Pulsatile Stretch Research Articles

Effect of pulsatile stretch on unfolded protein response in a new model of the pulmonary hypertensive vascular wall

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by hypoxemia and arterial remodeling. Dynamic stretch and recoil of the arterial wall during pulsation (in normal conduit arteries, stretch 20% above diastolic diameter) maintains homeostasis; a static arterial wall is associated with remodeling. PPHN is diagnosed by echocardiography as decreased pulmonary artery wall displacement during systole, causing decreased pulmonary arterial pressure acceleration time in a stiff artery.We hypothesized that a ‘normal’ amplitude of pulsatile stretch is protective against ER stress, while the loss of stretch is a trigger for hypoxia-induced stress responses. Using a novel in vitro model of pulmonary arterial myocytes subject to repetitive stretch-relaxation cycles within a normoxic or hypoxic environment, we examined the relative impact of hypoxia (pulmonary circuit during unresolved PPHN) and cyclic mechanical stretch (diminished in PPHN) on myocyte homeostasis, specifically on signaling proteins for autophagy and endoplasmic reticulum (ER) stress.Stretch induced autophagosome abundance under electron microscopy. Hypoxia, in presence or absence of pulsatile stretch, decreased unfolded protein response (UPR) hallmark BIP (GRP78) in contractile phenotype pulmonary arterial myocytes. Inositol requiring enzyme-1 α (IRE1α) was not activated; but hypoxia induced eif2α phosphorylation, increasing expression of ATF4 (activating transcription factor-4). This was sensitive to inhibition by autophagy inhibitor bafilomycin A1.We conclude that in the pulmonary circuit, hypoxia induces one arm of the UPR pathway and causes ER stress. Pulsatile stretch ameliorates the hypoxic UPR response, and while increasing presence of autophagosomes, does not activate canonical autophagy signaling pathways. We propose that simultaneous application of hypoxia and graded levels of cyclic stretch can be used to distinguish myocyte signaling in the deformable pulmonary artery of early PPHN, versus the inflexible late stage PPHN artery.

The molecular mechanism of mechanotransduction in vascular homeostasis and disease

Blood vessels are constantly exposed to mechanical stimuli such as shear stress due to flow and pulsatile stretch. The extracellular matrix maintains the structural integrity of the vessel wall and coordinates with a dynamic mechanical environment to provide cues to initiate intracellular signaling pathway(s), thereby changing cellular behaviors and functions. However, the precise role of matrix-cell interactions involved in mechanotransduction during vascular homeostasis and disease development remains to be fully determined. In this review, we introduce hemodynamics forces in blood vessels and the initial sensors of mechanical stimuli, including cell-cell junctional molecules, G-protein-coupled receptors (GPCRs), multiple ion channels, and a variety of small GTPases. We then highlight the molecular mechanotransduction events in the vessel wall triggered by laminar shear stress (LSS) and disturbed shear stress (DSS) on vascular endothelial cells (ECs), and cyclic stretch in ECs and vascular smooth muscle cells (SMCs)-both of which activate several key transcription factors. Finally, we provide a recent overview of matrix-cell interactions and mechanotransduction centered on fibronectin in ECs and thrombospondin-1 in SMCs. The results of this review suggest that abnormal mechanical cues or altered responses to mechanical stimuli in EC and SMCs serve as the molecular basis of vascular diseases such as atherosclerosis, hypertension and aortic aneurysms. Collecting evidence and advancing knowledge on the mechanotransduction in the vessel wall can lead to a new direction of therapeutic interventions for vascular diseases.

Endothelial Cell Mechanotransduction in the Dynamic Vascular Environment.

The vascular endothelial cells (ECs) that line the inner layer of blood vessels are responsible for maintaining vascular homeostasis under physiological conditions. In the presence of disease or injury, ECs can become dysfunctional and contribute to a progressive decline in vascular health. ECs are constantly exposed to a variety of dynamic mechanical stimuli, including hemodynamic shear stress, pulsatile stretch, and passive signaling cues derived from the extracellular matrix. This review describes the molecular mechanisms by which ECs perceive and interpret these mechanical signals. The translational applications of mechanosensing are then discussed in the context of endothelial-to-mesenchymal transition and engineering of vascular grafts.

