Muscle Cell Proliferation Research Articles

Low-power laser irradiation inhibits PDGF-BB-induced migration and proliferation via apoptotic cell death in vascular smooth muscle cells.

Vascular restenosis after injury of blood vessel has been implicated in various responses including apoptosis, migration, and proliferation in vascular smooth muscle cells (VSMCs) stimulated by diverse growth factors underlying platelet-derived growth factor (PDGF). Previous studies evaluated the effects of low-power laser (LPL) irradiation over various wavelength ranges on VSMC events in normal and pathologic states. However, whether VSMC responses are affected by LPL irradiation remains unclear. The purpose of this study is to explore the effects of LPL (green diode laser 532-nm pulsed wave of 300mW at a spot diameter of 1mm) irradiation on the responses, apoptosis, migration, and proliferation of VSMCs. The effect of LPL irradiation was tested on VSMCs through cytotoxicity, proliferation, migration, and apoptotic assays. Aortic ring assay was used to assess the effect of LPL irradiation on aortic sprout outgrowth. Protein expression levels were determined by western blotting. LPL irradiation did not affect VSMC viability but slightly attenuated PDGF-BB-induced proliferation in VSMCs. In addition, LPL irradiation inhibited PDGF-BB-evoked migration of VSMCs. Aortic sprout outgrowth in response to PDGF-BB was diminished in cells treated with LPL. In contrast, LPL irradiation evoked apoptosis in VSMCs in the presence of PDGF-BB. Similarly, activation of caspase-3 and Bax, as well as p38 mitogen-activated protein kinase (MAPK), in VSMCs treated with PDGF-BB was enhanced by exposure to LPL. These findings indicate that LPL irradiation induces vascular apoptosis via p38 MAPK activation and simultaneously inhibits VSMC proliferation and migration in response to PDGF-BB.

Serum Response Factor Promotes Cigarette Smoke-Induced Proliferation of Rat Pulmonary Artery Smooth Muscle Cells Through Upregulating Cyclin D1 Expression

Serum Response Factor Promotes Cigarette Smoke-Induced Proliferation of Rat Pulmonary Artery Smooth Muscle Cells Through Upregulating Cyclin D1 Expression

GW28-e0937 Effect of ANO1 Inhibitor on Proliferation and Migration of Vascular Smooth Muscle Cells in Spontaneously Hypertensive Rats

GW28-e0937 Effect of ANO1 Inhibitor on Proliferation and Migration of Vascular Smooth Muscle Cells in Spontaneously Hypertensive Rats

Effects of short-term starvation on the rhythmic expression of microRNAs in skeletal muscle of goldfish (Carassius auratus )

Molecular oscillators exist in peripheral tissues such as in skeletal muscles and diet is a dominant factor to affect Zeitgeber for peripheral clocks. MicroRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of starvation effects on miRNA rhythmic expression remains limited in teleost. In this study, the circadian expression pattern of miRNAs was investigated in goldfish muscle upon restricting feeding treatment. The data showed that 15 miRNAs exhibited a daily rhythmicity among the 70 miRNAs assayed in muscles of normally fed goldfish. While after 7-day and 15-day fasting treatment, 23 and 18 miRNAs showed circadian rhythmicity in goldfish muscles respectively. Only 4 miRNAs (miR-23a, miR-29, miR-199a-3p and miR-455) exhibited a daily rhythmicity in all three groups of different nutrient treatments. Correlation analysis of the circadian-miRNAs indicates different feeding stimulation could alter the circadian-miRNA components and their expression profile in skeletal muscle upon short-term feed deprivation. These miRNAs may play important roles in regulating circadian expression of genes involved in muscle cell differentiation and growth by nutritional alteration, thus having a potential application in fish aquaculture.

DR1 activation reduces the proliferation of vascular smooth muscle cells by JNK/c-Jun dependent increasing of Prx3.

