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Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells.

Moriya J, Ferrara N - Cardiovasc Diabetol (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Inhibition of PKC promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic endothelial cells. A, B: Photographs show tube formation in HUVECs exposed to 5.5 mM (Low) (A) or 30 mM (High) (B) glucose in the presence of Bis I (4 μΜ), PDGF-C (50 ng/mL), PDGF-C + Bis I, VEGF (50 ng/mL), VEGF + Bis I, and VEGF + Sulforaphane (5 μΜ). PBS treatment served as control (Control). Sulforaphane was used as a negative control for the assay. Scale bar = 300 μm. C: Total tube length was analyzed in the experiment of Figure 5A (left) and 5B (right). *P < 0.05, **P < 0.01 vs Control (VEGF(−), PDGF-C(−), Bis I(−), and Sulforaphane(−)) group. ##P < 0.01 vs VEGF(+), PDGF-C(−), Bis I (−), and Sulforaphane (−) group. †P < 0.05 vs VEGF(−), PDGF-C(+), Bis I (−), and Sulforaphane (−) group. n = 5 ~ 9 for each group. Data represent means ± standard error of the mean.
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Fig5: Inhibition of PKC promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic endothelial cells. A, B: Photographs show tube formation in HUVECs exposed to 5.5 mM (Low) (A) or 30 mM (High) (B) glucose in the presence of Bis I (4 μΜ), PDGF-C (50 ng/mL), PDGF-C + Bis I, VEGF (50 ng/mL), VEGF + Bis I, and VEGF + Sulforaphane (5 μΜ). PBS treatment served as control (Control). Sulforaphane was used as a negative control for the assay. Scale bar = 300 μm. C: Total tube length was analyzed in the experiment of Figure 5A (left) and 5B (right). *P < 0.05, **P < 0.01 vs Control (VEGF(−), PDGF-C(−), Bis I(−), and Sulforaphane(−)) group. ##P < 0.01 vs VEGF(+), PDGF-C(−), Bis I (−), and Sulforaphane (−) group. †P < 0.05 vs VEGF(−), PDGF-C(+), Bis I (−), and Sulforaphane (−) group. n = 5 ~ 9 for each group. Data represent means ± standard error of the mean.

Mentions: To investigate the effects of PKC inhibition on angiogenesis, we performed tube formation assays using HUVECs in normoglycemic and hyperglycemic conditions. Sulforaphane was used as a negative control for the assays. We found that both PDGF-C and VEGF-A treatment markedly increased tube formation in normoglycemic HUVECs, while this increase was blunted but still significant in hyperglycemic HUVECs (Figure 5A-C). Intriguingly, treatment with Bis I significantly augmented PDGF-C-induced tube formation in hyperglycemic HUVECs (Figure 5B, C). However, treatment with Bis I did not increase or even significantly decreased VEGF-A-induced tube formation (Figure 5A-C). Treatment with Bis I alone did not affect tube formation, both in normoglycemic and hypeglycemic HUVECs (Figure 5A-C). These results suggest that PKC inhibition promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic HUVECs possibly by upregulating PDGF-C/PDGFR-α axis.Figure 5


Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells.

Moriya J, Ferrara N - Cardiovasc Diabetol (2015)

Inhibition of PKC promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic endothelial cells. A, B: Photographs show tube formation in HUVECs exposed to 5.5 mM (Low) (A) or 30 mM (High) (B) glucose in the presence of Bis I (4 μΜ), PDGF-C (50 ng/mL), PDGF-C + Bis I, VEGF (50 ng/mL), VEGF + Bis I, and VEGF + Sulforaphane (5 μΜ). PBS treatment served as control (Control). Sulforaphane was used as a negative control for the assay. Scale bar = 300 μm. C: Total tube length was analyzed in the experiment of Figure 5A (left) and 5B (right). *P < 0.05, **P < 0.01 vs Control (VEGF(−), PDGF-C(−), Bis I(−), and Sulforaphane(−)) group. ##P < 0.01 vs VEGF(+), PDGF-C(−), Bis I (−), and Sulforaphane (−) group. †P < 0.05 vs VEGF(−), PDGF-C(+), Bis I (−), and Sulforaphane (−) group. n = 5 ~ 9 for each group. Data represent means ± standard error of the mean.
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Fig5: Inhibition of PKC promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic endothelial cells. A, B: Photographs show tube formation in HUVECs exposed to 5.5 mM (Low) (A) or 30 mM (High) (B) glucose in the presence of Bis I (4 μΜ), PDGF-C (50 ng/mL), PDGF-C + Bis I, VEGF (50 ng/mL), VEGF + Bis I, and VEGF + Sulforaphane (5 μΜ). PBS treatment served as control (Control). Sulforaphane was used as a negative control for the assay. Scale bar = 300 μm. C: Total tube length was analyzed in the experiment of Figure 5A (left) and 5B (right). *P < 0.05, **P < 0.01 vs Control (VEGF(−), PDGF-C(−), Bis I(−), and Sulforaphane(−)) group. ##P < 0.01 vs VEGF(+), PDGF-C(−), Bis I (−), and Sulforaphane (−) group. †P < 0.05 vs VEGF(−), PDGF-C(+), Bis I (−), and Sulforaphane (−) group. n = 5 ~ 9 for each group. Data represent means ± standard error of the mean.
Mentions: To investigate the effects of PKC inhibition on angiogenesis, we performed tube formation assays using HUVECs in normoglycemic and hyperglycemic conditions. Sulforaphane was used as a negative control for the assays. We found that both PDGF-C and VEGF-A treatment markedly increased tube formation in normoglycemic HUVECs, while this increase was blunted but still significant in hyperglycemic HUVECs (Figure 5A-C). Intriguingly, treatment with Bis I significantly augmented PDGF-C-induced tube formation in hyperglycemic HUVECs (Figure 5B, C). However, treatment with Bis I did not increase or even significantly decreased VEGF-A-induced tube formation (Figure 5A-C). Treatment with Bis I alone did not affect tube formation, both in normoglycemic and hypeglycemic HUVECs (Figure 5A-C). These results suggest that PKC inhibition promotes angiogenesis induced by PDGF-C, but not VEGF-A, in hyperglycemic HUVECs possibly by upregulating PDGF-C/PDGFR-α axis.Figure 5

Bottom Line: 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.

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus