Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCα and the MAPK/ERK cascade

Little is known about the regulatory mechanisms of endothelial cell (EC) proliferation by retinal pericytes and vice versa. In a model of coculture with bovine retinal pericytes lasting for 24 h, rat brain ECs showed an increase in arachidonic acid (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A2 (cPLA2) and its phosphorylated form and calcium-independent intracellular phospholipase A2 (iPLA2). No activation of the same enzymes was seen in companion pericytes. In ECs, the protein level of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was also enhanced significantly, a finding not observed in cocultured pericytes. The expression of protein kinase C-α (PKCα) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA2, PKCα, and ERK1/2. By confocal microscopy, activation of PKCα in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA2 in ECs taken from a model of mixed culture were clearly observed. However, no increased expression of both enzymes was found in cocultured pericytes. Our findings indicate that a sequential activation of PKCα contributes to endothelial ERK1/2 and cPLA2 phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCα-cPLA2 pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation. Little is known about the regulatory mechanisms of endothelial cell (EC) proliferation by retinal pericytes and vice versa. In a model of coculture with bovine retinal pericytes lasting for 24 h, rat brain ECs showed an increase in arachidonic acid (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A2 (cPLA2) and its phosphorylated form and calcium-independent intracellular phospholipase A2 (iPLA2). No activation of the same enzymes was seen in companion pericytes. In ECs, the protein level of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was also enhanced significantly, a finding not observed in cocultured pericytes. The expression of protein kinase C-α (PKCα) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA2, PKCα, and ERK1/2. By confocal microscopy, activation of PKCα in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA2 in ECs taken from a model of mixed culture were clearly observed. However, no increased expression of both enzymes was found in cocultured pericytes. Our findings indicate that a sequential activation of PKCα contributes to endothelial ERK1/2 and cPLA2 phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCα-cPLA2 pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation. The structural basis of the blood-brain barrier (BBB) is not only a specialized brain capillary endothelial cell (EC) but encompasses a highly organized complex consisting of an endothelium, pericytes, and astrocytic foot processes. For the endothelium to form a selective barrier between the blood stream and tissues, vascular maturation requires the formation of tight EC-to-cell contacts, the downregulation of endothelial proliferation, the deposition of a basal lamina to which the endothelium tightly adheres, and the recruitment of supporting cells to the vessel walls such as pericytes or smooth muscle cells (1Risau W. Mechanisms of angiogenesis. Nature. 1997; 386: 671-674Google Scholar, 2Hirschi K.K. Rohovsky S.A. D'Amore P.A. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 1998; 141: 805-814Google Scholar, 3Vestweber D. Molecular mechanisms that control endothelial contacts. J. Pathol. 2000; 190: 281-291Google Scholar). Pericytes have received much less attention than the other components of microvascular beds. These contractile cells of mesenchymal lineage have long processes paralleling the capillary axis and short processes encircling the capillary. The current view of the role of pericytes in early stages of angiogenesis states that the initial endothelial tubes form without pericyte contact and that subsequent acquisition of pericyte coverage leads to vessel remodeling and maturation (4Gerhardt H. Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003; 314: 15-23Crossref PubMed Scopus (832) Google Scholar). This view has been challenged because it was shown that pericytes invest in actively sprouting and remodeling vessels (4Gerhardt H. Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003; 314: 15-23Crossref PubMed Scopus (832) Google Scholar, 5Witmer A.N. van Blijswijk B.C. van Noorden C.J. Vrensen G.F. Schlingemann R.O. In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A. J. Histochem. Cytochem. 2004; 52: 39-52Google Scholar). Retinal pericytes have a mitogenic effect on retinal microvascular ECs (6Wong H.C. Boulton M. Marshall J. Clark P. Growth of retinal capillary endothelia using pericyte conditioned medium. Invest. Ophthalmol. Vis. Sci. 1987; 28: 1767-1775PubMed Google Scholar), which may have important implications in new vessel formation. In an in vitro model of BBB established by coculturing immortalized ECs with brain pericytes that expressed tumor growth factor (TGF)-β1, the permeability of ECs was decreased significantly (7Dohgu S. Takata F. Yamauchi A. Nakagawa S. Egawa T. Naito M. Tsuruo T. Sawada Y. Niwa M. Kataoka Y. Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production. Brain Res. 2005; 1038: 208-215Google Scholar). Furthermore, in mixed cultures, pericytes have an antiproliferative effect on ECs (8Orlidge A. D'Amore P.A. Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J. Cell Biol. 1987; 105: 1455-1462Google Scholar, 9Sato Y. Rifkin D.B. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of latent transforming growth factor-β1-like molecule by plasmin during co-culture. J. Cell Biol. Scholar, T. S. M. S. T. by retinal pericyte conditioned retinal endothelial cell J. 2004; Scholar). However, the basis of the induction is not soluble factors or or cell-to-cell and the vascular maturation the control of phosphorylated protein kinase and phosphorylated protein kinase upstream of and the that activated in angiogenic ECs and in endothelial cells. Sci. 2004; PubMed Scopus Google Scholar). 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M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar, A. M. M. M. phospholipase A2 in immortalized rat brain endothelial cells by Scholar). pericyte were from bovine as M. M. and in retinal pericytes. Scholar). The cells were in with bovine and and were on the with of a of rat The was for and with and with and in medium. ECs or pericytes were and with or the same the cell were on the with the same ECs or pericytes on were the pericytes or ECs on the the and in the a for 24 in cells were whereas cells on the were with and with a were as A. C. M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar, A. M. M. M. phospholipase A2 in immortalized rat brain endothelial cells by Scholar). or pericyte were established in as an in vitro model of the BBB on direct contact of pericytes were on the of the of the and in the well culture pericytes the were and and ECs were on the of the for 24 h, cells grown on both of the were with a and of ECs or pericytes were established on and with in or and for 24 The cells were with and without direct cell-to-cell contact with pericytes or ECs in as a for AA in the or of or in the of inhibitors such as or in for 24 The of or inhibitors and or or on AA release were also in the cells in the was and for The was by to and an was taken to the in by to it was with to the to and for subsequent was in of A. M. M. M. phospholipase A2 in immortalized rat brain endothelial cells by Scholar). The was and the was in In AA and were on with the phase of The were with or by and and the was by of AA and was on a of their with of the expression and of the PKCα as well as cPLA2, confocal was on ECs or pericytes on on a for as of ECs and pericytes were also were ECs and pericytes were mixed and in of pericyte medium. 24 of cells were by in and for as A. C. M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; using either or of PKCα and cPLA2 was observed by confocal using a confocal were by of cells in to The in cell was the was on a of from from 24 of ECs and pericytes were as A. C. M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar). The protein of the cell was by and were as A. C. M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar). were with cPLA2 PKCα or The were with for and the were by enhanced was from and cells and pericytes using to the and in of and were by and was in a with of of and The for was for was in of of and of in were as initial for for for for and a for The of was established as for rat cPLA2, and by were used in RT-PCR on using a and a was for rat using a and a was for The used for were and and their was for bovine cPLA2, and by were used in RT-PCR on pericytes. using a and a was for using a and a was for For the used were and and their was and were used as control to of in RT-PCR on and pericytes, For to and for the phase of was such that formation was to was with and to the which the phase of The were that of the a the of the were in the phase of and of for cPLA2, and were to the increase of in The same were also by and of for cPLA2, and in on pericytes. a control using RT-PCR of to Furthermore, of the of were by the the of The were on in and with The on the were and their were with as was to for between The was was to indicate In ECs in with pericytes for 24 h, a increase in AA release grown no was observed of ECs in with or induced a and in AA with with or inhibitors significantly AA release, that kinase may in AA production. 