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A novel role for calpains in the endothelial dysfunction of hyperglycemia ¹

Department of Physiology, Jefferson Medical College, Thomas Jefferson University Philadelphia, Pennsylvania, USA

²Correspondence: Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107-6799, USA. E-mail: Rosario.Scalia{at}mail.tju.edu

SPECIFIC AIMS

The calpains are a family of calcium-dependent proteases that have been implicated in a number of inflammatory disorders of the cardiovascular system. Increased calpain activity has also been associated with decreased nitric oxide (NO) production in endothelial cells. Hyperglycemia is associated with vascular inflammation characterized by the loss of endothelial NO. Therefore, we sought to examine the role of calpains in the endothelial dysfunction and vascular inflammation induced by hyperglycemia.

PRINCIPAL FINDINGS

1. Calpain activity is increased in rat mesentery exposed to elevated ambient glucose
Sprague-Dawley rats were injected intraperitoneally (i.p.) 16 h before experiments with either 25 mmol/L D-glucose alone or 25 mmol/L D-glucose plus one of two calpain inhibitors: calpain inhibitor I (25 µmol/L) or ZLLal (5 µmol/L). Activated calpains cleave the cytoskeletal protein II spectrin (240 kDa). Production of the calpain-specific 145 kDa spectrin breakdown product was increased 2.2-fold in highly vascularized mesenteric segments of rats injected with 25 mmol/L D-glucose, which was prevented by treatment of the rats with the calpain inhibitor ZLLal (Fig. 1 ). Thus, acute hyperglycemia increases calpain activity in highly vascularized tissues.

View larger version (22K): [in this window] [in a new window]   Figure 1. Hyperglycemia increases calpain activity. Mesenteric tissue was harvested and II spectrin breakdown products were analyzed by SDS-PAGE. A) Immunoblot illustrates increased 145 kDa spectrin breakdown products in response to hyperglycemia. B) Densitometric analysis of the calpain-specific 145 kDa spectrin breakdown product obtained from 3 to 5 rats in each group.

2. Calpains up-regulate leukocyte–endothelium interactions in response to acute elevations in ambient glucose via a NO synthase-dependent mechanism
Intraperitoneal injection of 25 mmol/L D-glucose caused a seven- and sixfold increase in leukocyte rolling and adhesion, respectively, in rat mesenteric postcapillary venules (P<0.01 vs. control rats), as quantified by intravital microscopy. Intraperitoneal administration of calpain inhibitor-I or ZLLal prevented the increase in leukocyte–endothelium interactions induced by D-glucose (P<0.01 vs. D-glucose alone). Similar results were obtained for the number of extravasated leukocytes. Twenty-five mmol/L D-glucose increased leukocyte extravasation from 1.05 ± 0.4 to 6.25 ± 0.629 cells/100 µm² (P<0.01), and this increase was blocked by administration of either calpain inhibitor-I or ZLLal (2.80±0.3 and 3.17±0.3 cells/100 µm², respectively, P<0.01 vs. D-glucose alone). Thus, inhibition of calpain activity in vivo attenuates glucose-induced leukocyte trafficking.

To test whether functional endothelial NO synthase (eNOS) is necessary for the anti-inflammatory action of calpain inhibition, we studied the anti-inflammatory effect of ZLLal in rat mesenteries superfused with 50 µmol/L L-NAME. Calpain inhibition failed to prevent leukocyte–endothelium interactions in response to pharmacological blockade of eNOS activity with L-NAME. These results clearly indicate that residual eNOS function is necessary for the anti-inflammatory effects of calpain inhibition in vivo.

3. Calpains impair eNOS activity during hyperglycemia by disrupting the association of hsp90 and eNOS
To study whether calpains regulate endothelial NO availability during hyperglycemia, we measured release of NO directly in mesenteric venules in vivo using a NO-specific microelectrode (Fig. 2 ). Control rats exhibited basal NO levels of 202.8 ± 34.0 nM/1000 µm². Intraperitoneal injection of D-glucose caused a 70% decrease in basal NO levels in rat postcapillary venules (P<0.05). Treatment with ZLLal significantly attenuated glucose-induced loss of endothelial NO, restoring NO levels to control values. Thus, hyperglycemia depresses release of endothelial NO in the microcirculation via a calpain-dependent mechanism.

