Harvey Wolinsky

Response of the Rat Aortic Media to Hypertension: Morphological and Chemical Studies

Clinical and experimental studies indicate that hypertension accelerates the development of arteriosclerosis. Morphological and chemical studies of the distended rat thoracic aorta were undertaken to define the structural and compositional alterations of the media which accompany hypertension and to relate these changes to increases in calculated medial stress. An 8-week period of hypertension was associated with significantly greater diameter, medial thickness, and cross-sectional area of the media of the thoracic aorta than in normotensive animals. Calculated wall tension was significantly higher in hypertensive animals, but the number of medial lamellar units was not greater than that usual in normotensive animals; this resulted in a strikingly elevated value for calculated tension per medial lamellar unit for the aortas of hypertensive animals. A highly significant linear relation was found between total tension and cross-sectional area of the media of the same segment. In addition, the absolute amounts of both medial elastin and collagen were increased in hypertensive animals; however, the percent of these elements remained essentially constant, indicating little change in the composition of aortic tissue. Increments in both fibrous proteins were linearly related to increases in calculated mural stress, and medial accumulations of elastin and collagen proceeded at similar rates. These findings demonstrate a linear relation among vessel dimensions and amounts of medial elastin and collagen and calculated wall tension.

Long-Term Effects of Hypertension on the Rat Aortic Wall and Their Relation to Concurrent Aging Changes: MORPHOLOGICAL AND CHEMICAL STUDIES

The effects of long-term (16 months) hypertension on the thoracic aorta of male rats were compared to previously reported short-term (2.5 months) changes and to concurrent aging changes. Hypertension was produced by clipping a renal artery. Although short-term hypertension was characterized by a disproportionate increase in noncollagenous alkali-soluble proteins, which have been attributed primarily to vascular smooth muscle, with long-term hypertension there was no further increase in these proteins but instead there were striking increases in mural accumulations of elastin and collagen. Chronically elevated wall tension in hypertensive vessels was associated with a progressive increase in wall thickness which resulted in a value for wall stress no different from that of control vessels. Concurrent aging changes were qualitatively similar to, but much less pronounced than, those seen with hypertension and were attributed to an increase in wall tension in controls resulting from a combination of significant increases in diameter and systolic blood pressure with age. This study of the interaction of vessel structure and function has revealed common features of what appears to be a diverse group of vascular alterations.

Effects of Hypertension and Its Reversal on the Thoracic Aorta of Male and Female Rats: Morphological and Chemical Studies

Effects of hypertension and its reversal on the wall of the thoracic aorta of male and female rats were studied. Hypertension for 10 weeks caused increased diameter, wall thickness, tangential tension, wall stress, and medial area in both sexes. In addition, absolute amounts of elastin, collagen, and noncollagenous, alkali-soluble protein were significantly increased in the walls of all hypertensive vessels. Associated with the decrease of calculated tension and wall stress to normotensive levels after reversal of hypertension, wall thickness and medial area in males remained significantly greater than in controls and similar to the values found in hypertensive vessels, whereas in females these parameters returned fully to control levels. Absolute amounts of noncollagenous, alkali-soluble proteins did return to control levels after hypertension reversal, but elastin and collagen remained elevated in amounts similar to those in vessels with sustained hypertension; in females, this resulted in an above-normal concentration of these fibrous elements in the vessel wall. Compositional changes of the female vessel wall after reversal of hypertension were thus similar to but more clearcut and complete than those found in the male aorta. Recognition of irreversible changes in vessels subjected to hypertension may have implications for the benefits to be gained from blood pressure reduction on the progression of cardiovascular disease.

Arterial lysosomes and connective tissue in primate atherosclerosis and hypertension.

