Muscle Cell Proliferation Research Articles

Histopathological and proteomic analyses identify integrin-β1 as a potential mediator of phlebosclerosis in uremic patients.

Patients with uremia have an excessive mortality from cardiovascular disease (CVD). Arterial remodeling is mainly responsible for uremia-induced CVD and has been well studied, yet venous remodeling is poorly understood. Here we investigate the histopathology and proteomic profiles of venous remodeling in uremic patients. Forearm cephalic veins were isolated from nine uremic patients during surgeries for arteriovenous fistula, and from nine healthy controls when applying surgical debridement. Hematoxylin-eosin, Masson's trichrome, von Kossa, and immunohistochemistry (IHC) against proliferating cell nuclear antigen were stained for histopathology. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was executed to explore the proteome of the veins. The core regulatory protein was validated by western blot, IHC, and immunofluorescence. Phlebosclerosis, characterized by intimal rarefaction and medial thickening with disordered proliferation of vascular smooth muscle cells (VSMCs), was the prominent pathological manifestation of peripheral veins in uremic patients, while inflammatory cell infiltration, atherosclerosis or calcification were not obviously detected. iTRAQ analysis showed that 350 proteins were significantly changed in phlebosclerosis of uremic patients compared with healthy controls, of which integrin-β1 (ITGβ1) exhibited the strongest regulatory ability by intermolecular interaction network analysis. The enhanced ITGβ1 expression was mainly co-expressed with the disordered proliferation of VSMCs while a little with vascular endothelial cells in the forearm cephalic veins of uremic patients. Phlebosclerosis is the prominent pathological manifestation in peripheral veins of uremic patients. This pathological alteration mainly attributes to the disordered proliferation of VSMCs, which is potentially mediated by ITGβ1.

Inhibitory effect of irisin on vasular smooth muscle cell inflammation and proliferation by regulating autophagy through adenosine monophosphate-actived protein kinase/mammalian target of rapamycin pathway

Objective To explore the underlining mechanisms of irisin inhibiting inflammation and proliferation of vascular smooth muscle cells (VSMCs). Methods The cultured primary rat aortic VSMCs were pre-incubated with irisin and then treated with platelet derived growth factor-BB (PDGF-BB). Enzyme linked immunosorbent assay (ELISA) and methyl thiazol tetrazolium (MTT) assay were used to measure the VSMCs inflammation and proliferation, respectively. The protein expression and phosphorylation of adenosine monophosphate-actived protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway related molecule AMPK/p-AMPK (Thr172), acetyl-CoA carboxylase (ACC)/phosphorylated (p)-ACC (Thr308), mTOR/p-mTOR (Ser2448), ribosomal protein S6 kinase (p70SK6)/p-70SK6 (Thr389), and autophay related molecule microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3Ⅱ and nucleoporin p62 (P62) were detected by Western blotting. Results After treatment with PDGF-BB, VSMCs proliferation dramatically increased, and the inflammatory cytokines IL-1β (37.9±8.5 vs. 10.8±2.9, t=18.620, P<0.01), IL-6 (57.4±14.1 vs. 32.4±8.7, t=6.250, P<0.01), MCP-1 (158.3±39.1 vs. 57.2±12.5, t=14.280, P<0.01), ICAM-1 (371.8±77.3 vs. 160.4±39.8, t=10.850, P<0.01), tumor necrosis factor-α (TNF-α) (16.3±3.8 vs. 7.8±2.4, t=7.390, P<0.01) in the cell supernatant were significantly up-regulated. The protein expression levels of AMPK (0.39±0.11 vs. 0.83±0.18, t=14.250, P<0.01), p-AMPK (0.25±0.03 vs. 0.49±0.12, t=12.370, P<0.01), ACC (0.54±0.16 vs. 1.07±0.24, t=10.670, P<0.01), p-ACC (0.38±0.07 vs. 0.97±0.30, t=18.130, P<0.01), LC3Ⅰ (0.67±0.21 vs. 0.67±0.21, t=5.780, P<0.01) and LC3Ⅱ (0.42±0.13 vs. 0.93±0.38, t=6.820, P<0.01) were down-regulated, mTOR (2.57±0.53 vs. 1.23±0.25, t=21.350, P<0.01), and p-mTOR (1.35±0.0.37 vs. 0.57±0.12, t=8.870, P<0.01), p70SK6 (1.85±0.39 vs. 0.81±0.19, t=7.170, P<0.01), p-70SK6 (0.69±0.22 vs. 0.38±0.11, t=9.260, P<0.01) and P62 (1.24±0.38 vs. 0.58±0.14, t=5.270, P<0.01) were up-regulated. However, pre-incubation with irisin and then stimulation with PDGF-BB could significantly decrease VSMCs proliferation [(122.14±9.42)%, t=4.970, P<0.01], and significantly down-regulate the inflammatory cytokines IL-1β (17.38±4.10, t=6.740, P<0.01), IL-6 (34.9±8.9, t=2.760, P<0.05), MCP-1 (47.28±9.20, t=16.270, P<0.01), ICAM-1 (169.2±55.8, t=7.780, P<0.01) and TNF-α (6.9±2.0, t=8.180, P<0.01) in the cell supernatant. The protein expression levels of AMPK (0.72±0.26, t=2.670, P<0.05), p-AMPK (0.46±0.13, t=3.280, P<0.05), ACC (0.95±0.28, t=5.870, P<0.01), p-ACC (0.78±0.25, t=3.140, P<0.01), LC3Ⅰ (1.34±0.35, t=1.960, P<0.05) and LC3Ⅱ (1.03±0.19, t=7.260, P<0.01) were up-regulated, and mTOR (1.16±0.33, t=6.910, P<0.01), p-mTOR (0.66±0.18, t=6.830, P<0.01), p70SK6 (1.12±0.34, t=2.360, P<0.05), p-70SK6 (0.37±0.12, t=3.120, P<0.01) and P62 (0.68±0.18, t=3.360, P<0.05) were down-regulated. Conclusion Irisin could inhibit the inflammation and proliferation of VSMCs possibly by up-regulating autophagy through activation of the AMPK/mTOR signaling pathway. Key words: Irisin; Adenosine monophosphate-actived protein kinase/mammalian target of rapamycin signaling pathway; Autophagy; Vascualar smooth muscle cell proliferation; Vein graft stenosis

