Inhibition of Protein Kinase Cα Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo
Although protein kinase C (PKC) activation is required for endothelial cell (EC) growth, migration, adhesion, and vessel formation, the role of individual PKC isoenzymes in these events is not defined. Because PKCα has been previously linked with enhanced EC migration and response to angiogenic growth factors, we characterized a specific phosphorothioate-modified 21-mer antisense PKCα (AS-PKCα). AS-PKCα (500 nmol/L) prevented the expression of PKCα protein by 90% in human ECs and did not reduce the expression of any other PKC isoenzyme. AS-PKCα reduced human EC migration by 64% compared with its control oligonucleotide in a “scratch” wounding assay, and AS-PKCα reduced human EC adhesion to the extracellular matrix protein vitronectin by 18%. Phosphorylation of mitogen-activated protein kinase (extracellular signal–regulated kinase 1/2) induced by vascular endothelial growth factor was inhibited by 30% in human ECs transfected with AS-PKCα. Compared with control, AS-PKCα also reduced the number of EC tubes formed in a 3D type I collagen gel assay by 37.5%. Finally, using an osmotic minipump, we infused AS-PKCα into mice in which myocardial infarction was induced by coronary ligation and found that the oligonucleotide was primarily taken up by intramyocardial blood vessels. Compared with the results with control oligonucleotide, AS-PKCα oligonucleotide inhibited the number of anti-PKCα–stained blood vessels by 48% and reduced the total vessel number by 72% as well. In conclusion, the expression of PKCα is required for full EC migration, adhesion to vitronectin, vascular endothelial growth factor–induced extracellular signal–regulated kinase activation, and tube formation and is likely to be of importance in myocardial angiogenesis in vivo after ischemia.
Angiogenesis, the formation of new blood vessels, is an important response to myocardial ischemia. In established blood vessels, the endothelial cells (ECs) remain in a quiescent state. After local ischemia, as well as wounding or inflammation, new blood vessels develop.1 Protein kinase C (PKC), an intracellular serine-threonine kinase, has been reported to have a pivotal role in angiogenesis and associated processes.2–4⇓⇓ PKC activation is required for EC proliferation after fibroblast growth factor (FGF)-2 stimulation, angiogenesis after vascular endothelial growth factor (VEGF) stimulation, and capillary tube formation after thrombin stimulation.5 Multiple studies have demonstrated that PKC is not a single entity but is instead a family of at least 12 isoenzymes,2,6,7⇓⇓ of which at least 5 are expressed in cultured human ECs2,8⇓ and in angiogenic vessels in vivo.9 The PKC isoenzymes are derived from different genes, have different activating cofactors, and vary in tissue and cellular distribution. Because of these different properties, these isoenzymes have distinct effects in cells in vitro.10–12⇓⇓ To date, no one has examined whether there is similar heterogeneity concerning the expression or role of individual PKC isoenzymes in endothelial function either in intact blood vessels or in angiogenesis in vivo, and very little work has been done regarding the detailed molecular mechanism of the effects of PKC in ECs. Such experiments in vascular cells and tissues are critical to an understanding of the role of PKC in angiogenesis. Removal of the individual PKC isoenzymes in knockout mice by homologous recombination has been reported for PKCγ, PKCβ, PKCε, and PKCθ.13–16⇓⇓⇓ The phenotype of these mice has been partially assessed, and abnormalities in the neural, immunological, and endocrine systems have been noted. Although these mice are viable, suggesting that embryonal microvascular formation is relatively intact, none have been tested for disturbance in vascular function or for neovascularization after myocardial ischemia. Furthermore, even in the absence of an abnormality, it is possible that the congenital absence of an individual gene product could produce a compensatory response during development that would obscure its real pathophysiological role. We have shown that overexpression of PKCα in rat ECs promotes their migration,11 but the specificity of that function for PKCα and whether PKCα has a function in angiogenesis in vivo have not been established. In the present study, we show, with the use of a PKCα-specific antisense (AS-PKCα) oligonucleotide, that inhibition of PKCα inhibits EC migration, adhesion, and extracellular signal–regulated kinase (ERK) phosphorylation and tube formation in vitro. Most important, we show in the present study that AS-PKCα oligonucleotide can be taken up by the intramyocardial endothelium and can inhibit neovascularization after infarction.