Vascular smooth muscle cells (VSMCs) proliferation is a key process in atherosclerosis. However, little is known about the underlying mechanisms, leading to a lack of effective therapy. This study was to investigate whether dopamine receptor 1 (DR1) is involved in the VSMCs proliferation and related mechanisms. A7r5 cells were treated with oxidized low-density lipoprotein (ox-LDL, 10, 20, 50, 100, 200µg/mL) in the presence or absence of the SKF38393 (DR1agonist), SCH23390 (DR1antiagonist), SP600125 (JNK inhibitor), PD98059(ERK1/2 inhibitor) or NAC (ROS inhibitor). Cell proliferation and related signaling pathway were evaluated. The expression of DR1 was negatively correlated with increasing of cell proliferation caused by ox-LDL. Cell proliferation and ROS generation in response to ox-LDL were prevented by DR1 agonist or over-expression. The peroxiredoxins protein (Prx1, 2, 3, 5, 6) were increased in A7r5 cells treated with ox-LDL; however, only Prx3 dramatically increased after activation of DR1 compared with ox-LDL group, which is related to activation of JNK/c-Jun pathway. In addition, ERK is associated with the restraining effects of DR1 activation. DR1 activation inhibits VSMCs proliferation primarily by JNK/c-Jun dependent increasing of Prx3, suggesting DR1 a potential target for the prevention of vascular proliferation disease.

A newly synthesized Ligustrazine stilbene derivative inhibits PDGF-BB induced vascular smooth muscle cell phenotypic switch and proliferation via delaying cell cycle progression

Vascular Smooth muscle cells (VSMCs) possess remarkable phenotype plasticity that allows it to rapidly adapt to fluctuating environmental cues, including the period of development and progression of vascular diseases such as atherosclerosis and restenosis subsequent to vein grafting or coronary intervention. Although VSMC phenotypic switch is an attractive target, there is no effective drug so far. Using rat aortic VSMCs, we investigate the effects of Ligustrazine and its synthetic derivatives on platelet-derived growth factor-BB (PDGF-BB) induced proliferation and phenotypic switch by a cell image-based screening of 60 Ligustrazine stilbene derivatives. We showed that one of the Ligustrazine stilbene derivatives TMP-C4a markedly inhibited PDGF-BB-induced VSMCs proliferation in a time and dose-dependent manner, which is more potent than Ligustrazine. Stimulation of contractile VSMCs with PDGF-BB significantly reduced the contractile marker protein α-smooth muscle actin expression and increased the synthetic marker proteins osteopontin expression. However, TMP-C4a effectively reversed this phenotypic switch, which was accompanied by a decreased expression of Matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and cell cycle related proteins, including cyclin D1 and CDK4. In conclusion, the present study showed that a new Ligustrazine stilbene derivative TMP-C4a suppressed PDGF-induced VSMC proliferation and phenotypic switch, indicating that it has a potential to become a promising therapeutic agent for treating VSMC-related atherosclerosis and restenosis.

4803Differential implication of PAR signaling in thrombin generation and proliferation of vascular smooth muscle cells from human thoracic and abdominal aortic aneurysms

4803Differential implication of PAR signaling in thrombin generation and proliferation of vascular smooth muscle cells from human thoracic and abdominal aortic aneurysms

Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study.

Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to drug-induced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting post-transcriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in humans. Thus, it may be important to avoid statin treatment in pathologies characterized by energy deficit and chloride channel malfunction. This study validates the measure of ClC-1 expression as a reliable clinical test for assessing statin-dependent risk of myopathy.

