Determining the significance of humoral versus hemodynamic factors as stimuli for monocyte infiltration of arteries in hypertensive humans is the objective of substantial current research (1-4). Although blood pressure and shear stress are clearly triggers for monocyte-endothelium interactions (5-7), signals for monocyte activation, recruitment, and migration through an activated endothelium may originate from the plasma, circulating leukocytes, and cells of the vascular wall in the absence of increased blood pressures (8-10). One potential humoral regulator of the vascular monocytic response is angiotensin II (Ang II), the major vasoregulatory peptide of the renin-angiotensin system (RAS; 11,12). Regulation of immune and endothelial cell signaling pathways by Ang II AT1-receptor stimulation (13,14) suggests that Ang II may promote activation and transendothelial migration of monocytes in the absence of increased pressures. We (15) proposed that Ang II AT1-receptor blockade may exert a beneficial effect on hypertension-associated cardiovascular disease because vascular cells and monocytes contain the major components of the RAS (16-19). This hypothesis is supported by evidence that Ang II upregulates adhesion molecules and chemokines within circulating monocytes and cellular elements of the vascular wall (20,21), exacerbates reperfusion injury in animal models of myocardial ischemia and hypertension (22), and induces pressure-independent cardiovascular remodeling (23) via AT1-receptor stimulation.
The adverse effects of an activated RAS on vascular function were demonstrated in the hypertensive (mRen2)27 transgenic rat, a model in which insertion of the mouse Ren-2 gene into the rat genome causes enhanced vascular production and release of Ang II (24,25). Local tissue RAS activation is an inherent mechanism potentially responsible for vascular and immune cell dysfunction in the mRen2 model (26). Endothelial dysfunction in the (mRen2)27 model is Ang II-dependent, because treatment with losartan but not the direct vasodilator hydralazine normalized blood pressure and restored endothelium-dependent relaxation (27). Mononuclear immunocytes from hypertensive mRen2 rats are functionally altered, as reflected by reduced sodium-proton exchange activity compared with normotensive rats (28). Our study used mRen2 rats and their normotensive Hannover Sprague-Dawley (SD) controls to investigate the differential effects of hypertension and prolonged AT1-receptor stimulation on monocyte activity and endothelial function. The number of monocytes adhered to endothelium and the extent of endothelial cell injury in the thoracic aorta were compared in mRen2 and normotensive SD rats by using an en face double-immunostaining technique. Injured and dying aortic endothelial cells permeable in situ to plasma immunoglobins were immunostained to evaluate the extent of endothelial injury (29). Monocytes adhered to the endothelium were immunodetected by their expression of the rat-specific surface glycoprotein ED-1 (30). Quantification of monocyte spontaneous superoxide production was estimated by tetrazolium nitroblue reduction to assess the status of circulating monocyte activation (31). Both Ang II AT1 receptor-dependent and -independent antihypertensive mechanisms were used to distinguish AT1-receptor-mediated processes from those associated with high blood pressure.
Male 14-week-old, hemizygote (mRen2)27 rats (410 ± 12 g body weight), derived from homozygote founder animals developed by Mullins et al. (32), and SD rats (429 ± 11 g body weight), derived from founder animals from the Zentralinstitut für Versuchstierkunde, Hannover, Germany, were obtained from the colony of animals bred and maintained at the Hypertension and Vascular Disease Center Transgenic Animal Facility. Hemizygote mRen2 male rats were the offspring of matings between mRen2 homozygote males and SD females. Animals were individually caged in cubicles at 21°C with a 12 h: 12 h light/dark cycle, and fed a standard rat chow (Rodent Laboratory Chow 5001; Purina Mills, Richmond, IN, U.S.A.) with tap water ad libitum.
