Laminar shear stress-induced GRO mRNA and protein expression in endothelial cells

BACKGROUND: The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS: The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS: The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.     

Flow modulates endothelial regulation of smooth muscle cell proliferation: a new model

BACKGROUND: With a co-culture model, we have previously demonstrated that endothelial cells (ECs) exert regulatory control over smooth muscle cell (SMC) behavior. ECs appeared to stimulate SMC proliferation in static culture. This study was performed to test the hypothesis that the EC stimulation of SMC proliferation was effected by shear stress. METHODS: Bovine SMCs were cultured on a thin semipermeable membrane either alone or opposite ECs in co-culture (SMC/EC). A novel parallel-plate flow device was developed and used for exposing the EC side of the co-culture to shear stress. EC and SMC proliferation rates were determined after 24 hours' exposure to 0, 1, or 10 dynes/cm2 of shear stress. RESULTS: SMC proliferation decreased significantly from 362 +/- 65 cpm/microgram DNA (control, mean +/- SEM) to 68 +/- 43 cpm/microgram (1 dyne/cm2) and 99 +/- 18 cpm/microgram (10 dynes/cm2)(P < .05). EC proliferation after flow decreased as compared with no-flow controls 71 +/- 15 cpm/micrograms DNA (control, mean +/- SEM) to 29 +/- 5 cpm/microgram (1 dyne/cm2) and 21 +/- 4 cpm/microgram (10 dynes/cm2)(P < .05). CONCLUSIONS: In a model designed to study SMC/EC interactions in a flow environment, it was seen that EC exposure to shear stress alters the growth characteristics of SMCs. This suggests that hemodynamic mechanical forces may be sufficient to alter the EC regulation of SMC behavior.     

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

OBJECTIVE: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. METHODS: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with beta-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. RESULTS: The pretreatment with 25 dyne/cm2 but not with 0 or 1 dyne/cm2 shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that beta-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained alpha-smooth muscle actin were present. The thickness of this neointima averaged 41 +/- 12 micrometer and 60 +/- 23 micrometer in endothelial cell-seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 +/- 46 micrometer in grafts that were not seeded with endothelial cells. CONCLUSION: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness.     

Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes

Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia. Recently, it was shown that pulsatile myocardial stretch in vivo markedly enhanced VEGF mRNA level in the heart. To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using an in vitro preparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch. We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes. We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-beta. These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia.     

Balantidiasis in Aymara children from the northern Bolivian Altiplano

Different forms of mechanical stimulation are among the physiological factors constantly acting on the vessel wall. We previously demonstrated that subjecting vascular smooth muscle cells (VSMCs) in culture to cyclic stretch increased the expression of high-molecular-weight caldesmon, a marker protein of a differentiated, contractile, VSMC phenotype. In the present work the effects of mechanical factors, in the form of circumferential stress and shear stress, on the characteristics of SM contractile phenotype were studied in an organ culture of rabbit aorta. Application of an intralumininal pressure of 80 mm Hg to aortic segments cultured in Dulbecco's modified Eagle's medium containing 20% fetal calf serum for 3 days prevented the decrease in high-molecular-weight caldesmon content (70+/-4% of initial level in nonpressurized vessel, 116+/-17% at 80 mm Hg) and filamin content (80+/-5% in nonpressurized vessel, 100+/-2% at 80 mm Hg). SM myosin and low-molecular-weight caldesmon contents showed no dependence on vessel pressurization. Neither endothelial denudation nor alteration of intraluminal flow rates affected marker protein content in 3-day vessel culture, thus excluding the possibility of any shear or endothelial effects. Maintenance of high high-molecular-weight caldesmon and filamin levels in the organ cultures of pressurized and stretched vessels demonstrates the positive role of mechanical factors in the control of the VSMC differentiated phenotype.     

Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells

We investigated the involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in human endothelial cells in response to shear stress and alterations of these kinases in in vitro-propagated endothelial cells (ECs). Potent activation (10-fold) of extracellular signal-regulated kinase (ERK2), a member of the MAPK family, occurred within 10 min of shear stress (5 dynes/cm2), whereupon rapid inactivation ensued. Shear stress also induced activation of stress-activated protein kinase (SAPK) or c-Jun NH2-terminal protein kinase (JNK) in ECs. Suramin pretreatment completely inhibited shear stress stimulation of ERK2, but not SAPK/JNK, highlighting a role for growth factor receptors in ERK activation. Translocation of ERK2 from the cytoplasm to the nucleus was observed in shear-stressed endothelial cells. In addition, we compared activities of MAPKs in shear-stressed cells derived from passages 4 and 10 (older). The magnitude of ERK2 activation was significantly lower in aged ECs compared to those of passage 4, while SAPK/JNK was not altered in the in vitro aged ECs. A similar level of ERK2 activation was found in both young and older cells stimulated with phorbol-12-myristate-13-acetate (PMA), indicating an age-related alteration of the plasma membrane. Taken together, these findings suggest that MAP kinase activation may be crucial for the expression of many genes in ECs stimulated by shear stress, and that an alteration in MAPK activities could contribute to the age-related decline in proliferative capacity.     

Pediatric emergency medicine--legal briefs

BACKGROUND: The lung expresses large amounts of endothelin-converting enzyme-1 (ECE-1), which catalyzes a step in the biosynthesis of potent vasoactive endothelin-1 (ET-1) from the inactive intermediate big ET-1. Because there has been no report concerning a possible relationship between ET-1 and ECE-1, we investigated the effects of ET-1 on ECE-1 expression in cultured rat pulmonary endothelial cells. METHODS AND RESULTS: ECE-1 messenger RNA (mRNA) and protein expression in cultured endothelial cells were assayed by Northern and Western blotting, respectively. Incubation with ET-1 for 6 hours caused a significant decrease in ECE-1 mRNA expression. The action of ET-1 on ECE-1 mRNA expression was antagonized by pretreatment with BQ788, a specific ETB receptor antagonist, but not by pretreatment with BQ123, a specific ETA receptor antagonist. The expression of ECE-1 protein was also inhibited at 6 hours after incubation with ET-1. The effects of ET-1 on ECE-1 mRNA and protein expression were shown to be mimicked by ionomycin, a calcium ionophore, but not by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator. CONCLUSIONS: The present results demonstrate that ET-1 suppressed ECE-1 protein levels by inhibiting ECE-1 mRNA expression through the ETB receptor, suggesting the existence of a feedback action of ET-1 on ECE-1 in pulmonary endothelial cells.     

Shear stress-induced release of PGE2 and PGI2 by vascular smooth muscle cells

This study addresses the direct effect of fluid flow shear stress on production of the vascular mediators, PGE2 and PGI2 by vascular smooth muscle cells (SMC). Results indicate that shear stress increases PGE2 and PGI2 release in SMC. The production patterns, however, differ between PGE2 and PGI2. For PGE2, the rate of production is moderate for the first three hours after the onset of shear stress, then dramatically increases between the fourth and fifth hours, returning to basal levels in the sixth hour. On the other hand, the rate for PGI2 production is maximal right after the onset of shear and remains elevated for the first three hours. The rate then plateaus and remains at a moderate level during the next three hours. The results also indicate that SMC production of PGI2 is more sensitive to shear stress than PGE2 production since a level of 0.5 dynes/cm2 produces a maximal PGI2 release whereas 1 dyne/cm2 produces only 1/4 the response seen at 20 dynes/cm2 for PGE2. The physiological implications of fluid shear stress regulation of SMC are discussed.     

Endothelin-1 secretion from cultured vascular endothelial cells of DOCA-salt hypertensive rats

The profile of endothelin-1 (ET-1) release from cultured vascular endothelial cells (ECs) obtained from deoxycorticosterone acetate (DOCA)-salt hypertensive rats, was examined and compared with that from normotensive sham rats. ET-1 release from ECs was increased in a time-dependent manner, and the level of DOCA-salt hypertensive rats was higher than that of sham rats. Incubation of ECs with transforming growth factor (TGF)-beta 1 or thrombin resulted in a significant increase in the ET-1 release, while FK409, a novel nitric oxide donor, produced a dose-dependent decrease in the release. In the case of ECs from DOCA-salt hypertensive rats, the potencies of TGF-beta 1- or thrombin-induced action was much less than that seen with sham rats, while the difference of reactivity to FK409 was not observed between ECs of DOCA-salt rats and sham rats. Thus, ET-1 production in ECs appears to be up-regulated in DOCA-salt hypertensive rats. In addition, there seems to be an abnormalities in the signaling pathway via TGF-beta 1- or thrombin-induced enhancement of ET-1 production in ECs of DOCA-salt hypertensive rats.     

