Intimal changes in the aorta of prehypertensive rats

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

49, 171-184 (1988)

lntimal Changes in the Aorta of Prehypertensive M. C. KOWALA,~'~ H. F. CUBNOUD,~ R. NICOLOSI,~~. Department

of Pathology,

Received

University of Massachusetts Worcester, Massachusetts January

20, 1988,

JORIS,AND G. MAJNO

Medical School, 01605

and in revised

form

March

Rats’

55 Lake

Avenue

North,

21, 1988

Intimal changes were quantitated in several rat models of arterial hypertension. One kidney-one clip rats drinking water (lK-lC-water), one-kidney rats treated with deoxycorticosterone acetate and drinking 1% NaCl (lK-DOCA-salt), and two-kidney rats drinking 1% NaCl(2K-salt) were studied after 1 to 8 weeks. The thoracic aorta was examined enface and by electron microscopy. Surprisingly, all 2K-salt, most lK-DOCA-salt (17 out of 19), and two-thirds of lK-lC-water rats (12 out of 18) had normal arterial pressure at sacrifice. In these normotensive 2K-salt, lK-lC-water, and lK-DOCA-salt animals, intimal mononuclear cells (which emigrated from the blood) increased between three- and ninefold. In these same normotensive lK-lC-water and lK-DOCA-salt rats, endothelial cell mitoses increased three- to sixfold with a corresponding increase in endothelial cell numbers. In the latter two groups, there was no evidence of endothelial cell denudation or changes in aortic circumference, and the subendothelial space widened mainly with reticular basement membrane presumably synthesized by the endothelium. In normotensive lK-DOCA-salt rats, most of the endothelial cells were thick and there were several intercellular gaps. Endothelial proliferation, synthesis of macromolecules, and gap formation, as well as increased mononuclear cell emigration, indicate functional changes in mononuclear cells and in endothelial cells. We suggest that the experimental procedures designed to produce hypertension also generate factor(s) which activates mononuclear cells and/or endothelial cells. This cellular activation leads to intimal changes independent of hypertension. o 1988AWICIICC press, IK.

INTRODUCTION With either genetic, salt-induced, renovascular, or mineralocorticoid hypertension, the intima of large arteries undergoes qualitatively similar changes. There is an increase in the number of subendothelial mononuclear cells as a result of increased emigration from the blood. Endothelial cells divide more frequently and incur morphologic changes, and the subendothelial space is widened with extracellular material (Esterly and Glagov, 1963; Still, 1967, 1968;Todd and Friedman, 1972; Schwartz and Benditt, 1977; Haudenschild et al., 1980, 1981; Limas et al., 1980, 1982; Daniel et al., 1982; De Chastonay et al., 1983; Chobanian et al., 1984; Suzuki et al., 1984; Kowala et al., 1986). These intimal changes associated with hypertension are generally presumed to be a consequence of increased arterial pressure. Our initial aim was to quantitate over time some of the cellular aspects of mineralocorticoid hypertension and to compare them with the intimal changes of renovascular and salt-induced hypertension. Although most experimental animals were not hypertensive between 1 and 8 weeks, we observed intimal changes in the aorta identical to those occurring with hypertension. Here we report these ’ Supported in part by Grants HL 25973 and HL 33529 from The National Institutes of Health. ’ Present address: Department of Clinical Sciences, University of Lowell, One University Avenue, Lowell, MA, 01854. 3 To whom correspondence and reprint requests should be addressed at Department of Clinical Sciences, College of Health Professionals, University of Lowell, One University Avenue, Lowell, MA 01854. 4 H.F.C. is a Clinician-Scientist of the American Heart Association. 171 0014-4800/88 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

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ET AL.

findings; the data suggest that some factor(s) other than elevated arterial pressure alters the intima before and presumably also during hypertension. MATERIALS Experimental

