Smooth Muscle Protein Research Articles

Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification.

Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive, precluding mechanism-based therapies. Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5). In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca2+. Phenotypic switching was accompanied by increased levels of ROS and Ca2+-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca2+-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca2+. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca2+ uptake via EVs and show that Ca2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodeling and calcification in the context of mineral imbalance. Graphic Abstract: A graphic abstract is available for this article.

Tau Protein in Human Brain Vascular Smooth Muscle

BackgroundIschemic stroke is a leading cause of long‐term disability and death worldwide. Thickening of the vascular wall and the reduction of lumen increase the incidence of ischemic stroke. Thus, therapeutic strategies targeting brain vascular narrowing should reduce the mortality and morbidity associated with ischemic stroke. Vascular smooth muscle cell (SMC) proliferation contributes to the development of vascular narrowing, however, cell signaling mechanisms of brain vascular SMC proliferation has not been defined. Tau is a microtubule‐associated protein indicated in various neurological pathologies, including Alzheimer’s disease. Although Tau protein is known to be expressed in neuronal cells, the role of Tau in brain vasculature is unknown. The present study tested the hypothesis that Tau is expressed in brain vascular SMCs.Methods & ResultsImmunohistochemistry of postmortem brain tissues from human patients died of ischemic stroke demonstrated the expression of Tau protein in the vascular smooth muscle media layer. Western blotting of cell lysates from cultured human brain vascular SMCs revealed the occurrence of a Tau isoform with a molecular weight of ~45 kD that was knocked down by Tau siRNA. Treatment of cells with a potent mitogen of SMCs, platelet‐derived growth factor (PDGF), promoted the phosphorylation of Tau at Thr181, but not, Ser198, Ser199, Ser202, Thr205, Thr212, Ser214, Ser356, Ser404, or Ser422. A Tau protein aggregation inhibitor, methylene blue, inhibited human brain vascular SMC growth.ConclusionsThis is the first demonstration that Tau protein is expressed in any kind of vascular SMCs. Moreover, results showing that PDGF causes the phosphorylation of Tau at a specific amino acid and a Tau aggregation inhibitor suppresses cell growth suggest that Tau may play a role in cell growth signaling in brain vascular SMCs.Support or Funding InformationSupported by NIH

Sex‐Differences in Gastric Smooth Muscle Function: Role of G Protein‐Coupled Estrogen Receptor

The regulation of gastrointestinal (GI) motility by estrogen and its receptors has been a topic of great interest owing to sexual dimorphism that exists in motility disorders. Scientific and clinical evidence establishes the fact of GI motility disturbances during estrous cycle but, there is a gap in the knowledge and understanding of estrogen and its receptors role in GI motility physiology during these cycles. Estrogen activates classical nuclear receptors ERα, ERβ and atypical 7‐transmembrane G protein‐coupled estrogen receptor (GPER or GPR30) to initiate both genomic and non‐genomic effects. GPER is involved in the several rapid cellular signaling events. The pleiotropic effects manifested by GPER action include regulation of body weight, glucose homeostasis, anti‐inflammation, anti‐nociception, and vascular muscle relaxation. Ablation of GPER leads to increase in mean arterial pressure in female mice, whereas treatment with GPER‐specific agonist G‐1 lowers arterial blood pressure in hypertensive and ovariectomized female rats. The role of GPER in mediating estrogen effects on GI smooth muscle is unknown.AimTo understand the role of GPER in gastric smooth muscle function in male and female mice.MethodsMuscle strips and muscle cells were isolated from the stomachs of control and GPER knockout mice. Expression of GPER was analyzed by qRT‐PCR and western blot. cAMP levels in response to estrogen and G‐1 was measured by radioimmunoassay. Relaxation was measured in organ bath studies in muscle strips and by scanning micrometry in isolated muscle cells.ResultsExpression of GPER (mRNA and protein) in the gastric smooth muscle was higher in female mice compared to male mice. 17β‐estradiol and G‐1 caused an increase in cAMP levels and inhibited acetylcholine‐induced muscle contraction (i.e., caused muscle relaxation) in gastric muscle strips and isolated muscle cells from both female and male mice, however, the increase in cAMP levels and the amount of relaxation was significantly greater in gastric muscle from female mice compared to male mice. The effect of 17β‐estradiol and G‐1 on muscle relaxation was concentration‐dependent. Relaxation was blocked by the GPER specific antagonist G15 (10 μM) in gastric muscle from control mice and absent in gastric muscle from GPER knockout mice. Relaxation was higher in proestrus and estrus phases compared to diestrus phase in female mice and this is consistent with higher expression of GPER in proestrus and estrus phases compared to diestrus phase.ConclusionActivation of GPER causes relaxation via cAMP/PKA pathway in gastric smooth muscle from female and male mice. Greater relaxation in gastric smooth muscle from female mice is due to higher expression of GPER in female mice compared to male mice. Pilot studies suggests that higher expression of GPER in the stomach of female mice is due to differential regulation of GPER expression by epigenetic mechanisms.Support or Funding InformationSupported by grants from VETAR (SM); IACUC approval AD10001201

