Activity Of Smooth Muscle Cells Research Articles

Suppression of Lysophosphatidylcholine-Induced Human Aortic Smooth Muscle Cell Calcification by Protein Kinase A Inhibition.

Lysophosphatidylcholine (lysoPtdCho) is produced mainly by the phospholipase A2-dependent hydrolysis of phosphatidylcholine (PtdCho) and can induce inflammatory activation and osteogenic gene expression in vascular smooth muscle cells. However, the mechanisms mediating these processes have not been fully elucidated. In this study, we investigated whether inhibition of protein kinase A (PKA) signaling suppressed lysoPtdCho-induced calcification of human aortic smooth muscle cells (HASMC). Calcium levels and alkaline phosphatase activity were significantly increased in HASMC treated with lysoPtdCho, but not PtdCho, compared with those in phosphate-buffered saline-treated HASMC. However, the addition of a PKA inhibitor (H-89) or PKA siRNA blocked lysoPtdCho-induced HASMC calcification. These results showed that lysoPtdCho could activate PKA-mediated HASMC calcification and that PKA may be a therapeutic target for lysoPtdCho-mediated vascular smooth muscle cell calcification.

Role of Carbon Monoxide in the Mechanisms of Action of Extracellular ATP on Contractile Activity of Vascular Smooth Muscle Cells.

We studied the role of carbon monoxide (CO) in the effect of P2X and P2Y receptor agonist ATP on the tone of rat aorta segments with intact endothelium. ATP (1-1000 μM) and P2X receptor agonist α,β-MeATP (100 μM) relaxed segments precontracted with phenylephrine (10 μM), while UTP (100-1000 μM) increased the amplitude of phenylephrine-induced contraction. The relaxing effect of ATP was enhanced by CORM II (100 μM), NO synthase inhibitor L-NAME, and guanylate cyclase inhibitor ODQ and attenuated by ZnPP IX (100 μM). The constrictive effect of UTP was weakened by CORM II (100 μM), but was not changed by ZnPP IX (100 μM). ZnPP IX (100 μM) weakened the relaxation response to α,β-MeATP. Thus, ATP involves the CO-dependent signaling cascade through P2X receptors.

The Role of 4-hydroxynonenal for Activation of Vascular Smooth Muscle Cell

The Role of 4-hydroxynonenal for Activation of Vascular Smooth Muscle Cell

Suv39h1 downregulation inhibits neointimal hyperplasia after vascular injury

Background and aimsNeointimal hyperplasia resulting from pathological vascular smooth muscle cells (VSMCs) activation is a common pathophysiological basis for numerous proliferative vascular diseases, such as restenosis. Suv39h1, an important transcription suppressor, may be involved in this process. Herein, we investigated the role of Suv39h1 in pathological intimal hyperplasia and its possible mechanisms in vitro and in vivo. MethodsAn adenovirus vector for Suv39h1 overexpression and a lentiviral vector for its downregulation were constructed and used to transfect cultured VSMCs in vitro. The functional changes in VSMCs stimulated by angiotensin II (Ang II) were observed and the possible mechanism was investigated. Additionally, rat carotid arteries with balloon injury were locally transfected with these viral vectors and changes in neointima formation, proliferating cell nuclear antigen (Pcna) expression and collagen deposition were examined. ResultsUpon Ang II stimulation, the expression of Suv39h1 and inhibitor of DNA binding 3 (Id3) was significantly increased. Suv39h1 downregulation inhibited Ang II-stimulated migration and proliferation of VSMCs, antagonized the production of Id3 and promoted p21 and p27Kip1 expression. In contrast, Suv39h1 overexpression had the opposite effects. Suv39h1 regulated the transcription of p21 and p27Kip1 by controlling H3K9me3 in the proximal promoter regions. Consistent with the VSMCs results, Suv39h1 and Id3 expression was significantly increased in blood vessels after balloon injury. Suv39h1 downregulation inhibited intimal hyperplasia, and attenuated Pcna expression and collagen synthesis in the intima, while Suv39h1 overexpression had the opposite effects. ConclusionsSuv39h1 downregulation effectively inhibited neointimal hyperplasia after vascular injury.

