Smooth Muscle Cell Differentiation Research Articles

MO441CALPROTECTIN IS A NOVEL CONTRIBUTING FACTOR IN VASCULAR CALCIFICATION AND A PREDICTOR OF CARDIOVASCULAR OUTCOME IN CKD PATIENTS*

Abstract Background and Aims Vascular calcification, leading to aortic stiffening and heart failure, is decisive risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). Promoted by bone mineral disorder and systemic inflammation in CKD patients, vascular calcification is a complex mechanism involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. Despite intensive research efforts in recent years, available treatments have limited effect and none of them prevent or reverse vascular calcification. The aim of this study was to analyse the serum proteome of CKD stage 3-4 patients in order to unravel new molecular changes associated to CV morbid-mortality and to decipher the role of novel candidates on vascular calcification to provide potential new therapeutic agents. Method In this study we used serum samples from two independent cohorts: 112 CKD stage 3-4 patients with a 4 years follow-up for CV events and 222 CKD stage 5 patients exhibiting a broad range of calcification degree determined by histological quantification in the epigastric and/or iliac artery. Serum proteome analysis was performed using tandem mass-spectrometry in a subcohort of 66 CKD3-4 patients and validation of protein candidates was performed using ELISA in the two full cohorts. Human primary vascular smooth muscle cells and mouse aortic rings were used for calcification assays. Calcium content was quantified using QuantiChrom calcium assay kit and calcium deposition was visualized by Alizarin Red and Von Kossa staining. Results Among 443 proteins detected in the serum of CKD3-4 patients, 134 displayed significant modified abundance in patients with CV events (n=32) compared to patients without (n=34). One of the most prominent changes was increased level of calprotectin (up to 8.6 fold, P<.0001). Using ELISA, we validated that higher serum calprotectin levels were strongly associated with higher probability of developing CV complications and increased mortality in CKD stage 3-4 patients (Figure A). Moreover, we showed that higher serum calprotectin was associated with increased vascular calcification levels in CKD stage 5 patients (Figure B). In vitro, calprotectin promoted calcification of human VSMCs (p<0.0001) (Figures C-D) and in mouse aortic rings (p<0.0001) (Figure E-F). Interestingly, these effects were significantly attenuated by paquinimod, a calprotectin inhibitor (Figures C-F). Conclusion Circulating calprotectin is a novel predictor of CV outcome and mortality in CKD patients. Calprotectin also shows calcification-inducing properties and its blockade by paquinimod alleviates its effects. Future experiments will consist in deciphering the signalling pathways involved in the regulation of calcification by calprotectin and evaluating in vivo the therapeutic potential of paquinimod on the development of medial vascular calcification lesions associated with CKD.

