Endothelial Injury in Transgenic (mRen-2)27 Hypertensive Rats

AJH 1997; 10:51 – 57

Endothelial Injury in Transgenic ( m Ren-2 ) 27 Hypertensive Rats William B. Strawn, Patricia Gallagher, Richard H. Dean, Detlev Ganten, and Carlos M. Ferrario

Transgenic [ ( mRen-2 ) 27 ] rats develop severe hypertension as the result of transfection with the mouse Ren-2 gene. This study tested the hypothesis that hypertensive [ ( mRen-2 ) 27 ] rats have increased endothelial dysfunction by examining the extent of vascular endothelial cell injury and turnover within the thoracic aorta of age-matched female transgene positive [ Tg ( " ) ] and transgene negative [ Tg ( Ï ) ] littermates. Transgenic hypertensive rats had arterial pressures significantly higher than Tg ( Ï ) animals, but no differences in heart rate or body weight. The extent of endothelial cell injury was estimated in Hau¨ tchen preparations of thoracic aorta endothelium by counting cells immunostained for the presence of cytoplasmic immunoglobulin G ( IgG ) at sites with or without intercostal artery branches. Both Tg ( " ) and Tg ( Ï ) littermates had a greater percentage of injured endothelial cells at branch sites than at nonbranch aorta ( P õ .01 ) . However, the number of vascular endothelial cells staining positively for IgG was significantly higher in hypertensive rats both at sites away from ( P õ .05 ) and in the immediate vicinity of ( P õ .1 ) the orifices of intercostal arteries. En face preparations of the thoracic aorta were also examined for cells

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incorporating 5-bromo-2 *-deoxyuridine ( BrdU ) to estimate the percentage of endothelial cells undergoing replication. There was no difference in endothelial cell replication at either branch or nonbranch sites between hypertensive and normotensive rats. However, the percentage of endothelial cells undergoing replication at branch sites in both Tg ( " ) and Tg ( Ï ) rats was significantly greater than at nonbranch sites ( P õ .01 ) . These data provide the first demonstration for the effects of high blood pressure on the vascular endothelium of a monogenetic model of hypertension produced by increased activity of the renin-angiotensin system. The divergent effects of this form of hypertension on vascular endothelial injury and endothelial turnover suggest that the decrease in the reparative capacity of the vascular endothelium induced by the combination of hypertension and associated angiotensinemia may contribute to the endothelial dysfunction accompanying vascular remodeling. q 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997; 10:51 – 57 KEY WORDS: Endothelial cell injury, endothelial cell replication, aorta, transgenic rat, TGR ( mRen2 ) 27, hypertension.

ncreasing evidence suggests the participation of the vascular endothelium in the pathophysiology of hypertension.1,2 The presence of injured endothelial cells accompanying hypertension heralds

a dysfunction of the endocrine role of the vascular endothelium in the modulation of vascular smooth muscle cell function and growth.3 In many models of genetic hypertension, endothelial injury and replica-

Received April 1, 1996. Accepted July 1, 1996. From The Hypertension Center, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina. The results of this study were presented in part at the Tenth Scientific Sessions of The American Society of Hypertension, May 1995.

This study was supported by a grant from the National Institutes of Health, HL-51952. Address correspondence and reprint requests to William B. Strawn, The Hypertension Center, The Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1032.

q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.