2079-P: Association of Serum FAM19A5 with Metabolic and Vascular Risk Factors in Human Subjects With or Without Type 2 Diabetes

Introduction: A recent in-vitro and in-vivo experimental study has revealed that family with sequence similarity 19 [chemokine (C-C motif)-like] member A5 (FAM19A5), a novel secreted adipokine has inhibitory effects on proliferation and migration of vascular smooth muscle cells, and neointima formation of injured arteries. However, the relationship between serum FAM19A5 level and cardio-metabolic risks has not been evaluated in humans. For the first time, we investigated the association between serum FAM19A5 concentration with various metabolic and vascular risk factors in human subjects with or without type 2 diabetes. Methods: Circulating FAM19A5 concentration was measured in 223 individuals (45 without diabetes and 178 with type 2 diabetes), and the association of FAM19A5 level with cardio-metabolic risk factors including body composition, plasma glucose, glycated hemoglobin (HbA1c), and atherosclerosis was explored. Results: Serum FAM19A5 (pg/ml) was higher in patients with type 2 diabetes [172.70 (116.19, 286.42)] compared with that in individuals without diabetes [92.09 (70.32, 147.24)] (p&amp;lt;0.001). Advance in serum FAM19A5 tertile was associated with a trend of increase in waist hip ratio (WHR), fasting plasma glucose (FPG), and mean brachial-ankle pulse wave velocity (baPWV) values. Serum FAM19A5 was positively correlated with waist circumference, WHR, alanine aminotransferase, FPG, HbA1c, and mean baPWV values. Multiple stepwise regression analysis identified WHR, low density lipoprotein cholesterol, and baPWV as determining factors for log-transformed serum FAM19A5 concentration (R2=0.0689). Discussion: A novel adipokine FAM19A5 was closely related to various metabolic and vascular risk factors in human subjects, suggesting its potential as a biomarker of cardio-metabolic disease in humans. Disclosure Y. Lee: None. J.A. Kim: None. E. Roh: None. S. Hong: None. K. Choi: None. S. Baik: None. H. Yoo: None.

