Differentiation Of Vascular Smooth Muscle Cells Research Articles

Epigenetic Upregulation of H19 and AMPK Inhibition Concurrently Contribute to S-Adenosylhomocysteine Hydrolase Deficiency-Promoted Atherosclerotic Calcification.

S-adenosylhomocysteine (SAH) is a risk factor of cardiovascular disease; inhibition of SAH hydrolase (SAHH) results in SAH accumulation and induces endothelial dysfunction and atherosclerosis. However, the effect and mechanism of SAHH in atherosclerotic calcification is still unclear. We aimed to explore the role and mechanism of SAHH in atherosclerotic calcification. The relationship between SAHH and atherosclerotic calcification was investigated in patients with coronary atherosclerotic calcification. Different in vivo genetic models were used to examine the effect of SAHH deficiency on atherosclerotic calcification. Human aortic and murine vascular smooth muscle cells (VSMCs) were cultured to explore the underlying mechanism of SAHH on osteoblastic differentiation of VSMCs. The expression and activity of SAHH were decreased in calcified human coronary arteries and inversely associated with coronary atherosclerotic calcification severity, whereas plasma SAH and total homocysteine levels were positively associated with coronary atherosclerotic calcification severity. Heterozygote knockout of SAHH promoted atherosclerotic calcification. Specifically, VSMC-deficient but not endothelial cell-deficient or macrophage-deficient SAHH promoted atherosclerotic calcification. Mechanistically, SAHH deficiency accumulated SAH levels and induced H19-mediated Runx2 (runt-related transcription factor 2)-dependent osteoblastic differentiation of VSMCs by inhibiting DNMT3b (DNA methyltransferase 3b) and leading to hypomethylation of the H19 promoter. On the contrary, SAHH deficiency resulted in lower intracellular levels of adenosine and reduced AMPK (AMP-activated protein kinase) activation. Adenosine supplementation activated AMPK and abolished SAHH deficiency-induced expression of H19 and Runx2 and osteoblastic differentiation of VSMCs. Finally, AMPK activation by adenosine inhibited H19 expression by inducing Sirt1 (sirtuin-1)-mediated histone H3 hypoacetylation and DNMT3b-mediated hypermethylation of the H19 promoter in SAHH deficiency VSMCs. We have confirmed a novel correlation between SAHH deficiency and atherosclerotic calcification and clarified a new mechanism that epigenetic upregulation of H19 and AMPK inhibition concurrently contribute to SAHH deficiency-promoted Runx2-dependent atherosclerotic calcification.

Application of OpenArray RT-qPCR for identification of microRNA expression signatures of lower extremity artery disease.

Lower extremity artery disease (LEAD) is an underestimated chronic vascular disease caused by the formation of atherosclerotic plaques in the lower limb arteries. The pathological processes underlying this disease are regulated by many various factors, including microRNAs (miRNAs). miRNAs constitute a pool of small, non-coding RNAs with a gene expression modulatory function. In the presented study, differentially expressed miRNAs were identified in peripheral blood mononuclear cells from 18 patients with LEAD compared to 10 healthy volunteers using OpenArray RT-qPCR. Sixteen miRNAs were significantly differentially expressed in the LEAD group. Four out of them, hsa-miR-138-5p, -34a-3p, -34a-5p, and -766-3p, were consistent with a previous next-generation sequencing study. The in silico analysis performed for these four miRNAs showed associations with vascular smooth muscle cells differentiation, inflammation, and apoptosis, potentially resulting from modulation of genes involved in cell cycle, cellular adhesion, and Notch signaling. The presented study expands our knowledge on the role of miRNA in the pathology of LEAD, providing potential candidates for biomarkers of this disease.