Application of laser scanning cytometry in vascular smooth muscle remodeling.

Pulmonary artery hyperplasia is the result of proliferation of the pulmonary arterial smooth muscles (PASM). Hypoxia-induced PASM proliferation in the fetus and the newborn is the primary cause of persistent pulmonary hypertension of the newborn (PPHN). This study was performed to characterize the utility of the Laser Scanning Cytometry (LSC) method in elucidating arterial cytoskeletal remodeling in an in vitro model of PPHN. The aim was to demonstrate the following: (a) LSC is a valid method for the analysis of nuclear and cytosolic fluorescence and (b) the cumulative effects of mechanical stretch together with hypoxia promote reactive oxygen species (ROS) formation. The molecular events in response to hypoxia and the mechanical overload of the pulmonary circuit were demonstrated in vitro by subjecting hypoxic cultured primary PASM or human airway smooth muscles (hASM) to repetitive stretch-relaxation cycles at rates comparable to dynamic stretch in vivo. The altered cytoskeleton in the form of filamentous to globular actin (F:G actin) ratio was imaged and quantified at the cellular level by LSC as an endpoint. LSC can remove the nuclear G-actin fluorescence from the total G-actin fluorescence. Pulsatile stretch was found to significantly increase the total endogenous ROS and superoxide anion release in normoxic and hypoxic conditions in primary PASM fibers. The effect of stretch was predominant in increasing superoxide anion release, only under hypoxic conditions. These findings, obtained by LSC in vitro are amenable to validation in any in vivo model of interest. The in vitro model is clinically relevant to human pulmonary vascular remodeling.

Pulsatile stretch as a novel modulator of amyloid precursor protein processing and associated inflammatory markers in human cerebral endothelial cells

Amyloid β (Aβ) deposition is a hallmark of Alzheimer’s disease (AD). Vascular modifications, including altered brain endothelial cell function and structural viability of the blood-brain barrier due to vascular pulsatility, are implicated in AD pathology. Pulsatility of phenomena in the cerebral vasculature are often not considered in in vitro models of the blood-brain barrier. We demonstrate, for the first time, that pulsatile stretch of brain vascular endothelial cells modulates amyloid precursor protein (APP) expression and the APP processing enzyme, β-secretase 1, eventuating increased-Aβ generation and secretion. Concurrent modulation of intercellular adhesion molecule 1 and endothelial nitric oxide synthase (eNOS) signaling (expression and phosphorylation of eNOS) in response to pulsatile stretch indicates parallel activation of endothelial inflammatory pathways. These findings mechanistically support vascular pulsatility contributing towards cerebral Aβ levels.

OP.8A.06] PULSATILE STRETCH ALTERS AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO GLYCOSPHINGOLIPID INHIBITION

Objective: Amyloid β (Aβ) deposits that arise from amyloid precursor protein (APP) are a hallmark of Alzheimer's disease (AD). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are associated with AD. Glycosphingolipid inhibitors that inhibit the production of the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release. This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). Design and method: hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n = 7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). Results: APP expression increased significantly in response to stretch in ECs pre-treated with D-PDMP (170.2 ± 27%, p < 0.01) compared to cells stretched without pre-treatment (114.5 ± 23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9 ± 4%) compared to vehicle control (100.0 ± 0%, p < 0.0001) and D-PDMP-treated-static control (107.0 ± 17%, p < 0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels decreased significantly (40.3 ± 15%) compared to vehicle control (100.0 ± 0%, p < 0.05) and PDMP-treated-static control (107.0 ± 17%, p < 0.01). Conclusions: APP expression increased in cerebral ECs pre-treated with D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on phospho-eNOS, although it did not statistically significance. Glycosphingolipid inhibition with D-PDMP and cyclic stretch may lead to ceramide accumulation thereby increasing the Aβ load. This can affect NO production and modulation of Aβ deposits.