Materials and Methods
Human ECs were isolated from umbilical veins as previously reported.17 These human umbilical vein ECs (HUVECs) between passages 1 and 3 were grown in medium 199 (M199, Life Technologies, Inc) supplemented with 20% newborn calf serum, 5% human serum, heparin (Sigma Chemical Co), EC growth factor (Sigma), bovine brain extract, 50 U/mL penicillin G, and 50 μg/mL streptomycin. Anti-PKCα was obtained from Transduction Laboratories.
Antisense Oligonucleotides for PKCα
The sequence of the antisense oligonucleotides for human PKCα that was used in the present study is as follows: AS-PKC-α, 5′-GGGAAAACGTCAGCCATGGTC-3′. This 21-mer was designed to hybridize with position 24–44 of the PKCα mRNA. The random sequence for PKC-α, 5′-GGCCTGACGTAAGATCGAGAC-3′ (also a 21-mer oligonucleotide), was generated as a control. The AS-PKCα for the mouse sequence is 5′-AACGTCAGCCATG-GTTCCCCC-3′. This 21-mer oligonucleotide was designed to hybridize with both mouse and human mRNA.
Transfection of AS-PKCα Oligonucleotide by GenePorter
HUVECs were grown to 80% to 90% confluence in 100-mm tissue culture dishes for transfection. GenePorter (Gene Therapy Systems) was prepared in 1 mL M199 for 20 minutes. Oligonucleotides were also diluted in 1 mL M199 for 20 minutes. Then, GenePorter medium and oligonucleotide M199 were mixed gently and equilibrated for 35 minutes. Another 3 mL of M199 was added to produce a final concentration of 250 nmol/L and 500 nmol/L oligonucleotide. The cells were washed with M199 twice and treated with the GenePorter-oligonucleotide mixture for 4 hours at 37°C in 5% CO2. M199 with serum containing the same concentrations of oligonucleotides was added to the plates for another 4 hours. After a total of 8 hours, the medium was returned to M199 with 20% human serum. Cells were harvested 3 days later.
HUVECs were transfected with AS-PKCα, random sequence, and control without oligonucleotides for 3 days according to the method mentioned. HUVECs were rinsed with ice-cold PBS and lysed in RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/mL phenylmethylsulfonyl fluoride, 30 μL/ml aprotinin, and 1 mmol/L sodium vanadate). Total protein (20 μg) from each sample was subjected to SDS-PAGE. The concentration of the protein was assessed by using the Bradford assay (Bio-Rad Laboratories) before loading. After transfer, the blotting membrane was incubated for 1 hour at room temperature in Tween 20–PBS buffer with 5% (wt/vol) dry milk to block nonspecific binding sites. Then, the membrane was incubated with primary antibody at a 1:1000 or 1:2000 (α-tubulin) dilution in PBS containing 0.05% Tween 20 with 5% (wt/vol) dry milk. The protein levels were quantified by densitometry with Bio-Rad gel molecular analysis software. The membrane was then stripped with stripping buffer and reblotted with monoclonal anti–α-tubulin antibody (Sigma) for protein loading control.
Endothelial “Wounding” Assays to Assess Migration
HUVECs were transfected with AS-PKCα oligonucleotides or their controls for 3 days. Cells were then treated with trypsin and cultured on 6-well plates in HUVEC medium until confluent. The monolayers were wounded by scraping ≈600 μm with a 1- to 200-μL pipette tip. The distance of the gap was measured under a ×4 phase objective of a light microscope and captured with a Sony video graphic system (monitor, PVM97; printer, UP890 MD).
