Response Of Smooth Muscle Cells Research Articles

Syndecan-1 in mechanosensing of nanotopological cues in engineered materials

The cells of the vascular system are highly sensitive to biophysical cues from their local cellular microenvironment. To engineer improved materials for vascular devices and delivery of cell therapies, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment. Syndecans are heparan sulfate proteoglycans (HSPGs) that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are well poised to serve as mechanosensors of the cellular microenvironment. Nanotopological cues have become recognized as major regulators of cell growth, migration and phenotype. We hypothesized that syndecan-1 could serve as a mechanosensor for nanotopological cues and can mediate the responsiveness of vascular smooth muscle cells to nanoengineered materials. We created engineered substrates made of polyurethane acrylate with nanogrooves using ultraviolet-assisted capillary force lithography. We cultured vascular smooth muscle cells with knockout of syndecan-1 on engineered substrates with varying compliance and nanotopology. We found that knockout of syndecan-1 reduced alignment of vascular smooth muscle cells to the nanogrooves under inflammatory treatments. In addition, we found that loss of syndecan-1 increased nuclear localization of Yap/Taz and phospho-Smad2/3 in response to nanogrooves. Syndecan-1 knockout vascular smooth muscle cells also had elevated levels of Rho-associated protein kinase-1 (Rock1), leading to increased cell stiffness and an enhanced contractile state in the cells. Together, our findings support that syndecan-1 knockout leads to alterations in mechanosensing of nanotopographical cues through alterations of in rho-associated signaling pathways, cell mechanics and mediators of the Hippo and TGF-β signaling pathways.

DJ-1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-like Cells in Response to Sphingosylphosphorylcholine.

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox-sensitive molecules including a multifunctional protein DJ-1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)-stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α-smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC-stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ-1 in hMSCs in response to SPC. The increased abundance of oxidized DJ-1 in SPC-stimulated hMSCs was validated by immunoblot analysis. The SPC-induced increase in the expression of α-SMA was stronger in DJ-1-knockdown hMSCs than in control cells. Moreover, the expression of α-SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ-1-overexpressing hMSCs. Exogenous H2 O2 mimicked the responses induced by SPC treatment. These results indicate that the ROS-related DJ-1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.

A novel patterned magnetic micropillar array substrate for analysis of cellular mechanical responses

Traction forces generated at cellular focal adhesions (FAs) play an essential role in regulating various cellular functions. These forces (1–100 nN) can be measured by observing the local displacement of a flexible substrate upon which cells have been plated. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces. A tool capable of applying a force to FAs independently, by actively moving the micropillars, should become a powerful tool to delineate the cellular mechanotransduction mechanisms. Here, we developed a patterned magnetic micropillar array PDMS substrate that can be used for the mechanical stimulation of cellular FAs and the measurement of associated traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Iron particles were embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field, which also controlled their location on the substrate. Applying a magnetic field of 0.3 T bent the pillars by ∼4 µm and allowed transfer of external forces to the actin cytoskeleton through FAs formed on the pillar top. Using this approach, we investigated the traction force changes in cultured aortic smooth muscle cells (SMCs) after local compressive stimuli to release cell pretension. The mechanical responses of SMCs were roughly classified into two types: almost a half of the cells showed a little decrease of traction force at each pillar following compressive stimulation, although cell area increased significantly; and the rest showed the opposite, with increased forces and a simultaneous decrease in area. The traction forces of SMCs fluctuated markedly during the local compression. The root mean square of traction forces significantly increased during the compression, and returned to the baseline level after its release. These results suggest that the fluctuation of forces may be caused by active reorganization of the actin cytoskeleton and/or its dynamic interaction with myosin molecules. Thus, our magnetic micropillar substrate would be useful in investigating the mechanotransduction mechanisms of cells.

Incrementing the Frequency of Dynamic Strain on SMC-Cellularised Collagen-Based Scaffolds Affects Extracellular Matrix Remodeling and Mechanical Properties.