The cellular events that occur in the vessel wall consequent to changes in endothelial permeability result in the progression of vascular disease, particularly atherosclerosis. Female rhesus monkeys were fed an atherogenic diet or were made hypertensive for 6-8 months; and their vessels were then compared with vessels from control monkeys. Length-defined segments of coronary vessels, the thoracic aorta, and the abdominal aorta showed significant increases in total connective tissue in the atherosclerotic and hypertensive groups; pulmonary vessels did not. The diseased aortic segments had increased levels of two lysosomal enzymes, acid phosphatase and beta-N-acetylglucosaminidase; pulmonary vessels were not diseased and did not show these changes. Coronary vessels from the atherosclerotic and hypertensive groups did not show an increase in enzyme levels on biochemical measurements, but focal accumulations of lysosomes were identified by cytochemical techniques. In atherosclerotic lesions, a doubling of cholesterol and more than a tenfold increase in cholesterol ester were found. These connective tissue and lysosomal changes are early features of primate vascular disease and may result from the accumulation of excessive substrate (cholesterol ester) in the lysosomes of vascular smooth muscle cells.

A Method for the Isolation of Intima-Media Samples from Arteries

Summary A method is described for the preparation of aortic intima-media samples which are free of adventitia. Advantages of its use over the usual method of isolation are described in two types of application.

Hydrolase activities in the rat aorta. I. Effects of diabetes mellitus and insulin treatment.

SUMMARY Vascular disease in diabetics could arise in part from altered vessel wall catebolism. Specific activities of hydrolases in aortic smooth muscle cells from rats with streptozotocin-induced diabetes were measured. Enzymes included: neutral α-glucosidase, α-mannosidase, and lysosomal /V-acetyl β-glucosaminidase, β-galactosidase, cathepsin C, acid α-glucosidase, and acid cholesteryl esterase. After 4,8, and 11 weeks of diabetes, activities of all enzymes studied were decreased significantly in diabetic vessels, decreases ranging from 15% for cathepsin C to 62% for amannosidase. After 3 weeks of diabetes, insulin treatment for 1 week restored enzyme levels to normal. After 7 weeks of diabetes, 1 week of insulin treatment did not restore enzyme levels fully to normal (acid cholesteryl esterase was unchanged); 4 weeks of insulin did. Acid phosphatase and JV-acetyl β-glucosaminidase activities were reduced markedly in histochemical studies of diabetic aortas at all time periods and were restored by insulin treatment. AUoxan-induced diabetes gave results similar to those with streptozotocin. Significant decreases of aortic hydrolase activities, including those of lysosomes, occur in experimental diabetes mellitus and could contribute to accumulation of substrates in vascular smooth muscle cells.

Comparison of Medial Growth of Human Thoracic and Abdominal Aortas

Recent morphologic studies of adult mammalian thoracic and abdominal aortic segments have shown that the adult human abdominal aorta deviates significantly from the usual pattern of medial lamellar architecture. In the present study medial growth of these two aortic segments from prenatal life to adulthood was compared in terms of medial architecture and calculated tangential tension levels. During prenatal life, these parameters were very similar in the two segments. However, the postnatal increase in the medial thickness of the thoracic segment was due mainly to the addition of lamellar units which increased in number from 35 to 56; only a minor contribution was made by increased thickness of each unit which changed from 0.012 to 0.017 mm. The converse was true for the abdominal segment; the number of lamellar units increased only from 25 to 28, but lamellar unit thickness increased strikingly from 0.012 to 0.026 mm. Calculated wall stress was similar in the two segments throughout growth, but tension per lamellar unit became disparate in the segments during the first decade of life, culminating in unusually elevated values in the adult human abdominal aortic media.

Modification of the Effects of Hypertension on Lysosomes and Connective Tissue in the Rat Aorta

