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Cardiovascular Research Advance Access first published online on December 4, 2007
This version [Corrected Proof] published online on January 17, 2008
Cardiovascular Research, doi:10.1093/cvr/cvm085
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Protein kinase C {alpha} promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor

Hui Xu1,2,{dagger}, Petra Czerwinski1,{dagger}, Marcus Hortmann1, Hae-Young Sohn3, Ulrich Förstermann1 and Huige Li1,*

1 Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany
2 Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
3 Institute of Cardiology, Medical Policlinic, Ludwig Maximilians University, Munich, Germany

* Corresponding author. Tel: +49 6131 39 36929; fax: +49 6131 39 36611. E-mail address: huigeli{at}uni-mainz.de

Received 31 August 2007; revised 14 November 2007; accepted 23 November 2007

Time for primary review: 36 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Protein kinase C (PKC) plays an important role in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined.

Methods and results: mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms {alpha}, {delta} and {varepsilon} in HUVEC. Knockdown of PKC {alpha} diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC {alpha} activation and PKC-dependent VEGF upregulation.

Conclusions: PKC {alpha} activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC {alpha}-dependent positive feedback mechanism.

KEYWORDS Angiogenesis; Gene expression; Growth factors; Protein kinase C


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Angiogenesis, the process of postnatal neovascularization, is a critical component of several human diseases, including ischaemic heart disease, cancer, and diabetic microvascular disease.1 Diabetes, for example, is characterized by impaired angiogenesis in ischaemic limbs, but enhanced retinal neovascularization.1 Angiogenesis has also been shown to play a central role in the recovery of the myocardium following ischaemia and infarction.2 All the abovementioned diseases are associated with activation of protein kinase C (PKC).3,4

PKC is a family of serine/threonine kinases that comprises at least 10 isozymes grouped into three classes: conventional PKC (cPKC) ({alpha}, {gamma}, and the alternatively spliced βI and βII), novel PKC (nPKC) ({delta}, {varepsilon}, {eta}/L, {theta}), and atypical PKC (aPKC) ({eta}, {iota}/{lambda}). In addition, PKC µ and {nu} are considered by some to constitute a fourth class, and by others to comprise a distinct family called protein kinase D.5,6

Indeed, PKC has been reported to have a pivotal role in angiogenesis. PKC-activating phorbol esters were reported to induce angiogenesis.7–9 However, different PKC isoforms may have unique and even opposing functions during vessel assembly.10 Overexpression of PKC {delta} in rat epididymal fat pad endothelial cells inhibited endothelial differentiation in a matrigel assay,11 whereas inhibition/downregulation of PKC {alpha} in human umbilical vein endothelial cells (HUVEC) inhibited vessel formation in vitro and myocardial neovascularization in vivo.12 Molecular targets downstream of PKC in endothelial cells for angiogenesis remain elusive.

Vascular endothelial growth factor (VEGF) is a crucial angiogenesis factor.13,14 PKC-induced VEGF expression has been shown previously in non-vascular cells. In human glioblastoma cells, phorbol ester induced VEGF expression via a post-transcriptional mRNA stabilization mechanism by activation of PKC {alpha} and {zeta}.15 Ischaemic preconditioning upregulated VEGF mRNA via nuclear translocation PKC {varepsilon} in the rat ischaemic myocardium.2 In retinal capillary pericytes, stretch-induced VEGF expression via PI3K-mediated activation of PKC {zeta} that is independent of Erk1/2 or cPKC/nPKC isoforms.16 Also in retinal epithelial cells, high glucose-stimulated VEGF expression was prevented by a specific inhibitor of PKC {zeta}.17

According to the traditional concept, PKC induces VEGF in non-vascular cells under conditions such as tumorgenesis, ischaemia, or diabetic retinopathy. This in turn enhances angiogenesis of endothelial cells via a paracrine mechanism. In this classical model, endothelial cells function as targets and effector cells of the PKC–VEGF axis. The present study provides evidence for an additional mechanism operative in this scenario. We show for the first time that PKC induces VEGF in vascular endothelial cells. Endothelial cells actively produce VEGF in response to PKC {alpha} activation. VEGF, in turn, promotes its sustained release via an autocrine positive feedback loop.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Cell culture
HUVEC were isolated by collagenase digestion as described.18 HUVEC-derived EA.hy 926 endothelial cells19 were kindly provided by Dr Cora-Jean Edgell (Chapel Hill, NC, USA). EA.hy 926 endothelial cells were grown under 10% CO2 in Dulbecco's modified Eagle’s medium (DMEM, Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1x HAT (hypoxanthine, aminopterin and thymidine) (Invitrogen, Karlsruhe, Germany).18 During the last 12 h prior to experiment, HUVEC were kept in medium with 1% FBS.

