Effects of hypoxia and hyperosmosis on the expression of matrix metalloproteinases in broiler lung fibroblasts Ahmet Akkoc a , M. Mufit Kahraman a* , Mehmet Cansev b and M. Ozgur Ozyigit a a Department of Pathology, Faculty of Veterinary Medicine, University of Uludag, 16059 Gorukle, Bursa, Turkey b Department of Clinical Pharmacology, Faculty of Medicine, University of Uludag, 16059 Gorukle, Bursa, Turkey *E-mail: mufitk@uludag.edu.tr ABSTRACT This study investigated the effects of hypoxic and hyperosmotic stresses on the matrix metalloproteinases 2 and 9 (MMP-2, MMP-9) and their tissue inhibitors 2 and 1 (TIMP-2, TIMP-1) respectively in cultured lung fibroblasts from broiler chickens. Isolated and sub-cultured primary lung fibroblasts from 1-day-old broilers were exposed to hypoxia and hyperosmosis. The presence of the enzymes and the effects of hypoxia and hyperosmosis on the enzymes in cultured lung fibroblasts, and in the culture media, were evaluated with the streptavidin-biotin-peroxidase method and western blotting using commercially available primary monoclonal antibodies. Short (6 hours) and long term (12 hours) hypoxia, hypoxia þ hyperosmosis (6 hours) and hyperosmosis (6 hours) triggered the release of both MMP-2 and TIMP-1 in experimental groups when compared with their controls. Neither MMP-9 nor TIMP-2 antibodies stained the fibroblast cells kept in normoxic, hypoxic or hyperosmotic conditions. Keywords: broiler lung fibroblast, hypoxia, hyperosmosis, matrix metalloproteinases 1. INTRODUCTION Pulmonary hypertension syndrome (PHS), or Ascites Syndrome, is a common metabolic disorder asso- ciated with right ventricular failure and fluid accumu- lation in the abdominal cavity in fast-growing meat- type chickens. In the acute form of AS, broilers die without showing the classical signs of severe lung oedema (Julian, 1993, 2005). Although there is an important genetic component to susceptibility to PHS environmental factors, such as cold, high altitude and diet play significant roles in development of the syndrome (Julian, 2000). Such conditions impose difficulties on broilers in fulfilling tissue demands for oxygen. The close relationships between the increased oxygen demand, pulmonary hypertension and extra workload on the heart have also been investigated in AS (Julian, 1993, 2000; Hassanzadeh et al., 1997; Olkowski et al., 1999; Olkowski et al., 2005). The role of hypoxia, as a secondary cause, in the pathogenesis of PHS has been shown in both experimental studies and naturally occurring cases (Witzel et al., 1990; Wideman et al., 1997; Wideman and French, 1999; Wideman, 2001; Hassanzadeh et al., 2004; Julian, 2005). In experi- mental models, sodium chloride (salt) has been used to induce PHS in broiler chickens (Julian, 1987, 2005; Mirsalimi et al., 1992; Mirsalimi and Julian, 1993; Ozyigit et al., 2005). It has been postulated that the broilers have a more rigid lung with small, less expandable blood capillaries (Mirsalimi et al., 1993). Furthermore, large nucleated erythrocytes in birds treated with salt might cause a further increase in the resistance to blood flow and thus to the hypoxia (Mirsalimi et al., 1992; Mirsalimi and Julian, 1993). The contribution of high levels of sodium ions in the formation of increased interstitial fluid accumulation in the lung of broilers has also been reported (Julian et al., 1992). Primary (spontaneous) PHS is the result of ineffi- ciency of small lungs to cope with increasing oxygen demands of the tissues and organs in fast growing broiler chickens raised in low altitude. At high alti- tudes, hypoxia is thought to have a central role in the pathogenesis of avian ascites (Julian, 2005). Dramatic vascular adventitial changes, mostly of fibroblastic nature, have been documented during the vascular remodelling in hypoxia-induced pulmonary hyperten- sion in mammals (Siu et al., 1998; Stenmark et al., 2000; Das et al., 2002). Early and dramatic changes also occur in the extracellular matrix proteins and matrix metalloproteinase (MMP-2) expression during www.avaianbiologyresearch.co.uk doi: 10.3184/175815511X13000117548916 AVIAN BIOLOGY RESEARCH 4 (1), 2011 6–16