Enhancement of Migration by Protein Kinase Cα and Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase Cδ in Capillary Endothelial Cells

Abstract

Activation of protein kinase C (PKC) induces angiogenesis, migration, and proliferation of endothelial cells (EC), but can also prevent growth factor-induced EC proliferation. To determine whether these disparate effects are mediated by substrates of individual PKC isoenzymes, PKCα and PKCδ were overexpressed in rat microvascular EC. Basal and stimulated migration were enhanced in PKCα EC compared with either PKCδ or control EC. Serum-induced growth of PKCδ EC was decreased, while that of PKCα cells was similar to control EC. Phorbol ester markedly inhibited PKCδ EC growth but enhanced growth of PKCα and control EC. To determine possible causes for this altered proliferation, the effect of PKCδ on adhesion, mitogen-activated protein kinase activity, and cell cycle progression was measured. Adherence of PKCδ EC to vitronectin was significantly enhanced. Serum-induced extracellular signal-regulated kinase-2 activity was increased equally in both PKCα and PKCδ EC above that of control, while extracellular signal-regulated kinase-1 activity was similar in all EC. Cell cycle analysis suggested that PKCδ EC entered S phase inappropriately and were delayed in passage through S phase. Thus, PKCα may mediate some proangiogenic effects of PKC activation; conversely, PKCδ may direct antiangiogenic aspects of overall PKC activation, including slowing of the cell cycle progression. Activation of protein kinase C (PKC) induces angiogenesis, migration, and proliferation of endothelial cells (EC), but can also prevent growth factor-induced EC proliferation. To determine whether these disparate effects are mediated by substrates of individual PKC isoenzymes, PKCα and PKCδ were overexpressed in rat microvascular EC. Basal and stimulated migration were enhanced in PKCα EC compared with either PKCδ or control EC. Serum-induced growth of PKCδ EC was decreased, while that of PKCα cells was similar to control EC. Phorbol ester markedly inhibited PKCδ EC growth but enhanced growth of PKCα and control EC. To determine possible causes for this altered proliferation, the effect of PKCδ on adhesion, mitogen-activated protein kinase activity, and cell cycle progression was measured. Adherence of PKCδ EC to vitronectin was significantly enhanced. Serum-induced extracellular signal-regulated kinase-2 activity was increased equally in both PKCα and PKCδ EC above that of control, while extracellular signal-regulated kinase-1 activity was similar in all EC. Cell cycle analysis suggested that PKCδ EC entered S phase inappropriately and were delayed in passage through S phase. Thus, PKCα may mediate some proangiogenic effects of PKC activation; conversely, PKCδ may direct antiangiogenic aspects of overall PKC activation, including slowing of the cell cycle progression. INTRODUCTIONThe formation of new blood vessels and the repair of those damaged by disease or injury depend upon endothelial migration and proliferation (1Yang E.Y. Moses H.L. J. Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (420) Google Scholar, 2Ausprunk D.H. Folkman J. Microvasc. Res. 1977; 14: 53-65Crossref PubMed Scopus (1047) Google Scholar). Several external agents that promote or inhibit proliferation and migration have been identified (3Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar, 4Grant D.S. Kleinman H.K. Goldberg I.D. Bhargava M.M. Nickoloff B.J. Kinsella J.L. Polverini P. Rosen E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1937-1941Crossref PubMed Scopus (645) Google Scholar), but the intracellular messengers that mediate these processes are less clear.Activation of the serine-threonine kinase protein kinase C (PKC) 1The abbreviations used are: PKCprotein kinase CRFPECrat fat pad epididymal endothelial cell(s)ECendothelial cell(s)PBSphosphate-buffered salineBSAbovine serum albuminMAPmitogen-activated proteinERKextracellular signal-regulated kinasePMAphorbol 12-myristate 13-acetateHGFhepatocyte growth factor. by phorbol esters induces migration, proliferation (5Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (68) Google Scholar), and tube formation of cultured endothelial cells (6Kinsella J.L. Grant D.S. Weeks B.S. Kleinman H.K. Exp. Cell Res. 1992; 199: 56-62Crossref PubMed Scopus (48) Google Scholar, 7Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar) and causes angiogenesis in vivo (7Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 8Hu D.E. Fan T.P. Inflammation. 