536 R.M.Nerem ber,and these results suggest that the inhibition of cell proliferation by shear stress is at the level of DNA synthesis,and not due to endothelial cells rounding up during mitosis and being removed by flow(30).Furthermore,to determine whether the ef- fect of shear stress on endothelial cell growth was related to an alteration in cell cy- cle kinetics,both cell size and DNA content were analyzed by flow cytometry (33). For sub-confluent cells and following exposure to a shear stress of 30 dynes/cm2 for 24 hours,the percentage of sub-confluent cells in S phase was reduced significantly, with there simultaneously being an increase in the percentage of cells in Go/G phase.When these experiments were repeated with confluent cells,similar results were obtained.Thus,shear stress appears to inhibit the transition of endothelial cells from the Go/G phase to S phase in the cell cycle. In separate experiments,cells in both sheared and static cultures were videotaped at 400x magnification in order to visualize and quantify the process of mitosis and cytokinesis(46).Whereas in static culture a cell rounds up as part of the process of division,elongated endothelial cells in the presence of flow preserve their elongated, flattened shape over the entire duration of cell division.In some cases,the temporal nature of the intracellular mitotic events was observed to be altered.Thus,the influ- ence of flow is not only to hold up entry into S phase,but also to affect the cell di- vision process itself. Finally,alterations in the endothelial cell's growth state were further studied by analysis of the cell cycle-dependent accumulation of the messenger RNA(mRNA)for the proto-oncogene c-myc(8).Both control and shear stress treated endothelial cells were exposed to the potent agonist a-thrombin,and total cellular RNA was prepared and analyzed on northern blots.Using a probe for the proto-oncogene c-myc,we ob- served that c-myc levels at one hour increased 28 fold in endothelial cells in static cul- ture,but only 4 fold in endothelial cells which had been exposed to flow.Further experiments with other a-thrombin signal transduction events will need to be per- formed in order to delineate the mechanism involved;however,it is clear that the in- fluence of flow and the associated shear-stress level is manifest at the gene expression level. An obviously critical question is how does an endothelial cell recognize a mechan- ical stimulus,and having done so,how does it transduce that signal into the types of change in cell replication which we have observed?The intracellular events that reg- ulate the endothelial cell's growth program in response to shear stress are not known However,in many cell types,it is thought that the signaling mechanisms important to alterations in cell shape and cytoskeletal structure also are involved in growth con- trol.If this is true in regard to endothelial cell growth,then the recognition is perhaps a membrane event,possibly linked to the control of Ca++metabolism.There are re- ports of a transient elevation in intracellular calcium in response to flow (4),and we have seen this recently in our own laboratory (21).An important biochemical path- way controlling Ca+mobilization is the phospholipase C-mediated hydrolysis of polyphosphoinositides.In this pathway,activation of a cell surface receptor stimu- lates a phosphoinositide-specific phospholipase C to hydrolyze phosphatidylinositol 4,5 bisphosphate (PIP2)to form inositol 1,4,5,trisphosphate (IP3),a potent mobilizer of intracellular Ca++,and diacylglycerol,a stimulator of the Ca++/phos- pholipid-dependent enzyme,protein kinase C.Recently,it has been demonstrated that there is shear-stress stimulation of this pathway,with a peak in IP;being measured shortly after the onset of flow(37).Furthermore,studies in our laboratory have im-536 R.M. Nerem ber, and these results suggest that the inhibition of cell proliferation by shear stress is at the level of DNA synthesis, and not due to endothelial cells rounding up during mitosis and being removed by flow (30). Furthermore, to determine whether the ef￾fect of shear stress on endothelial cell growth was related to an alteration in cell cy￾cle kinetics, both cell size and DNA content were analyzed by flow cytometry (33). For sub-confluent cells and following exposure to a shear stress of 30 dynes/cm 2 for 24 hours, the percentage of sub-confluent cells in S phase was reduced significantly, with there simultaneously being an increase in the percentage of cells in G0/G1 phase. When these experiments were repeated with confluent cells, similar results were obtained. Thus, shear stress appears to inhibit the transition of endothelial cells from the Go/GI phase to S phase in the cell cycle. In separate experiments, cells in both sheared and static cultures were videotaped at 400• magnification in order to visualize and quantify the process of mitosis and cytokinesis (46). Whereas in static culture a cell rounds up as part of the process of division, elongated endothelial cells in the presence of flow preserve their elongated, flattened shape over the entire duration of cell division. In some cases, the temporal nature of the intracellular mitotic events was observed to be altered. Thus, the influ￾ence of flow is not only to hold up entry into S phase, but also to affect the cell di￾vision process itself. Finally, alterations in the endothelial cell's growth state were further studied by analysis of the cell cycle-dependent accumulation of the messenger RNA (mRNA) for the proto-oncogene c-myc (8). Both control and shear stress treated endothelial cells were exposed to the potent agonist c~-thrombin, and total cellular RNA was prepared and analyzed on northern blots. Using a probe for the proto-oncogene c-myc, we ob￾served that c-myc levels at one hour increased 28 fold in endothelial cells in static cul￾ture, but only 4 fold in endothelial cells which had been exposed to flow. Further experiments with other a-thrombin signal transduction events will need to be per￾formed in order to delineate the mechanism involved; however, it is clear that the in￾fluence of flow and the associated shear-stress level is manifest at the gene expression level. An obviously critical question is how does an endothelial cell recognize a mechan￾ical stimulus, and having done so, how does it transduce that signal into the types of change in cell replication which we have observed? The intracellular events that reg￾ulate the endothelial cell's growth program in response to shear stress are not known. However, in many cell types, it is thought that the signaling mechanisms important to alterations in cell shape and cytoskeletal structure also are involved in growth con￾trol. If this is true in regard to endothelial cell growth, then the recognition is perhaps a membrane event, possibly linked to the control of Ca ++ metabolism. There are re￾ports of a transient elevation in intracellular calcium in response to flow (4), and we have seen this recently in our own laboratory (21). An important biochemical path￾way controlling Ca ++ mobilization is the phospholipase C-mediated hydrolysis of polyphosphoinositides. In this pathway, activation of a cell surface receptor stimu￾lates a phosphoinositide-specific phospholipase C to hydrolyze phosphatidylinositol 4,5 bisphosphate (PIP2) to form inositol 1, 4, 5, trisphosphate (IP3), a potent mobilizer of intracellular Ca ++, and diacylglycerol, a stimulator of the Ca++/phos - pholipid-dependent enzyme, protein kinase C. Recently, it has been demonstrated that there is shear-stress stimulation of this pathway, with a peak in IP3 being measured shortly after the onset of flow (37). Furthermore, studies in our laboratory have im-