2.2 Isolation and characterization of human endothelial progenitor cells
Mononuclear cells were isolated by density gradient centrifugation with Histopaque-1077 (Sigma–Aldrich) from 20 mL of peripheral blood and grown in endothelial basal medium (Clonetics) supplemented with endothelial growth medium SingleQuots and 20% FBS on fibronectin-coated six-well culture plates (Becton Dickinson). After four days in culture, non-adherent cells were removed by washing with PBS and adherent cells cultured for another three days.20,21 To detect the uptake of 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (DiLDL, Invitrogen), cells were incubated with DiLDL (2.4 µg/mL) at 37°C for 1 h. Cells were then fixed with 2% paraformaldehyde for 10 min and incubated with FITC-labelled Ulex europaeus agglutinin I (lectin, 10 µg/mL, Sigma–Aldrich) for 1 h. Dual-stained cells, positive for both lectin and DiLDL, were considered endothelial progenitor cells (EPC).20

2.3 Angiogenesis assay on matrix gels
Growth factor-reduced Matrix gels (Matrigel, Becton Dickinson) were allowed to polymerize in a 96-well plate for 1 h at 37°C. HUVEC were seeded at 1x104 per well and grown for 18 h in a humidified 37°C, 5% CO2 incubator. In some experiments, cells were cultured in the presence or absence of different kinds of reagents.

Some Matrigel assays were performed with a mixture of 1x104 HUVEC and 3x103 DiLDL-labelled EPC.22,23 For fluorescent labelling of EPC, cells were incubated with DiLDL (2.4 µg/mL) for 3 h. Tube formation was documented using an inverted fluorescence microscope and pictures were captured with a digital camera and stored in a computer system.24

2.4 Real-time RT-PCR for mRNA expression analyses
mRNA expression of VEGF or basic fibroblast growth factor (bFGF) was analysed with quantitative Real-Time RT-PCR using an iCyclerTM iQ System (Bio-Rad Laboratories, Munich, Germany). One hundred nanograms of total RNA from EPC, HUVEC, or EA.hy 926 cells were used for real-time RT-PCR analysis with the QuantiTectTM Probe RT-PCR kit (Qiagen, Hilden, Germany). TaqMan Gene Expression Assays (pre-designed probe and primer sets) were obtained from Applied Biosystems (Foster City, CA, USA) for analysing the mRNA expression of VEGF and bFGF (assay ID Hs00173626_m1 and Hs00246601_m1, respectively). mRNA expression levels of target genes were normalized to TATA box-binding protein mRNA (TBP, Applied Biosystems, assay ID Hs00427620_m1).

2.5 Western blot analysis for protein kinase C expression and activation
Soluble (cytosolic) and particulate (membrane) protein fractions, or total protein were isolated from HUVEC or EA.hy 926 cells as previously described.25 Western blotting was performed using 20 µg of protein and a monoclonal anti-PKC {alpha} antibody (Santa Cruz Biotechnology), a polyclonal anti-PKC {delta} antibody (Santa Cruz Biotechnology), a polyclonal anti-PKC {varepsilon} antibody (P8458, Sigma–Aldrich), or a polyclonal antibody against phosphorylated myristolated alanine-rich C-kinase substrate (p-MARCKS, Santa Cruz Biotechnology).

2.6 Knockdown of protein kinase C {alpha}, {delta}, {varepsilon}, and VEGF with siRNA
HiPerformance Validated siRNAs for PKC {alpha} (SI00605927), PKC {delta} (SI02660539), and PKC {varepsilon} (SI02622088), VEGF (SI02757643) and negative control siRNA (sense UUCUCCGAACGUGUCACGUdTdT and antisense ACGUGACACGUUCGGAGAAdTdT) were obtained from Qiagen.