1995; 19: 39-54Crossref PubMed Scopus (26) Google Scholar, 9Wright P.S. Cross-Doersen D. Miller J.A. Jones W.D. Bitonti A.J. J. Cell. Physiol. 1992; 152: 448-457Crossref PubMed Scopus (22) Google Scholar). In addition, chemical inhibitors of PKC or the down-regulation of PKC by prolonged treatment with phorbol esters abrogates the proliferative effects induced by growth factors and mitogens (10Presta M. Tiberio L. Rusnati M. Dell'Era P. Ragnotti G. Cell Regul. 1991; 2: 719-726Crossref PubMed Scopus (57) Google Scholar, 11Daviet I. Herbert J.M. Maffrand J.P. FEBS Lett. 1990; 259: 315-317Crossref PubMed Scopus (35) Google Scholar) and also enhances endothelial permeability (12Lynch J.J. Ferro T.J. Blumenstock F.A. Brockenauer A.M. Malik A.B. J. Clin. Invest. 1990; 85: 1991-1998Crossref PubMed Scopus (238) Google Scholar, 13Nagpala P.G. Malik A.B. Vuong P.T. Lum H. J. Cell Physiol. 1996; 166: 249-255Crossref PubMed Scopus (87) Google Scholar) and alters the expression level of several fibrinolytic enzymes and their inhibitors (14Langer D.J. Kuo A. Kariko K. Ahuja M. Klugherz B.D. Ivanics K.M. Hoxie J.A. Williams W.V. Liang B.T. Cines D.B. et al.Circ. Res. 1993; 72: 330-340Crossref PubMed Scopus (34) Google Scholar). In contrast, treatment of endothelial cells with direct activators of PKC alters some responses that are usually associated with stimulation by physiologic agonists (15Murphy H.S. Maroughi M. Till G.O. Ward P.A. Am. J. Physiol. 1994; 267: L145-L151PubMed Google Scholar) and, under some conditions, can prevent growth factor-induced proliferation (16Ahmed A. Plevin R. Shoaibi M.A. Fountain S.A. Ferriani R.A. Smith S.K. Am. J. Physiol. 1994; 266: C206-C212Crossref PubMed Google Scholar). This apparent paradox might be explained by the fact that the PKC family is composed of related but structurally distinct isoenzymes, each a product of separate genes and with discrete cofactor requirements, substrate specificity, and tissue distribution (16Ahmed A. Plevin R. Shoaibi M.A. Fountain S.A. Ferriani R.A. Smith S.K. Am. J. Physiol. 1994; 266: C206-C212Crossref PubMed Google Scholar, 17Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar, 18Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Since phorbol esters activate multiple isoenzymes of PKC, the possibility is raised that each PKC isoenzyme may selectively mediate separate, and perhaps opposing, effects within stimulated endothelial cells.Preliminary studies in our laboratory revealed that rat capillary endothelial cells expressed several isoenzymes of PKC, including PKCα, -δ, -η, -θ, and -ζ. Of these isoenzymes, previous investigations have found that overexpression of PKCα or PKCδ in various cultured cells could affect their proliferation (19Eldar H. Zisman Y. Ullrich A. Livneh E. J. Biol. Chem. 1990; 265: 13290-13296Abstract Full Text PDF PubMed Google Scholar, 20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 21Watanabe T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar). Overexpression of PKCα in fibroblasts promoted their proliferation, while proliferation was inhibited in human breast cancer cells and other cell lines (20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 22Kindregan H.C. Rosenbaum S.E. Ohno S. Niles R.M. J. Biol. Chem. 1994; 269: 27756-27761Abstract Full Text PDF PubMed Google Scholar, 23Persons D.A. Wilkison W.O. Bell R.M. Finn O.J. Cell. 1988; 52: 447-458Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 24Ways D.K. Kukoly C.A. deVente J. Hooker J.L. Bryant W.O. Posekany K.J. Fletcher D.J. Cook P.P. Parker P.J. J. Clin. Invest. 