CREG ameliorates embryonic stem cell differentiation into smooth muscle cells by modulation of TGF-β expression

Vascular smooth muscle cell (SMCs) differentiation is critical for cardiovascular development, but the mechanisms remain largely unknown. The overall aim of this study was to investigate the functional impact and mechanism of cellular repressor of E1A-stimulated genes (CREG) in SMC differentiation. Two embryonic stem cell (ESC) models were generated (1) the overexpression of CREG (CREG-OE), by transfection with Pcreg-IRECS2-EGFP vector, and (2) the knockout of CREG, by transfection with CREG shRNA (CREG-KO). Interesting, SMC-marker levels (SM α-actin, SM22, Calponin, and SM-MHC) dramatically increased in CREG-OE ESCs into the SMC while significantly decreased in CREG-KO ESCs during differentiation. After 14 days, and calcium ion concentrations in angiotensin II-stimulated embryoid bodies were increased in CREG-OE ESCs but reduced in CREG-KO ESCs. Consistently, the contractile capacity of SMC from CREG-OE ESC was increased, while the contractile capacity of SMC CREG1 from CREG-KO ESCs was significantly reduced. Furthermore, we demonstrated that CREG promotes differentiation of ESCs to SMCs and maturation of their function through the transforming growth factor-β -smad2/3 pathway.

Relationships between Indicators of Lower Extremity Artery Disease and miRNA Expression in Peripheral Blood Mononuclear Cells.

Lower extremity artery disease (LEAD) is an underdiagnosed and globally underestimated vascular disease caused by the progressive and chronic formation of atherosclerotic plaques in the arteries of the lower limbs. Much evidence indicates that the abnormal course of pathophysiological processes underlying LEAD development is associated with altered miRNA modulatory function. In the presented study, relationships between miRNA expression and clinical indicators of this disease (ABI, claudication distance, length of arterial occlusion, Rutherford category, and plaque localization) were identified. MiRNA expression profiles were obtained using next-generation sequencing in peripheral blood mononuclear cells (PBMCs) of 40 LEAD patients. Correlation analysis performed using the Spearman rank correlation test revealed miRNAs related to ABI, claudication distance, and length of arterial occlusion. In the DESeq2 analysis, five miRNAs were found to be dysregulated in patients with Rutherford category 3 compared to patients with Rutherford category 2. No miRNAs were found to be differentially expressed between patients with different plaque localizations. Functional analysis performed using the miRNet 2.0 website tool determined associations of selected miRNAs with processes underlying vascular pathology, such as vascular smooth muscle cell differentiation, endothelial cell apoptosis, response to hypoxia, inflammation, lipid metabolism, and circadian rhythm. The most enriched functional terms for genes targeted by associated miRNAs were linked to regulation of the cell cycle, regulation of the transcription process, and nuclear cellular compartment. In conclusion, dysregulations of miRNA expression in PBMCs of patients with LEAD are indicative of the disease and could potentially be used in the prediction of LEAD progression.

Tissue engineered in-vitro vascular patch fabrication using hybrid 3D printing and electrospinning.

Three-dimensional (3D) engineered cardiovascular tissues have shown great promise to replace damaged structures. Specifically, tissue engineering vascular grafts (TEVG) have the potential to replace biological and synthetic grafts. We aimed to design an in-vitro patient-specific patch based on a hybrid 3D print combined with vascular smooth muscle cells (VSMC) differentiation. Based on the medical images of a 2 months-old girl with aortic arch hypoplasia and using computational modelling, we evaluated the most hemodynamically efficient aortic patch surgical repair. Using the designed 3D patch geometry, the scaffold was printed using a hybrid fused deposition modelling (FDM) and electrospinning techniques. The scaffold was seeded with multipotent mesenchymal stem cells (MSC) for later maturation to derived VSMC (dVSMC). The graft showed adequate resistance to physiological aortic pressure (burst pressure 101 ​± ​15 ​mmHg) and a porosity gradient ranging from 80 to 10 ​μm allowing cells to infiltrate through the entire thickness of the patch. The bio-scaffolds showed good cell viability at days 4 and 12 and adequate functional vasoactive response to endothelin-1. In summary, we have shown that our method of generating patient-specific patch shows adequate hemodynamic profile, mechanical properties, dVSMC infiltration, viability and functionality. This innovative 3D biotechnology has the potential for broad application in regenerative medicine and potentially in heart disease prevention.

Differentiation and molecular characterization of endothelial progenitor and vascular smooth muscle cells from induced pluripotent stem cells.

Pluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period. Induced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied. The optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period. EPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.

H19 Promotes Osteoblastic Transition by Acting as ceRNA of miR-140-5p in Vascular Smooth Muscle Cells.

Arterial medial calcification is a common disease in patients with type 2 diabetes, end-stage renal disease and hypertension, resulting in high incidence and mortality of cardiovascular event. H19 has been demonstrated to be involved in cardiovascular diseases like aortic valve diseases. However, role of H19 in arterial medial calcification remains largely unknown. We identified that H19 was upregulated in ß-glycerophosphate (β-GP) induced vascular smooth muscle cells (VSMCs), a cellular calcification model in vitro. Overexpression of H19 potentiated while knockdown of H19 inhibited osteogenic differentiation of VSMCs, as demonstrated by changes of osteogenic genes Runx2 and ALP as well as ALP activity. Notably, H19 interacted with miR-140-5p directly, as demonstrated by luciferase report system and RIP analysis. Mechanistically, miR-140-5p attenuated osteoblastic differentiation of VSMCs by targeting Satb2 and overexpression of miR-140-5p blocked H19 induced elevation of Satb2 as well as the promotion of osteoblastic differentiation of VSMCs. Interestingly, over-expression of Satb2 induced phosphorylation of ERK1/2 and p38MAPK. In conclusion, H19 promotes VSMC calcification by acting as competing endogenous RNA of miR-140-5p and at least partially by activating Satb2-induced ERK1/2 and p38MAPK signaling.

Legumain Is an Endogenous Modulator of Integrin αvβ3 Triggering Vascular Degeneration, Dissection, and Rupture.

The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive. Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn-/-), macrophage-specific Lgmn knockout mice (LgmnF/F;LysMCre), and RR-11a-treated C57BL/6 mice were given BAPN (β-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvβ3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses. Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvβ3 in VSMCs and blocked integrin αvβ3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection. LGMN signaling may be a novel target for the prevention and treatment of TAD.

RIP2 Knockdown Attenuates Vascular Smooth Muscle Cells Activation via Negative Regulating Myocardin Expression.

RIP2 is an adaptor protein contributing to the activation of nuclear factor-κB induced by TNF receptor-associated factor (TRAF) and nucleotide oligomerization domain (NOD)-dependent signaling implicated in innate and adaptive immune response. Beyond regulation of immunity, we aimed to elucidate the role of RIP2 in vascular smooth muscle cell (VSMC) phenotypic modulation. In the current study, we observed that RIP2 showed an increased expression in VSMCs with PDGF-BB stimulation in a dose-dependent manner. Knockdown of RIP2 expression mediated by adenovirus dramatically accelerated the expression of VSMC-specific differentiation genes induced by PDGF-BB. Silencing of RIP2 inhibited proliferative and migratory ability of VSMCs. Additionally, we demonstrated that RIP2 knockdown can promoted myocardin expression. Furthermore, RIP2 inhibition also can attenuate the formation of intimal hyperplasia. These findings suggested that RIP2 played an important role in regulation of VSMCs differentiation, migration, and proliferation that may due to affect myocardin expression. Our results indicated that RIP2 may be a novel therapeutic target for intimal hyperplasia.

The effects of mechanical trapping of cells into the concave micro-grooves on the cell nucleus and differentiation of the vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) in arterial walls in vivo have contractile phenotype with an elongated shape and significant alignment to the circumference of the arterial wall. On the other hand, VSMCs spread randomly and form irregular shapes during cultivation on a rigid dish, and dedifferentiated to synthetic phenotype. To clarify the mechanisms of such morphological and phenotypic changes in vascular smooth muscle cells, it is essential to develop a cell culture model that consider the mechanical environment of aortic tissue. In this study, we fabricated silicone rubber (PDMS) micro-grooved substrates with three different groove widths (5, 10, 20 μm) using microfabrication technology to promote cell elongation and alignment, and optimized the groove size to improve contractile differentiation. We found that VSMCs adhering into the concaves of grooves allowed remarkable elongation of the cell nucleus and the 5 μm micro-grooved substrates significantly suppressed cell proliferation and promoted vascular smooth muscle cells differentiation.