Before carrying out the adhesion assay, HUVECs were transfected with AS-PKCα or control for 3 days. Cell adhesion was then performed as previously described with minor modifications.18,19⇓ Briefly, 96-well polystyrene plates were coated with 10 μg/mL vitronectin in PBS. HUVECs were harvested and suspended in M199 (GIBCO-BRL) medium with 2% BSA at a concentration of 106 cells/mL. Cells (50 000) were added to each well of a 96-well plate. After incubation at 37°C for 1 hour, nonadherent cells were removed with gentle washing by sterile PBS. The remaining cells in wells of control plates without oligonucleotide treatment were used as the standard (100%). Quantification of cell attachment was determined by staining the cells with 0.5% crystal violet in 20% methanol, washing the cells, and then eluting the dye with 0.1 mol/L sodium citrate, pH 4.2, and measuring the absorbency at 595 nm.
ERK Activity Assay
HUVECs were first transfected with AS-PKCα oligonucleotides, random oligonucleotides, or control without oligonucleotides for 3 days. Then, HUVECs were grown in 1% serum-starvation medium at 37°C for 18 hours. Afterward, cells were stimulated by VEGF or FGF-2 in serum-free medium for specified times. Phosphorylation of ERK1/2 was detected by immunoblotting with the use of polyclonal phospho-ERK12 antibody (New England Biolabs) with horseradish peroxidase–conjugated goat complexes on nitrocellulose, which were visualized by an enhanced chemoluminescence detection system (Amersham). Density of the bands was analyzed by Bio-Rad gel molecular analysis software.
Cell Proliferation Analysis
Cell proliferation was determined by counting the cells with a Coulter counter. Subconfluent cells were synchronized in medium without EC growth factor for 24 hours and then seeded at 2.0×104 per 35-mm plate in 2 mL of medium without EC growth factor or with 20 ng/mL VEGF or 20 ng/mL basic FGF (bFGF). After being cultured for indicated periods, the HUVECs were washed with PBS, treated with trypsin, and suspended in the medium for counting.
3D In Vitro Tube Formation
A 3D collagen gel assay for angiogenesis was performed in vitro as described.20 Briefly, rat tail collagen type I from Upstate Biotech Inc (1.75 mg/mL) and human fibronectin at a final concentration of 90 μg/mL were mixed with 1.8 mg/mL NaHCO3, 1× M199, and 0.075 mol/L HEPES and placed on ice. HUVECs were then mixed with the collagen mixture at a final concentration of 2×106 cells/mL. Approximately 400 μL of the above cell-collagen mixture was added to a 12-well plate and incubated for 10 minutes after the addition of 1 mL HUVEC medium. After 2 days, tube length was measured by microscope. To confirm that tubes were actually formed in the collagen gel, the gels were fixed and embedded in paraffin and cut into 5-μm sections and stained with hematoxylin and eosin. Tubular structures complete with lumina that were formed in the collagen gel were identified under high-magnification microscopy.
In Vivo Experiments
Eight- to 9-week-old male C57BL/6 mice were used in all experiments. The day before the induction of myocardial infarction, the mice were anesthetized with methoxyflurane, and a miniosmotic pump (Alzet) was implanted between the scapulae to infuse the oligonucleotides into the mice. The rate of infusion of the pump was 0.25 μL/h for 14 days. Each oligonucleotide was diluted in saline solution to a defined concentration; the dosage for AS-PKCα and random sequence oligonucleotides was 1 or 2 mg/kg per day for 14 days.
Myocardial infarction was created by ligating the left coronary artery in the mouse heart at open surgery as described.21 Briefly, the mice were anesthetized initially with methoxyflurane and ventilated with a rodent respirator attached to a nose cone. The heart was exposed through a left-sided thoracotomy at the 4th intercostal space. The myocardial infarction was produced by permanent ligation of the left coronary artery with an 8.0 monofilament polypropylene suture with a tapered needle. The suture was passed underneath the left coronary artery 1 to 3 mm from the tip of the left atrium. Successful ligation of the coronary was identified by the regional discoloration resulting from ischemia. The chest was closed; after 14 days, the mice were euthanized, and the hearts were taken for immunohistochemistry to measure the PKC gene expression and vessel formation.