Abstract 321: Protein Tyrosine Phosphatase 1B: a Novel Regulator of Proliferation and Apoptosis in the Development of Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling that leads to an increase in pulmonary arterial pressure resulting in right ventricle failure and death. PAH is driven by pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. Protein Tyrosine Phosphatase 1B (PTP1B), a negative regulator for platelet-derived growth factor (PDGF) and BCL-2, has recently been implicated in PAH in humans. While PDGF and BCL-2 are increased in PAH patients, the pathway for regulating BCL-2 and PDGF is poorly understood. We aim to investigate if PTP1B has a role in proliferation and resistance to apoptosis in PAH in human PACMCs and in the Sugen/Hypoxia/Normoxia (Su/Hx/Nx) PH rat model. Method: Adult male Sprague-Dawley rats were treated with single intraperitoneal dose of SU5416 (20 mg/kg) and kept in Hx for 3 weeks followed by Nx for 2 weeks. Saline treated rats kept in Nx for 5 weeks served as control (n=4/group). RV catheterization was performed terminally for recording RV systolic pressure (RVSP). RV, LV, and interventricular septum (IVS) were isolated for Fulton index (FI, RV/IVS+LV). We analyzed gene expression in lungs via qPCR. Healthy hPASMCs were incubated with a PTP1B inhibitor (Ethyl-3,4-dephostatin) at IC50=0.58ug/ml for 24hrs under Nx conditions and cells were stained with Ki67 to assess proliferation. Results: Su/Hx/Nx rats had severe PH evidenced by a significantly elevated RVSP compared to control (88.97+/- 13.67 vs 28.47+/- 2.22 mmHg, p<0.05). PH rats also showed severely reduced RV function and increased RV hypertrophy (FI= 0.7+/- 0.063 vs 0.274 +/-0.01, p<0.05). PH lungs exhibited severe pulmonary vascular remodeling with excessive growth of the PASMCs. PTP1B was significantly decreased in PH lungs compared to controls (0.158+/-0.0647 vs 1+/-0.06, P<0.05). BCL-2 expression was significantly increased in PAH compared to control (2.01+/-0.162 vs 1 +/-0.1, P<0.01). Inhibition of PTP1B in cultured hPASMCs increased proliferation by ~2 fold as assessed by Ki67 positive cells (n=3). Conclusion: Severe angioproliferative PH in rats is associated with a downregulation of PTP1B and increased expression of BCL-2 and PASMC proliferation.

The inhibitory effect of Isoliquiritigenin on the proliferation of human arterial smooth muscle cell

BackgroundIsoliquiritigenin (ISL) has various biological activities including as antioxidant and an inhibitor of PI3K/AKT signaling pathway. However, both oxidative stress and activated PI3K/AKT signaling contribute to the aberrant proliferation of vascular smooth muscle cells (VSMCs). This study is aimed to explore the effect of ISL on the proliferation of human arterial smooth muscle cells (HASMCs) and to investigate the underlying mechanisms.MethodsBrdU incorporation, cell cycle and reactive oxygen species (ROS) in normal or ISL treated HASMCs were analyzed by flow cytometry. Cell viablity was measured by CCK-8. Protein expression levels were examined by Western blot, and superoxide dismutase (SOD) activity was detected by using commercial kit.ResultsWe observed that ISL could inhibit the proliferation of HASMCs in a dose and time dependent manner. Cell cycle of ISL treated HASMCs arrested mainly in G1/S phase and accompanied with elevated expression of p27 and decreased expression of CyclinD1 and CyclinE. In addition, ISL could down-regulated the expression of p-PI3K and p-AKT, alleviated oxidative stress and enhanced the SOD activity in HASMCs. Furthermore, H2O2 treatment partly improved cell viability and up regulated p-PI3K and p-AKT in HASMCs.ConclusionsTherefore, we concluded that ISL inhibited the proliferation of HASMCs via attenuating oxidative stress and suppressing PI3K/AKT signaling pathway. The inhibitory effect of ISL on PI3K/AKT signaling pathway, at least partly, was mediated by ROS.