Shear stress gradients remodel endothelial monolayers in vitro via a cell proliferation-migration-loss cycle

Wall shear stress has been implicated in the genesis of atherosclerosis because a strong correlation exists between the location of developing arterial lesions and regions where particular gradients in stress occur. Studying the behavior of endothelial cells in such regions may contribute to our understanding of the disease etiology. We report the detailed migratory history of endothelial cells subjected to large shear stress gradients caused by a surface protuberance in an in vitro model system. The history of cell migration, cell division, and cell loss from the surface was continuously monitored in confluent human umbilical vein endothelial cell monolayers for 48 hours after the onset of flow. Individual cells were tracked using time-lapse video microscopy. In contrast to a uniform laminar flow field in which cells were observed to continually rearrange their relative position with no net migration, in a disturbed flow field there was a net migration directed away from the region of high shear gradient. This organized migration pattern under disturbed flow conditions was accompanied by more than a twofold increase in cell motility. In addition, cell division increased in the vicinity of the flow separation (maximum shear stress gradient of 34 dyne/cm2 per mm) whereas cell loss was increased upstream and downstream in the regions where the shear gradient diminishes. These data suggest a steady cell proliferation-migration-loss cycle and indicate that local shear stress gradient may play a key role in the morphological remodeling of the vascular endothelium in vivo.     

Characterization of a sudden expansion flow chamber to study the response of endothelium to flow recirculation

In order to simulate regions of flow separation observed in vivo, a conventional parallel plate flow chamber was modified to produce an asymmetric sudden expansion. The flow field was visualized using light reflecting particles and the size of the recirculation zone was measured by image analysis of the particles. Finite element numerical solutions of the two and three-dimensional forms of the Navier-Stokes equation were used to determine the wall shear stress distribution and predict the location of reattachment. For two different size expansions, numerical estimates of the reattachment point along the centerline of the flow chamber agreed well with experimental values for Reynolds numbers below 473. Even at a Reynolds number of 473, the flow could be approximated as two-dimensional for 80 percent of the chamber width. Peak shear stresses in the recirculation zone as high as 80 dyne/cm2 and shear stress gradients of 2500 (dyne/cm2)/cm were produced. As an application of this flow chamber, subconfluent bovine aortic endothelial cell shape and orientation were examined in the zone of recirculation during a 24 h exposure to flow at a Reynolds number of 267. After 24 h, gradients in cell orientation and shape were observed within the recirculation zone. At the location of reattachment, where the wall shear stress was zero but the shear stress gradients were large, cells plated at low density were still aligned with the direction of flow. No preferred orientation was observed at the gasket edge where the wall shear stress and shear stress gradients were zero. At higher cell densities, no alignment was observed at the separation point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the stability of technetium-labeled peptides to challenge with cysteine

-Tacrolimus (FK 506) is a powerful, widely used immunosuppressant. The clinical utility of FK 506 is complicated by substantial hypertension and nephrotoxicity. To clarify the mechanisms of FK 506-induced hypertension, we studied the chronic effects of FK 506 on the synthesis of endothelin-1 (ET-1), the expression of mRNA of ET-1 and endothelin-converting enzyme-1 (ECE-1), the endothelial nitric oxide synthase (eNOS) activity, and the expression of mRNA of eNOS and C-type natriuretic peptide (CNP) in rat blood vessels. In addition, the effect of the specific endothelin type A receptor antagonist FR 139317 on FK 506-induced hypertension in rats was studied. FK 506, 5 mg. kg-1. d-1 given for 4 weeks, elevated blood pressure from 102+/-13 to 152+/-15 mm Hg and increased the synthesis of ET-1 and the levels of ET-1 mRNA in the mesenteric artery (240% and 230%, respectively). Little change was observed in the expression of ECE-1 mRNA and CNP mRNA. FK 506 decreased eNOS activity and the levels of eNOS mRNA in the aorta (48% and 55%, respectively). The administration of FR 139317 (10 mg. kg-1. d-1) prevented FK 506-induced hypertension in rats. These results indicate that FK 506 may increase blood pressure not only by increasing ET-1 production but also by decreasing NO synthesis in the vasculature.     

