Protection of the arterial internal elastic lamina by inhibition of the renin-angiotensin system in the rat.

Spontaneous rupture of the internal elastic lamina (IEL) occurs in some arteries of the rat during growth and aging. Inbred, normotensive, Brown Norway (BN) rats are particularly susceptible to rupture of the IEL, especially in the abdominal aorta (AA). Preliminary experiments showed that different angiotensin-converting enzyme (ACE) inhibitors protect against rupture of the IEL in the BN rat to a greater extent than hydralazine, suggesting a role of the renin-angiotensin system (RAS) in this phenomenon. To explore this possibility, we have treated male BN rats from 4.5 to 14 weeks of age with either enalapril or losartan (both at 1, 3, and 10 mg x kg(-1) x d(-1)) or with the calcium antagonists mibefradil (at 3, 10, 30, and 45 mg x kg(-1) x d(-1)) and amlodipine (at 30 mg x kg(-1) x d(-1)). Systolic blood pressure (SBP) was measured weekly, and at the end of treatment we (1) recorded body and heart weights, (2) measured various parameters of the RAS in plasma, (3) quantified interruptions in the IEL on "en face" preparations of AA, and (4) quantified elastin, collagen, and cell proteins in the media of the thoracic aorta. Results showed that enalapril and losartan similarly decrease SBP and rupture of the IEL in the AA, suggesting that enalapril inhibits the latter via a decrease in the production of angiotensin II (Ang II) and not via another effect on ACE. The decrease in IEL rupture and in SBP, as well as the modifications in the parameters of the RAS, were all dose dependent. Mibefradil had little effect on the RAS and, at the highest doses, decreased SBP to an extent similar to that for enalapril at 3 mg x kg(-1) x d(-1) but did not significantly inhibit IEL rupture. Amlodipine decreased SBP, increased plasma renin concentration, and was without effect on IEL rupture. All treatments at the highest doses had a hypotrophic effect on the aortic media but differed in their effects on the heart, with enalapril and losartan decreasing and mibefradil and amlodipine increasing heart weight, suggesting that the inhibition of IEL rupture may be related to a cardiac hypotrophic effect. All these results, taken together, suggest that Ang II plays a role in the rupture of the IEL that is, in part, independent of SBP.

The variability of blood pressure in mice selected for low (MGL) and high (MGH) blood magnesium levels.

Arterial blood pressure has been determined using a tail cuff method in 164 unanaesthetized, adult mice with genetically low (MGL) or high (MGH) erythrocyte magnesium levels. The mice came from the 9th, 10th, 11th and 12th generations of a bidirectional selective breeding undertaken at the CSAL-CNRS (Orleans). The two lines differed significantly by the magnesium contents of their erythrocytes, plasma, kidney and bone. Ten successive measurements of systolic blood pressure were recorded from each animal, without habituation, within a single period of about 2 min. All mice had elevated blood pressures presumably due to the stress of the experimental procedure. The first, the tenth and the average values of these 10 measurements yielded similar results in both sexes and in both lines. Younger animals (4 months of age) had significantly higher values (180 mmHg) than older ones (10-13 months, 161 mmHg), and this difference was more pronounced in the MGL than in the MGH strain. In both age-groups and lines, about two-thirds of all mice tested showed an increasing arterial pressure during the testing period while the remaining third exhibited decreasing values. Whatever the age-group or the variation pattern during the course of the blood pressure measurements. MGL mice had median values (mean of the fifth and sixth measurements) higher than those of MGH mice. The difference observed between the two strains can be attributed to a greater sensitivity and/or reactivity of the MGL mice to the stress induced by the manipulation, heating and immobilization required for blood pressure measurement.

Induction of aneurysms in the rat by a stenosing cotton ligature around the inter-renal aorta.

Aortic aneurysms have been induced in the rat by combining the chemotactic property of cotton for inflammatory cells and the resulting granuloma formation with the mechanical and haemodynamic stress of aortic coarctation. A stenosing cotton ligature was placed around the aorta, between the renal arteries, in male Wistar rats under standardized conditions. Three months later, 7 out of 12 rats (58%) had developed saccular aneurysms of the inter-renal aorta. The aneurysmal wall consisted of a collagenous shell with a few newly formed elastic fibrils on its luminal side. Detailed histological studies of the inter-renal aorta at different times after placing stenosing or non-stenosing cotton or nylon ligatures between the renal arteries, together with studies using anti-hypertensive therapy (cilazapril) and immunohistochemical studies using an anti-macrophage antibody (ED1) were performed to try to establish cellular events involved in this aneurysmal remodelling. We conclude that in this model aneurysm formation requires (i) deep mechanical injury to the aortic wall, (ii) the presence of hypertension upstream to the stenosis and (iii) an inflammatory response to the cotton ligature. The early inflammatory reaction was less in the case of nylon and although macrophages were present in both cases the most striking difference was the greater incidence of PMNs in the case of cotton.

