Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis Heidi A. Eriksen, Jari Satta, Juha Risteli, Mikko.

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Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis Heidi A. Eriksen, Jari Satta, Juha Risteli, Mikko Veijola, Päivi Väre, Ylermi Soini Atherosclerosis Volume 189, Issue 1, Pages 91-98 (November 2006) DOI: 10.1016/j.atherosclerosis.2005.11.034 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 In situ hybridization of aortic valves. (A) With a type I collagen antisense probe, clear signals (arrows) can be detected in fusiform cells around the calcified nodules (asterisk) of a calcified valve. (B) A negative control with a sense probe shows no signals in a calcified valve. (C) In a normal valve, no detectable signals are present for collagen type I mRNA. (D) For type III collagen mRNA, weak signals (arrows) can be seen in cells adjacent to degenerative calcified tissue (asterisk). Atherosclerosis 2006 189, 91-98DOI: (10.1016/j.atherosclerosis.2005.11.034) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Type I and type III collagen structures and total collagen content of the insoluble matrix. (A) The total collagen content assessed by hydroxyproline is expressed as per total protein content. (B) The amount of ICTP is expressed as moles per mole of collagen and (C) the amount of IIINTP as moles per mole of collagen. *p<0.001, †p<0.01. Atherosclerosis 2006 189, 91-98DOI: (10.1016/j.atherosclerosis.2005.11.034) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Size exclusion chromatography of (A) one control, (B) the SOFT, and (C) the CALC fraction of one AS sample. Trivalently cross-linked ICTP is indicated by open circles, and the elution position is marked with bars. The relative SP4 immunoreactivity (closed circles) at the position of ICTP elution, was about two-fold in the healthy sample compared to the SOFT or CALC fractions. The SP4 assay revealed a shoulder in the ICTP peak (big arrow) that was absent in both the mineralized and the non-mineralized fractions of the AS sample. The divalently cross-linked and non-cross-linked C-terminal telopeptide structures present in both SOFT and CALC fraction of AS sample are indicated by small arrow. Atherosclerosis 2006 189, 91-98DOI: (10.1016/j.atherosclerosis.2005.11.034) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Effect of age in healthy control samples. (A) ICTP decreased over age (r=−0.908, p<0.001). (B) The SP 4/ICTP ratio correlated positively with age (r=0.538, p<0.01). (C) IIINTP had correlated negatively with age (r=−0.753, p<0.001). Atherosclerosis 2006 189, 91-98DOI: (10.1016/j.atherosclerosis.2005.11.034) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Immunohistochemical stainings of the aortic valves. (A) In a calcified valve, PINP immunoreactivity (arrows) can be detected adjacent to the calcified nodules (asterisk). (B) In a control valve, no PINP immunoreactivity is present. (C) In ICTP staining of a calcified valve, strong immunoreactivity (arrows) is present around the calcific nodules (asterisk). Immunoreactivity is linear and irregularly present. (D) In a normal valve, linear immunostaining is seen with ICTP. (E) Strong immunoreactivity (arrows) for PIIINP can be detected in areas adjacent to the calcific nodules (asterisk), suggesting either synthesis of type III collagen or presence of type III pN-collagen in the diseased valve. (F) In IIINTP immunostaining of calcified valves, immunoreactivity is linear and not so clearly concentrated around the calcified nodules (asterisk). Atherosclerosis 2006 189, 91-98DOI: (10.1016/j.atherosclerosis.2005.11.034) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions