Targeting the Calpain/Calpastatin System as a New Strategy to Prevent Cardiovascular Remodeling in Angiotensin II–Induced Hypertension

Abstract

Abstract-In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation.

Critical role of the calpain/calpastatin balance in acute allograft rejection

“…In addition, in vivo, endothelial cell calpains could be implicated in lymphocyte transendothelial migration, as they participate in the assembly of docking structures involved in diapedesis process [27]. Thus, evidence is accumulating to suggest that the calpain inhibition by calpastatin is sufficient to limit lymphocyte recruitment, as we previously demonstrated in a model of peritonitis [13].…”

“…In addition, in vivo, endothelial cell calpains could be implicated in lymphocyte transendothelial migration, as they participate in the assembly of docking structures involved in diapedesis process [27]. Thus, evidence is accumulating to suggest that the calpain inhibition by calpastatin is sufficient to limit lymphocyte recruitment, as we previously demonstrated in a model of peritonitis [13].Besides the observed decrease in T-cell migration, mechanisms underlying delayed rejection could involve reduced proliferative responses. But in vitro experiments showed conclusively that the calpain inhibition by calpastatin transgene rather increased T-cell proliferation.…”

“…by both measuring the calpain-specific cleavage of fluorescent AMC substrate and by measuring the accumulation of 145/150-kDa spectrin BDP by Western blot analysis, as previously described [12,13].…”

“…All procedures involving these animals were conducted in accordance with national guidelines and institutional policies. CalpTG mice were created in the laboratory using the cDNA clone of rabbit calpastatin inserted on the PCI expression vector, which includes a viral promoter (CMV immediate-early enhancer/ promoter region) [12,13]. The presence and the expression of the transgene were identified in founder CalpTG mice by PCR and RT-PCR analysis, respectively [12].…”

“…These data are consistent with a model in which calpains and calpastatin are not co-localized within the cell at rest. Calpastatin diffusion after calcium-related cell stimulation allows calpastatin to interact with calpains, thereby modulating its activity [13]. Given that the calpain activity is involved in the activation of NF-kB and NFATc1 [6,9], two pathways leading to the generation of effector T cells [16], the nuclear expression of these transcription factors was also determined in T cells from WT and CalpTG.…”