Characterization of protein kinase C β isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization

Abstract

Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKC␤2 isoform and a significant decrease in retinal neovascularization in PKC␤ isoform null mice. The mitogenic action of VEGF, a potent hypoxiainduced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKC␤1 or ␤2 isoforms and inhibited significantly by the overexpression of a dominantnegative PKC␤2 isoform but not by the expression of PKC ␣, ␦, and isoforms. Association of PKC␤2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKC␤2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKC␤-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

The PKCβ/HuR/VEGF pathway in diabetic retinopathy

“…In fact, hyperglycemia induces, through various routes, an enhanced generation of diacylglycerol, [10,13] the physiological activator of PKC, that at the retinal level seems to predominantly stimulate the beta isoform of the kinase [15], although, as already mentioned, other PKC isoenzymes are also activated [16,17]. The crucial involvement of PKCβ is further emphasized, on the one hand, by a A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 dramatic increase in the angiogenic response to retinal ischemia in transgenic mice overexpressing PKCβ and, on the other hand, by a significant decrease in retinal neovascularization in PKCβ null mice [38]. Moreover, besides the retina, it has been observed that the lack of PKCβ can protect against diabetic-induced dysfunctions and augment VEGF expression in other tissues affected by this pathology [39].…”

Leptin Stimulates Ischemia-Induced Retinal Neovascularization

“…Ten-week-old male and female heterozygous ob/ϩ mice on the C57BL/6 background (Charles River Japan, Yokohama, Japan) were used to produce wild-type ϩ/ϩ, heterozygous ob/ϩ, and homozygous ob/ob newborn mice. Ischemia-induced retinal neovascularization model was performed as described (1,16). Briefly, litters of 7-day-old (postnatal day 7 or P7) mice, along with their nursing mothers, were exposed to 75 Ϯ 2% oxygen for 5 days and then returned to room air on P12.…”

“…Analysis of neovascularization. Mice were perfused from the left ventricle with high-molecular-weight (2,000 kDa) fluorescein-conjugated dextran (Sigma, St. Louis, MO) in PBS solution (1,16). Eyes were enucleated and fixed in 4% paraformaldehyde.…”

“…After staining with periodic acid-Schiff, 10 intact sections of equal length, each 30 m apart, were evaluated for a span of 300 m. All retinal vascular cell nuclei anterior to the internal limiting membrane were counted in each section by a fully masked protocol. The mean of all 10 counted sections yielded average neovascular cell nuclei per 6-m section per eye (1,16). Cell cultures.…”

Growth factors and protein kinase C inhibitors as novel therapies for the medical management diabetic retinopathy

“…When PKC b knockout mice are exposed to similar retinal ischaemia, retinal neovascularization is reduced. 69 These data and the safety and pharmocologic results of PKC b inhibitors to date have supported the undertaking of two Phase II/III studies and a large Phase III study. These two Phase II/III studies have just recently been completed and data analysis should begin soon.…”