Erratum to “Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension. [Archives of Biochemistry and Biophysics 664 (2019) 68–75

Erratum to “Mitochondrial Rieske iron-sulfur protein in pulmonary artery smooth muscle: A key primary signaling molecule in pulmonary hypertension. [Archives of Biochemistry and Biophysics 664 (2019) 68–75

Functional cell phenotype induction with TGF-β1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor β1 (TGF-β1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-β1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-β1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-β1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-β1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-β1-containing collagen-PU constructs could induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Non-activatable mutant of inhibitor of kappa B kinase α (IKKα) exerts vascular site-specific effects on atherosclerosis in Apoe-deficient mice

Background and aimsIKKα is an important regulator of gene expression. As IKKα kinase inactivity in bone marrow-derived cells does not affect atherosclerosis, we here investigate the impact of a whole body-IKKα kinase inactivity on atherosclerosis. MethodsApolipoprotein E (Apoe)-deficient mice homozygous for an activation-resistant Ikkα-mutant (IkkαAA/AAApoe−/−) and Ikkα+/+Apoe−/− controls received a Western-type diet. Atherosclerotic lesion size and cellular content were analyzed using histology and immunofluorescence. Vascular protein expression and IKKα kinase activity were quantified by Luminex multiplex immuno-assay and ELISA. ResultsA vascular site-specific IKKα expression and kinase activation profile was revealed, with higher total IKKα protein levels in aortic root but increased IKKα phosphorylation, representing activated IKKα, in the aortic arch. This was associated with a vascular site-specific effect of IkkαAA/AA knock-in on atherosclerosis: in the aortic root, IkkαAA/AA knock-in decreased lesion size by 22.0 ± 7.7% (p < 0.01), reduced absolute lesional smooth muscle cell numbers and lowered pro-atherogenic MMP2. In contrast, IkkαAA/AA knock-in increased lesion size in the aortic arch by 43.7 ± 20.1% (p < 0.001), increased the abundance of lesional smooth muscle cells in brachiocephalic artery as main arch side branch and elevated MMP2. A similar profile was observed for MMP3. No effects were observed on necrotic core or collagen deposition in atherosclerotic lesions, nor on absolute lesional macrophage numbers. ConclusionsA non-activatable IKKα kinase differentially affects atherosclerosis in aortic root vs. aortic arch/brachiocephalic artery, associated with a differential vascular IKKα expression and kinase activation profile as well as with a vascular site-dependent impact on lesional smooth muscle cell accumulation and protein expression profiles.

