Charles R. White

Temporal Gradients in Shear, but Not Spatial Gradients, Stimulate Endothelial Cell Proliferation

Background—The effect of temporal and spatial gradients in shear on primary human endothelial cell (HUVEC) proliferation was investigated. The sudden-expansion flow chamber (SEFC) model was used to differentiate the effect of temporal gradients in shear from that of spatial gradients. With a sudden onset of flow, cells are exposed to both temporal and spatial gradients of shear. The temporal gradients can be eliminated by slowly ramping up the flow. Methods and Results—HUVEC proliferation in the SEFC remained unstimulated when the onset of flow was slowly ramped. Sudden onset of flow stimulated a 105% increase of HUVEC proliferation (relative to ramped onset) within the region of flow reattachment. To further separate temporal and spatial gradients, a conventional parallel-plate flow chamber was used. A single 0.5-second impulse of 10 dyne/cm2 increased HUVEC proliferation 54±3% relative to control. When flow was slowly ramped over 30 seconds, HUVEC proliferation was not significantly different from controls. Steady laminar shear over 20 minutes inhibited HUVEC proliferation relative to controls regardless of step (36±8%) or ramp (21±5%) onsets of flow. Conclusions—The results indicate that temporal gradients in shear stress stimulate endothelial cell proliferation, whereas spatial gradients affect endothelial proliferation no differently than steady uniform shear stress.

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endotheliums ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell–cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.

PECAM-1 Mediates NO-Dependent Dilation of Arterioles to High Temporal Gradients of Shear Stress

Objective—In response to changes in wall shear stress (WSS) the vascular endothelium releases several factors, among others nitric oxide. On the basis of studies of endothelial cells in culture, suggesting that platelet endothelial cell adhesion molecule-1 (PECAM-1) is specifically involved in sensing and coupling high temporal gradients of fluid shear stress with activation of eNOS, we hypothesized that dilations of isolated skeletal muscle arterioles from PECAM-1 knockout mice (PECAM-KO) will be reduced to rapid increases in WSS elicited by increases in perfusate flow. Methods and Results—Small and large step increases in flow resulted in substantial dilations in arterioles of WT mice (45±4%), but they were markedly reduced in arterioles of PECAM-KO mice (22±5%). The initial slope of dilations, when WSS increased rapidly, was greater in vessels of WT than those of PECAM-KO mice (slopes: 0.378 and 0.094, respectively), whereas the second phase of dilations, when flow/shear stress was steady, was similar in the 2 groups (slopes: 0.085 and 0.094, respectively). Inhibition of eNOS significantly reduced the initial phase of dilations in arterioles from WT, but not from those of PECAM-KO mice. The calcium ionophore A23187 elicited similar NO-mediated dilation in both WT and PECAM-KO mice. Conclusions—In isolated arterioles of PECAM-KO mice activation of eNOS and consequent dilation by agonists is maintained, but the dilation to high temporal gradients of wall shear stress elicited by increases in perfusate flow is reduced. Thus, we propose that PECAM-1 plays an important role in the ability of the endothelium to sense and couple high temporal gradients of wall shear stress to NO-mediated arteriolar dilation during sudden changes in blood flow in vivo.

Rapid Activation of Ras by Fluid Flow Is Mediated by Gαq and Gβγ Subunits of Heterotrimeric G Proteins in Human Endothelial Cells

Objective—Temporal gradients in fluid shear stress have been shown to induce a proatherogenic phenotype in endothelial cells. The biomechanical mechanism(s) that enables the endothelium to respond to fluid shear stress requires rapid activation and signal transduction. The small G protein Ras has been identified as an early link between rapid mechanotransduction events and the effects of shear stress on downstream signal-transduction cascades. The aim of this study was to elucidate the upstream mechanotransduction signaling events mediating the rapid activation of Ras by fluid shear stress in human endothelial cells. Methods and Results—Direct measurement of Ras-bound GTP and GDP showed that fluid-flow activation of Ras was rapid (10-fold within 5 seconds) and dose dependent on shear stress magnitude. Treatment with protein tyrosine kinase inhibitors or pertussis toxin did not significantly affect flow-induced Ras activation. However, activation was inhibited by transient transfection with antisense to G&agr;q or the G&bgr;&ggr; scavenger &bgr;-adrenergic receptor kinase carboxy terminus. Transfection with several G&bgr;&ggr; subunit isoforms revealed flow-induced Ras activation was most effectively enhanced by G&bgr;1&ggr;2. Conclusions—These results suggest that the rapid, shear-induced activation of Ras is mediated by G&agr;q through the activity of G&bgr;&ggr; subunits in human vascular endothelial cells.

