Effects of pulsatile flow on cultured vascular endothelial cell morphology

Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.     

Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells

Placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression increase during pregnancy. Shear stress, the frictional force exerted on endothelial cells by blood flow, stimulates vessel dilation, endothelial NO production, and eNOS expression. In order to study the effects of pulsatile flow/shear stress, we adapted Cellco CELLMAX artificial capillary modules to study ovine fetoplacental artery endothelial (OFPAE) cells for NO production and eNOS expression. OFPAE cells were grown in the artificial capillary modules at 3 dynes/cm2. Confluent cells were then exposed to 10, 15, or 25 dynes/cm2 for up to 24 h. NO production by OFPAE cells exposed to pulsatile shear stress was inhibited to nondetectable levels by the NOS inhibitor l-NMMA and reversed by excess NOS substrate l-arginine. NO production and expression of eNOS mRNA and protein by OFPAE cells were elevated by shear stress in a graded fashion (P < 0.05). The rise in NO production with 25 dynes/cm2 shear stress (8-fold) was greater (P < 0.05) than that observed for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). The acute shear stress-induced rise in NO production by OFPAE cells was via eNOS activation, whereas the prolonged NO rise occurred by elevations in both eNOS expression and enzyme activation. Thus, elevations of placental blood flow and physiologic shear stress may be partly responsible for the increases in placental arterial endothelial eNOS expression and NO production during pregnancy.     

Theoretical study of intracellular stress fiber orientation under cyclic deformation

We studied stress fiber orientation under a wide range of uniaxial cyclic deformations. We devised and validated a hypothesis consisting of two parts, as follows: (1) a stress fiber aligns to avoid a mechanical stimulus in the fiber direction under cyclic deformation. This means that, among all allowable directions, a stress fiber aligns in the direction which minimizes the stimulus, i. e., the summation of the changes in length of the stress fiber over one stretch cycle; and (2) there is a limit in the sensitivity of the cellular response to the mechanical stimulus. Due to this sensing limit, the orientation angle in stress fibers is distributed around the angle corresponding to the minimum stimulus. To validate this hypothesis, we approximated an anisotropic deformation of the membrane on which cells were to be cultured. We then obtained the relationships between the stretch range and the fiber angle in the undeformed state which minimize the mechanical stimuli, assuming that the membrane on which stress fibers and cells adhered was homogeneous and incompressible. Numerical simulation results showed that the proposed hypothesis described our previous experimental results well and was consistent with the experimental results in the literature. The simulation results, taking account of the second part of the hypothesis with a small value for the limit in sensitivity to the mechanical stimulus, could explain why cell orientation is distributed so widely with cyclic stretch ranges of <10%. The proposed hypothesis can be applied to various types of deformation because the mechanical stimulus is always sensed and accumulates under cyclic deformation without the necessity of a reference state to measure the stimulus.     

Conditioned media from mesenchymal stromal cells and periodontal ligament fibroblasts under cyclic stretch stimulation promote bone healing in mouse calvarial defects

Background aimsWhen cells are exposed to stresses such as mechanical stimuli, they release growth factors and adapt to the surrounding environment H ere, we demonstrated that mechanical stimulation during culture affects the production of osteogenic and angiogenic factors.MethodsHuman bone marrow derived mesenchymal stromal cells (hMSCs) and human periodontal ligament fibroblasts (HPLFs ) were cultured under cyclic stretch stimulation for 24 h. Collected of the cells and conditioned media (CM), the gene and protein expression levels of osteogenic and angiogenic factors were evaluated. CM was also evaluated for angiogenic activity and calc ification ability. In in vivo study, CM was administered to a mouse calvarial defect model and histologically and radiologically evaluated.ResultsQuantitative real time polymerase chain reaction results showed that the expression of bone morphogenetic pro tein 2, 4 (BMP 2, 4), vascular endothelial growth factor A (VEGF A), and platelet derived growth factor AA (PDGF AA) was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in each cell type. Enzyme linked immunosor bent assay results revealed that the expression of BMP 2,4, VEGF A was upregulated in the cyclic stretch group in comparison with the non stretch group in each cell type. Only HPLFs showed significant difference in PDGF AA expression between the cyclic str etch and the non stretch group. Tube formation assay and Alizarin Red S staining results showed that angiogenic activity and calcification ability of CM was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in eac h cell type. CM was administered to the mouse calvarial defect model. Histological and radiological examination showed that the bone healing was promoted by CM from the cyclic stretch culture group. Immunohistological staining revealed that CM from cyclic stretch group have greater angiogenic effect than CM from the non stretch group.ConclusionsThese results indicate that osteogenesis was promoted by CM obtained under cyclic stretch stimulation through the increase of angiogenesis in the mouse calvarial defect model.     

