Proceedings of The Physiological Society

University of Edinburgh (2011) Proc Physiol Soc 25, C21 and PC21

Oral Communications

Use of a novel in vitro technique to investigate needle-induced vascular injury

P. Coats1, S. Humza1, D. Kingsmore2, R. M. Wadsworth1

1. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom. 2. Vascular Surgery, Western Infirmary, Glasgow, United Kingdom.

The arteriovenous fistula (AVF) undergoes a structural adaptation which in the long term does not favour patency (Ross, 2005). AVF failure is attributed primarily to luminal occlusion due to neointimal hyperplasia (Maya et al, 2004). The arterial wall response to injury and the relationship between inflammation and the cardiovascular system has not been considered in this type of vascular disease. To date there is no clear mechanistic understanding of the neointimal hyperplasia that develops in the AVF. Specifically there is an absence of data of the cumulative effect of needle cannulation injury and its possible association with neointimal growth and consequent AVF failure. Thus the aim of this study was to identify the effect of repeated needle cannulation on vascular structure in our established in-vitro organ culture model. Aorta were dissected from Male, Sprague-Dawley rats, (12 weeks, 250-300g) under tissue culture conditions and cultured in growth medium 50:50 (V:V) mix of Ham’s nutrient mix F12 (Gibco), Waymouths MB 752/1 (Gibco), 10% fetal bovine serum (FBS) (Biosera) and 1% pen-strep (Invitrogen). Intact aorta was cultured for 28 days under the following conditions:- cultured non-injured (control), cultured-injured and cultured-injured-drug treated (flurbiprofen; 10uM). Tissues were needle cannulated through the side wall following 24 hours culture and thereafter every 72 hours over the 28 day culture period. Vascular smooth muscle (VSM) cells were isolated and cultured for cell proliferation and western blot analysis. Planimetry of wax embedded and sectioned tissues using haematoxylin and eosin staining: Needle injury produced significant hyper-proliferative structural remodelling. Media: lumen ratios were 0.47 ± 0.033 and 0.64 ± 0.047 in the control and needle injured aortas respectively (n=6; p<0.05). The cyclooxgenase inhibitor (flurbiprofen) reduced needle injury-dependent hypertrophy by 18 ± 3.2% when compared with cultured non-injured tissues n=6, p<0.05). VSM cell proliferation assay confirmed flurbiprofen’s (10 uM)anti-proliferative effect on VSM cells reducing proliferation by 8-fold when compared with control (n=8; p<0.05). Western blot analysis, established that flurbiprofen (10 uM) reduces the phosphorylation of the MAP Kinase Erk-1/2. Presently there is no prophylactic intervention to postpone or reduce occlusive failure in the forearm AVF graft. We have identified for the first time that needle injury can evoke hyperproliferative adaption in vascular tissues. Moreover we have identified that cyclooxygenase inhibition can reduce this hyperproliferative response and this may provide insight to a potential therapeutic advance in promoting the functional survival of the AVF in patients undergoing haemodialysis.

Where applicable, experiments conform with Society ethical requirements