth muscle (SM) 22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signalling cascades in vascular smooth muscle cells (VSMCs). Sm22α knockout results in increased expression of pro-inflammatory genes in the aortas which are controlled by NF-κB. This study aimed to investigate the relationship between SM22α and SIRT1 in the control of vascular inflammation.
UNASSIGNED: The ligation injury model of Sirt1-Tg/Sm22α-/- mice displayed an increased level of the inflammatory molecules in the carotid arteries compared with Sirt1-Tg mice, accompanied with aggravating neointimal hyperplasia. In the in vitro study, on the one hand, we showed that TNF-α induced the epigenetic silencing of SM22α transcription via EZH2-mediated H3K27 methylation in the SM22α promoter region, contributing to inflammatory response. On the other hand, TNF-α simultaneously induced SIRT1 phosphorylation via CKII and thereby protected against inflammation. Phosphorylated SIRT1 interacted with and deacetylated EZH2 and, subsequently, promoted SM22α transcription by inhibiting EZH2 activity. Increased SM22α in turn facilitated the phosphorylation and activation of SIRT1 via recruitment of CKII to SIRT1, which amplified the anti-inflammatory effect of SIRT1.
UNASSIGNED: Our findings demonstrate that, in response to TNF-α stimulation, CKII-SIRT1-SM22α acts in a loop to reinforce the expression of SM22α, which limits the inflammatory response in VSMCs in vivo and in vitro. The anti-inflammatory effect of SIRT1 may be dependent on SM22α to some extent. Our data point to targeted activation of SIRT1 in VSMCs as a promising therapeutic avenue in preventing cardiovascular diseases.

The insulin-sensitive glucose transporter 4 (GLUT4) is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs) and is significantly upregulated in rabbit neointima. This study investigated the role of GLUT4 in VSMC proliferation, the cellular mechanism underlying PDGF-BB-stimulated GLUT4 translocation, and effects of SM22α, an actin-binding protein, on this process. Chronic treatment of VSMCs with PDGF-BB significantly elevated GLUT4 expression and glucose uptake. PDGF-BB-induced VSMC proliferation was dependent on GLUT4-mediated glucose uptake. Meanwhile, the response of GLUT4 to insulin decreased in PDGF-BB-stimulated VSMCs. PDGF-BB-induced GLUT4 translocation partially rescued glucose utilization in insulin-resistant cells. Immunofluorescence and western blot analysis revealed that PDGF-BB induced GLUT4 translocation in an actin dynamics-dependent manner. SM22α disruption facilitated GLUT4 translocation and glucose uptake by promoting actin dynamics and cortical actin polymerization. Similar results were observed in VSMCs of SM22α -/- mice. The in vivo experiments showed that the glucose level in the neointima induced by ligation was significantly increased in SM22α -/- mice, accompanied by increased neointimal thickness, compared with those in wild-type mice. These findings suggest that GLUT4-mediated glucose uptake is involved in VSMC proliferation, and provide a novel link between SM22α and glucose utilization in PDGF-BB-triggered proliferation.
• GLUT4-mediated glucose uptake is required for the VSMC proliferation. • PDGF-BB-induced GLUT4 translocation partially rescues glucose uptake in insulin resistance. • SM22α disruption enhances PDGF-BB-induced GLUT4 translocation. • Glucose level in injured vascular tissue is positively correlated with neointimal hyperplasia.