PKCβ promotes vascular inflammation and acceleration of atherosclerosis in diabetic ApoE null mice

Abstract

Objective: Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)β, it was logical to investigate the role of PKCβ in modulation of atherosclerosis in diabetes mellitus.

Approach and results: ApoE(-/-) and PKCβ(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCβ(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCβ reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCβ and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1β in U937 macrophages.

Conclusions: These data link enhanced activation of PKCβ to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCβ in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCβ activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Keywords: PKCβ; antigens, CD11c; atherosclerosis; diabetes mellitus; inflammation.

Figure 1. Accelerated activation of PKCβII in aorta of diabetic ApoE^−/− mice

Membranous protein from aortas of the indicated mice at age 10 weeks was subjected to Western blot for detection of phosphor-PKCβII, phosphor-PKCδ and β-actin. Representative of N=3 mice/condition.

Figure 2. Impact of PKCβ deletion on diabetic atherosclerosis

Shown are representative images of aortic root sections stained with Oil Red O at age 14 weeks (A) and 20 weeks (B) and Sudan IV stained aortic en face at age 20 weeks (C). Scale bar =200 μm. Mean atherosclerotic lesion areas (μm²) were determined in ND ApoE^−/− (n=13), D ApoE^−/− (n=14) and D PKCβ ^−/−/ApoE^−/− male mice (n=15 and n= 13) at age 14 weeks (A) and 20 weeks (B). Lesion complexity index was calculated in D ApoE^−/− and D PKCβ ^−/−/ApoE^−/− male mice at age 20 weeks (n=10 and n=13) (D). D: diabetic; ND: nondiabetic.

Figure 3. Impact of PKCβ inhibitor on diabetic atherosclerosis

Shown are representative images of aortic root sections stained with Oil Red O at age 20 weeks (A). Scale bar =200 μm. Mean atherosclerotic lesion areas (μm²) were determined in D ApoE^−/− male mice fed vehicle chow (VEH, n=14) or fed ruboxistaurin (RBX, n=13) at age 20 weeks (A). Lesion complexity index was calculated in D ApoE^−/− male mice fed VEH chow or fed RBX at age 20 weeks (n=10 and n=14) (B). D: diabetic.

Figure 4. Impact of diabetes and PKCβ on the expression of inflammatory mediators and activation of MAP kinase

Gene expression profiling was validated by real-time PCR in aortic RNA of D ApoE^−/− mice vs. ND ApoE^−/− mice (n≥3, ^#p ≤0.05, *p≤0.01 and ^p≤0.001) or D PKCβ ^−/−/ApoE^−/− mice (n≥3, ##p≤0.05, **p≤0.01 and ^^p≤0.001) at age 10 weeks (A). Shown are some representative genes not altered in D ApoE^−/− or D PKCβ ^−/−/ApoE^−/− mice (B). Aortic RNA was subjected to real-time PCR for detection of Egr-1 RNA (C). Aortic protein was subjected to Western blot for detection of phosphor (P)-ERK1/2 and total (T)-ERK (D), P-JNK and T-JNK (E). D: diabetic; ND: nondiabetic.

Figure 5. Impact of PKCβ on the inflammatory cells in atherosclerotic lesions in diabetes

The sections of aortic root from D PKCβ ^−/−/ApoE^−/− mice (A, 10x magnification, Scar bar=200μm) and D ApoE^−/− mice (B, 10x magnification, Scar bar=200μm; & C, 40x magnification, scar bar= 50μm) at age 20 weeks were stained with anti-MOMA-2 and anti-CD11c, or isotype-matched antibodies. Nuclei were counterstained with DAPI. The merging of MOMA-2 and CD11C or merging of isotype-matched antibodies is shown. To determine the relative amounts of MOMA-2 positive macrophages (D) or CD11-expressing cells (E) in an atherosclerotic lesion, the positive area of MOMA-2 or CD11c was divided by the total lesion area of atherosclerosis. D: Diabetic.

Figure 6. Impact of high glucose and PKCβ-dependent signaling pathways on the expression of inflammatory mediators in U937 macrophages

U937 cells were seeded and exposed to 5 mM (low) or 25 mM (high) glucose in the absence or presence of siRNA to Egr-1 or the inhibitors of PKCβ, ERK1/2, JNK and p38. Cells were harvested followed by RNA isolation. Real-time PCR analysis of gene expression was performed. Data are represented as the relative gene expression of Egr-1 (A), CD11c (B), CCL2 (C ) and IL-1β (D) normalized to 18s rRNA. * p<0.001, the level of gene expression in U937 macrophages exposed to high glucose vs. low glucose; ^p<0.001, ^^p<0.01 and # N.S. (Not significant), the level of gene expression in U937 macrophages exposed to high glucose with the treatment of inhibitors or siRNA vs. non-treatment.