The mice receiving either AS-PKCα oligonucleotide or control were euthanized 14 days after myocardial infarction. The heart was then removed, fixed in 4% paraformaldehyde, and embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin and monoclonal antibody against PKCα or with mouse platelet-EC adhesion molecule-1 (PECAM-1, BD Pharmingen). Immunohistochemical experiments were performed by using the Vectastain Elite ABC kit (Vector Laboratories). Negative controls were performed by omitting the primary antibodies. Double staining of sections was carried out by first staining the sections for PKCα and subsequently staining them for PECAM-1, as detailed above. The results were viewed with the use of a Nikon microscope and were photographed by a Nikon digital camera. The numbers of vessels in the sections were counted, and the mean and probability values were calculated by using the Student t test. Ten visual fields were quantified in sites of active angiogenesis in three different areas. For each group of mice, the ratios of the number of anti-PKCα–staining vessels to the total number of vessels were also calculated.
Data are presented as mean±SE. Significant differences were determined by the Student t test (P<0.05).
Specificity and Efficacy of AS-PKCα
To determine the specificity of the AS-PKCα oligonucleotide, the oligonucleotide, a random sequence oligonucleotide, and control were transfected into HUVECs by using the GenePorter reagent at 250 nmol/L and 500 nmol/L (final concentration). A 5′-fluorescein–labeled AS-PKCα was also used to test the transfection efficiency, which was found to be nearly 100%. Western blot analyses were performed to measure level of the PKCα protein expression level in these treated cells. Figure 1 shows experimental results at an oligonucleotide concentration of 500 nmol/L. The results show that AS-PKCα inhibited PKCα protein expression by 90% and 50% at 500 nmol/L and at 250 nmol/L concentrations, respectively. At 500 nmol/L, AS-PKCα did not inhibit the other nontargeted PKC isoenzymes. Thus, the AS-PKCα synthesized in these experiments is a specific and potent inhibitor of PKCα.
Inhibition of EC Migration by AS-PKCα
To determine the role of PKCα in the migration of ECs, HUVECs were first transfected with AS-PKCα oligonucleotides and controls for 3 days, and then the transfected cells were subcultured in 6-well plates at a 5×105 cell density. The endothelial wounding assay was performed to test cell migration. The result is shown in Figure 2; AS-PKCα inhibited HUVEC migration by 64% compared with control. These experiments show that PKCα is required for full EC migration.
Inhibition of EC Adhesion by AS-PKCα
EC adhesion via the αvβ3 and αvβ5 integrins is a critical step in angiogenesis. To determine the effect of PKCα on the function of these integrins, we measured the ability of ECs to adhere to the matrix protein vitronectin. Cells transfected for 3 days were seeded on 96-well plates. The results from the experiments are shown in Figure 3. Compared with control, AS-PKCα inhibited EC adhesion; adhesion of the cells treated with AS-PKCα decreased by 18% compared with that seen with the untreated and control oligonucleotide–treated cells. Thus, these experiments demonstrate that inhibition of PKCα may affect adhesion to vitronectin, a process important in angiogenesis.
Effect of AS-PKCα on ERK Activity
In preliminary experiments, we found that ERK1/2 activity increased after stimulation by 50 ng/mL VEGF or 50 ng/mL FGF-2, with a peak at 20 minutes, followed by a gradual decline in activity. Thus, the 20-minute point was chosen for use in subsequent ERK1/2 experiments (Figure 4). ECs were transfected with AS-PKCα and random sequence oligonucleotides or no oligonucleotide control for 3 days. Then, ECs were stimulated by VEGF or FGF-2. The ERK1/2 activity was measured by immunoblotting. We found that AS-PKCα inhibited 30% of the phosphorylation of ERK1/2 induced by VEGF but not any of the ERK phosphorylation resulting from FGF-2. Thus, PKCα appears to contribute to VEGF-induced ERK phosphorylation, with the majority of this phosphorylation being induced by other VEGF-stimulated mediators.
Effects of AS-PKCα on Cell Proliferation
Figure 5 shows the result on AS-PKCα cell proliferation in different conditions. In Figure 5A, HUVECs grown in the medium without endothelial growth supplements exhibited no changes with AS-PKCα compared with control. In contrast, the growth rate of HUVECs treated with 20 ng/mL VEGF was inhibited 18% to 20% by AS-PKCα (Figure 5B). AS-PKCα did not affect the growth rates, which were similar in HUVECs stimulated by 20 ng/mL bFGF (Figure 5C).