622-P: Mechanism of Lin28a Promoting Vascular Smooth Muscle Cell Proliferation and Migration in Restenosis

Background: Restenosis (RS) after percutaneous transluminal angioplasty severely affects the curative effect of patients with lower extremity peripheral arterial disease. Our previous research indicated that abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) was the key factor in the RS plaque formation. The process of Lin28a regulating cell proliferation demonstrates similarities with the proliferation of VSMCs in RS. Here, we aim to explore the specific role of Lin28a in RS. Methods: To distinguish from atherosclerotic lesions, RS and atherosclerosis (AS) models were simultaneously established in type 2 diabetic rats. Lin28a level in VSMCs was detected by immunofluorescence double staining. The changes of VSMCs were determined when modulating Lin28a in vivo and vitro and downstream mechanism was further explored. Results: Thirty-two folds higher level of Lin28a was found in VSMCs in RS compared with AS (P&amp;lt;0.05). Overexpression of Lin28a significantly promoted proliferation and migration of VSMCs in vitro. Let7 was identified as the direct target of Lin28a; screening for Let7 subtypes by in vivo and vitro showed that Let7c, let7d, let7g, miR98 were negatively regulated by Lin28a in VSMCs. Among them, downregulation of Let7c markedly suppressed the VSMCs’ proliferation and migration accompanied by upregulation of Lin28a expression; while these effects were alleviated by overexpression of Let7c. Conclusion: The present study demonstrated that Lin28a is critically involved in the progression of RS via facilitating the proliferation and migration of VSMCs. A double negative feedback loop is often present in Let7 and Lin28a, thus Let7 subtypes were detected and Let7c could be the direct downstream of Lin28a in regulating VSMCs in RS. Combined with the fact that Lin28a was also regulated by Let7c, we inferred a feedback loop could be formed between Let7c and Lin28a. Above all, the interaction in Lin28a and Let7c may contribute to rapid formation of RS. Disclosure Q. Zhang: None. X. Zhou: None. X. Shen: None. S. Jiang: None. T. Xie: None. Z. Zou: None. C. Xu: None. R. Zhang: None. Y. Cui: None. P. Gong: None. J. Dong: None. L. Liao: None. Funding National Natural Science Foundation of China (81670757, 81570742, 81770822, 81800732); Shandong Provincial Natural Science Foundation of China (ZR2017LH025); Shandong Provincial Medicine and Health Science and Technology Development Program (2017WS461)

476-P: Pemafibrate, a PPAR Alpha Agonist, Attenuates Neointima Formation after Vascular Injury in Nondiabetic and Diabetic Mice

Recently, the prevention of cardiovascular events has become one of most important purpose of diabetes care. We have previously reported that GLP-1 receptor agonist exendin-4 (BBRC 2011) and DPP-4 inhibitor linagliptin (Cardiovasc Diabetol 2014) attenuate neiontima formation after vascular injury. On the other hand, PPAR agonist is also reported its vascular protective effect. Then, we examined whether selective PPAR alpha agonist pemafibrate could attenuate neointima formation after vascular injury and vascular smooth muscle cell (VSMC) proliferation. We performed endothelial denudation injury in mice treated with high fat diet (HFD) or normal chow. Orally administered pemafibrate significantly attenuated neointima formation after vascular injury in both HFD and normal chow mice. Interestingly, pemafibrate increased serum FGF-21 concentration and decreased serum insulin concentration in HFD mice. In addition, body weight was slightly but significantly decreased by pemafibrate in HFD mice. In vitro experiments, pemafibrate attenuated VSMC proliferation in growth curve in a dose dependent manner, but not another PPAR alpha agonist bezafibrate. This suggested that the attenuation of VSMC proliferation by pemafibrate could be the drug effect, not class effect of PPAR alpha agonist. BrdU assay revealed that pemafibrate inhibited DNA synthesis in VSMC dose-dependently. In cell cycle distribution, G1 to S phase entry was significantly inhibited by pemafibrate in flow cytometry analysis. However, apoptosis was not induced by pemafibrate in VSMC in TUNEL assay. These data suggested that pemafibrate attenuates neiontima formation after vascular injury and VSMC proliferation inhibiting cell cycle progression. Disclosure T. Horikawa: None. T. Nomiyama: None. H. Takahashi: None. T. Kawanami: None. Y. Hamaguchi: None. T. Tanaka: None. T. Yanase: None.

