Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells
© Moriya and Ferrara; licensee BioMed Central. 2015
Received: 28 August 2014
Accepted: 20 January 2015
Published: 7 February 2015
Diabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) were downregulated in ischemic limbs of diabetic mice, although the underlying mechanisms remained elusive. Protein kinase C (PKC) is a family of serine/threonine kinases and is known to be involved in angiogenesis. The purpose of this study is to elucidate the mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes.
Human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) cultured in normoglycemic or hyperglycemic conditions were examined. We also examined the effects of PKC inhibition on the PDGF-C/PDGFR-α axis in endothelial cells exposed to hyperglycemia.
Hyperglycemia inhibited proliferation and decreased viability of both HUVECs and HMVECs. Hyperglycemic endothelial cells exhibited decreased PDGFR-α expression both at messenger RNA (mRNA) and protein levels, while there was no significant change in expression of PDGF-C. We also found that expression of PKC-α, one of the PKC isoforms, was increased in hyperglycemic endothelial cells and that inhibition of PKC upregulated PDGFR-α expression in these cells. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by PDGF-C was significantly attenuated in hyperglycemic endothelial cells, whereas inhibition of PKC effectively reversed these inhibitory effects. Moreover, inhibition of PKC also promoted angiogenesis induced by PDGF-C in hyperglycemic endothelial cells, which was not observed in vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis.
These findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of hyperglycemia through upregulation of PKC. Targeting PKC to restore PDGF-C signaling might be a novel therapeutic strategy for the treatment of vascular complications in diabetes.
There are increasing numbers of patients who suffer from ischemic cardiovascular diseases, and most of such patients have some risk factors. Among them, diabetes is known to have strong association with the development of cardiovascular diseases [1,2]. Indeed, vascular complications associated with diabetes may result from atherosclerosis of large vessels, causing cardiac, cerebral and peripheral vascular diseases [3,4]. Moreover, these macrovascular atherosclerotic diseases are often refractory to conventional therapies, leading to increased morbidity and mortality among patients of diabetes [5-7]. Thus, it is important to establish novel therapeutic modalities for these patients.
Therapeutic angiogenesis is a relatively new and promising concept for treating patients with ischemic cardiovascular diseases [8-10]. It involves the use of angiogenic growth factors to promote development of collateral arteries. Vascular endothelial growth factor-A (VEGF-A) has been used as a major tool of this therapy since it is considered as the key regulator of angiogenesis [11-14]. However, several lines of evidence have shown that the benefits of this treatment are limited . Indeed, the angiogenic response after ischemia is attenuated in patients with diabetes, interfering with the response to treatment [16,17]. Moreover, it has recently been reported that a ligand-independent VEGF receptor 2 (VEGFR2) signaling pathway is activated in diabetic endothelial cells, leading to impaired responses to exogenous VEGF-A and limited angiogenic events [18,19]. Collectively, these studies suggest that further basic research is needed to elucidate the mechanisms of angiogenesis, especially in diabetic state, to improve the overall outcome of therapeutic angiogenesis.
Platelet-derived growth factors (PDGFs) are potent mitogenic and migratory factors for many cell types of mesenchymal origin . The PDGF family consists of four different polypeptide chains encoded by four different genes: PDGF-A, PDGF-B, and the more recently discovered PDGF-C and PDGF-D [21,22]. Among these, PDGF-C has been known to promote angiogenesis independently of VEGF-A . Actually, inhibition of PDGF-C leads to reduced angiogenesis in experimental tumors refractory to anti-VEGF treatment  or to suppression of both choroidal and retinal neovascularization . Moreover, PDGF-C has been reported to revascularize ischemic tissues by mobilizing endothelial progenitor cells or stimulating migration of endothelial cells . We also have recently found that expression of PDGF-C and its receptor, PDGF receptor α (PDGFR- α) was downregulated in ischemic limb tissues of diabetic mice, possibly contributing to impaired angiogenesis of diabetes. Moreover, introduction of PDGF-C significantly promoted revascularization in these mice . Collectively, these studies suggest that PDGF-C may be an interesting candidate for therapeutic angiogenesis. However, the underlying mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes still remain to be elucidated.
Therefore, we sought to investigate such mechanisms by using human endothelial cells (ECs) exposed to hyperglycemic condition. Hyperglycemia inhibited cell proliferation and decreased cell viability, which is in agreement with a previous study . Moreover, we found that the expression of PDGFR-α was downregulated in hyperglycemic ECs both at mRNA and protein levels. We also found that upregulation of protein kinase C (PKC)-α expression was involved in decreased PDGFR-α expression in hyperglycemic ECs. Consistent with this finding, inhibition of PKC led to augmentation of intracellular signaling induced by PDGF-C, resulting in promotion of angiogenesis in hyperglycemic ECs. These findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of diabetes through upregulation of PKC. Targeting PDGF-C and PKC might be a novel strategy for therapeutic angiogenesis in the diabetic state.
Materials and methods
Recombinant human VEGF-A and PDGF-C were from Peprotech (Rocky Hill, NJ) and Sigma-Aldrich (St. Louis, MO), respectively. D-mannitol and d-glucose were from Sigma-Aldrich. Trypan blue solution was from GE Healthcare Life Sciences (Little Chalfont, UK). Bisindolylmaleimide I (Bis I) was purchased from Cell Signaling Technology (Danvers, MA). Sulforaphane was from Trevigen (Gaithersburg, MD).
Human umbilical vein endothelial cells (HUVECs), human cardiac microvascular endothelial cells (HMVECs), and human aortic smooth muscle cells (AoSMCs) were purchased from Lonza (Walkersville, MD). HUVECs were grown in EGM-2 medium (Lonza), HMVECs in EGM-2-MV medium (Lonza), and AoSMCs in SmGM-2 medium (Lonza) on gelatin-coated tissue culture dishes (BD Bioscience, Becton, NJ), respectively. Cells from fewer than 5 generations were used for all experiments. High-glucose cultures were grown in the presence of 30 mM glucose for a minimum of 5 days and then subjected to further analysis. The total cell numbers were calculated by a Countess automated cell counter (Life Technologies, Carlsbad, CA). For signaling pathway analysis and tube formation analysis, HUVECs were serum-starved for 15 hours in EBM-2 medium (Lonza) supplemented with 0.1% fetal bovine serum (Omega Scientific, Tarzana, CA) before being treated with test substances.
Total RNA was extracted by an RNeasy Plus Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. cDNA was prepared using High Capacity cDNA Reverese Transcription Kits (Life Technologies). Quantitative real-time polymerase chain reaction (PCR) was performed by the ViiA7 Real-Time PCR system (Life Technologies) with the Taqman Gene Expression Assays and the Taqman Fast Advanced Master Mix (Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA was used as an endogenous control for quantitative real-time PCR analyses. At least three biological replicates were included in each analysis.
Western blot analysis
Whole cell lysates were prepared in RIPA buffer (Thermo Scientific, Rockford, IL) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). The lysates (20 to 40 μg) were resolved by SDS-PAGE. The proteins were then transferred into a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and incubated with the primary antibody followed by anti-rabbit IgG-horseradish peroxidase antibody or anti-mouse IgG-horseradish peroxidase antibody (GE Healthcare, Little Chalfont, UK). Specific proteins were detected by the enhanced chemiluminescent substrate (Thermo Scientific). The primary antibodies used for Western blotting were as follows: anti-PDGFRα antibody (951, sc-431), anti-PKCα antibody (C-20, sc-208), anti-PKCβΙΙ antibody (C-18, sc-210), anti-PKCδ antibody (C-17, sc-213) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-extracellular signal-regulated kinase (ERK) antibody (20G11, #4376), anti-ERK antibody (137F5, #4695), anti-phospho-Akt antibody (D9E, #4060), anti-Akt antibody (C67E7, #4691) (Cell Signaling Technology), and anti-β-actin antibody (AC-74, A2228) (Sigma-Aldrich).
Tube formation assay
The tube formation assay was performed using a commercially available kit (In Vitro Angiogenesis Tube Formation Assay Kit, Trevigen, #3470-096-K) according to the manufacturer’s instructions. Briefly, growth factor-reduced basement membrane extract solution in a 96-well plate was allowed to form a reconstituted matrix for one hour at 37°C. HUVECs were seeded at 1.5 × 104 per well and cultured for up to 24 hours in the presence or absence of different kinds of test substances. Capillary-like tube formation was assessed by photography under a stereoscopic microscope (Zeiss, Oberkochen, Germany) at a ×80 magnification. Total tube length was analyzed by using Image J software (NIH, Bethesda, MD).
The data are shown as means ± standard error of the mean. Differences between two groups were analyzed by a two-sided Student t-test. One-way analysis of variance (ANOVA) was used for multiple group comparison followed by the Bonferroni procedure for comparison of means. All experiments were repeated at least three times. In all analyses, P values of < 0.05 were considered statistically significant.