Burn-Induced Impairment of Ileal Muscle Contractility Is Associated with Increased Extracellular Matrix Components

IntroductionSevere burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown. MethodsMale Sprague Dawley rats approximately 3 months old and weighing 300–350 g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 h after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externa was isolated from the ileal segment to prepare smooth muscle protein extracts for Western blot analysis. ResultsCompared with sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin and laminin were significantly up-regulated in burned rats compared with sham rats. TGF-β signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-β signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats’ ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues. ConclusionDeposition of fibrosis-related proteins after severe burn is contributors to decreased small intestinal motility.

Biomarkers for acute mesenteric ischemia diagnosis: state of the art and perspectives

Acute mesenteric ischemia (AMI) is one of the most severe diagnostic and therapeutic vital emergencies. This affection is characterized by the insufficient blood supply to the gastrointestinal tract, related to an occlusive or non-occlusive mechanism, resulting in an ischemic and inflammatory injury that may progress to necrosis of the intestinal wall. The clinical picture is nonspecific, dominated by acute abdominal pain. At present, no early biological marker is commonly used in clinical practice for diagnostic purposes. The purpose of this review was to review the markers that have been evaluated in this condition. Among the biological blood markers which have shown a diagnostic interest in the IMA, there are notably the two stereoisomers of lactate (D and L), D-dimers, and alpha glutathione transferase. More specific markers include the intestinal fatty acid binding protein or I-FABP, which is a marker of enterocyte necrosis, citrulline, a marker of enterocyte mass, or Smooth muscle protein 22 (SM22) marker for muscle damage. The early diagnosis of intestinal ischemia remains a challenge. It is likely that in the future IMA's biomarker research will be better customized and adapted to the physiopathological mechanism. More global approaches (proteomics, metabolomics) should also make it possible to identify new biomarkers.

Peritubular cells of the human testis: prostaglandin E2 and more.

Several layers of slender, smooth muscle-like, peritubular cells and extracellular matrix (ECM) form the peritubular compartment of the human testis. Peritubular cells are the least explored testicular cells. Human testicular peritubular cells (HTPCs) can be isolated from small testicular fragments of patients and studied in vitro. We have used this cellular model, in combination with human testicular samples, to examine how peritubular cells may contribute to male (in)fertility. Human testicular peritubular cells(HTPCs) retain contractile abilities in vitro and secrete many proteins. Among them are factors, which serve intra-testicular roles, for example, glial cell line-derived neurotrophic factor (GDNF), thought to be important for the renewal of spermatogonial stem cells (SSCs). Studies in mutant mice indicated that peritubular cell-derived GDNF is crucial for lifelong spermatogenesis. Thus, peritubular cells are a functional part of the SSC niche. Peritubular cells of mice and men express androgen receptors (AR). In mouse peritubular cells, androgens enhanced GDNF production, but not in HTPCs. Rather, AR activation increased the levels of AR and smooth muscle proteins and thereby enhanced the smooth muscle-like phenotype. Following the lead of a proteomic analysis, which identified the key prostaglandin (PG)-synthesizing enzyme (PTGS1=COX1), we found that HTPCs secrete PGE2 . COX1, and PGE2 receptors (EP1, 2, and 4) were identified in peritubular cells in situ, supporting in vivo relevance. In HTPCs, activation of EP1/4 increased GDNF and a smooth muscle protein. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), which blocks PG synthesis. Added to HTPCs it reduced PGE2 and GDNF production and lowered smooth muscle protein levels. If applicable to the in vivo situation, the results suggest that ibuprofen and possibly other NSAIDs may impair important peritubular cell functions and consequently testicular functions. The few examples highlighted, together with others not mentioned here, indicate that HTPCs provide an experimental window into the human testis.

Neural Crest Cells Differentiate Into Brown Adipocytes and Contribute to Periaortic Arch Adipose Tissue Formation.

Periaortic arch adipose tissue (PAAT) plays critical roles in regulating vascular homeostasis; however, its anatomic features, developmental processes, and origins remain unclear. Approach and Results: Anatomic analysis and genetic lineage tracing of Wnt1 (wingless-type MMTV [mouse mammary tumor virus] integration site family member 1)-Cre+;Rosa26RFP/+ mice, Myf5 (myogenic factor 5)-Cre+;Rosa26RFP/+ mice, and SM22α-Cre+;Rosa26RFP/+ mice are performed, and the results show that PAAT has unique anatomic features, and the developmental processes of PAAT are independent of the others periaortic adipose tissues. PAAT adipocytes are mainly derived from neural crest cells (NCCs) rather than from Myf5+ progenitors. Most PAAT adipocyte progenitors expressed SM22α+ (smooth muscle protein 22-alpha) during development. Using Wnt1-Cre+;PPARγflox/flox mice, we found that knockout of PPAR (peroxisome proliferator-activated receptor)-γ in NCCs results in PAAT developmental delay and dysplasia, further confirming that NCCs contribute to PAAT formation. And we further indicated PAAT dysplasia aggravates Ang II (angiotensin II)-induced inflammation and remodeling of the common carotid artery close to aorta arch. We also found that NCCs can be differentiated into both brown and white adipocytes in vivo and in vitro. RNA sequencing results suggested NCC-derived adipose tissue displays a distinct transcriptional profile compared with the non-NCC-derived adipose tissue in PAAT. PAAT has distinctive anatomic features and developmental processes. Most PAAT adipocytes are originated from NCCs which derive from ectoderm. NCCs are progenitors not only of white adipocytes but also of brown adipocytes. This study indicates that the PAAT is derived from multiple cell lineages, the adipocytes derived from different origins have distinct transcriptional profiles, and PAAT plays a critical role in Ang II-induced common carotid artery inflammation and remodeling.Visual OvervieW: An online visual overview is available for this article.

Smooth muscle‐selective ablation of A‐kinase anchoring protein 150 (AKAP150) mitigates diabetes‐induced cardiac dysfunction

Inadequate blood flow to the myocardium due to microvascular dysfunction may play a role in the development and progression of diabetic cardiomyopathy, although contributing mechanisms are poorly understood. Hyperglycemia, a hallmark of diabetes mellitus, promotes enhanced arterial tone via altered excitation‐contraction and ‐transcription coupling that is dependent upon kinase/phosphatase targeting to the membrane of vascular smooth muscle by the A‐kinase anchoring protein AKAP150. In this study, we tested the hypothesis that AKAP150‐dependent signaling in smooth muscle contributes to cardiac dysfunction in an experimental model of diabetes. We generated smooth muscle selective AKAP150‐null animals by crossing AKAP5‐floxed mice (Akap150fl/fl) with knock‐in mice expressing Cre recombinase under the control of SM22α (smCre). Offspring (AKAP150fl/fl‐smCre) were viable and demonstrated genetic recombination and loss of AKAP150 protein in vascular smooth muscle (aortic smooth muscle, mesenteric arteries, coronary arteries), but not in heart or brain tissues. Male AKAPfl/fl and AKAPfl/fl‐smCre animals (8–12 weeks) were maintained on a high fat/high sucrose (HFHS) diet for 20 weeks. Body weight and blood glucose (fasting and non‐fasting) were significantly elevated in HFHS groups compared to control chow‐fed animals at time points after 8 weeks. At 20 weeks, echocardiography revealed that AKAP150fl/fl animals on HFHS diet had significantly greater end systolic volume, as well as reduced ejection fraction and fractional shortening when compared to AKAP150fl/fl mice maintained on control diet for the same period. However, no significant differences in cardiac function or chamber dimensions were observed between AKAP150fl/fl‐smCre animals on HFHS versus AKAP150fl/f‐smCre animals on control diet. Our results suggest that AKAP150‐mediated signaling in vascular smooth muscle may contribute to altered cardiac function in a HFHS model of diabetes.Support or Funding InformationThis work was supported in part by grants from the National Institutes of Health (GM103492, HL142710) and American Heart Association (16SDG27260070),This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