Role of Nitric Oxide in Hypoxic Cerebral Vasodilatation in the Ovine Fetus

To investigate the role of nitric oxide (NO) in fetal cerebral circulatory responses to acute hypoxia, near‐term fetal sheep were instrumented with laser Doppler probes placed in the parasagittal parietal cortices and vascular catheters in the sagittal sinus and brachiocephalic artery. After a 3 day recovery period, responses of cortical blood flow (CBF) to hypoxia were compared with and without inhibition of nitric oxide synthase (NOS). After an initial 30 min baseline period, fetuses were given a bolus followed by a continuous infusion of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), or saline vehicle as control. After administration of l‐NAME, CBF decreased by 14 ± 6 % (P < 0.01) despite increases in arterial blood pressure of 15 mmHg, resulting in an ∼60 % increase in cerebrovascular resistance. Thirty minutes following initiation of l‐NAME or vehicle infusion, fetal systemic hypoxia was induced by allowing the ewes to breathe 10–11 % oxygen. In control fetuses CBF increased progressively to 145 ± 9 % of baseline (P < 0.01) after 30 min, while cortical release of cyclic guanylate cyclase (cGMP), an index of NOS activity, increased 26 ± 8 % (P < 0.05). In contrast, CBF in l‐NAME‐treated fetuses increased to only 115 % of the reduced CBF baseline, whereas cortical release of cGMP did not change significantly. In summary, basal levels of NO lower resting cortical vascular resistance by ∼15 % in the fetal sheep. Inhibition of NO synthesis attenuates hypoxic cerebral relaxation but does not completely prevent the characteristic increases in CBF. Hypoxic increases in NO directly increase cortical production of cGMP and inhibition of NO synthesis ablates these changes in cGMP.

Analysis of Temporal Shear Stress Gradients During the Onset Phase of Flow Over a Backward-Facing Step

Endothelial cells in blood vessels are exposed to bloodflow and thus fluid shear stress. In arterial bifurcations and stenoses, disturbed flow causes zones of recirculation and stagnation, which are associated with both spatial and temporal gradients of shear stress. Such gradients have been linked to the generation of atherosclerotic plaques. For in-vitro studies of endothelial cell responses, the sudden-expansion flow chamber has been widely used and described. A two-dimensional numerical simulation of the onset phase of flow through the chamber was performed. The wall shear stress action on the bottom plate was computed as a function of time and distance from the sudden expansion. The results showed that depending on the time for the flow to be established, significant temporal gradients occurred close to the second stagnation point of flow. Slowly ramping the flow over 15 s instead of 200 ms reduces the temporal gradients by a factor of 300, while spatial gradients are reduced by 23 percent. Thus, the effects of spatial and temporal gradients can be observed separately. In experiments on endothelial cells, disturbed flow stimulated cell proliferation only when flow onset was sudden. The spatial patterns of proliferation rate match the exposure to temporal gradients. This study provides information on the dynamics of spatial and temporal gradients to which the cells are exposed in a sudden-expansion flow chamber and relates them to changes in the onset phase of flow.

