Normand Vigneault

Novel Fibrogenic Pathways Are Activated in Response to Endothelial Apoptosis: Implications in the Pathophysiology of Systemic Sclerosis

Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-xL overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-xL protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of α-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.

Apoptosis of endothelial cells triggers a caspase-dependent anti-apoptotic paracrine loop active on VSMC

Increased endothelial apoptosis and decreased apoptosis of vascular smooth muscle cells (VSMC) are central to initiation of myo‐intimal thickening. We hypothesized that apoptosis of endothelial cells (EC) induces the release of anti‐apoptotic mediator(s) active on VSMC. We found that serum‐free medium conditioned by apoptotic EC decreases apoptosis of VSMC compared with fresh serum‐free medium. Inhibition of endothelial apoptosis during conditioning with a pan‐caspase inhibitor ZVAD‐FMK blocked the release of the anti‐apoptotic factor(s) active on VSMC. VSMC exposed to serum‐free medium conditioned by apoptotic EC showed increased ERK 1/2 phosphorylation, enhanced Bcl‐xl expression, and inhibition of p53 expression. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as a C‐terminal fragment of the domain V of perlecan. Serum‐free medium supplemented with either a synthetic peptide containing the EGF motif of the domain V of perlecan or chondroitin 4‐sulfate, a glycosaminoglycan anchored on the domain V of perlecan, increased ERK 1/2 phosphorylation and Bcl‐xl protein levels while inhibiting apoptosis of VSMC. These results suggest that a proteolytic activity developing downstream of activated caspases in apoptotic EC initiates degradation of pericellular proteoglycans and liberation of bioactive fragments with a robust impact on inhibition of VSMC apoptosis.

Micropuncture study of renal phosphorus transport in hypophosphatemic vitamin D resistant rickets mice.

SummaryA micropuncture study of inorganic phosphorus (Pi) transport was performed in 6 mice presenting hypophosphatamic vitamin D resistant rickets (Hyp) and results compared to those obtained in 13 normal (N) mice. The mean plasma (P Pi) and fractional excretion of Pi (FE Pi) in N and Hyp mice were 85.10±2.27 mg/l and 15.81±1.90 vs 48.43±3.29 mg/l and 35.34±5.26%, respectively.In N mice, tubular fluid over plasma Pi ratio (TF/P Pi) progressively decreases along the proximal tubule to reach approximately 0.6 in the late accessible part. The fraction of filtered Pi (% E Pi) remaining in this segment of the nephron is therefore 20–25%. In Hyp mice the TF/P Pi in proximal tubule remains relatively high and does not significantly vary with TF/P inulin (mean TF/P Pi=1.19±0.12 S.E., compared to 0.73±0.04 in N mice). Precise conclusions concerning % E Pi at the end of the proximal tubule of Hyp mice are not available because of the scattering of the data. However, all the values of % E Pi (mean % E Pi: 78.68±7.39 compared to 49.10±4.59 in N mice) are far above the total urinary FE Pi (35.34 ±5.26), suggesting a relatively greater distal fractional Pi reabsorption in Hyp mice than in N mice. Since the P Pi, and therefore the amount of filtered Pi is lower in Hyp mice, it is probable that the absolute amount of Pi reabsorbed distally is comparable in the two series of animals.

Blockade of the apoptotic machinery by cyclosporin A redirects cell death toward necrosis in arterial endothelial cells: regulation by reactive oxygen species and cathepsin D.

Blockade of the mitochondrial permeability transition pore (mPTP) by cyclosporin A (CsA) inhibits apoptosis in various cell types. However, use of CsA in humans is associated with damage to the arterial endothelium. We evaluated whether inhibition of the apoptotic machinery by CsA promotes other forms of cell death in arterial endothelial cells (EC).

Soluble Fas: a novel predictor of atherosclerosis in dialysis patients

BACKGROUND Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Disregulation of apoptosis within the vessel wall and upregulation of the Fas/Fas-ligand (Fas-L) system contribute to the development of atherosclerosis. Cross-sectional studies have suggested that elevated plasma levels of the soluble form of Fas (sFas) are associated with CVD. However, the role of sFas and sFas-L in predicting future cardiovascular events has yet to be defined. METHODS We evaluated the role of plasma sFas and sFas-L levels as predictors of CVD in a prospective cohort of 107 chronic hemodialysis patients. RESULTS During the study period (27 months), 53 patients (49.5%) presented with at least one cardiovascular end point. On univariate analysis, baseline sFas levels were significantly associated with the occurrence of cardiovascular end points, whereas sFas-L levels were not. Using Cox proportional hazards, increased sFas levels were associated with a significantly greater risk for cardiovascular end points (P = 0.03). This effect was independent of baseline CVD history, classic risk factors for atherosclerosis (diabetes, hypercholesterolemia, hypertension, and smoking), and markers of inflammation (C-reactive protein [CRP], soluble intercellular adhesion molecule-1). Increased CRP levels also were associated with cardiovascular end points (P = 0.04). In addition, increased cardiovascular mortality was found in patients in the highest sFas tertile compared with those in the lowest tertile (27.8% versus 8.6%; P = 0.04). CONCLUSION Increased plasma sFas levels are predictive of future CVD. These results suggest that sFas is a novel and independent predictor of active atherosclerotic disease in patients with ESRD.

