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J Thorac Cardiovasc Surg 2000;120:427
© 2000 The American Association for Thoracic Surgery

Letters to the editor

Osteopontin expression and calcium content in human aortic valves

Cambridge, United Kingdom, and Paris, France

To the editor:

The cause or causes of calcific aortic stenosis (CAS) remain unknown. A number of studies have suggested genetic factors, preexisting rheumatic inflammation, or osteopontin expression. ^1,2 Osteopontin is a calcium-binding glycoprotein ligand of CD44 surface receptor also present in fetal bone, teeth, and exocrine endothelial cells. The present study was undertaken to test the hypothesis that osteopontin and valve calcium content could be related to CAS.

We studied aortic valves from patients undergoing aortic valve replacement using monoclonal antibodies against osteopontin and -smooth muscle actin. In a prospective study, we evaluated 15 unselected patients aged 22 to 84 years (mean 58.4 years), of whom 10 were women, undergoing aortic valve replacement for valvular stenosis or regurgitation. Valve specimens were received in the operating room and preserved in ice during transportation; they were then stored at –70°C. A portion of the tissue was removed and fixed in aqueous Bouin solution for immunohistochemical study. Total calcium content (in micrograms per gram of dry tissue) was determined by 7N HNO₃ digestion and atomic absorption spectroscopy.

After fixation in Bouin solution, the samples were embedded in paraffin. Four-micrometer transverse sections cut on a microtome at room temperature were stained for osteopontin (MPIIIB10 from the Developmental Studies Hybridoma Bank, University of Iowa, diluted 1:100, and Actin 1A4 from Sigma Chemical Company, St Louis, Mo) with an ABC-peroxidase kit (Vector Laboratories, Burlingame, Calif) and counterstained with hematoxylin. Sections were evaluated without knowledge of the tissue osteopontin expression or calcium content. Ten revealed calcification. Of these, 5 had fibrous thickening with calcification; the remainder were calcific AS of the senile type, but the presence of thick-walled vessels suggested postinflammatory valve disease. Five patients with uncalcified valves had fibrotic disruption of cusp architecture, associated with neovessels with thick muscular walls and few mononuclear inflammatory cells, consistent with postinflammatory (rheumatic) valve disease. Calcium content was 3091 µmol/g dry tissue (SE = 1048 µmol/g dry tissue) versus 359 µmol/g dry tissue (SE = 297 µmol/g dry tissue) (Wilcoxon signed-rank ², P = .0004).

Osteopontin expression was present in 9 of 10 calcified valves and 1 positive fetal femur in a control subject. It was restricted to the cytoplasm of fibroblast-like spindle cells, with a paranuclear granular locoalization, probably in the Golgi area. No osteopontin expression was noted in the 5 patients with uncalcified valves (² = 8.02, P < .005). Mohler and associates ¹ reported indentification of osteopontin in CAS (n = 5) and in noncalcified regurgitant valves (n = 3). Our results show that osteopontin and Ca²⁺ are directly associated with CAS, results differing from those of Mohler and colleagues. ¹ O’Brien and coworkers ² have shown that osteopontin messenger RNA can be produced by macrophages.

We thank Professor Philippe Menaché, Dr Philippe Chappuis of the Department of Biochemistry, and Dr Karen Jensen of the University of Iowa for their help in this study. The mouse osteopontin antibody was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, and the University of Iowa, Iowa City, Iowa, under contract NO1-HD-6-2915 from the National Institute of Child Health and Human Development (NICHD).

12/8/106968 doi:10.1067/mtc.2000.106968

References

1. Mohler ER, McClelland P, Adam LP, Hathaway DR. Identification of bone morphogenic protein-2 and osteopontin in calcified aortic valves. Circulation 1994;90:1-52. [Free Full Text]
   
2. O’Brien KD, Kuusisto J, Reichenbach DD, Ferguson M, Giachelli CM, Alpers CE, et al. Osteopontin is expressed in human aortic valvular lesions. Circulation 1995;92:2163-8. [Abstract/Free Full Text]
   

This Article Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): John H. Kennedy Permission Requests Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kennedy, J. H. Articles by Tedgui, A. Search for Related Content PubMed PubMed Citation Articles by Kennedy, J. H. Articles by Tedgui, A.