This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kent, K. C. Articles by Ware, J. A. Search for Related Content PubMed PubMed Citation Articles by Kent, K. C. Articles by Ware, J. A.

(Circulation Research. 1995;77:231-238.)
© 1995 American Heart Association, Inc.

Articles

Requirement for Protein Kinase C Activation in Basic Fibroblast Growth Factor–Induced Human Endothelial Cell Proliferation

K. Craig Kent, Shinsuke Mii, Elizabeth O. Harrington, James D. Chang, Sheila Mallette, J. Anthony Ware

From the Departments of Surgery (Division of Vascular Surgery) (K.C.K., S.M., S.M.) and Medicine (Cardiovascular Division) (E.O.H., J.D.C., J.A.W.) and the Harvard Thorndike Laboratories and Charles A. Dana Research Institute (E.O.H., J.D.C., J.A.W.), Beth Israel Hospital, Harvard Medical School, Boston, Mass.

Correspondence to K. Craig Kent, MD, Division of Vascular Surgery, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)–induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca²⁺ increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca²⁺ and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca²⁺, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca²⁺). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-, -, -, and -) as well as small amounts of the conventional (Ca²⁺-regulated) isoenzymes PKC- and -ß. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca²⁺, suggests that one or more of these Ca²⁺-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.

Key Words: protein kinase C • endothelial cell • growth factors • proliferation • isoenzymes • signal transduction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A prominent feature of arterial injury is disruption of the luminal endothelial surface, resulting in exposure of the formed elements of the blood to the thrombogenic subintimal layer. Migration and proliferation of endothelial cells across the area of injury are necessary to restore antithrombotic capability to the vessel wall. During this process, endothelial cells at the margin of the injured segment transform from their usual quiescent and differentiated state to a proliferative phenotype that serves to restore the integrity of the endothelial surface in the segment of injured vessel.^{1 2} Release of endothelial cells from contact inhibition in situ, which is required for proliferation, is mediated by growth factors released by vascular cells or adherent platelets. One of these growth factors, bFGF, is a potent endogenous mitogen for endothelial and other cells^{3 4} and is integrally involved in the reparative process that follows arterial injury. The addition of bFGF not only induces endothelial proliferation and capillary tube formation in vitro⁵ but also, when administered to animals, dramatically increases the rate of reendothelialization of an injured arterial segment.⁶

The receptor for bFGF has been identified and found to be a transmembrane homodimer with intrinsic tyrosine kinase activity⁷ ; however, little is known about the intracellular mechanisms by which attachment of bFGF to its receptor leads to endothelial proliferation. In particular, the role of PKC, a family of intracellular serine-threonine kinases, in bFGF-induced mitogenesis of HEC is not yet clear. Exposure to bFGF causes activation of PKC and proliferation in several cell types; however, the relative importance of phosphoinositide metabolism, Ca²⁺ mobilization, and PKC activation in bFGF-induced proliferation varies greatly, even among closely related cell types. For example, PKC activation is critical for bFGF-induced growth of Swiss (rat) 3T3 fibroblasts,⁸ but not for that of Chinese hamster lung fibroblasts.⁹ Furthermore, some studies have found that tumor-promoting phorbol esters, which activate PKC, are mitogenic for endothelial cells^{10 11} and induce angiogenesis,^{12 13} whereas others have found that phorbol esters prevent the action of some endothelial growth factors.¹⁴ Also, a dual proliferative–antiproliferative effect of PKC has been described, which may depend on the source of the endothelial cells¹⁵ or the conditions of stimulation.¹⁶ Although there are several potential hypotheses for this discrepancy among cell types, a particularly attractive explanation lies in the diversity of the PKC-related gene products.^{17 18} In virtually all mammalian tissues and cells, PKC activity does not reflect that of a single enzyme but rather of a family of structurally related isoenzymes that are products of different genes and have been classified on the basis of their Ca²⁺ and phorbol ester sensitivities into three subfamilies termed cPKC (-, -ß, and -), nPKC (-, -, -, and -), and aPKC (- and, recently, - and -µ).¹⁸ Although cPKCs, which have been the most extensively studied, can be activated by an increase in Ca²⁺ and the addition of phorbol esters, nPKCs are not regulated by Ca²⁺, and aPKCs are not regulated by Ca²⁺ or phorbol esters. The differences among the PKCs in tissue distribution and in regulation of their activity are well established, and it is increasingly evident that specific cellular functions are mediated by individual isoenzymes. For example, recent studies in Chinese hamster ovary cells¹⁹ and in NIH-3T3 fibroblasts²⁰ reveal that overexpression of nPKC-, but not of PKC-, -,¹⁹ or -,²⁰ inhibits cell proliferation, possibly by arresting cell cycle progression at the end of the S-phase.¹⁹ The foregoing results make it clear that an understanding of the role of PKC in vascular repair after injury or angiogenesis will be achieved only through studies conducted in HEC in which the diversity of the PKC family is considered. Thus, the purposes of the present study were to determine whether PKC becomes activated in HEC after bFGF, whether such activation is required for bFGF-induced HEC proliferation, and which isoenzymes of PKC might be candidates to mediate bFGF-induced endothelial proliferation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Triton X-100, aprotinin, leupeptin, thrombin, heparin, EGTA, Mn²⁺, and protein assay kit were obtained from Sigma Chemical Co; human recombinant bFGF and EGF, UBI; bovine hypothalamus endothelial mitogen, fibronectin, and acetylated LDL, Biomedical Technologies, Inc; PBS, FBS, penicillin G, streptomycin, PMA, PDBu, M-199, L-glutamine, and rabbit polyclonal-, GIBCO BRL; gelatin, Difco Laboratories; collagenase type II, Worthington; fura 2-AM, Molecular Probes; ionomycin, Calbiochem; PMSF, Fluka; sodium orthovanadate, Fisher Scientific Co; anti-mouse alkaline phosphatase–conjugated IgG, BCIP, and NBT, Promega; random priming kit (used to label cDNA probes), Boehringer Mannheim; rabbit polyclonal PKC-, -ß, and -, Santa Cruz Biotech, Inc; and nylon transfer membrane (Genescreen) and dCTP-[-³²P], New England Nuclear. Rabbit polyclonal PKC- was a generous gift from Dr Peter M. Blumberg (National Cancer Institute, Bethesda, Md). The polyclonal anti–PKC- antibody was raised in rabbits injected with a peptide specific to PKC- and was affinity purified in our laboratory.