CYCLIC STRETCH MEDIATES CHANGES IN AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO A GLYCOSPHINGOLIPID INHIBITOR

Amyloid β (Aβ) deposits, a hallmark of Alzheimer's disease (AD), are aggregates of Aβ peptides that are cleaved of amyloid precursor protein (APP). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are independently associated with AD (Nation et al. 2013; Takasugi et al. 2015; Austin et al. 2013). Glycosphingolipid inhibitors that inhibit the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release (Takasugi et al. 2015). This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n=7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). APP expression significantly increased in response to stretch in ECs pre-treated with D-PDMP (170.2±27%, p<0.01) compared to those that have been stretched without pre-treatment (114.5±23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9±4%) compared to vehicle control (100.0±0%, p<0.0001) and PDMP-treated-static control (107.0±17%, p<0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels significantly decreased (40.3±15%) compared to vehicle control (100.0±0%, p<0.05) and PDMP-treated-static control (107.0±17%, p<0.01). APP expression increased in cerebral ECs pre-exposed to D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on peNOS, although was not statistically significant. Glucosyl and/or lactosyl ceramide synthases may have regulatory roles in modulating APP expression and NO signalling in cerebral ECs and may have implications on Aβ load, NO production and related vascular pathologies in the context of AD.

Abstract 256: Differential Biomechanical Regulation of Vein Graft Endothelial CD39 by Pulsatile Radial Forces

Saphenous veins are frequently used bypass grafts in coronary &amp; peripheral artery bypass surgery, but are more prone to failure than arterial grafts due to adverse remodeling. The endothelium senses radial and linear biomechanical forces exerted by blood flow. Thin-walled venous blood flow in situ is non-pulsatile until transposed as an arterial graft. The impact of cyclic stretch from pulsatile distention on the vein wall is not well elucidated. CD39 is a potent ecto-enzyme at the blood:vessel interface regulating vascular thrombo-inflammation by dissipating extracellular ATP &amp; ADP. We recently identified that vascular CD39 expression can be induced by laminar shear stress. However, the response of CD39 to cyclic stretch during vein graft arterialization remains unknown. We hypothesized that the vasculo-protective enzyme CD39 is induced by the venous endothelium in response with pulsatile stretch. Methods: Primary human saphenous vein endothelial cells (HSVEC) were exposed to cyclic stretch in vitro , mimicking arterial &amp; vein graft patterns. CD39 transcript &amp; protein expression were quantified. Ecto-ATPase and -ADPase activity of CD39 on HSVEC were quantified with a malachite green assay. CD39 haploinsufficient ( Cd39 +/- ) &amp; WT mice underwent carotid bypass surgery with a vein graft, &amp; subsequent neointimal hyperplasia was compared. Results: High (16%, 1 Hz) and low (5%, 1 Hz) levels of cyclic stretch of HSVEC elicited distinct patterns of CD39 expression. Cd39 mRNA remained unchanged after 48h of low or high stretch (n=6,each). Low-level cyclic stretch did not alter CD39 protein expression (n=6,each). In stark contrast, 16% cyclic stretch induced increases in CD39 protein expression after 24h (60%) and 48h (130%) compared with static controls (n=6 each, P &lt;0.05). CD39-dependent ATP &amp; ADP hydrolysis increased by 60% &amp; 100% in HSVEC following high-level cyclic stretch (n=4, p&lt;0.005). Preliminary murine vein graft model studies suggest that CD39-deficiency exacerbates vein graft neointimal hyperplasia compared to WT controls. Conclusions: We have identified CD39 as a novel, stretch-responsive endothelial ecto-enzyme in human saphenous veins, which may represent a protective response to venous distention from arterial stretch patterns.