Structural changes in the wall of the abdominal aorta in modeling of long-term cannabinoid receptors CB1 blockade in laboratory animals

The purpose of the work is to detect the effect of long-term cannabinoid receptors CB1 blockade on the abdominal aorta wall structure in experimental rats. Materials and methods. The study was performed on two groups of 100-day-old male Wistar rats. The first group was made up of intact animals. The second group consisted of animals that were administrated with rimonabant hydrochloride solution (10 mg/kg -1 of body weight) per os with drinking water per day. The experiment duration was 10 days, and then the abdominal aorta of each animal was isolated for the histological measurements. Computer morphometry, the study of the subendothelial layer thickness with the internal elastic lamina and media, the ratio of the lumen diameter to the vessel wall were conducted in the abdominal aorta wall structure assessment. Results. It has been established that long-term cannabinoid CB1 receptors blockade in 100-day-old male Wistar rats resulted in morphological changes in all layers of the abdominal aorta wall that manifested in alterations of endothelial cells structure and their desquamation in the intima. The inner elastic lamina became thin and discontinuous. The thickness of the media was not uniform and there was a decrease in the number of elastic laminae as well as an increase in the number of chaotically oriented smooth muscle cells. Changes in adventitia manifested in swelling and loosely packed fibers with their increased dispersion. The number of collapsed vasa vasorum was decreased to single and hypertrophy of the nerve ganglia was observed. There was a thinning of the internal lamina and a decreased percentage of the lumen and vessel components of the wall at the expense of other tissues. Conclusions . The study results indicate that the long-term cannabinoid CB1 receptors blockade is a factor that damages endothelium of the abdominal aorta and leads to a decrease in elastin and proliferation of smooth muscle cells in the media, as well as a decrease in the percentage of the lumen and wall of the abdominal aorta at the expense of other tissues.

Changes in expression of Talin1 and F-actin during serum-induced proliferation of pulmonary artery smooth muscle cells of rats with hepatopulmonary syndrome

Objective To evaluate the changes in the expression of Talin1 and F-actin during serum-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) of rats with hepatopulmonary syndrome (HPS). Methods Twenty healthy male Sprague-Dawley rats, weighing 220-250 g, were used for producing HPS by chronic ligation of the common bile duct.Blood samples from the abdominal aorta were collected to prepare serum.Primarily cultured PASMCs obtained from rats were seed in 6- or 96-well plates and divided into 2 groups using a random number table method: control group (group C) and HPS group (group HPS), with 24 wells in each group (for 6-well plates) or with 30 wells in each group (for 96-well plates). In C and HPS groups, normal rat serum or HPS rat serum were added, respectively, with the final concentration of 5%.At 24, 48 and 72 h of incubation, the expression of Talin1, F-actin and G-actin was determined by Western blot, the F-actin/G-actin ratio was calculated, and the proliferation of PASMCs was measured by 3H-TdR incorporation and CCK-8 assays. Results Compared with group C, the proliferation of PASMCs was significantly enhanced, the expression of Talin1 was up-regulated, and the F-actin/G-actin ratio was increased in group HPS (P<0.05). The proliferation of PASMCs was gradually enhanced, the expression of Talin1 was gradually up-regulated, and the F-actin/G-actin ratio was gradually increased with the prolonged incubation time in group HPS (P<0.05). Conclusion The mechanism by which the HPS rat serum induces proliferation of PASMCs may be related to up-regulating the expression of Talin1 and F-actin. Key words: Hepatopulmonary syndrome; Myocytes, smooth muscle; Pulmonary artery; Cell proliferation; Talin; Actins

An electrospun citric acid modified polyvinyl alcohol scaffold for vascular tissue engineering

The main aim of this study is to fabricate an electrospun citric acid modified polyvinyl alcohol polyester that is biodegradable with non-toxic by-products and can be used for the culture of vascular smooth muscle cells. In this study, we have optimized the conditions for the electrospinning process of this polyester. The fibre morphology was studied by scanning electron microscopy which indicated that the fibre diameter was optimum at a range of 200 to 700 µm at 5% concentration and flow rate of 0.3 mL/h. The membranes were characterized for the change in structural aspects at the molecular level. The results showed development of more crystalline domains on electrospinning. The surface characteristics were also explored. Cell culture studies confirmed that the electrospun scaffold supported the attachment and proliferation of smooth muscle cells, which was evident from the cell proliferation assay. Hence, the electrospun polyester scaffolds are non-toxic and biocompatible with vascular smooth muscle cells, and find promising potential as scaffolds for vascular tissue engineering.