Blood pressure measurement and antihypertensive treatment
Thirty mRen2 and 30 SD rats, accustomed for 2 weeks to the restraint conditions of conscious tail-cuff blood pressure measurements, had their systolic blood pressures (SBPs) and heart rates (HRs) measured 3 days before implantation of osmotic minipumps (5 ml/h infusion rate, Alzet model 2ML2; Alza Corp., Palo Alto, CA, U.S.A.) in a subcutaneous space between the scapulae. Osmotic pumps were primed by submersion in sterile saline for 4 h at 37°C before implantation to assure immediate drug delivery. Ten rats of each strain received long-term subcutaneous infusions of saline (120 μl/24 h), hydralazine (3 mg/kg/24 h), or losartan (10 mg/kg/24 h) for 12 days. Measurements of SBP and HR by the tail-cuff technique were repeated after 11 days of infusion.
Plasma angiotensin peptide levels and circulating cell counts
Rats were anesthetized by intramuscular injection of a combination of ketamine hydrochloride (40 mg/kg Ketaset; Fort Dodge Laboratories, Fort Dodge, IA, U.S.A.), xylazine (4.0 mg/kg Rompun; Miles Inc., Shawnee Mission, KS, U.S.A.), and acepromazine maleate (0.5 mg/kg PromAce; Fort Dodge) 12 days after pump implantation. The abdominal aorta, exposed through a ventral midline incision, was catheterized with an 18-gauge angiocatheter (Angiocath; Beckton Dickinson Vascular Division, Sandy, UT, U.S.A.). The plasma concentrations of the angiotensin peptides, Ang II and angiotensin-(1-7) [Ang-(1-7)], were evaluated by radioimmunoassay, as described previously (33). Blood (7 ml) for the assay of plasma angiotensin peptides was withdrawn from the aorta and placed into tubes containing EDTA (25 mM, final concentration) and protease inhibitors (0.44 mM o-phenanthroline; 0.12 mM pepstatin A; 1 mM 4-chloromercuribenzoic acid, final concentration) to prevent peptide degradation (34). After centrifugation of blood samples at 4°C, the plasma was frozen on dry ice and stored at −20°C until analyzed.
The following blood cell estimates were obtained from fresh, unseparated arterial blood samples: (a) total white blood cell count by use of a hemocytometer (Fisher Scientific, Fair Lawn, NJ, U.S.A.); (b) differential count for neutrophils, monocytes, and lymphocytes from a counterstained blood smear; and (c) the number of activated monocytes that reduced nitroblue tetrazolium (NBT).
Monocyte activation determined by nitroblue tetrazolium reduction
The NBT assay, which reflects spontaneous superoxide formation within leukocytes (35), was used to identify activated circulating monocytes. Arterial blood (0.01 ml) was transfered into a 0.5-ml microfuge tube and mixed with an equal amount of 0.1% NBT (Boehringer Mannheim, Mannheim, Germany) solution. The blood-NBT mixture was incubated at 37°C for 30 min followed by 15 min at room temperature. After gentle mixing, blood smears were made, stained with Wright's stain, and 30 monocytes were counted at ×100 oil-objective magnification. NBT-positive monocytes showed a stippled cytoplasmic distribution of formazan or a single dense formazan deposit.
After obtaining blood samples, heparin (1,000 U; Elkins-Sinn, Cherry Hill, NJ, U.S.A.) was immediately administered through the arterial catheter and allowed to circulate for 10 min. A bolus injection of pentobarbital (50 mg/kg; The Butler Co., Columbus, OH, U.S.A.) was administered through the catheter immediately before fixation perfusion. The thoracic aorta was retrograde perfused and fixed in situ at 110 mm Hg (measured at carotid artery) with 150 ml heparinized (2 units/ml) phosphate-buffered saline (PBS; 50 mM NaPO4, 0.15 M NaCl, pH 7.5), followed by 4% phosphate-buffered formalin for 10 min. Perfusate egress was through incisions in the jugular veins. The thoracic aorta was excised, immersed in 4% formalin for 72 h, and stored in PBS at 4°C until processed for immunocyto-chemistry.