OP.2D.03] PULSATILE STRETCH ALTERS EXPRESSION AND PROCESSING OF AMYLOID PRECURSOR PROTEIN IN HUMAN CEREBRAL ENDOTHELIAL CELLS

Amyloid β (Aβ) deposition is a hallmark of Alzheimer's disease (AD). Aβ is generated via enzymatic cleavage of amyloid precursor protein (APP). Increased APP expression is associated with elevated amyloid burden. Elevated pulsatility of arterial pressure, as a vascular factor indicative of vascular stiffness, is associated with AD. This study investigates whether increased magnitude of pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) alters expression and processing of APP.hCMECs were cultured on flexible silicone chambers and subjected to 5%, 10% or 15% cyclic stretch (ShellPA stretch system, Menicon Life Science) for 18 hours at 1 Hz (n = 5-11 for each stretch magnitude). Comparison was made with controls (0% stretch) to evaluate the effect of pulsatility on APP. APP expression was quantified using western blots with glyceraldehyde 3-phosphate dehydrogenase as the loading control. APP processing was evaluated in the cell culture supernatants by ELISA for secreted Aβ42. Results were analyzed using one way ANOVA (mean ± SEM, % control) or Pearson correlation.The densitometric analysis (expressed as a percentage of control values) of protein expression of APP showed that it was significantly higher at 10% of cyclic stretch (160.9 ± 21%) compared to both the static control (100%, p < 0.05) and 5% cyclic stretch magnitude (107.8 ± 12%, p < 0.05). The APP protein expression at 15% stretch magnitude (138.3 ± 12%) was slightly, but not significantly higher than the static control and 5% cyclic stretch. There was a significant correlation between the % stretch magnitude and secretion of A β42 (r = 0.4132, p < 0.0001).Increasing cyclic stretch of cerebral vascular ECs altered the expression of APP and the secretion of A β42. Prolonged cyclic stretch may induce APP expression and A β formation. Findings mechanistically support the association of elevated haemodynamic pulsatility and arterial stiffness with AD.

Increased Expression of Lamin A/C Correlate with Regions of High Wall Stress in Abdominal Aortic Aneurysms

Since aortic diameter is the most -significant risk factor for rupture, we sought to identify stress-dependent changes in gene expression to illuminate novel molecular processes in aneurysm rupture. We constructed finite element maps of abdominal computerized tomography scans (CTs) of seven abdominal aortic aneurysm (AAA) patients to map wall stress. Paired biopsies from high- and low-stress areas were collected at surgery using vascular landmarks as coordinates. Differential gene expression was evaluated by Illumina Array analysis, using the whole genome DNA-mediated, annealing, selection, extension, and ligation (DASL) gene chip (n = 3 paired samples). The sole significant candidate from this analysis, Lamin A/C, was validated at the protein level, using western blotting. Lamin A/C expression in the inferior mesenteric vein (IMV) of AAA patients was compared to a control group and in aortic smooth muscle cells in culture in response to physiological pulsatile stretch. -Areas of high wall stress (n = 7) correlate to those -regions which have the thinnest walls [778 µm (585-1120 µm)] in comparison to areas of lowest wall stress [1620 µm (962-2919 µm)]. Induced expression of Lamin A/C -correlated with areas of high wall stress from AAAs but was not significantly induced in the IMV from AAA patients compared to controls (n = 16). Stress-induced expression of Lamin A/C was mimicked by exposing aortic smooth muscle cells to prolonged pulsatile stretch. Lamin A/C protein is specifically increased in areas of high wall stress in AAA from patients, but is not increased on other vascular beds of aneurysm patients, suggesting that its elevation may be a compensatory response to the pathobiology leading to aneurysms.