Ultrastructure study of the transgenic REN2 rat aorta – part 2: media, external elastic lamina, and adventitia

Background The renin-angiotensin-aldosterone system (RAAS) plays an important role in the development and progression of vascular stiffness, hypertension and accelerated atherosclerosis, which are associated with the metabolic syndrome (MetS) and type 2 diabetes mellitus. In addition to the intima, RAAS plays an important role in vascular media and adventitial remodeling. Methods Descending thoracic aortas of young male transgenic heterozygous (mRen2) 27 (Ren2) rats were utilized for ultrastructural study. This lean model of hypertension, insulin resistance, and oxidative stress harbors the mouse renin gene and is known to have increased aortic tissue levels of angiotensin II, angiotensin type 1 receptors, and elevated plasma aldosterone levels. Results Ultrastructural observations substantiate known and novel findings in the tunica media, internal and external elastic lamina, and tunica adventitia, which includes: increased media collagen - proteoglycan matrix expansion, increased secretory and proliferative activity and migration of vascular smooth muscle cells (VSMCs) into a newly developing subendothelial neointima, increased VSMC caveolae, mitochondria degeneration, apoptosis; and lipid retention at the elastin lamellar interface. Openings in the external elastic lamina allow pericyte-to-VSMC contacts. The tunica adventitia exhibits stromal pericyte hyperplasia with actively synthetic phenotype and pericyte-pericyte connections. Conclusion While these studies only represent a single snapshot in time, they provide an evaluation of early abnormal ultrastructural vascular remodeling in Ren-2 models of the conduit-elastic thoracic aorta.

Nerve growth factor receptor expression marks activated human perivascular adventitial fibroblasts in distinct perturbed states

Abstract Vascular alterations are common in human autoimmune skin diseases, including systemic sclerosis and discoid lupus. Much is known about endothelial cell, pericyte and vascular smooth muscle cell activation. In contrast, the adventitial layer of the vascular unit is less well explored. This compartment serves as a reservoir for leukocytes and mesenchymal stem cells, and appears to play important sentinel and remodeling roles. We recently demonstrated that the compartment, as well as the perivascular adventitial fibroblasts, expand in the skin of systemic sclerosis, discoid lupus, and dermatitis patients. Vascular cell adhesion molecule-1 (VCAM) display marked activated adventitial fibroblasts in discoid lupus and dermatitis, and correlated with perivascular T cell infiltration. Here we demonstrate elevated expression of Nerve growth factor receptor (NGFR) by perivascular adventitial fibroblasts in the skin of systemic sclerosis patients but not control subjects. Adventitial NGFR expression occurred without elevated VCAM indicating a distinct activation state. Conversion to the NGFR+ state was also observed in the absence of large, perivascular leukocyte infiltrates. In systemic sclerosis, NGFR was more common than VCAM on perivascular adventitial fibroblasts. Conversely, in discoid lupus lesions, VCAM was more common than NGFR on adventitial fibroblasts suggesting two distinct activation programs. Similar adventitial NGFR staining was seen in both pathological (e.g. hypertrophic and keloid) and normal reparative skin wounds. Our data indicate that elevated NGFR expression can identify an activated perivascular adventitial fibroblast in disease.