Transforming Growth Factor β1 Increases Expression of Contractile Genes in Human Pulmonary Arterial Smooth Muscle Cells by Potentiating Sphingosine-1-Phosphate Signaling.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and carries a very poor prognosis. Understanding of PAH pathogenesis is needed to support the development of new therapeutic strategies. Transforming growth factor β (TGF-β) drives vascular remodeling and increases vascular resistance by regulating differentiation and proliferation of smooth muscle cells (SMCs). Also, sphingosine-1-phosphate (S1P) has been implicated in PAH, but the relation between these two signaling mechanisms is not well understood. Here, we characterize the signaling networks downstream of TGF-β in human pulmonary arterial smooth muscle cells (HPASMCs), which involves mothers against decapentaplegic homolog (SMAD) signaling as well as Rho GTPases. Activation of Rho GTPases regulates myocardin-related transcription factor (MRTF) and serum response factor (SRF) transcription activity and results in upregulation of contractile gene expression. Our genetic and pharmacologic data show that in HPASMCs upregulation of α smooth muscle actin (αSMA) and calponin by TGF-β is dependent on both SMAD and Rho/MRTF-A/SRF transcriptional mechanisms.The kinetics of TGF-β-induced myosin light chain (MLC) 2 phosphorylation, a measure of RhoA activation, are slow, as is regulation of the Rho/MRTF/SRF-induced αSMA expression. These results suggest that TGF-β1 activates Rho/phosphorylated MLC2 through an indirect mechanism, which was confirmed by sensitivity to cycloheximide treatment. As a potential mechanism for this indirect action, TGF-β1 upregulates mRNA for sphingosine kinase (SphK1), the enzyme that produces S1P, an upstream Rho activator, as well as mRNA levels of the S1P receptor (S1PR) 3. SphK1 inhibitor and S1PR3 inhibitors (PF543 and TY52156/VPC23019) reduce TGF-β1-induced αSMA upregulation. Overall, we propose a model in which TGF-β1 activates Rho/MRTF-A/SRF by potentiating an autocrine/paracrine S1P signaling mechanism through SphK1 and S1PR3. SIGNIFICANCE STATEMENT: In human pulmonary arterial smooth muscle cells, transforming growth factor β depends on sphingosine-1-phosphate signaling to bridge the interaction between mothers against decapentaplegic homolog and Rho/myocardin-related transcription factor (MRTF) signaling in regulating α smooth muscle actin (αSMA) expression. The Rho/MRTF pathway is a signaling node in the αSMA regulatory network and is a potential therapeutic target for the treatment of pulmonary arterial hypertension.

Proteoglycan 4 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification.

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE−/− mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE−/− mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers’ expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.

Nano-Sized Hydroxyapatite Induces Apoptosis and Osteogenic Differentiation of Vascular Smooth Muscle Cells via JNK/c-JUN Pathway.

PurposeThe deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.MethodsTransmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.ResultsWe found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.ConclusionJNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.

Rho/ROCK-MYOCD in regulating airway smooth muscle growth and remodeling.

Abnormal airway remodeling is a common pathological change seen in chronic respiratory diseases. Altered proliferation and differentiation of airway smooth muscle cells (ASMCs) are the major components of airway remodeling, and the resultant structural abnormalities are difficult to restore. Understanding of airway smooth muscle regulation is urgently needed to identify potential intervention targets. MYOCD (or myocardin) and myocardin-related transcription factors (MRTFs) are key cotranscription factors in muscle growth, which have not been extensively investigated in airway smooth muscle cells. In addition, the RhoA/ROCK signaling pathway is known to play an important role in airway remodeling partly through regulating the proliferation and differentiation of ASMCs, which may be connected with MYOCD/MRTF cotranscription factors [Kumawat et al. (Am J Physiol Lung Cell Mol Physiol 311: L529-L537, 2016); Lagna et al. (J Biol Chem 282: 37244-37255, 2007)]. This review focuses on this newly recognized and potentially important RhoA/ROCK-MYOCD/MRTFs pathway in controlling airway smooth muscle growth and remodeling.

Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis.

Endothelial dysfunction, and the differentiation of smooth muscle cells (SMCs) into proliferative, secretory phenotypes, are two major pathophysiological processes in atherosclerosis. SMCs have the potential to recruit macrophages in atherosclerotic plaques, in which macrophages drive inflammatory responses. In this study, we found that microRNA-503-5p (miR-503-5p) was enriched in either extracellular vesicles (EVs), secreted by oxidized low-density lipoprotein-treated macrophages, or the EVs from peripheral blood mononuclear cells of atherosclerosis patients. miR-503-5p was transferred intercellularly from macrophages to the co-cultured human coronary artery endothelial cells (HCAECs) and HCASMCs via EVs, thus reducing the proliferative and angiogenic abilities of HCAECs and accelerating the proliferative and migrating abilities of HCASMCs. Smad family members 1, 2 and 7 were negatively regulated by miR-503-5p in HCAECs and HCASMCs. miR-503-5p was verified as an enhancer of inflammatory cytokines and adhesion molecules released by macrophages, in part via the down-regulation of smad family members 1, 2 and 7. The inhibition of miR-503-5p by lentivirus reduced atherosclerotic lesion formations in the aorta of atherosclerotic mice. Our work demonstrated a miR-503-5p- and EV-mediated mechanism for macrophage communication with HCAECs and HCASMCs in atherosclerosis. miR-503-5p is pro-atherosclerotic stimuli that may be a therapeutic target for atherosclerosis treatment.

