Celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase-1.

BACKGROUND AND PURPOSE: Celastrol, a triterpene from plants, has been used in traditional oriental medicine to treat various diseases. Here, we investigated the cardioprotective effects of celastrol against ischaemia. EXPERIMENTAL APPROACH: Protective pathways induced by celastrol were investigated in hypoxic cultures of H9c2 rat cardiomyoblasts and in a rat model of myocardial infarction, assessed with echocardiographic and histological analysis. KEY RESULTS: In H9c2 cells, celastrol triggered reactive oxygen species (ROS) formation within minutes, induced nuclear translocation of the transcription factor heat shock factor 1 (HSF1) resulting in a heat shock response (HSR) leading to increased expression of heat shock proteins (HSPs). ROS scavenger N-acetylcysteine reduced expression of HSP70 and HSP32 (haeme oxygenase-1, HO-1). Celastrol improved H9c2 survival under hypoxic stress, and functional analysis revealed HSF1 and HO-1 as key effectors of the HSR, induced by celastrol, in promoting cytoprotection. In the rat ischaemic myocardium, celastrol treatment improved cardiac function and reduced adverse left ventricular remodelling at 14 days. Celastrol triggered expression of cardioprotective HO-1 and inhibited fibrosis and infarct size. In the peri-infarct area, celastrol reduced myofibroblast and macrophage infiltration, while attenuating up-regulation of TGF-ß and collagen genes. CONCLUSIONS AND IMPLICATIONS: Celastrol treatment induced an HSR through activation of HSF1 with up-regulation of HO-1 as the key effector, promoting cardiomyocyte survival, reduction of injury and adverse remodelling with preservation of cardiac function. Celastrol may represent a novel potent pharmacological cardioprotective agent mimicking ischaemic conditioning that could have a valuable impact in the treatment of myocardial infarction.

Regulation of experimental peritonitis: a complex orchestration.

Experimental peritonitis is a frequently used inflammatory model to evaluate leukocyte recruitment. By the intrinsic characteristics of the peritoneal cavity, the various resident cell populations have a role to play in the initiation, the modulation and the resolution of peritoneal inflammation. Through various manipulations of these cell populations, we gained important knowledge on their respective roles in peritoneal inflammation. In this brief review, we will focus on the cellular regulation of leukocyte recruitment in experimental peritonitis.

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication.

The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.

Chronic transplant vasculopathy microenvironment present in the renal allograft reprograms macrophage phenotype.

Macrophages and monocytes are important in chronic allograft dysfunction. Chronic transplant vasculopathy is an important cause of chronic renal allograft dysfunction. It is characterized by the presence of apoptotic endothelial cells, which generate an apocrine loop that leads to typical myointimal proliferation. However, the exact nature of the reprogramming induced by this microenvironment on recruited monocyte and macrophage phenotypes is ill defined. Human umbilical vein endothelial cells with a serum-starved condition (SSC) for 4 hours were used as a model of apoptotic endothelial cells. This conditioned medium was centrifuged to isolate the apoptotic bodies. Monocytes were harvested from healthy donors using Ficoll gradient and immunomagnetic selection. Blood monocyte-derived macrophages (5-7 days of maturation) were exposed to centrifuged apoptotic cell-conditioned media for 24 hours. Cells were harvested for immunoblotting, and supernates were retained for enzyme-linked immunosorbent assay to determine cytokine and chemokine production. Macrophages generated less IL-6 and IL-8 when cultured in SSC compared with control. Immunoblotting for STAT3 (signal transducer and activator of transcription 3) in macrophages revealed that SSC increased STAT3 levels, which persisted after lipopolysaccharide stimulation. These results suggest that SSC attenuates the pro-inflammatory phenotype in macrophages, through a STAT3-dependent mechanism.

Endothelial apoptosis and chronic transplant vasculopathy: recent results, novel mechanisms.

Chronic transplant vasculopathy (CTV) is a progressive form of vascular obliteration affecting the arteries, arterioles and capillaries of solid organ transplants. It is characterized by intimal accumulation of mononuclear cells, vascular smooth muscle cells (VSMC), myofibroblasts and connective tissue. Mounting evidence, based on animal models and human biopsy results, suggests that acute and persistent rejection triggering apoptosis of endothelial cells (EC) plays a pivotal role in CTV. The precise mechanisms that underlie the induction of fibroproliferative changes in association with endothelial apoptosis have yet to be clearly delineated. Recent observations in the field of apoptosis research provide some important mechanistic clues. First, endothelial apoptosis creates a state of hyperadhesiveness for mononuclear cells, thus facilitating sustained leukocyte infiltration. Second, phosphatidylserine-dependent engulfment of apoptotic cells by infiltrating mononuclear leukocytes promotes transforming growth factor-beta1 production. Third, apoptosis of EC triggers extracellular matrix (ECM) proteolysis thus initiating the production of fibroproliferative/fibrogenic ECM fragments. The relative importance of these mechanisms in the pathophysiology of CTV will need to be addressed in vivo. Yet, these recent developments provide a new mechanistic framework that will help better define the importance of immune-mediated EC apoptosis in the regulation of vascular repair.

Exclusion mapping of major crystallization inhibitors in idiopathic calcium urolithiasis.

PURPOSE: We determined whether genetic variation at 3 loci coding for putative crystallization inhibitors is linked to calcium urolithiasis. MATERIALS AND METHODS: We studied a cohort of 64 French-Canadian sibships including multiple recurrent calcium stone formers, comprising 154 independent pairs of siblings with at least 1 stone episode. Physical and meiotic mapping of the genes coding for osteopontin and uromodulin (Tamm-Horsfall protein) as well as the osteocalcin related gene (ORG or putative nephrocalcin) was performed and microsatellite markers were identified. We used nonparametric linkage analysis in the whole affected sib pair cohort as well as in affected pairs without hypercalciuria, that is concordant for 24-hour urine calcium excretion in the first quartile (mean plus or minus standard deviation 0.053 +/- 0.020 mmol./kg. or 3.4 +/- 1.3 mmol. daily), and in the first and second quartiles (mean 0.064 +/- 0.027 mmol./kg. or 4.9 +/- 2.1 mmol. daily, respectively). RESULTS: Lod scores were less than 0.3 for all 3 loci using these affection statuses. Further analysis enabled the exclusion of uromodulin at (relative risk or lambda = 3.9 and 2.5), osteopontin (lambda = 2.7 and 1.6) and ORG (lambda = 5.5 and 3.7) for affected sib pairs concordant for urine calcium excretion in the lowest and 2 lowest quartiles, respectively. CONCLUSIONS: Loci encoding 3 crystallization inhibitors are unlikely to be major genes involved in calcium stone formation in our population.