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tion are positively correlated and augmented during the accelerating phase of arterial hypertension.4 – 6 When Schwartz and Lombardi 7 compared the replicative activity of the vascular endothelium in spontaneously hypertensive rats ( SHR ) receiving antihypertensive agents, the decreased cell replication in the vascular endothelium of treated animals suggested that cell replication was elevated during chronic hypertension. In agreement with these findings, Wu et al 8 reported that both endothelial injury and replication were significantly increased in the aortas of adult SHR compared to Wistar-Kyoto ( WKY ) rats. Their study suggested that areas with elevated endothelial replication may be predisposed to increased transendothelial transport of plasma macromolecules. The relative contribution of hormonal and hemodynamic factors to the production of hypertension-mediated vascular endothelial dysfunction is receiving greater attention. The introduction of an animal model in which chronic hypertension results from the insertion of the mouse Ren-2 gene into the genome of normotensive rats [ ( m Ren-2 ) 27 ] afforded us the opportunity to assess the impact of the hypertensive stress and increased angiotensin II ( Ang II ) production on the vascular endothelium. To the extent that innate differences in endothelial function are present in prehypertensive SHR, 9,10 investigation of these mechanisms in this form of transgenic hypertension could clarify the contribution of genetic influences to the adaptive vascular remodeling process associated with high blood pressure. Germane to this idea, Schiffer et al 11 showed that endothelial denudation did not change medial DNA synthesis in the arteries of SHR, whereas medial DNA synthesis was significantly higher in the aorta of WKY rats following aortic coarctation-induced hypertension. These investigators suggested that the lack of response in SHR was a prehypertensive modification unique to this strain and unrelated to the vascular adaptation mediated by the elevated blood pressure. With this in mind, the objective of the present study was to test the hypothesis that elevated blood pressures characteristic of transgenic [ ( m Ren-2 ) 27 ] rats increase endothelial dysfunction. As a marker of endothelial cell dysfunction, the extent of aortic endothelial cell injury in transgenic [ ( m Ren-2 ) 27 ] rats was examined by determining the percentage of endothelial cells with cytoskeleton-bound plasma immunoglobulin G ( IgG ) .13 To explore the effects of hypertension on the proliferative ability of the vascular endothelium, nuclear incorporation of the thymidine analog, 5-bromo-2 *-deoxyuridine ( BrdU ) , into replicating endothelial cells was similarly measured.

MATERIALS AND METHODS Animals Ten female, 16-week-old transgene positive [ Tg ( / ) ] rats ( 309 { 7 kg body weight ) were compared

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to 10 age-matched transgene-negative [ Tg ( 0 ) ] littermates ( 312 { 7 kg body weight ) . Littermates were obtained from the Hypertension Center Transgenic Animal Resource through the cross-breeding of hemizygous [ ( m Ren-2 ) 27 ] transgenic rats with Hannover Sprague-Dawley rats. Founder homozygote [ ( m Ren2 ) 27 ] rats were provided by Dr. Ganten from stock developed in Germany.12 Hannover Sprague-Dawley rats were acquired from the Zentralinstitut fu¨r Versuchstierkunde, Hannover, Germany. Tg ( / ) and Tg ( 0 ) littermates were identified by polymerase chain reaction analysis of ear-punch biopsies using primers specific for the mouse Ren-2 gene.12 All animals were housed in identical conditions with a 12 h:12 h light – dark cycle in an environment with controlled temperature and humidity. A standard rat chow diet ( Rodent Laboratory Chow 5001, Purina Mills, Richmond, IN ) containing 17 mEq of Na / and 28 mEq of K / / 100 g solid weight was provided with tap water ad libitum. Twenty-four hours before experiments, one tablet containing 50 mg of BrdU ( Boehringer-Mannheim, Indianapolis, IN ) was implanted subcutaneously in all animals under light ether anesthesia.