The relationship of Iysosomes to the vascular effects of hypertension and the possible modification of these effects by anti-inflammatory agents (methylprednisolone and aspirin), vitamin E, and estrogen were studied. Each of these agents was given to a group of hypertensive rats; untreated hypertensive rats and normotensive rats served as controls. Vessel wall morphology and dimensions of aortas from hypertensive rats were unaffected by treatment. Usual connective tissue accumulations seen in hypertensive vessels were suppressed to normotensive levels in the methylprednisolone- and estrogen-treated hypertensive groups. Two lysosomal enzymes, acid phosphatase and N-acetyl-β-d-glucosaminidase (NAGA), increased over normal levels in hypertensive vessels from 1.03 ± 0.08 (SE) to 2.04 ± 0.19 μmoles/aortic pair hour−1 and from 1.10 ± 0.24 to 4.57 ± 0.38 μmoles/aortic pair hour−1 for the respective enzymes. Normal enzyme levels in estrogen-treated hypertensive rats (1.14 ± 0.15 μmoles/aortic pair hour−1 for acid phosphatase and 2.04 ± 0.44 μmoles/aortic pair hour−1 for NAGA) and intermediate levels in methylprednisolone-treated hypertensive rats (1.35 ± 0.10 μmoles/ aortic pair hour−1 for acid phosphatase and 2.48 ± 0.54 μmoles/aortic pair hour−1 for NAGA) were found. Other treated groups showed the usual elevations associated with hypertension. These group differences were also seen after cytochemical staining for lysosomal acid phosphatase and NAGA. The parallel changes in aortic connective tissue and lysosomal enzymes in hypertension and their modification by two drugs suggest that these events are related.

Hypertensive diabetic cardiomyopathy in the rat: ultrastructural features

We previously described a cohort of diabetic patients with typical congestive cardiomyopathy, in whom myocardial lesions were related to concommitant high blood pressure. To evaluate the association of diabetes mellitus and hypertension in more detail, we studied 4 groups of rats with either no disease, streptozotocin-induced diabetes mellitus, renovascular hypertension, or a combination of hypertension and diabetes. Analysis revealed significant myocardial fibrosis and degeneration in the hypertensive-diabetic group when compared to controls, without an obvious relationship to small vessel lesions. The myocardial alterations appeared similar to those observed in patients with hypertension and diabetes mellitus. Of note, although hypertensive animals had focal moderate lesions, diabetic animals had no pathological changes. To further characterize these histological changes, we performed electron microscopy on the 4 animal groups, which we are reporting in this study. Our analysis of the ultrastructural alterations confirms the previous histological observations. Diabetic animals only had increased cellular lipid, and mild, focal areas of myofibrillolysis, with no significant increases in perivascular and perisarcolemmal basal lamina. Consistent with our light microscopic finding that PAS positive material was associated with interstitial or replacement fibrosis, we noted basal lamina proliferation in the hypertensive and hypertensive-diabetic groups, particularly in areas of scarring. Pericapillary basal lamina was increased to the greatest extent in the hypertensive-diabetics. Qualitative alterations of myocardial cells and muscular blood vessels were similar in both the hypertensive and hypertensive-diabetic animals; however, there were more extensive changes in the latter group.

Hydrolase activities in the rat aorta. II. Effects of hypertension alone and in combination with diabetes mellitus.

SUMMARY Hypertension is an important risk factor for atherosclerosis and often occurs in association with diabetes mellitus. Specific activities of hydrolases in homogenates of aortas from rats with renal-clip hypertension, normotension following a period of hypertension, and hypertension combined with streptozotocin-induced diabetes mellitus were measured. Enzymes included: neutral α-glucosidase, and lysosomal JV-acetyl-β-gIucosaminidase, β-galactosidase, cathepsin C, acid α-glucosidase, and acid cholesteryl esterase. After 6 or 12 weeks of hypertension, specific activities of all enzymes measured were significantly increased, levels ranging from 24% above normal for cathepsin C to 351% above normal forAr-acetyl-β-glucosaminidase. Six weeks of normotension following 6 weeks of hypertension resulted in restoration to normal of four of the six enzyme activities; the remaining two enzymes were significantly below normal levels. Combined hypertension and diabetes mellitus showed smooth muscle cell levels of four of the five hydrolases measured to be significantly lower than those present with hypertension alone. In every instance, histochemical studies of aortas showed acid phosphatase and N-acetyβglucosaminidase activities which corresponded to the biochemical findings. These findings indicate profound and discrete effects of two clinical risk factors on vascular smooth muscle cell lysosomes.