Apoptosis and expression of Bcl-2 family (Bcl-2 and Bax) mrna and protein associated with transforming growth factor-β1 (TGF-β1) signaling protein in canine myxomatous mitral valve disease

Abstract

Canine myxomatous mitral valve disease (MMVD) causes severe congestive heart failure. The pathogenesis mainly involves with atypical proliferation of valvular interstitial cells (VICs) and accumulation of mucopolysaccharides. There is a strong evidence of TGF-β1 signaling in naturally-occurring MMVD. Additionally, the cell proliferation is unlikely a major mechanism of increased cell density in canine MMVD. Thereby, monitoring of TGF- β1 signaling related to anti-apoptotic and apoptotic responses of VICs in canine MMVD may play a beneficial role in determining the pathogenesis. This study presented that age and valve thickness of dogs affected MMVD were statistically significant difference when compare with normal dogs. TUNEL and immunohistochemical assays were applied to detect apoptotic marker (TUNEL), TGF- β1 signaling protein (p-Smad2/3), cleaved caspase-3, pro-apoptotic (Bax) and anti-apoptotic protein (Bcl-2) in VICs of normal and MMVD valves. This study showed that MMVD valves had down-regulated level of p-Smad2/3 and Bcl-2, whereas the level of Bax was up-regulated compared to normal valves. Cleaved caspase-3 remained unchanged. There were statistically significant correlation between valve thickness and expression of TUNEL, p-Smad2/3, Bcl-2 and Bax. Furthermore, mRNA expression of bax and bcl-2 was used to determine the fold change and ratio of bax to bcl-2. There was not statistically significant different between bax and bcl-2 mRNA expression and stage of MMVD. The ratio of bax to bcl-2 tends to decrease with the late staged MMVD. In conclusion, the TGF β1-Smad signaling pathway is down-regulated in canine MMVD, which related to decrease in DNA damage in VICs. An imbalance of Bax and Bcl-2 may reflect the shift of pro-apoptotic and anti-apoptotic responses in MMVD. The VICs seem not to undergo apoptosis based on the constant expression of cleaved caspase-3 and the absence of apoptotic bodies in normal and MMVD valves. Further studies are warranted to specify the exact roles of apoptosis and anti-apoptosis involved in MMVD. This may lead to new strategies in the management of canine MMVD to prevent or stop the disease progression.