Abstract 15922: Role of Polo-Like Kinase-1 in Intimal Hyperplasia in Saphenous Vein Graft: Potential Implication in Vein-Graft Disease

Coronary revascularization by coronary artery bypass grafting (CABG) is the choice of procedure in patients with multi-vessel or left main coronary artery disease. Concerns have been raised on the long term result of CABG using saphenous vein graft (SVG) as its patency significantly declines following surgery, compared to internal mammary artery (IMA), which is almost immune to restenosis. Proliferation of smooth muscle cells (SMCs) is the key event in the pathogenesis of intimal hyperplasia leading to SVG failure. PDGF-BB is a major growth factor released at the site of pulsatile stretch- and shear stress-induced graft injury. Here, we examined, for the first time, the expression of PLK1 and its phosphorylation/activation in isolated human bypass graft conduits. Human SV and IMA vessels were freshly collected, SMCs isolated and cultured up to 5th passage. In cultured SMCs, effect of PDGF-BB was examined on total and phosphorylated PLK1 (pPLK1) by Western blot analysis. Cell proliferation was measured using thymidine incorporation, MTT method and cell count. We found significantly higher expression of pPLK1 and total PLK1 in PDGF-stimulated SV SMCs than IMA. SV SMCs had 5-fold increase in the density of pPLK1 and had 2-fold increase in the density of total PLK1. While in the IMA SMCs, increase in pPLK1 was significantly lower than in SV SMCs. Also, this increase was not sustained. These data suggest a greater and sustained sensitivity of SV SMCs to PDGF-BB induced PLK1 activity than that of IMA. A PLK1 blocker inhibited PDGF-induced proliferation in both IMA and SV SMCs. These data demonstrate differential activity of PDGF-induced PLK1 activation, which was greater in SV SMCs than in IMA. This could explain the development of intimal hyperplasia in SV conduits than the IMA following CABG. Thus, inhibition of PLK1 could be a target in developing better therapeutic approach to prevent vein-graft disease.

Cyclic stretch and hypertension increase retinal succinate: potential mechanisms for exacerbation of ocular neovascularization by mechanical stress.

We investigated succinate metabolism in cells undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. We seeded ARPE-19 cells on 6-well BioFlex collagen I-coated, silicone elastomer-bottomed culture plates. Cells then were subjected to pulsatile stretch using a computer-controlled vacuum stretch apparatus. A physiologic stretch frequency of 60 cycles per minute and 5% to 15% prolongation of the elastomer-bottomed plates were used. Succinate concentration was assessed by enzymatic analysis and high-performance liquid chromatography-mass spectrometry. The VEGF was measured using enzyme-linked immunosorbent assays. The 12-week-old male SHRs and weight-matched Wistar-Kyoto (WKY) control rats were treated with or without 100 mg·kg(-1)·day(-1) captopril for 1 week. The vitreous body and retina of each rat were extracted after 1 week of therapy, and the vitreoretinal succinate concentration was measured. Cells exposed to cyclic stretch accumulated intracellular succinate in a time- and magnitude-dependent manner, and also accumulated VEGF protein levels. Moreover, BAPTA/AM, an intracellular calcium chelate reagent, significantly inhibited the stretch-induced succinate increase. After cyclic stretch, levels of intracellular fumarate, a citric acid cycle intermediate, also were significantly increased compared to controls. The BAPTA/AM inhibited this increase. For the in vivo experiments, hypertension increased vitreoretinal succinate and fumarate in SHRs compared to the normotensive WKY controls. When hypertension was reduced using captopril, vitreoretinal succinate returned to baseline levels. These findings suggest that cyclic stretch and hypertension increased intracellular succinate in cultured retinal pigment epithelial cells and the vitreoretinal succinate of SHRs through a calcium-dependent pathway.