Experimental Procedure Experiments were performed while the rats were anesthetized with sodium pentobarbital ( 50 mg / kg, intraperitoneally ) . The abdominal aorta was cannulated with an 18-gauge angiocatheter (Angiocath, Beckton Dickinson Vascular Division, Sandy, UT ) and their arterial pressure was recorded with a solid state pressure transducer ( Uniflow, Baxter Health Care Co., Irvine, CA ) connected to a polygraph recorder ( Model 7, Grass Instruments, Quincy, MA ) . Heart rate was displayed on the chart from the output of an electronic tachometer triggered by the rising stroke of the arterial pressure pulse waveform. At the completion of this recording period, the rats were given a bolus injection of sodium pentobarbital ( 10 mg / kg, intravenously ) , and a solution of saline connected to a pressure reservoir was infused into the aorta at a pressure of 100 mm Hg and a flow rate of 20 mL /min. The perfusion continued until clear fluid appeared at the egress site in a jugular vein. The perfusate was changed to 10% phosphate-buffered formalin administered at the same pressure and flow rate for an additional 10 min of perfusion fixation. The thoracic segment of the aorta was excised and immersed in 10% formaldehyde for 1 h before processing for detection of injured and replicating endothelial cells. Assessment of Endothelial Cell Injury The immunocytochemical technique to detect cytoplasmic vimentinbound IgG developed by Hansson and Schwartz 17 was used to quantify the presence of injured vascular endothelial cells in the thoracic aortic. Irreversibly injured endothelial cells were identified by combining immunohistological staining of autologous IgG and Hau¨tchen preparation of the thoracic aorta endothelium, as de-

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scribed previously.18 After fixation, the thoracic aorta containing the first two pairs of intercostal arteries was cut longitudinally and pinned flat on a square of Teflon with the luminal side exposed. The endothelial surface was incubated with rabbit anti-rat IgG ( 1:1000 dilution, Vector Laboratories, Burlingame, CA ) as the primary antibody, followed by incubation with biotin-labeled goat anti-rabbit IgG ( Vector ) , and, lastly, a peroxidaseconjugated avidin:biotin complex ( Vectastain Rabbit IgG Elite ABC kit, Vector ) . The peroxidase reaction was visualized with 0.02% hydrogen peroxide added to an equal volume of 0.1% diaminobenzidine tetrachloride ( DAB ) in 0.1 M Tris buffer, pH 7.2. Analysis of Endothelial Cell Replication The section of the thoracic aorta containing the third and fourth intercostal branch pairs was used to assess endothelial replication. The presence of endothelial nuclei in S-phase was identified by incorporation of BrdU.19 The section was pinned flat, immersed in 4N HCl and then rinsed in 0.1 mol / L Na-tetraborate, pH 8.5. After rinsing several times in PBS, the endothelial surface was incubated with a primary mouse monoclonal antibody to BrdU ( 1:10 dilution, Boehringer-Mannheim, Indianapolis, IN ) , followed by incubation with a biotinylated horse anti-mouse IgG ( Vector ) and, finally, an avidin:biotin peroxidase complex ( Standard Vectastain Elite ABC kit, Vector ) . Peroxidase conjugate was visualized with hydrogen peroxide and DAB, as described previously.

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counted in nonbranch regions of the aorta, and at intercostal artery branches. In all cases Tg ( / ) rats showed increased IgG staining at aortic intercostal artery branches when compared with Tg ( 0 ) littermates. Results from quantification of the Hau¨tchen preparations are summarized in Figure 3. In Tg ( 0 ) animals, IgG-positive endothelial cells represented 0.22 { 0.06% and 2.32 { 0.57% in nonbranch and branch sites, respectively. In contrast, Tg ( / ) rats showed a statistically significant increase in the occurrence of endothelial injury at both nonbranch ( 0.79 { 0.08%, P õ .05 ) and branch sites ( 8.28 { 0.89%, P õ .01 ) . Differences in the number of IgG positive endothelial cells between nonbranch and branch regions of the thoracic aorta of Tg ( / ) rats were also statistically significant ( P õ .01 ) . En face preparations of the proximal aorta were used to estimate endothelial cell replication by counting BrdU-positive endothelial nuclei at branch and nonbranch aorta ( Figure 4 ) . In contrast to the differences observed in endothelial cell injury, no significant differences in endothelial replication were observed between Tg ( / ) and Tg ( 0 ) littermates, though endothelial replication was greater at branch ( 0.100 { 0.018% and 0.073 { 0.015%, respectively ) than at nonbranch aorta ( 0.032 { 0.014% and 0.029 { 0.012%, respectively ) in both groups ( Figure 3 ) . Moreover, a significant positive correlation was observed at branch aorta sites between endothelial injury and replication in both Tg ( / ) ( r Å

Quantification of Endothelial Injury and Replication The number of IgG-positive cells determined in the thoracic aorta at both intercostal artery branches and at sites without branches ( Figure 1 ) was expressed as a percentage of the total number of cells investigated, as described previously.18 The results obtained from each animal were averaged, and the average value was used in the statistical analysis.