1995; 95: 1906-1915Crossref PubMed Scopus (266) Google Scholar); similar alterations of cell growth have been observed in cells overexpressing PKCδ (20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 21Watanabe T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar, 25Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar). Thus, the possibility exists that activation of either of these isoenzymes mediates the inhibitory component of PKC activation on endothelial growth, while the other promotes one or more processes essential to endothelial repair and angiogenesis. Such an effect would be presumably mediated by isoenzyme-specific substrates, of which a few have been identified (e.g. elongation factor eEF-1α (26Kielbassa K. Muller H.-J. Meyer H.E. Marks F. Gschwendt M. J. Biol. Chem. 1995; 270: 6156-6162Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)). In the present study, we began by testing the effect of overexpression of PKCα and PKCδ on endothelial migration and proliferation, both of which are essential processes for angiogenesis and wound healing. When initial experiments revealed an inhibitory effect of PKCδ on endothelial proliferation, we then examined potential underlying causes.DISCUSSIONThe two major findings of this study are that overexpression of two different PKC isoenzymes normally expressed in microvascular endothelial cells exert distinct effects on endothelial proliferation, migration, and adhesion to extracellular matrix and that PKCδ-mediated inhibition of endothelial growth results from a defect in S phase of the endothelial cell cycle. The observation that overexpression of PKCδ, but not PKCα, prevents proliferation of microvascular endothelial cells, while PKCα enhances their migration in response to HGF (scatter factor), suggests that these isoenzymes phosphorylate different substrates in these cells with different physiologic effects. The disparity between the effects of overexpression of the two isoenzymes is heightened by PKC-activating phorbol esters in that treatment of PKCα EC exerts a mitogenic effect similar to that in control cells, while PKCδ EC were even more strongly inhibited by similar treatment. Thus, these data suggest that activation of each of these isoenzymes by angiogenic stimuli, such as HGF or those contained in serum, may mediate distinct aspects of several processes that are required for vascular repair and angiogenesis. Stimulation of endothelium with phorbol esters, which activate both PKCα and PKCδ, as well as several other isoenzymes expressed in endothelium (5Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (68) Google Scholar, 38Bussolino F. Silvagno F. Garbarino G. Costamagna C. Sanavio F. Arese M. Soldi R. Aglietta M. Pescarmona G. Camussi G. Bosia A. J. Biol. Chem. 1994; 269: 2877-2886Abstract Full Text PDF PubMed Google Scholar), has been noted to have both stimulatory and inhibitory effects on endothelial proliferation and angiogenesis (5Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (68) Google Scholar, 10Presta M. Tiberio L. Rusnati M. Dell'Era P. Ragnotti G. Cell Regul. 1991; 2: 719-726Crossref PubMed Scopus (57) Google Scholar, 39Kosaka C. Sasaguri T. Zen K. Masuda J. Shimokado K. Ogata J. Ann. N. Y. Acad. Sci. 1995; 748: 538-540Crossref PubMed Scopus (1) Google Scholar, 40Sozeri O. Vollmer K. Liyanage M. Frith D. Kour G. Mark G.E. d. Stabel S. Mark G.E. Oncogene. 