Preventing Calpain Externalization by Reducing ABCA1 Activity with Probenecid Limits Melanoma Angiogenesis and Development.

Calpains, intracellular proteases specifically inhibited by calpastatin, play a major role in neoangiogenesis involved in tumor invasiveness and metastasis. They are partly exteriorized via the ATP-binding cassette transporter A1(ABCA1) transporter, but the importance of this process in tumor growth is still unknown. The aim of our study was to investigate the role of extracellular calpains in a model of melanoma by blocking their extracellular activity or exteriorization. In the first approach, a B16-F10 model of melanoma was developed in transgenic mice expressing high extracellular levels of calpastatin. In these mice, tumor growth was inhibited by ∼ 3-fold compared with wild-type animals. In vitro cytotoxicity assays and in vivo tumor studies have demonstrated that this protection was associated with a defect in tumor neoangiogenesis. Similarly, in wild-type animals given probenecid to blunt ABCA1 activity, melanoma tumor growth was inhibited by ∼ 3-fold. Again, this response was associated with a defect in neoangiogenesis. In vitro studies confirmed that probenecid limited endothelial cell migration and capillary formation from vascular explants. The observed reduction in fibronectin cleavage under these conditions is potentially involved in the response. Collectively, these studies demonstrate that probenecid, by blunting ABCA1 activity and thereby calpain exteriorization, limits melanoma tumor neoangiogenesis and invasiveness.

Stiripentol protects against calcium oxalate nephrolithiasis and ethylene glycol poisoning.

Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

Calpain 1 in bronchoalveolar lavage fluid is associated with poor prognosis in lepidic predominant pulmonary adenocarcinoma.

Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.

Specific calpain inhibition protects kidney against inflammaging.

Calpains are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Transgenic mice over-expressing rabbit calpastatin (CalpTG) are protected against vascular remodelling and angiotensin II-dependent inflammation. We hypothesized that specific calpain inhibition would protect against aging-related lesions in arteries and kidneys. We analysed tissues from 2-months and 2-years-old CalpTG and wild-type mice and performed high throughput RNA-Sequencing of kidney tissue in aged mice. In addition, we analysed inflammatory response in the kidney of aged CalpTG and wild-type mice, and in both in vivo (monosodium urate peritonitis) and in vitro models of inflammation. At two years, CalpTG mice had preserved kidney tissue, less vascular remodelling and less markers of senescence than wild-type mice. Nevertheless, CalpTG mice lifespan was not extended, due to the development of lethal spleen tumors. Inflammatory pathways were less expressed in aged CalpTG mice, especially cytokines related to NF-κB and NLRP3 inflammasome activation. CalpTG mice had reduced macrophage infiltration with aging and CalpTG mice produced less IL-1α and IL-1ß in vivo in response to inflammasome activators. In vitro, macrophages from CalpTG mice produced less IL-1α in response to particulate activators of inflammasome. Calpains inhibition protects against inflammaging, limiting kidney and vascular lesions related to aging.

Calcium and vitamin D have a synergistic role in a rat model of kidney stone disease.

Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm(3)). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.

Extracellular Calpain/Calpastatin Balance Is Involved in the Progression of Pulmonary Hypertension.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression.

Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.

Vasopressin regulates renal calcium excretion in humans.

Antidiuretic hormone or arginine vasopressin (AVP) increases water reabsorption in the collecting ducts of the kidney. Three decades ago, experimental models have shown that AVP may increase calcium reabsorption in rat kidney. The objective of this study was to assess whether AVP modulates renal calcium excretion in humans. We analyzed calcium, potassium, and sodium fractional excretion in eight patients affected by insipidus diabetes (nephrogenic or central) under acute vasopressin receptor agonist action and in 10 patients undergoing oral water load test affected or not by inappropriate antidiuretic hormone secretion (SIADH). Synthetic V2 receptor agonist (dDAVP) reduced significantly calcium fractional excretion from 1.71% to 0.58% (P < 0.05) in patients with central diabetes insipidus. In patients with nephrogenic diabetes insipidus (resistant to AVP), calcium fractional excretion did not change significantly after injection (0.48-0.68%, P = NS). In normal subjects undergoing oral water load test, calcium fractional excretion increased significantly from 1.02% to 2.54% (P < 0.05). Patients affected by SIADH had a high calcium fractional excretion at baseline that remained stable during test from 3.30% to 3.33% (P = NS), possibly resulting from a reduced calcium absorption in renal proximal tubule. In both groups, there was a significant correlation between urine output and calcium renal excretion. In humans, dDAVP decreases calcium fractional excretion in the short term. Conversely, water intake, which lowers AVP concentration, increases calcium fractional excretion. The correlation between urine output and calcium excretion suggests that AVP-related antidiuresis increases calcium reabsorption in collecting ducts.

Calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation.

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

Demographics and characterization of 10,282 Randall plaque-related kidney stones: a new epidemic?

Renal stone incidence has progressively increased in industrialized countries, but the implication of Randall plaque in this epidemic remains unknown. Our objectives were to determine whether the prevalence of Randall plaque-related stones increased during the past decades after having analyzed 30,149 intact stones containing mainly calcium oxalate since 1989 (cross-sectional study), and to identify determinants associated with Randall plaque-related stones in patients (case-control study). The proportion of Randall plaque-related stones was assessed over 3 time periods: 1989-1991, 1999-2001, and 2009-2011. Moreover, we analyzed clinical and biochemical parameters of 105 patients affected by calcium oxalate stones, with or without plaque. Of 30,149 calcium oxalate stones, 10,282 harbored Randall plaque residues (34.1%). The prevalence of Randall plaque-related stones increased dramatically during the past years. In young women, 17% of calcium oxalate stones were associated with Randall plaque during the 1989-1991 period, but the proportion rose to 59% 20 years later (P < 0.001). Patients with plaques experienced their first stone-related event earlier in life as compared with those without plaque (median age 26 vs 34 years, P = 0.02), had increased ionized serum calcium levels (P = 0.04), and increased serum osteocalcin (P = 0.001) but similar 25-hydroxyvitamin D levels. The logistic regression analysis showed that age (odds ratio [OR] 0.96, confidence interval [CI] 0.926-0.994, P = 0.02), weight (OR 0.97, CI 0.934-0.997, P = 0.03), and osteocalcin serum levels (OR 1.12, CI 1.020-1.234, P = 0.02) were independently associated with Randall plaque. The prevalence of the FokI f vitamin D receptor polymorphism was higher in patients with plaque (P = 0.047). In conclusion, these findings point to an epidemic of Randall plaque-associated renal stones in young patients, and suggest a possible implication of altered vitamin D response.

An animal model of type A cystinuria due to spontaneous mutation in 129S2/SvPasCrl mice.

Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b0,+AT (type B cystinuria). Here, we evidenced in a commonly used congenic 129S2/SvPasCrl mouse substrain a dramatically high frequency of kidney stones that were similar to those of patients with cystinuria. Most of 129S2/SvPasCrl exhibited pathognomonic cystine crystals in urine and an aminoaciduria profile similar to that of patients with cystinuria. In addition, we observed a heterogeneous inflammatory infiltrate and cystine tubular casts in the kidney of cystinuric mice. As compared to another classical mouse strain, C57BL/6J mice, 129S2/SvPasCrl mice had an increased mortality associated with bilateral obstructive hydronephrosis. In 129S2/SvPasCrl mice, the heavy subunit rBAT of the tetrameric transporter of dibasic amino acids was absent in proximal tubules and we identified a single pathogenic mutation in a highly conserved region of the Slc3a1 gene. This novel mouse model mimicking human disease would allow us further pathophysiological studies and may be useful to analyse the crystal/tissue interactions in cystinuria.

Prevalence and correlates of metabolic acidosis among patients with homozygous sickle cell disease.