α1D-adrenoceptor involves the relaxation effect of farrerol in rat aortic vascular smooth muscle cells

The aim of this study was to investigate the relaxation effect of farrerol on rat aortic vascular smooth muscle cells (VSMCs) and its underlying mechanism. VSMCs were cultured primarily and were used to examine the relaxation effect of farrerol. Cells surface and length were measured by dynamic observation, or by rhodamine-phalloidin labeling and hematoxylin-eosin staining. Cells contractive activity were tested using collagen gel contraction assay. The [Ca2+]in was measured with molecular probe fluo-4-AM. The mRNA and protein expression of regulatory proteins for contraction were measured. In addition, rat aortic VSMCs were transfected with lentivirus-mediated α1D-adrenoceptor gene-shRNA, then the effect of farrerol were detected by the above experimental methods. The results revealed that 10 μΜ AngⅡ promoted cell contraction, increased [Ca2+]in and enhanced collagen contraction in rat aortic VSMCs. 10 μΜ AngⅡ not only increased expression of myosin light chain kinase (MLCK) and smooth muscle protein 22α (SM22α), but also increased phosphorylation level of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1). The above effects induced by AngⅡ could be significantly inhibited by farrerol in a concentration dependent manner. When the cells were transfected with lentivirus mediated α1D-adrenoceptor gene-shRNA, the effects of farrerol on changes induced by AngⅡ in rat aortic VSMCs were markedly reversed. In conclusion, farrerol could produce relaxtion effect in rat aortic VSMCs precontracted by 10 μΜ AngⅡ, which was involved in downregulation expression of MLCK and SM22α, and inhibition phosphorylation level of MYPT1 and MLC via activating α1D-adrenoceptor gene.

Nucleolin promotes AngII‑induced phenotypic transformation of vascular smooth muscle cells via interaction with tropoelastin mRNA.

The current study aimed to clarify the role of nucleolin in the phenotypic transformation of vascular smooth muscle cells (VSMCs) and to preliminarily explore its underlying mechanism. The spatial and temporal expression patterns of nucleolin, and the effects of angiotensin II (Ang II) on the expression of VSMC phenotypic transformation markers, α-smooth muscle-actin, calponin, smooth muscle protein 22α and osteopontin were investigated. The effects of nucleolin on VSMC phenotypic transformation and the expression of phenotypic transformation-associated genes, tropoelastin, epiregulin and fibroblast growth factor 2 (b-FGF), were determined. Protein-RNA co-immunoprecipitation was used to investigate the potential target genes regulated by the nucleolin in phenotypic transformation of VSMCs. Finally, the stability of tropoelastin mRNA and the effects of nucleolin on the expression of tropoelastin were assayed. The results revealed that Ang II significantly promoted the phenotypic transformation of VSMCs. The expression of nucleolin was gradually upregulated in VSMCs treated with Ang II at different concentrations for various durations. Ang II induced nucleolin translocation from the nucleus to cytoplasm. Additionally, Ang II significantly promoted the phenotypic transformation of VSMCs. Overexpression and silencing of nucleolin regulated the expressions of tropoelastin, epiregulin and b-FGF. There was an interaction between tropoelastin mRNA and nucleolin protein, promoting the stability of tropoelastin mRNA and enhancing the expression of tropoelastin at the protein level. Upregulation of nucleolin had an important role in Ang II-induced VSMC phenotypic transformation, and its underlying mechanism may be through interacting with tropoelastin mRNA, leading to its increased stability and protein expression. The findings provide a new perspective into the regulatory mechanism of VSMC phenotypic transformation.

Hyperglycemia induces vascular smooth muscle cell dedifferentiation by suppressing insulin receptor substrate-1–mediated p53/KLF4 complex stabilization

Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.

