William E. Stehbens

An appraisal of cholesterol feeding in experimental atherogenesis

D IETARY LIPIDS are widely considered to be the major environmental agent responsible for severe atherosclerosis in populations of technologically developed countries, and the arterial lipid deposition induced in rabbits by the chronic administration of cholesterol or egg yolk’** is thought to support this view. Though lipid lesions can be produced by a high-fat diet alone and even by diets containing 14% to 20% vegetable fat3’4 or specific proteins associated with hypercholesterolemia,5-7 cholesterol-rich diets remain the principal means of inducing hypercholesterolemia, with the vascular lesions so produced accepted as atherosclerotic*-‘3 and the diets themselves regarded as atherogenic. However, with further knowledge and improved ultrastructural techniques, review of the model and its applicability is advisable to alert those interested in atherogenesis to the differences between diet-induced and spontaneous lesions despite known similarities. The Henle-Koch postulates,i4-i6 which stressed the importance of reproducing the same morbid condition in experimental animals to validate an etiologic agent, has been regarded as the epitome of scientific logic and method. A few authors have alleged that cholesterol-fed animals comply with the principles of Koch’s postulates,“.l’.‘8 but many acknowledge that feeding rabbits a cholesterol-rich diet does not reproduce atherosclerosis as it occurs in rnan.19-” Nevertheless the diet-induced lipid-containing lesions are still considered atherosclerotic by very many investigators.‘0~23~27-5’ In the light of new knowledge the Henle-Koch postulates have required modification for some diseases, and specific criteria applicable to experimental atherogenesis have been proposed27,52: (1) The experimental procedure must reproduce or be comparable to those conditions existing in man. (2) The sequence of pathologic changes in the vessel wall must be similar to that in man. (3) The experimental lesion must be similar to human atherosclerosis morphologically and topographically. (4) The complications of the disease, ie, intimal tears, ulceration, mural dissection, thrombosis, and aneurysmal dilatation, must result when severe lesions are produced. Application of these criteria will ensure maintenance of scientific standards and failure to comply, invalidation of the experimental model. Heedful of these criteria, this review is concerned with the pathology of diet-induced lesions with particular reference to the rabbit.

Pathology and pathogenesis of intracranial berry aneurysms.

Berry aneurysms are peculiarly common to the cerebral circulation of man. They are acquired lesions related to the large calibre and atherosclerosis of the cerebral arteries. The aetiology and pathogenesis are intimately concerned with haemodynamic stresses at arterial forks without scientific evidence of maldevelopmental errors. Whilst connective tissue disorders may predispose to their development, they are not essential aetiological factors. There is a need to identify the particular haemodynamic stresses and biophysical responses of the mural connective tissues in their development.

Vascular complications in experimental atherosclerosis

A THEROSCLEROSIS, “the inevitable fate of all human arteries,“’ has two distinct stages. There is a quiescent or developmental stage and then a stage occurring mainly in the sixth decade and beyond when the complications become manifest pathologically and clinically primarily due to hemorrhage or tissue ischemia.2 The difference between individuals is not in the presence or absence of the disease but rather in the variation in disease severity because this determines the onset of complications. According to the Henie-Koch postulates, the specific pathogenic agent must reproduce the same morbid condition in the experimental animal. Modified criteria for experimental atherosclerosis3*4 specify that in reproducing the disease, complications of atherosclerosis must develop in the advanced experimental lesions. Though several authors have alleged that dietary-induced lesions reproduce the complications of atherosclerosis,5-‘6 most admit that the complications of atherosclerosis are not regularly produced even in the advanced stages of the dietary-induced lesions.4.‘7-24 For this reason, a comprehensive appraisal of the vascular complications in experimental atherogenesis is reported with special emphasis on the hypercholesterolemic rabbit. In any consideration of the alleged production of complications, the sequential pathologic changes in the vessel wall must be similar to those occurring spontaneously in man and the experimental procedure must reproduce or be comparable to conditions existing in man.3’4

The epidemiological relationship of hypercholesterolemia, hypertension, diabetes mellitus and obesity to coronary heart disease and atherogenesis

Coronary heart disease (CHD) is an imprecise, inappropriate monitor of atherosclerosis severity and by inapplicable extrapolation CHD risk factors are incorrectly assumed to be causes of atherosclerosis. Taking into account (1) the misuse and substantial diagnostic error of CHD, (2) errors in determining the prevalence of risk factors, (3) the use of a young non-representative minority of sufferers of CHD, (4) bias posed by inclusion of familial hypercholesterolemia (FH) in clinical studies and (5) mutual inter-relationships, genetic influence and age dependence of hypercholesterolemia, hypertension, diabetes mellitus and body mass or obesity, it is unlikely that multivariate statistical analyses can adequately differentiate between their effects. These factors are age dependent and so are CHD and atherosclerosis. The importance of hypercholesterolemia in atherogenesis is suspect particularly since the vascular pathology of familial hypercholesterolemia and of cholesterol-fed animals has been misrepresented and does not provide support for the role of hypercholesterolemia in atherogenesis.

The relationship of hypercholesterolemia to atherosclerosis with particular emphasis on familial hypercholesterolemia, diabetes mellitus, obstructive jaundice, myxedema, and the nephrotic syndrome

ARLY THIS CENTURY, clinical reports E indicated that premature severe atherosclerosis occurred frequently in a group of diseases associated with hypercholesterolemia, viz, familial hypercholesterolemia (FH), diabetes mellitus, obstructive jaundice, hy~thyroidism* and the nephrotic syndrome. i4 The observations suggested an association between endogenous hypercholesterolemia and atherogenesis and were used to support the “lipid hy~thesis” of the etiology of atherosclerosis. Oliver’ asserted that the epidemiologic association of FH with coronary heart disease was the strongest clinical evidence in support of a relationship between plasma cholesterol concentration and coronary heart disease.6 Although extrapolation from the cause of coronary heart disease to the etiology of atherosclerosis is invafid, some researchers have considered atherosclerosis a disorder of lipid metabolism’ despite its universal presence in humans, its widespread presence in lower animals, and its existence in most individuals in the absence of any known lipid disorder. Furthermore, the vascular lesions in FH are said to differ from atherosclerosis in normoli~proteinemic individIlaIs.“” For these reasons an appraisal of the pathology of atherosclerosis in FH, diabetes mellitus, obstructive jaundice, hypothyroidism, and the nephrotic syndrome is presented.

An improved immunofluorescence techniquefor the histological examination of blood vessel tissue

Autofluorescence of elastic fibres in blood vessel samples is a common interference with the specific fluorescence of FITC-conjugated antibodies. Counterstaining with eriochrome black T changed the yellow-green colour of elastic fibres to dark red, thus turning a disturbing feature into a useful reference background. A second counterstain, p-phenylenediamine, visualized cell nuclei as an amber colour. To demonstrate the improvement of this staining technique, cryosections from blood vessel samples, derived from control veins, arteries and experimental aneurysms of different ages (15 to 99 month old) in 5 sheep, were stained with antibodies against procollagen III, collagen type IV, laminin, and nidogen. The specific distribution of these connective tissue components could now be related to the location of the elastic fibres and the cells (cell nuclei).

Scanning electron microscopic investigation of the afferent arteries of experimental femoral arteriovenous fistulae in rabbits.

&NA; Scanning electron microscopy of the intimal surface of anastomosed arteries of experimental femoral arteriovenous fistulae in rabbits was conducted to determine whether the muscular femoral artery differed from the elastic common carotid artery in its response to the hemodynamic stress of the arteriovenous shunt. Control femoral arteriotomies were performed in an additional five rabbits. The animals were sacrificed at varying intervals from 2 days to more than nine months postoperatively. Within two days postoperatively transverse and longitudinal tears involving the internal elastic lamina and also the endothelium appeared in the afferent artery as far proximal to the fistula as the lower abdominal aorta. Tears distal to the fistula were fewer and later in appearance. The tears healed rapidly. The response as demonstrated by this technique was similar to that of the common carotid artery of carotid‐jugular fistulae except that tears appeared earlier postoperatively with longitudinal as well as transverse disposition. Similar tears were found in four of the five control arteriotomies in the vicinity of the suture or at the site of clamping. The experiments reveal the readiness with which hemodynamic stress induces intimal tears.

Scanning electron microscopic study of hemodynamically induced tears in the internal elastic lamina of rabbit arteries

&NA; A scanning electron microscopic investigation of tears in the internal elastic lamina of the afferent artery of experimental arteriovenous fistulae was undertaken to determine their site of initiation and to elucidate their nature. Carotid‐jugular arteriovenous fistulae and contralateral common carotid arteriotomies were performed in rabbits sacrificed from two to 63 days postoperatively. Following de‐endothelialization, scanning electron microscopy of the internal elastic lamina revealed predominantly transverse straight tears with sharp margins. Even in chronic fistulae the tears retained their sharp margins despite their propagation. No evidence of repair was observed. The tears commenced by rupture of the elastic trabeculae traversing the fenestrae. Enzymatic digestion of the internal elastic lamina did not resemble the hemodynamically induced tears and no evidence of elastolytic enzyme activity was detected. The tears suggest that the abnormally stressed elastic tissue has undergone some structural alteration leading to a reduction in its tensile strength.

Early berry aneurysm formation in marfan's syndrome

A detailed study of cerebral arterial forks by serial sectioning was made in a 33-year-old woman with typical Marfans syndrome who died of septicemia following cardiac surgery. Two forks of the right middle cerebral artery exhibited the atrophic changes associated with early berry aneurysm formation indicating that the mode of development of these aneurysms is similar in both Marfans syndrome and in non-Marfan subjects.

The lipid hypothesis and the role of hemodynamics in atherogenesis

A THEROSCLEROSIS , the inevitable fate of all human arteries, 1 is responsible for almost half the mortality of western populations and for a considerable and variable degree of incapacitation prior to the fatal outcome. It can thus be regarded as the major medical problem of our aging populations. Adequate control and management await elucidation of the etiology and underlying pathogenetic mechanisms. Etiology of the arthrosclerotic process is one of the most controversial areas in medical science. In order to achieve progress in such perplexing, controversial fields of scientific study, periodic review of the current state of knowledge is essential. This survey aims to indicate the areas in which our knowledge is deficient or misleading and to emphasize certain facts that could be helpful and even crucial in determining the role of lipids and hemodynamics in atherogenesis.