John A. Frangos

Mechanism of shear-induced prostacyclin production in endothelial cells

Human umbilical vein endothelial cells at confluence were subjected to steady shear flow. It was previously shown that flow induced a burst in prostacyclin production followed by a sustained stimulation of production several fold higher than basal levels (1). In the presence of EGTA, prostacyclin production was inhibited in the steady state phase by 74%. Preincubation of endothelial cells with quin2/AM, used here as an intracellular calcium chelator, also inhibited the production of prostacyclin (83%). Inhibition of intracellular calcium mobilization had no significant effect. Incubation of cells with nifedipine, a voltage operated channel blocker, had no effect on shear induced prostacyclin production, whereas ibuprofen decreased shear induced prostacyclin production. RHC-80267, a diacylglycerol lipase inhibitor, inhibited 66% of shear induced PGI2 production. Our results suggest that both extracellular and intracellular Ca2+ are necessary and the phospholipase C pathway may be the main source for liberating arachidonic acid in shear induced prostacyclin production.

Fluid Shear Stress Stimulates Membrane Phospholipid Metabolism in Cultured Human Endothelial Cells

There is evidence suggesting that fluid shear stress activates phospholipid turnover in endothelial cells, but it is not clear which phospholipids are involved in the transduction of the flow signal. Cultured human umbilical-vein endothelial cells were prelabeled with [14C]-arachidonic acid and subjected to laminar shear stresses of 0.4, 1.4 and 22 dyn/cm2 for times up to 30 min, after which the distribution of the radioactivity in the phospholipids was determined. We observed decreases in labeled phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid at 10-30 s, and increases in labeled diacylglycerol (DG) and free arachidonate, as well as a simultaneous elevation in inositol 1,4,5-triphosphate (IP3) levels. A second peak in IP3 levels was observed 10 min after the onset of shear. This is in contrast with agonist-stimulated endothelial cells, where IP3 levels go back to initial values within a few minutes after stimulation. The flow-induced IP3 response was the same in the presence or absence of ATP and serum in the perfusing medium. These results are consistent with the activation of phospholipase C, phospholipase A2 and DG lipase by shear stress. This suggests that several phospholipids are involved in the production of free arachidonic acid and DG, which are likely to be important mediators of the shear stress signal. In addition, flow may lead to a chronic stimulation of endothelial-cell metabolism.

Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein

Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells, G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDPβS and pertussis toxin inhibited flow‐induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine‐induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin‐sensitive G protein and modulates the stimulus‐response coupling of other agonists.

Exogenous, basal, and flow-induced nitric oxide production and endothelial cell proliferation

The role of nitric oxide (NO) from endogenous and exogenous sources in regulating large vessel and microvascular endothelial cell proliferation was investigated. Exogenous NO liberated from five different chemical donors inhibited bovine aortic, bovine retinal microvascular, and human umbilical vein endothelial cell proliferation in a dose‐dependent manner as determined by 3H‐thymidine incorporation. The potency of the donors varied as a function of the donors half‐lives. Donors with half‐lives greater than 30 min were more effective than donors with significantly shorter half‐lives. Coincubation of endothelial cells with 0.4 mM deoxyadenosine and 0.4 mM deoxyguanosine reduced the percentage of inhibition due to an NO donor. These data are consistent with a ribonucleotide reductase‐dependent mechanism of inhibition. Inhibition of basal NO production with four different inhibitors of nitric oxide synthase (NOS) did not modify proliferation. Laminar flow with a wall shear stress of 22 dyn/cm2inhibited the proliferation of subconfluent bovine aortic endothelial cells. The addition of a NOS inhibitor did not abrogate the flow‐induced inhibition of proliferation, suggesting that flow‐stimulated release of NO from endothelial cells did not account for flow‐induced inhibition of proliferation. Taken together, these data suggest that relatively large concentrations of exogenous NO inhibit endothelial cell proliferation, while endogenous levels of NO are inadequate to inhibit proliferation. J. Cell. Physiol. 171:252–258, 1997.

Protein kinase C mediates flow-induced prostaglandin E2 production in osteoblasts

SummaryInterstitial fluid flow generated by skeletal loading may be responsible for load-induced bone remodeling. Production of prostaglandin E2 (PGE2), a potent mediator of bone remodeling, is augmented in osteoblasts exposed to fluid flow. Exposure to fluid flow resulted in a slight initial increase in PGE2 production (1–2 hour), followed by a dramatic increase (2–8 hours). The initial phase of only slightly increased PGE2 production was dependent on substrate availability. H7, a protein kinase C inhibitor, strongly inhibited flow-induced prostaglandin E2 production at all time points examined without effecting production in stationary cultures. Blocking protein synthesis with cycloheximide resulted in a 56% reduction in long-term flow-induced PGE2 production. Thus, the later phase appeared to be the result of an increased number of enzymes as well as increased activity of existing enzymes or increased substrate availability. In conclusion, fluid flow increases PGE2 production in osteoplasts via a protein kinase C-dependent pathway involvingde novo protein synthesis.