Statistical Analysis Values are expressed as means { SEM. Analysis of variance ( ANOVA ) was used to test whether the hemodynamic effects of blood pressure or the presence of the mRen-2 gene were statistically significant. Student’s t test for paired observations were used to test whether there were significant differences between branch and nonbranch regions. RESULTS Table 1 summarizes the characteristics of the hypertension in the group of [ ( m Ren-2 ) 27 ] transgenic rats. As in other studies, 25,35 hypertension was associated with no changes in heart rate when compared to Tg ( 0 ) rats.

Endothelial Cell Injury and Replication in [(mRen2)27] Rats Representative examples of Hau¨tchen preparations of the thoracic aorta endothelium from Tg ( 0 ) and Tg ( / ) littermates are shown in Figure 2. Endothelial cells staining positively for IgG were

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FIGURE 1. IgG-positive and BrdU-positive endothelial cells were counted at branch and nonbranch regions of the aorta in Tg ( / ) and Tg ( 0 ) littermates after perfusion fixation and immunohistochemistry. Branch aorta was defined as an area 1.0 mm in diameter from the center of the lumen of a branching intercostal artery of the aorta. For this purpose, an eyepiece with a calibrated grid and circle was used. A calibrated square grid was used to define the area to be counted in the unbranched aorta. IgG-positive cells were counted for each of the four branches of the two most proximal pairs of intercostal arteries. BrdU-positive cells were similarly counted for the next two pairs of intercostal arteries. Cell density ( cells / field ) was determined from the average cell number obtained from four fields in the branch areas and 10 fields in the non-branch areas. IgG- and BrdU-positive cells were counted until a total of 100 cells of each were identified, or a minimum of 100,000 cells were investigated. The cells were counted at 1200 magnification.

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TABLE 1. HEMODYNAMIC CHARACTERISTICS OF HEMIZYGOUS [(mRen-2)27] RATS Transgene Negative

Transgene Positive

129 { 8 110 { 7

169 { 6* 145 { 7*

92 { 8 335 { 6

123 { 8* 338 { 5

Systolic blood pressure, mm Hg Mean blood pressure, mm Hg Diastolic blood pressure, mm Hg Heart rate, beats/min

tritiated thymidine ( 3H-TdR ) , was not different between SHR and WKY rats. In their studies, however, an increase in endothelial replication was observed after discontinuation of antihypertensive therapy in hypertensive, but not normotensive, strains. In contrast, Wu et al 8 found that endothelial replication, estimated by endothelial cells in the mitotic phase of cell division, was increased in adult untreated SHR com-

Values are means { SEM for arterial pressure and heart rate in the two groups of transgenic negative (n Å 10) and positive (n Å 10) rats after anesthesia. *P ° .001 of differences between groups.

0.69, P õ .05 ) and Tg ( 0 ) littermates ( r Å 0.48, P õ .05 ) . No correlation was found between injury and replication at nonbranch sites in either type of littermate.