Notwithstanding the efforts injected in vascular tissue engineering in the past 30 years, the clinical translation of engineered artery constructs is far from being successful. One common approach to improve artery regeneration is the use of cyclic mechanical stimuli to guide cellular remodeling. However, there is a lack of information on the effect of cyclic strain on cells within a 3D environment. To this end, this work explored the effect of gradual increase in stimulation frequency on the response of human umbilical artery smooth muscle cells (HUASMCs) embedded in a 3D collagen matrix. The results demonstrate that, with an applied strain of 5%, the gradual increase of frequency from 0.1 to 1 Hz improved collagen remodeling by HUASMCs compared to samples constantly stimulated at 1 Hz. The expression of collagen, elastin and matrix metalloproteinase-2 (MMP-2) genes was similar at 7 days for gradual and 1 Hz samples which showed lower amounts than static counterparts. Interestingly the mechanical properties of the constructs, specifically the amplitude of the time constants and the elastic equilibrium modulus, were enhanced by gradual increase of frequency. Taken together, these results show an increase in collagen remodeling by the HUASMCs overtime under incremental cyclic mechanical strain. This work suggests that only the in-depth investigation of the effects of stimulation parameters on the behavior of vSMC under cyclic strain in a 3D environment could lead to the design of optimized control strategies for enhanced vascular tissue generation and maturation in bioreactors.

Chemical imaging of fresh vascular smooth muscle cell response by epi-detected stimulated Raman scattering.

An understanding of deformation of cardiovascular tissue under hemodynamic load is crucial for understanding the health and disease of blood vessels. In the present work, an epi-detected stimulated Raman scattering (epi-SRS) imaging platform was designed for in situ functional imaging of vascular smooth muscle cells (VMSCs) in fresh coronary arteries. For the first time, the pressure-induced morphological deformation of fresh VSMCs was imaged with no fixation and in a label-free manner. The relation between the loading pressure and the morphological parameters, including angle and length of the VSMCs, were apparent. The morphological responses of VMSCs to drug treatment were also explored, to demonstrate the capability of functional imaging for VSMCs by this method. The time-course imaging revealed the drug induced change in angle and length of VSMCs. The present study provides a better understanding of the biomechanical framework of blood vessels, as well as their responses to external stimulations, which are fundamental for developing new strategies for cardiovascular disease treatment.

The influence of low level laser irradiation on vascular reactivity

PurposeThe mechanism of action of low level laser irradiation on tissues is unclear. Authors of publications present the positive clinical impact of low and medium power laser irradiation on vascular reactivity. The purpose of this study was to analyze the role of vascular endothelium in laser-induced constricted by endothelin-1 and phenylephrine. Materials and methodsExperiments were performed on isolated and perfused rat tail arteries of weighing 250–350g male Wistar rats. Contractility of arteries as a response to endothelin-1 and phenylephrine was measured after exposure to laser stimulation (10, 30 and 110mW). ResultsLaser irradiation inhibits vascular smooth muscle contraction induced by endothelin-1 and an alpha-adrenergic receptor agonist, phenylephrine proportionally to the laser power. Concentration-response curves were shifted to the right with significant reduction in maximal response. Laser irradiation at the power of 10mW, 30mW, and 110mW reduced the maximum response of arteries stimulated with phenylephrine sequentially to 88%, 72%, and 52%. Similar findings were observed during stimulation of endothelin-1. Laser irradiation at the power of 10mW, 30mW and 110mW resulted in maximal response respectively reduced to 94%, 62% and 38%. ConclusionOur results strongly suggest that during low level laser irradiation vascular smooth muscle cells reactivity is reduced, this effect is present in arteries with normal endothelium. The mechanism of action of laser biosimulation on tissues is unclear. Authors of publications present the positive clinical impact of low level laser irradiation on vascular reactivity.

Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock.

We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin 43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed that AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser262 and gap junctional communication in VSMCs. Activation of RhoA with U-46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junction independent. Gap junctions between VSMCs, gap junctional communication, and Cx43 phosphorylation at Ser262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process.

