Herta M. Crauwels

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta.

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′‐[γ‐thio] triphosphate (ATPγS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4‐deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53±0.07) was compatible with literature, but the pKb for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non‐P2Y2 receptor subtype(s). Further, pyridoxal‐phosphate‐6‐azophenyl‐2′‐4′‐disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2′‐deoxy‐N6‐methyladenosine3′,5′‐bisphosphate (MRS2179) inhibited ADP‐evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP‐evoked vasodilatation.

Heterogeneity in relaxation mechanisms in the carotid and the femoral artery of the mouse

The participation of prostanoids, nitric oxide and non-prostanoid non-nitric oxide factors in endothelium-dependent relaxations was investigated in phenylephrine (PE)-constricted carotid and femoral arteries of C57BL6 mice. The carotid artery was more sensitive to acetylcholine as compared to the femoral artery, and cyclooxygenase inhibition did not influence the relaxation in either vessel. In the carotid artery, high doses of acetylcholine caused transient constrictions, which were abolished by indomethacin or piroxicam. In the carotid but not the femoral artery, N(omega)-nitro-L-arginine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced PE-induced contractions enormously, suggesting that endogenous nitric oxide production is much higher in the carotid artery. While in the carotid artery all relaxation was abolished by N(omega)-nitro-L-arginine or ODQ, a residual response (34+/-5% and 74+/-4%, respectively) but with a different shape, was maintained in the femoral artery. This N(omega)-nitro-L-arginine-resistant relaxation was abolished by the combination of apamin and charybdotoxin. In both arteries, ODQ abolished relaxation to S-nitroso-N-acetyl-D-penicillamine, while N(omega)-nitro-L-arginine enhanced the sensitivity to this donor of exogenous nitric oxide. In 30 mM KCl, the relaxation to acetylcholine was abolished by N(omega)-nitro-L-arginine or ODQ in either artery. In conclusion, in the carotid artery endothelium-dependent relaxation is mediated predominantly by nitric oxide acting via cyclic GMP-dependent pathways, while in the femoral artery part of the relaxation can be attributed to a non-prostanoid non-nitric oxide factor operating via apamin/charybdotoxin-sensitive potassium channels.

Transcription Profiles of Aortic Smooth Muscle Cells from Atherosclerosis-Prone and -Resistant Regions in Young Apolipoprotein E-Deficient Mice before Plaque Development

Background/Aims: Site-specific atherosclerosis is generally attributed to differential gene expression in endothelial cells. We investigated whether the transcriptome of smooth muscle cells is different between atherosclerosis-prone and atherosclerosis-resistant regions in apolipoprotein E-deficient (apoE–/–) mice before plaque development, and in C57Bl/6 mice. Methods: De-endothelialized aortas (both strains: 3 males, 3 females, age 4 months) were divided into atherosclerosis-prone (AA: ascending aorta, aortic arch and proximal 2 mm of thoracic aorta) and -resistant (CTA: central thoracic aorta, i.e. 6 mm distal from the proximal 2 mm) regions. The transcriptome of these two regions was compared using whole-genome mouse microarrays. Results: Microarray analysis revealed differential expression (>2-fold difference) of 70 and 244 genes in C57Bl/6 and apoE–/– mice. This was confirmed for 6 genes using the real-time quantitative polymerase chain reaction. Up- or downregulation in the AA was observed for 33 and 37 genes in C57Bl/6, and for 186 and 58 genes in apoE–/– mice, respectively. The 201 genes that showed exclusively differential expression in apoE–/– mice were related to atherosclerotic processes, such as cell adhesion, proliferation, differentiation, motility, cell death, lipid metabolism and immune responses. Conclusion: Our findings indicate that smooth muscle cells display an altered transcriptome at atherosclerosis-prone locations before actual lesion development.

Local application of advanced glycation end products and intimal hyperplasia in the rabbit collared carotid artery

OBJECTIVE Accumulation of advanced glycation end products (AGEs) in the vessel wall has been implicated in atherogenesis. The aim of our study was to examine the effects of local application of glycated bovine serum albumin (AGE-BSA) on collar-induced intimal hyperplasia in a diabetes-free setting. METHODS Intimal thickening was induced by placing a collar around the carotid artery of rabbits. Via a catheter attached to an osmotic minipump, control BSA or AGE-BSA was administered locally to the vessel wall in a dose of 1.5 or 15 microg h(-1) during 14 days. Vessels receiving phosphate buffered saline (PBS, 5 microl h(-1)) were used as controls. RESULTS Infusion of AGE-BSA 15 microg h(-1) significantly enhanced intimal thickening as compared to control BSA or PBS. Positive remodelling, measured as an increase in the area comprised by the external elastic lamina and preservation of lumen size, was only significant after treatment with the higher dose of AGE-BSA. In all other groups, intimal thickening was accompanied by a decrease of the lumen without outward displacement. Infusion of control BSA or AGE-BSA changed the cell composition of the neointima, with a significant enhancement in the number of T-lymphocytes and macrophages and a reduction in the percentage of intimal area occupied by smooth muscle cells. These effects were however similar for control BSA as well as AGE-BSA. CONCLUSIONS It is concluded that infusion of control BSA or AGE-BSA may aggravate collar-induced intimal thickening by evoking an inflammatory response. This supports the concept that inflammation contributes to atherogenesis. Further, the significant enhancement in intimal hyperplasia by AGE-BSA suggests that glycated proteins provide an additional stimulus for the development of atherosclerotic lesions.

Abstract no.: 6 Endothelium-dependent relaxation by purinergic receptors in the aorta of apolipoprotein E-deficient mice

Previously we reported that the acetylcholine‐induced relaxation in the isolated aorta of apolipoprotein E‐deficient (apoE‐/‐) mice deteriorates after the development of atherosclerotic plaques, but remains normal in adjacent, plaque‐free segments. The present study investigated the presence of functional purinergic receptors in the murine aorta, and whether their function changes before or after the development of atherosclerosis. Endothelium‐dependent relaxation was measured in aorta segments of apoE‐/‐, C57BL6 (WT) and human apoAI‐overexpressing apoE‐/‐ mice (apoAI/apoE‐/‐) on regular chow. Rings were isometrically contracted with phenylephrine to 50% of their maximum force before performing cumulative concentration‐response curves to different nucleotides or their stable analogues. After the functional study, the cross‐sectional area of the plaque was determined in every segment. The nucleotides induced complete (UTP, UDP, ATP) or partial (ADP) relaxation that was abolished by endothelial cell removal or nitric oxide (NO) synthase inhibition. The responses pointed to the presence of functional P2y1, P2y2 or P2y4 receptors on endothelial cells. RT‐PCR confirmed the presence of P2y1 and P2y4 mRNA in the aorta of WT mice. Nucleotide responses were unaltered in lesion‐free apoE‐/‐ mice (5 months). However, in atherosclerotic segments of apoE‐/‐ mice (18 months), the relaxation to ATP was impaired compared to age‐matched WT controls (maximum amplitude (Emax) 25 ± 14%, n = 6 vs. 90 ± 3%, n = 5, P < 0.01). A similar defect was seen for the stable analogue ATP‐gamma‐S (Emax 36 ± 12% vs. 86 ± 3%, P < 0.01). Atherosclerotic apoE‐/‐ segments were less sensitive to the NO donor spermineNONOate (pD2 6.74 ± 0.18) than WT segments (7.25 ± 0.20), but maximum relaxation was unaltered. In non‐atherosclerotic aorta segments of the same apoE‐/‐ mice all relaxation responses remained normal and were not different from WT. Strong negative correlations (P < 0.001) existed between lesion size and the Emax for ATP (rs = −0.82) and ATP‐gamma‐S (rs = −0.73) in apoE‐/‐ mice. ApoAI overexpression improved the purinergic responses (Emax ATP 64 ± 9%, ATP‐gamma‐S 64 ± 10%, n = 5) and these were not different from WT (P > 0.05). An analysis of covariance with plaque size as covariate suggested that this benefit was secondary to the strongly reduced plaque formation in apoAI/apoE‐/‐ mice. It is concluded that functional P2 y receptors are present on murine aortic endothelium. Furthermore, endothelium‐dependent purinergic relaxation declines after plaque development. This deterioration involves decreased bioavailability of NO rather than enhanced ATP degradation. The defect is, however, not systemic since the responses remain unaltered in plaque‐free segments of atherosclerosis‐prone apoE‐/‐ mice.