Cell Culture
HUVEC were isolated from pooled umbilical cords. The cells were harvested with 0.1% collagenase as described previously²¹ and maintained in M-199, 20 U/mL porcine intestinal heparin, 100 µg/mL L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, and 30 µg/mL bovine hypothalamus growth factor, supplemented with 20% FBS with the use of standard cell culture techniques. The cells were serially passaged at a ratio of 1:3. All cultured cells were identified as endothelial by their typical cobblestone appearance, uptake of fluorescent acetylated LDL, and production of von Willebrand factor as measured by enzyme-linked immunosorbent assay.²¹ In some of the reverse-transcription PCR experiments (discussed later), the source of RNA was one of several well-characterized endothelial cell lines derived from primary cultures—these include the PY4.1 cell line, derived from mouse hemangioma²² and characterized and generously provided by Dr Victoria Bautch, University of North Carolina at Chapel Hill; EAhy.926 cell line, derived from HUVEC fused with a carcinoma cell line²³ and characterized and generously provided by Dr Cora-Jean Edgell, University of North Carolina at Chapel Hill; the RFPEC, which is derived from rat microvascular cells²⁴ and generously provided by Dr Robert Rosenberg and Dr Michael Simons, Massachusetts Institute of Technology and Beth Israel Hospital, Boston, Mass; and the AG4762 cell line, which is derived from bovine aorta and was purchased from ATCC.

Proliferation Assay and Downregulation of PKC
For the proliferation assay, first- and second-passage HEC were plated onto 2-cm² wells at a density of 5x10³ cells per well in culture medium with 10% FBS and were permitted to attach for 6 hours. Seventy-two hours after the administration of agonists and inhibitors, cell numbers were determined with a Coulter counter (Coulter Electronics, Inc). Ten percent serum was found in preliminary experiments to maintain the viability of HEC for the 3-day period without increasing their number (nonproliferative conditions).

In some experiments, PKC was downregulated by treating HEC with 1 µmol/L PMA in culture medium with 10% FBS for 72 hours; nondownregulated cells were maintained in 10% serum for an equal period of time. PKC-downregulated cells displayed diminished adhesiveness for the substratum and therefore were seeded at a higher initial density. Sample wells were counted at the beginning of each assay to ensure that the starting numbers of control and downregulated cells initially were the same.

In Situ PKC Assay
HEC cultures were grown to confluence on gelatinized 96-well plates. After a 24-hour incubation with culture medium containing 10% serum, cells were washed with M-199 and then incubated with bFGF (10 ng/mL) for the indicated time periods. Cells were then washed with PBS, and PKC was assayed with the use of [Ac-MBP(4-14)], which acts as a specific substrate of PKC.^{25 26} To each well, we added 100 µL total volume of the following: lysis buffer (final concentrations: 0.137 mol/L NaCl, 5.4 mmol/L KCl, 0.3 mmol/L Na₂HPO₄, 0.4 mmol/L K₂HPO₄, 1 mg/mL glucose, 20 mmol/L HEPES, 10 mmol/L MgCl₂, 50 µg/mL digitonin, and 25 mmol/L B-glycerophosphate, pH 7.2), 100 µmol/L [³²P]ATP, 2.5 mmol/L CaCl₂, 2 µg/mL phosphatidylserine, and 100 µmol/L Ac-MBP.^{4 5 6 7 8 9 10 11 12 13 14} After 10 minutes' incubation, 50 µL from each well was spotted onto phosphocellulose disks, washed with 1% (vol) concentrated H₃PO₄ in H₂O, and counted. In parallel experiments, HUVEC were stimulated for the various time periods with carrier lacking FGF, and PKC activity was expressed as percent increase in activity in FGF versus in carrier-stimulated cells.

Immunoblotting
Confluent second-passage HUVEC were lysed in Laemmli buffer²⁷ and then boiled for 5 minutes. Total protein (30 µg, as determined by the Lowry method) from each sample was subjected to SDS-PAGE. The electrophoretically separated proteins were transferred to a nylon membrane and labeled by incubation with antibodies to the various PKC isoenzymes.

Calcium Determination
Changes in [Ca²⁺]_i of the HUVEC were measured after exposure of the cells to thrombin, endoperoxide analogue U46619, and bFGF by methods previously described.²¹ Briefly, HEC were plated onto 10x30-mm glass slides coated with human fibronectin (1 µg/cm²). After the HEC reached confluence, fresh medium containing fura 2-AM (5 µmol/L) was added, and the HEC were incubated for 45 minutes at 37°C. Slides containing the fura 2-AM–loaded cells were then rinsed in PBS buffer and placed in modified HEPES-Tyrode buffer containing 1 mmol/L Ca²⁺ at room temperature. For [Ca²⁺]_i determination, the glass slides were placed diagonally in polycarbonate cuvettes containing HEPES-Tyrode buffer at 37°C and supported above a magnetic stir bar with the use of a paraffin wedge. Fluorescent measurements were performed with a temperature-controlled, dual-excitation wavelength spectrofluorimeter with stirring (SPEX Fluorolog-2) as described previously.²¹

RNA Extraction
RNA was extracted from HEC and the endothelial cell lines EAhy.926, PY4.1, RFPEC, and AG4762. Confluent endothelial cells were treated with trypsin, subjected to centrifugation, and washed. The resultant cell pellet was mixed for 30 seconds with the use of a Vortex mixer and incubated on ice for 10 minutes in 200 µL lysis buffer (140 mmol/L NaCl, 1.4 mmol/L MgCl₂, 0.5% Triton X-100, 10 mmol/L vanadyl-ribonucleoside, and 10 mmol/L Tris, pH 8.3). The lysate was then spun in a microcentrifuge at 15 000 rpm for 10 minutes, and the supernatant fraction was transferred to a fresh microcentrifuge tube containing an equal volume of proteinase K buffer (25 mmol/L EDTA, 300 mmol/L NaCl, 2% SDS, 200 µg/mL proteinase K, and 200 mmol/L Tris, pH 7.5) and incubated at 65°C for 1 hour. The sample was extracted once with phenol-chloroform, and total RNA was precipitated with ethanol and redissolved in 100 µL diethyl pyrocarbonate–treated water. Polyadenylated RNA was obtained from total RNA with the use of the Poly A Tract mRNA Isolation Systems (Promega).

Northern Transfer and PCR Analysis
For Northern transfer analysis, each RNA sample (either 20 µg of total RNA or 2 µg of polyadenylated RNA) was fractionated by electrophoresis through a 1.5% agarose formaldehyde gel. RNA was transferred to a nylon membrane (Gene Screen) by positive pressure (Posiblot, Stratagene), and the blots were crosslinked by ultraviolet irradiation. cDNA fragments specific for each human PKC isoenzyme (corresponding to base pair [bp] 184-1536 [PKC-], 22-1536 [PKC-ß], 877-1884 [PKC-], 725-1711 [PKC-], 574-1393 [PKC-], 525-1631 [PKC-], and 739-1897 [PKC-]) were radiolabeled with ³²P-dCTP by random primer extension, achieving an average specific activity of 8.5x10⁸ cpm/µg. Prehybridization and hybridization conditions were carried out as previously described²⁸ ; when the blots are washed under stringent conditions, these probes do not detect other PKC isoenzymes (James D. Chang, J. Anthony Ware, unpublished data).

For analysis with PCR, 1 ng of total RNA extracted from HEC or various endothelial cell lines was converted to cDNA with the use of Moloney murine leukemia virus reverse transcriptase and amplified according to a PCR protocol described previously.²⁸ In brief, degenerate oligonucleotide primers were used that were designed to amplify, in a single reaction, all cPKC and nPKC isoenzymes. The sequences of oligonucleotide primers used to direct first-strand cDNA synthesis and the first round of PCR amplification have been described previously.²⁸ One tenth (10 µL) of the products of the initial amplification reaction were then subjected to a subsequent round of PCR amplification directed with the use of a pair of internally nested degenerate primers, thereby greatly increasing the ability of this method to detect rare transcripts. An aliquot of 10 µL of each reaction mixture was subjected to agarose gel electrophoresis, stained with ethidium bromide, and photographed.

Statistical Analysis
All values are given as mean±SEM. Statistical analysis was performed with Student's t test as adjusted for multiple comparisons, and P<.05 was considered to be a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In initial experiments, we sought to determine whether bFGF increased PKC activity and [Ca²⁺]_i in HEC. Total PKC activity in HEC incubated with bFGF at 1 and 5 minutes was minimally elevated but became significantly elevated after 10 minutes of stimulation. Enzymatic activity reached a maximum (115% increase above baseline) at 30 minutes (Fig 1) and then diminished to a 20% to 30% increase at 1 to 4 hours. In comparison, after 10 minutes of stimulation with PMA, PKC activity increased by 70% (data not shown). In parallel experiments, HEC displayed no increase in [Ca²⁺]_i after stimulation with bFGF for at least 30 minutes (ie, beyond the period of peak activation of PKC) (Fig 2). In comparison, incubation of HEC with thrombin (1 U/mL) or the thromboxane A₂ mimetic U46619 (data not shown) produced a rapid increase in [Ca²⁺]_i that peaked at 30 seconds and persisted for 3 minutes (Fig 2). Thus, the addition of bFGF induces an increase in total PKC activity in HEC that is not preceded or accompanied by a measurable increase in [Ca²⁺]_i.

View larger version (33K): [in this window] [in a new window]   Figure 1. Bar graph showing effect of bFGF on PKC activity. HECs were incubated for 24 hours under nonproliferative conditions and stimulated with FGF (10 ng/mL). Incorporation of 32P into Ac-MBP(4-14) was measured as described in "Materials and Methods." Results for the prescribed time periods are expressed as the mean±SEM of the percent increase in PKC activity in FGF-stimulated HECs compared with cells incubated with carrier. Baseline phosphorylation ranged from 2.4 to 6.9 pmol/min per 104 cells.

View larger version (11K): [in this window] [in a new window]   Figure 2. Plot showing lack of Ca2+ increase induced by bFGF in HEC. Tracings are a direct recording of the fluorescence ratio from fura 2-AM–loaded HEC that were stimulated with bFGF (10 ng/mL) (solid line) or thrombin (1 U/mL) (broken line) added at the point indicated by the arrow in each tracing. These tracings are from single determinations that are representative of three similar experiments. In separate experiments, no appreciable increase in [Ca2+]i was observed for as long as 30 minutes in FGF-stimulated cells; the slight rise in fluorescence observed at times beyond 200 seconds is similar to that seen in unstimulated HEC and represents a small time-dependent leak of the fluorescent fura 2-AM dye from the cytoplasm of surrounding cells.

To determine whether inhibition of PKC eliminates bFGF-induced HEC proliferation, we incubated the HEC with either of two partially specific PKC antagonists that have differing mechanisms of inhibition. The first, chelerythrine, binds to the catalytic domain of PKC and appears to have no effect on protein tyrosine kinases, cAMP-dependent protein kinase, or calcium/calmodulin–dependent protein kinase.^{29 30} Incubation of HEC with chelerythrine at a concentration of 3.2 µmol/L produced half-maximal inhibition of bFGF-induced proliferation. The more widely used PKC inhibitor, H-7, which interferes with ATP binding to PKC,³¹ also suppresses proliferation of HEC induced by bFGF; an H-7 concentration of 26 µmol/L produces half-maximal inhibition (Fig 3).

View larger version (17K): [in this window] [in a new window]   Figure 3. Plots showing effect of PKC inhibitors on HEC proliferation by bFGF. Equal numbers of HEC (5x103) were seeded onto 2-cm2 wells and then maintained in nonproliferative conditions (incubated in 10% serum). They were treated with bFGF (10 ng/mL) for 72 hours in medium containing the indicated concentrations of H-7 (A) or chelerythrine (B) before the cells were counted. Percent inhibition was determined by comparing cell counts from cultures treated with H-7 and chelerythrine with those of control cells treated with bFGF only. Stimulation of control cells with bFGF (10 ng/mL) produced an approximately threefold increase in number of cells. Experiments were performed in triplicate with five different cell lines and reported as mean±SEM.

A third method of inhibiting PKC isoenzymes that are sensitive to phorbol ester is to induce downregulation of PKC through prolonged (72-hour) incubation of HEC with high concentrations (1 µmol/L) of PMA.³² Such cells did not proliferate further when additional PMA was added. Downregulated cells proliferated significantly less than did control cells when stimulated with increasing concentrations of bFGF (Fig 4). Thus, bFGF-induced HEC proliferation requires the presence of a phorbol ester–sensitive PKC isoenzyme. This requirement does not extend to all growth factors that use receptor tyrosine kinases; in parallel experiments, we found that neither the chemical inhibitors nor PKC downregulation prevents HEC mitogenesis induced by EGF (50 ng/mL) (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 4. Bar graph showing effect of PKC downregulation on bFGF-induced HEC proliferation. Nonproliferating HEC were incubated in 10% serum only (closed bars) or serum with PMA (1 µmol/L; hatched bars) for 72 hours before stimulation with bFGF in the concentrations indicated. Differences between control and downregulated cells were significant for all concentrations of FGF except 0 ng/mL. Experiments were performed in triplicate with five different cell lines; other conditions were as reported in Fig 3.