1992; 7: 2259-2262PubMed Google Scholar) that can be temporally dispersed within the same cells (41Zhou W. Takuwa N. Kumada M. Takuwa Y. J. Biol. Chem. 1993; 268: 23041-23048Abstract Full Text PDF PubMed Google Scholar). The results from this study suggest that PKCδ might mediate those aspects of PKC activation that are inhibitory for endothelial repair and angiogenesis, while an component of the proangiogenic endothelial migration, might be mediated by of in endothelium are by endothelial migration, proliferation (3Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar, Bell L. J.A. J. Cell Biol. 1990; PubMed Scopus Google Scholar). is an component of angiogenesis as well (3Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar). The present that endothelial migration is enhanced in PKCα EC might from of in response to stimuli, which is a component of cell overall PKC stimulation has been associated with of of endothelial cells (3Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar, C.A. 1993; PubMed Scopus Google Scholar). results suggest that PKCα, but not PKCδ, may be one of migration response to HGF (scatter factor), a angiogenic that is present with in a in the D.S. Kleinman H.K. Goldberg I.D. Bhargava M.M. Nickoloff B.J. Kinsella J.L. Polverini P. Rosen E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1937-1941Crossref PubMed Scopus (645) Google Scholar, F. M. E. M. L. G. L. A. P.M. J. Cell Biol. 1992; PubMed Scopus Google Scholar, J.J. J. Clin. Invest. 1993; PubMed Scopus Google Scholar, Y. R. J. I. M. A. K. S. Y. K. et Res. 1995; PubMed Scopus Google several possible for the PKCδ-mediated inhibition of endothelial growth, including a in endothelial adhesion to a to activate such as or and a defect in progression of endothelial cells through the cell cycle. Of these the to be the adhesion, both endothelial cell growth and migration are to of the cell to matrix Bell L. J.A. J. Cell Biol. 1990; PubMed Scopus Google Scholar, M.A. M. D.A. J. Cell Biol. 1993; PubMed Scopus Google Scholar, L. Circ. Res. 1995; 77: PubMed Scopus Google Scholar). the vitronectin or in the or M. J.A. D.A. Science. 1995; 270: PubMed Scopus Google Scholar), the of these two for endothelial cell proliferation. In this study, microvascular endothelial cells in which PKCδ was overexpressed markedly enhanced adhesion to vitronectin or and a in mediated adhesion was not the of the in proliferation of PKCδ EC. This of adhesion could from either a of the of these or an in by either a direct effect on Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar) or as an cell adhesion that prevents Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar). PKC activation has been with of for and in several cell N. P. 1996; PubMed Google Scholar, M. Smith M. Ware J.A. PubMed Google Scholar). PKCα EC PKCδ EC increased expression of these compared with a PKCδ-mediated effect on of these for vitronectin is a for our of the kinase family of and is in of the intracellular that to endothelial cell growth, migration, and adhesion G. G. M. Y. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Activation of overall PKC to activation of PKCα, has been to phosphorylate kinase O. Vollmer K. Liyanage M. Frith D. Kour G. Mark G.E. d. Stabel S. Mark G.E. Oncogene. 1992; 7: 2259-2262PubMed Google Scholar), an of kinase Frith D. Stabel S. Oncogene. 1994; Google Scholar). In our both PKCα and PKCδ and kinase activity with a of activity in endothelial The by which PKCδ proliferation and cell cycle be or to that to or In addition, our results suggest that activation of activity by PKCδ is not for endothelial cell proliferation. This those of et S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar), found that PKCδ inhibited growth of cells a component of the from of endothelial cell proliferation by PKCδ to from a defect in endothelial cell cycle in which the cells S phase inappropriately and to this phase. of the of this defect and the of PKCδ, that the isoenzyme with one of the that and of S phase. S phase has been associated with of vascular cells with growth factor J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), but such was not found in PKCδ EC. O. Harrington and J. A. of PKC activity has not been associated with this S phase defect and with of cell cycle Overexpression of PKCδ, but not PKCα or in fibroblasts causes an in phase of the cell but activation with T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar); were in S phase. In that study, inhibition of cell cycle progression by PKCδ was to an isoenzyme-specific the PKC activity was in the cells in the other is possible that the observed effect an in overall In this study, the of activity that the defect in cell cycle as well as in adhesion and migration, from of PKCδ with isoenzyme-specific Thus, in to isoenzyme-specific this in S phase to be for endothelial effects of individual PKC isoenzymes on proliferation are to to cell overexpression of PKCα in cells proliferation and not to cell C. I. R. 1991; PubMed Scopus Google Scholar, C. M. S. Parker P.