Construction of cell-containing, anisotropic, three-dimensional collagen fibril scaffolds using external vibration and their influence on smooth muscle cell phenotype modulation

Numerous methods have been developed for preparing guiding channels/tracks to promote the alignment of highly oriented cell types. However, these manufacture methods cannot fabricate interconnected guiding channels within three-dimensional (3D) scaffolds. Providing a suitable architectural scaffold for cell attachment could lead cells to more rapidly display a desired phenotype and perform their unique functions. Previously, we developed a simple device composed of a pneumatic membrane that can generate a tunable vibration frequency to apply physical stimulation for fabricating a 3D aligned collagen fibril matrix with the characteristic D-period structure in one step. In the present study, we aimed to evaluate the cellular responses of thoracic aortic smooth muscle cells (A7r5) incorporated during the fabrication of 3D-aligned collagen fibrils with D-periods and compared these cells with those incorporated in a 3D, randomly distributed collagen matrix and in a two-dimensional (2D) aligned substrate after up to 10 days of culture. The results consistently demonstrated that A7r5 cells cultured within the 3D and 2D anisotropic matrices were aligned. Cells cultured in the 3D aligned scaffolds exhibited a higher proliferation rate as well as higher F-actin and smoothelin expression levels compared with cells cultured in 3D randomly distributed scaffolds. Together, these results indicate that a 3D-reconstituted, anisotropic collagen matrix fabricated by our process provides synergistic effects of tension stimulation and matrix stiffness on encapsulated cells and can direct A7r5 cells to transform from a synthetic phenotype into a contractile state.

Mechanisms of Smooth Muscle Cell Differentiation Are Distinctly Altered in Thoracic Aortic Aneurysms Associated with Bicuspid or Tricuspid Aortic Valves.

Cellular and molecular mechanisms of thoracic aortic aneurysm are not clear and therapeutic approaches are mostly absent. Thoracic aortic aneurysm is associated with defective differentiation of smooth muscle cells (SMC) of aortic wall. Bicuspid aortic valve (BAV) comparing to tricuspid aortic valve (TAV) significantly predisposes to a risk of thoracic aortic aneurysms. It has been suggested recently that BAV-associated aortopathies represent a separate pathology comparing to TAV-associated dilations. The only proven candidate gene that has been associated with BAV remains NOTCH1. In this study we tested the hypothesis that Notch-dependent and related TGF-β and BMP differentiation pathways are differently altered in aortic SMC of BAV- vs. TAV-associated aortic aneurysms. SMC were isolated from aortic tissues of the patients with BAV- or TAV-associated aortic aneurysms and from healthy donors used as controls. Gene expression was verified by qPCR and Western blotting. For TGF-β induced differentiation SMC were treated with the medium containing TGF-β1. To induce proosteogenic signaling we cultured SMC in the presence of specific osteogenic factors. Notch-dependent differentiation was induced via lentiviral transduction of SMC with activated Notch1 domain. MYOCD expression, a master gene of SMC differentiation, was down regulated in SMC of both BAV and TAV patients. Discriminant analysis of gene expression patterns included a set of contractile genes specific for SMC, Notch-related genes and proosteogenic genes and revealed that control cells form a separate cluster from both BAV and TAV group, while BAV- and TAV-derived SMC are partially distinct with some overlapping. In differentiation experiments TGF-β caused similar patterns of target gene expression for BAV- and TAV derived cells while the induction was higher in the diseased cells than in control ones. Osteogenic induction caused significant change in RUNX2 expression exclusively in BAV group. Notch activation induced significant ACTA2 expression also exclusively in BAV group. We show that Notch acts synergistically with proosteogenic factors to induce ACTA2 transcription and osteogenic differentiation. In conclusion we have found differences in responsiveness of SMC to Notch and to proosteogenic induction between BAV- and TAV-associated aortic aneurysms.

Inhibition of ARC promoting the apoptosis of rat pulmonary arterial smooth muscle cells after serum deprivation invitro.