The effect of endoplasmic reticulum stress on detrusor muscle apoptosis in diabetic cystopathy

Objective To observe the effect of Endoplasmic Reticulum Stress (ERS) on detrusor muscle apoptosis in Streptozotocin (STZ)-induced diabetic rats. Methods At 4, 8, 12, and 16 weeks after induction of diabetic rat models, the sections of detrusor muscle tissue were made and were stained with Van Gieson solution, the collagen fibers of each group were observed and their proportions were calculated; the sub-cellular ultrastructure was observed by Transmission Electron Microscopy (TEM). Proliferating Cell Nuclear Antigen (PCNA) assay kit and Strept Avidin-Biotin Complex (SABC) staining were used for the evaluation of Detrusor Smooth Muscle (DSM) cell proliferation. Moreover, the expression of three hallmarks of ERS-associated apoptosis, including Glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and cysteinyl aspartate-specific protease (Caspase)-12, was detected by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results The proportion of collagen fibers showed a significant climbing trend [(13.38±2.65)%, (18.14±1.93)%, (19.71±3.05)% vs. (7.86±0.72)%, P<0.05] since DM8w group, except DM4w group [(8.02±0.43)%]. With the time prolonged, the swelled and fused cisternaes started to show; the mild particle fusion and degranulation also could be seen in the zone of ER; the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed. Compared with the control group, the increased PCNA-positive percentages [(20.34±4.55)%, (46.88±7.12)%, (39.78±1.93)%, (22.15±5.64)% vs. (16.26±2.43)%, P<0.05] and apoptosis indexes [(17.12±2.78)%, (28.12±4.09)%, (28.12±4.09)%, (43.39±6.82)% vs. (6.32±1.23)%, P<0.05] can be seen in model groups. Elevated expression of GRP78, CHOP, and Caspase-12 at both protein and mRNA levels in a time-dependent fashion were detected (P<0.05). Conclusion Morphological and ultrastructural damages to the DSM as well as the cell proliferation and apoptosis indicate the time-dependent alterations. The occurrence of ERS may be involved in the development of diabetic cystopathy (DCP) and may contribute to the diabetic detrusor muscle apoptosis. Key words: Endoplasmic reticulum stress; Diabetic cystopathy; Detrusor; Apoptosis

Role of interleukin 1 in the development of atherosclerosis

Atherosclerosis is now considered as chronic inflammatory vascular disease connected to «pathological» activation of innate and adaptive immunity, characterized by lipid deposition, leukocyte infiltration and proliferation of vascular smooth muscle cells. Subclinical (low grade) inflammation plays fundamental role at all stages of atherosclerotic process progression and determines cardiovascular catastrophes development and mortality. Proinflammatory cytokines including interleukin (IL) 1, IL6, tumor necrosis factor α (TNFα), IL17, IL18, IL27, IL33, IL37 tightly interacting within cytokine network occupy an important place among numerous mediators participating in immunopathogenesis of atherosclerosis and rheumatoid arthritis. IL1β playing an important role in the development of many acute and chronic immunoinflammatory diseases attracts particular attention. IL1β significance in the development of atherosclerosis is determined by many mechanisms including procoagulant activity, enhancement of monocytes and leucocytes adhesion to vascular endothelium, vascular smooth muscle cells growth and others. Fundamental role of inflammation in the development of atherosclerosis is well proved in investigations of anti-atherosclerotic effect of canakinumab. Randomized placebo-controlled trial CANTOS (Canakinumab ANti-inflammatory Thrombosis Otcomes Study) assessing efficacy of canakinumab as new tool for secondary prophylaxis cardiovascular complications in general population of patients with severe atherosclerotic vascular damage. CANTOS results in combination with accumulated in rheumatology data on cardiovascular effects of anti-inflammatory drugs are of great importance for personification of approach to secondary prophylaxis of caused by atherosclerosis cardiovascular complications. They also contribute to the development of inflammatory theory of atherosclerosis pathogenesis in the whole.