HUVEC or EA.hy 926 cells were plated on six-well plate 24 h prior to transfection and were 50–80% confluent when siRNA was added. Transfection was performed with 50 nM siRNA duplexes using the amphiphilic delivery system SAINT-RED (Synvolux Therapeutics, Groningen, The Netherlands) according to the manufacturer’s instructions.26 Briefly, siRNA was complexed with 15 nmol of transfection reagent, diluted with M199-HSA to 1 mL, and added to the cells for 4 h. Subsequently, 2 mL of culture medium was added and incubation proceeded for 72–96 h. Preliminary experiments indicated that the most effective knockdown of PKC {alpha} and {varepsilon} were achieved 96 h after siRNA transfection. Downregulation of PKC {delta} was most effective after 72 h, and downregulation of VEGF after 48 h. Therefore, experiments were performed under these conditions.

2.7 Statistics
Statistical differences between mean values were determined by analysis of variance (ANOVA) followed by Fisher’s protected least-significant difference test for comparison of different means.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Protein kinase C activation improves endothelial cell morphology and angiogenic activity
When freshly isolated HUVEC were treated with 10 nM phorbol-12-myristate-13-acetate (PMA) for 24 h, their morphology changed towards to a more typical endothelial phenotype (Figure 1A). This could be prevented by simultaneous treatment of the cells with the PKC inhibitor Gö 6983 (1 µM, Figure 1A). Changes in endothelial cell morphology were paralleled by changes in angiogenic activity. In Matrigel experiments, PMA (10 nM, 18 h) also markedly enhanced vascular tube formation of HUVEC in a Gö 6983-inhibitable, PKC-dependent manner (Figure 1B).


Figure 1
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  		Figure 1 Activation of protein kinase C (PKC) promotes cobblestone morphology and angiogenic activity of endothelial cells. (A) Human umbilical vein endothelial cells (HUVEC) were kept in 1% fetal bovine serum-containing medium for 12 h and then treated with 10 nM phorbol-12-myristate-13-acetate (PMA), 1 µM Gö 6983, a PKC inhibitor, or both compounds in combination for another 24 h. (B) Tube formation of HUVEC in Matrigel in the presence of 10 nM PMA and/or 1 µM Gö 6983 (18 h). The pictures shown are representative of three to six independent experiments.

 
3.2 Protein kinase C activation enhances vascular endothelial growth factor expression but downregulates basic fibroblast growth factor expression in human endothelial cells
Because VEGF and bFGF play important roles in angiogenesis,14 we analysed the effect of PMA on the expression of these growth factors. Treatment of HUVEC or EA.hy 926 cells with 10 nM PMA for 6 or 24 h resulted in a marked upregulation of VEGF mRNA expression (Figure 2A and C). The VEGF upregulation by PMA (24 h) could be prevented by the PKC inhibitors GF109203X (1 µM), Gö 6983 (1 µM), or Gö 6976 (1 µM), or by the MEK1/2 inhibitor U0126 (10 µM) (Figure 2B).


Figure 2
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  		Figure 2 Protein kinase C activation enhances the expression of vascular endothelial growth factor (VEGF). Human umbilical vein endothelial cells (HUVEC) (panels A and B) were left untreated (control, Co) or treated with 10 nM of PMA, either alone or in combination with PKC inhibitors GF109203X (GFX, 1 µM), Gö 6983 (1 µM), Gö 6976 (1 µM) or the MEK1/2 inhibitor U0126 (10 µM). (C) HUVEC-derived EA.hy 926 cells were treated with PMA (10 nM). (D) EA.hy 926 cells were pretreated with PMA (10 nM) for 24 h. Then, actinomycin D (Act-D, 5 µg/mL) was added to stop transcription and RNA was prepared at different time points after Act-D. (E) EA.hy 926 cells were pretreated with Act-D and PMA was added 30 min thereafter. VEGF mRNA expression was analysed with quantitative real-time RT-PCR. Columns represent mean ± SEM, n = 9–15 (**P < 0.01).

 
To determine the effect of PMA on the stability of VEGF mRNA, EA.hy 926 cells were pretreated with 10 nM PMA for 24 h. Then, actinomycin D (Act-D, 5 µg/mL) was added to stop gene transcription. VEGF mRNA was analysed at 4 or 18 h after Act-D. VEGF mRNA showed a half-life of about 3 h and PMA did not change VEGF mRNA stability (Figure 2D). When gene transcription was first stopped by Act-D pretreatment and PMA added 30 min after Act-D, PMA could no longer increase VEGF mRNA expression (Figure 2E), indicating that PMA-induced VEGF upregulation is a result of transcription activation.