Effects of chronic absorption of dietary supplements of methionine and cystine on arterial morphology in the rat.

Male Wistar rats were fed diets containing supplements of either methionine or cystine from 10 weeks of age and compared to rats fed a control diet or a high protein diet kept under identical conditions. At 11-16 months of age, the aorta and the renal, iliac and caudal arteries of all rats were fixed and examined by light and electron microscopy. Cystine-fed rats showed arterial morphology similar to that of control rats and of those having received a high protein diet. Methionine-fed rats showed marked thickening of the arterial wall which was due, on the one hand, to massive intimal thickening, as a result of accumulation of granular material in the subendothelial region and, on the other hand, to marked thickening of the media as a result of increased extracellular material around smooth muscle cells. Zones of early phases of chondroid metaplasia were also observed in the media. Thus cystine and methionine, despite their interrelated metabolism, have very different effects on the morphology of the arterial wall. However, cystine and methionine both inhibited the spontaneous rupture of the internal elastic lamina in the renal artery. This latter result is discussed in the light of the similarities between spontaneous rupture of the internal elastic lamina and beta-aminopropionitrile-induced aortic aneurysm and rupture.

Role of elastase and lysyl oxidase activity in spontaneous rupture of internal elastic lamina in rats.

Rupture of the internal elastic lamina may occur spontaneously with age in certain arteries of the rat and to various extents in different strains. This phenomenon may have some bearing on certain aspects of arterial pathology. For this study, we investigated biochemically the mechanisms of formation of interruptions in the internal elastic lamina (IIEL) by comparing aortas of Brown Norway (BN) rats, which develop numerous IIEL in the abdominal aorta, with those of Long-Evans (LE) rats, which develop none. We isolated aortic elastin from BN and LE rats and determined its amino acid composition and its susceptibility to different elastases. No differences were found between the two strains, but the quantity of elastin isolated per aorta was lower in the BN than in the LE rats. Elastase-like activity (ELA) of whole aortic extracts, measured with Suc(Ala)3NA as a substrate, was greater in the BN rats than in the LE rats of both sexes. The assay of ELA in endothelium, media, and adventitia extracted separately showed very low levels in the media compared to the endothelium and adventitia. The endothelium accounts for about one-half of the total aortic ELA, but a difference between the two strains was detected only in the adventitia. With 3H-insoluble elastins prepared from BN and LE aortas as substrates, elastinolytic activity (EA) was detected only in extracts of endothelium after prior exposure to trypsin. Extracts from BN endothelium on BN elastin were more active than were those from LE endothelium on LE elastin. The assay of lysyl oxidase activity in aortic extracts from the two strains with 3H-collagen from chick embryo calvaria as the substrate showed a lower activity in the BN than in the LE rats. Taken together, these results suggest that increased elastase activity and decreased lysyl oxidase activity may be involved in the formation of IIEL.

Horseradish peroxidase as a permeability marker in injured rat caudal and iliac arteries.

The permeability to Horseradish Peroxidase (HRP) of the rat caudal artery at the level of spontaneous lesions was evaluated by electron microscopy and compared with that of lesions experimentally induced by pinching or internal scraping of the caudal and iliac arteries. No HRP reaction product is observed in the extracellular space of the arterial wall when (i) the internal elastic lamina (IEL) and the endothelium are absent, (ii) the IEL is maintained and the endothelium is absent and (iii) the IEL is absent and the endothelium has regenerated. That HRP does enter the arterial wall in cases of gross endothelial damage is shown by its selective retention in damaged smooth muscle cells in such cases. In contrast, HRP reaction product is detected in the subendothelial space when the IEL is maintained and is covered by a regenerating or recently regenerated endothelium. Furthermore, the amount of tracer visualized under the same experimental conditions is greater in the iliac than in the caudal artery. We conclude that the detection of HRP in the subendothelial space of the artery wall requires the presence of regenerating or recently regenerated endothelial cells lying on an intact IEL. It is thus not simply related to endothelial permeability but depends also upon the retention of HRP by extracellular substances. In addition, the quantity of marker retained varies between different sites in the arterial tree.

