Effects of mechanical stress and high glucose on pericyte proliferation, apoptosis and contractile phenotype Elena Beltramo * , Elena Berrone, Sara Giunti, Gabriella Gruden, Paolo Cavallo Perin, Massimo Porta Department of Internal Medicine, University of Turin, Corso AM Dogliotti 14, I-10126 Turin, Italy Received 15 March 2006; accepted in revised form 12 May 2006 Available online 5 July 2006 Abstract Pericyte loss is an early step of diabetic retinopathy. High glucose induces apoptosis in retinal pericytes, but systemic and capillary hypertension are also believed to be important in the onset and progression of diabetic retinopathy. The haemodynamic insult of retinal capillary hypertension can be mimicked by exposing pericytes to mechanical stretch. We investigated the effect of stretch combined with high glucose on pericyte proliferation/apoptosis and morphology. Bovine retinal pericytes, cultured in either normal or high glucose concentrations in flexible-base plates, were exposed to mechanical stretch for 48/72 h. Cell replication was determined by both cell counting and DNA synthesis, apoptosis by ELISA, cell morphology and actin cytoskeleton distribution by immunofluorescence. Both reduction in cell proliferation and increase in apoptosis were confirmed in high glucose alone. When cells were subjected to stretch, proliferation was reduced and apoptosis increased in both normal and high glucose in comparison with unstretched controls. In both cases, a synergistic effect of hyperglycaemia combined with stretch was shown. Cell morphology showed modifications of cytoskeleton in all experimental conditions; in particular, cells subjected to stretch showed a clear elongation and translocation of actin fibres. In conclusion, our results show that stretch, alone or combined with high glucose, reduces cell pro- liferation, increases apoptosis and induces morphological changes in pericyte cytoskeleton. Further elucidations of the mechanisms on the basis of reduced proliferation of pericytes subjected to high glucose and stretch could help to clarify the effects of combined hyperglycaemia and hypertension in the pathogenesis of diabetic retinopathy. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: pericytes; mechanical stress; high glucose; replication; apoptosis; cytoskeleton; diabetic retinopathy 1. Introduction Diabetic retinopathy (DR), the main cause of blindness among adults of working age in the developed countries, is characterised clinically by the development of proliferative DR (PDR) and macular edema (Aiello et al., 1998). Clinical studies have conclusively demonstrated that hyper- glycaemia is the primary pathogenetic factor in the develop- ment of DR. In addition, there is evidence that systemic hypertension also contributes to both the pathogenesis and the progression of DR (Klein et al., 1998; Klein and Klein, 2002; Wan Nazaimoon et al., 1999) and anti-hypertensive therapy has been shown to reduce the risk of clinical compli- cations from diabetic eye disease (Matthews et al., 2004). Pericyte loss is one of the earliest changes that take place in the retinal capillaries in DR. Ultrastructural analysis of retinal microaneurysms reveals a consistent dropout of pericytes, which may render capillaries vulnerable to microaneurysms (Hammes et al., 2002). Moreover, in PDR there is a correlation between absence of pericytes and retinal neovascularization Abbreviations: DR, diabetic retinopathy; PDR, proliferative diabetic retinopathy; BRP, bovine retinal pericytes; FCS, fetal calf serum; BrdU, 5-bromo-2 0 -deoxyuridine; FITC, fluorescein-isothiocyanate; SMC, smooth muscle cells. * Corresponding author. Tel.: þ39 011 633 6487/4303; fax: þ39 011 663 4751. E-mail address: elena.beltramo@unito.it (E. Beltramo). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.05.008 Experimental Eye Research 83 (2006) 989e994 www.elsevier.com/locate/yexer