Effects of flow on the synthesis and release of fibronectin by endothelial cells.

SummaryHuman umbilical vein endothelial cells at confluence were subjected to steady shear flow. The effect of flow on the synthesis of fibronectin, its release into the medium, and incorporation into the extracellular matrix were investigated. The total content of fibronectin in endothelial cells exposed to flow was found to be lower than that in static controls after periods of 12 to 48 h. In the presence of cycloheximide there was no difference in the fibronectin content of sheared and unsheared cells. Our results suggest that the synthesis of fibronectin is inhibited by the flow-induced perturbation of endothelial cells.

Fluid flow increases membrane permeability to merocyanine 540 in human endothelial cells.

Fluid shear stress is a ubiquitous stimulus of mammalian cell metabolism; however, its signal transduction pathway is unknown. We hypothesized that shear stress may alter some physical properties of the cell membrane. Using primary human umbilical vein endothelial cells (HUVECs), we investigated the effects of shear on the cell membrane by monitoring flow-induced changes in the uptake of the amphipath merocyanine 540 (MC540). Under static conditions, MC540 was rapidly internalized by HUVECs at 37 degrees C, and so was the membrane impermeant dye lucifer yellow, suggesting that the MC540 uptake was partly due to endocytosis. However, exposure to steady flow for 5 min at 37 degrees C induced an increase in MC540 uptake while that of lucifer yellow was unchanged, suggesting that the flow-induced increase in MC540 uptake was not endocytosis-related. The increase in MC540 uptake was significant for levels of steady shear of 6 dyne/cm2 and above. Pulsatile flow was more stimulatory than steady flow at 2 dyne/cm2, but no significant difference between the two was seen at higher shear stress levels. We conclude that fluid shear stress enhanced the uptake of MC540 by a mechanism other than endocytosis, suggesting an increase in plasma membrane permeability during exposure of the cells to shear stress.

Flow Effects on Endothelial Cell Signal Transduction, Function, and Mediator Release

The circulation in animals has evolved to reduce the diffusional distance between the nutrient supply and the cells constituting the organism. Depending on environmental changes and the activity performed by the animal, the metabolic demand by the different tissues can vary significantly. For a long time, it was generally accepted that there were two different control mechanisms for controlling blood flow within the circulation in animals: a systemic control by the central nervous system via the sympathetic neuronal network and by hormones from the adrenal gland, and a local control mediated by the metabolism of the organs. The role of flow as a potential factor for local regulation of vascular function has been recognized more recently. There is now considerable evidence that the size of blood vessels and vascular tone are dependent on the local level of wall shear stress in the vasculature, and that endothelial cells mediate the response of vessels to changes in flow conditions (74, 89, 98, 91, 125, 149, 150, 173, 183). In other words, endothelial cells, which are in direct contact with the flowing blood, act as flow sensors and generate signals to trigger the appropriate response by the vessels. Given the large amount of data showing that various agonists stimulate endothelial cells to secrete various vasoactive and mitogenic substances (for review, see [57]), it appears likely that flow-induced effects on vessel size and tone, which are generally effected by the underlying smooth muscle cells, may also be mediated by substances released by the endothelium.

Shear sensitivity in animal cell culture

Over the past year, considerable progress has been made in understanding shear sensitivity in animal cell culture as a result of extensive theoretical and experimental work. Here we review this progress, paying special attention to the physical and biological mechanisms by which mechanical forces act upon cells, and the effects of such forces.

Shear Stress-Induced Gene Expression in Human Endothelial Cells

Cells are surrounded by an extracellular fluid which contains nutrients essential for maintenance of cellular life. The transport of nutrients therefore significantly influences the metabolic function of cells. A single prokaryotic cell can readily obtain enough nutrients from the environment via diffusional transport. For large, multicellular organisms, such as humans, transport by diffusion alone, however, is unable to deliver sufficient nutrients to and remove metabolic wastes from cells. A circulatory system thus exists to provide rapid, convective mass transport throughout the body, supplemented by the diffusional transport between the blood capillaries and the cells via extracellular fluid. In vertebrates the circulatory system supplies the body with nutrients and removes metabolites via blood flow. It is therefore indispensable in maintaining a normal physiological state. Dysfunction of the circulatory system may result in many diseases such as atherosclerosis, heart failure, and hypertension.