AND METHODS

Animals

One hundred four outbred Wistar male rats (8-10 weeks old, 280-340 g; Charles River Breeding Laboratories, Wilmington, MA) were maintained in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals prepared by the ILAR, NRC (DHEW Publication No. NIH 85-23, 1985), and the guidelines of the Animal Care Advisory Committee of the University of Massachusetts Medical School. The induction of renovascular hypertension was attempted by placing a stainless steel U-shaped clip (Ethicon, Sommerville, NJ; 0.2 mm internal diameter) on the left renal artery and then removing the right kidney. These one kidney-one clip rats (n = 18) drank tap water (i.e., lK-lC-water). The model of mineralocorticoid hypertension was essentially that of Gavras et al. (1975) and Yamamoto et al. (1983). Nineteen rats were unilaterally nephrectomized, and then injected subcutaneously with 30 mg/kg BW/week of deoxycorticosterone acetate (DOCA, D-7000 Sigma, St Louis, MO) mixed in sesame oil (S-3171 Sigma). These animals drank 1% NaCl (i.e., IK-DOCA-salt). Other experimental groups were (a) two-kidney animals treated with DOCA (30 mg/kg BW/week) and drinking water (2K-DOCA-water), this group (n = 7) was established to determine the effect of DOCA alone, and (b) two-kidney rats drinking 1% NaCl (2K-salt) to assess the effect of drinking salt water (n = 22). The control group (n = 38) consisted of untreated and sham operated rats with two kidneys drinking tap water (2K-water-controls). These animals were pooled together since there were no differences in the results. All animals were fed standard Purina Formula chow No. 5008 ad lib. Animals were sacrificed at the following time intervals after surgery or treatment; lK-lC-water, 1 week; 2K-salt and IKDOCA-salt, 2, 4, and 8 weeks; 2K-DOCA-water, 8 weeks; 2K-water-controls, 1, 2, 4, and 8 weeks. Mean Arterial

Pressure

The mean arterial pressure (MAP) and heart rate of each rat were determined once before sacrifice as previously described (Kowala et al., 1986). Under ether anesthesia, a polyethylene catheter (PE-50, Clay Adams, Parsippany, NJ; internal diameter, 0.58 mm; outer diameter, 0.96 mm) containing heparinized saline was inserted into the right carotid artery. The nose cone containing ether was removed, and the MAP and heart rate were continuously displayed on a Parametron 4 monitor (Roche, Cranbury, NJ) while the rat gradually regained consciousness. The MAP and heart rate were recorded when the hind leg first twitched following gentle pinching of the foot. These measurements were made either in the morning or in the afternoon. Light Microscopy

The thoracic aorta was studied en face. After MAP measurement, the thorax was opened and a 1Cgauge needle was pushed through the apex of the left ven-

INTIMAL

CHANGES

IN

PREHYPERTENSIVE

RATS

173

tricle and 3% glutaraldehyde (in 0.1 M sodium cacodylate buffer) was perfused at 110 mm Hg pressure for 5 min. Severed jugular veins provided the outflow. Thereafter the aorta was dissected out and immersed in fixative for 4 more hr and stored in 0.1 M sodium cacodylate buffer at 4°C. Four-millimeter segments of aorta were cut at the first intercostal branch, then stained with hematoxylin, and mounted as previously described (Joris et al., 1983). Three-millimeter rings of thoracic aorta (adjacent to the en face segments) were embedded in glycol methacrylate, cross sectioned at 1.5 pm, and then stained with toluidine blue. Electron

Microscopy

Rings of perfusion-fixed thoracic aorta (just distal of the en face samples) were postfixed in 1.3% osmium tetroxide, dehydrated in graded alcohols, and embedded in Epon 812 (Fullam, Latham, NY). Thin cross sections were stained with uranyl acetate and lead citrate and examined with a JEM 100s transmission electron microscope. Quantitating

Leukocyte

Emigration

and Endothelial

Proliferation

The numbers of leukocytes attached to the endothelial surface (adherent cells) and of those lying between the endothelium and the internal elastic lamella (intima1 cells) were used to gauge the extent of leukocyte emigration into the aortic intima. The aorta was examined enfuce at 400~ magnification, and adherent cells and endothelial mitoses were counted and then divided by the area of the whole specimen to give adherent cells or endothelial mitoses per square millimeter. Intimal cells in nine fields (three rows of three fields evenly positioned at 40x) were counted at 400x, added up, then divided by the total area of the nine fields (1.2 mm2) to give intimal cells per square millimeter. Endothelial nuclei in nine fields (three rows of three fields positioned at 40x) were counted at 1000x, summed, and then divided by the total area (0.2 mm2) to give endothelial cells per square millimeter. Smooth muscle cells in the intimal cushions were not counted. The circumference of the aorta (between the first and second intercostal branches) was obtained by averaging three measurements of the width of each en face specimen. For normotensive rats, an analysis of variance was applied to each variable (MAP, heart rate, intimal cells/mm2, etc.), and was followed by a Newman-Keuls test to determine significant differences among the means of control and experimental groups. RESULTS experimental rats gained weight equally except at 8 rats (410 + 30 g) were lighter than their age-matched