Paraadventitial tissues were removed, and the aortas were cut into rings, each containing two sets of intercostal artery branches. Double-labeling immunocytochemistry (36) detected injured endothelial cells containing cytoplasmic immunoglobulin G (IgG) and adherent monocytes expressing ED-1. Aortic rings were placed into a 1.5-ml microcentrifuge tube, washed 3 times with PBS, rinsed in 3% hydrogen peroxide/methanol to block endogenous peroxidase activity, and proteolytically digested with 0.01% type XXIV protease (Sigma Chemical Co., St. Louis, MO, U.S.A.). Sections were washed 3 times with PBS, and incubated in PBS containing 5% normal rabbit serum (Vector Laboratories, Burlingame, CA, U.S.A.) for 20 min at room temperature. In each of the following steps, sections were incubated for 10 min at 37°C and washed 3 times with PBS at room temperature between incubations. Antibodies were diluted in 0.1% bovine serum albumin (Sigma Chemical) in PBS. Plasma-derived IgG bound to vimentin within the cytoplasm of injured and permeable endothelial cells (37) was immunolabeled by incubation with rabbit anti-rat IgG (1:1,000 dilution), biotinylated goat anti-rabbit IgG (Vector), and peroxidase-conjugated avidin-biotin complex (Rabbit IgG Peroxidase Vectastain Elite ABC Kit, Vector). Peroxidase 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, and then rinsed in tap water for 5 min. Monocytes adhered to the endothelium were labeled by incubation with a primary biotinylated mouse anti-rat antibody (1:500 dilution ED-1; Serotec, Oxford, England) that recognizes the single-chain glycoprotein ED-1 selectively expressed on the lysosomal membrane of rat monocytes (38). After incubation with the primary antibody, tissue was incubated with a peroxidase-conjugated avidin-biotin complex (Standard Peroxidase Vectastain Elite ABC Kit; Vector), and peroxidase activity was visualized as described earlier. Aortic rings were cut longitudinally, exposing the luminal surface, and pinned flat with the adventitia against Teflon sheeting. Haütchen preparations of the endothelial monolayers was performed on immunostained aortas as described elsewhere (39,40). The aorta segments were dehydrated in progressive ethanol concentrations, coated with ether-ethanol (1:5, vol/vol) and placed with the luminal side down on glass slides coated with nitrocellulose (20% nitrocellulose in ethanol-ether, 3:2, vol/vol). After adherence of the endothelium to the glass slide, the aorta segments were rehydrated, and the nonendothelial tissues were removed. The endothelium and nitrocellulose film were detached from the slide, placed on a clean slide with the luminal side of the endothelium up, and the nitrocellulose was dissolved in ether-alcohol (4:1, vol/vol). Cell nuclei were counterstained with Mayer's hematoxylin.
Quantification of endothelial cell injury and monocyte adherence
Estimates of the numbers of cells immunolabeled for IgG and ED-1 were made by identifying and counting cells from the Haütchen preparations of thoracic aorta. The entire endothelial surface of each aortic segment was systematically scanned by using light microscopy combined with an imaging system (MCID; Imaging Research, St. Catherine's, Ontario, Canada) calibrated for area allowing analysis of cells per square millimeter (mm2). The numbers of endothelial cells in each Haütchen preparation were estimated by sampling instead of counting each cell, because a large number of endothelial cells are present within the endothelium of the entire rat thoracic aorta. To estimate the total number of endothelial cells in the segments of thoracic aorta examined, hematoxylin-stained endothelial cell nuclei satisfying the criteria for size, shape, and staining pattern were counted at a magnification of ×100 in 10 0.15-mm2 randomly selected areas within a 5 × 5-mm square superimposed on the seven areas between each of the first eight intercostal artery pairs. Only the endothelium within the square was examined to avoid artifacts located at edges where the aorta was transected and the complicated topography at artery bifurcations. Dark-staining, polymorphic, pyknotic nuclei, or a combination of these, or dividing nuclei with chromatin bodies were excluded from the count as not representative of normal, mature endothelial cells. The counts from 10 fields within each square were summed, averaged, and used to calculate the cell density (number of endothelial cells/25 mm2). Endothelial cells with immunodetectable IgG within their cytoplasm were discriminated from ED-1-positive monocytes by size and shape of hematoxylin-stained nuclei. Injured endothelial cells had typically ovoid nuclei and were consistently larger than the round, mononuclear monocytes. The relatively large cytoplasm of endothelial cells contrasted with the relatively small cytoplasm-to-nucleus ratio of monocytes. Monocytes often had darker-staining nuclei than endothelial cells. Endothelial cell injury represented the total number of IgG-containing cells counted in all seven aortic segments from each aorta, expressed as a percentage of the estimated number of endothelial cell nuclei present. Monocyte adherence (monocytes/mm2) represented the total number of ED-1-positive monocytes counted within each of the aortic segments divided by the surface area of the aorta. The results from each aorta were averaged, and the average value for each treatment group was used for statistical analysis. Cell counting was performed on Haütchen preparations by a single investigator under blind conditions to ensure consistent interpretation of immunocytochemical staining and to avoid outcome bias.