The Hypothesis of “Embryonic Recall”: Mechanotransduction as Common Denominator Linking Normal Cardiogenesis to Recovery in Adult Failing Hearts

Cardiac regeneration remains a clinical target regardless of numerous therapeutic concepts. We formulated a hypothesis claiming that periodic coronary venous pressure elevation (PICSO; Pressure controlled Intermittent Coronary Sinus Occlusion) initiates embedded, but dormant developmental processes in adult jeopardized hearts. Hemodynamics in the primitive beating heart tube is sensed transducing “mechanical” epigenetic information during normal cardiac development. In analogy mechanotransduction via shear stress and pulsatile stretch induced by periodic elevation of blood pressure in cardiac veins reconnects this dormant developmental signal, setting regenerative impulses in the adult heart. Significant increase of hemeoxygenase-1 gene expression (p &lt; 0.001) and vascular endothelial growth factor (VEGF) (p &lt; 0.002) as well as production of VEGRF2 in experimental infarction underscores the resurgence of developmental stimuli by PICSO. Molecular findings correspond with risk reduction (p &lt; 0.0001) in patients with acute coronary syndromes as well as observations in heart failure patients showing substantial risk reduction up to 5 years endorsing our hypothesis and preclinical experience that PICSO via hemodynamic power activates regenerative processes also in adult human hearts. These results emphasize that our proposed hypothesis “embryonic recall” claiming revival of an imbedded albeit dormant “epigenetic” process is able not only to sculpture myocardium in the embryo, but also to redesign structure in the adult and failing heart.

Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% (“physiologic”) or 20% (“supraphysiologic”) at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.

Vascular lumen simulation and highly-sensitive nitric oxide detection using three-dimensional gelatin chip coupled to TiC/C nanowire arrays microelectrode

Reproducing the physiological environment of blood vessels for the in vitro investigation of endothelial cell functions is very challenging. Here, we describe a vascular-like structure based on a three-dimensional (3D) gelatin chip with good compatibility and permeability which is also cost-effective and easy to produce. The controllable lumen diameter and wall thickness enable close mimicking of blood vessels in vitro. The 3D gelatin matrix between adjacent lumens is capable of generating soluble-factor gradients inside, and diffusion of molecules with different molecular weights through the matrix is studied. The cultured human umbilical vein endothelial cells proliferate on the gelatin lumen linings to form a vascular lumen. The hemodynamic behavior including adhesion, alignment of endothelial cells (ECs) under shear stress and pulsatile stretch is studied. Furthermore, a microelectrode comprising TiC/C nanowire arrays is fabricated to detect nitric oxide with sub-nM detection limits and NO generation from the cultured ECs is monitored in real time. This vascular model reproduces the surrounding parenchyma of endothelial cells and mimics the hemodynamics inside blood vessels very well, thereby enabling potential direct investigation of hemodynamics, angiogenesis, and tumor metastasis in vitro.

Tissue Factor Pathway Inhibitor

To investigate in women older than 60 whether aortic stiffness or pulse pressure (PP) is associated with selected procoagulant or anticoagulant factors and to examine whether pulsatile stretch influences these factors in human vascular smooth muscle cells (VSMCs) in vitro. Aortic pulse wave velocity (PWV) and carotid PP were studied in 123 apparently healthy postmenopausal women. PWV, PP, von Willebrand factor, and free tissue factor pathway inhibitor (TFPI), but not mean arterial pressure, increased with age. Free TFPI and PWV were positively correlated, even after adjustment for age and PP and other confounding parameters. In vitro, 5% or 10% pulsatile stretch (at 1 Hz) enhanced TFPI synthesis and secretion by VSMCs in a time-independent manner (1 to 48 hours) without changes in protein level of smooth muscle myosin heavy chain. Application of 5% static stretch had no effect. In postmenopausal women, free TFPI increases as vascular wall function deteriorates and PP increases. These findings are supported by the increase in TFPI synthesized by VSMCs in response to cyclic stress in vitro. They suggest that VSMCs require pulsatility to interfere with the coagulation process and highlight the relevance of plasma free TFPI levels to cardiovascular diseases.

