ROLE OF APOPTOSIS IN SUPRASPINATUS TENDINOSIS

Participants: R.E. Hughes, J. Carpenter, M. Moalli, B. Nolan, R. Taylor, C. Hadgis

Keywords: shoulder, tendon, overuse, apoptosis, hypoxia

Introduction

Rotator cuff tendon pathology is an important source of musculoskeletal morbidity, especially in athletic and working populations. Intrinsic and extrinsic factors have been hypothesized to cause rotator cuff disorders. Epidemiologic studies have linked repetitive physical exertions to rotator cuff disorders. Anatomic observations have demonstrated a hypovascular region of the supraspinatus tendon near its insertion. It has also been demonstrated that tension applied to tendon, which occurs during physical exertions, impairs the blood flow within tendon. Therefore, it has been speculated that local hypoxic conditions lead to tenocyte cell death.

Apoptosis has been shown to be induced in vitro by hypoxia (Stempien-Otero et al., 1999). Hypoxia-induced apoptosis may be a critical step in the development of supraspinatus tendinosis. The long term goal of this research is to investigate the role of apoptosis in the development of tendinosis in vivo. Studies of tendon hypoxia will be conducted in tissue culture prior to animal experimentation to determine whether hypoxic conditions induce apoptosis in the tissue of interest. There are four specific aims of this project: (1) to develop a tissue culture system for assessing the effect of mechanical loading and tissue hypoxia on tendon; (2) to determine if hypoxia induces apoptosis of rat supraspinatus tendon fibroblasts; (3) to determine if mechanical stress induces apoptosis in rat supraspinatus tendon fibroblasts; and (4) to determine if there is a synergistic effect of hypoxia and mechanical loading on apoptosis in rat supraspinatus tendon fibroblasts.

Materials and Methods

Supraspinatus tendons will be harvested from Sprague-Dawley rats and placed in culture. Tendons will be placed in normoxic, hypoxic, and mechanically loaded conditions. Apoptosis will be assessed using light microscopy and immunohistochemistry with antibodies to tissue transglutaminase. Fragmentation of DNA will be assessed using the TdT-mediated dUTP biotin nick end-labeling (TUNEL) assay. The project will be conducted in the cell culture and molecular biology facilities of the Orthopaedic Research Laboratory.

Progress

This project is an extension of the rat overuse model developed by Drs. Soslowsky and Carpenter. Proposals have been submitted to obtain funding for this project. The protocols and test systems area being established.

References

1. Stempien-Otero, A., Karsan, A., Cornejo, C.J., Xiang, H., Eunson, T., Morrison, R.S., Kay, M., and Harlan, J. (1999) Mechanisms of hypoxia-induced endothelial cell death. J. Biological Chemistry 274: 8039-8045.