Apoptosis and abundance of Bcl-2 family and transforming growth factor β1 signaling proteins in canine myxomatous mitral valves.

Abstract

OBJECTIVES: To determine the percentage of cells undergoing apoptosis within canine myxomatous valves and to evaluate whether TGFβ1 can be implicated as an anti-apoptosic signal through the Bcl-2 family of signaling proteins. ANIMALS: Post-mortem mitral valve leaflets harvested from 5 normal dogs, 5 dogs with early-stage myxomatous mitral valve disease (MMVD), and 5 dogs with late-stage MMVD. MATERIALS AND METHODS: The number of cells expressing cleaved caspase-3, DNA fragmentation (TUNEL marker) and apoptotic bodies were evaluated as a measure of apoptosis. To evaluate the relationship between TGFβ1 signaling and apoptosis, the abundance of activated TGFβ1 signaling protein, phosphorylated Smad 2/3 (p-Smad 2/3), and Bcl-2 family proteins (pro-apoptotic Bax and anti-apoptotic Bcl-2) was determined by immunohistochemistry. RESULTS: Cells in normal and both stages of MMVD expressed the TUNEL marker and cleaved caspase-3, but not apoptotic bodies. The percentage of TUNEL marker and cleaved caspase-3 positive nuclei was not significantly different between groups of dogs (p > 0.05). P-Smad 2/3 and Bax were more abundant in myxomatous mitral valves while Bcl-2 was less abundant. P-Smad 2/3 primarily increased in the atrialis layer and was abundantly increased only in late-stage MMVD. CONCLUSIONS: These data suggest that interstitial cells in MMVD are in a pro-apoptotic condition; however, they do not execute apoptosis. Thus, apoptosis does not explain differences in cellular density in canine MMVD. TGFβ1 signaling through the canonical SMAD pathway is increased in myxomatous mitral valves, but does not apparently mediate interstitial cell apoptosis in canine MMVD.