TGF-\u03b2-SNAIL axis induces M\xfcller glial-mesenchymal transition in the pathogenesis of idiopathic epiretinal membrane

The epithelial-mesenchymal transition (EMT) is a key process in fibrogenic diseases where transdifferentiated myofibroblasts produce excessive amounts of extracellular matrix, resulting in organ dysfunction. Idiopathic epiretinal membrane (iERM) is a vision-threatening disorder characterized by fibrocellular proliferation and contraction on the central retina. Müller glial cells, which regulate retinal physiology and structure, are the major cellular components in the iERM tissue; however, the pathological role of this cell type remains incompletely understood. Here we revealed the involvement of Müller glial-mesenchymal transition (GMT), as an alternative to EMT, in the pathogenesis of iERM lacking epithelial contribution in nature. Of various pro-fibrotic cytokines, transforming growth factor (TGF)-β1 stimulation to human Müller glial cells exclusively increased mRNA and protein levels of several EMT-related molecular markers, together with the transcription factor SNAIL but not SLUG or TWIST. TGF-β1-stimulated Müller cells also exhibited EMT-related cell motility, while reducing the expression of glutamine synthetase (GS), a Müller glial marker. Notably, all of these TGF-β-induced EMT features were reversed by SNAI1 knockdown in Müller cells. iERM patient specimens demonstrated co-immunolocalization of SNAIL with TGF-β1, GS, and smooth muscle protein 22. Our data implicated a critical role of the TGF-β-SNAIL axis in Müller GMT to promote iERM formation.

Teniposide regulates the phenotype switching of vascular smooth muscle cells in a miR-21-dependent manner

The switch of vascular smooth muscle cells (SMCs) from the contractile phenotype to proliferative one can make contributions to atherosclerosis and neointima formation. MiR-21 can prevent the rupture of advanced lesion plaques. We previously reported the protection of DNA topoisomerase II (Topo II) inhibitors against atherosclerosis and vascular calcification. However, it remains unknown if Topo II inhibitors can change SMC phenotypes. Herein, we show that teniposide protected SMC phenotype switching during atherosclerosis by enhancing expression of smooth muscle α-actin (SMA) while reducing osteopontin (OPN) expression in aortic lesion plaques. In vitro, teniposide induced expression of smooth muscle protein 22-α and calponin 1, but inhibited expression of OPN and epiregulin in human aortic SMCs (HASMCs). Moreover, teniposide attenuated platelet derived growth factor-BB-induced HASMC proliferation and migration. Mechanistically, the effect of teniposide on SMC phenotypes was completed, at least in part, by activating miR-21 expression. In addition, teniposide ameliorated ligation-induced carotid artery remodeling in C57BL/6J mice by regulating SMA and OPN expression. Taken together, our study demonstrates that teniposide regulates SMC phenotype switching by upregulating expression of contractile genes in a miR-21-dependent manner, and this function is an important anti-atherogenic mechanism of teniposide.

PO-235 Endurance training increased the expression of myocardin in thoracic aorta of T2DM rats