Values are expressed as means ± standard error of the mean (SEM). Analyses of variance (ANOVA) or analyses of covariance (ANCOVAs) were used for all comparisons. Where appropriate, a square-root transformation was used to yield distributions suitable for parametric analysis. All tests of significance were two-tailed. Values of p < 0.05 were considered statistically significant.
Blood pressure and heart rate measurements
SBPs of mRen2 rats were significantly higher than those of SD rats 3 days before treatment. Table 1 summarizes the hemodynamic changes on day 11 of prolonged infusion in mRen2 and SD rats of saline, hydralazine, or losartan. Treatment with either hydralazine or losartan reduced SBPs of mRen2 rats to levels comparable to those in treated SD rats. SBPs in all antihypertensive-treated rats and saline-treated SD rats were not significantly different from those measured in SD rats before treatment. Only saline-treated mRen2 rats had SBPs significantly higher than other treatment groups on day 11. Treatment with losartan or hydralazine had no effect on HRs of SD or mRen2 rats.
Plasma angiotensin peptide measurements
The changes in plasma Ang II and Ang-(1-7) concentrations with administration of hydralazine or losartan are summarized in Table 2. Plasma concentrations of Ang II in anesthetized hypertensive mRen2 rats were not different from those in anesthetized SD rats, nor did hydralazine affect angiotensin peptide levels in either strain. Blockade of AT1 receptors with losartan resulted in a significant (p < 0.05) elevation in plasma Ang II concentrations in both mRen2 and SD rats. This change was accompanied by a twofold increase in plasma levels of Ang-(1-7) in mRen2 rats (p < 0.05), but not in SD rats.
Hematocrit and circulating cell counts
Hematocrit and total and differential white blood cell counts are shown in Table 3. Treatment had no effect on hematocrits in SD or mRen2 rats, nor were hematocrits different between the two strains. Saline-, losartan-, and hydralazine-treated hypertensive mRen2 rats had significantly increased circulating total white blood cell counts that were about twofold higher than those in similarly treated SD rats (p < 0.05). The number of monocytes, neutrophils, and lymphocytes, determined by differential counting, were about two- to fourfold increased in mRen2 rats. Treatment with hydralazine or losartan had no significant effect on the differential cell counts in SD or mRen2 rats, although a downward trend was noted in mRen2 rats treated with hydralazine or losartan.