Apoptosis has important pathophysiological consequences contributing to pulmonary arterial hypertension (PAH). However, the mechanism underlying apoptosis in PAH remains unknown. Apoptosis repressor with caspase recruitment domain (ARC) is an essential factor in cell apoptosis, and regulates intrinsic and extrinsic apoptosis signaling pathways. It is hypothesized that ARC may be involved in the apoptotic responses of pulmonary arterial smooth muscle cells (PASMCs) following mild chronic injury. In the present study, serum deprivation (SD) was used to induce apoptosis of PASMCs. It was demonstrated that the expression of ARC in PASMCs was significantly increased following SD stimulation within 24 h, and ARC downregulation using small interfering RNA significantly enhanced the apoptosis of PASMCs following SD stimulation. In addition, the results demonstrated that ARC downregulation significantly increased the expression of proapoptotic factors and the level of reactive oxygen species (ROS), and decreased the mitochondrial membrane potential following SD exposure, suggesting ARC regulates the apoptosis of PASMCs via modulating mitochondrial function and ROS accumulation. The results of the present study revealed that ARC inhibition promotes the apoptosis of PASMCs following SD stimulation, and that ARC expression increases in the early stages of SD injury.

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca2+) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca2+-release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca2+ signals in PS1dE9 mutant mice.

Smooth muscle cells of human veins show an increased response to injury at valve sites

ObjectiveVenous valves are essential but are prone to injury, thrombosis, and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs nonvalve sites to elucidate biologic differences associated with vein valves. MethodsTissue explants of fresh human saphenous veins were prepared, and the migration of SMCs from explants of valve sinus vs nonvalve sinus areas was measured. Proliferation and death of SMCs were determined by staining for Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling. Proliferation and migration of passaged valve vs nonvalve SMCs were determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs nonvalve intima-media was determined by RNA sequencing. ResultsValve SMCs demonstrated greater proliferation in tissue explants compared with nonvalve SMCs (19.3% ± 5.4% vs 6.8% ± 2.0% Ki67-positive nuclei at 4 days, respectively; mean ± standard error of the mean, five veins; P < .05). This was also true for migration (18.2 ± 2.7 vs 7.5 ± 3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P < .0001). Cell death was not different (39.6% ± 16.1% vs 41.5% ± 16.0% terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, respectively, at 4 days, five veins). Cultured valve SMCs also proliferated faster than nonvalve SMCs in response to platelet-derived growth factor subunit BB (2.9 ± 0.2-fold vs 2.1 ± 0.2-fold of control, respectively; P < .001; n = 5 pairs of cells). This was also true for migration (6.5 ± 1.2-fold vs 4.4 ± 0.8-fold of control, respectively; P < .001; n = 7 pairs of cells). Blockade of fibroblast growth factor 2 (FGF2) inhibited the increased responses of valve SMCs but had no effect on nonvalve SMCs. Exogenous FGF2 increased migration of valve but not of nonvalve SMCs. Unlike in the isolated, cultured cells, blockade of FGF2 in the tissue explants did not block migration of valve or nonvalve SMCs from the explants. Thirty-seven genes were differentially expressed by valve compared with nonvalve intimal-medial tissue (11 veins). Peptide-mediated inhibition of SEMA3A, one of the differentially expressed genes, increased the number of migrated SMCs of valve but not of nonvalve explants. ConclusionsValve compared with nonvalve SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathologic lesion formation in valves. Whereas FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in the valve wall tissue remain unclear but may be among the differentially expressed genes discovered in this study. One of these genes, SEMA3A, mediates a valve-specific inhibitory effect on the injury response of valve SMCs.