In contrast, a 6 h treatment of HUVEC with 10 nM PMA resulted in a marked downregulation of bFGF mRNA expression, which could be completely prevented by the PKC inhibitor Gö 6983 (10 nM PMA: 19.5 ± 4.0%, P < 0.01; 1 µM Gö 6983: 98.2 ± 10.1%; PMA+Gö 6983: 81.9 ± 13.2% of control, respectively, n = 6).

3.3 Protein kinase C-induced angiogenesis is mediated by vascular endothelial growth factor
We then analysed the functional role of VEGF in PMA-induced angiogenesis. PMA-induced HUVEC angiogenesis could be largely reduced by ZM 323881, an inhibitor of VEGF receptor 2 (VEGFR2). Treatment of HUVEC with 50 nM validated VEGF siRNA for 48 h resulted in a downregulation of VEGF mRNA from control level (100%) to 33.0 ± 6.2% (P < 0.01, n = 6). Similar downregulation of VEGF mRNA expression was achieved in EA.hy 926 cells (32.4 ± 10.0% of control, 50 nM VEGF siRNA, 48 h, P < 0.01, n = 6). siRNA-mediated knockdown of VEGF expression markedly blocked PMA-induced angiogenesis, whereas a control siRNA had no effect (Figure 3). Thus, PMA-induced angiogenesis is VEGF-dependent.


Figure 3
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  		Figure 3 Phorbol-12-myristate-13-acetate (PMA)-induced angiogenesis is mediated by VEGF. Tube formation on Matrigel was performed using human umbilical vein endothelial cells (HUVEC) treated with PMA (10 nM, 18 h) in the absence or presence of VEGFR2 inhibitor ZM 323881 (ZM, 1 µM). Some other HUVEC transfected with control siRNA or VEGF siRNA were used in tube formation experiments (48 h after transfection) in the absence or presence of 10 nM PMA (18 h). Pictures shown are representative of three to six independent experiments.

 
3.4 Protein kinase C {alpha} is responsible for phorbol-12-myristate-13-acetate-induced vascular endothelial growth factor expression and angiogenesis
Treatment of HUVEC with 10 nM PMA for 2 or 6 h resulted in a translocation of PKC {alpha}, {delta}, and {varepsilon} from cytosol to the membrane, indicating an activation of all three PKC isozymes (Figure 4A).


Figure 4
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  		Figure 4 Protein kinase C {alpha} (PKC {alpha}) is responsible for phorbol-12-myristate-13-acetate (PMA)-induced VEGF expression. (A) Human umbilical vein endothelial cells (HUVEC) were treated with 10 nM PMA. Subcellular fractions (cytosolic and membrane fractions) were isolated and protein expression of PKC {alpha}, {delta}, and {varepsilon} determined by western blotting. β-Tubulin was shown as internal control. Blots shown are representative of three experiments. (B) siRNA-mediated PKC knockdown. EA.hy 926 cells were treated with control siRNA, PKC {alpha} siRNA, PKC {delta} siRNA, or PKC {varepsilon} siRNA (50 nM). Protein levels of PKC isoforms after siRNA treatment was analysed by western blotting. (C) EA.hy 926 cells pretreated with siRNA were incubated with 10 nM PMA for 24 h and VEGF mRNA expression was analysed with quantitative real-time RT-PCR. Columns represent mean ± SEM, n = 6 (**P < 0.01).

 
Treatment of the cells with siRNA to PKC {alpha}, {delta}, and {varepsilon} resulted in a significant downregulation of the respective PKC isoforms (Figure 4B). A marked upregulation of VEGF could still be observed in PKC {delta} or {varepsilon}-downregulated cells (Figure 4C). However, when PKC {alpha} was knocked down, PMA-induced VEGF expression was largely blocked (Figure 4C), indicating that PKC {alpha} was responsible for VEGF upregulation by PMA. PMA-induced angiogenesis of HUVEC was markedly attenuated by Gö 6976, an inhibitor of cPKC isoforms, and by PKC {alpha} knockdown (Figure 5). The control siRNA had no effect (Figure 5).