In the next set of experiments, we investigated whether PKC activation was sufficient to induce HEC proliferation. In contrast to the results achieved with long-term PMA incubation, short-term incubation of HEC with PMA induced a concentration-dependent increase in endothelial cell number that reached significance (P<.05) at a PMA concentration of 10 nmol/L and increases to a maximum at a concentration of 1 µmol/L. A second phorbol ester, PDBu, also stimulated endothelial proliferation in a concentration-dependent manner that was maximal at a concentration of 100 nmol/L (Fig 5). A proliferative effect was observed when HUVEC were stimulated with phorbol esters for as long as 72 hours. The lack of a proliferative effect after 72 hours presumably was due to downregulation and depletion of PKC by phorbol esters. Thus, short-term activation of phorbol ester–sensitive PKC was sufficient to induce HEC proliferation.

View larger version (26K): [in this window] [in a new window]   Figure 5. Bar graph showing effects of PMA and PDBu on HEC proliferation. Second-passage HEC were incubated in nonproliferative concentrations (10%) of serum for 72 hours before the addition of PMA (closed bars) or PDBu (hatched bars) in the indicated concentrations for an additional 72 hours, and the number of cells was determined.

Because PKC is activated after bFGF stimulation in the absence of increased [Ca²⁺]_i, we considered the possibility that non–Ca²⁺-dependent isoenzymes of PKC represented a substantial proportion of the PKCs expressed in HEC. To define the isoenzyme distribution on a protein level, a series of antibodies directed against the individual isoenzymes of PKC were tested. Whole HEC lysates were subjected to SDS-PAGE and probed with polyclonal antibodies to PKC- and PKC-ß, the two cPKCs (Ca²⁺-regulated PKC isoenzymes) that are expressed in nonneurological mammalian tissues,¹⁷ as well as polyclonal consensus antibodies that recognize both isoenzymes. PKC- and PKC-ß were both identified in HEC. We next probed HEC with polyclonal anti-peptide antibodies directed against the nPKCs (Ca²⁺-independent PKC isoenzymes). A pronounced 78-kDa band representing aPKC- readily disappeared after the addition of blocking peptide. Also readily identifiable were nPKC-, -, and - isoenzymes (Fig 6); another novel isoenzyme, nPKC-, could not be identified in HEC.

View larger version (14K): [in this window] [in a new window]   Figure 6. Immunoblot analysis of PKC isoenzymes. Total cellular protein was subjected to electrophoresis through a 10% SDS-polyacrylamide gel and transferred to nitrocellulose support. Membranes were probed with antibodies specific for each of the PKC isoenzymes in the presence and absence of a blocking peptide. Immunoblot analysis was made of HUVEC for PKC-, -ß, -, -, -, and - isoenzymes with antibodies specific for each isoenzyme. Immunoblotting was repeated a minimum of three times with three separate cell lines; a representative example is displayed.

To confirm these findings, we performed Northern transfer analysis on RNA extracted from HEC, which revealed expression of nPKC-, -, and - (Fig 7) and aPKC-. Hybridization of large amounts of polyadenylated mRNA with cDNA probes specific for the Ca²⁺-dependent isoenzymes, followed by prolonged autoradiographic exposure, revealed only trace amounts of mRNA corresponding to cPKC- and -ß (not shown).

View larger version (37K): [in this window] [in a new window]   Figure 7. Northern transfer analysis of total RNA isolated from HEC. RNA was extracted from confluent second-passage cells, and Northern transfer analysis was performed with 32P-labeled probes for individual PKC isoenzymes. Hybridization signals are shown for PKC-, -, and -; each lane contains 2 µg of polyadenylated RNA.

Finally, to assess further the distribution of PKC with a method that can detect expression below the limits of detection by these methods, we performed PCR amplification of reverse-transcribed RNA encoding PKC isoenzymes expressed in HEC and, for comparison, platelets, which we have previously shown by these nonquantitative methods to contain mRNA transcripts corresponding to both the cPKC and nPKC isoenzymes in approximately equal amounts.²⁸ The HEC, in contrast, were found to express mainly mRNA transcripts corresponding to nPKC isoenzymes (Fig 8). To determine whether this paucity of cPKCs was characteristic of other endothelial cell lines or was unique to the primary culture of HUVEC used for our HEC cultures, we performed a similar analysis on four cell lines derived from endothelial cells (PY4.1, murine hemangioma; RFPEC, rat microvascular cells; EAhy.926, a fusion of HEC with tumor cells; and AG4762, bovine aorta derived). None of the four endothelium-derived cell lines revealed robust expression of the cPKC isoenzymes; AG4762 and PY4.1 both, however, expressed faint bands that correspond to that subfamily. All four lines, in contrast, revealed easily detectable transcripts consistent with expression of nPKC isoenzymes. (The band just above the nPKC band corresponds to the size expected for nPKC-.²⁷ ) Thus, these methods establish that HEC express at least three phorbol ester–sensitive, Ca²⁺-independent isoenzymes (nPKC-, -, and -) that therefore are candidates to mediate bFGF-induced HEC proliferation.