Abstract 241: An Increased Response to Injury by Smooth Muscle Cells of Human Veins at Valve Sites

Objective: Venous valves are prone to injury, thrombosis and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs non-valve sites to elucidate biological differences associated with vein valves. Methods: SMC migration was measured using 2.5 mm 2 explants of the intima/media of valve sinus segments (without valve leaflets) vs. non-valve segments of human saphenous veins. Proliferation and death of SMCs was determined by staining for Ki67 and TUNEL, respectively. Proliferation and migration of passaged valve vs non-valve SMCs was determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs non-valve intima/media was determined by RNA-Seq. Results: Valve SMCs demonstrated greater proliferation within tissue explants compared to non-valve SMCs (19.3±5.4% vs. 6.8±2.0% Ki67 positive nuclei at 4 days, respectively; mean ± SEM, 5 veins; P&lt;.05). This was also true for migration (18.2±2.7 vs. 7.5±3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P&lt;.0001). Cell death was not different (39.6±16.1% vs. 41.5±16.0% TUNEL positive cells, respectively, at 4 days, 5 veins). Cultured valve SMCs also proliferated faster than non-valve SMCs in response to PDGF-BB (2.9±0.2 vs. 2.1±0.2 fold of control, respectively; P&lt;.001; N=5 vein’s paired cells). This was also true for migration (6.5±1.2 vs. 4.4±0.8 fold of control, respectively; P&lt;.001; N=7 vein’s paired cells). Blockade of FGF2 inhibited the increased responses of valve SMCs, but had no effect on non-valve SMCs. Exogenous FGF2 increased migration of valve, but not non-valve SMCs. Unexpectedly, blockade of FGF2 did not block migration of valve or non-valve SMCs from tissue explants. 37 genes were differentially expressed by valve compared to non-valve intimal/medial tissue (11 veins). Conclusions: Valve, compared to non-valve, SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathological lesion formation in valves. While FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in valve wall tissue remain unidentified. Here, the newly identified differentially expressed genes may play a role.

Vasculo-protective effect of BMS-309403 is independent of its specific inhibition of fatty acid-binding protein 4.

Fatty acid-binding protein (FABP) 4 is an adipocytokine mainly expressed in adipocyte and macrophage. Blood FABP4 is related not only to metabolic disorders including insulin resistance and atherosclerosis but also increased blood pressure. We tested the hypothesis that FABP4 plays roles in pathogenesis of hypertension development including proliferation, migration, and inflammation of vascular smooth muscle cells (SMCs) as well as contractile reactivity. FABP4 alone had no influence on proliferation, migration, and inflammation of rat mesenteric arterial SMCs, while it significantly enhanced smooth muscle contraction and increases of systolic blood pressure (SBP) induced by noradrenaline (NA). BMS-309403, an FABP4 inhibitor, significantly inhibited platelet-derived growth factor-BB-induced DNA synthesis and migration via preventing p38 and HSP27 activation. Further, BMS-309403 significantly inhibited tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 as well as monocyte adhesion via preventing NF-κB activation. Interestingly, SMCs do not express FABP4. Long-term treatment of spontaneously hypertensive rats (SHR) with BMS-309403 significantly inhibited impaired relaxation in isolated mesenteric arteries and left ventricular hypertrophy, while it had no influence on SBP. We for the first time showed that FABP4 acutely enhances NA-induced increases of SBP possibly through the enhancement of peripheral arterial contractility. BMS-309403 prevents proliferation, migration, and inflammatory responses of SMCs, although exogenous application of FABP4 has no influence on the cellular responses. Furthermore, we demonstrated that long-term treatment with BMS-309403 partially improves the pathological conditions of SHR. These results indicate that BMS-309403 would be useful for developing a new pharmacotherapeutic agent against obesity-associated hypertension and complications.

Morphological and growth responses of vascular smooth muscle and endothelial cells cultured on immobilized heparin and dextran sulfate surfaces.