Effect of vasopressin antagonism on structure and mechanics of small arteries and vascular expression of endothelin-1 in deoxycorticosterone acetate salt hypertensive rats

The structural and mechanical properties of small arteries are altered in rat models of hypertension. The precise role of humoral factors in these changes has not been determined. In deoxycorticosterone acetate (DOCA) salt hypertension, endothelin-1 (ET-1) peptide content and gene expression are enhanced in mesenteric resistance arteries. These vessels also present augmented vasoconstrictor responsiveness to vasopressin versus control uninephrectomized rats. To determine whether an interaction exists between vasopressin and ET-1 in the pathogenesis of small-artery structural alterations in DOCA-salt rats, we examined the effect of chronic V1 vasopressin receptor antagonism (OPC-21268, 30 mg/kg BID) on the structure and mechanical properties of mesenteric resistance arteries using a pressure myograph and the effect on preproendothelin-1 (preproET-1) gene expression, determined by Northern blot analysis of preproET-1 mRNA. Tail-cuff systolic pressures were elevated in DOCA-salt (200+/-11 mm Hg) versus uninephrectomized rats (109+/-4 mm Hg) and decreased slightly but significantly by OPC-21268 to 187+/-7 mm Hg (P<0.01). Treatment with DOCA-salt increased vascular media-lumen ratios and media cross-sectional areas and reduced both stress and incremental elastic modulus for a given pressure. However, there was no change in distensibility or incremental elastic modulus versus media stress. OPC-21268 partially attenuated the vascular growth in DOCA-salt rats. PreproET-1 mRNA was increased 2-fold in mesenteric arteries of DOCA-salt rats versus uninephrectomized rats, an effect abrogated by OPC-21268. Thus, DOCA-salt hypertension is associated with altered morphology of the small-arterial wall, without altering stiffness of the arterial wall components. OPC-21268 regressed in part these changes, suggesting the involvement of vasopressin. The concomitant attenuation of enhanced ET-1 expression by OPC-21268 suggests that ET-1 may be involved in mediating in part the vascular effects of vasopressin in DOCA-salt hypertensive rats.     

A biomathematical model of intracranial arteriovenous malformations based on electrical network analysis: theory and hemodynamics

Hemodynamics play a significant role in the propensity of intracranial arteriovenous malformations (AVMs) to hemorrhage and in influencing both therapeutic strategies and their complications. AVM hemodynamics are difficult to quantitate, particularly within or in close proximity to the nidus. Biomathematical models represent a theoretical method of investigating AVM hemodynamics but currently provide limited information because of the simplicity of simulated anatomic and physiological characteristics in available models. Our purpose was to develop a new detailed biomathematical model in which the morphological, biophysical, and hemodynamic characteristics of an intracranial AVM are replicated more faithfully. The technique of electrical network analysis was used to construct the biomathematical AVM model to provide an accurate rendering of transnidal and intranidal hemodynamics. The model represented a complex, noncompartmentalized AVM with 4 arterial feeders (with simulated pial and transdural supply), 2 draining veins, and a nidus consisting of 28 interconnecting plexiform and fistulous components. Simulated vessel radii were defined as observed in human AVMs. Common values were assigned for normal systemic arterial pressure, arterial feeder pressures, draining vein pressures, and central venous pressure. Using an electrical analogy of Ohm's law, flow was determined based on Poiseuille's law given the aforementioned pressures and resistances of each nidus vessel. Circuit analysis of the AVM vasculature based on the conservation of flow and voltage revealed the flow rate through each vessel in the AVM network. Once the flow rate was established, the velocity, the intravascular pressure gradient, and the wall shear stress were determined. Total volumetric flow through the AVM was 814 ml/min. Hemodynamic analysis of the AVM showed increased flow rate, flow velocity, and wall shear stress through the fistulous component. The intranidal flow rate varied from 5.5 to 57.0 ml/min with and average of 31.3 ml/min for the plexiform vessels and from 595.1 to 640.1 ml/min with an average of 617.6 ml/min for the fistulous component. The blood flow velocity through the AVM nidus ranged from 11.7 to 121.1 cm/s with an average of 66.4 cm/s for the plexiform vessels and from 446.9 to 480 dyne/cm2 with an average of 463.5 dyne/cm2 for the fistulous component. The wall shear stress ranged in magnitude from 33.2 to 342.1 dyne/cm2 with an average of 187.7 dyne/cm2 for the plexiform vessels and from 315.9 to 339.7 cm/s with an average of 327.8 cm/s for the fistulous component. The described novel biomathematical model characterizes the transnidal and intranidal hemodynamics of an intracranial AVM more accurately than was possible previously. This model should serve as a useful research tool for further theoretical investigations of intracranial AVMs and their hemodynamic sequelae.