Promyelocytic leukemia protein promotes the phenotypic switch of smooth muscle cells in atherosclerotic plaques of human coronary arteries.

Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.

Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and Rho.

SUMMARYReversible differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in vascular biology and disease. Changes in VSMC differentiation correlate with stiffness of the arterial extracellular matrix (ECM), but causal relationships remain unclear. We show that VSMC plasticity is mechanosensitive and that both the de-differentiated and differentiated fates are promoted by the same ECM stiffness. Differential equations developed to model this behavior predicted that a null VSMC state generates the dual fates in response to ECM stiffness. Direct measurements of cellular forces, proliferation, and contractile gene expression validated these predictions and showed that fate outcome is mediated by Rac-Rho homeostasis. Rac, through distinct effects on YAP and TAZ, is required for both fates. Rho drives the contractile state alone, so its level of activity, relative to Rac, drives phenotypic choice. Our results show how the cellular response to a single ECM stiffness generates bi-stability and VSMC plasticity.

MiR-30b-5p Downregulation as a Predictive Biomarker of Coronary In-Stent Restenosis

In-stent restenosis (ISR) is one of the main limitations of percutaneous coronary intervention (PCI) therapy with drug-eluting stents (DES) implantation. The aim of this study was to determine if circulating microRNAs (miRNAs) have diagnostic capability for determining ISR in a cohort of matched patients. Blood samples were collected from 55 patients who underwent previously PCI and were readmitted for a new coronary angiography. Patients were divided into subgroups comprising patients who presented ISR or not (non-ISR). A microarray analysis determined that up to 49 miRNAs were differentially expressed between ISR and non-ISR patients. Of these, 10 miRNAs are related to vascular smooth muscle and endothelial cells proliferation, migration, and differentiation, well-known hallmarks of vascular remodeling. Additionally, we identified that the expression of miR-30b-5p is significantly lower in serum samples of ISR patients, as compared to non-ISR. A further analysis demonstrated that miR-30b-5p provides better values of the receiver operator characteristic curve than other miRNAs and biochemical parameters. Finally, the in-silico analysis suggests that miR-30b-5p is predicted to target 62 genes involved in different signaling pathways involved in vascular remodeling. In conclusion, we determined for the first time that circulating mi-R30b-5p can reliably prognose restenosis in patient with implanted DES, which could be potentially helpful in the establishment of an early diagnosis and therapy of ISR.

Integrin-Linked Kinase (ILK) Regulates Urinary Stem Cells Differentiation into Smooth Muscle via NF-κB Signal Pathway.

Objectives Urinary stem cells (USCs) have the capacity for unlimited growth and are promising tools for the investigations of cell differentiation and urinary regeneration. However, the limited life span significantly restricts their usefulness. This study is aimed at exploring the effect of integrin-linked kinase (ILK) on the smooth muscle cells (SMCs) differentiation of the dog USCs and investigating its molecular mechanism. Methods An immortalized USCs cell line with the molecular markers and biological functions was prepared. After successfully inducing the differentiation of USCs into SMCs, the expression level of the unique key factor and its mechanisms in this process was determined through real-time polymerase chain reaction, Western blot, or Immunofluorescence staining. Results We found that high cell density promoted USCs differentiation SMCs, and ILK was necessary for USCs differentiation into SMCs. Knocking down ILK decreased the expression of SMCs specific-marker, while using a selective ILK agonist increased the expression of SMCs specific-marker. Furthermore, ILK regulated SMCs differentiation in part through the activation of NF-κB pathway in USCs. A NF-κB activity assay showed overexpression of ILK could significantly upregulate NF-κB p50 expression, and NF-κB p50 acts as downstream signal molecular of ILK. Conclusion High cell density induces the differentiation of USCs into SMCs, and ILK is a key regulator of myogenesis. Furthermore, NF-κB signaling pathway might play a crucial role in this process.