Heparin has shown promise as a component of various biomaterial formulations, but its variable properties and inhibitory effects on some cell types have raised interest in use of dextran sulfate as an alternative. In this study, we characterized the interactions of vascular smooth muscle (SMC) and endothelial cells (EC) with heparin and dextran sulfate immobilized onto chitosan-based films. Films were modified by blending chitosan with type I collagen and covalently attaching heparin or dextran sulfate at various levels. Cell-material interactions were evaluated by quantifying cell spreading, shape and proliferation rate. ECs proliferated well on chitosan, but the polymer was a mediocre substrate for SMC growth. Immobilizing heparin on chitosan further inhibited SMC proliferation. However, blending collagen reversed the heparin inhibition of SMC growth, resulting in a pro-proliferative effect of heparin immobilized on chitosan-collagen films. Dextran sulfate surfaces supported both SMC and EC proliferation with or without the presence of collagen. The results indicate that inhibitory effects of heparin on SMC are reversed by immobilization in the presence of collagen, and that dextran sulfate may be superior to heparin as a biomaterial additive for promoting vascular cell growth in chitosan-based scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1725-1735, 2017.

Osteopontin regulates the proliferation of rat aortic smooth muscle cells in response to gingipains treatment

ObjectiveThe present study aimed to explore the possible effects of osteopontin (OPN) in the proliferation of rat aortic smooth muscle cells (RASMCs) stimulated by gingipains. MethodsThe proliferation of RASMCs in response to active gingipains treatment was evaluated by CCK-8 assay. OPN siRNA was designed, constructed and transfected into RASMCs at different concentrations. The cell cycle of RASMCs was analyzed by flow cytometry. OPN, α-SMA and calponin expression were examined by real-time PCR and western blot analysis. ResultsGingipains promoted the proliferation of RASMCs and OPN expression. With siRNA-mediated OPN expression knockdown, the cell cycle of RASMCs was blocked in the G0/G1 phase. Furthermore, the expression of specific differentiation markers, α-SMA and calponin, also decreased. ConclusionsThese results demonstrate that OPN has an impact on the proliferation and differentiation of RASMCs stimulated by gingipains.

Altered vasoreactivity in neonatal rats with pulmonary hypertension associated with bronchopulmonary dysplasia: Implication of both eNOS phosphorylation and calcium signaling.

Bronchopulmonary dysplasia (BPD) consists of an arrest of pulmonary vascular and alveolar growth, with persistent hypoplasia of the pulmonary microvasculature and alveolar simplification. In 25 to 40% of the cases, BPD is complicated by pulmonary hypertension (BPD-PH) that significantly increases the risk of morbidity. In vivo studies suggest that increased pulmonary vascular tone could contribute to late PH in BPD. Nevertheless, an alteration in vasoreactivity as well as the mechanisms involved remain to be confirmed. The purpose of this study was thus to assess changes in pulmonary vascular reactivity in a murine model of BPD-PH. Newborn Wistar rats were exposed to either room air (normoxia) or 90% O2 (hyperoxia) for 14 days. Exposure to hyperoxia induced the well-known features of BPD-PH such as elevated right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and decreased pulmonary vascular density. Intrapulmonary arteries from hyperoxic pups showed decreased endothelium-dependent relaxation to acetylcholine without any alteration of relaxation to the NO-donor sodium nitroprusside. This functional alteration was associated with a decrease of lung eNOS phosphorylation at the Ser1177 activating site. In pups exposed to hyperoxia, serotonin and phenylephrine induced exacerbated contractile responses of intrapulmonary arteries as well as intracellular calcium response in pulmonary arterial smooth muscle cells (PASMC). Moreover, the amplitude of the store-operated Ca2+ entry (SOCE), induced by store depletion using a SERCA inhibitor, was significantly greater in PASMC from hyperoxic pups. Altogether, hyperoxia-induced BPD-PH alters the pulmonary arterial reactivity, with effects on both endothelial and smooth muscle functions. Reduced activating eNOS phosphorylation and enhanced Ca2+ signaling likely account for alterations of pulmonary arterial reactivity.

