Characterization of the extracellular matrix metabolism in normal mitral valves and chronic valve disease (CVD) (syn. Endocardiosis) in dogs
Morphology, immunohistochemistry and mRNA analyses
Chronic heart valve disease (CVD) in dogs and the mitral valve prolapse (MVP) in man have similar characteristics but some clinical and pathological differences have been noted. The pathogenesis of CVD in dogs is still unclear but alterations of the extracellular matrix (ECM), enzymatic imbalances and atypical differentiation of valvular stromal cells (VSC) are discussed. The present study characterized the composition and distribution of the ECM components, the expression patterns of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the expression of different isoforms of the transforming growth factor-v (TGF-v). This was performed in mitral valves (MV) of unaffected dogs, as well as in dogs suffering from abnormal mitral valves due to CVD. MVs of 85 dogs (normal (n=22); mild (n=26), moderate (n=21), severe (n=16) CVD) were investigated macroscopically, histologically (H.-E., picrosirius red) and immunohistochemically (collagen I, III, VI, laminin, fibronectin, MMP-2,-9,-14, TIMP-2,-3, TGF-v1,-v2,-v3, actin). Samples from normal (n=15) or diseased (n=10) canine MVs were subject to real-time polymerase chain reaction (PCR) for quantification of mRNA encoding MMP-2,-9,-14 and TIMP-2,-3. In the normal MV, ECM components were expressed in a typical layered pattern. MMP-2,-9,-14, and TIMP-2 were detected in single VSC. TIMP-3 showed moderate intra-and extracellular expression. The mRNA encoding MMP-2,-9 and-14 and TIMP-3 was low. The VSC showed no expression of actin but displayed mild expression of TGF-v1 and TGF-v2 and moderate expression of TGF-v3. Advanced CVD was characterized by myxomatous nodular lesions, displaying a marginal and a central region containing mainly collagen I, VI and fibronectin in the former and collagen I and III in the latter. Immunohistochemical labelling intensity of MMP-2 and MMP-9 decreased significantly. However, mRNA values encoding MMP-2 and-9 were not decreased. In contrast, immunohistochemical MMP-14, TIMP-2 and TIMP-3 expression was increased. This was confirmed by significantly increased transcription of mRNA encoding MMP-14, TIMP-2, and TIMP-3. The activated subendothelial VSCs strongly expressed actin, TGF-v1 and-v3. Inactive VSCs within the centre of the nodules had significantly less labelling for TGF-v1 and-v3. In conclusion, in canine CVD an accumulation of proteoglycans and a distinctly altered expression of ECM components were detected. MMPs and TIMPs are involved in ECM metabolism in normal canine MV. During CVD, a decreased expression of proteolytic MMPs and an increased expression of profibrotic TIMPs probably resulted in a depressed metabolism and an accumulation of ECM components. These processes are regulated by TGF-v isoforms which also induce myofibroblast-like differentiation of VSC. Changed haemodynamic forces and expression of MMPs may in turn regulate TGF-v expression.