Identification and Characterization of Protein Phosphatase Methylesterase 1 (Ppme1) in Skeletal Muscle

Skeletal muscle atrophy results from a range of physiological conditions, including immobilization, denervation, glucocorticoid treatment, cancer, and aging. To provide a better understanding of the pathway of skeletal muscle atrophy, a previous study conducted a microarray analysis of muscle from mice undergoing neurogenic atrophy in response to denervation as well as dexamethasone exposure. The data generated from this microarray identified a suite of several hundred predicted genes that displayed differential expression under conditions of muscle wasting, including protein phosphatase methylesterase‐1 (Ppme1). The Ppme1 gene was observed to be transcriptionally upregulated in response to neurogenic skeletal muscle atrophy (i.e. denervation). To confirm that Ppme1 is expressed in muscle, qPCR was performed using RNA isolated from cultured muscle cells at different stages of proliferation and differentiation and expression was found to be relatively constant in both proliferating and differentiated cells. In addition, Ppme1 protein levels were analyzed by Western blot analysis and found to be expressed at a constant level at different time points throughout muscle cell proliferation and differentiation. Additionally, the cDNA of Ppme1 was fused in frame with GFP and subcellular localization was analyzed by confocal fluorescent microscopy in proliferating myoblasts. To assess the transcriptional activity of the Ppme1 gene, fragments of the promoter were cloned, fused to a reporter gene, transfected into C2C12 cells, and found to be highly transcriptionally active. Interestingly, the Ppme1 reporter gene constructs were not significantly affected by ectopic expression of myogenic regulatory facts such as MyoD1 and myogenin. Finally, mouse myoblast cells treated with an inhibitor of Ppme1 displayed reduced AP‐1 reporter activity as well as delayed muscle cell differentiation. The results presented here help contribute new insights into the molecular mechanisms of muscle atrophy and may provide a better understanding of the genome‐wide transcriptional changes and cellular events that occur during skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leukotriene B4 Contributes to Development of Cardiac Allograft Vasculopathy

Purpose Cardiac allograft vasculopathy (CAV) is a highly prevalent vaso-occlusive disease that is a leading cause of graft failure and mortality after heart transplantation. While the pathogenesis of CAV remains incompletely understood, histologic evidence suggests that macrophages, which constitute an important part of the innate immune response, may play an important role. Prior studies in pulmonary hypertension have shown that macrophage-derived leukotriene B4 (LTB4) induces proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We hypothesized that LTB4 plays a similar role in the development of CAV. Methods Human studies: We performed immunofluorescence staining of sections from human CAV+ and CAV- coronary arteries with CD68 (macrophage marker) and 5-LO (5-lipoxygenase, LTB4-producing enzyme), and measured serial LTB4 plasma levels in 14 patients with angiographic CAV and 14 matched CAV- controls. Rat model: We performed aortic transplants in a rat CAV model (Fischer into Lewis orthotopic aortic transplants, n=11) with allogeneic transplants as controls (n=6), and performed CD68 and 5-LO staining of aortic cross-sections of mice with and without aortic intimal hyperplasia. We then treated rat allogeneic transplants with bestatin (protease inhibitor that blocks LTB4 production) to determine whether LTB4 inhibition can prevent the intimal proliferation seen in this rat CAV model. Results Immunofluorescence staining showed that 5-LO co-localized with CD68+ macrophages in the intimal layer of CAV+ human arterioles and in allogeneic rat aortic transplant models. Plasma LTB4 levels were higher in CAV+ cases compared to CAV- controls (n=14 per group, p Conclusion Macrophage-derived leukotriene B4 may contribute to development of CAV. Blockade of LTB4 synthesis has the potential to prevent CAV development after heart transplantation.

Abstract P035: Habitual Aerobic Exercise-Induced Decrease in Circulating C1q Level is Associated With Reduction of Cardiovascular Diseases Risks in the Elderly

Introduction: Habitual aerobic exercise leads to reduction of arterial wall thickness and arterial stiffness with aging. Recently, C1q has been identified as a novel myokine, and the expression of muscle C1q mRNA increases with aging. Additionally, in vivo and in vitro studies, C1q induces proliferation of vascular smooth muscle cells. However, it remains unclear whether the secretion of myokines, C1q, TNF-alpha and IL-6 are related to the changes in cardiovascular disease risk by aging and regular exercise. PURPOSE: This study aimed to clarify whether serum C1q, TNF-alpha and IL-6 levels are associated with the changes in cardiovascular disease risks with aging and regular aerobic exercise. Methods: In a cross-sectional study, 127 healthy male and female subjects (18-81 years) were enrolled in this study. Subjects were divided into two groups; young (&lt;40 year) and middle-aged and older (≥40 year) groups. Serum C1q, TNF-alpha, IL-6 levels were determined by ELISA. Risks of cardiovascular disease were estimated by using carotid-femoral pulse wave velocity (cfPWV) and common carotid intima-media thickness (ccIMT). Additionally, in an intervention study, thirteen older male subjects were randomly divided into two groups: aerobic training group (AT: n=6) and a sedentary control group (Con: n=7). subjects in the AT group completed 8 wk of aerobic exercise training (60-70% peak oxygen uptake for 45 min, 3 days/wk). Results: In the cross-sectional study, serum C1q, TNF-alpha and IL-6 levels, cfPWV and ccIMT were significantly higher in middle-aged and older group as compared with young group (respectively p&lt;0.05). Positive correlations between cfPWV and serum C1q (r=0.41, p&lt;0.05), TNF-alpha (r=0.28, p&lt;0.05) or IL-6 (r=0.26, p&lt;0.05) levels were seen. Additionally, ccIMT was positively correlated with only serum C1q (r=0.67, p&lt;0.05). After adjusting for TNF-alpha and IL-6, the association between the serum C1q and cfPWV (ß=0.44, p&lt;0.05) or ccIMT (ß=0.83, p&lt;0.05) remained statistically significant. Additionally, in the intervention study, cfPWV and serum C1q level significantly decreased after AT intervention (each P&lt;0.05). Furthermore, the AT-induced change in serum C1q level was positively correlated with AT-induced change in cfPWV (r=0.65, P&lt;0.05) and ccIMT (r=0.59, P&lt;0.05). Conclusion: These results suggest that circulating C1q level may reflect the changes of arterial stiffness and thickness with aging and regular exercise, and the reduction of serum C1q level in response to regular aerobic exercise may contribute to the reduction in cardiovascular disease risks in the elderly.

Calcium-Sensing Receptor Arbitrates Hypoxia-Induced Proliferation of Pulmonary Artery Smooth Muscle Cells via the G Protein-PLC-IP3 Pathway.

Pulmonary arterial hypertension (PAH), also known as broilers ascites syndrome, is characterized by hypoxia, pulmonary artery pressure, and right heart failure. However, less information is available about the molecular mechanisms of PAH. We evaluated the mediation of calcium-sensing receptor by inducing hypoxia for the possible proliferation of pulmonary artery smooth muscle cells via the G protein pathway. For this purpose, we used an in vitro trial of chicken cell culture and confirmed our results by using immunohistochemistry, immunofluorescence staining, quantitative real-time polymerase chain reaction assay, and Western blotting analysis. Our results showed that the mRNA and protein expression levels of calcium-sensing receptor (CaSR) were significantly upregulated in cells when co-incubated with CaCl2. However, the levels of mRNA and protein were obviously decreased when supplemented with blocking agents (NiCl2, 2-APB, and D609). Furthermore, the experimentally induced hypoxia also upregulated the expression of CaSR gene as compared to CaSR gene expression in control cells. Together, these results indicate that hypoxia plays an important role in the expression of CaSR gene in pulmonary artery smooth muscle cells and reveals new targets for the CaSR excited hypothesis to prevent and control PAH in chickens.

The relationship of arterial hypertension with the disturbance of glucose tolerance

Arterial hypertension and impaired glucose tolerance are in many ways interdependent and often precede a more pronounced pathology, the metabolic syndrome. In this regard, it is an actual problem of modern medicine, as the timely diagnosis and treatment of this combination can reduce the disability and mortality in this cohort of patients, prolonging their life and preserving their working capacity. Clinical features of hypertension with concomitant metabolic disorders are: frequent formation of refractory hypertension, early damage to target organs-development of LVH (left ventricular hypertrophy), quickly leading to myocardial dysfunction, renal hyper filtration and MAU, reduction of aortic and arteries elasticity. It has been established that impairment of insulin's ability to suppress glucose production in the liver and/or stimulate glucose uptake by peripheral tissues can underlie a decrease in insulin sensitivity. Since in healthy people 75–80% of glucose is utilized by skeletal muscles, it is more likely that its main cause of IR is impaired insulin stimulated glucose utilization [8]. A certain value in the development of hypertrophy and proliferation of smooth muscle cells and fibroblasts of the vascular wall in hypertension has a stimulating effect of insulin on collagen synthesis. It has been established that the thickness of the intima media complex of the carotid artery and the number of desquamated endotheliocytes circulating in the blood of patients with hypertension with IGT closely correlate with elevated insulin levels [5]. Thus, hypertension and IGT are largely interdependent conditions, significantly affect health and often precede a more pronounced pathology-the metabolic syndrome. Timely diagnosis and correction of this combination can reduce disability and premature death, prolong and improve their quality of life, and maintain their efficiency.