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (110) Google Scholar), but in and human breast cancer cell in PKCα expression altered the cell enhanced proliferation, and increased D.A. Wilkison W.O. Bell R.M. Finn O.J. Cell. 1988; 52: 447-458Abstract Full Text PDF PubMed Scopus (242) Google Scholar, G. D. H. Cell. 1992; PubMed Scopus Google Scholar). Thus, the effects of individual PKC isoenzymes are to be mediated by substrates or with tissue or within an individual cell the substrates with which each isoenzyme of the by which these two individual PKC isoenzymes mediate either enhanced adhesion or migration or proliferation of endothelial cells of their Such with the of endothelial to individual PKC isoenzymes by this study, would essential to our of and angiogenesis. INTRODUCTIONThe formation of new blood vessels and the repair of those damaged by disease or injury depend upon endothelial migration and proliferation (1Yang E.Y. Moses H.L. J. Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (420) Google Scholar, 2Ausprunk D.H. Folkman J. Microvasc. Res. 1977; 14: 53-65Crossref PubMed Scopus (1047) Google Scholar). Several external agents that promote or inhibit proliferation and migration have been identified (3Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar, 4Grant D.S. Kleinman H.K. Goldberg I.D. Bhargava M.M. Nickoloff B.J. Kinsella J.L. Polverini P. Rosen E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1937-1941Crossref PubMed Scopus (645) Google Scholar), but the intracellular messengers that mediate these processes are less clear.Activation of the serine-threonine kinase protein kinase C (PKC) 1The abbreviations used are: PKCprotein kinase CRFPECrat fat pad epididymal endothelial cell(s)ECendothelial cell(s)PBSphosphate-buffered salineBSAbovine serum albuminMAPmitogen-activated proteinERKextracellular signal-regulated kinasePMAphorbol 12-myristate 13-acetateHGFhepatocyte growth factor. by phorbol esters induces migration, proliferation (5Kent K.C. Mii S. Harrington E.O. Chang J.D. Mallette S. Ware J.A. Circ. Res. 1995; 77: 231-238Crossref PubMed Scopus (68) Google Scholar), and tube formation of cultured endothelial cells (6Kinsella J.L. Grant D.S. Weeks B.S. Kleinman H.K. Exp. Cell Res. 1992; 199: 56-62Crossref PubMed Scopus (48) Google Scholar, 7Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar) and causes angiogenesis in vivo (7Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 8Hu D.E. Fan T.P. Inflammation. 1995; 19: 39-54Crossref PubMed Scopus (26) Google Scholar, 9Wright P.S. Cross-Doersen D. Miller J.A. Jones W.D. Bitonti A.J. J. Cell. Physiol. 1992; 152: 448-457Crossref PubMed Scopus (22) Google Scholar). In addition, chemical inhibitors of PKC or the down-regulation of PKC by prolonged treatment with phorbol esters abrogates the proliferative effects induced by growth factors and mitogens (10Presta M. Tiberio L. Rusnati M. Dell'Era P. Ragnotti G. Cell Regul. 1991; 2: 719-726Crossref PubMed Scopus (57) Google Scholar, 11Daviet I. Herbert J.M. Maffrand J.P. FEBS Lett. 1990; 259: 315-317Crossref PubMed Scopus (35) Google Scholar) and also enhances endothelial permeability (12Lynch J.J. Ferro T.J. Blumenstock F.A. Brockenauer A.M. Malik A.B. J. Clin. Invest. 1990; 85: 1991-1998Crossref PubMed Scopus (238) Google Scholar, 13Nagpala P.G. Malik A.B. Vuong P.T. Lum H. J. Cell Physiol. 