Spontaneous rupture of the internal elastic lamina in the rat: the manifestation of a genetically determined factor which may be linked to vascular fragility.

The spontaneous rupture of the internal elastic lamina (IEL) in various arteries occurs to different extents in different rat strains. We have quantified this phenomenon in the caudal and renal arteries and abdominal aorta in two normotensive inbred strains: the Brown Norway (BN) and Long Evans (LE) strains. At 5 weeks of age, BN rats of both sexes exhibited small numbers of interruptions in the IEL of the caudal artery, whereas LE rats did not. Postpubertal male and female BN rats presented large numbers of IEL interruptions in the caudal artery and significant numbers in the renal artery and abdominal aorta, whereas LE rats showed few in the caudal artery and none in the other arteries. Treatment with beta-aminopropionitrile (BAPN, an inhibitor of lysyl oxidase, the enzyme involved in the formation of cross-links in elastin and collagen) increased the formation of IEL ruptures in both strains in the caudal and renal artery and in the abdominal aorta in BN rats, but not in the abdominal aorta of LE rats. Apart from IEL ruptures, which were more prevalent in BN rats, no differences were observed in the ultrastructure of the aortic elastic fibers between the two strains, either in controls or in BAPN-treated rats. When male rats of both strains were made hypertensive by unilateral nephrectomy and administration of deoxycorticosterone and salt, mortality was more precocious in the BN strain although blood pressure was significantly higher in the BN strain at only one time point. The incidence of cerebrovascular hemorrhage was 48% in BN rats and 0% in LE rats. Hypertension increased the formation of ruptures in the IEL in some arteries - to a greater extent in the BN than in the LE rats. These results raise the possibility that the propensity to spontaneous rupture of the IEL, which is in part genetically determined, may reflect a latent form of vascular fragility which becomes significant in hypertension, resulting in poor survival and susceptibility to cerebrovascular accidents.

In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus.

The binding sites for [3H]corticosterone (3HB) and [3H]aldosterone (3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a "pure glucocorticoid' RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.

Spontaneous arterial lesions involving breaks in the internal elastic lamina in the rat: effects of beta-aminopropionitrile and familial distribution.

We have studied the effects of beta-aminopropionitrile (BAPN) administration on the formation of spontaneous arterial lesions, characterized principally by a rupture in the internal elastic lamina (IEL) in the caudal and renal arteries of the Wistar rat. Treatment with BAPN (an inhibitor of lysyl oxidase) increased the formation of these lesions in rats up to 12 weeks of age but had differential effects on the caudal and renal artery in older rats. Administration of the nitrile to weanling rats led to the premature formation of lesions in caudal arteries of both male and female rats which morphologically resemble lesions which form spontaneously later in life. Dietary supplements of copper or pyridoxine were without effect on the formation of spontaneous caudal artery lesions when given from 5 wks of age but a copper supplement from midgestation slightly inhibited lesion formation only in male rats. This suggests that if copper deficiency is involved in spontaneous lesion formation, it is only a contributory factor. Quantification of either caudal or renal artery lesions within different litters of Wistar rats showed that there exists a familial aggregation in the frequency of spontaneous lesion formation, certain litters showing significantly higher levels of lesions than others. Adult Sprague-Dawley rats also appear to be more susceptible to the development of renal artery IEL defects than Wistar rats. The possibility of a hereditary disorder leading to a minor defect in elastic fibre structure which could be responsible for the spontaneous lesions is discussed.

"Spontaneous" endothelial injury and lipid accumulation in the rat caudal artery.

We have studied the role of endothelial injury and subsequent repair in the accumulation of plasma lipids within lesions of the rat caudal artery where spontaneous endothelial damage occurs. Lesions from diet-induced hypercholesterolemic rats were classified both according to the morphology of their luminal cell covering and the extent of medial lipid deposits. Lesions covered with regenerating and newly regenerated cells accumulated more lipids than deendothelialized lesions or those covered with cells resembling normal endothelium, which suggests that the lipid accumulation consecutive to spontaneous endothelial injury occurs during the early phase of repair. In addition, lesions located in the distal part of the artery accumulated less lipids than those in the proximal part, although endothelial damage occurred with equal frequency in both locations. This suggests that even in the presence of severe hypercholesterolemia, favorable local conditions are required in addition to endothelial injury for maximal arterial lipid accumulation to occur.

Effect of contralateral nephrectomy alone or in association with hypertension on rat renal artery lesions.