Hyperglycemia inhibits cell proliferation and decreases cell viability of endothelial cells
Expression of PDGFR-α is downregulated in hyperglycemic endothelial cells
Upregulation of PKC leads to decreased PDGFR-α expression in hyperglycemic endothelial cells
Inhibition of PKC leads to increased intracellular signaling induced by PDGF-C in endothelial cells
Inhibition of PKC promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic endothelial cells
PDGFs have a variety of effects in many cell types. Among the four members of the PDGF family, PDGF-B and PDGF-C have been extensively characterized for their role in vessel maturation and growth. While PDGF-B requires other angiogenic factors like VEGF-A to induce neovascularization, PDGF-C has the potential to promote revascularization after ischemia, independently of other factors [23,26]. Moreover, we recently reported that expression of PDGF-C or PDGFR-α was downregulated in ischemic tissues of diabetic mice, resulting in the impaired blood flow recovery after ischemia . Also, a recent study reported that PDGF signaling is critically involved in the pathogenesis of diabetes . On the other hand, a clinical study revealed that there were no significant differences in the serum concentrations of VEGF between control subjects and diabetic patients . Taken together, these findings support the hypothesis that PDGF-C might be a promising new target for the treatment of vascular complications related to diabetes.
The present study sought to investigate how the PDGF-C/PDGFR-α axis is downregulated using cultured human ECs exposed to hyperglycemia. We found that hyperglycemia led to inhibition of cell proliferation, decreased cell viability, and reduced angiogenic responses to VEGF-A in ECs (Figures 1A-B, 5A-C). These results are consistent with our previous in vivo studies in diabetic mice showing reduced blood flow recovery and neovascularization after ischemia even with introduction of VEGF-A [27,31].
We also show here that expression of PDGFR-α was significantly downregulated in hyperglycemic ECs, while expression of PDGF-C was almost unchanged between normoglycemic and hyperglycemic ECs (Figure 2A). On the other hand, we found that expression of PDGF-C was significantly downregulated both at baseline and after ischemia in limb tissues of diabetic mice in our previous in vivo study . Since in vivo murine limb tissues contain various cell types besides ECs such as vascular smooth muscle cells, fibroblasts, and skeletal muscle cells, it is possible that decreased PDGF-C expression occurs in cell types other than ECs in diabetic limbs. Indeed, we observed that the expression of PDGF-C was decreased in hyperglycemic human aortic smooth muscle cells (AoSMCs) in vitro (data not shown), suggesting that SMCs might be more responsible for the decreased expression of PDGF-C in vivo as well. Further studies are required to identify the cellular localization of the decreased PDGF-C and/or PDGFR-α expression in vivo.
Our results also suggest that PKC is critically involved in the decreased PDGFR-α expression in hyperglycemic ECs. We found that expression of PKC-α was significantly upregulated in hyperglycemic ECs (Figure 3A, B). PKC is a member of a large family of serine/threonine kinases and is known to be involved in angiogenesis . Moreover, PKC is also implicated in the increased risk of atherosclerosis in diabetes . However, the role of PKC for angiogenesis still remains elusive because the effect of PKC on angiogenesis seems to be different or even opposite depending on PKC isoforms . Moreover, there are some conflicting reports indicating that PKC-α promotes [40,41] or inhibits  angiogenic activity of ECs, and it has been known that inhibition of PKC leads to suppression of VEGF release from platelets . We show here that inhibition of PKC by Bis I leads to increased PDGFR-α expression, resulting in potentiation of intracellular signaling, and augmentation of angiogenesis induced by PDGF-C, but not by VEGF-A (Figures 3, 4, 5). Based on these findings, we speculate that the complexity and apparent discrepancies in the effects of PKC on angiogenesis stem not only from different PKC isoforms but also from downstream molecular targets of PKC in ECs. Further studies are needed to confirm whether PKC differentially regulates angiogenesis induced by PDGF-C and VEGF-A signaling, in addition to the effects of specific inhibition or activation of PKC-α on angiogenesis induced by PDGF-C in the diabetic state.
In summary, the present study shows that downregulation of PDGF-C/PDGFR-α axis is involved in impaired endothelial cell functions in hyperglycemia at least in part through upregulation of PKC. Therefore, targeting PKC to restore PDGF-C signaling might be anovel therapeutic option for the treatment of vascular complications in diabetic patients.
We thank H. Xin, C. Zhong, W. Liang and N. Protopsaltis for helpful discussions and advice. This work was supported by start-up funds of University of California, San Diego (to N.F.) and by grants from the Uehara Memorial Foundation, Kanae Foundation for the Promotion of Science, and the NOVARTIS Foundation for Gerontological Research (to J.M.).
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