To examine further the function of PKCα in tube formation, AS-PKCα transfected into ECs was assayed in a 3D collagen gel experiment. Figure 6 shows that compared with control, AS-PKCα oligonucleotide inhibited 3D collagen tube formation by 37.5% in complete medium (Figure 6B). In Figure 6C, AS-PKCα slightly inhibited tube formation by 25% compared with control in HUVECs stimulated by 50 ng/mL VEGF. Thus, PKCα appears to be important mediator for tube formation as well as EC migration in vitro.
In Vivo Experiments
On the basis of these in vitro experimental results, we tested the importance of AS-PKCα in angiogenesis in vivo. In initial experiments, we tested the feasibility of delivering AS-PKC oligonucleotides to intramyocardial blood vessels with an implanted osmotic minipump. In this experiment, an FITC-labeled AS-PKCα oligonucleotide was loaded into a micro-osmotic pump and implanted subcutaneously between the scapulae of the mice. After 10 days, the mice were euthanized, and the hearts were examined to determine whether the oligonucleotide entered the heart microvasculature by looking at a heart tissue sample under a fluorescence microscope. These results (shown in Figure 7) indicated that PKCα could circulate to the microvasculature of the heart. Figure 8A shows the immunohistochemical staining for PKCα and staining for PKCε and PKCβ1 for the AS-PKCα–treated sample to determine the specificity of AS-PKCα. Mα is the mouse sample treated with AS-PKCα oligonucleotide designed to block both human and mouse PKCα protein synthesis. PKCα and PECAM-1 double staining are shown in Figure 8B at 2 mg/kg per day of AS-PKCα oligonucleotide or control. In Figure 8A, PKCα gene expression is shown to be inhibited significantly compared with the saline and random sequence controls in heart tissue samples at the oligonucleotide dosage of 2 mg/kg per day. To further establish the identity of the vessels, double staining was performed, and the results are shown in Figure 8B. The vessels stained brown indicate those that are double-stained with black (anti-PKCα) and red (anti–PECAM-1). In Figure 8C, the inhibitory effect of antisense PKCα is represented as the bar graph of the total vessel number in the three groups of mice. Figure 8C demonstrates that AS-PKCα inhibited vessel formation by 72%. In Figure 8D, the effect of the inhibition of AS-PKCα is present as the ratio of the anti-PKCα–stained vessel number to the total vessel number in each group itself. The ratios for saline-treated and random sequence–treated mouse hearts are 0.97±0.01 and 0.99±0.01, respectively; however, in the antisense-treated mouse heart sample, the ratio is 0.51±0.07 (all are mean±SEM, n=5). Thus, compared with control, AS-PKCα oligonucleotide inhibited PKCα expression by 48%. Staining results show the dosage-dependent inhibition to PKCα expression. At 1 mg/kg body wt per day, there are no inhibitions for PKCα expression (data not shown). The other PKC isoenzymes were not inhibited by AS-PKCα oligonucleotide.
The major finding of the present study is that PKCα is a necessary component of the signal transduction pathway that is involved in EC migration, adhesion, and tube formation in vitro and angiogenesis after myocardial ischemia. In addition, the present study also demonstrated that delivery of an AS-PKCα oligonucleotide could selectively inhibit PKCα gene expression in vivo, along with preventing myocardial vessel formation, in a murine model of myocardial infarction.
PKC is a family of at least 12 kinases, some of which have been shown to be essential for EC growth and angiogenesis. Previously, we have demonstrated that overexpression of PKCα in rat ECs promotes migration, a key component of angiogenesis, whereas overexpression of PKCδ to a similar degree does not affect migration but, instead, slows cell cycle progression by delaying the passage of the cells through S phase.11 PKCθ overexpression has neither of these effects.12 These findings suggest that PKC isoenzymes phosphorylate different substrates in these cells with different physiological effects. There are several limitations in the interpretation of overexpression studies because of the potential lack of specificity. Thus, we chose to inhibit an individual PKC isoenzyme to determine its effects on angiogenesis after ischemia. The results of the present study demonstrate that AS-PKCα inhibited EC migration significantly, thus confirming our previous conclusion11 and expanding those findings markedly to show implications for angiogenesis in vivo.