Cyclic stretch downregulates arterial vascular connexin43 protein expression: an ex vivo study

Vascular cells may communicate through gap junctions that are formed by connexin (Cx) proteins. We investigated differential regulation of arterial gap junctions by steady and cyclic stretch and the underlying mechanotransduction pathways. Ex vivo culture of rabbit thoracic aortas was used to investigate regulation of Cx43 by cyclic stretch. After culturing for 6 or 24 h, Cx43 protein levels were quantified using Western blot. Cultures under a pulsatile pressure (mean 80 mmHg, pulse 30 mmHg) decreased Cx43 protein at both 6 and 24 h as compared with cultures under a steady pressure (80 mmHg). The regulation of Cx43 protein was mediated by pulsatile pressure-induced cyclic stretch, not by cyclic stress. Protein levels of active and total Src were also decreased by cyclic stretch at 24 h. The Src- specific inhibitor PP1 in steady culture only or in both steady and pulsatile culture conditions eliminated the difference in Cx43 protein levels between the two culture conditions. Addition of reactive oxygen species inhibitor apocynin to the pulsatile culture abolished the differences in Src and Cx43 protein levels between the two cultures. Thus, Src and reactive oxygen species appear to play a role in cyclic stretch-mediated regulation of Cx43 protein. These results are likely to have important implications in cardiovascular physiology and pathophysiology under conditions wherein significant alterations in the level of cyclic stretch are present.

Mechanotransduction of pulsatile stretch in a cell culture model of cardiac hypertrophy connexin 43 (Cx43) expression

In the heart gap junction intercellular coupling represents the fundament of proper electrical propagation. In cardiac hypertrophy up-regulation of Cx43 has been observed and was suggested as being related to sudden cardiac death. It has been found, that expression and distribution of Cx43, which is the main gap junction forming connexin in the ventricle, is altered in many pathological conditions. We therefore wanted to elucidate whether exposure of cardiomyocytes to chronic pulsatile stretch affects Cx43 and whether mechanotransduction pathways interfere with adrenergic regulation of Cx43, already shown in former experiments.

Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-κB activation

This study investigated interactions between the effects of mechanical stretch and thrombin on RhoA activation in rat aortic smooth muscle cells (RASMC). Equibiaxial, pulsatile stretch, or thrombin produced a significant increase in RhoA activation. Surprisingly, in combination, 30 min of stretch inhibited the ability of thrombin to activate RhoA. NO donors and 8-bromo-cGMP significantly inhibited thrombin-induced RhoA activation. Interestingly, the nitric oxide synthase (NOS) inhibitor l-NAME increased basal RhoA activity, suggesting that NOS activity exerts a tonic inhibition on RhoA. Stretching RASMC increases nitrite production, consistent with the idea that NO contributes to the inhibitory effects of stretch. Thrombin stimulates MAP kinase and NF-κB pathways through Rho and these responses were blocked by 8-bromo-cGMP or stretch and restored by l-NAME. These data suggest that stretch, acting through NO and cGMP, can prevent the ability of thrombin to stimulate Rho signaling pathways that contribute to pathophysiological proliferative and inflammatory responses.

Induction of NEDD4 under simulated microgravity

Vascular endothelial cells are exposed to mechanical forces e.g. wall shear stress, wall tension, and pulsatile stretch, which are modulated by the gravitational field. The effects of microgravity may influence endothelial cell function including protein synthesis and degradation.Therefore, this study was designed to investigate microgravity dependent effects on the ubiquitin‐ligase NEDD4 in endothelial cells. In order to produce microgravity endothelial cells (EA hy926, HUVEC) were seeded on microcarriers and cultured in the Rotating Wall Vessel for 24 h and 48 h. Controls were kept under normal gravity or exposed to shear stress using a cone and plate apparatus (6 dyn\cm2, 24 h). Gene expression and protein concentration were analysed by real‐time RT‐PCR and Western blotting, respectively. Our results indicate that NEDD4 is upregulated to 200% under microgravity and downregulated to 70% by shear stress relative to controls (1G, no shear). The present data suggest that NEDD4 is sensitive to mechanical forces; the induction under microgravity may be a mechanism to enhance protein degradation in endothelial cells.