VasoTracker: An open access pressure myography platform

Pressure myography has long been the gold standard technique for assessing the function of blood vessels ex vivo. The technique utilizes diameter measurement of isolated, pressurized arteries to report on the functional activity of smooth muscle and/or endothelial cells. However, despite the capability of the pressure myograph to assess vascular function, its widespread adoption has been hampered by the cost and inflexibility of the available commercial systems.Here, we present a complete, open source platform for performing pressure myography, based on Arduino hardware and μManager software. VasoTracker includes all components that would be expected of a commercial pressure myograph system (heated vessel chamber, temperature controller, pressure monitor, computer, camera, microscope, and acquisition/analysis software), but can be built for a fraction of the cost ($4000 for VasoTracker versus >$40,000 for commercial alternatives). As part of the open source movement, the design of VasoTracker is extensively documented on our website (http://www.vasotracker.com/), where we provide full descriptions of the required hardware and software, and provide parts lists and instructions to enable anyone to build their own pressure myograph. The fully integrated system enables the investigation of artery function using a wide array of protocols, offering the ability to study myogenic responses and pressure‐diameter relationships as well as smooth muscle contraction and endothelium‐dependent dilation.The VasoTracker software is a data acquisition and blood vessel diameter measurement interface that, although designed to complement the VasoTracker hardware, can be used to acquire images of blood vessels using the large array of cameras and microscopes supported by μManager. As such, the software can be used to provide existing imaging set‐ups with the capability to measure blood vessel diameter online. The software acquires, displays and records video images, and automatically measures the diameter of mounted blood vessels. In addition, temperature and pressure data can be recorded and user interventions (such as the addition of pharmacological agents) can be logged. Each parameter is conveniently displayed, and blood vessel diameter is graphed in real time.In releasing VasoTracker, we provide the vascular community with an inexpensive, transparent and fully customizable tool for studying blood vessel function. We believe that the low entry barrier and the potential for future developments of VasoTracker provides researchers with a platform to expand and enhance the pressure myography technique with tailored vascular reactivity experiments.Support or Funding InformationThis work was funded by the Wellcome Trust (204682/Z/16/Z; 202924/Z/16/Z) and the British Heart Foundation (PG/16/54/32230; PG16/82/32439), whose support is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of Gut Bacterial Translocation Ameliorates Vascular Calcification through Inhibiting Toll-Like Receptor 9-Mediated BMP-2 Expression.

Aims Vascular calcification (VC) is a primary risk factor for cardiovascular mortality in chronic renal failure (CRF) patients; thus, effective therapeutic targets are urgently needed to be explored. Here, we identified the role of intestinal bacterial translocation in CRF-related VC. Methods and Results Antibiotic supplementation by oral gavage significantly suppressed intestinal bacterial translocation, CRF-related VC, and aortic osteogenic gene and Toll-like receptor (TLR) gene expression in CRF rats. Furthermore, TLR4 and TLR9 activation in vascular smooth muscle cells (VSMCs) aggravated inorganic phosphate- (Pi-) induced calcification. TLR9 inhibition, but not TLR4 inhibition, by both a pharmacological inhibitor and genetic methods could significantly reduce CRF rats' serum or CRF-induced VC. Interestingly, bone morphogenic protein-2 (BMP-2) levels were increased in the aorta and sera from CRF rats. Increased BMP-2 levels were also observed in VSMCs treated with TLR9 agonist, which was blocked by NF-κB inhibition. Both siRNA knockdown of BMP-2 and NF-κB inhibitor significantly blocked TLR9 agonist-induced VSMC calcification. Conclusions Gut bacterial translocation inhibited by oral antibiotic significantly reduces CRF-related VC through inhibition of TLR9/NF-κB/BMP-2 signaling.

Notch3 signaling activation in smooth muscle cells promotes extrauterine growth restriction-induced pulmonary hypertension

Background and aimsEarly postnatal life is a critical developmental period that affects health of the whole life. Extrauterine growth restriction (EUGR) causes cardiovascular development problems and diseases, including pulmonary arterial hypertension (PAH). PAH is characterized by proliferation, migration, and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs). However, the role of PASMCs in EUGR has not been studied. Thus, we hypothesized that PASMCs dysfunction played a role in EUGR-induced pulmonary hypertension. Methods and resultsHere we identified that postnatal nutritional restriction-induced EUGR rats exhibited an elevated mean pulmonary arterial pressure and vascular remodeling at 12 weeks old. PASMCs of EUGR rats showed increased cell proliferation and migration features. In EUGR-induced PAH rats, Notch3 signaling was activated. Relative mRNA and protein expression levels of Notch3 intracellular domain (Notch3 ICD), and Notch target gene Hey1 in PASMCs were upregulated. We further demonstrated that pharmacological inhibition of Notch3 activity by using a γ-secretase inhibitor DAPT, which blocked the cleavage of Notch proteins to ICD peptides, could effectively inhibit PASMC proliferation. Specifically knocked down of Notch3 in rat PASMCs by shRNA restored the abnormal PASMC phenotype in vitro. We found that administration of Notch signaling inhibitor DAPT could successfully reduce mean pulmonary arterial pressure in EUGR rats. ConclusionsThe present study demonstrated that upregulation of Notch3 signaling in PASMCs was crucial for the development of EUGR-induced PAH. Blocking Notch3-Hey1 signaling pathway in PASMCs provides a potential therapeutic target for PAH.