Regulation of stretch-induced JNK activation by stress fiber orientation

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.     

An apparatus to study the response of cultured endothelium to shear stress

An apparatus which has been developed to study the response of cultured endothelial cells to a wide range of shear stress levels is described. Controlled laminar flow through a rectangular tube was used to generate fluid shear stress over a cell-lined coverslip comprising part of one wall of the tube. A finite element method was used to calculate shear stresses corresponding to cell position on the coverslip. Validity of the finite element analysis was demonstrated first by its ability to generate correctly velocity profiles and wall shear stresses for laminar flow in the entrance region between infinitely wide parallel plates (two-dimensional flow). The computer analysis also correctly predicted values for pressure difference between two points in the test region of the apparatus for the range of flow rates used in these experiments. These predictions thus supported the use of such an analysis for three-dimensional flow. This apparatus has been used in a series of experiments to confirm its utility for testing applications. In these studies, endothelial cells were exposed to shear stresses of 60 and 128 dynes/cm2. After 12 hr at 60 dynes/cm2, cells became aligned with their longitudinal axes parallel to the direction of flow. In contrast, cells exposed to 128 dynes/cm2 required 36 hr to achieve a similar reorientation. Interestingly, after 6 hr at 128 dynes/cm2, specimens passed through an intermediate phase in which cells were aligned perpendicular to flow direction. Because of its ease and use and the provided documentation of wall shear stress, this flow chamber should prove to be a valuable tool in endothelial research related to atherosclerosis.     

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.     

Collective influence of substrate chemistry with physiological fluid shear stress on human umbilical vein endothelial cells

In the treatment of cardiovascular diseases, vascular scaffold materials play an extremely important role. The appropriate substrate chemistries and 15 dynes/cm² physiological fluid shear stress (FSS) are both required to ensure normal physiological activity of human umbilical vein endothelial cells (HUVECs). The present study reported the collective influence of substrate chemistries and FSS on HUVECs in the sense of its biological functions. The CH₃, NH₂, and OH functional groups were adopted to offer a variety of substrate chemistries on glass slides by the technology of self-assembled monolayers, whereas FSS was generated by a parallel-plate fluid flow system. Substrate chemistries on its own by no means had noticeable effects on eNOS, ATP, NO, and PGI₂ expressions, while FSS stimuli enhanced their production. While substrate chemistries, as well as FSS, were both exerted, the releases of ATP, NO, and PGI₂ were dependent on substrate chemistries. Study of F-actin organization and focal adhesions (FAs) formation of HUVECs before FSS exposure proves that F-action organization and FAs formation followed similar chemistry-dependence. Hereby proposed a feasible mechanism, that is, the F-actin organization and FAs formation of HUVECs are controlled by substrate chemistries, further advancing the modulation of FSS-triggered responses of HUVECs.     

Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch

Hemodynamic forces affect endothelial cell morphology and function. In particular, circumferential cyclic stretch of blood vessels, due to pressure changes during the cardiac cycle, is known to affect the endothelial cell shape, mediating the alignment of the cells in the direction perpendicular to stretch. This change in cell shape proceeds a drastic reorganization at the internal level. The cellular scaffolding, mainly composed of actin filaments, reorganize in the direction which later becomes the cell’s long axis. How this external mechanical stimulus is ’sensed’ and transduced into the cell is still unknown. Here, we develop a mathematical model depicting the dynamics of actin filaments, and the influence of the cyclic stretch of the substratum based on the experimental evidence that external stimuli may be transduced inside the cell via transmembrane proteins which are coupled with actin filaments on the cytoplasmic side. Based on this view, we investigate two approaches describing the formulation of the transduction mechanisms involving the coupling between filaments and the membrane proteins. As a result, we find that the mechanical stimulus could cause the experimentally observed reorganization of the entire cytoskeleton simply by altering the dynamics of the filaments connected with the integral membrane proteins, as described in our model. Comparison of our results with previous studies of cytoskeletal dynamics reveals that the cytoskeleton, which, in the absence of the effect of stretch would maintain its isotropic distribution, slowly aligns with the precise direction set by the external stimulus. It is found that even a feeble stimulus, coupled with a strong internal dynamics, is sufficient to align actin filaments perpendicular to the direction of stretch.     

Melanocytes Respond to Mechanical Stretch by Activation of Mitogen‐Activated Protein Kinases (MAPK)

Cells of human epidermis are permanently targeted by mechanical stimuli. Besides mechanical forces from external sources the body itself generates mechanical forces via muscle contractions and growth processes. Recently, it was demonstrated that mechanical stretch is connected to enhanced proliferation in epidermal cells. The underlying biochemical events are still a matter of debate. Here we show that mechanical stretch leads to activation of both ERK1/2 and SAPK/JNK in human melanocytes and keratinocytes. In response to a 5 min single stretch ERK1/2 becomes moderately induced in melanocytes and peaked 30 min after the stimulus. In keratinocytes strong activation of ERK1/2 is present directly after the stimulus. SAPK/JNK shows the same activation pattern in both cell species – a slow but steady activation. The different kinetics of both MAPK suggest that different signalling cascades were activated. Future studies should evaluate the relevance of stretch‐dependent MAPK activation in triggering the cell proliferation.     

Arterial shear stress stimulates surface expression of the endothelial glycoprotein Ib complex

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.     

Lamin A/C Regulates Endothelial Glucocorticoid Receptor Nuclear Translocation in Response to Cyclic Stretch

The glucocorticoid receptor (GR) has multiple phosphorylation sites that can be activated by MAPKs, which have been previously shown to be activated in response to cyclic stretch in endothelial cells. It is possible therefore that physiological and/or pathological degree of cyclic stretch may also initiate phosphorylation-induced changes in GR subcellular localization as we previously showed with shear stress. However, little is known about the effects of cyclic stretch on glucocorticoid receptor (GR) activity in endothelial cells. We used control and lamin shRNA BAECs and subjected them to ligand (dexamethasone) treatment, physiological stretch (10% at 1 Hz), or pathological stretch (20% at 1 Hz or 10% at 2 Hz), in order to evaluate GR nuclear translocation in endothelial cells with and without lamin A/C as well as potential upstream protein regulators of GR subcellular movement during cyclic stretch. Upon exposure to pathological degrees of stretching, control shRNA BAECs showed greater nuclear concentration of GR at each time point compared to when they were stretched at physiological parameters. The response of GR in lamin-deficient cells to cyclic stretching was relatively non-existent compared to that observed in control shRNA cells. Our results suggest that in cells with lamin A/C, cyclic stretch activates GR through the JNK pathway, and ERK has some inhibitory role on GR nuclear translocation. DUSP proteins become upregulated in response to stretch as a result of GR activation (DUSP1) or by stretch-induced MAPK signaling. In lamin-deficient cells, only the combination of cyclic stretch and p38 inhibition was able to induce marginal nuclear translocation. Increased MAPK phosphorylation due to lamin A/C absence could drive DUSP expression as a negative feedback mechanism. Upregulation of the cytoplasmic DUSP6 suggests a significant role of ERK in reducing GR sensitivity to mechanical strain.     

Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

The elongation and orientation of cultured endothelial cells in response to shear stress

Vascular endothelial cells appear to be aligned with the flow in the immediate vicinity of the arterial wall and have a shape which is more ellipsoidal in regions of high shear and more polygonal in regions of low shear stress. In order to study quantitatively the nature of this response, bovine aortic endothelial cells grown on Thermanox plastic coverslips were exposed to shear stress levels of 10, 30, and 85 dynes/cm2 for periods up to 24 hr using a parallel plate flow chamber. A computer-based analysis system was used to quantify the degree of cell elongation with respect to the change in cell angle of orientation and with time. The results show that (i) endothelial cells orient with the flow direction under the influence of shear stress, (ii) the time required for cell alignment with flow direction is somewhat longer than that required for cell elongation, (iii) there is a strong correlation between the degree of alignment and endothelial cell shape, and (iv) endothelial cells become more elongated when exposed to higher shear stresses.     

Mechanisms of coronary angiogenesis in response to stretch: role of VEGF and TGF-beta

To test the hypotheses that cyclic stretch of 1) cardiac myocytes produces factors that trigger angiogenic events in coronary microvascular endothelial cells (CMEC) and 2) CMEC enhances the expression of growth factors, cardiac myocytes and CMEC were subjected to cyclic stretch in a Flexercell Strain Unit. Vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor mRNA and protein levels increased approximately twofold in myocytes after 1 h of stretch. CMEC DNA synthesis increased approximately twofold when conditioned medium from stretched myocytes or VEGF protein was added, and addition of VEGF neutralizing antibody blocked the increase. CMEC migration and tube formation increased with the addition of conditioned media but were markedly attenuated by VEGF neutralizing antibody. Myocyte transforming growth factor-beta [corrected] (TGF-beta) increased 2.5-fold after 1 h of stretch, and the addition of TGF-beta neutralizing antibodies inhibited the stretch-induced upregulation of VEGF. Stretch of CMEC increased VEGF mRNA in these cells (determined by Northern blot and RT-PCR) and increased the levels of VEGF protein (determined by ELISA analysis) in the conditioned media. Therefore, cyclic stretch of cardiac myocytes and CMEC appears to be an important primary stimulus for coronary angiogenesis through both paracrine and autocrine VEGF pathways. These data indicate that 1) CMEC DNA synthesis, migration, and tube formation are increased in response to VEGF secreted from stretched cardiac myocytes; 2) VEGF in CMEC subjected to stretch is upregulated and secreted; and 3) TGF-beta signaling may regulate VEGF expression in cardiac myocytes.     

Oscillatory shear alters endothelial hydraulic conductivity and nitric oxide levels

This study addresses the role of nitric oxide (NO) and downstream signaling pathways in mediating the influences of oscillatory shear stress on the hydraulic conductivity (L(p)) of bovine aortic endothelial cell (BAEC) monolayers. Exposure of BAEC monolayers to 20 dyne/cm2 steady shear stress for 3 h induced a 3.3-fold increase in L(p). When an oscillatory shear amplitude of 10 dyne/cm2 was superimposed on a steady shear of 10 dyne/cm2 to produce a non-reversing oscillatory shear pattern (10+/-10 dyne/cm2), L(p) increased by 3.0-fold within 90 min. When the amplitude was increased to 15 dyne/cm2, resulting in a reversing oscillatory shear pattern (10+/-15 dyne/cm2), the increase in L(p) over 3 h was completely suppressed. Twenty and 10+/-10 dyne/cm2 induced 2.9- and 2.6-fold increases in NO production above non-sheared controls, respectively, whereas 10+/-15 dyne/cm2 stimulated a 14-fold increase in NO production. The inhibition of L(p) with reversing oscillatory shear may be associated with alterations in cyclic guanosine monophosphate (cGMP) production downstream of NO which is up-regulated by reversing oscillatory shear, but is unaffected by steady shear.