Ion Channels and Transporters in Muscle Cell Differentiation.

Investigations on ion channels in muscle tissues have mainly focused on physiological muscle function and related disorders, but emerging evidence supports a critical role of ion channels and transporters in developmental processes, such as controlling the myogenic commitment of stem cells. In this review, we provide an overview of ion channels and transporters that influence skeletal muscle myoblast differentiation, cardiac differentiation from pluripotent stem cells, as well as vascular smooth muscle cell differentiation. We highlight examples of model organisms or patients with mutations in ion channels. Furthermore, a potential underlying molecular mechanism involving hyperpolarization of the resting membrane potential and a series of calcium signaling is discussed.

LncRNA H19 sponges miR-103-3p to promote the high phosphorus-induced osteoblast phenotypic transition of vascular smooth muscle cells by upregulating Runx2

Elucidating the mechanism of the osteogenic phenotypic transdifferentiation of vascular smooth muscle cells (VSMCs) is the key to determining the diagnosis and treatment of arterial medial calcification (AMC). Long noncoding RNAs (lncRNAs) have been reported to participate in the regulation of vascular physiology and pathology. Here, we investigated the effect and mechanism of the lncRNA H19 on the osteoblastic differentiation of VSMCs induced by high phosphorus. H19 was expressed at high levels in high phosphorus-induced primary rat VSMCs. Further experiments indicated that H19 played a positive role in the osteoblast phenotypic transition by suppressing miR-103-3p expression and subsequently promoting osteoblast-specific marker expression, including bone morphogenetic protein 2 (BMP-2) and osteopontin (OPN). Mechanistically, we recognized RUNX family transcription factor 2 (Runx2) as a direct target of miR-103-3p. Moreover, H19 directly interacted with miR-103-3p, and overexpression of miR-103-3p reversed the upregulation of Runx2 induced by H19. Therefore, H19 positively regulated Runx2 expression by sponging miR-103-3p and promoted the osteoblast phenotypic transition in VSMC calcification. Collectively, the lncRNA H19 promoted osteogenic differentiation by modulating the miR-103-3p/Runx2 axis in the process of VSMC calcification induced by a high phosphorus concentration. The current study provided new insights into an important role for the lncRNA H19 as a miRNA sponge in VSMCs and supplied novel insights into lncRNA-directed diagnostics and therapeutics for vascular calcification.

Narrative review of exosomes: novel players in vascular calcification of chronic kidney disease.

Vascular calcification (VC) has been proved the main pathophysiology basis that results in the cardiovascular disease which become the leading cause of death in patients with chronic kidney disease. The disordered mineral metabolism, hyperphosphatemia, overuse of calcium binders, occurred in the setting of uremia, have become the critical risk factors of vascular calcification in chronic kidney disease, in which the vascular smooth muscle cells undergo differentiation to osteoblast-like cells. The mechanisms involved in vascular calcification are complicated and exosomes, as novel players, are proven to play a vital role in VC and function as initializers for mineral deposition. Exosomal miRNAs play an important role in the regulation of cellular functions in vascular calcification. In this review, we focused on the roles of exosomes during VC, especially on their effects on regulating vascular calcification through initializing mineral deposition as a nidus, transporting microRNAs to the recipient cells and mediating the vascular smooth muscle cells differentiation to osteoblast-like cells.