Mir-455-3p-1 represses FGF7 expression to inhibit pulmonary arterial hypertension through inhibiting the RAS/ERK signaling pathway

ObjectiveTo analyze the effects of miR-455-3p-1 and its possible mechanisms in pulmonary arterial hypertension (PAH). MethodsA microarray assay was used to examine the expressed genes between normal and PAH. The expressed genes in PAH was assessed by qRT-PCR. The targeted interaction between miRNAs and FGF7 was confirmed using a dual luciferase reporter assay. A CCK-8 assay and cell count were used to analyze the pulmonary artery smooth muscle cells (PASMCs) activity and proliferation level, respectively. Apoptotic PASMCs were detected by flow cytometry. In addition, the mRNA and protein expression levels of RAS/ERK signaling pathway were determined by qRT-PCR and a Western blot assay, respectively. A PAH rat model was used to identify the effects of miR-455-3p-1 in vivo. ResultsFGF7 was upregulated in PAH. MiR-455-3p-1 was downregulated in PAH. MiR-455-3p-1 targeted FGF7. MiR-455-3p-1 decreased the expression of FGF7. Moreover, the effect of FGF7 on PASMCs was suppressed by miR-455-3p-1. MiR-455-3p-1 upregulation was associated with reduced mRNA and protein levels of core RAS/ERK signal genes, suggesting the inhibition of the RAS/ERK pathway. Furthermore, miR-455-3p-1 upregulation improved the RVSP, mPAP, ratio of RV/LV + S, CO and RV function of PAH rat model in vivo. ConclusionOur findings illustrate a role for miR-455-3p-1 in modulating FGF7-RAS/ERK signaling and suggest that an agomir of miR-455-3p-1 could inhibit the proliferation of PASMCs and mitigate PAH in vivo.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteinsand a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

VasoTracker, a Low-Cost and Open Source Pressure Myograph System for Vascular Physiology.

Pressure myography, one of the most commonly used techniques in vascular research, measures the diameter of isolated, pressurized arteries to assess the functional activity of smooth muscle and endothelial cells. Despite the widespread adoption of this technique for assessing vascular function, there are only a small number of commercial systems and these are expensive. Here, we introduce a complete, open source pressure myograph system and analysis software, VasoTracker, that can be set-up for approximately 10% of the cost of commercial alternatives. We report on the development of VasoTracker and demonstrate its ability to assess various components of vascular reactivity. A unique feature of the VasoTracker platform is the publicly accessible website (http://www.vasotracker.com/) that documents how to assemble and use this affordable, adaptable, and expandable pressure myograph.

Thromboresistant semi-IPN hydrogel coating: Towards improvement of the hemocompatibility/biocompatibility of metallic stent implants

Here we developed a semi-interpenetrating network (IPN) hydrogel obtained by free radical polymerization to fabricate a coated stent with the aim of incorporating a natural topography present in the human body to improve biological activity. The method involves sandwiching a bare metal stent in the semi-IPN hydrogel via solution cast molding. The bio-functionality of the membrane could be tuned by incorporating Polydopamine into the matrix, and also the mechanical property was optimized by choosing an adequate concentration of acrylamide. The coating containing polydopamine hydrogel showed good mechanical stability under continuous flow condition, as demonstrated by crimping and deployment into a catheter without damage. Stent polymer bonding was enhanced via polydopamine incorporation in the matrix. The non-thrombogenicity of the coating containing hydrogel was confirmed through dynamic hemocompatibility studies in vitro. Vascular simulations, including other biomechanical performance, like durability testing, radial strength, and recoil, were demonstrated. The dopamine containing hydrogel membrane (DCHM) was found to promote cell material interaction due to the ability of the catechol to bind protein and induce HUVECs cytoplasmic spreading, proliferation, and migration, with reduced smooth muscle cell (SMCs) activity. SMCs inhibition correlated well with the amount of incorporated catechol in the matrix. Our results show that this material used as coated stent could be more effective in suppressing platelet aggregation with improved haemocompatibility/biocompatibility for faster re-endothelialization than bare metal stent (BMS).