Oct-1 Mediates ACTH-Induced Proliferation of Vascular Smooth Muscle Cells

Oct-1 Mediates ACTH-Induced Proliferation of Vascular Smooth Muscle Cells

Hypoxia-induced ARHGAP26 deficiency inhibits the proliferation and migration of human ductus arteriosus smooth muscle cell through activating RhoA-ROCK-PTEN pathway.

The Rho family plays crucial roles in O2 -induced vasoconstriction, cell proliferation, and migration. Rho GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein for the small GTPases of the Rho family. Our previous studies have demonstrated that ARHGAP26 expression was significantly downregulated in patent human ductus arteriosus (DA) tissue. However, its role underlying the maintenance of DA patency is unclear. In this study, patent (fetal) and constricted (newborn) mouse DA tissues were harvested to confirm the differences in the levels of expression of ARHGAP26. Human DA smooth muscle cells (DASMCs) were isolated and cultured in vitro and used to test the function of ARHGAP26. The expression of ARHGAP26 was significantly lower in patent (fetal) than constricted (newborn) mouse DA. ARHGAP26-knocked-down human DASMCs showed reduced proliferation and migration, which are both crucial to anatomic closure of DA. Moreover, after culturing under hypoxic conditions, the expression of ARHGAP26 in human DASMCs was significantly lower and hypoxia-induced ARHGAP26 deficiency activated the phosphorylation level of phosphatase and tensin homolog (PTEN) in DASMCs by mediating the activity of RhoA and RhoA-associated kinase 1 (ROCK1). Use of Y27632, an inhibitor of ROCK which further reduces the phospholipid activity of PTEN can reverse the inhibitory effect of PTEN on the proliferation and migration of human DASMCs. This provides insight into the molecular regulation of the RhoA-ROCK-PTEN pathway in DA smooth muscle cells, which may be a suitable therapeutic target or diagnostic biomarker for perinatal DA tone management.

Treatment of horses with asthma syndrome

Treatment of horses with asthma syndrome

Fer1l6 is essential for the development of vertebrate muscle tissue in zebrafish.

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein–based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.

GLP-1 attenuates Ang II-induced proliferation and migration in rat aorta smooth muscle cells via inhibition of the RhoA/ROCK2 signaling pathway.

Glucagon-like peptide 1 (GLP-1), a neuroendocrine hormone produced by the gastrointestinal tract, plays a significant role in blood glucose regulation; drugs derived from GLP-1 are currently used for the treatment of type 2 diabetes. In addition to regulating glucose homeostasis, the protective effects of GLP-1 on the cardiovascular system are also of interest. However, the vascular protective mechanisms of GLP-1 remain unclear. The present study was designed to evaluate the role of GLP-1 in the proliferation and migration of vascular smooth muscle cells, and the underlying mechanisms. In this study, proliferation, migration, cyclin D1 expression, and phosphorylation of MLC, as well as RhoA and Rho-associated coiled-coil forming protein kinase 2 (ROCK2) expression, were increased in rat aorta smooth muscle cells (RASMCs) following incubation with angiotensin II (Ang II). These effects were significantly attenuated by GLP-1, forskolin (a cAMP activator) and Y-27632 (a ROCK2 inhibitor). However, H89 (a PKA inhibitor) inhibited the action of GLP-1, both in terms of inhibition of RASMC proliferation and migration, and RHOA/ROCK2 expression. These results indicate that GLP-1 inhibits Ang II-induced RASMC proliferation and migration via the cAMP/PKA/RhoA/ROCK2 signaling pathway. Our data suggest that GLP-1 should be considered for use in the clinical treatment of cardiovascular diseases, in addition to its current use in the treatment of diabetes mellitus.