Figure 5
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  		Figure 5 Phorbol-12-myristate-13-acetate (PMA)-induced angiogenesis depends on protein kinase C {alpha} (PKC {alpha}). Tube formation was performed on Matrigel in the presence of PMA (10 nM, 18 h) and/or Gö 6976 (1 µM). Some other cells were pretreated with control siRNA, or siRNA to PKC {alpha} (see Figure 4). Pictures shown are representative of three to six independent experiments.

 
3.5 Protein kinase C activation enhances vascular endothelial growth factor expression and angiogenic activity of endothelial progenitor cells
In order to investigate whether the PKC {alpha}-VEGF-angiogenesis pathway also exists in EPC, human EPC were isolated from peripheral blood and characterized by DiLDL- and lectin double staining (Figure 6A). Incubation of EPC with 10 nM PMA for 24 h also resulted in a six-fold upregulation of VEGF mRNA expression (Figure 6B). PMA-treatment enhanced the ability of EPC to incorporate into the vascular network when co-cultured with HUVEC in Matrigel angiogenesis assay (Figure 6C).


Figure 6
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  		Figure 6 Protein kinase C (PKC) activation enhances vascular endothelial growth factor (VEGF) expression and angiogenic activity of endothelial progenitor cells (EPC). (A) Characterization of EPC. Human EPC were isolated from peripheral blood. DiLDL uptake and lectin binding was determined by fluorescence microscopy. (B) EPCs were treated with 10 nM PMA for 24 h and VEGF mRNA expression analysed with quantitative real-time RT-PCR. (C) Tube formation (10 nM PMA, 18 h) performed on Matrigel with a co-culture of human umbilical vein endothelial cells (HUVEC) and DiLDL-labelled EPC. Columns represent mean ± SEM, n = 6 (**P < 0.01, compared with control).

 
3.6 Autocrine feedback loop for vascular endothelial growth factor in human umbilical vein endothelial cells
Treatment of HUVEC with VEGF for 10 min resulted in a phosphorylation of the putative PKC substrate MARCKS (Figure 7A) and a translocation of PKC {alpha} to the membrane (Figure 7B), indicating activation of this PKC isoform. A 24 h treatment of HUVEC with VEGF resulted in a doubling of VEGF mRNA expression, which could be prevented by PKC inhibitor Gö 6983 (Figure 7C).


Figure 7
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  		Figure 7 Vascular endothelial growth factor (VEGF)–protein kinase C (PKC) feedback loop in human umbilical vein endothelial cells (HUVEC). (A) HUVEC was treated with VEGF (or PMA, as a positive control) for 10 min. Phosphorylation of the putative PKC substrate MARCKS (A) or translocation of PKC {alpha} from cytosol to membrane fractions (B) were analysed by western blotting. (C) HUVEC was treated with VEGF (100 ng/mL) for 24 h and/or Gö 6983 (1 µM) and VEGF mRNA expression analysed with quantitative real-time RT-PCR. Columns represent mean ± SEM, n = 6 (*P < 0.05, compared with control, Co).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
When freshly isolated HUVEC were treated with the PKC activator PMA, they developed the typical cobblestone morphology, characteristic for functional endothelial cells (Figure 1A). One important aspect of endothelial cells’ functionality is the ability to form new blood vessels. Indeed, PMA markedly enhanced tube formation of HUVEC in a Matrigel assay (Figure 1B). This improved angiogenic ability was completely blocked by the PKC inhibitor Gö 6983 (Figure 1B). These results indicate that PMA-induced angiogenesis is PKC-dependent.

This phenomenon as such has been reported previously.27,28 Using antisense technology, a recent paper demonstrated that inhibition of PKC {alpha} prevented endothelial cell tube formation.12 However, no downstream targets of PKC have been defined in this study. In the current study, we now demonstrate that PKC promotes angiogenesis via induction of VEGF in endothelial cells.

Numerous factors are crucial in the angiogenic process of endothelial cells. These include VEGF and bFGF.14 In HUVEC and in HUVEC-derived EA.hy 926 cells, we observed a marked induction of VEGF mRNA expression in response to PMA (Figure 2A and C). In contrast, bFGF expression was reduced by PMA treatment. The PMA-induced VEGF expression was PKC-dependent (based on results with PKC inhibitors and PKC knockdown experiments, Figures 2 and 4). VEGF induction by PMA was also reduced by U0126, a specific inhibitor of MEK1/2,29 indicating that Erk1/2 lies downstream of PKC activation (Figure 2B). The induction of VEGF mRNA could be a result of an increased gene transcription and/or enhanced mRNA stability. Our results indicate that PKC-induction of VEGF expression is purely transcriptional.