Endothelium-derived relaxing factor and cyclic GMP-dependent vasorelaxation in human chorionic plate arteries

Endothelium derived relaxing factor (EDRF), now widely believed to be nitric oxide (NO), may play an important part in the control of fetoplacental vascular tone. To further explore this role we have determined the relaxation responses to exogenous NO and examined the temporal relationship between intracellular concentrations of cyclic GMP and vascular tone in isolated ring segments of human chorionic plate arteries. We have also determined the dose relations for the contractile agonists serotonin and the thromboxane analog U46619. Lastly, we have explored the relaxation responses to a wide range of agents known to elicit EDRF release in other vascular beds. Chorionic plate arteries relaxed significantly to exogenous NO with concomitant increases in cyclic guanosine monophosphate over basal values. ED50s for serotonin and U46619 were 1.48 x 10(-6) M and 3.39 x 10(-8) M respectively. The ED50 for NO derived from S-nitroso-N-acetyl-penicillamine was 1.28 x 10(-6) M. Endothelium-intact segments of chorionic plate arteries pre-contracted with either serotonin or U46619 failed to relax significantly to acetylcholine, adenosine diphosphate, A23187, bradykinin, and histamine and only minimally to substance P. We suggest that EDRF is likely to be important in the control of placental vascular tone, but that it is not possible to demonstrate its action in an unperfused experimental system.

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule‐1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule‐1 (PECAM‐1) expression is pro‐atherogenic. PECAM‐1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane‐bound G proteins Gαq/11 also mediate flow‐induced responses. Here, we investigated whether PECAM‐1 and Gαq/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co‐localization of Gαq/11 and PECAM‐1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gαq/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM‐1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gαq/11–PECAM‐1 complex within 30 s and a partial relocalization of the Gαq/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow‐induced Gαq/11–PECAM‐1 dissociation. These results allow us to conclude that Gαq/11–PECAM‐1 forms a mechanosensitive complex and its localization suggests the Gαq/11–PECAM‐1 complex is a critical mediator of vascular diseases.

Effects of Maturation on Cyclic GMP-Dependent Vasodilation in Ovine Basilar and Carotid Arteries

ABSTRACT: The present experiments examine the effects of maturation on cyclic GMP (cGMP)-mediated vasodilation in 688 segments of common carotid (COM) and basilar (BAS) arteries taken from newborn (3− to 7-d-old) and nonpregnant adult sheep. The main finding is that maximum efficacy for relaxation decreased with maturation in both artery types for the nitric oxide releasing vasodilators S-nitroso-N-acetyl-penicillamine and nitroglycerin. These decreases could not be explained by changes in the -log ED50 concentrations for either vasodilator. Determination of the time course of cGMP responses to S-nitroso-N-acetyl-penicillamine or nitroglycerin at 10 μM revealed that the peak cGMP responses to these agents (range: 5.3 ± 0.8 to 8.3 ±1.6 pmol/mg of protein) also did not vary significantly with age. However, cGMP attained peak values more rapidly in adult (COM: 50 s; BAS 30 s) than in newborn (COM: 60–80 s: BAS, 40–60 s) segments and returned to baseline more slowly in newborn than in adult segments, suggesting that maturation accelerates cGMP turnover. Correspondingly, baseline levels of cGMP were higher in newborn (COM: 1.0 ± 0.1; BAS: 3.3 ± 0.5 pmol/mg of protein) than in adult (COM: 0.3 ±0.1; BAS: 1.7 ± 0.2 pmol/mg of protein) segments. Despite these differences in cGMP time course, rates of relaxation in response to S-nitroso-N-acetyl-penicillamine and nitroglycerin did not vary significantly with age, indicating that the temporal relation between cGMP and relaxation is different in newborn and adult arteries. Together, these results suggest that the capacity of the cGMP pathway to produce relaxation is attenuated by maturation through changes possibly related to cGMP turnover.

Extracellular signal‐regulated kinase activation and endothelin‐1 production in human endothelial cells exposed to vibration

Hand–arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynauds phenomenon. Chronic exposure to hand‐held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low‐volume fluid oscillation. Rapid low‐volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal‐regulated kinase (ERK1/2) phosphorylation and endothelin‐1 (ET‐1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET‐1 release compared to steady flow, and endothelin‐1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium.