Organ Culture: Impact On Agonist- And Hypoxia-Induced Responses Of Porcine Inferior Pulmonary Veins

A Arnold, H Widmer, I Rowe, SF Cruickshank. Robert Gordon University, Aberdeen, Scotland, UK

Hypoxaemia in human inferior pulmonary veins (IPVs) has been positively correlated with body mass index suggesting a role for IPVs in obesity-related disorders including sleep apnoea(1). Despite an established link between metabolic syndrome (MS) and pulmonary venous hypertension (PVH)(2) the direct effect of MS and obesity on IPV function is unclear. Blood vessel culture may be used to investigate the effects of a range of conditions (e.g. high glucose) on vessel function. However organ culture itself affects contractile function in blood vessels including pulmonary arteries(3). The effect of vessel culture on IPV function remains to be established.

Adult porcine lungs (Sus scrofa domesticus) of either sex were obtained from a local abattoir and 4th- 5th order IPVs were dissected free (mean external diameter 4.0±0.6 mm, n=10-11). Vessels were used either fresh (FIPVs) or after 24 hours in culture (CIPVs). For organ culture, unpressurised IPVs were placed in Eagle Minimum Essential Medium supplemented with 2% penicillin-streptomycin in 6-well plates and kept in a humidified incubator at 37oC under 95%air/5%CO2. Myography methods have been described elsewhere(4). Bath solutions were gassed with 95%O2/5%CO2 (hyperoxia) or 95%N2/5%CO2 (hypoxia). All drugs were applied cumulatively. Concentration ranges used were 10-7-3x10-5M for 5HT, 10-9-10-4M for histamine, 10-8-10-5M for PGF2 α and 10-11-10-6M for U46619. KCl, histamine, 5HT and carbamoylcholine were dissolved in aqueous solution, PGF2 α in ethanol and U46619 in DMSO. Data are expressed as % tension change (normalised to average final KCl response in FIPVs) and presented as mean ± S.E.M. Statistical significance was determined at P<0.05 using Student’s unpaired t test or 2-way ANOVA with Bonferroni’s post-test.

Maintaining IPVs in culture for 24 hours increased KCl-induced contractions by 35% (CIPVs= 5.0±0.6 (n=10); FIPVs= 3.7±0.4g (n=11); P<0.001). Similar relaxation with 10µM carbamoylcholine following constriction with 2µM PGF2 α suggested comparable endothelial function. Spontaneous contractions were seen more commonly in CIPVs than FIPVs (59% and 41%, respectively). CIPVs showed increased histamine-induced contraction over the concentration range 10-6-10-4M (P<0.001; n=6-7). An increased contraction in CIPVs vs. FIPVs to U46619 only occurred at the highest concentration (10-6M) (P<0.05; n=7) but no effect was seen in 5-HT induced contractions (P>0.05; n=5-7). Hypoxia produced contraction in 100% and 90% of FIPVs and CIPVs, respectively. The AUC for hypoxic contraction was increased in CIPVs compared to FIPVs (2324±406 (n=10) and 970±60 (n=9), respectively; P<0.0001).

Previous research suggests blood vessel culture mimics chronic hypoxia(3) and in the rat pulmonary vein, chronic hypoxia has been shown to blunt agonist-induced contraction(5). Here we report that 24 hours organ culture enhanced IPV contractility to a range of stimuli, including hypoxia. The effect of organ culture on contractile function should be considered when using the technique for IPV studies.

(1) Yamane T et al, Am J Respir Crit Care Med 178:3, 2008

(2) Robbins IM et al, Chest 136:1, 2009

(3) Manoury B et al, Br J Pharmacol 158:3, 2009

(4) Dospinescu C et al, Am J Physiol Lung Mol Physiol 303:5, 2012

(5) Zhao Y et al, Resp Physiol 100:1, 1995