DISCUSSION This study is the first report examining the extent and location of endothelial injury in the thoracic aorta of transgenic hypertensive [ ( m Ren-2 ) 27 ] rats. Endothelial cell injury was significantly increased at both aortic branches and nonbranch sites in transgene-positive animals compared to transgene-negative littermates. This finding both confirms and extends previous reports showing increased endothelial injury in other rodent models of genetic hypertension.4 – 6 The demonstration that endothelial turnover is not significantly augmented in Tg ( / ) rats in the presence of vascular endothelial injury is a novel finding which requires further investigation. A previous report by Bachmann et al 20 suggested that hypertension in [ ( m Ren-2 ) 27 ] rats was not associated with histological evidence of aortic endothelial damage. The techniques used in our study detected endothelial cell injury of Tg ( / ) rats with a greater sensitivity than the morphological analysis of the vascular wall used by these authors. Using the same techniques employed in the present study, Hansson and Schwartz 17 found that injured endothelial cells comprised 0.19 { 0.04% of total endothelial cells in the aortas of normotensive Sprague-Dawley rats. These data agree closely with the 0.23 { 0.11% we found in normotensive Tg ( 0 ) littermates. The percentages of injured cells in Tg ( / ) littermates were similar to those reported by Strawn et al 18 in cynomolgus monkeys exposed to a sustained social stress and by Pettersson et al 21 in hypertensive rabbits. Although hypertension in this genetic model of high blood pressure was accompanied by increased endothelial injury, the replicative activity of the endothelium in the same regions of the thoracic aorta was not augmented. This finding agrees with the observations of Schwartz and Lombardi, 7 who found that endothelial replication, estimated by incorporation of

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FIGURE 2. Details of an IgG-positive cell ( a ) in the proximal thoracic aorta ( original magnification 2001 ) and intercostal artery branch ostia and nonbranch aorta in Tg ( 0 ) ( b ) and Tg ( / ) ( c ) littermates ( original magnification 201 ) . Endothelial injury was markedly increased in this transgene-positive littermate.

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The marked concentration of endothelial cell injury in the branch regions in the aortas of both Tg ( / ) and Tg ( 0 ) littermates is in agreement with the distribution and frequency of endothelial injury occurring at branches in SHR and WKY rats.7,8 Although Wu et al 8 were unable to show any difference in injury between SHR and WKY at nonbranch regions, there is evidence for altered endothelial structure occurring early in life 29,30 and independently of blood pressure in SHR.10

FIGURE 3. Endothelial Cell Injury: Data are expressed as means { SEM of the frequency of injured endothelial cells as a percentage of total number of cells in branch and nonbranch aorta in Tg ( 0 ) and Tg ( / ) littermates. The percentage of injured endothelial cells was greater at both branch and nonbranch aorta in Tg ( / ) littermates than in Tg ( 0 ) littermates. Endothelial Cell Turnover: Endothelial replication was represented as the percentage of BrdU-positive endothelial nuclei counted at branch and nonbranch aorta in Tg ( / ) and Tg ( 0 ) littermates. There were no differences in the percentages of BrdU-positive cells between Tg ( / ) and Tg ( 0 ) littermates, although the percentage of BrdUpositive cells was greater at branch aorta than nonbranch aorta in both groups.

pared to WKY rats. As commented by Schwartz and Lombardi, 7 the relation between endothelial injury and turnover is not a linear phenomena. The morphological and morphometrical techniques employed by Wu et al 8 to assess endothelial cell replication are not readily comparable to the techniques used by us. In our study, we used nuclear incorporation of BrdU instead of 3H-TdR to indicate replicating endothelial cells in the S-phase of mitosis. While the accuracy of the BrdU immunohistochemical technique is equivalent to that of 3H-TdR autoradiography, 19 the morphological technique employed by Wu et al 8 may account for endothelial cells in phases of mitosis other than the S-phase. Whereas our study defined branch regions as the area within a circle 1 mm in diameter, Wu et al 8 used a 2.5 mm square. By comparison, the smaller diameter used to assess injury at branches in the present study reduced the number of potential BrdU-positive cells at intercostal branches, and correspondingly increased those in nonbranch areas.

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FIGURE 4. Detail ( original magnification 2001 ) of a BrdUpositive endothelial cell nucleus ( a ) in the proximal thoracic aorta ( b ) of a Tg ( 0 ) littermate ( original magnification 401 ) , and branch aorta ( c ) in a Tg ( / ) littermate ( original magnification 401 ) .