BACKGROUND AND OBJECTIVES: Very few studies report acid base disorders in homozygous patients with sickle cell anemia (SCA) and describe incomplete renal acidosis rather than true metabolic acidosis, the prevalence of which is unknown and presumably low. This study aimed to assess the prevalence of metabolic acidosis and to identify its risk factors and mechanisms. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study retrospectively analyzed 411 homozygous patients with SCA with a GFR ≥ 60 ml/min per 1.73 m(2), referred in a single center between 2007 and 2012. Acidosis and nonacidosis groups were compared for clinical and biologic data including SCA complications and hemolytic parameters. A subgroup of 65 patients with SCA, referred for a measured GFR evaluation in the setting of sickle cell-associated nephropathy, was further analyzed in order to better characterize metabolic acidosis. RESULTS: Metabolic acidosis was encountered in 42% of patients with SCA, with a higher prevalence in women (52% versus 27% in men; P<0.001). Several hemolytic biomarkers, such as lactate dehydrogenase, were different between the acidosis and nonacidosis groups (P=0.02 and P=0.03 in men and women, respectively), suggesting higher hemolytic activity in the former group. To note, fasting urine osmolality was low in the whole study population and was significantly lower in men with SCA in the acidosis group (392 versus 427 mOsm/kg; P=0.01). SCA subgroup analysis confirmed metabolic acidosis with a normal anion gap in 14 patients, characterized by a lower urinary pH (P<0.02) and no increase in urinary ammonium. Serum potassium, plasma renin, and aldosterone were similar between the two groups and thus could not explain impaired urinary ammonium excretion. CONCLUSIONS: These results suggest that the prevalence of metabolic acidosis in patients with SCA is underestimated and related to impaired ammonium availability possibly due to an altered corticopapillary gradient. Future studies should evaluate whether chronic metabolic acidosis correction may be beneficial in this population, especially in bone remodeling.

The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma.

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

Calpain activity is essential in skin wound healing and contributes to scar formation.

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFß-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

Ureteral obstruction promotes proliferation and differentiation of the renal urothelium into a bladder-like phenotype.

The renal urothelium, the monolayered epithelium that covers the papilla, is the direct target of increased pressure during obstruction, yet most studies have mainly focused on tubules, fibroblasts, and inflammatory cells. We studied this epithelium in a unilateral ureteral obstruction mouse mode land found that it was disrupted and had broken tight junctions, enlarged intercellular space, with loss of apicaluroplakins, and marginal lumen desquamation. Shortly after obstruction these urothelial cells proliferated, peaking at day 2. By day 14, the renal urothelium was transformed into a multilayered barrier with newly synthesized uroplakins including the de novo induction of uroplakin II. This proliferation was found to be fibroblast growth factor (FGF)dependent. Renal urothelial cells constitutively express the FGF receptor 2, and obstruction activated the receptor by phosphorylation. Treatment with FGF receptor 2-antisense or vitamin A (an inhibitor of the MAP kinase in the FGFR2 pathway) decreased renal urothelial cell proliferation. Among known FGF receptor 2 ligands, only FGF7 was upregulated.Infusion of FGF7 into control mice caused the formation of a multilayered structure at 7 days, resembling the urothelium 14 days following obstruction. Thus, the pressure/stretching of renal monolayered urothelial cells is a very efficient trigger for proliferation, causing the formation of a bladder-like multistratified barrier with enhanced apical uroplakin plaques. Presumably, this ensures efficient barrier protection and repair.

The role of calpains in myocardial remodelling and heart failure.

Calpains are cytosolic calcium-activated cysteine proteases. Recently, they have been proposed to influence signal transduction processes leading to myocardial remodelling and heart failure. In this review, we will first describe some of these molecular mechanisms. Calpains may contribute to myocardial hypertrophy and inflammation, mainly through the activation of transcription factors such as NF-κB. They play an important role in the fibrosis process partly by activating transforming growth factor ß. They are also implicated in cell death as they cause the breakdown of sarcolemma and sarcomeres. Nevertheless, a key to understanding the molecular basis of calpain-mediated myocardial remodelling likely lies in the identification of mechanisms involved in calpain secretion, since cytosolic and extracellular proteases would have different functions. Finally, we will provide an overview of the available evidence that calpains are indeed actively involved in the common causes of heart failure, including hypertension, diabetes, atherosclerosis, ischaemia-reperfusion, atrial fibrillation, congestive failure, and mechanical unloading.

Calpastatin controls polymicrobial sepsis by limiting procoagulant microparticle release.

RATIONALE: Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release. OBJECTIVES: We investigated the contribution of calpain in eliciting tissue damage during sepsis. METHODS: To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. MEASUREMENTS AND MAIN RESULTS: In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy. CONCLUSIONS: These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.

Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization.

OBJECTIVE: Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. METHODS AND RESULTS: A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. CONCLUSIONS: This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.