2015 ISAKOS Biennial Congress ePoster #301

Acid-Sensing Ion Channel in Healthy and Injured Human ACL

Antonio Maestro, MD, PhD, Gijon, Asturias SPAIN
Jose A. Vega, PhD, Oviedo SPAIN
Luis Rodriguez, MD, Gijon SPAIN
Olivia Garcia Suarez, PhD, Oviedo SPAIN
Angel Gago, MD, Oviedo SPAIN
Ivan Pipa Muñiz, MD, Gijon SPAIN
Nicolas Rodriguez Garcia, MD, El Berron, Asturias SPAIN

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

FDA Status Not Applicable

Summary: The occurrence and distribution of ASIC2, 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 ASIC2, with is gated by pH and mechanical forces, is involved in the biological mechanisms underlining the rupture of ACL, due to mechanical or inflammatory processes. The expression of ASIC2

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Abstract:

Introduction

In recent years several ion channels have been identified as responsible for detecting a range of thermal, chemical and mechanical stimuli. In particular, the acid-sensing ion channels (ASICs) are H+-gated voltage-insensitive, amiloride-sensitive ion channels belonging to the degenerin/epithelial Na+ channel (DEG/ENaC) superfamily of cation channels. They monitor moderate deviations from the physiological values of extracellular pH, but also participate in mechanoception and nociception. All these mechanisms, especially mechanoreception and pH detecting, are also present in fibroblasts but they are poorly understood, and no data exist in fibroblasts from joint ligaments, in spite of the special mechanical roles of these structures.
Objectives: Since ASIC2 is expressed in a broad range of musculoskeletal tissues where it might play chemosensory or mechanosensory functions, we try to know its role on ACL injury.

Methods

the occurrence and distribution of TRPV4, at the mRNA and protein levels, in the healthy and injured human anterior cruciate ligament was investigated. The research was aimed to better known whether or not ASIC2 participates in the biological 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 sequence was based upon the published sequence for Homo sapiens ASIC2 - Gene Bank accession number NM_214788-, and was: sense 5'-ttgttggccagcttagtcct-3', reverse 5'-gggtcggtagttagtgaagt-3'), Westernblot and immunohistochemistry (anti-ASIC2 rabbit polyclonal antibody, conjugated to an immunogenic carrier protein). Results: ASIC2 mRNA was detected in normal tissue and was up-regulated five-folds in injured ACL. Westernblot in both normal and pathological tissues identified two protein bands: a robust band of with a molecular weight of ~72 kDa and a faint band of ~68 kDa, both consistent with ASIC2, but we cannot determine what ASIC2 isoform it detects. In healthy tissues immunohistochemistry revealed presence of ASIC2 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 ASIC2 positive cells significantly increased in the injured ligaments, and the increase of ASIC2 positive chondrocyte-like cells throughout the ligament was noticeable. Also the intensity of the immunostaining in the blood vessel walls was augmented. All together these results provide evidence for a regulatory role for ASIC2 in human anterior cruciate ligament, and its involvement in the injury of ACL.

Conclusions

present results strongly suggest an up-regulation of ASIC2 in response to mechanical stress and changes in local pH due to partial ligament disruption. Moreover a sensory role, especially mechanosensory, of ASIC2 can is suggested by its presence in nerves and blood vessels due to biomechanical or inflammatory processes.