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As discussed by Schiffers, 11 multiple factors contribute to the vascular remodeling associated with hypertension. The hemodynamic stress accompanying the adaptive response may be modulated by neurohormones, growth factors, extracellular matrix components, cell density, and cellular phenotypes. Germane to this interpretation, it was shown that the expression of Ang II receptor subtypes is regulated differently in the aorta of SHR than in WKY rats.28 It is therefore likely that alterations in function of the SHR endothelium are represented in part by a genetic alteration preceding the accelerated phase of hypertension rather than a response of the endothelium to sustained hypertension. It was suggested that the breeding schemes used in a variety of facilities to produce the inbred SHR are likely responsible for these endothelial adaptations.4 In contrast to inbred rodent models of hypertension, [ ( m Ren-2 ) 27 ] hypertensive rats were created by the introduction of multiple copies of the Ren-2 gene into an outbred rat stock.12 Therefore, changes in endothelial function due to inbreeding may be excluded from further consideration. The role of the RAS in endothelial injury and replication in hypertension is not well understood, though Ang II can induce increased endothelial permeability in small arteries and the aorta.22 The majority of studies implicating the RAS in the vascular response to endothelial injury have addressed the proliferative effects of Ang II on vascular smooth muscle cells.23,24 Few studies have addressed the effect of Ang II on endothelial injury and function in hypertension. Recently, Stoll et al 16 demonstrated in cultures that Ang II induced proliferation of endothelial cells in the coronary artery of Sprague-Dawley rats pretreated with an AT 2 receptor antagonist, and that this effect was reversed by additional pretreatment with an AT 1 receptor antagonist. Their results suggest that the growth modulating effects of Ang II are dependent of the receptor types located on a particular cell, and that AT 2 receptor stimulation has an antimitogenic effect on endothelial cells. Though plasma and tissue Ang II levels were not measured in the present study, we 25 previously reported that this form of transgene hypertension is associated with increased levels of circulating Ang II. Campbell et al 15 recently reported increased Ang II levels in all tissues studied, including the aorta of [ ( m Ren-2 ) 27 ] homozygous hypertensive rats. Although Hilgers et al 26 found no increases in plasma Ang II, vascular formation of Ang II was significantly augmented. We assessed the possibility that the low replication percentages observed in our animals contributed to the absence of a significant difference in replication between Tg ( / ) and Tg ( 0 ) littermates. To provide an accurate estimate of endothelial replication, the number of cells counted must be large enough to provide

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a statistically significant measurement. Since it is apparent that the number of BrdU-labeled cells appearing after immunostaining is typically small, a large number of endothelial cells must be available for counting, an advantage of the Hau¨ tchen technique employed in our study. From the binomial distribution, with a BrdU percentage of 0.3% and a cell count of 100,000 cells, the 95% confidence limits are {0.02%. This suggests that the total cell counts in this study were large enough to give accurate estimates of the occurrence of labeled cells, even with the low BrdU values observed. It is clear that endothelial cell function is altered in vivo by changes in blood pressure and blood flow dynamics 29 and that the RAS is involved.30,31 Exposure of endothelial cells to increased fluid flow in vivo, 32 in perfused vessels 33 or in vitro 34 stimulated the production of nitric oxide. Whether endothelial injury results in diminished endothelial release of nitric oxide is not known, though in rats with hypertension induced by aortic coarctation, the depression of endothelium-dependent, nitric oxide-mediated dilation of large distensible arteries is strictly localized to areas exposed to the enhanced blood pressure.32 A specific endothelial role for the RAS in the [ ( m Ren2 ) 27 ] rats was reported by us in previous studies. 35,36 In addition, Tschudi et al 37 found a dramatic decrease in basal endothelium-derived nitric oxide release in the coronary arteries of [ ( m Ren-2 ) 27 ] transgenic rats. In summary, the major finding of the present study is that endothelial cell injury in transgenic hypertensive rats is increased without a concomitant increase in endothelial cell replication. These data suggest that in this rat model of genetic hypertension, components of either the local or circulating RAS contribute in part to growth modulation of the aortic endothelium, and that Ang II may have an antimitogenic action on injured endothelium in hypertension.

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