Serum from asthmatic rat treated with acupuncture inhibits acetylcholine-induced contractile responses of airway smooth muscle cells

OBJECTIVETo investigate the influence of serum from asthmatic rat treated with acupuncture (acupuncture serum) on the contractile responses of airway smooth muscle cells (ASMCs). METHODSOVA-induced asthmatic rats were subject to acupuncture treatment at the points of Dazhui (GV 14), Fengmen (BL 12), and Feishu (BL 13). The resulting serum was collected, as well as serum from normal animals. Contraction of ASMCs was induced with 10 µM acetylcholine (Ach), and acupuncture serum or normal serum was supplemented 15 min later. The effects of the sera on the changes in cell length, width, and surface area were monitored in real time via a live cell imaging system. RESULTSThe contraction rates of ASMCs 15 min and 30 min after Ach stimulation reached 38.2% ± 3.8% and 49.7% ± 4.6%, both of which were significantly higher than unstimulated control cells (P < 0.01). Acupuncture serum at the 1: 100 dosage reduced the contraction rate of ASMCs from 40.1% ± 5.1% to 17.6% ± 6.5% (P < 0.01). Thus, the treatment significantly inhibited the Ach-induced contractile responses of ASMCs and yielded a relaxation efficiency of 58%, which was greater than the effects obtained with a 1: 250 dosage. Treatment with acupuncture serum at 1: 500 dosage or normal serum displayed no clear effect of suppressing Ach-induced contraction of ASMCs. CONCLUSIONOur data suggest that acupuncture serum has the effect of inhibiting Ach-induced contraction of ASMCs, thereby promoting relaxation of the cells.

Ursolic acid suppresses leptin-induced cell proliferation in rat vascular smooth muscle cells.

Accumulating lines of evidence indicate that high leptin levels are associated with adverse cardiovascular health in obese individuals. Proatherogenic effects of leptin include endothelial cell activation and vascular smooth muscle cell proliferation and migration. Ursolic acid (UA) has been reported to exhibit multiple biological effects including antioxidant and anti-inflammatory properties. In this study, we investigated the effect of UA on leptin-induced biological responses in rat vascular smooth muscle cells (VSMCs). A-10 VSMCs were treated with leptin in the presence or absence of UA. Intracellular reactive oxygen species (ROS) was probed by 2',7'-dichlorofluorescein diacetate. The expression of extracellular signal-regulated kinase (ERK)1/2, phospho-(ERK)1/2, nuclear factor-kappa B (NF-κB) p65 and p50, and matrix metalloproteinase-2 (MMP2) was determined by Western blotting. Immunocytochemistry and confocal laser scanning microscopy were also used for the detection of NF-κB. The secretion of MMP2 was detected by gelatin zymography. UA exhibited antioxidant activities in vitro. In rat VSMCs, UA effectively inhibited cell growth and the activity of MMP2 induced by leptin. These suppressive effects appeared by decreasing the activation of (ERK)1/2, the nuclear expression and translocation of NF-κB, and the production of ROS. UA appeared to inhibit leptin-induced atherosclerosis, which may prevent the development of obesity-induced cardiovascular diseases.

Therapeutics Targeting Drivers of Thoracic Aortic Aneurysms and Acute Aortic Dissections: Insights from Predisposing Genes and Mouse Models.

Thoracic aortic diseases, including aneurysms and dissections of the thoracic aorta, are a major cause of morbidity and mortality. Risk factors for thoracic aortic disease include increased hemodynamic forces on the ascending aorta, typically due to poorly controlled hypertension, and heritable genetic variants. The altered genes predisposing to thoracic aortic disease either disrupt smooth muscle cell (SMC) contraction or adherence to an impaired extracellular matrix, or decrease canonical transforming growth factor beta (TGF-β) signaling. Paradoxically, TGF-β hyperactivity has been postulated to be the primary driver for the disease. More recently, it has been proposed that the response of aortic SMCs to the hemodynamic load on a structurally defective aorta is the primary driver of thoracic aortic disease, and that TGF-β overactivity in diseased aortas is a secondary, unproductive response to restore tissue function. The engineering of mouse models of inherited aortopathies has identified potential therapeutic agents to prevent thoracic aortic disease.