Therapeutic effect of mesenchymal stem cell on pulmonary hypertension and its influence on tumor necrosis factor α/nuclear factor of activated T cells

Objective To illustrate that tumor necrosis factor-α(TNF-α)/ nuclear factor of activated T cells pathway is the main function way that mesenchymal stem cells(MSC) inhibit proliferation of pulmonary vascular smooth muscle cells during treating pulmonary hypertension(PH). Methods MSC from human umbilical cord was used to treat PH rat models induced by monocrotaline.Rats were divided into the control group, the PH model group and the MSC group.The general conditions of the rats were observed.Haemodynamics was detected.Pathological sections and immunohistochemistry method were used to detect the lung structure and tissue changes.Changing conditions of TNF-α/NFAT were detected. Results Compared with rats in the PH model group, the general conditions of the MSC group tended to be normal evidently: the right ventricular systolic pressure (RVSP) dropped [(30.37±3.13) mmHg vs.(47.90±3.45) mmHg, 1 mmHg=0.133 kPa], the aortic pressure (MAoP) increased [(115.03±16.01) mmHg vs.(92.78±16.28 mmHg)], the thickening condition of arterial intima-media was evidently relieved [(17.22±1.21)% vs.(31.68±2.26)%], the plasma TNF-α level decreased obviously [(842±76) ng/L vs.(245±24) ng/L], and the lung tissue TNF-α level decreased (0.172±0.024 vs.0.248±0.051), and all the differences were statistically significant (all P<0.05). The activation of pulmonary artery NFATc2 in the MSC treatment group was apparently inhibited. Conclusions MSC therapy may perform the treating effect in PH by inhibiting the over-proliferation of inflammation related pulmonary vascular smooth muscle cells via TNF-α/NFAT pathway. Key words: Mesenchymal stem cell; Pulmonary hypertension; Tumor necrosis factor; Nuclear factor of activated T cells

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products

BackgroundAirway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated.MethodsRat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay.ResultsResults showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 μg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE.ConclusionsOur study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

Downregulation of Talin-1 expression associates with increased proliferation and migration of vascular smooth muscle cells in aortic dissection

BackgroundThis study aimed to assessed whether Talin-1 is involved in the pathogenesis of aortic dissection via regulating vascular smooth muscle cell (VSMC) biological function.MethodsHuman aortic samples were obtained from organ donors who died from nonvascular diseases as normal controls and from patients undergoing surgical repair of thoracic aortic dissection. The expression level and distribution of Talin-1 were detected using westernblot analysis and immunohistochemistry in each sample. We inhibited the expression of Talin-1 via RNA interference in VSMCs. VSMC proliferation was detected by Cell-counting Kit-8 analyses. Scratch test and flow cytometry were used to identify the migration and apoptosis ability. Antibody microarray analysis and qRT-PCR were used to detect some protein and mRNA changes which were induced by Talin-1 downregulation.ResultsTalin-1 was significantly downregulated in the media of aortic dissection samples compared with controls (P < 0.05). Talin-1 knockdown significantly induced VSMC proliferation and migration in vitro. Proteins which involved in cell cycle can be regulated by downregulating Talin-1. Down regulation of Talin-1 can significanly increased the expression of anaphase-promoting complex subunit 2 (APC2) and decreased p19 alternative reading frame (p19ARF), Cullin-3, and beta actin’s expression.ConclusionsTalin-1 induces VSMCs proliferation and migration. It downregulated in aortic dissection, which might play a potential role in the development of aortic dissection.

Effects of small interfering RNA silencing in epidermal growth factor receptor on proliferation and migration of vascular smooth muscle cells