1996; 166: 249-255Crossref PubMed Scopus (87) Google Scholar) and alters the expression level of several fibrinolytic enzymes and their inhibitors (14Langer D.J. Kuo A. Kariko K. Ahuja M. Klugherz B.D. Ivanics K.M. Hoxie J.A. Williams W.V. Liang B.T. Cines D.B. et al.Circ. Res. 1993; 72: 330-340Crossref PubMed Scopus (34) Google Scholar). In contrast, treatment of endothelial cells with direct activators of PKC alters some responses that are usually associated with stimulation by physiologic agonists (15Murphy H.S. Maroughi M. Till G.O. Ward P.A. Am. J. Physiol. 1994; 267: L145-L151PubMed Google Scholar) and, under some conditions, can prevent growth factor-induced proliferation (16Ahmed A. Plevin R. Shoaibi M.A. Fountain S.A. Ferriani R.A. Smith S.K. Am. J. Physiol. 1994; 266: C206-C212Crossref PubMed Google Scholar). This apparent paradox might be explained by the fact that the PKC family is composed of related but structurally distinct isoenzymes, each a product of separate genes and with discrete cofactor requirements, substrate specificity, and tissue distribution (16Ahmed A. Plevin R. Shoaibi M.A. Fountain S.A. Ferriani R.A. Smith S.K. Am. J. Physiol. 1994; 266: C206-C212Crossref PubMed Google Scholar, 17Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar, 18Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Since phorbol esters activate multiple isoenzymes of PKC, the possibility is raised that each PKC isoenzyme may selectively mediate separate, and perhaps opposing, effects within stimulated endothelial cells.Preliminary studies in our laboratory revealed that rat capillary endothelial cells expressed several isoenzymes of PKC, including PKCα, -δ, -η, -θ, and -ζ. Of these isoenzymes, previous investigations have found that overexpression of PKCα or PKCδ in various cultured cells could affect their proliferation (19Eldar H. Zisman Y. Ullrich A. Livneh E. J. Biol. Chem. 1990; 265: 13290-13296Abstract Full Text PDF PubMed Google Scholar, 20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 21Watanabe T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar). Overexpression of PKCα in fibroblasts promoted their proliferation, while proliferation was inhibited in human breast cancer cells and other cell lines (20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 22Kindregan H.C. Rosenbaum S.E. Ohno S. Niles R.M. J. Biol. Chem. 1994; 269: 27756-27761Abstract Full Text PDF PubMed Google Scholar, 23Persons D.A. Wilkison W.O. Bell R.M. Finn O.J. Cell. 1988; 52: 447-458Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 24Ways D.K. Kukoly C.A. deVente J. Hooker J.L. Bryant W.O. Posekany K.J. Fletcher D.J. Cook P.P. Parker P.J. J. Clin. Invest. 1995; 95: 1906-1915Crossref PubMed Scopus (266) Google Scholar); similar alterations of cell growth have been observed in cells overexpressing PKCδ (20Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechtle C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar, 21Watanabe T. Ono Y. Taniyama Y. Hazama K. Igarashi K. Ogita K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10159-10163Crossref PubMed Scopus (297) Google Scholar, 25Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar). Thus, the possibility exists that activation of either of these isoenzymes mediates the inhibitory component of PKC activation on endothelial growth, while the other promotes one or more processes essential to endothelial repair and angiogenesis. Such an effect would be presumably mediated by isoenzyme-specific substrates, of which a few have been identified (e.g. elongation factor eEF-1α (26Kielbassa K. Muller H.-J. Meyer H.E. Marks F. Gschwendt M. J. Biol. Chem. 1995; 270: 6156-6162Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)). In the present study, we began by testing the effect of overexpression of PKCα and PKCδ on endothelial migration and proliferation, both of which are essential processes for angiogenesis and wound healing. When initial experiments revealed an inhibitory effect of PKCδ on endothelial proliferation, we then examined potential underlying