ERK1/2, from the mitogen-activated protein kinase (MAPK) family, is a key component of the cascade of intracellular signaling pathways that lead to EC growth, migration, and adhesion. Previous studies have shown that inhibition of PKCε in ECs prevents MAPK (ERK) activity in response to shear stress.22 In the present study, AS-PKCα oligonucleotide inhibited a small portion of ERK-1 activity induced by VEGF but not by FGF-2, suggesting that PKCα is involved in the pathway to ERK1/2 induced by VEGF. Others have found that the mitogenic effects of VEGF on ECs are mediated by PKCα.23 In the present research, the slight inhibitory effects of AS-PKCα on VEGF-stimulated HUVEC proliferation and collagen tube formation are consistent with the reduction in ERK activity and thus confirm previous results.23 In other studies, Kent et al24 also demonstrated that, although FGF-2 increased overall PKC activity, the changes in cellular function were correlated with changes in Ca2+-independent PKC isoenzymes rather than PKCα. The results in the present study confirm that AS-PKCα oligonucleotide inhibited a component of the ERK1/2 activity induced by VEGF but not by FGF-2 and that it also inhibited the EC adhesion to vitronectin. Previously, we had shown that overexpressing PKCα did not affect vitronectin adhesion,11 raising the possibility that such adhesion is regulated by a threshold amount of PKCα, which is not achieved in AS-PKCα–treated ECs. Because of the association of αvβ5 with VEGF-mediated angiogenesis (in contrast to the association of FGF-2 with αvβ3),25 we speculate that the PKCα isoenzyme is activated after αvβ5 adhesion to vitronectin.
Because of the lack of specific chemical inhibitors for PKC isoenzymes, assessment of the function of individual PKC isoenzymes in vivo has not been definitive. In cardiovascular research, antisense oligonucleotides (predominantly phosphorothioate oligodeoxynucleotides) have been used in ischemia and other disease models. For example, antisense oligodeoxynucleotides inhibited PKCα expression in porcine aortic ECs and were used to demonstrate that the Ca2+ antagonist nifedipine influences ischemia-induced endothelial permeability via PKCα inhibition.26 In addition, antisense oligonucleotides have also been successfully used to inhibit PKC protein synthesis by vascular smooth muscle cells.27,28⇓ The advantage of antisense strategy is its high selectivity29; a sequence of 15 bases is sufficient to ensure the required selectivity.30 The present studies show the feasibility of this method to investigate gene expression in the myocardial vascular tube.
An antisense oligonucleotide to PKCα has been used in a phase I study in patients with cancer31 and was found to prevent tumor growth in vivo, but the mechanism for this effect is not known. In the present study, the dosages used in the clinical trial were used as guides for treatment after myocardial infarction. Because it used a myocardial infarction angiogenesis model to study the function of AS-PKCα, the present study cannot be compared directly with that clinical observation, but it suggests that one cause of that effect may be the inhibition of angiogenesis. The present study treated mice for 14 days to maintain the concentration of antisense oligonucleotide circulating in the heart of each mouse and, thus, to prolong the inhibition of PKCα. The length of time necessary for treatment likely remains to be established. In summary, our determination that inhibition of PKCα gene expression was feasible in vivo and could prevent vessel formation in an animal model is well correlated with the effects of this gene product in vitro. Thus, we conclude that PKCα is a key mediator of angiogenesis and a potential target in the development of selective agonists (cardiovascular disease) or antagonists (cancer). Finally, the method used in the present study will provide a useful tool for investigating the roles of individual PKC isoenzymes in postinfarction collateral formation in vitro and in vivo.
This work was supported by NIH grant HL-65980-05 (Dr Ware).
Original received October 23, 2001; revision received January 25, 2002; accepted January 28, 2002.
- ↵Ware JA, Simons M. Angiogenesis and Cardiovascular Disease. New York, NY: Oxford University Press; 1999: 30–59.