View larger version (46K): [in this window] [in a new window]   Figure 2. Calpain inhibition preserves basal NO release from rat mesenteric venules in vivo. A) Photomicrographs show representative mesenteric venule from a control rat (top panel) and a D-glucose-treated rat (bottom panel). The microelectrode used for NO measurements is shown, and leukocytes can be seen interacting with the endothelium (arrows indicate rolling cells, arrowheads adherent cells). Large black dots are the optical projection of the optical Doppler velocimeter used to measure shear rates. B) Graph summarizing the effect of 25 mmol/L D-glucose and 5 µmol/L ZLLal on basal NO levels. Data are expressed as nanomolar NO/1000 µm2. Numbers at the base of the bars indicate the number of rats in each group.

We also examined the association of eNOS with hsp90, since hsp90 binding to eNOS enhances eNOS activity. Immunoprecipitation studies demonstrated reduced association of eNOS and hsp90 in the mesenteric tissue of rats treated with 25 mmol/L D-glucose. Treatment of rats with ZLLal preserved the eNOS/hsp90 association in the face of hyperglycemia, demonstrating that loss of eNOS/hsp90 association during hyperglycemia is calpain dependent.

4. Calpains have a role in the up-regulation of cell adhesion molecule expression during hyperglycemia
To dissect the anti-inflammatory mechanism of calpain inhibition, we studied surface expression of the proatherogenic adhesion molecules ICAM-1 and VCAM-1. A low percentage of ileal venules stained positive for ICAM-1 and VCAM-1 in control rats. Rats injected i.p. with 25 mmol/L D-glucose exhibited a 3.3-fold and 5.6-fold increase in the number of ileal venules staining for ICAM-1 and VCAM-1, respectively. ZLLal prevented up-regulation of both ICAM-1 and VCAM-1 on the endothelial cell surface (P<0.01 vs. glucose alone).

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates that increased calpain activity contributes to vascular inflammation in hyperglycemia. We provide direct evidence that hyperglycemia reduces physiologic levels of endothelial NO in the microcirculation via calpain-dependent decreased association of the regulatory protein hsp90 with eNOS. We demonstrate that inhibition of calpain activity during hyperglycemia attenuates leukocyte–endothelium interactions and preserves endothelial NO release.

The mechanisms by which hyperglycemia causes endothelial dysfunction remain only partially understood. Increased oxidative stress and loss of endothelial NO are largely considered a key mechanism by which high glucose disrupts the homeostasis of endothelial cells. During diabetes, loss of endothelial NO leads to increased endothelial adhesiveness and subsequent sequestration of leukocytes in the microvasculature. The resulting microvascular inflammation likely contributes to the pathogenesis of atherosclerosis, a cardiovascular disease highly prevalent among diabetic patients.

Recent in vivo studies have reported that calpain inhibition exerts anti-inflammatory effects during several cardiovascular disorders such as ischemia/reperfusion, hemorrhagic shock, and endotoxic shock, demonstrating a role for calpains in vascular inflammation. Activated calpains decrease NO production by endothelial cells. Accordingly, we investigated the role of calpains in the vascular inflammatory signal of hyperglycemia.

We found evidence that acute hyperglycemia increases calpain activity and causes endothelial dysfunction leading to pathologic leukocyte trafficking in the microcirculation. One possible mechanism responsible for leukocyte–endothelium interactions during hyperglycemia is loss of endothelial NO release. We measured NO levels in vivo and found that elevated ambient glucose significantly reduces basal NO levels in the microcirculation directly at the site of inflammation. This effect of hyperglycemia on endothelial NO release was prevented by inhibition of calpain activity, which further supports a role for calpains in the endothelial dysfunction and vascular inflammation of diabetes.

A mechanism that may explain the relationship between inhibition of calpain activity and preservation of endothelial NO in hyperglycemia is altered post-translational regulation of eNOS. The molecular chaperone heat shock protein 90 is an important regulator of eNOS function that has been shown to be a substrate for calpains. Hsp90 increases NO production by stabilizing the eNOS complex. We found evidence that the hsp90/eNOS association is decreased in rat mesenteric tissue exposed to elevated ambient glucose and that calpain inhibition preserves the association of hsp90 with eNOS. Thus, increased calpain activity in response to hyperglycemia may inhibit endothelial NO production by disrupting the post-translational regulation of eNOS (Fig. 3 ).

View larger version (61K): [in this window] [in a new window]   Figure 3. Schematic illustrating the proposed mechanism by which calpains contribute to inflammatory signals in the hyperglycemic vasculature. Hyperglycemia increases calpain activity leading to increased proteolysis of the eNOS regulatory protein hsp90. The loss of hsp90 from the eNOS complex results in decreased NO production, with subsequent up-regulation of inflammatory cell adhesion molecules and increased leukocyte–endothelium interactions.

In conclusion, our study uncovers a novel signaling pathway implicated in the pathophysiology of diabetic vascular disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1213fje; doi: fj.02-1213fje

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