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M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar), the AA release by whereas was to the AA release by In the AA release by These that AA release of in ECs was by both cPLA2 activation may the The of in to calcium-independent in cell-induced AA to in coculture to the role of both cell in ECs and pericytes, protein expression by Western blot Our in a coculture that contact but the of soluble factors for 24 h, ECs significantly expressed cPLA2 and protein than ECs in cultures, by and in was by of These that in ECs, by both In pericytes in the same coculture showed no increase in either cPLA2 or protein shown in the of pericytes in the coculture induced an increase in the endothelial phosphorylated forms of No in ERK1/2 protein expression were observed. using for pericytes not These indicate that in ECs, the kinase phosphorylation and activation In as shown in ECs for 24 with pericytes in of the pericytes not in direct contact with ECs but from by the and a that by soluble showed in the phosphorylated forms of cPLA2, PKCα, and ERK1/2 by and with control cells grown in was by of In ECs, the phosphorylated form of cPLA2 is seen the whereas were as shown A. 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M. of phospholipase A2 and kinases by in immortalized endothelial cells. 2005; Scholar). the increase of cPLA2 and phosphorylation from to may an increase in cPLA2 the mechanisms by which pericytes may mediate the phosphorylation of endothelial cPLA2, used inhibitors of phosphorylation upstream of of endothelial PKCα and ERK1/2. both inhibitors of upstream which were to significantly from to the expression of phosphorylated cPLA2 in the of cocultured ECs for 24 in the of both of ECs and pericytes with or or a of both inhibitors for 24 not in in the of ECs of with not The also to the basal level the expression of the phosphorylated form of endothelial PKCα from to whereas the same inhibitors were to significantly ERK1/2 phosphorylation by is in the of intracellular in cell The strong effect of upstream inhibitors observed in ECs may to the of in the control by basal phosphorylation of regulatory the cell which also in both early and for and and indicate that immortalized ECs of cPLA2 significantly increased by 24 of to conditioned by the of pericytes. of ECs with kinase inhibitors the cell of cPLA2 phosphorylated protein to the control that soluble pericyte factors induced cPLA2 protein increase by PKCα and were used as control cells for with ECs. in by no to pericytes not the of pericytes in coculture increased cPLA2 and protein in ECs, cPLA2 and by RT-PCR pericyte for 24 in pericytes induced a increase of endothelial cPLA2 with control to expression of shown in indicate in ECs grown in in the of retinal pericytes, the was expressed and that factors an increase with basal in In for cPLA2 and in cocultured pericytes not with cell endothelial PKCα is in phosphorylation by pericytes the that activation in ECs is by PKCα, the effect on PKCα expression of contact and between cells in shown in in either the model or the contact model ECs in contact with pericytes grown on the of processes contact with cell through the the of endothelial PKCα protein expression the same to in was by of the pericyte PKCα not that the of ECs was without effect on pericyte protein in of mixed of ECs and pericytes grown in contact on the same showed the formation of by the of PKCα in ECs and pericytes in coculture for 24 PKCα ECs, with a to a and with a PKCα to were with the and and the of was and for in of cells. in of confocal PKCα in the of control ECs grown as shown by in PKCα for 24 with pericytes, confocal revealed of with and with PKCα or in the perinuclear and the PKCα increased protein and a of the of the PKCα to the perinuclear and of ECs. the cell and with pericytes in in of a of in the perinuclear of the cell of and were also with confocal and the cells on a of in the for cPLA2 expression in ECs and is shown in were with a to a and with a cPLA2 to of was with the and and the shown The that the of cPLA2 by of and is significantly in with pericytes than in ECs grown of cPLA2 enhanced expression of the in cocultured ECs. expression of PKCα and cPLA2 was not induced in pericytes in the of ECs with control pericytes in the of ECs. analyses of PKCα and cPLA2 were in and in pericytes in no activation of both pericyte enzymes by ECs not In mixed of ECs and pericytes grown for 24 on the same in the well model with cells in direct to PKCα an for PKCα in ECs with a in pericytes not In a confocal of ECs and pericytes in grown on the same showed that the of cPLA2 was significantly in ECs than in pericytes not cells such as smooth muscle cells and pericytes important for EC-to-cell because may vessel of the endothelial on the in vitro BBB has been with ECs with or without interactions and blood-brain barrier J. 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