Protein kinase C upregulates intercellular adhesion molecule-1 and leukocyte-endothelium interactions in hyperglycemia via activation of endothelial expressed calpain

Abstract

Objective: We tested the hypothesis of a role for the calcium-dependent protease calpain in the endothelial dysfunction induced by hyperglycemic activation of protein kinase C (PKC).

Methods and results: Chronic hyperglycemia with insulin deficiency (type 1 diabetes) was induced in rats by streptozotocin. Total PKC and calpain activities, along with activity and expression level of the 2 endothelial-expressed calpains isoforms, μ- and m-calpain, were measured in vascular tissue homogenates by enzymatic assays and Western blot analysis, respectively. Intravital microscopy was used to measure and correlate leukocyte-endothelium interactions with calpain activity in the microcirculation. Expression levels and endothelial localization of the inflammatory adhesion molecule intercellular adhesion molecule-1 were studied by Western blot analysis and immunofluorescence, respectively. The mechanistic role of hyperglycemia alone in the process of PKC-induced calpain activation and actions was also investigated. We found that in the type 1 diabetic vasculature, PKC selectively upregulates the activity of the μ-calpain isoform. Mechanistic studies confirmed a role for hyperglycemia and PKCβ in this process. The functional implications of PKC-induced calpain activation were upregulation of endothelial expressed intercellular adhesion molecule-1 and leukocyte-endothelium interactions.

Conclusions: Our results uncover the role of μ-calpain in the endothelial dysfunction of PKC. Calpain may represent a novel molecular target for the treatment of PKC-associated diabetic vascular disease.

Figure 1. Calpain and PKC activities are increased in the hyperglycemic, insulin-deficient mesenteric microcirculation

Calpain (panel A) and PKC (Panel B) activities in all experimental groups of rats were measured in protein extracts of the vascularized mesentery using the fluorogenic substrate Succ-LLVY-AMC and by the incorporation of γ-phosphate from [γ-³²P]ATP into the substrate QKRPSQRSKYL, respectively. The calpain inhibitor ZLLal effectively blocked calpain activity (Panel A) but failed to attenuate PKC activity (Panel B) in the STZ-diabetic microcirculation. Activity levels are expressed as fold change from nondiabetic control values. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of rats studied in each group.

Figure 2. PKC inhibition attenuates calpain activity in the hyperglycemic, insulin-deficient microcirculation

Following superfusion of the rat mesentery with the fluorogenic calpain substrate t-BOC-Leu-Met-CMAC, active calpains in the venular endothelium were visualized by fluorescent intravital microscopy and measured by densitometry. Control post-capillary venules (V) had a very low basal level of calpain activity indicated by a low level of fluorescent staining (Panel A). Calpain activity was markedly increased in the post-capillary venular endothelium of STZ-diabetic rats, as demonstrated by intense fluorescent staining (Panel B, white arrows). The PKC inhibitor BIM-I reduced fluorescent staining for calpain activity in the STZ-diabetic microcirculation (Panel C). The calpain inhibitor PD150606 also attenuated fluorescent staining for calpain activity. White line in Panel A represents 10 µm scale. Yellow circle in panel B depicts a representative region of interest (ROI) used for densitometry. The bar graph shows densitometry quantification of calpain activity in all experimental groups of rats. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of rats studied in each group.

Figure 3. RHMEC experience a PKCβ-dependent increase in calpain activity in response to high glucose

Calpain activity was measured in attached RHMEC using the fluorogenic substrate Succ-LLVY-AMC and expressed as fold change from control. Genetic inhibition of PKCβ by expression of a kinase-inactive dominant-negative (dn) PKCβ, and pharmacological inhibition of either total PKC activity with BIM-I or selective PKCβ activity with LY379196 prevented upregulation of calpain in response to high glucose. The mutant protein constructed on rabbit PKCβ-II cDNA could be distinguished from the endogenously expressed PKCβ-I by antibodies raised against c-terminus of PKCβ-I and PKCβ-II. Expression of PKCα was not affected by the dnPKCβ over-expression (Immunoblot). The dnPPKCβ used in this study has been shown to inhibit select functions mediated by both PKCβ-I and PKCβ-II, but not by other PKCs such as PKCα. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of experiments.

Figure 4. Leukocyte-endothelium interactions in mesenteric post-capillary venules

Leukocyte rolling (A) and adhesion (B) were studied in all experimental groups of rats by brightfield intravital microscopy and expressed as the number of leukocytes per minute and the number of leukocytes per 100 µm vessel length, respectively. Calpain or PKC inhibition reduced leukocyte-endothelium interactions to a similar extent in the STZ-diabetic microcirculation. In contrast, the inactive PKC inhibitor, BIM-V, failed to attenuate leukocyte rolling and adhesion in STZ-diabetic rats. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of rats studied in each group.

Figure 5. PKC-calpain signaling regulates ICAM-1 expression in the hyperglycemic, insulin-deficient vasculature

ICAM-1 expression in the thoracic aorta was studied by western blot analysis. Expression of ICAM-1 was increased above control levels in STZ diabetic rats. Treatment of STZ diabetic rats with the calpain inhibitor ZLLal or with the PKC inhibitor BIM-I returned ICAM-1 expression levels to control values. The bar graph shows quantification by densitometry of the signal from western blot analysis. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of rats studied in each group.

Figure 6. Acute hyperglycemia upregulates leukocyte-endothelium interactions and calpain activity in mesenteric post-capillary venules in a PKC-dependent manner

Post-capillary venules from all experimental groups of rats were viewed under brightfield microscopy. Following superfusion of the rat mesentery with the fluorogenic calpain substrate t-BOC-Leu-Met-CMAC, active calpains in the venular endothelium were visualized by fluorescent microscopy and measured by densitometry, as shown in Figure 2. Elevated ambient glucose increased calpain activity (panel A) and leukocyte adhesion in post-capillary venules (Panel B). Treatment of rats with BIM-I prevented both calpain activity and leukocyte adhesion in post-capillary venules exposed to elevated ambient glucose. Panel A shows densitometry quantification of calpain activity in all experimental groups of rats. Panel B is leukocyte adhesion expressed as the number of cells per 100 µm in all experimental groups of rats. Bars represent mean ± SEM, and numbers at the base of the bars represent the number of rats studied in each group.