Dominant Role for Calpain in Thromboxane-Induced Neuromicrovascular Endothelial Cytotoxicity

Abstract

Thromboxane A 2 (TXA 2 ) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA 2 -evoked neural microvascular endothelial cell death. Stable TXA 2 mimetics 5-heptenoic acid, 7--5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA 2 dependence of these effects was corroborated by antagonism using the selective TXA 2 receptor blocker (Ϫ)-6,8-difluoro-9-p-methylsulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA 2 (mimetics) augmented generation of known active m-calpain (but not -calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of ␣-spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17-and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17␤-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolylcarbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA 2 -induced neurovascular endothelial cell death.

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli

“…4); (iii) generation of calpain-2 active fragments and cleavage of cytoskeletal protein a-spectrin (relevant substrate of this enzyme) (Supplementary Material, Fig. S1), in agreement with previous studies (Quiniou et al, 2006;Zhang et al, 2009);and (iv) reduction in the number of microvessels anatomically present in cortical brain regions (Fig. 6).…”

The Human Host Defense Peptide LL-37 Induces Apoptosis in a Calpain- and Apoptosis-Inducing Factor–Dependent Manner Involving Bax Activity

“…Jurkat cells were exposed to LL-37 (200 μg/mL) in the absence or presence of calpain inhibitor (Ac-LLnL-CHO, 1 μmol/L) to analyze the involvement of calpains in Bax translocation following LL-37 treatment. Calpains have been previously shown to trigger Bax activation downstream of death induction (38,41). Figure 7B shows that Bax localizes to the mitochondrial fraction in Jurkat cells exposed to LL-37, whereas this relocation is blocked by pretreatment of cells with calpain inhibitor.…”

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo

“…In addition, m-calpain, but not μ-calpain, seems to mediate β-catenin degradation in ASCs, which is analogous to previous observations in neurovascular ECs. 77 There are many different calpain targets in different cells, such as β-catenin in colon cancer cells, 36,37 CD31 in platelets, 78,79 and caspase 12 in cardiomyocytes and neurons. 80,81 We found that the activation of TP receptors promotes calpain cleavage of β-catenin in ASCs on VEGF treatment, without overt influence of CD31 and caspase 12.…”