benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Calcinosis

Aortic valve stenosis characteristically progresses due to cuspal calcification, often necessitating valve replacement surgery. The present study investigated the hypothesis that TGF-beta1, a cytokine that causes calcification of vascular smooth muscle cells in culture, initiates apoptosis of valvular interstitial cells as a mechanistic event in cuspal calcification.. Noncalcified and calcified human aortic valve cusps were obtained at autopsy or at the time of cardiac surgery. The distributions within cusps of TGF-beta1, latent-TGF-beta1-associated peptide, and TGF-beta receptors were studied using immunohistochemistry. The effects of TGF-beta1 on mechanistic events contributing to aortic valve calcification were also investigated using sheep aortic valve interstitial cell (SAVIC) cultures.. Immunohistochemistry studies revealed that calcific aortic stenosis cusps characteristically contained within the extracellular matrix qualitatively higher levels of TGF-beta1 than noncalcified cusps. Noncalcified normal valves demonstrated only focal intracellular TGF-beta1. Addition of TGF-beta1 to SAVIC cultures led to a cascade of events, including: cellular migration, aggregation, formation of apoptotic-alkaline phosphatase enriched nodules, and calcification of these nodules. The time course of these events in the SAVIC culture system was rapid with nodule formation with apoptosis by 72 hours, and calcification after 7 days. Furthermore, ZVAD-FMK, an antiapoptosis agent (caspase inhibitor), significantly inhibited calcification and apoptosis induced by TGF-beta1, but had no effect on nodule formation. However, cytochalasin D, an actin-depolymerizing agent, inhibited nodule formation, but not calcification.. TGF-beta1 is characteristically present within calcific aortic stenosis cusps, and mediates the calcification of aortic valve interstitial cells in culture through mechanisms involving apoptosis.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Amino Acid Chloromethyl Ketones; Animals; Aortic Valve; Aortic Valve Stenosis; Apoptosis; Calcinosis; Caspase Inhibitors; Cells, Cultured; Disease Progression; Female; Humans; Immunohistochemistry; Male; Sheep; Transforming Growth Factor beta; Transforming Growth Factor beta1

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Topics: Amino Acid Chloromethyl Ketones; Anthraquinones; Apoptosis; Calcinosis; Calcium; Calcium Radioisotopes; Cell Division; Cells, Cultured; Coloring Agents; Cycloheximide; Electron Probe Microanalysis; fas Receptor; Humans; Immunoglobulin M; In Situ Nick-End Labeling; Ki-67 Antigen; Muscle, Smooth, Vascular; Protein Synthesis Inhibitors