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Activation of PKC-Δ and SHP-1 by Hyperglycemia Causes Vascular Cell Apoptosis and Diabetic Retinopathy

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Activation of PKC-Δ and SHP-1 by Hyperglycemia Causes Vascular Cell Apoptosis and Diabetic Retinopathy A RTICLES Activation of PKC-δ and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy Pedro Geraldes1, Junko Hiraoka-Yamamoto1, Motonobu Matsumoto1, Allen Clermont1, Michael Leitges3, Andre Marette4, Lloyd P Aiello1,2,5, Timothy S Kern6 & George L King1,7 Cellular apoptosis induced by hyperglycemia occurs in many vascular cells and is crucial for the initiation of diabetic pathologies. In the retina, pericyte apoptosis and the formation of acellular capillaries, the most specific vascular pathologies attributed to hyperglycemia, is linked to the loss of platelet-derived growth factor (PDGF)-mediated survival actions owing to unknown mechanisms. Here we show that hyperglycemia persistently activates protein kinase C- (PKC-, encoded by Prkcd) and p38 mitogen-activated protein kinase (MAPK) to increase the expression of a previously unknown target of PKC- signaling, Src homology-2 domain–containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase. This signaling cascade leads to PDGF receptor- dephosphorylation and a reduction in downstream signaling from this receptor, resulting in pericyte apoptosis independently of nuclear factor-B (NF-B) signaling. We observed increased PKC- activity and an increase in the number of acellular capillaries in diabetic mouse retinas, which were not reversible with insulin treatment that achieved normoglycemia. Unlike diabetic age-matched wild-type mice, diabetic Prkcd–/– mice did not show activation of p38 MAPK or SHP-1, inhibition of PDGF signaling in vascular cells or the presence of acellular capillaries. We also observed PKC-, p38 MAPK and SHP-1 activation in brain pericytes and in the renal cortex of diabetic mice. These findings elucidate a new signaling pathway by which hyperglycemia can induce PDGF resistance and increase vascular cell apoptosis to cause diabetic vascular complications. A common cellular pathology for many diabetic vascular com- pericyte and endothelial cell death, recapitulating the early changes plications is an enhanced rate of cellular apoptosis as observed in of diabetic retinopathy10. However, it is unclear whether pericyte retinal pericytes, renal podocytes and vascular endothelial cells1,2. loss results from decreased PDGF-B abundance, as the expression of Hyperglycemia, the major risk factor for diabetic microvascular com- PDGF-B in the retina is increased in diabetic compared to nondia- plications3, is responsible for inducing apoptosis in these vascular betic rats11. In addition, although oxidative stress and activation of 4 5,6 © All rights reserved. 2009 Inc. Nature America, tissues directly or indirectly by altering cytokine expression and NF-κB have been reported to be involved in hyperglycemia-induced leading to the accumulation of toxic products in organs affected by cell apoptosis, the interrelationship between PDGF-B inhibition and diabetes. Although intensive insulin treatment in individuals with oxidative stress in mediating pericyte apoptosis and the initiation of diabetes can delay the onset and progression of diabetic complica- diabetic retinopathy is not known. In investigating the mechanisms by tions3, initiation of intensive glycemic control after periods of poor which hyperglycemia induces pericyte apoptosis, we have uncovered glycemia does not substantially decrease the rate of progression of a new signaling pathway by which hyperglycemia induces vascular retinopathy and other microvascular diseases, suggesting that hyper- damage: activation of PKC-δ and p38α MAPK, leading to increased glycemia-induced chronic cellular changes are difficult to reverse7. expression of the protein tyrosine phosphatase (PTP) SHP-1, which, To clarify the biochemical mechanisms involved in hyperglycemia- in turn, leads to the dephosphorylation of PDGFR-β. induced cellular apoptosis, we characterized the signaling cascade of hyperglycemia leading to pericyte apoptosis in diabetic retinopathy, RESULTS the most specific vascular pathology attributed to diabetes, which may PKC- affects PDGF resistance and acellular capillary formation represent a common pathway for other microvascular abnormalities As PKC activation is associated with retinal and vascular pathologies in diabetes8,9. of diabetes12, we studied the effects of PKC activation on PDGF sign- Noteworthy recent findings implicate a role of PDGF receptor-β aling inhibition and pericyte apoptosis. In situ PKC activity increased (PDGFR-β) in pericyte apoptosis. PDGF-B or PDGFR-β deficient by 40% (P = 0.028) in mouse retina after 3 months of diabetes mice show retinal pericyte depletion associated with the develop- (Fig. 1a). Immunoblot analyses showed that the amounts of both the ment of microaneurysms and acellular capillaries as a result of both PKC-β2 and PKC-δ isoforms were increased (P = 0.022 and P = 0.043, 1Dianne Nunnally Hoppes Laboratory for Diabetes Complications and 2Beetham Eye Institute of Joslin Diabetes Center, Boston, Massachusetts, USA. 3The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway. 4Department of Anatomy and Physiology, Université Laval, Québec, Québec, Canada. 5Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA. 6Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. 7Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. Correspondence should be addressed to G.L.K. ([email protected]). Received 25 March; accepted 22 September; published online 1 November 2009; doi:10.1038/nm.2052 1298 VOLUME 15 | NUMBER 11 | NOVEMBER 2009 NATURE MEDICINE A RTICLES P = 0.028 a 1,500 c d PDGF-BB ––+ +–– + + ) p-Akt –1 n y Akt 1,000 mi p-ERK –1 ERK 500 PKC assa NDMDMNDM DM +/+ –/– (pmol mg Prkcd Prkcd ) 0 ) NDM DM Prkcd+/+ NDM Prkcd+/+ DM Prkcd+/+ DM + Ins b 3 3 Prkcd+/+Prkcd–/– rsus saline PKC-α xpression 2 2 es ersus saline 20 xpression ve ri PKC- 2 ) * * β 2 15 * * 1 1 PKC-δ 10 5 0 0 p-Akt/Akt e PKC-ε (per mm p-ERK/ERK e +/+ –/– +/+ –/– old increase 0 Prkcd Prkcd old increase v Prkcd Prkcd (f (f PKC-ζ M M 25 µM Acellular capilla DM DM ND ND –/– –/– NDM DM NDM DM DM+Ins Prkcd NDM Prkcd DM g Cytosolic Membrane + 1.2 4 4 +/ NDM DM e f * d U) VEGF PKC-β 1.1 * * kc 1.0 P = 0.043 PDGF PKC-δ Pr P = 0.028 ) 3 * ) 3 0.4 xpression pression (A xpression 2 2 ex 0.2 old increase old increase – (f (f PKC 1 1 –/ 0 mRNA e d kc PKC mRNA e 0 0 Pr Cytosolic Cytosolic NDM DM NDM DM NDM DM NDM DM Membrane Membrane 50 µm +/+ Prkcd Prkcd–/– Prkcd+/+ Prkcd –/– PKC-β PKC-δ NDM DM Figure 1 PKC-δ activation causes PDGF-B inhibition and the formation of acellular capillaries. (a) In situ P = 0.038 PKC activity assay, as measured in retinas of mice diabetic for 3 months (DM mice, n = 6) as compared to 50 ) NDM 40 nondiabetic mice (NDM mice, n = 6). (b) Immunoblot analyses of PKC isoforms in the cytosolic and membrane –1 DM h 30 fractions of retinal isolated microvessels of NDM and DM mice (top) and densitometric quantification (bottom) –1 ue leakage (n = 4). (c) Hematoxylin and periodic acid-Schiff staining of retinal vasculature indicating acellular capillaries bl 20 (ml mg +/+ –/– ans (red arrows). Bottom left, quantification of acellular capillaries in Prkcd and Prkcd mouse retinas (NDM 10 +/+ +/+ +/+ –/– –/– Ev Prkcd , n = 22; DM Prkcd , n = 19; DM + insulin (Ins) Prkcd , n = 12; NDM Prkcd , n = 13; DM Prkcd , 0 n = 13). (d) Immunoblot analyses of Atk and ERK phosphorylation after intravitreous injection of saline Prkcd+/+ Prkcd–/– or PDGF-BB into NDM and DM mouse retinas (top) and densitometric quantification (bottom) (n = 3). (e,f) Expression of VEGF and PDGF (e) and PKC-β and PKC-δ (f) mRNA in isolated microvessels from NDM and DM Prkcd+/+ or Prkcd–/– mice (n = 6). (g,h) Flat mounts of retinas infused with FITC-dextran (g) and permeability assay using Evans blue dye (n = 8) (h). Results are shown as means ± s.d. of three or four independent experiments. *P < 0.05 versus NDM Prkcd+/+ mice. respectively) in membrane fractions of isolated retinal microvessels intravitreous injection of PDGF-BB (dimeric PDGF composed of (Fig. 1b) and whole retina (Supplementary Fig. 1a). PKC-δ activa- two B chains), the levels of phospho-ERK and phospho-Akt in the © All rights reserved. 2009 Inc. Nature America, tion induces apoptosis in other cell types13. Therefore, we evaluated retina of nondiabetic Prkcd+/+ mice and nondiabetic and diabetic retinal vascular changes in nondiabetic and diabetic PKC-δ–null Prkcd–/– mice increased by 1.6- to 2.1-fold (P < 0.05) but remained (Prkcd–/–) and age-matched control (Prkcd+/+) mice with or without unchanged in diabetic Prkcd+/+ mice (Fig. 1d). Vascular endothelial interventional glycemic control with insulin implants. Six months growth factor (VEGF), PDGF-B, PKC-β and PKC-δ mRNA or pro- after the streptozotocin treatment used to induce diabetes, Prkcd+/+ tein expression increased in isolated retinal microvessels (Fig. 1e,f) and Prkcd–/– diabetic mice had lower body weights and plasma and in whole retina (Supplementary Fig. 2a–c) of diabetic as com- insulin levels but increased glucose concentrations compared with pared with nondiabetic Prkcd+/+ mice by 2.4- to 2.9-fold (0.001 < nondiabetic mice with a similar genetic background (Supplementary P < 0.05). Normalization of blood glucose concentrations did not Fig. 1b). Treatment of the diabetic mice using insulin implants for the prevent the increases in PDGF-B or PKC-δ protein or mRNA expres- last 3 months of the experiment normalized their body weight and sion induced by diabetes (Supplementary Fig. 2a–c). Consistent with glucose level but not their hyperinsulinemia (Supplementary Table 1). the histological retinal findings, PDGF-B, PKCβ and VEGF protein Histological analyses of the retina showed that diabetes increased the and mRNA expression were not higher in diabetic Prkcd–/– mice as number of acellular capillaries by 59% (P < 0.05) (Fig. 1c) and the compared to nondiabetic Prkcd–/– mice (Fig. 1e,f and Supplementary combined numbers of acellular capillaries and pericyte loss (ghosts) Fig.
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