The burden of atherosclerosis and the frequency of resultant occlusive coronary artery disease (CAD) in the population are increasing together with an alarming increase in the incidence of metabolic syndrome and obesity in the industrialized world. Occlusive CAD places individuals at risk of myocardial infarction and heart failure. While substantial progress has been made in our understanding of the pathogenesis of atherosclerosis most models of diet-induced atherosclerosis in mice do not develop occlusive CAD like humans. Recently, Zhang et al (2005) reported on the development of a mouse model (SR-BI KO/ApoER61h/h) through exhibit diet-induced hypercholesterolemia through the breeding of mice deficient in the HDL receptor SR-BI with hypomorphic apolipoprotein E mice. The same authors have established that this new model displays the characteristic profiles of hypercholesterolemia, cardiac hypertrophy, spontaneous myocardial infarctions and progressive cardiac dysfunction leading to death within 8 weeks after initiating a high cholesterol and high folate diet. However, it has not been established whether this new model also develops endothelial dysfunction during the progression of CAD as found in humans.
In this proposal our aim is to investigate whether endothelial dysfunction contributes significantly to the pathogenesis of diet-induced occlusive CAD in SR-BI KO/ApoER61h/h male mice (referred to hereafter as HypoE mice). To do this we will use our recent experience in synchrotron contrast microangiography in mice to compare arterial vessel internal diameters (30-250 micrometres) and vessel number during administration of vasodilators before and after NOS/COX blockade in mice fed normal chow or a high fat – high folate diet for 1 week followed by 2 weeks on a normal chow diet. On the experiment day, mice were anaesthetized with pentobarbital sodium (50 mg/kg i.p.) and artificially ventilated. A jugular vein was cannulated for a drug infusion. The right carotid artery was cannulated for high speed injection of contrast agent and intermittent recording of arterial pressure. Contrast medium was injected as small boli at 15 min intervals (0.08-0.15 ml bolus, pure Iomeron 350, Bracco-Eisai). The same imaging and high-speed shutter system as described in earlier studies was used here. Images (1024 x 1024 pixels) were stored in 10-bit format. In all images a 50 micron tungsten wire was included for calibration. The input field of the SATICON camera was 9.5 mm x 9.5 mm. Shutter open time was ~1.0 ms. Monochromatic X-ray energy was adjusted to 33.2 keV, just above the iodine K-edge energy for maximal contrast. Images from each animal were recorded during baseline conditions and during 1) ACh infusion (3 micrograms /kg/min), 2) SNP infusion (5 micrograms/kg/min), 3) 15 min after L-NAME (NOS inhibitor, 10mg/kg ip.) + sodium meclofenamate (COX inhibitor, 2mg/kg ip), and during 4) post blockade ACh infusion (3 micrograms/kg/min).
We found that in contrast to the consistent dilatory responses of vessel segments in control (heterozygous HypoE, Figure 1B typical example) mice the coronary vessels across all branching orders in hearts of homozygous HypoE mice were either constricted during ACh stimulation (Figure 1E) or showed weak dilation relative to baseline conditions. Likewise, post NOS/COX blockade HypoE homozygous mouse hearts did not demonstrate consistent dilatory responses to repeated ACh stimulation, unlike heterozygous mice (Fig.1C and F). Importantly, occlusive stenoses were clearly visible in regions of the heart that were confirmed to be infarcted or steatotic (arrows in Fig.1F). ACh stimulation in stenotic segments of hearts in HypoE homozygous mice evoked constriction or total occlusion of flow in some cases. These data suggest that occlusive CAD in this mouse model results in endothelial dysfunction characterized by altered vasodilator production and diminished vessel responsitivity due to apolipoprotein E accumulation.
Zhang S, Pichard MH et al. Circulation 111:3457-3464, 2005
Figure 1. Coronary angiograms from a representative HypoE heterozygous (control, A-C) and homozygous (D-F) mice during baseline conditions (A & D), acetylcholine stimulation before (B & E) and after (C & F) combined NOS and COX blockade. Average relative vessel segment internal diameter changes from baseline are shown also in relation to vessel branching order. Arrow heads indicate occluded blood flow around a suspected plaque. Tungsten wire in lower right corner is 50 micrometer diameter for calibration purposes. |