- ↵Haralabopoulos GC, Grant DS, Kleinman HK, Maragoudakis ME. Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Am J Physiol. 1997; 273(1 pt 1): C239–C245.
- ↵Hofman J. The potential for isoenzyme-selective modulation of protein kinase C. FASEB J. 1997; 11: 649–669.
- ↵Strasser RH, Simonis G, Schon SP, Braun MU, Ihl-Vahl R, Weinbrenner C, Marquetant R, Kubler W. Two distinct mechanisms mediate a differential regulation of protein kinase C isozymes in acute and prolonged myocardial ischemia. Circ Res. 1999; 85: 77–87.
- ↵Ashton AW, Watanabe G, Albanese C, Harrington EO, Ware JA, Pestell RG. Protein kinase Cδ inhibition of S-phase transition in capillary endothelial cells involves the cyclin dependent kinase inhibitor p27Kip1. J Biol Chem. 1999; 274: 20805–20811.
- ↵Harrington EO, Loffler J, Nelson PR, Kent KC, Simons M, Ware JA. Enhancement of migration by protein kinase Cα and inhibition of proliferation and cell cycle progression by protein kinase Cδ in capillary endothelial cells. J Biol Chem. 1997; 272: 7390–7397.
- ↵Tang S, Morgan KG, Parker C, Ware JA. Requirement for protein kinase C θ for cell cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells. J Biol Chem. 1997; 272: 28704–28711.
- ↵Leitges M, Schmedt C, Guinamard R, Davoust J, Schaal S, Stabel S, Tarakhovsky A. Immunodeficiency in protein kinase Cβ-deficient mice. Science. 1996; 273: 788–791.
- ↵Shizukuda Y, Helisch A, Yokota R, Ware JA. Down-regulation of protein kinase C δ activity enhances endothelial cell adaptation to hypoxia. Circulation. 1999; 100: 1909–1916.
- ↵Leavesley DI, Ferguson GD, Wayner EA, Cheresh DA. Requirement of the integrin β3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. J Cell Biol. 1992; 117: 1101–1107.
- ↵Sierra-Honigmann MR, Nath AK. Murakami C, Garcia-Cardena G, Papapetropoulos A, Sessa WC, Madge LA, Schechner JS, Schwabb MB, Polverini PJ, Flores-Riveros JR. Biological action of leptin as an angiogenic factor. Science. 1998; 281: 1683–1686.
- ↵Traub O, Monia BP, Dean NM, Berk BC. PKC-ε is required for mechano-sensitive activation of ERK1/2 in endothelial cells. J Biol Chem. 1997; 272: 31251–31257.
- ↵Wellner M, Maasch C, Kupprion C, Lindschau C, Luft FC, Haller H. The proliferative effect of vascular endothelial growth factor protein kinase C-α and protein kinase C-ζ. Arterioscler Thromb Vasc Biol. 1999; 19: 178–185.
- ↵Kent KC, Mii S, Harrington EO, Chang JD, Mallette S, Ware JA. Requirement for protein kinase C activation in basic fibroblast growth factor-induced human endothelial cell proliferation. Circ Res. 1995; 77: 231–242.
- ↵Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct α v integrins. Science. 1995; 270: 1500–1502.
- ↵Hempl A, Lindschau C, Massch C, Mahn M, Bychkov R, Noll T, Luft FC, Haller H. Calcium antagonists ameliorate ischemia-induced endothelial cell permeability by inhibiting protein kinase C. Circulation. 1999; 99: 2523–2529.
- ↵Haller H, Lindschau C, Maasch C, Olthoff H, Kurscheid D, Luft FC. Integrin-induced protein kinase Cα and Cε translocation to focal adhesions mediates vascular smooth muscle cell spreading. Circ Res. 1998; 82: 157–165.
- ↵Fisher TL, Terhorst T, Cao X, Wagner RW. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 1993; 21: 3857–3865.
- ↵Donis KH. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979; 7: 179–192.
- ↵Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, Dorr A, Sikic BI. Phase I study of an antisense oligonucleotide to protein kinase C-α (ISIS 3521/CGP64128A) in patients with cancer. Clin Cancer Res. 1999; 5: 3357–3363.