PKC-induced angiogenesis seems to be mediated mainly by induction of endothelial VEGF formation, because PMA-induced angiogenesis was largely reduced by VEGFR2 inhibitor ZM 323881 or by knockdown of VEGF expression with siRNA (Figure 3).

Previous work from our laboratory has indicated that EA.hy 926 cells express a total of eight PKC isoforms, namely {alpha}, βI, {delta}, {varepsilon}, {eta}, {zeta}, {lambda}, and µ.25 The hallmark of PKC activation in cells is its translocation from the cytosol to the membrane, followed by downregulation after prolonged activation.5 Indeed, a long-term incubation (30 h) of EA.hy 926 cells with 100 nM PMA led to a marked downregulation of PKC {alpha} and {varepsilon}, and a slight downregulation of PKC {delta},25 indicating activation of these PKC isoforms. Short-term PMA incubation resulted in a redistribution (from cytosol from membrane) and activation of PKC {alpha}, {delta}, and {varepsilon} in EA.hy cells. PKC {alpha}, {delta}, and {varepsilon} are also expressed in HUVEC, and PMA also activated these PKC isozymes in these cells (Figure 4A). siRNA-mediated knockdown of PKC {alpha}, but not PKC {delta} or {varepsilon}, prevented PMA-induced VEGF expression (Figure 4B and C). These results demonstrate that PKC {alpha} is the PKC isoform mediating the PMA-induced VEGF expression. Because the siRNA knockdown of PKC {varepsilon} was not as efficient as that of PKC {alpha}, we cannot completely rule out the (minor) participation of PKC {varepsilon} in VEGF expression.

Our results indicate that PKC {alpha} is also the PKC isoform responsible for PMA-induced angiogenesis. As shown in Figure 5, angiogenic activity of HUVEC in response to PMA was largely reduced by Gö 6976, a selective inhibitor of cPKC isoforms, and by siRNA-mediated knockdown of PKC {alpha}.

Translocation and activation of PKC {alpha}, {delta}, and {varepsilon} isoforms have been observed in ischaemic rat heart.30 Moreover, heart ischaemia triggers a rapid recruitment of EPC to the myocardium. The recruited EPC expresses an array of potentially cardioprotective factors (including VEGF) and act as reservoirs and producers of molecules contributing to neovascularization.31 The data in the present study indicate that VEGF induction in recruited EPC may be mediated by PKC activation (Figure 6). Similar to endothelial cells, PMA treatment also increased the expression of VEGF in cultured EPC (Figure 6B). VEGF from these EPC may stimulate their own angiogenic activity (as shown in Figure 6C) and may also stimulate the angiogenic activity of neighbouring endothelial cells.

Interestingly, the reverse effect has also been demonstrated. VEGF has been shown to activate PKC in endothelial cells and PKC activity seems to be important for the angiogenic effect of VEGF.32–34 Indeed, treatment of HUVEC with VEGF (Figure 7A), led to enhanced phosphorylation of MARCKS, a putative substrate of PKC, as well as translocation (and activation) of PKC {alpha} (Figure 7B). The induction of VEGF mRNA expression by VEGF itself, was PKC-dependent (Figure 7C), demonstrating a VEGF–PKC positive feedback loop in human endothelial cells. Also in this case, PKC {alpha} seems to be the responsible PKC isoform, because VEGF does not activate PKC {delta} or {varepsilon} in HUVEC.32

In conclusion, the current study demonstrates an autocrine feedback loop in which PKC enhances VEGF, and VEGF in turn stimulates PKC activity. Given the importance of VEGF in endothelial angiogenesis, this positive feedback loop may be crucial in maintaining the angiogenic process. Human EPC show the same pathway and can thus contribute to angiogenesis in autocrine and paracrine fashion.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by the Collaborative Research Center SFB 553 (project A1 to H.L. and U.F.) from the DFG (Deutsche Forschungsgemeinschaft), Bonn, Germany. P.C. is a member of the Neuroscience Graduate School at the Johannes Gutenberg University of Mainz supported by DFG Research Training Group (GRK 1044).


   Acknowledgements
 
We thank Ursula Wollscheid for excellent technical assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} Both authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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