Neuromedin B modulates phosphate-induced vascular calcification

Vascular calcification is the heterotopic accumulation of calcium phosphate salts in the vascular tissue and is highly correlated with increased cardiovascular morbidity and mortality. In this study, we found that the expression of neuromedin B (NMB) and NMB receptor is upregulated in phosphate-induced calcification of vascular smooth muscle cells (VSMCs). Silencing of NMB or treatment with NMB receptor antagonist, PD168368, inhibited the phosphate-induced osteogenic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling and VSMC apoptosis. PD168368 also attenuated the arterial calcification in cultured aortic rings and in a rat model of chronic kidney disease. The results of this study suggest that NMB–NMB receptor axis may have potential therapeutic value in the diagnosis and treatment of vascular calcification.

IL-18 Mediates Vascular Calcification Induced by High-Fat Diet in Rats With Chronic Renal Failure.

Objective: Vascular calcification (VC) is an important predictor of cardiovascular morbidity and mortality in patients with chronic renal failure (CRF). It is well-known that obesity and metabolic syndrome (OB/MS) predicts poor prognosis of CRF patients. However, the influence of OB/MS on VC in CRF patients isn't clear. IL-18 mediates OB/MS-related inflammation, but whether IL-18 is involved in OB/MS -mediated VC in CRF patients hasn't been studied. In this study, it was explored that whether OB/MS caused by high-fat diet (HFD) can affect the level of serum IL-18 and aggravate the degree of VC in CRF rats. Furthermore, it was studied that whether IL-18 induces rat vascular smooth muscle cells (VSMCs) calcification by activating the MAPK pathways.Approach: The rats were randomly assigned to the sham-operated, CRF and CRF + HFD groups. CRF was induced by 5/6 nephrectomy. Serum IL-18 levels and aortic calcification indicators were compared in each group. Primary rat VSMCs calcification were induced by β-glycerophosphate and exposed to IL-18. VSMCs were also treated with MAPK inhibitors.Results: The weight, serum levels of hsCRP, TG and LDL-C in CRF + HFD group were significantly higher than those in sham-operated and CRF groups (p < 0.05). Compared with the sham-operated group, the calcium content and the expression of BMP-2 of aorta in CRF and CRF + HFD groups were significantly increased (p < 0.05). Moreover, the calcium content and the expression of BMP-2 of aorta in CRF + HFD group was significantly higher than those in CRF group (p < 0.05). And the serum IL-18 level was positively correlated with aortic calcium content. It was also found that p38 inhibitor SB203580 can suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18. But the JNK inhibitor SP600125 can't suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18.Conclusions: These findings suggest that obesity-related inflammation induced by high-fat diet could exacerbate VC in CRF rats. Furthermore, serum IL-18 level had a positive correlation with the degree of VC. It is also found that IL-18 promoted osteogenic differentiation and calcification of rat VSMCs via p38 pathway activation.

MiR-145 attenuatesphenotypic transformation of aortic vascular smooth muscle cells to prevent aortic dissection.

Background miR‐145 is closely related to vascular smooth muscle cells (VSMC) phenotype transformation; however, the regulatory mechanisms through which miR‐145 regulates the VSMC phenotype transformation under mechanical stretching are unclear. In this study, we evaluated the roles of miR‐145 in VSMCs subjected to mechanical stretching in aortic dissection (AD).MethodsThe expression of miR‐145 in the aortic vessel wall of model animals and patients with AD was analyzed by quantitative polymerase chain reaction. miR‐145‐related protein‐protein interaction networks and Wikipathways were used to analyze VSMC phenotypic transformation pathways regulated by miR‐145. We used gain‐ and loss‐of‐function studies to evaluate the effects of miR‐145 on VSMC differentiation under mechanical stretch induction and assessed whether Krüppel‐like factor 4 (KLF4) was regulated by miR‐145 in the aorta under mechanical stretch conditions.Results miR‐145 was abundantly expressed in the walls of the normal human aorta, but was significantly downregulated in animal models and the walls of patients with dissection. We found that contractile phenotype‐related proteins were downregulated in VSMCs subjected to mechanical stretching, whereas the expression of secreted phenotype‐related proteins increased. miR‐145 overexpression also downregulated contractile phenotype‐related proteins in VSMCs and suppressed upregulation of phenotype‐related proteins. Finally, under mechanical stretching, KLF4 expression was significantly increased in VSMCs, and overexpression of miR‐145 blocked this effect.ConclusionOur results confirmed that mechanical stretch‐induced phenotypic transformation of VSMCs to promote AD via upregulation of KLF4; this mechanism was regulated by miR‐145, which directly modulated KLF4 expression and VSMC differentiation.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

A Loss of Nuclear-Cytoskeletal Interactions in Vascular Smooth Muscle Cell Differentiation Induced by a Micro-Grooved Collagen Substrate Enabling the Modeling of an In Vivo Cell Arrangement.