Objective To investigate the effect of silencing epidermal growth factor receptor (EGFR) by small interfering RNA (siRNA) interference on the proliferation and migration of vascular smooth muscle cells (VSMCs), and the related mechanisms. Methods VSMCs of rats were isolated and cultured. VSMCs were infected with EGFR siRNA lentiviral vectors. The cells were divided into siRNA-EGFR group, siRNA-negative control group and blank control group. The cell proliferation abilities were detected by 5’-bromo-2’-deoxyuridine (5-BrdU) method. The cell migration abilities in different transfection groups were examined by Transwell chamber and scratch assays. The expression levels of EGFR, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 genes in different transfection groups were detected using real-time quantitative polymerase chain reaction (Real-time PCR) technology. The expression levels of EGFR, p-ERK, p-JNK and p-p38 proteins in different transfection groups were detected by Western blotting. Results The number of migration cells in the siRNA-EGFR group was 48.5±6.1, which were significantly lower than the siRNA-negative control group and blank control group, which were 86.2±7.2 and 88.3±7.6; the difference was statistically significant (F=37.261, P=0.000). Compared with 0 h, after 24 h, the migration rate of VSMC in the siRNA-EGFR group was (35.7±3.8)%, which was significantly lower than that of siRNA-EGFR group and blank group, which were (74.2±5.7)% and (75.5±6.1)%; the differences were statistically significant (F=40.315, P=0.000). The relative expression levels of ERK, JNK and p38 mRNA in the siRNA-EGFR group (0.53±0.09, 0.47±0.11, 0.41±0.07) were lower than the siRNA-negative control group (0.82±0.12, 0.64±0.10, 0.68±0.09) and blank control group (0.85±0.14, 0.66±0.13, 0.70±0.12), the differences were statistically significant (F=15.391, 12.817, 11.264, P=0.000, 0.000, 0.000). The relative expression levels of p-ERK, p-JNK and p-p38 proteins in the siRNA-EGFR group (0.46±0.09, 0.38±0.06, 0.33±0.06) were lower than the siRNA-negative control group (0.71±0.12, 0.57±0.08, 0.52±0.07) and blank control group (0.73±0.13, 0.56±0.07, 0.54±0.09), the differences were statistically significant (F=20.152, 14.375, 13.843, P=0.000, 0.000, 0.000). Conclusion Specific inhibition of EGFR could effectively reduce proliferation and migration capacities of VSMCs, which might be related to inhibition of mitogen-activated protein kinase signaling pathway. Key words: Vascular smooth muscle cells; Epidermal growth factor receptor; Small interference; Cell proliferation; Cell migration

Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

Abstract 210: Milk Fat Globule Epidermal Growth Factor VIII Mediates Vascular Remodeling by Increasing Inflammation and Smooth Muscle Activation

Vascular remodeling, defined as a change in the geometry of the vessel wall, occurs in the pathological process of vascular diseases, like atherosclerosis, hypertension and restenosis. The resulting neointimal formation is a part of a reparative response including thrombosis, inflammatory cell infiltration, vascular smooth muscle cell (VSMC) proliferation and migration, which lead to the stenosis of blood vessels and the restricted blood flow. Milk fat globule epidermal growth factor VIII (Mfge8), a secreted glycoprotein, is well-characterized for its capacity of assisting the clearance of apoptotic cells in vascular system. Recently, Mfge8 has been identified as a pivot relay between pro-inflammatory signals and activated VSMCs, contributing to intima-media thickening of the vessel wall by promoting VSMC proliferation and migration in aged arteries. We have noted intense Mfge8 expression in the endothelial cells and VSMCs of the carotid artery following ligation injury in mice, suggesting that Mfge8 may regulate the two characteristics of vascular remodeling, inflammatory cell infiltration and VSMC activation, in response to low blood flow. To elucidate the functions of Mfge8 in a flow-induced model of vascular remodeling, a complete carotid ligation was conducted in wild-type (WT) or Mfge8 knockout (KO) mice. Morphometric analysis demonstrated that genetic deletion of Mfge8 in mice reduces carotid intima and media thickening compared to WT mice. Deficiency of Mfge8 prevented VSMC phenotypic modulation, as evidenced by the decreased expression of smooth muscle myosin heavy chain and attenuated cell proliferation in tunica media after ligation injury. VSMCs transfected with SiRNA against Mfge8 migrated slower than in controls as early as 0.5 days post-platelet-derived growth factor (PDGF) stimulation. Further, Mfge8-null mice showed a dramatic decrease in leukocyte infiltration into the vessel wall. Collectively, in a flow-induced model of vascular remodeling, Mfge8 plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating neointimal formation.