Circ_Lrp6, a Circular RNA Enriched in Vascular Smooth Muscle Cells, Acts as a Sponge Regulating miRNA-145 Function.

microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Previous work has established a role for miRNAs in regulating vascular smooth muscle cell (VSMC) activity. Whether circular RNAs are involved in the modulation of miRNA activity in VSMCs is unknown. We aimed to identify circular RNAs interacting with miRNAs enriched in VSMCs and modulating the cells' activity. RNA sequencing and bioinformatics identified several circular RNAs enriched in VSMCs; however, only one, possessing multiple putative binding sites for miR-145, was highly conserved between mouse and man. This circular RNA gemmed from alternative splicing of Lrp6 (lipoprotein receptor 6), a gene highly expressed in vessels and implicated in vascular pathologies and was thus named circ_Lrp6. Its role as a miR-145 sponge was confirmed by determining reciprocal interaction through RNA immunoprecipitation, stimulated emission depletion microscopy, and competitive luciferase assays; functional inhibition of miR-145 was assessed by measuring expression of the target genes ITGβ8 (integrin-β8), FASCIN (fascin actin-bundling protein 1), KLF4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). The interaction was preferentially localized to P-bodies, sites of mRNA degradation. Using loss- and gain-of-function approaches, we found that circ_Lrp6 hindered miR-145-mediated regulation of VSMC migration, proliferation, and differentiation. Differential expression of miR-145 and circ_Lrp6 in murine and human vascular diseases suggests that the ratio of circ_Lrp6 bound to miR-145 versus unbound could play a role in vascular pathogenesis. Viral delivery of circ_Lrp6 shRNA prevented intimal hyperplasia in mouse carotids. circ_Lrp6 is an intracellular modulator and a natural sponge for miR-145, counterbalancing the functions of the miRNA in VSMCs.

P116 PERIOSTIN PROMOTES ENDOPLASMIC RETICULUM STRESS-MEDIATED FIBROSIS IN CROHN’S DISEASE

Fibrostenosis complicates Crohn’s disease in the 30-50% of patients with a Montreal B2 phenotype. Current medical therapies can delay but no prevent critical fibrostenosis and the need for surgery. Transforming growth factor-β1 (TGF-β1)-induced excess extracellular matrix (ECM) protein production in ileal subepithelial myofibroblasts (SEMF) are a major structural component of fibrosis. The role of endoplasmic reticulum (ER) stress in the pathogenesis of Crohn’s disease has been delineated in epithelial and lamina propria mononuclear cells and more recently we have delineated the role of ER stress in SEMF of fibrostenotic ileum. ER stress marker and chaperone GRP78 can bind to type I collagen in a stimulated extracellular environment, which suggests the potential link between ER stress and fibrosis formation. The secreted matricellular protein periostin, typically promotes fibrosis in a TGF-β1-dependent fashion and interacts with plasma membrane integrin family members, including αVβ3. We have previously shown αVβ3 integrin is crucial for LAP-TGF-β1 activation in ileal mesenchymal cells, SEMF and smooth muscle cells. Our RNAseq data identified TGFB1, ITGAV, ITGB3 COL1A1 and all as significantly upregulated (25-200 fold) in SEMF of strictured ileum. However, the role of periostin in SEMF during the ER stress-mediated component of intestinal fibrosis in Crohn’s disease remains unknown. To determine the role of a periostin during the development of intestinal fibrosis in fibrostenotic Crohn’s disease, SEMF were isolated from the patients with each Crohn’s disease phenotype and place them into primary culture or 3D culture. Periostin expression was determined by qRT-PCR and immunoblot analysis. Binding of periostin to αvβ3 integrin was determined by proximity ligation hybridization assay. Knockdown of periostin in cells was accomplished with siRNA. Active and total TGF-β1 protein levels were measured by ELISA. Our data show that periostin expression and production are increased in SEMF from strictured ileum compared to normal ileum in the same patient. IL-6 promotes the interaction between secreted periostin and integrin αvβ3 on the SEMF cell membrane. This was associated with increased levels of active TGF-β1 and increased the resulting production of collagen 1α1. Induction of ER stress in SEMF from normal ileum with tunicamycin elicited increased expression of periostin and αVβ3. The ER stress-induced increase in αVβ3, however, was inhibited by siRNA-mediated knockdown of periostin. The increase in active TGF-β1 seen following ER stress was also decreased in the SEMF in which periostin was knocked down. Our data suggest ER stress-induced periostin links increased αVβ3 integrin and αVβ3-dependent activation of latent TGF-β1 and the resulting increase in collagen production (Figure 1).