Abstract no.: 5 Endothelium‐dependent relaxation and impact of a Western type diet in the aorta of apolipoprotein E‐deficient mice

Previously we reported that even in old (18 months) apolipoprotein E‐deficient mice (apoE‐/‐) on regular chow (RC), all nitric oxide (NO)‐mediated responses are preserved in plaque‐free vessels. Only when plaques develop, localised vasomotor dysfunction occurs. The impact of increased oxidative stress on relaxations was evaluated by feeding apoE‐/‐ mice either a Western type diet (WD; 0.15% cholesterol, 41% fat) or RC (no cholesterol, 4% fat) for 8, 16 and 24 weeks. The aorta was systematically sectioned and segments of the aortic root (1 mm) and the thoracic aorta (5 × 2 mm, covering whole vessel, varying degrees of atherosclerosis) were studied in organ baths followed by morphometry. The WD enhanced plasma levels of total cholesterol (1382 ± 109 vs. 425 ± 21 mg/dl) and soluble ICAM‐1 (μg/ml; 8 weeks 43 ± 3 vs. 28 ± 1; 16 weeks 37 ± 2 vs. 25 ± 1; 24 weeks 41 ± 4 vs. 29 ± 1). In RC mice, relaxations to exogenous NO and acetylcholine (ACh) were preserved in all segments up to 24 weeks. When fed the WD however, maximum relaxation (Emax) to exogenous NO (acidified NaNO2) was impaired in the aortic root at 8 (83 ± 3 vs. 92 ± 1%), 16 (71 ± 4 vs. 87 ± 3%) and 24 weeks (59 ± 7 vs. 83 ± 1%); shifts in the pD2 values pointed to decreased sensitivity. ACh‐relaxation was unaltered at 8 weeks, but deteriorated in the aortic root at 16 (74 ± 6 vs. 99 ± 1%) and 24 weeks (51 ± 6% vs. 95 ± 3%). Similar changes occurred in the most distal thoracic aorta segment of WD mice. However, in all those segments plaques were detectable, and average lesion size was increased 6‐fold by the WD. Furthermore, responses to ACh (Emax rs = −0.54; pD2 rs = −0.36, P < 0.001) and exogenous NO (Emax rs = −0.59; pD2 rs = −0.30, P < 0.01) inversely correlated to plaque size in WD mice, as previously reported for RC apoE‐/‐ mice. The dysfunction was apparent only after plaque development. Indeed, segments of the central thoracic aorta were largely free of atherosclerosis and showed unaltered relaxation, even after the onset of atherogenesis at 24 weeks WD. In conclusion, the elevation of soluble ICAM‐1 points to enhanced oxidative stress in WD apoE‐/‐ mice. Yet, endothelium‐dependent relaxation is still maintained until plaques develop. As opposed to the human situation, endothelial dysfunction in hypercholesterolemic apoE‐/‐ mice is not a systemic but a local process, strictly associated with lesion formation. Finally, the preservation of vasomotor responses in plaque‐free segments, and the correlation between lesion size and dysfunction, further emphasise that it is crucial to document plaque dimensions in vasomotor studies in atherosclerosis research.