View larger version (24K): [in this window] [in a new window]   Figure 8. Expression of PKC subfamilies in platelets and endothelial cells. Both panels depict agarose gel electrophoresis of products of reverse-transcribed (RT) RNA encoding PKC isoenzymes amplified by PCR with the use of degenerate, PKC-specific oligonucleotide primers capable of amplifying all discovered members of the cPKC and nPKC subfamilies. DNA marker lane (HindIII digested lambda phage) is shown (M), as are the anticipated positions of the cPKC subfamily (top arrow) expressed in platelet RNA and of the nPKC subfamily (bottom arrow) expressed in all cell types investigated. In A, amplified RT RNA derived from human platelets (P) and HUVEC (EC) is shown after electrophoresis through a 1% agarose gel. In B, amplified RT RNA derived from the EAhy.926 (EA), RFPEC (RF), PY4.1 (PY), and AG4762 (AG) endothelium-derived cell lines and HUVEC (HU) is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study demonstrate that growth-promoting effects of bFGF on HEC are mediated through activation of PKC. This conclusion is supported by two discrete observations. First, stimulation of HEC with bFGF produces an increase in PKC activity that is maximal at 30 minutes; such a sustained increase in PKC activity has been shown to be necessary for the induction of cell growth in transformed bovine aortic endothelial cells.¹⁰ Second, depletion of PKC by prolonged incubation of endothelial cells with high concentrations of PMA almost completely eliminates the growth-promoting effect of bFGF. Stimulation of HEC with two different tumor-promoting phorbol esters produced a concentration-dependent increase in their proliferation; however, if phorbol esters were incubated with cells more than 72 hours, the rate of growth rapidly diminished, and by 72 hours, cells were depleted of PKC, thus precluding any potential for further endothelial proliferation. (Although this intervention has been shown to also reduce binding of FGF to its receptor by 50%,¹⁴ it is clear that the total inhibitory effect on DNA synthesis¹⁴ and proliferation [present study] are much greater than can be explained by that effect.) In addition, both H-7 and the more-selective PKC inhibitor chelerythrine inhibited HEC proliferation in a concentration-dependent manner. Although the use of chemical inhibitors and of downregulation has been criticized,^{17 33 34} the fact that similar results were obtained with all three maneuvers bolsters our conclusion that PKC is required for bFGF-induced HEC proliferation. This conclusion cannot be generalized to all receptor tyrosine kinases, since EGF-induced mitogenesis of HEC was not affected by PKC inhibitors or by downregulation. However, the results of the present study suggest that PKC activation is sufficient to induce HEC proliferation; whether PKC is the only "upstream" mediator necessary for bFGF-induced mitogenesis is not resolved by these experiments.

The classic model for PKC activation in vivo is one in which the enzyme is activated by diacylglycerol and inositol triphosphate–mediated Ca²⁺ release, resulting from phosphatidylinositide metabolism as a consequence of phosphoinositidase-specific phospholipase C.^{35 36} Although phospholipase C- can be activated by FGF, thus triggering this pathway, experiments in which the receptor for bFGF was mutated so that it could no longer bind phospholipase C-, thus preventing phosphatidylinositide turnover, revealed that diacylglycerol and inositol triphosphate generation is not required for bFGF to induce mitogenesis in transfected myoblasts.^{37 38} These findings are compatible with the lack of Ca²⁺ increase and/or phosphatidylinositide turnover noted in bovine lens epithelium³⁹ and fibroblasts⁴⁰ after bFGF stimulation. Thus, PKC activated through this mechanism is not necessary for bFGF-induced proliferation of nonendothelial cells. These observations, however, do not diminish the possibility that PKC is necessary for bFGF-induced proliferation, as it has become clear that PKC can be activated by mediators other than diacylglycerol derived from phosphoinositide-specific phospholipase C, including arachidonate and other free fatty acids,^{41 42} lipoxygenase products,^{43 44} and phosphatidylcholine-derived diacylglycerol.⁴⁵ In particular, products of the action of phosphatidyl-inositol 3 kinase, which is activated after exposure to several mitogens,⁴⁶ including bFGF, have been shown to activate individual isoenzymes of PKC.⁴⁷ Activation of PKC by any of these mediators could, in some instances, occur in the absence of an increase in [Ca²⁺]_i, since Ca²⁺-mobilizing mediators, such as inositol triphosphate, are not always generated concomitantly, which is in contrast to the situation with phosphatidylinositol turnover.^{35 36} Although cPKCs, which are those most often studied, depend absolutely on an increase in [Ca²⁺]_i for their activation, neither of the two other groups of PKCs (nPKCs and aPKCs) require Ca²⁺ for activation.^{17 18} If one postulates that PKC is activated by one or more of the aforementioned alternate (or nonclassic) mechanisms in human endothelium, then one of the requirements is that it can occur in the absence of an increase in [Ca²⁺]_i. Thus, our finding that an increase in [Ca²⁺]_i did not precede PKC activation is compatible with this hypothesis and obviates a role for phosphatidylinositide turnover in the initial PKC activation induced by bFGF in HEC.

Also compatible with this model is the reduced expression in endothelium of cPKCs and the presence of at least four isoenzymes that are independent of Ca²⁺ in their activity. The diversity of the PKC family may also provide an explanation for the differences between some of the results of the present study and those reported previously. For example, endothelial cells from bovine aorta, a commonly used model for endothelial cell regulation, might be expected to exhibit different physiological responses arising from PKC activation than those of the cells in the present study, since the predominant isoenzymes that have been identified in those cells are the Ca²⁺-dependent, cPKC isoenzymes.⁴⁸ A related variable is the difference among isoenzymes in the completeness and time course of downregulation with prolonged incubation with phorbol ester,³² which might explain the differing effects of phorbol esters noted in other studies.¹⁵ Although a recent report has also found both cPKC and nPKC expression in HEC,⁴⁹ the exact distribution and relative abundance of these isoenzymes, as assessed in that report only through immunoblotting, differ from our findings. The reason for this discrepancy is not clear but cannot be explained by passage number, which was similar in the two studies, or by state of confluence, since we did not find that the PKC distribution changes with achievement of confluence.

We found that three nPKCs (-, -, and -) are highly expressed in HEC and are candidates for the gene product(s) that mediate bFGF-induced mitogenesis. Another isoenzyme that was highly expressed, aPKC-, is unlikely to mediate bFGF-induced proliferation in HEC, as this isoenzyme is unresponsive in most systems to downregulation by prolonged exposure to phorbol ester, which in the present study prevented proliferation induced by further addition of either phorbol ester or bFGF. If the inhibitory effect of nPKC- on proliferation of Chinese hamster ovary cells and fibroblasts^{19 20} can be generalized to other cell types, then nPKC- is most likely not the isoenzyme mainly responsible for bFGF-induced HEC proliferation, leaving nPKC- and - as the major candidates. Our finding that one or more of a limited subset of PKC isoenzymes present in HEC appear to be critical mediators of their proliferation after exposure to bFGF raises the possibility that these isoenzymes may represent selective targets for novel pharmacological agents to promote angiogenesis and vascular repair after injury.