Vascular smooth muscle cells (VSMCs) remodel vascular walls actively owing to mechanical cues and dedifferentiate to the synthetic phenotype from contractile phenotype in pathological conditions. It is crucial to clarify the mechanisms behind the VSMC phenotypic transition for elucidating their role in the vascular adaptation and repair and for designing engineered tissues. We recently developed novel micro-grooved collagen substrates with “wavy wrinkle” grooves to induce cell–substrate adhesion, morphological polarization, and a tissue-like cell arrangement with cytoskeletal rearrangements similar to those in vascular tissue in vivo. We found that cultivation with this micro-grooved collagen significantly induced VSMC contractile differentiation. Nonetheless, the detailed mechanism underlying the promotion of such VSMC differentiation by micro-grooved collagen has not been clarified yet. Here, we investigated the detailed mechanism of the cell arrangement into a tissue and contractile-differentiation improvement by our micro-grooved collagen substrates in terms of nuclear–cytoskeletal interactions that possibly affect the nuclear mechanotransduction involved in the activation of transcription factors. We found that VSMCs on micro-grooved collagen manifested significant cell arrangement into a tissue and nucleus slimming with a volume reduction in response to the remodeling of the actin cytoskeleton, with consequent inhibition of nuclear shuttling of a transcriptional coactivator, Yes-associated protein (YAP), and improved contractile differentiation. Furthermore, VSMC nuclei rarely deformed during macroscopic cell stretching and featured a loss of nesprin-1–mediated nuclear–cytoskeletal interactions. These results indicate that our micro-grooved collagen induces a cell alignment mimicking in vivo VSMC tissue and promotes contractile differentiation. In such processes of contractile differentiation, mechanical interaction between the nucleus and actin cytoskeleton may diminish to prevent a nuclear disturbance from the excess mechanical stress that might be essential for maintaining vascular functions.

β-Hydroxybutyric Inhibits Vascular Calcification via Autophagy Enhancement in Models Induced by High Phosphate.

Background: Vascular calcification (VC) is a landmark of aging, while β-hydroxybutyric acid (BHB) induced by calorie restriction has been identified as a promising factor to extend the lifespan. However, the effect of BHB on VC and the potential mechanism remain unknown.Methods: A total of 160 subjects with or without metabolic abnormalities (MAs) were assigned to four groups according to different calcification severities. The association between BHB, MAs, and VC was investigated via mediation analysis. Then, with high phosphate-induced calcification models, the effect of BHB on arterial ring calcification and osteogenic phenotypic differentiation of vascular smooth muscle cells (VSMCs) was investigated. Hereafter the expressions of autophagy biomarkers, autophagy flux, and effects of autophagy inhibitors on VC were detected.Results: Severe VC was observed in the elderly, accompanied with a higher proportion of hypertension, chronic kidney disease, and lower estimated glomerular filtration rate. The serum BHB level was an independent influencing factor of VC severities. With mediation analysis, BHB was determined as a significant mediator in the effects of MAs on VC, and the indirect effect of BHB accounted for 23% of the total effect. Furthermore, BHB directly inhibited arterial ring calcification and osteogenic phenotypic differentiation in VSMCs, accompanied with autophagy enhancement in VSMCs. In accordance, the inhibition of autophagy counteracted the protective effect of BHB on VC.Conclusion: The present study demonstrated that BHB mediated the effects of MAs on VC; then, it further elucidated that BHB could inhibit arterial and VSMC calcification via autophagy enhancement.