Purine-rich element binding protein B attenuates the coactivator function of myocardin by a novel molecular mechanism of smooth muscle gene repression.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purβ) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purβ to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purβ specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purβ homodimer. DNA binding analyses demonstrated that Purβ interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purβ associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purβ point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purβ repressor and the myocardin-serum response factor-CArG complex.

Disease-Relevant Single Cell Photonic Signatures Identify S100\u03b2 Stem Cells and their Myogenic Progeny in Vascular Lesions

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100β+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- β1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100β+ vSCs and identified the presence of S100β+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.Graphical abstract

RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/\u03b2-Catenin Signaling Pathway

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

Fibrin and Transforming Growth Factor Alpha Affect Prostatic Smooth Muscle Cell's Phenotype and Motility.

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFβ1 or TGFβ3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

Brain-Derived Neurotrophic Factor Expression and Signaling in Different Perivascular Adipose Tissue Depots of Patients With Coronary Artery Disease.

BackgroundBrain‐derived neurotrophic factor (BDNF) is expressed in neuronal and nonneuronal cells and may affect vascular functions via its receptor, tropomyosin‐related kinase B (TrkB). In this study, we determined the expression of BDNF in different perivascular adipose tissue (PVAT) depots of patients with established coronary atherosclerosis.Methods and ResultsSerum, vascular tissue, and PVAT surrounding the proximal aorta (C‐PVAT) or internal mammary artery (IMA‐PVAT) was obtained from 24 patients (79% men; mean age, 71.7±9.7 years; median body mass index, 27.4±4.8 kg/m2) with coronary atherosclerosis undergoing elective coronary artery bypass surgery. BDNF protein levels were significantly higher in C‐PVAT compared with IMA‐PVAT, independent of obesity, metabolic syndrome, or systemic biomarkers of inflammation. mRNA transcripts of TrkB, the BDNF receptor, were significantly reduced in aorta compared with IMA. Vessel wall TrkB immunosignals colocalized with cells expressing smooth muscle cell markers, and confocal microscopy and flow cytometry confirmed BDNF receptor expression in human aortic smooth muscle cells. Significantly elevated levels of protein tyrosine phosphatase 1B, a negative regulator of TrkB signaling in the brain, were also observed in C‐PVAT. In vitro, inhibition of protein tyrosine phosphatase 1B blunted the effects of BDNF on smooth muscle cell proliferation, migration, differentiation, and collagen production, possibly by upregulation of low‐affinity p75 neurotrophin receptors. Expression of nerve growth factor or its receptor tropomyosin‐related kinase A did not differ between C‐PVAT and IMA‐PVAT.ConclusionsElevated expression of BDNF in parallel with local upregulation of negative regulators of neurotrophin signaling in perivascular fat and lower TrkB expression suggest that vascular BDNF signaling is reduced or lost in patients with coronary atherosclerosis.

Phenotypic switch of smooth muscle cells in paediatric chronic intestinal pseudo-obstruction syndrome.

Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo‐obstruction (CIPO) syndrome, a life‐threatening intestinal motility disorder. We showed that CIPO‐SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet‐Derived Growth Factor Receptor‐alpha (PDGFRA) expression. We showed that this modulation occurs without origin‐related differences in CIPO circular and longitudinal‐derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO‐SMC towards an undifferentiated stage. The development of CIPO‐SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

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

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

Efficient endothelial and smooth muscle cell differentiation from human pluripotent stem cells through a simplified insulin-free culture system