P116 PERIOSTIN PROMOTES ENDOPLASMIC RETICULUM STRESS-MEDIATED FIBROSIS IN CROHN’S DISEASE

Fibrostenosis complicates Crohn’s disease in the 30-50% of patients with a Montreal B2 phenotype. Current medical therapies can delay but no prevent critical fibrostenosis and the need for surgery. Transforming growth factor-β1 (TGF-β1)-induced excess extracellular matrix (ECM) protein production in ileal subepithelial myofibroblasts (SEMF) are a major structural component of fibrosis. The role of endoplasmic reticulum (ER) stress in the pathogenesis of Crohn’s disease has been delineated in epithelial and lamina propria mononuclear cells and more recently we have delineated the role of ER stress in SEMF of fibrostenotic ileum. ER stress marker and chaperone GRP78 can bind to type I collagen in a stimulated extracellular environment, which suggests the potential link between ER stress and fibrosis formation. The secreted matricellular protein periostin, typically promotes fibrosis in a TGF-β1-dependent fashion and interacts with plasma membrane integrin family members, including αVβ3. We have previously shown αVβ3 integrin is crucial for LAP-TGF-β1 activation in ileal mesenchymal cells, SEMF and smooth muscle cells. Our RNAseq data identified TGFB1, ITGAV, ITGB3 COL1A1 and all as significantly upregulated (25-200 fold) in SEMF of strictured ileum. However, the role of periostin in SEMF during the ER stress-mediated component of intestinal fibrosis in Crohn’s disease remains unknown. To determine the role of a periostin during the development of intestinal fibrosis in fibrostenotic Crohn’s disease, SEMF were isolated from the patients with each Crohn’s disease phenotype and place them into primary culture or 3D culture. Periostin expression was determined by qRT-PCR and immunoblot analysis. Binding of periostin to αvβ3 integrin was determined by proximity ligation hybridization assay. Knockdown of periostin in cells was accomplished with siRNA. Active and total TGF-β1 protein levels were measured by ELISA. Our data show that periostin expression and production are increased in SEMF from strictured ileum compared to normal ileum in the same patient. IL-6 promotes the interaction between secreted periostin and integrin αvβ3 on the SEMF cell membrane. This was associated with increased levels of active TGF-β1 and increased the resulting production of collagen 1α1. Induction of ER stress in SEMF from normal ileum with tunicamycin elicited increased expression of periostin and αVβ3. The ER stress-induced increase in αVβ3, however, was inhibited by siRNA-mediated knockdown of periostin. The increase in active TGF-β1 seen following ER stress was also decreased in the SEMF in which periostin was knocked down. Our data suggest ER stress-induced periostin links increased αVβ3 integrin and αVβ3-dependent activation of latent TGF-β1 and the resulting increase in collagen production (Figure 1).

Functional pathways associated with human carotid atheroma: a proteomics analysis.