We have studied the effect of unilateral nephrectomy alone or in association with experimentally-induced (DOCA-salt treatment) or genetic (SHR) hypertension on the formation of lesions in the contralateral renal artery. These lesions, which form spontaneously with age in small numbers in the renal artery of the control Wistar rat, are characterized principally by an interruption of the internal elastic lamina (IEL) over part or all of the vessel circumference. Such lesions are also present in small numbers in renal arteries of intact WKY and SHR rats. Unilateral nephrectomy increases lesion formation in the remaining renal artery in all groups of rats and this increase is greatest when hypertension accompanies nephrectomy. The increase in lesion formation is accompanied in most cases by a significant increase in length of the remaining renal artery and in all cases by compensatory renal hypertrophy. The possibility that the increased flow in the renal artery irrigating the hypertrophied kidney may be responsible for the increased lesion formation is discussed. Lesions in renal arteries of control and nephrectomized normotensive rats and SHR did not differ greatly morphologically, and lesions containing lipid deposits were observed in all groups. Only in the DOCA-salt hypertension group were severe arterial alterations observed.

Natural incidence of interruptions in the internal elastic lamina of caudal and renal arteries of the rat.

Interruptions in the internal elastic lamina (IEL) have been quantified by light microscopy in the caudal arteries of different strains of rats and in caudal and renal arteries of (i) male and female virgin rats up until 2 years of age, (ii) repeatedly bred rats and (ii) hypertensive rats. Results showed that gaps in the IEL exist in caudal arteries of adult males in all strains studied, but to a lesser extent in the hairless mutant. In the virgin Wistar rat these interruptions in the IEL form with age in both caudal and renal arteries, and are more numerous (i) in the male than in the female in both arteries, and (ii) in the caudal than in the renal artery in both sexes. Repeated breeding in the Wistar rat abolishes the sex difference in incidence of IEL gaps in both renal and caudal arteries. The Sprague-Dawley breeder rat is more susceptible than the Wistar breeder to their formation in the renal artery. Hypertension in the male consistently increases the formation of IEL gaps in the renal but not in the caudal artery. The importance of local factors, e.g. hemodynamics, in the formation of these defects in the IEL, and their possible relationship with the development of arteriosclerosis is discussed.

"Spontaneous" endothelial injury in the rat caudal artery.

Numerous spontaneous lesions, characterized principally by a gap in the internal elastic lamina, form with age in the caudal artery of the young, male Wistar rat. Such newly formed lesions were studied, with special reference to the state of the endothelium, using three techniques: "en face" examination of silver-stained caudal artery, [3H]thymidine autoradiography, and light and electron microscopy. In many, but perhaps not all cases, areas of endothelium are damaged or removed either simultaneously or shortly after the formation of the break in the internal elastic lamina and some underlying smooth muscle cells are damaged. Polymorphonuclear cells and monocytes are attracted to the site of injury. A repair process rapidly ensues including rapid regeneration of the endothelium and proliferation of smooth muscle cells in the underlying media and of some adventitial fibroblasts. This process results in the restitution of the endothelial monolayer and the laying down of one or several layers of longitudinal smooth muscle and collagen and elastic fibers in the subendothelial space. This demonstration of naturally occurring damage to fairly large areas of endothelium and the events which occur in the repair process is of interest in view of the "response-to-injury" theory of atherogenesis; observations are discussed in the light of previous studies performed on experimentally removed endothelium.

Decreased dietary lipid deposition in spontaneous lesions distal to a stenosis in the rat caudal artery.

When male Wistar rats are fed a high fat and cholesterol diet, the small lesions in the caudal artery which form spontaneously with age exhibit, to various degrees, focal accumulation of lipids. Lesions in the proximal part of the artery show more intense lipid deposits than those in the distal part. In order to determine the influence of changes in hemodynamic factors on lipid accumulation within these lesions, a stenosis was experimentally induced in the proximal part of the caudal artery in 15, 5-week old rats. These rats, plus 15 age-matched controls, were then fed a high fat and cholesterol diet for 8 weeks and thereafter their caudal arteries were taken for histological examination and quantification of lipid deposits within lesions. Results showed that in the stenosed arteries, the mean percentage of highly fatty lesions was significantly decreased and that of fat-free lesions was significantly increased distal to the stenosis compared to control arteries, indicating the importance of hemodynamic factors in the deposition of nutritional lipids within spontaneous arterial lesions. However, within the stenosed group, there was apparently no correlation between the degree of stenosis and the extent of fat deposits within lesions.

Spontaneous lesions in the rat caudal artery. Effects of diet-induced hypercholesterolaemia alone and in association with hypertension.

Caudal arterial lesions form spontaneously with age in the male Wistar rat and are characterized by a break in the internal elastic lamina (IEL), with associated damage to endothelium and underlying smooth muscle cells followed by rapid repair. Such lesions have been studied under conditions of hypercholesterolaemia. Diet-induced hypercholesterolaemia did not enhance the formation of these lesions compared to age-matched control rats. Lesioned areas of caudal arteries from hypercholesterolaemic rats showed marked or slight focal lipid deposits depending upon their location along the artery. Studies in rats of different ages suggest that the majority of lipid probably accumulates during the phase of lesion repair and that the absence of the IEL per se does not appear to be responsible for the lipid accumulation. The effect of hypertension associated with hypercholesterolaemia on caudal arterial lesions was studied using DOCA salt-Wistar and spontaneously hypertensive rats. The association of these two factors did not increase lesions either in the caudal artery or in the renal artery studied for comparison. In caudal artery the number of fatty lesions appeared to be related more to the level of cholesterolaemia than to blood pressure. The extent of lipid accumulation within lesions in the caudal artery appears not to correlate with that in the renal artery, demonstrating the importance of local factors.

Autoradiographic localization of 3H-angiotensin II in rat adrenal glands.

Autoradiographic studies of rat adrenal gland approximately 1 min after intra-aortic injection of 3H-angiotensin II show that radioactivity concentrates in the zona glomerulosa. This concentration is reduced by concomitant administration of excess unlabelled angiotensin II.

Autoradiographic localization of 3H-glucocorticoids and 3H-cortexolone in mouse pituitary.

Autoradiograms of mouse pituitaries were prepared 30 min after injection of 3H-dexamethasone (3HDM), 3H-corticosterone (3HB) and 3H-cortexolone (3HS) either alone or in the presence of competing unlabelled steroids. 3H-dexamethasone accumulated in cell nuclei of both the pars distalis and the pars nervosa but not in those of the pars intermedia. This preferential accumulation (nuclear/cytoplasmic grain density, 4 : 1) was abolished by the concurrent administration of excess dexamethasone. 3H-corticosterone, to a much less marked extent than 3H-dexamethasone, accumulated in cell nuclei of the pars distalis but not in those of the pars intermedia and the pars nervosa. Excess unlabelled corticosterone diminished nuclear grain density in the pars distalis. After 3-h-cortexolone injection, preferential nuclear uptake was not observed. In a second series of experiments, excess dexamethasone (10 x, 100 x), corticosterone (100 x, 300 x) and cortexolone (100 x, 300 x) administered with 3H-dexamethasone were without effect on cytoplasmic grain density but totally abolished preferential nuclear accumulation. Parallel biochemical studies on kidney cytoplasmic preparations from the same animals showed no differences in total cytoplasmic radioactivity between treatments but marked differences in cytoplasmic bound 3H-dexamethasone. The results demonstrate: i) that dexamethasone binds specifically to cell nuclei of the pars distalis and the pars nervosa and that this nuclear concentration is abolished by competing corticosterone and cortexolone as well as dexamethasone; ii) that corticosterone localizes in cell nuclei of the pars distalis but much less markedly than dexamethasone; iii) that cortexolone fullfils the criteria of a glucocorticoid antagonist at the pituitary cell level.

Autoradiographic studies of a glucocorticoid agonist and antagonist: localization of 3H-corticosterone and 3H-cortexolone in mouse brain.

The localization in the mouse brain of corticosterone, the natural glucocorticoid in the mouse, and cortexolone, reported to be a glucocorticoid antagonist, was studied by autoradiography 30 min after in vivo administration of the tritiated compounds. After 3H-corticosterone (3HB) injection, radioactivity was preferentially concentrated in cell nuclei of several structures within the limbic system, and in nuclei of certain neurones of the cerebral cortex and medullar oblongata. This nuclear concentration was abolished after injection of 3H-corticosterone with an excess of unlabelled corticosterone. After 3H-cortexolone (3HS) injection, a diffuse radioactivity was observed throughout the brain. However, a higher concentration of grains was present in the ventral nucleus arcuatus and in the infundibulum. When excess unlabelled cortexolone was administered with 3H-cortexolone this preferential accumulation of grains was abolished. The accumulation of 3H-cortexolone in the medial basal hypothalamic region suggests that cortexolone concentrates preferentially in dexamethasone (DM) target regions, and in addition the autoradiographic results show that the cortexolone-receptor complex does not accumulate in the cell nucleus.