Abstract 317: Satellite Cells Influence Collateral Vessel Formation via Paracrine Effects

Satellite cells are myogenic cells that play a critical role in skeletal muscle repair. They serve as stem cells for muscles, remaining dormant in healthy muscle but activating upon injury resulting in increased proliferation and differentiation into myoblasts. Another key aspect of muscle regeneration is reestablishing vascular supply, but the role of satellite cells in this process is not well established though they are known to produce a number of potential paracrine signals. Thus we hypothesized that satellite cells promote vascular growth through paracrine signaling induced by activation following muscle injury or ischemic damage from diseases such as peripheral artery disease. Using a murine model of hind limb ischemia, we showed that satellite cells increased 3.4 fold (p&lt;0.01) in response to ischemia. To determine if satellite cells produce paracrine factors, we used a co-culture system for migration and proliferation. Satellite cells freshly isolated from the ischemic limb led to a 3.5 fold increase in smooth muscle migration (p&lt;0.0001) and a 1.3 fold increase (p&lt;0.01) in smooth muscle proliferation. Additionally, cultured satellite cells increased endothelial cell migration 2.8 fold. These results demonstrate the satellite cells produce paracrine factors which can drive both smooth muscle and endothelial cell migration and proliferation which are required for the development of collateral vessels. To test the potential therapeutic capability of satellite cells, alginate encapsulated satellite cells were delivered in the hind limb ischemic model. Using a whole animal in vivo imager to track luciferase expression of the cells, we found the encapsulated cells were viable for up to 2 weeks. The mice that received satellite cells also had significantly increased perfusion (28%, p&lt;0.05) at 2 weeks as measured by Laser Doppler imaging. In conclusion our studies have shown that satellite cells increase in response to ischemia, produce paracrine factors that increase vascular cell migration in vitro, and lead to functional increases in perfusion in vivo. We believe these results demonstrate the critical role satellite cells play in collateral vessel formation and may be a potential new therapeutic approach for treating peripheral artery disease.

Abstract 428: Role of Thrombospondin-1 in Atherosclerotic Complications Associated With Metabolic Syndrome

Atherosclerosis is the leading cause of increased morbidity and mortality in metabolic syndrome (MetS), a constellation of risk factors that include hyperglycemia and visceral obesity. Individuals with MetS have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a key characteristic of VSMC phenotypic transition. We previously reported that high glucose and high leptin, mimicking hyperglycemia and obesity, independently upregulate a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), expression in VSMCs. The goal of the present study was to investigate the role of TSP-1 in development of atherosclerosis in MetS. We generated a mouse model of combined MetS and atherosclerosis ( KKAy +/- /ApoE -/- ) by crossing obese hyperglycemic agouti yellow KKAy +/- mice with atherosclerotic ApoE -/- mice. Upon weaning, yellow KKAy +/- /ApoE -/- mice and age-matched black KKAy -/- /ApoE -/- littermates were maintained on regular chow diet from 4-18 wks of age. Mice were euthanized after an overnight fasting; blood, aortae and heart were collected for lipid profiling, immunoblotting and atherosclerotic lesion studies. Yellow KKAy +/- /ApoE -/- mice showed significant increase in body weight, blood glucose and plasma lipid levels vs. black KKAy -/- /ApoE -/- littermates. Aortic root morphometry demonstrated increased lesion burden (Oil red O) and neointimal thickening (H &amp; E) in MetS KKAy +/- /ApoE -/- vs. non-MetS KKAy -/- /ApoE -/- mice. Immunohistochemistry further revealed co-localization of α-SMA (SMC marker) and PCNA (proliferation marker) expression in aortic roots of KKAy +/- /ApoE -/- vs. KKAy -/- /ApoE -/- mice. Notably, lesion formation was associated with an increase in TSP-1 expression in aortic vessels. Consistently, TSP-1 and PCNA expression were elevated in aortic SMC (aSMC) primary cultures derived from obese diabetic KKAy +/- mice vs. lean non-diabetic KKAy -/- littermates. Finally, incubation of aSMC with a TSP-1 blocking peptide reduced PCNA and vimentin (SMC synthetic marker) expression coupled with attenuated SMC proliferation in obese diabetic KKAy +/- mice. Together, these results suggest a putative role of TSP-1 in accelerated atherosclerosis and VSMC phenotypic transition in MetS.

Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats.

Structural adaptation of arteries to weightlessness might lower the working ability or even threaten the physical health of astronauts, but the underlying mechanism is unclear. Acid sphingomyelinase (ASM) catalyzes ceramide (Cer) generation controlling arterial remodeling through multiple signaling pathways. In the present study, we aimed to investigate the contribution of ASM/Cer to the changes of common carotid artery intima-media thickness (CIMT) induced by simulated weightlessness. Hindlimb-unloaded tail-suspended (HU) rats were used to simulate the effect of weightlessness. Morphology of the carotid artery (CA) was examined by hematoxylin-eosin staining. Protein content of ASM or proliferating cell nuclear antigen (PCNA) was detected by Western blot. Cer level was measured by immunohistochemistry analysis. Apoptosis events were observed by transferase-mediated dUTP nick end labeling (TUNEL) staining. During 4weeks of tail suspension, CIMT was increased gradually in HU but not in their synchronous control rats (P<0.05). Correspondingly, the CA of HU rats had a lower apoptosis and higher proliferation of vascular smooth muscle cells (VSMCs). As compared to the control, both ASM protein expression and Cer content were reduced significantly in CA of HU rats (P<0.05), incubation of which with permeable Cer reversed the changes in apoptosis and proliferation substantially. Furthermore, when the ASM protein content as well as Cer level in CA of control rats was diminished by using an ASM inhibitor, an increase of CIMT along with reduced apoptosis and enhanced proliferation of VSMCs was found. Our results suggest that by controlling the balance between apoptosis and proliferation, ASM/Cer plays an important role in the regulation of CIMT during simulated weightlessness.

Airway smooth muscle cells from ovalbumin-sensitized mice show increased proliferative response to TGFβ1 due to upregulation of Smad3 and TGFβRII

ABSTRACTObjective: This study aimed to elucidate the role of Transforming growth factor (TGF)-β1 signaling in the proliferation of airway smooth muscle cells (ASMCs). Background: TGF-β1 is an important cytokine in airway remodeling in asthma. However, results of studies focusing on the effect of TGFβ1 on proliferation of ASMCs are controversial. Methods: An allergic model that mimics airway remodeling in chronic asthma was established and primary ASMCs were cultured. Cell proliferation was detected by viable cell counting and Cell Counting Kit (CCK)-8 analysis. Expression and phosphorylation of Smad3, type 1 TGFβ receptor (TGFβRI), type 2 TGFβ receptor (TGFβRII), extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), C-Jun N-terminal kinase (JNK) and AKT were detected by western blot. siRNAs were used to knock down Smad3 and TGFβRII. Results: Smad3 and TGFβRII were up-regulated in primary ASMCs isolated from ovalbumin (OVA)-sensitized mice as compared with ASMCs isolated from unsensitized control mice, which persisted for at least four passages. TGFβ1 stimulated proliferation of ASMCs isolated from OVA-sensitized mice, which was inhibited by specific siRNA targeting Smad3 or TGFβRII. However ASMCs from control mice showed no proliferative response to TGFβ1. TGFβ1-induced proliferation of ASMCs from OVA-sensitized mice was markedly attenuated by PD-98059, a specific ERK1/2 inhibitor. TGFβ1 induced ERK1/2 phosphorylation within 15 minute, which was partially blocked by specific inhibitor of Smad3 (SIS3). Conclusions: ASMCs isolated from OVA-sensitized mice showed hyper-proliferation upon TGFβ1 stimulation. This might have been associated with up-regulated Smad3 and TGFβRII and mediated by ERK1/2 downstream to Smad3.