Advances in large-scale analysis are becoming very useful in understanding health and disease. Here, we used high-throughput mass spectrometry to identify differentially expressed proteins between early and advanced lesions. Carotid endarterectomy samples were collected and dissected into early and advanced atherosclerotic lesion portions. Proteins were extracted and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Differentially expressed proteins were identified and verified using multiple reaction monitoring (MRM), on which advanced systems biology and enrichment analyses were performed. The identified proteins were further compared to the transcriptomic data of 32 paired samples obtained from early and advanced atherosclerotic lesions. A total of 95 proteins were upregulated, and 117 proteins were downregulated in advanced lesions compared to early atherosclerotic lesions (p < 0.05). The upregulated proteins were associated with proatherogenic processes, whereas downregulated proteins were involved in extracellular matrix organization and vascular smooth muscle cytoskeleton. Many of the identified proteins were linked to various "upstream regulators", among which TGFβ had the highest connections. Specifically, a total of 19 genes were commonly upregulated, and 30 genes were downregulated at the mRNA and protein levels. These genes were involved in vascular smooth muscle cell activity, for which enriched transcription factors were identified. This study deciphers altered pathways in atherosclerosis and identifies upstream regulators that could be candidate targets for treatment.

Oral health in patients with systemic sclerosis

The aim of this study was to conduct literature review about oral manifestations of scleroderma. Systemic sclerosis is a multisystem autoimmune disorder characterized by widespread fibrosis, vascular alterations and inflammation. Systemic sclerosis mainly affects people in age from 30 to 50 years, but the onset of disease can occur in any age. The disturbance of microcirculation with the activation and proliferation of endothelium and smooth muscle cells plays an important role in pathogenesis of sclerodrma. These changes lead to sclerosis and fibrosis of various part in human body. Orofacial manifestations of scleroderma include: reduced mouth opening, widening of periodontal ligament, teleangiectasia, bone lesions. Systemic scleroderma is often accompanied by Sjogren's syndrome. Oral manifestations of Sjogren's syndrome are recurrent parotitis, angular cheilitis, xerostomia and multiple caries. Sjogren's syndrome leads to negative impact on patients health and mental status. Orofacial manifestations of systemic sclerosis are still poorly known, that is why more researches should be made to improve dental treatment of patients with systemic sclerosis.

Vascular Effects of H2S-Donors: Fluorimetric Detection of H2S Generation and Ion Channel Activation in Human Aortic Smooth Muscle Cells.

Hydrogen sulfide (H2S) evokes vascular effects through several mechanisms including in wide part the activation of some ion channels such as ATP-sensitive potassium (KATP) channels and voltage-gated Kv7 potassium channels. Electrophysiological methods are very accurate, but they require high expertise and high specialized equipment. A more manageable fluorimetric technique which allows to record the membrane potential variations by the employment of an anionic bis-oxonol dye named DiBac4(3) with the administration of different blockers of several potassium channels could be useful to discover the targets of H2S-induced vascular hyperpolarization. Coupled with this technique, a fluorimetric detection (by the use of WSP-1 dye) of H2S generation in human vascular smooth muscle cells after H2S-donor administration could confirm the ability of these molecules to evoke the hyperpolarizing effect through the H2S release.

Hydroxysafflor yellow A (HSYA) targets the platelet-activating factor (PAF) receptor and inhibits human bronchial smooth muscle activation induced by PAF.

Hydroxysafflor yellow A (HSYA) is the main active ingredient of edible plant safflower. HSYA has demonstrated anti-inflammatory effects. The inflammatory response is the key mechanism responsible for asthma, and the pro-inflammatory platelet-activating factor (PAF) is known to play a role in the pathology of bronchial asthma. In this study, we stimulated human bronchial smooth muscle cells (HBSMCs) with PAF and examined the effects of HSYA on the resulting asthma-related process. PAF stimulation induced HBSMC activation, induced proliferation, increased expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor-α, and activated asthma-related signaling pathways. All these effects were significantly inhibited by treatment with HSYA (9, 27, 81 μmol L-1). The effects of HSYA were prevented by the addition of a PAF receptor (PAFR) antagonist or by PAFR gene silencing with small interfering RNA. These results suggest that HSYA may inhibit PAF-induced activation of HBSMCs by targeting the PAFR. Overall, these findings provide evidence that HSYA can be applied as a potential therapeutic agent in the treatment of bronchial asthma.