Enhanced development of caspase-independent cortical cell death during cold storage in kidneys of non-heart-beating donors.

Background. Understanding the mechanisms of injury associated with cardiac arrest is essential for defining strategies aimed at improving preservation and function of kidneys harvested in non-heart-beating (NHB) donors. Methods. We standardized a model of NHB donors in rats and studied the kinetics and types (apoptosis vs. necrosis) of renal cell death developing during cold storage. Using quantitative polymerase chain reaction, immunoblotting, and caspase inhibition, we also studied the molecular pathways regulating renal cell death in this model. Results. The kinetics and extent of cell death developing in cortical tubules during cold storage were found to be increased in non-heart-beating (NHB) kidneys. Apoptosis of cortical tubules predominated in NHB kidneys exposed to 10 hr of cold storage, whereas necrosis increased after longer periods of cold ischemia. Shortly after cardiac arrest, a rapid up-regulation of Bax and Hsp 70 was found at the protein level in NHB kidneys. After 24 hr of cold storage, induction of Bax was maintained, whereas protein levels of Hsp70 returned to levels comparable to heart-beating (HB) controls. Also, mRNA levels of Bax were found to increase during cold storage in NHB kidneys. Cortical cell death was found to be largely caspase-independent but responsive to hydroxyl-radical scavenging with dimethyl sulfoxide (DMSO). Conclusions. Cardiac arrest promotes activation of death-inducing molecules such as Bax and is associated with increased development of caspase-independent renal cell death during cold storage. Developing strategies, such as free radical scavenging, aimed at inhibiting cell death during cold storage, could prove useful for improving preservation of NHB kidneys.

Magnesium handling by the papilla of the young rat

In a recent study it was found that in Mg loaded rats, the fraction of filtered Mg (% E Mg) recovered in the bend of the loop of Henle of papilla was greater than the filtered load. However, the site of this Mg addition was unspecified and could be either the juxtamedullary proximal tubule, the pars recta, or in the papilla, the descending limb of the loop of Henle.In order to investigate the movement of Mg in the various structures of the papilla, we have studied: 1. The transport of this electrolyte along the collecting duct. 2. Its relative concentration in the loop of Henle and in the adjacent vasa recta. The experiments have been performed in hydropenic and Mg loaded rats.In the collecting duct, the inulin and Mg concentrations increase proportionally, indicating an absence of any transport of Mg along this part of this nephron.In the vasa recta of the accessible papilla, the capillary over peripheral plasma Mg ratio (C/UF Mg) in hydropenia and after Mg loading [1.88±0.15 (ES) and 2.89±0.24] were significantly lower than the corresponding TF/UF Mg in the adjacent loops of Henle (2.90±0.17 and 4.04±0.37). This finding reduces the possibilities of a Mg passive diffusion from the capillaries to the tubular lumen, unless the electrical potential of the descending limb is more negative than −5 mV. The hypothesis of an active secretion, or a passive diffusion of Mg in the deep proximal tubule, in the pars recta, or in the early non accessible descending limb constitutes the other alternative.

A new fluorometric method for determination of picomoles of inorganic phosphorus. application to the renal tubular fluid.

Abstract A new method for the determination of inorganic phosphorus (Pi) in 4 nl of renal tubular fluid is described. Phosphate is converted to hexadimolybdatophosphate, which is reacted with thiamine to produce a highly fluorescent thiochrome. The reaction is stable and reproducible. Problems of interference are minimal. Microdeterminations of Pi in the proximal tubules and glomerular filtrates of the rat yielded results similar to those published in the literature.

Micropuncture Study of Phosphorus Transport in Genetic Hypophosphatemic Mice

Altough hypophosphatemic vitamin D-resistant rickets was first described by Albright and coll. in 1937 (1) the pathogenesis of this disease is still unknown. Two opposing hypothesis exist regarding the primary defect responsible for the constant hypophosphatemia. The first suggests a faulty intestinal absorption of calcium with secondary hyperparathyroidism, whereas the second places the initial responsibility on a defect of renal transport of phosphorus. The measurement of serum immunoreactive PTH has been found normal or low by Rook and coli (9), and by Arnaud (2), slightly increased by Lewy (7), and definitely increased by Reitz (8). Since the hyperparathyroidism is not a constant finding and when present, it seems not pronounced enough to explain the biological trait of the disease the hypothesis of a primary renal defect seems more probable.