Endothelial injury and leukocyte adherence in Häutchen preparations from coronary arteries and aorta of cynomolgus monkeys.

The aim was to describe the production of en face preparations (Häutchens) of the endothelium from the aorta and the coronary arteries of cynomolgus monkeys (Macaca fascicularis), to describe the morphology of the endothelium and to estimate endothelial injury. The present study describes, for the first time, the application of this technique to the coronary arteries. Numerous leukocytes (peroxidase-positive and -negative cells) were observed on the endothelium and a positive correlation between the numbers of adherent leukocytes and injured endothelial cells was found. A relatively leukocyte-independent measure of endothelial injury in the aorta was obtained by counting areas with a restricted number of leukocytes. The results indicated an even distribution and a relatively low frequency of injured endothelial cells along the arterial tree. The percentage of injured endothelial cells in LAD and LCX was 0.18 and 0.16, and in non-branched and circumostial areas of the descending thoracic aorta the corresponding figures were 0.22 and 0.28. There were, on average, 344 and 236 adherent leukocytes/mm2 in the non-branched and circumostial areas of the descending thoracic aorta, respectively, compared to 13 in the LAD and 23 in the LCX. Proportionally more peroxidase-positive leukocytes (possibly granulocytes) were found on the coronary endothelium than on the aortic endothelium, and the peroxidase-positive cells were preferentially located around small artery branches in the coronary arteries. The foregoing data provide an estimate of the distribution and frequency of endothelial injury and adherent leukocytes at pathobiologically relevant arterial sites in unmanipulated cynomolgus monkeys, an animal frequently used in atherosclerosis research.

Psychosocial stress causes endothelial injury in cynomolgus monkeys via beta1-adrenoceptor activation.

Current evidence links psychosocial factors to exacerbation of diet-induced atherosclerosis in monkeys via activation of the sympathetic nervous system. However, it is uncertain whether these factors can potentiate initial lesion formation, and do so even in the absence of dietary provocation, and whether any such effects can be prevented by beta-adrenergic blockade. As endothelial injury has been considered an initiating event in atherogenesis, we studied the effect of psychosocial stress on endothelial integrity in 48 adult male cynomolgus monkeys (Macaca fascicularis). All animals were housed in 12 social groups of four monkeys each for 11 weeks. The monkeys in half of the groups were exposed to a socially unstable ('stressed') condition for 72 h and received saline (n = 8), a lipophilic beta1-blocker (metoprolol, 0.30 mg/kg per h; n = 8), or hydrophillic beta1-blocker (atenolol, 0.15 mg/kg per h; n = 8). The remaining six social groups were assigned to the socially stable (non-stressed) condition; for 72 h these animals all remained in their social groups and were similarly treated with saline (n = 8), metoprolol (n = 8), or atenolol (n = 8). The frequency of IgG-positive (injured) endothelial cells was estimated on en face (Häutchen) preparations from the thoracic aorta and coronary arteries. Psychosocial stress caused a significant increase in the number of injured endothelial cells in the circumostial areas of the descending thoracic aorta in the placebo group (0.3 vs. 0.8%, P < 0.02), an effect that had not been demonstrated previously. Moreover, beta-blockade significantly (P < 0.01) inhibited the stress effect, with no differences between the two beta-blocking agents. The number of injured endothelial cells in the non-branched portions of the aorta and coronary arteries were low and indistinguishable among groups; irregularities in the size and location of branching points in the coronary arteries precluded analysis of these sites. This study demonstrated that psychosocial stress induces endothelial injury, and that this effect is mediated via beta1-adrenoceptor activation.

Endothelial injury in vivo: a technical and statistical approach to the study of aortic integrity.

The endothelium can be a link connecting risk factors with the development of cardiovascular disease, and methods for studying endothelial integrity are therefore important. We describe a method of studying endothelial injury in vivo by combining immunohistochemistry with an improved technique of producing "enface" preparations (Häutchens) aortic endothelium of rabbits and guinea pigs. These Häutchens enabled the study of large numbers of endothelial cells and adherent cells (probably leukocytes) at different locations along the aorta. The statistical distributions of the number of injured endothelial cells and adherent cells in a visual field were also investigated, and both closely followed a log-normal distribution. Based on this distribution, a method to estimate endothelial injury by grouping the cell count data, instead of exact counting, was developed. The grouped cell count data were then used to calculate the grouped mean and grouped standard deviation for each animal. The improvements of the technical and statistical methods offer good opportunities to study various aspects of endothelial integrity in a time efficient manner.