A major obstacle for using human pluripotent stem cells (hPSCs) derived vascular cells for cell therapy is the lack of simple, cost-saving, and scalable methods for cell production. Here we described a simplified and chemically defined medium (AATS) for endothelial cells (ECs) and smooth muscle cells (SMCs) differentiation. AATS medium does not contain insulin, enabling the rapid and highly efficient vascular mesoderm formation through accelerating metabolic and autophagy-enhanced mesoderm induction. Transcriptome profiling confirmed that hPSC-derived ECs and SMCs in the AATS medium closely resembled primary ECs and SMCs formed in vivo. ECs appeared to adhere and grow better in the AATS medium over other cell types, which allowed the purification of CD31+CD144+ double-positive cells. Furthermore, the AATS medium was compatible with 3D microscaffold (MS) culture, which may facilitate large-scale bioproduction of ECs. HPSC-derived ECs and SMCs in the AATS medium exhibited strong revascularization potential in treating murine ischemic models. Our study provided a cost-effective and efficient medium system to manufacture GMP compatible, off-the-shelf ECs, and SMCs to model human diseases and vascular repair.

Interstitial Cells of Cajal and Neural Structures in the Human Fetal Appendix.

Background/AimsThe interstitial cells of Cajal (ICC) are located within and around the digestive tract’s muscle layers. They function as intestinal muscle pacemakers and aid in the modification of enteric neurotransmission. The appendix’s unique position requires an appropriate contraction pattern of its muscular wall to adequately evacuate its contents. We investigated the development and distribution of nervous structures and ICC in the human fetal appendix.MethodsSpecimens were exposed to anti-c-kit (CD117) antibodies to investigate ICC differentiation. Enteric plexuses were examined using anti-neuron-specific enolase, and the differentiation of smooth muscle cells was studied with anti-desmin antibodies.ResultsDuring weeks 13-14, numerous myenteric plexus ganglia form an almost uninterrupted sequence throughout the body and apex of the appendix. Fewer ganglia were present at the submucosal border of the circular muscle layer and within this layer. A large number of ganglia appear within the circular and longitudinal muscle layers in a later fetal period. The first ICC subtypes noted were of the myenteric plexus and the submucous plexus. In the later fetal period, the number of intramuscular ICC markedly rises, and this subtype becomes predominant.ConclusionsThe ICC and nervous structure distribution in the human fetal appendix are significantly different from all other parts of the small and large intestine. The organization of ICC and the enteric nervous system provides the basis for the specific contraction pattern of the muscular wall of the appendix.

Shuxuetong injection simultaneously ameliorates dexamethasone-driven vascular calcification and osteoporosis.

Osteoporosis (OP) and vascular calcification (VC) share a number of common risk factors, pathophysiological mechanisms and etiology, which are known as bone-vascular axis. The present study aimed to investigate the effects of Shuxuetong (SXT) injection on VC and osteoporosis. A rat model of VC and osteoporosis was induced by dexamethasone (DEX; 1 mg/kg/day for 4 weeks, intramuscularly). Simultaneously, 0.6 ml/kg/day SXT was intraperitoneally injected. Compared with control rats, DEX induced significantly more VC and OP, as determined by increased calcium deposition and alkaline phosphatase activity in the aorta, disturbed structure, decreased levels of cortical bone thickness and trabecular bone area, and increased apoptosis in the bone. SXT injection ameliorated DEX-induced VC and osteoporosis; furthermore, the osteoblastic differentiation of vascular smooth muscle cells and the activation of endoplasmic reticulum stress in the DEX group was also prevented by SXT injection. Compared with control rats, protein expression levels of sclerostin, a crucial crosslink of the bone-vascular axis, were significantly increased in the aorta and bone of rats with DEX, which was also attenuated by SXT injection. Thus, the present study suggested that SXT injection could ameliorate both VC and OP, and may be mediated by the regulation of sclerostin. The present study may provide the basis a novel strategy for the prevention and treatment of VC and OP, which emerge as side-effects of glucocorticoids.