Control and normotensive weeks, when lK-DOCA-salt controls (486 + 49 g; P < 0.05). Mean Arterial

Pressure

and Heart Rate

The MAP of semiconscious 2K-water-controls (1, 2, 4, and 8 weeks) ranged between 90 to 119 mm Hg, and therefore rats with a MAP of greater than 120 mm Hg were regarded as hypertensive. The 2K-DOCA-water and 2K-salt rats were normotensive. The lK-DOCA-salt rats had normal MAPS, except for two animals (2 and 8 weeks), which had MAPS of 127 and 137 mm Hg. Two thirds of the lK-lC-water rats (12 out of 18) were normotensive, while the remaining lK-

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ET AL.

IC-water animals were hypertensive with pressures ranging from 120 to 141 mm Hg. There was no evidence of altered pulse width (difference between systolic and diastolic pressure) in any of the groups. The average heart rates of 2Kwater-controls and all experimental animals were normal except for 8-week IKDOCA-salt rats, in which the average heart rate significantly decreased compared to that of age-matched 2K-water-control rats (Table I). Aortic Intima-En Face The aortic intima was mainly studied in controls and in normotensive experimental animals. We will also report briefly on the hypertensive animals, as most of these data were published previously (Kowala et al., 1986). 2K-water-controls. Preparations of the thoracic aorta demonstrated that a few mononuclear cells (with an occasional neutrophil) were attached to the endothelial surface, either individually or in clusters of up to 20 cells. Intimal cells (bloodborne mononuclear leukocytes which had migrated beneath the endothelium) were more common; they were individually scattered about (Fig. 1) or clustered in small groups of up to 50 cells (Table I). During the experimental period, endothelial mitoses were consistently low; the number of endothelial cells per square millimeter decreased, while the aortic circumference increased (Table II). Normotensive 2K-DOCA-water rats. The thoracic aorta of these animals resembled the 8-week 2K-water-controls. Adherent and intimal mononuclear cells were scattered in small groups, and the numbers of endothelial mitoses and cells per square millimeter were similar to those of controls (Tables I and II). Normotensive 2K-salt rats. There were few mononuclear cells attached to the endothelial surface; however, at 4 and 8 weeks, intimal cells increased threefold compared to controls, without alterations in MAP (Table I). These subendothelial mononuclear cells were grouped in patches. Endothelial mitoses and cells per

TABLE I Average Values (+-SD) of Mean Arterial Pressure (MAP), Heart Rate, Adherent and Intimal Cells (per mm’ of Aorta) in Control and Normotensive Experimental Rats Weeks after surgery or treatment

MAP (mm Hg)

Heart rate (beats/min)

Adherent cells/mm*

cells/mm2

No. rats

1 1

107 2 7 110 f 7

363 f 39 387 -+ 37

4?3 11 f 8

22 f 13 201 k 160”

10 12

2K-water-control 2K-salt IK-DOCA-salt

101 2 8 97 k 16 loo? 11

315 f 266 356 k 8’ 348 + 20

8*8 s-c4 s-t4

49 k 39 98 rt 50 221 2 98”

8 5 6

2K-water-control ZK-salt lK-DOCA-salt

106 + 7 982 11 95 2 6

345 k 40d 370 t sd 363 r 34

6+3 8?6 14-t 11

70 + 42 226 2 105” 330 _t 98”

6 5 5

2K-water-control 2K-DOCA-water ZK-salt lK-DOCA-salt

102 107 98 93

366 356 361 288

6?5 9a4 3-t2 30 k 26a

96 143 259 808

2K-water-control lK-IC-water

aP bn =n *n en

< = = = =

0.05 compared 6. 4. 3. 11.

to age-matched

k 2 5 2

10 9 12 13

ZK-water-controls.

2 k f +

28’ 69 30e 68”

Intimal

T 2 t k

58 35 65” 151n

14 7 12 6

INTIMAL

CHANGES

IN PREHYPERTENSIVE

RATS

175

FIG. 1. Control thoracic aorta (2 weeks) enface. Blood flow is from the left to right. Endothelial cell nuclei are large, pale, and oval, whereas nuclei of underlying medial smooth muscle cells are long and thin. There is one adherent mononuclear cell attached to the endothelial surface, and one subendothelial mononuclear cell (intimal cell) with an irregularly shaped nucleus (arrow head) (Hematoxylin, X545). FIG. 2. A lK-DOCA-salt rat (8 weeks) with normal MAP. There is a dramatic increase in the number of intimal cells in the aorta compared to that of the control in Fig. 1. (Hematoxylin, x545). FIG. 3. Thoracic aorta as seen enface from a normotensive lK-DOCA-salt rat treated for 8 weeks. There are three endothelial cells undergoing mitosis (arrow heads) and many intimal cells in the aorta. (Hemotoxylin, x545).

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ET AL.

TABLE II Average Values (&SD) of Mean Arterial Pressure (MAP), Endothelial Mitoses and Cells (per mm’ of Aorta) and the Aortic Circumference (mm) in Control and Normotensive Experimental Rats Weeks after surgery or treatment

MAP (mm Hg)

Endothelial mitoses/mm2

2K-water-control lK-IC-water

1 1

107 T 7 110 + 7

0.3 I- 0.3 1.1 It 0.4”

2.516 + 399 2859 k 313”

4.9 f 0.2 5.0 f 0.3

10 12

2K-water-control ZK-salt lK-DOCA-salt

2 2 2

101 r 8 97 k 16 look 11

0.2 +- 0.2 0.6 t 0.2 0.7 2 0.7

2260 + 229 2153 -+ 229 2459 f 62

5.5 f 0.46 5.3 + 0.3 5.6 2 0.4

8 5 6

ZK-water-control ZK-salt IK-DOCA-salt

4 4 4

106 + 7 982 11 95 -1- 6

0.2 + 0.3 0.5 t 0.3 0.6 k 0.6

2029 -t 206’ 2080 2 134 2364 t 132”

5.7 k 0.56 5.7 + 0.4 5.8 f 0.2

6 5 5

2K-water-control 2K-DOCA-water ZK-salt lK-DOCA-salt

8 8 8 8

102 107 98 93

0.2 0.1 0.3 1.2

18% 1987 2042 2475

6.1 6.2 5.7 6.0

(1P < 0.05 compared b P < 0.05 compared cl8 = 5.

+ f 2 ‘-

10 9 12 13

2 t + 2

0.3 0.1 0.2 0.5”

Endothelial cells/mm2

k + 2 2

126’ 191 128 163”

Circumference (mm)

2 ‘+ k

0.5* 0.4 0.3 0.2

No. rats

14 7 12 6

to age-matched ZK-water-controls to l-week ZK-water-controls.

square millimeter and aortic circumference remained within the control values (Table II). Normotensive IK-IC-water rats. The number and distribution of adherent mononuclear cells was unchanged compared to those of controls, while the number of intimal mononuclear cells increased ninefold at 1 week (Table I). The subendothelial mononuclear cells were grouped in patches in the thoracic aorta. Dividing endothelial cells increased threefold with a corresponding increase in the number of endothelial cells. The circumferences of the thoracic aortae were similar to those of controls and there was no endothelial denudation (Table II). Normotensive IK-DOCA-salt rats. In the thoracic aorta, the distribution and the average number of adherent cells were similar to those of controls at 2 and 4 weeks; however, by 8 weeks their number increased fivefold. These cells were mostly mononuclear (with a few neutrophils) and were attached either individually or in groups of up to 50 cells. Intimal mononuclear cells in the early stages were found in clusters, and later they were widespread (Fig. 2). At 2, 4, and 8 weeks cell counts revealed increases of five-, five-, and eightfold, respectively compared to age-matched 2K-water-controls (Table I). Five animals at 2, 4, and 8 weeks also had several adherent and intimal basophils. Initially, the number of dividing endothelial cells varied considerably between animals; by 8 weeks mitotic figures were six times greater compared to controls and the number of endothelial cells had increased significantly at 4 and 8 weeks (Fig. 3, Table II). The aortic circumferences were similar to those of controls and endothelial denudation was not observed. Aortic Intima-Sectioned Material In 2K-water-controls (l-8 weeks; Fig. 4), 2K-salt (2-8 weeks), and 2KDOCA-water rats (8 weeks), l.Q.~m sections of the thoracic intima showed that the endothelium rested for long segments directly on the internal elastic lamella. There were a few areas of focal widening in the intima due to “vacuoles” origi-

INTIMAL

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IN PREHYPERTENSIVE

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177

FIG. 4. Cross section of a control thoracic aorta (8 weeks) showing the endothelium resting on the internal elastic lamella. (Methacrylate section, toluidine blue, x690). FIG. 5. Thoracic aorta from a normotensive lK-DOCA-salt rat (8 weeks). The endothelial cells and their nuclei are thicker compared to those of the control in the previous figure. The subendothelial space is wide and contains large amounts of hyaline material as well as four intimal cells. (Methacrylate section, toluidine blue, x690).

nating from medial smooth muscle cells (myointimal herniae) or a rare subendothelial mononuclear cell (intimal cell). Occasionally, a small amount of hyaline material was present beneath the endothelium; by electron microscopy the material consisted of reticular basement membrane and a few collagen fibers. The normotensive lK-lC-water rats (1 week) showed a slight widening of the subendothelial space along the entire circumference. The intima contained hyaline material, mononuclear cells, and myointimal herniae. Electron microscopy revealed that the extracellular matrix consisted of multiple layers of basement membrane, scanty elastin, and occasional collagen fibers. A few endothelial cells were thick and contained large amounts of rough endoplasmic reticulum and ribosomes. In the normotensive lK-DOCA-salt rats, the intima was unevenly widened with hyaline material after 2 and 4 weeks of treatment. By 8 weeks, the entire subendothelial space had expanded and comprised small dark masses, hyaline material, mononuclear cells, and myointimal herniae. Most endothelial cells and their nuclei had thickened (Fig. 5). By electron microscopy, the wide subendothelial space contained large amounts of reticular basement membrane, elastin, a few collagen fibers, and crystalline masses probably corresponding to crystallized hemoglobin and fibrin with periodicities of either 90 A or 190 8, (Fig. 6). Endothelial cells had some nuclear folding, increased amounts of rough endoplasmic reticulum, and ribosomes. Sometimes stress fibers or microtendons were seen along the abluminal side of the endothelium. Several intercellular gaps were seen at 2 and 8 weeks (Fig. 7).

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INTIMAL

n

CHANGES

IN

PREHYPERTENSIVE

RATS

179

‘r ..*

FIG. 7. Electron micrograph of a lK-DOCA-salt rat (2 weeks) with normal MAP. There is a gap between two endothelial cells. Beneath the endothelium is reticular basement membrane and the endothelial basal lamina rests on the internal elastic lamella. (X 16,500).

Intimal Changes in Hypertensive Rats At 1 week, hypertensive lK-lC-water rats had an l&fold increase in the number of intimal mononuclear cells compared to age-matched controls. Endothelial mitoses was elevated g-fold and the number of endothelial cells increased significantly. Intimal mononuclear cells, and the number of endothelial mitoses and cells were also elevated in the two hypertensive lK-DOCA-salt rats. None of the hypertensive animals lost endothelium or had changes in the aortic circumference. By light and transmission electron microscopy, the aortae from hypertensive lK-lC-water rats and the two hypertensive lK-DOCA-salt animals were identical to their normotensive counterparts. DISCUSSION Monitoring MAP-A Critique Ideally MAP should be continuously monitored via indwelling catheters in conscious unrestrained rats. Recent evidence suggests that indwelling catheters in the abdominal aorta of normal rats induce an increase in the number of intimal mononuclear cells upstream in the thoracic segment (Gordon et al., 1981; Kowala and Nicolosi, 1987). It appears that temporary direct measurements or the indirect tail-cuff method are the best options to measure arterial pressure. Monitoring MAP in semiconscious 2K-water-controls indicated that our values were close to those of normal conscious rats (i.e., average, 108 mm Hg) (Gordon et al., 1981; Butiag and Butterfield, 1982; Badonnel et al., 1983; Kowala and Nicolosi, 1987). In addition, the same monitoring procedure was able to demonstrate hypertension in lK-lC-water animals, in lK-DOCA-salt rats, and in those with their aorta coarcted (Kowala et al., 1986). This validates the pressure levels obtained in the semiconscious experimental animals. The MAPS of 2K-DOCA-water, 2K-salt, and most lK-DOCA-salt rats were similar to those of controls; however, since pressure was monitored once per animal, it is possible that they were intermittently hypertensive. We feel that this is unlikely, as a total of 46 (out of 48) of these treated animals were recorded as normotensive. The lack of response to unilateral nephrectomy, salt, and DOCA treatment was unexpected since a similar protocol used by Gavras et al. (1975) and by Yamamoto et al. (1983) produced hypertension after 2, 4, and 8 weeks in Wistar rats. The only difference was that at the beginning of treatment our rats were older (starting weight, 280-340 g) than the animals used by the other authors

180

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ET Al.,.

(160-270 g). The reduced susceptibility of adult rats to IK-DOCA-salt-induced hypertension compared to weanling or prepubertal rats (Zicha et al., 1986) may explain the resistance of our animals. In the case of the IK-lC-water rats, our published (Kowala and Nicolosi, 1987) and unpublished observations in chronically catheterized lK-IC-water animals indicate that during the first week after surgery some rats rapidly develop hypertension, while others remained normotensive or had mild intermittent hypertension. This recent study suggests that the lK-lC-water rats whose MAPS were monitored once were either normotensive or had mild intermittent hypertension. The fact that all lK-1C rats become hypertensive by 4 weeks (Kowala et al., 1986) confirms the existence of a prehypertensive phase in this model. Emigration of Mononuclear Cells Increased mononuclear cell migration into the arterial intima is normally associated with hypertension (Esterly and Glagov, 1963; Still, 1967, 1968; Todd and Friedman, 1972; Haudenschild et al., 1980, 1981; Limas et al., 1980, 1982; Meairs et al., 1984; Chobanian et al., 1984; Suzuki et al., 1984; Kowala et al., 1986), yet in our normotensive 2K-salt, IK-lC-water, and IK-DOCA-salt rats this phenomenon also occurred. This suggests that mononuclear cell diapedesis occurred independently of chronic hypertension. In 2K-salt rats, the number of intimal cells stabilized by 8 weeks indicating that mononuclear cell emigration into the aorta was mild and transient. We have demonstrated that in the IK-1C model there is an early burst of leukocyte invasion, which ultimately levels off (Kowala et al., 1986). In IK-DOCA-salt rats, there was a rapid and continued increase of intimal cells; the number of adherent cells was elevated only at 8 weeks, suggesting that mononuclear cell invasion into the intima started early and eventually became heavy. As for the mechanisms behind increased mononuclear cell emigration in the normotensive experimental rats, data from the literature suggest several possibilities. Stimulated macrophages secrete interleukin 1 (Oppenheim and Gery, 1982) and endothelium treated with interleukin 1 is more sticky for leukocytes (Bevilacqua et al., 1985; Cavender et al., 1986). Basic fibroblast growth factor extracted from macrophages and from kidney homogenates stimulates arterial endothelial cells to divide in vitro (Baird et al., 1985a, b), and proliferating endothelial cells in coronary artery collaterals appear to facilitate monocyte adhesion (Schaper et al., 1976). Therefore, cell-derived factors may increase endothelial cell stickiness for mononuclear cells and enhance leukocyte emigration. Intimal Changes in the Aorta In 2K-water-controls, the rate of endothelial turnover is small and constant, while the diameter of the thoracic aorta increases gradually. This results in the reduction of endothelial cell density with time. During hypertension, there is an increase in endothelial mitoses (Schwartz and Benditt, 1977; Schwartz and Standaert, 1982; Daniel et al., 1982; De Chastonay, 1983) and an increase in their cell number (Haudenschild et al., 1981). In the normotensive IK-lC-water and lKDOCA-salt rats, there was also an increase in endothelial mitoses which in turn elevated the density of the endothelial cells. Since there was no evidence of endothelial denudation or cell death, and the aortic circumferences remained similar to those of controls, it is unlikely that endothelial proliferation was in

INTIMAL

CHANGES

IN PREHYPERTENSIVE

RATS

response to stretching of the arterial wall (Schwartz and Benditt, al., 1982) or as a result of hemodynamic damage to the endothelium Benditt, 1977; De Chastonay et al., 1983).

181 1977; Daniel et (Schwartz and

Another cellular event described in hypertension is thickening of the endothelial cells (Still, 1967; Htittner et al., 1982; Haudenschild et al., 1980). In our material, endothelial thickening was particularily apparent in the lK-DOCA-salt animals with normal MAP; the endothelial cells also contained increased amounts of rough endoplasmic reticulum and ribosomes, some of these cells had nuclear folding and there were several intercellular gaps. The presence of intercellular gaps in the endothelial lining may explain the increased permeability of the aorta to fibrinogen and red blood cells (Still, 1967,1968; Haudenschild et al., 1980,198l; Limas et al., 1980, 1982; Kowala et al., 1986). However the mechanism of gap formation remains unclear since electron micrographs did not suggest endothelial contraction. Widening of the subendothelial space with extracellular matrix is another histological feature of hypertension (Esterly and Glagov, 1963; Still, 1967, 1968; Todd and Friedman, 1972; Haudenschild et al., 1980, 1981; Limas et al., 1980, 1982; Meairs et al., 1984; Chobanian et al., 1984; Suzuki et al., 1984) and this change was also observed in the normotensive lK-lC-water and lK-DOCA-salt rats. Increased permeability (Esterly and Glagov, 1963; Htittner et al., 1970, 1973, 1979; Gabbiani et al., 1979; Haudenschild et al., 1980) via the intercellular gaps and elevated synthesis of macromolecules by the endothelium (McGuire and Twietmeyer, 1985; Kowala et al., 1986) are possible mechanisms behind the intimal thickening. In the literature there is further evidence of arterial changes occurring without hypertension. (a) Mononuclear cell emigration and widening of the subendothelial space progress with age in untreated pigs and rats (French et al., 1963; Gerrity and Cliff, 1972; Guyton et al., 1983; Haudenschild et al., 1981) with presumably normal blood pressure. (b) In Dahl salt-sensitive ruts, widening of the subendothelial space correlated better with the duration of the 8% NaCl diet than with blood pressure (Limas et al., 1982). (c) Normotensive one-kidney rats fed 8% NaCl(7 days) had increased endothelial turnover (De Chastonay et al., 1983). (d) In hypertensivelhypercholesterolemic baboons, it was suggested that some factor other than increased arterial pressure affects atherogenesis (McGill et al., 1985). (e) Intimal changes occurred in normotensive rats with chronic indwelling catheters (Gordon et al., 1981; Kowala and Nicolosi, 1987). Cellular

Changes-The

Mechanisms

The most dramatic changes occurred in one-kidney rats with renal ischemia, and in those animals subjected to the combination of unilateral nephrectomy, chronic salt consumption, and DOCA treatment. Salt water on its own mildly increased mononuclear cell emigration, while DOCA alone had no effect. We suggest that in salt-loaded, renovascular, and mineralocorticoid models, the manipulations designed to initiate hypertension may affect the kidney to generate factor(s) which “activate” the endothelium and/or the mononuclear cell. By “cellular activation” is meant a shift to a higher level of activity (Adams and Hamilton, 1984). Therefore activation of mononuclear and/or endothelial cells may lead to increased mononuclear cell emigration, endothelial synthesis of macromolecules which widens the intima (Kowala et al., 1986), and endothelial proliferation. The nature of the renal factor(s) is speculative. In the lK-lC-water model, the

182

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plasma levels of renin are elevated for at least 2 days after surgery (Bianchi and Ferrari, 1983; Norman er al., 1984) and coincide in part with some of the intimal events. With mineralocorticoidand salt-induced hypertension, plasma levels of renin, angiotensin II, and aldosterone are low (Haack et al., 1977; Gabbiani et al., 1979; Bianchi and Ferrari, 1983), although increased production of angiotensin II by the aorta (Okamura et al., 1986) may have local regulatory functions (Dzau, 1986), and also induce leukocyte chemotaxis (Goetzl et al., 1980). Elevated levels of antidiuretic hormone in the lK-DOCA-salt rat (Bianchi and Ferrari, 1983; Ouchi et al., 1987) may also have a stimulatory effect on endothelial cells (Brock et al., 1986).

In summary, we found the following changes in the aortic intima of normotensive 2K-salt, IK-lC-water, and lK-DOCA-salt rats: (1) mononuclear cell emigration, (2) endothelial proliferation, (3) altered endothelial structure, (4) interendothelial gaps, and (5) endothelial synthesis of macromolecules, and these observations are in line with other reports on pressure-independent events. The intimal changes reflect altered cell function; thus we cautiously hypothesize that the manipulations intended to cause hypertension initiate the release of renal factor(s) which activates the endothelium and/or the mononuclear cell. Cellular activation leads to intimal changes which are independent of chronic hypertension; therefore, it is suggested that lowering blood pressure may not entirely prevent the development of intimal lesions. ACKNOWLEDGMENTS We are indebted to Jean M. Underwood and Eva Moring for technical help, to Christopher D. Hebert for the photographic prints, and to Jane M. Manzi and Karen A. Ginese for the preparation of the manuscript.

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