Abstract
Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2, FN1, TGFB1, HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.
Highlights
Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot
We have detected a significant increase in the microvessel and arteriole density in the contracted M-side of the relapsed clubfoot tissue, which was connected to an increase in pro-angiogenic factors[8]
There was an increase in the percentage of the smooth muscle actin (SmActin) positive area (p = 0.0251), the TGF-β positive area (p = 0.0057), the hypoxia-inducible factor 1 alpha (HIF1A) positive area (p = 0.0071), the lysyl oxidase (LOX) positive area (p = 0.0221), the lysyl oxidase-like 2 (LOXL2) positive area (p = 0.0207), the tenascin C (TN-C) positive area (p = 0.0009), the MMP-2 positive area (p = 0.0097), the MMP-9 positive area (p = 0.0334) and the Fibronectin positive area (p = 0.0111) in the M-side compared to the L-side (Figs. 1, 2)
Summary
Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We have detected a significant increase in the microvessel and arteriole density in the contracted M-side of the relapsed clubfoot tissue, which was connected to an increase in pro-angiogenic factors[8] These pathways are identical with those described in other fibroproliferative and angioproliferative diseases, such as Dupuytren’s contracture and Peyronie’s d isease[9,10]. The findings can provide important information about pathologic conditions in clubfoot tissue and can help to unravel the etiology of clubfoot relapses. Such knowledge may contribute to better future outcomes and even to the development of therapeutic strategies
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