	Selected Abbreviations and Acronyms

 

[Ac-MBP(4-14)] = synthetic peptide from myelin basic protein aPKC = atypical protein kinase C BCIP = 5-bromo-4-chloro-3-indolyl phosphate [Ca2+]i = cytoplasmic intracellular calcium concentration cPKC = conventional protein kinase C EGF = epidermal growth factor FBS = fetal bovine serum FGF = fibroblast growth factor H-7 = 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine HEC = human endothelial cells HUVEC = human umbilical vein endothelial cells LDL = low-density lipoprotein M-199 = medium-199 NBT = nitro blue tetrazolium nPKC = novel protein kinase C PCR = polymerase chain reaction PDBu = phorbol 12,13-dibutyrate PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate RFPEC = rat fat pad endothelial cell line SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

	Acknowledgments

 
This work was supported by the Beth Israel Surgical Trust and by National Institutes of Health (NIH) grants HL-38820 and HL-47032. Dr Ware is the recipient of a research career award (HL-02271) from the NIH. The authors gratefully acknowledge the investigators who provided cell lines and reagents, including Drs Robert Rosenberg, Michael Simons, Victoria Bautch, Cora-Jean Edgell, and Peter M. Blumberg, and John Jaster for preparation of the manuscript.

Received March 17, 1995; accepted April 5, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Lindner V, Reidy MA, Fingerle J. Regrowth of arterial endothelium: denudation with minimal trauma leads to complete endothelial cell regrowth. Lab Invest. 1989;61:556-563. [Medline] [Order article via Infotrieve]

2. Reidy MA, Schwartz SM. Endothelial regeneration: time course of intimal changes after small defined injury to rat aortic endothelium. Lab Invest. 1981;44:301-308. [Medline] [Order article via Infotrieve]

3. Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575-606. [Medline] [Order article via Infotrieve]

4. Gospodarowicz D, Neufeld G, Schweigerer L. Fibroblast growth factor. Mol Cell Endocrinol. 1986;46:187-204. [Medline] [Order article via Infotrieve]

5. Sato Y, Shimada T, Takaki R. Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro. Biochem Biophys Res Commun. 1991;180:1098-1102. [Medline] [Order article via Infotrieve]

6. Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest. 1990;85:2004-2008.

7. Dionne CA, Crumley G, Bellot F, Kaplow JM, Searfoss G, Ruta M, Burgess WH, Jaye M, Schlessinger J. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors. EMBO J. 1990;9:2685-2692. [Medline] [Order article via Infotrieve]

8. Kaibuchi K, Tsuda T, Kikuchi A, Tanimoto T, Yamashita T, Taka Y. Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts. J Biol Chem. 1986;261:1187-1192. [Abstract/Free Full Text]

9. Magnaldo I, L'Allemain G, Chambard JC, Moenner M, Barritault D, Pouyssegur J. The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts. J Biol Chem. 1986;261:16916-16922. [Abstract/Free Full Text]

10. Presta M, Tiberio L, Rusnati M, Dell'Era P, Ragnotti G. Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells. Cell Regul. 1991;2:719-726. [Medline] [Order article via Infotrieve]

11. Moodie SA, Martin W. Effects of cyclic nucleotides and phorbol myristate acetate on proliferation of pig aortic endothelial cells. J Pharmacol. 1991;102:101-106.

12. Monsento R, Orci L. Phorbol esters induce angiogenesis in vitro from large vessel endothelial cells. J Cell Physiol. 1987;130:284-291. [Medline] [Order article via Infotrieve]

13. Kinsella JL, Grant DS, Weeks BS, Kleinman HK. Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, matrigel. Exp Cell Res. 1992;199:56-62. [Medline] [Order article via Infotrieve]

14. Hoshi H, Kan M, Mioh H, Chen JK, McKeehan WL. Phorbol ester reduces number of heparin-binding growth-factor receptors in human adult endothelial cells. FASEB J. 1988;2:2797-2800. [Abstract]

15. Doctrow SR, Folkman J. Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens. J Cell Biol. 1987;104:679-687. [Abstract/Free Full Text]

16. Zho W, Takuwa N, Kumada M, Takuwa Y. Protein kinase C-mediated bidirectional regulation of DNA synthesis, RB protein phosphorylation, and cyclin-dependent kinases in human vascular endothelial cells. J Biol Chem. 1993;268:23041-23048. [Abstract/Free Full Text]

17. Chang JD, Ware JA. Ca²⁺ and protein kinase C in platelets. In: Lapetina EG, ed. Advances in Molecular and Cell Biology. In press.

18. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992;258:607-614. [Abstract/Free Full Text]

19. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, Kikkawa U, Nishizuka Y. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies. Proc Natl Acad Sci U S A. 1992;89:10159-10163. [Abstract/Free Full Text]

20. Mischak H, Goodnight JA, Kolch W, Martiny-Baron G, Schaechtle C, Kazanietz MG, Blumberg PM, Pierce JH, Mushinski JF. Overexpression of protein kinase C- and - in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J Biol Chem. 1993;268:6090-6096. [Abstract/Free Full Text]

21. Kent KC, Collins LJ, Schwerin FT, Raychowdhury MK, Ware JA. Identification of functional PGH₂/TXA₂ receptors on human endothelial cells. Circ Res. 1993;72:958-965. [Abstract/Free Full Text]

22. Dubois NA, Kolpack LC, Wang R, Azizkhan RG, Bautch VL. Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas. Exp Cell Res. 1991;196:302-313.[Medline] [Order article via Infotrieve]

23. Edgell CJS, McDonald CC, Graham JB. Permanent cell line with expression human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A. 1983;80:3734-3737. [Abstract/Free Full Text]

24. de Agostini AI, Watkins SC, Slayter HS, Youssoufian H, Rosenberg RD. Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta. J Cell Biol. 1990;111:1293-1304. [Abstract/Free Full Text]

25. Hashimoto K, Kishimoto A, Aihara H, Yasuda I, Mikawa K, Nishizuka Y. Protein kinase C during differentiation of human promyelocytic leukemia cell line, HL-60. FEBS Lett. 1990;263:31-34. [Medline] [Order article via Infotrieve]

26. Koide H, Ogita K, Kikkawa U, Nishizuka Y. Isolation and characterization of the E subspecies of protein kinase C from rat brain. Proc Natl Acad Sci U S A. 1992;89:1149-1153. [Abstract/Free Full Text]

27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]

28. Chang JD, Xu Y, Raychowdhury MK, Ware JA. Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC): a new member of the nPKC gene family expressed in skeletal muscle, megakaryoblastic cells, and platelets. J Biol Chem. 1993;268:14208-14214. [Abstract/Free Full Text]

29. Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990;172:993-999. [Medline] [Order article via Infotrieve]

30. Ko F-N, Chen I-S, Wu S-J, Lee L-G, Haung T-F, Teng C-M. Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans. Biochim Biophys Acta. 1990;1052:360-365. [Medline] [Order article via Infotrieve]

31. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984;23:5036-5041. [Medline] [Order article via Infotrieve]

32. Blumberg PM. Complexities of the protein kinase C pathway. Mol Carcinog. 1991;4:339-344. [Medline] [Order article via Infotrieve]

33. Szallasi Z, Smith CB, Pettit GR, Blumberg PM. Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts. J Biol Chem. 1994;269:2118-2124. [Abstract/Free Full Text]

34. Wilkinson SE, Hallam TJ. Protein kinase C: is its pivotal role in cellular activation over-stated? Trends Pharmacol Sci. 1994;15:53-57. [Medline] [Order article via Infotrieve]

35. Meldrum E, Parker PJ, Carozzi A. The PtdIns-PLC superfamily and signal transduction. Biochim Biophys Acta. 1991;1092:49-71. [Medline] [Order article via Infotrieve]

36. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361:315-325. [Medline] [Order article via Infotrieve]

37. Peters K, Marie J, Wilson E, Ives H, Escobedo J, Del Rosario M, Mirda D, Williams LT. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca²⁺ but not mitogenesis. Nature. 1992;358:678-681. [Medline] [Order article via Infotrieve]

38. Mohammadi M, Dionne CA, Li W, Li M, Spivak T, Honegger AM, Jaye M, Schlessinger J. Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature. 1992;358:682-684.

39. Moenner M, Magnaldo I, L'Allemain G, Barritault D, Pouyssegur J. Early and late mitogenic events induced by FGF on bovine epithelial lens cells are not triggered by hydrolysis of polyphosphoinositides. Biochem Biophys Res Commun. 1987;146:32-40. [Medline] [Order article via Infotrieve]

40. Nanberg E, Morris C, Higgins T, Vara F, Rozengurt E. Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca²⁺ mobilization or inositol phosphate accumulation. J Cell Physiol. 1990;143:232-242. [Medline] [Order article via Infotrieve]

41. Sekiguchi K, Tsukuda M, Ogita K, Kikkawa U, Nishizuka Y. Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids. Biochem Biophys Res Commun. 1987;145:797-802. [Medline] [Order article via Infotrieve]

42. Nishikawa M, Hidaka H, Shirakawa S. Possible involvement of direct stimulation of protein kinase C by unsaturated fatty acids in platelet activation. Biochem Pharmacol. 1988;37:3079-3089. [Medline] [Order article via Infotrieve]

43. Shearman MS, Naor Z, Sekiguchi K, Kishimoto A, Nishizuka Y. Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. FEBS Lett. 1989;243:177-182. [Medline] [Order article via Infotrieve]

44. Grabarek J, Ware JA. Protein kinase C activation without membrane contact in platelets stimulated by bryostatin. J Biol Chem. 1993;268:5543-5549. [Abstract/Free Full Text]

45. Pessin MS, Baldassare JJ, Raben DM. Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts: comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor. J Biol Chem. 1990;265:7959-7966. [Abstract/Free Full Text]

46. Varticovski L, Harrison-Findik D, Keeler ML, Susa M. Role of PI3-kinase in mitogenesis. Biochim Biophys Acta. 1994;1226:1-11. [Medline] [Order article via Infotrieve]

47. Nakanishi H, Brewer KA, Exton JH. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993;268:13-16. [Abstract/Free Full Text]

48. Rosales OR, Isales C, Nathanson M, Sumpio BE. Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells. Biochem Biophys Res Commun. 1992;189:40-46. [Medline] [Order article via Infotrieve]

49. Bussolino F, Silvagno F, Garbarino G, Costamagna C, Savanio F, Arese M, Soldi R, Aglietta M, Pescarmona G, Camussi G, Bosia A. Human endothelial cells are targets for platelet-activating factor (PAF). J Biol Chem. 1994;269:2877-2886.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. D. Short, S. M. Fox, C. F. Lam, K. R. Stenmark, and M. Das Protein Kinase C{zeta} Attenuates Hypoxia-induced Proliferation of Fibroblasts by Regulating MAP Kinase Phosphatase-1 Expression Mol. Biol. Cell, April 1, 2006; 17(4): 1995 - 2008. [Abstract] [Full Text] [PDF]

				S.-L. Pan, J.-H. Guh, C.-Y. Peng, S.-W. Wang, Y.-L. Chang, F.-C. Cheng, J.-H. Chang, S.-C. Kuo, F.-Y. Lee, and C.-M. Teng YC-1 [3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl Indazole] Inhibits Endothelial Cell Functions Induced by Angiogenic Factors in Vitro and Angiogenesis in Vivo Models J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 35 - 42. [Abstract] [Full Text] [PDF]

				H. Tang, B. Low, S. A. Rutherford, and Q. Hao Thrombin induces endocytosis of endoglin and type-II TGF-{beta} receptor and down-regulation of TGF-{beta} signaling in endothelial cells Blood, March 1, 2005; 105(5): 1977 - 1985. [Abstract] [Full Text] [PDF]

				G. Pintus, B. Tadolini, A. M Posadino, B. Sanna, M. Debidda, C. Carru, L. Deiana, and C. Ventura PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation Cardiovasc Res, October 1, 2003; 59(4): 934 - 944. [Abstract] [Full Text] [PDF]

				Y. Zhang, J. Li, C. Partovian, F. W. Sellke, and M. Simons Syndecan-4 modulates basic fibroblast growth factor 2 signaling in vivo Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2078 - H2082. [Abstract] [Full Text] [PDF]

				Y. Zhang, L. J. Bloem, L. Yu, T. B. Estridge, P. W. Iversen, C. E. McDonald, J. P. Schrementi, X. Wang, C. J. Vlahos, and J. Wang Protein kinase C {beta}II activation induces angiotensin converting enzyme expression in neonatal rat cardiomyocytes Cardiovasc Res, January 1, 2003; 57(1): 139 - 146. [Abstract] [Full Text] [PDF]

				S. Hussain, J. W. Assender, M. Bond, L.-F. Wong, D. Murphy, and A. C. Newby Activation of Protein Kinase Czeta Is Essential for Cytokine-induced Metalloproteinase-1, -3, and -9 Secretion from Rabbit Smooth Muscle Cells and Inhibits Proliferation J. Biol. Chem., July 19, 2002; 277(30): 27345 - 27352. [Abstract] [Full Text] [PDF]

				I. Spyridopoulos, C. Luedemann, D. Chen, M. Kearney, D. Chen, T. Murohara, N. Principe, J. M. Isner, and D. W. Losordo Divergence of Angiogenic and Vascular Permeability Signaling by VEGF: Inhibition of Protein Kinase C Suppresses VEGF-Induced Angiogenesis, but Promotes VEGF-Induced, NO-Dependent Vascular Permeability Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 901 - 906. [Abstract] [Full Text] [PDF]

				M. Murakami, A. Horowitz, S. Tang, J. A. Ware, and M. Simons Protein Kinase C (PKC) delta Regulates PKCalpha Activity in a Syndecan-4-dependent Manner J. Biol. Chem., May 31, 2002; 277(23): 20367 - 20371. [Abstract] [Full Text] [PDF]

				A. Wang, M. Nomura, S. Patan, and J. A. Ware Inhibition of Protein Kinase C{alpha} Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo Circ. Res., March 22, 2002; 90(5): 609 - 616. [Abstract] [Full Text] [PDF]

				A. P. McLaughlin and G. W. De Vries Role of PLCgamma and Ca2+ in VEGF- and FGF-induced choroidal endothelial cell proliferation Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1448 - C1456. [Abstract] [Full Text] [PDF]

				H. Itoh, S. Yamamura, J. A. Ware, S. Zhuang, S. Mii, B. Liu, and K. C. Kent Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H359 - H370. [Abstract] [Full Text] [PDF]

				D. Corseaux, T. Meurice, I. Six, L. Rugeri, M. D. Ezekowitz, P. Rouvier, R. Bordet, C. Bauters, and B. Jude Basic Fibroblast Growth Factor Increases Tissue Factor Expression in Circulating Monocytes and in Vascular Wall Circulation, April 25, 2000; 101(16): 2000 - 2006. [Abstract] [Full Text] [PDF]

				D. Chen, K. Walsh, and J. Wang Regulation of cdk2 Activity in Endothelial Cells That Are Inhibited From Growth by Cell Contact Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 629 - 635. [Abstract] [Full Text] [PDF]

				K. Kuboki, Z. Y. Jiang, N. Takahara, S. W. Ha, M. Igarashi, T. Yamauchi, E. P. Feener, T. P. Herbert, C. J. Rhodes, and G. L. King Regulation of Endothelial Constitutive Nitric Oxide Synthase Gene Expression in Endothelial Cells and In Vivo : A Specific Vascular Action of Insulin Circulation, February 15, 2000; 101(6): 676 - 681. [Abstract] [Full Text] [PDF]

				Y. Shizukuda, A. Helisch, R. Yokota, and J. A. Ware Downregulation of Protein Kinase C{delta} Activity Enhances Endothelial Cell Adaptation to Hypoxia Circulation, November 2, 1999; 100(18): 1909 - 1916. [Abstract] [Full Text] [PDF]

				H. Yoshiji, S. Kuriyama, D. K. Ways, J. Yoshii, Y. Miyamoto, M. Kawata, Y. Ikenaka, H. Tsujinoue, T. Nakatani, M. Shibuya, et al. Protein Kinase C Lies on the Signaling Pathway for Vascular Endothelial Growth Factor-mediated Tumor Development and Angiogenesis Cancer Res., September 1, 1999; 59(17): 4413 - 4418. [Abstract] [Full Text] [PDF]

				R. Volk, J. J. Schwartz, J. Li, R. D. Rosenberg, and M. Simons The Role of Syndecan Cytoplasmic Domain in Basic Fibroblast Growth Factor-dependent Signal Transduction J. Biol. Chem., August 20, 1999; 274(34): 24417 - 24424. [Abstract] [Full Text] [PDF]

				E. Villard, A. Alonso, M. Agrapart, M. Challah, and F. Soubrier Induction of Angiotensin I-converting Enzyme Transcription by a Protein Kinase C-dependent Mechanism in Human Endothelial Cells J. Biol. Chem., September 25, 1998; 273(39): 25191 - 25197. [Abstract] [Full Text] [PDF]

				S. Tang, K. G. Morgan, C. Parker, and J. A. Ware Requirement for Protein Kinase C theta  for Cell Cycle Progression and Formation of Actin Stress Fibers and Filopodia in Vascular Endothelial Cells J. Biol. Chem., November 7, 1997; 272(45): 28704 - 28711. [Abstract] [Full Text] [PDF]

				J. C. Mason, H. Yarwood, K. Sugars, and D. O. Haskard Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1233 - C1240. [Abstract] [Full Text] [PDF]

				M. Yukawa, R. Yokota, R. T. Eberhardt, L. von Andrian, and J. A. Ware Differential Desensitization of Thromboxane A2 Receptor Subtypes Circ. Res., April 19, 1997; 80(4): 551 - 556. [Abstract] [Full Text]

				E. O. Harrington, J. Loffler, P. R. Nelson, K. C. Kent, M. Simons, and J. A. Ware Enhancement of Migration by Protein Kinase Calpha and Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase Cdelta in Capillary Endothelial Cells J. Biol. Chem., March 14, 1997; 272(11): 7390 - 7397. [Abstract] [Full Text] [PDF]

				Y.-C. Chai, P. H. Howe, P. E. DiCorleto, and G. M. Chisolm Oxidized Low Density Lipoprotein and Lysophosphatidylcholine Stimulate Cell Cycle Entry in Vascular Smooth Muscle Cells. EVIDENCE FOR RELEASE OF FIBROBLAST GROWTH FACTOR-2 J. Biol. Chem., July 26, 1996; 271(30): 17791 - 17797. [Abstract] [Full Text] [PDF]

				H. Haller, W. Ziegler, C. Lindschau, and F. C. Luft Endothelial Cell Tyrosine Kinase Receptor and G Protein–Coupled Receptor Activation Involves Distinct Protein Kinase C Isoforms Arterioscler Thromb Vasc Biol, May 1, 1996; 16(5): 678 - 686. [Abstract] [Full Text]

				A. Wang, M. Nomura, S. Patan, and J. A. Ware Inhibition of Protein Kinase C{alpha} Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo Circ. Res., March 22, 2002; 90(5): 609 - 616. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kent, K. C. Articles by Ware, J. A. Search for Related Content PubMed PubMed Citation Articles by Kent, K. C. Articles by Ware, J. A.