Objective In recent years, a large number of experimental studies have shown that the proliferation and migration of VSMC are the pathological basis of various vascular diseases, including AS, hypertension, and restenosis after angioplasty. It’s rather remarkable that Phenotypic Modulation of VSMC plays an important role in their proliferation and migration. Myocardin is a key transcription factor for the differentiation of VSMC by far, which can effectively activate the differentiation process of SMC. Many studies have shown that endurance training is an effective way to improve glucose and lipid metabolism. This study attempted to explore the effects of long-term external stress (endurance training) on arterial smooth muscle phenotype modulation and myocardin through secondary vascular disease, in order to provide theoretical support and practical basis for sports therapy in T2DM secondary diseases.&#x0D; Methods Male SD rats were randomly divided into the general feed group (pC) and the high-sugar and high-fat feed group (pD). After 7 weeks, rats in pD were injected a small doses of streptozotocin through abdominal cavity. Those Non-fasting Blood Glucose (NFBG) ≥16.7mmol/L after 72h and with insulin resistance were diagnosed as diabetes. Thereafter, the pC group was randomly divided into a blank control group (C), an endurance training group (E), a diabetes model group (D), and a diabetic+endurance training group (DE). No load platform training was conducted in E&amp;DE group, 5 days per week for 8 weeks. 8 weeks after, BP was measured through left common carotid artery intubation, blood sugar was test by enzyme chemical assay, α-SM-actin, SM-MHC , SM22α,Myocardin and KLF4 were measured through ELISA. &#x0D; Results 1. Compared with C, MCP (carotid blood pressure) of D increased significantly, FBG and FINS decreased significantly, expression of α-SM-actin, SM-MHC, SM22α and Myocardin Significantly dropped, KLF4 rose significantly. Compared with D, the expression of FBG in E was significantly down-regulated, FINS was significantly up-regulated, and the expression of α-SM-actin, SM22α and Myocardin was significantly up-regulated.&#x0D; Conclusions 8 weeks of endurance training significantly increased the expression level of contractile protein in the aorta smooth muscle of diabetic rats, making the smooth muscle phenotype changed from synthetic to contractile type, which effectively inhibited the excessive proliferation and migration of smooth muscle cells.Myocardin is one of the hot spots in the study of vascular differentiation in recent years. This study shows that the role of endurance training in improving plaque formation and lowering blood pressure may be produced by regulating myocardin.

Acute Hepatitis B versus Autoimmune Hepatitis: A Clinical Conundrum

A 41 year-old Hispanic man presented with 10-days of right upper quadrant pain and fatigue. Past medical history was significant for gallstone pancreatitis and papillary thyroid cancer, for which he had cholecystectomy and thyroidectomy. His only medication was levothyroxine. He regularly traveled to Mexico and denied tobacco, alcohol, or illicit drug use. Physical exam revealed normal vitals, jaundice, and right upper quadrant tenderness. Labs revealed ALT of 4136 U/L, AST 3609 U/L, Alk Phos 223 U/L, total bilirubin 17.3 mg/dl, direct bilirubin 15 mg/dl, and INR 2.2. MRI of the abdomen showed peri-portal edema and enlarged peri-portal lymph nodes consistent with acute hepatitis. Hepatitis serologies revealed positive HBV core IgM AB, positive HBV surface Ag, and negative HBV surface Ab, indicating acute HBV infection. HBV viral load was 44,844 IU/ml. Anti-smooth muscle (ASMA) Ab titers were elevated at 1:320. All other labs were normal. Liver biopsy revealed minimal portal inflammation with diffuse hepatocyte balloon degeneration with portal and peri-portal fibrosis (figures 1 and 2). The patient was subsequently started on Tenofovir and prednisone to treat both acute HBV and autoimmune hepatitis (AIH). With this dual therapy approach, his liver abnormalities corrected. At 6-month follow up, Tenofovir and prednisone were discontinued. The patient had repeat liver biopsy revealing portal inflammation with minimal interface activity and stage 1 portal fibrosis consistent with AIH. He was restarted on prednisone and Tenofovir for HBV reactivation prophylaxis. To date, his liver function remains normal. False-positive ASMA Ab in the setting of chronic HBV infection occurs due to homology between HBV-DNA polymerase and smooth muscle proteins. This antigenic mimicry results in positive ASMA assays in patients with chronic HBV. There are only sporadic reports of similar antigenic mimicry occurring in acute HBV infection. In a study of 31 patients with acute HBV infection, 22 had positive ASMA. Two had a titer greater than 1:200 and one had titer greater than 1:400. This leads to uncertainty about the diagnosis of acute HBV infection versus concurrent AIH.This patient had serologic diagnosis of acute HBV infection. However, given his ASMA titer of 1:320, age, and initial non-diagnostic liver biopsy, underlying AIH could not be ignored. To address both etiologies the patient was treated with Tenofovir and prednisone which successfully reversed his acute liver injury.2392_A Figure 1. Diffuse hepatocyte ballooning and degeneration2392_B Figure 2. Portal and periportal fibrosis