2015 ISAKOS Biennial Congress ePoster #316

Expression of Putative Mechanoproteins in Human ACL: TRPV4

Antonio Maestro, MD, PhD, Gijon, Asturias SPAIN
Jose A. Vega, PhD, Oviedo SPAIN
Olivia Garcia Suarez, PhD, Oviedo SPAIN
Luis Rodriguez, MD, Gijon SPAIN
Gorka Vazquez, MD, Oviedo SPAIN
Ivan Pipa Muñiz, MD, Gijon SPAIN
Nicolas Rodriguez Garcia, MD, Gijón, Asturias SPAIN

University of Oviedo. FREMAP, Oviedo. Gijón., SPAIN

FDA Status Not Applicable

Summary: The occurrence and distribution of TRPV4, at the mRNA and protein levels, was analyzed in the healthy and injured human anterior cruciate ligament (ACL). The research was aimed to better known whether or not TRV4 can be involved in the biological mechanisms underlining the rupture of ACL, due to mechanical or inflammatory processes. The expression of TRPV4 mRNA as well as the number of TRPV4 posit

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Abstract:

Introduction

Mechanotransduction represents the cellular and extracellular mechanisms by which cells modulate the rates of matrix synthesis and degradation, and therefore their resistance to mechanical requirements. The mechanisms involved in mechanotransduction in fibroblasts are poorly understood, and no data exist in fibroblasts from joint ligaments, in spite of the special mechanical roles of these structures. The Transient Receptor Potential (TRP) superfamily of ion channels has revealed the potential mechanisms by which cells sense diverse stimuli identifying ion channels that are gated by mechanical loading, and other stimuli, that it is important for sensation of several physical and chemical stimuli. Objectives: Since TRP vanilloid 4 (TRPV4) is expressed in a broad range of musculoskeletal tissues where appears to play a mechanosensory role, we try to know its role on ACL injury. Methods: Here we have analyzed the occurrence and distribution of TRPV4, at the mRNA and protein levels, in the healthy and injured human anterior cruciate ligament. The research was aimed to better known whether or not TRV4 can be involved in the mechanisms underlining the rupture of ACL, due to mechanical or inflammatory processes. Healthy (n = 5; obtained from human donors obtained during removal of organs for transplantation) and injured (n = 10; obtained during surgery of the knee due to traumatic injury) ACL were used in the present study an processed for quantitative rt-PCR (the oligonucleotide primer was based upon the published sequence for Homo sapiens TRPV4 -GenBank accession number NM_021625.4-, and was: 5'-taccaagatgtacgacctgc-3', reverse: 5'- ggagaccacgttgatgtaga-3'), Westernblot and immunohistochemistry (anti-TRPV4 rabbit polyclonal antibody, against a synthetic peptide derived from the cytoplasmic N-terminus conjugated to immunogenic carrier protein). Results: TRPV4 mRNA was detected in normal tissue and was up-regulated four-folds in injured ACL. Westernblot in both normal and pathological tissues identified a protein with an estimated molecular weight of 100 kDa (predicted 98.28) consistent with TRPV4. In healthy tissues immunohistochemistry revealed presence of TRPV4 in fibroblasts and chondrocyte-like cells (at the ligament insertion borders), as well as in the blood vessel walls and nerve profiles supplying the anterior cruciate ligament. The number of TRPV4 positive cells notably increased in the injured ligaments, and the increase of TRPV4 positive chondrocyte-like cells throughout the ligament was noticeable. All together these results provide evidence for a regulatory role for TRPV4 in human anterior cruciate ligament, in addition to the control of other musculoskeletal Conclusions: Probably this function is modulating the production or influence of pro-inflammatory molecules in response to mechanical stress and partial ligament disruption. Moreover, a sensory role, especially mechanosensory, of TRPV4 is suggested by its presence in nerves, before (in the healthy) and in the injured ACL.