Estimate of circulating monocyte activation by NBT assay
The number of NBT-positive monocytes (cells/mm3) and percentages of circulating monocytes that were NBT positive in arterial blood samples from saline-, hydralazine-, and losartan-treated mRen2 and SD rats are shown in Fig. 1. Spontaneous monocyte superoxide production, as detected by the NBT assay, was observed in all rats regardless of treatment. The distribution of NBT-positive monocytes was asymmetric, with a trend in saline- and hydralazine-treated mRen2 rats for activated monocyte counts to be >50 cells/mm3. Values for activated monocyte counts >40 cells/mm3 were rarely encountered in either losartan-treated mRen2 rats or any of the treated subgroups of SD rats. After transformation, these data satisfied the conditions for parametric evaluation. Hypertensive mRen2 rats administered saline or hydralazine had greater (p < 0.05) numbers of NBT-positive monocytes than did similarly treated SD rats. A significant (p < 0.05) effect was associated with treatment in mRen2 rats. Lower numbers and percentages of activated monocytes were observed in losartan-treated (37.6 ± 10.6/mm3 and 7.2 ± 2.0%), than in saline-treated (85.0 ± 15.2/mm3 and 13.7 ± 3.0%) mRen2 rats. Although hydralazine reduced blood pressure in mRen2 rats, it had no significant effect on the number of activated monocytes (63.0 ± 18.2/mm3). The estimated numbers and percentages of NBT-positive monocytes in SD rats administered saline (19.6 ± 6.9/mm3 and 7.0 ± 3.0%) were not affected by treatment with hydralazine (21.0 ± 9.8/mm3 and 8.6 ± 3.0%) or losartan (18.4 ± 7.9/mm3 and 7.0 ± 3.0%). The percentages of NBT-positive monocytes in mRen2 rats receiving saline were significantly greater (p < 0.05) than in saline-treated SD rats, whereas percentages in hydralazine-treated mRen2 (10.1 ± 3.0%) and SD rats were not different. Only losartan reduced both the number and percentage of NBT-positive monocytes in mRen2 rats equal to SD rats.
Endothelial injury and monocyte adherence
A representative Haütchen preparation of the thoracic endothelium from a hypertensive saline-treated mRen2 rat illustrates the commonly observed colocalization of IgG-positive endothelial cells and ED-1 monocytes- macrophages (Fig. 2, top). Injured endothelial cells and monocytes-macrophages often were clustered in patches (Fig. 2, bottom). In contrast, in all treated SD rats and hydralazine- and losartan-treated mRen2 rats, immunostained cells were usually single and widely scattered, and their location revealed no apparent relation between injured endothelium and monocytes-macrophages. The quantified data for endothelial injury and adherent monocytes-macrophages are shown in Fig. 3. In all cases, saline-treated hypertensive mRen2 rats had increased IgG- and ED-1-immunostained cells compared with SD rats. The average number of endothelial cells counted was 229 ± 26 in the 0.15-mm2 sampling areas of normotensive SD rats. Endothelial cell density did not vary significantly within the seven segments of each aorta, within groups of similarly treated SD or mRen2 rats, or between similarly treated SD and mRen2 rats. The estimated number of endothelial cells per aorta was calculated by multiplying the cell density within each aortic segment by the seven aortic segments investigated. By using this method, an average of 267,167 ± 11,242 endothelial cells were scanned per aorta. The average number of IgG-positive endothelial cells in saline-treated mRen2 rats (2,229 ± 202) was significantly greater (p < 0.05) than in SD rats treated with saline (1,129 ± 132). The numbers of injured endothelial cells in SD rats treated with hydralazine (1,076 ± 112) or losartan (1,229 ± 156) were not different from those in similarly treated mRen2 rats (1,156 ± 122 and 987 ± 132, respectively). The reduction in the absolute number of injured endothelial cells by antihypertensive treatment in mRen2 rats was reflected in a similar reduction in the percentage of endothelial cells with injury because the estimated number of endothelial cells present in the aorta was not different between animals. Analysis of these data revealed the percentage of endothelial cells with detectable injury in saline-treated mRen2 rats (0.98 ± 0.10%) was significantly (p < 0.05) reduced by hydralazine (0.47 ± 0.06%) or losartan (0.39 ± 0.05%). IgG-positive endothelial cells in aortas from SD rats administered saline (0.43 ± 0.07%) were significantly (p < 0.05) lower than in saline-treated mRen2 rats, but were not affected by hydralazine (0.41 ± 0.10%) or losartan (0.46 ± 0.10%), nor were they different from those of antihypertensive-treated mRen2 rats. The number of monocytes adhered to the endothelium of mRen2 rats was not altered by hydralazine (26.0 ± 6.0/mm2) but was reduced (p < 0.05) by losartan (9.2 ± 2.0/mm2) compared with saline-treatment (32.0 ± 10.0/mm2). By comparison, the numbers of monocyte adhered to the endothelium of the thoracic aorta of saline-treated (7.2 ± 3.0/mm2) and hydralazine-treated (8.1 ± 2.1/mm2) SD rats were significantly lower (p < 0.05) than those in similarly treated mRen2 rats. No treatment effect on monocyte adherence was observed in SD rats treated with hydralazine or losartan (6.9 ± 1.0/mm2).
The major aim of this study was to determine if the adherence of monocytes to aortic endothelium in male transgenic (mRen2)27 hypertensive rats was a function of blood pressure or the activated RAS inherent in this model. An additional aim was to determine if the extent of endothelial injury in the aorta was similarly dependent on humoral or hemodynamic parameters. The contribution of AT1-receptor stimulation to vascular endothelial injury and monocytic inflammation in chronic Ang II-dependent hypertension was evaluated by comparing the effects of AT1-receptor blockade with losartan and direct vasodilation with hydralazine. Blood pressures in saline-treated hypertensive (mRen2)27 male rats were significantly reduced by losartan and hydralazine without effect on HRs or body weights. Similar findings by others showed pressures in mRen2 rats equally depressed within 24 h by administration of either losartan or hydralazine (41) remained at the same values throughout continuous infusion (42). Injured IgG-containing endothelial cells were detected in ∼0.5% of the total endothelial cell population investigated in normotensive SD rats. In a similar study, Hansson and Schwartz (43) observed focal, spontaneous endothelial cell injury at a frequency of 0.20% in 5-month-old SD rats. This finding suggests that some low level of background endothelial cell injury is present in normotensive rat aortas. The higher level of background injury observed in SD rats in our study was probably due to genetic variations between rat stocks, age of the rats studied, and technique variability. Endothelial injury in the thoracic aorta of adult male hypertensive mRen2 rats was significantly greater than that in normotensive SD rats, confirming and extending our previous findings with the same techniques in adult female hypertensive mRen2 rats (44). Treatment with hydralazine reduced endothelial injury in mRen2 rats but had no effect on monocyte activation or adherence in mRen2 rats. In contrast, losartan reduced all measured indices of vascular injury and monocytic inflammation. Administration of hydralazine or losartan had no effect on these parameters in normotensive SD rats. Our results suggest that endothelial injury in mRen2 rats is primarily related to the hemodynamic effects of increased blood pressure, whereas monocyte activity is related to RAS activation and is mediated by AT1-receptor-associated pathways.
The concept of injury used in this experiment and detected by immunohistochemistry includes the generally accepted processes that follow altered plasma membrane permeability. The rationale for use of this technique in this experiment derived from studies by Hansson et al. (45), which demonstrated that endothelial cells injured in the aorta in situ do not exclude plasma proteins from their cytoplasmic compartments. Immunoelectron microscopy showed that IgG is deposited and bound to vimentin in the cytoplasmic matrix of cells with concurrent organelle swelling, chromatin changes, plasma membrane damage, and other signs of irreversible injury (46). Given the low frequency of injury in vivo and of IgG-positive cells in Haütchen preparations, it was necessary to screen large numbers of cells to reduce sampling error and obtain accurate estimates of the extent of injury. Although endothelial cell density was not different between normotensive and hypertensive strains, the number of injured endothelial cells with immunodetectable IgG injury was significantly reduced in mRen2 rats by antihypertensive treatment. The finding of equivalent endothelial cell density in normotensive SD and hypertensive mRen2 rats was consistent with findings in studies of other rat models of genetic chronic hypertension (47). The counting techniques and statistics used in this study and the values obtained for intact and injured endothelial cells corresponded favorably to work by other investigators using the same techniques (48).
The presence of injured endothelial cells unable to maintain their intracellular composition and residing in the arterial endothelium for extended periods may predispose the underlying tissue to pathologic development. Areas with such injured endothelial cells are highly permeable to plasma macromolecules (49). Injured and dying endothelial cells may be an important focus for pathophysiologic reactions in the aortas of hypertensive individuals. Pertinent to this study, the intracellular binding of IgG to the cytoskeleton activates complement, which may lead to formation and release of chemotactic factors that attract monocytes to endothelial cells (50). In humans and experimental models, monocytes occur frequently in areas with endothelial cell injury near atherosclerotic lesions (51), for example, and hypertension is a risk factor for the development of atherosclerosis (52).
Mechanisms other than hydralazine-induced vasodilation and AT1-receptor blockade by losartan may have reduced monocyte and endothelial activity in mRen2 rats in this study. Hydralazine directly inhibits arterial NADH-oxidase and the production of arterial wall oxygen free radicals that are attractants for monocytes (53). Losartan blocks the interaction of leukocyte-activating N-formyl peptides with monocytes through non-Ang II binding sites (54). Although our results do not allow us definitely to conclude that these and other pathways affecting monocyte-endothelium interactions were unaltered by hydralazine and losartan, only losartan reduced monocyte activation and adherence. This study demonstrated for the first time an increase in monocyte activation and adherence directly related to the activation of the RAS and the potential protective role of losartan in the vascular inflammatory process.
The clustered distribution of adherent monocytes and the degree of circulating monocyte activation observed in hypertensive mRen2 rats, and similar observations by investigators in other strains of genetically hypertensive rats, suggest that high blood pressure upregulates adhesion mechanisms in these hypertension models. Carotid arteries in spontaneously hypertensive rats (SHRs) release cytokines, express intracellular adhesion molecule-1 (ICAM-1), and support adhesion and subendothelial accumulation of monocytes-macrophages to a greater extent than carotid arteries in normotensive controls (55). Activation and adhesion of monocytes in hypertensive mRen2 rats was comparable to observations in other genetic models of hypertension (56,57). The initiating stimuli for adhesion molecule upregulation and the type of inflammatory activity in genetically hypertensive rats measured by spontaneous NBT reduction is not yet fully understood. Proinflammatory agents in tissue and plasma may induce these adhesive changes. Studies by others showed that Ang II upregulates endothelial cell-adhesion mechanisms through activation of adhesion molecules, nuclear activating factors, and chemokines (58). The monocytic inflammatory response observed in aortas of Ang II-infused rats was accompanied by a significant increase in monocyte chemoattractant protein-1 (MCP-1) messenger RNA (mRNA) expression (59). When blood pressures were normalized by either losartan or hydralazine administration, only losartan completely inhibited MCP-1 expression. Ang II upregulated AT1 receptors in human peripheral monocytes (60) and increased intracellular Ca2+ in cultured human mononuclear cells via AT1 receptors (61). Taken together with results from our study, these findings suggest that the RAS may exert a proinflammatory influence on the endothelium through Ang II AT1-receptor stimulation even after normalization of blood pressure.
Non-AT1 Ang II receptors were potentially stimulated concurrent with AT1-receptor blockade in our experiment because losartan administration resulted in increased plasma Ang II levels in both SD and mRen2 rats. Kim et al. (62) showed that Ang II treatment of human aortic endothelial cells induced the binding of human monocytes that was reduced by the AT1 antagonist losartan as well as the AT2-receptor antagonists, PD123177 and PD 123319, implicating both AT1 and AT2 receptors in Ang II-induced monocyte binding. Others investigators also suggested a role for non-AT1, non-AT2 receptors in the leukocyte-endothelium interaction. Gräfe et al. (63) found that increased expression of the endothelial adhesion molecule E-selectin in response to Ang II stimulation promoted leukocyte adhesion to human coronary endothelial cells by non-AT1, non-AT2 receptor-mediated mechanisms. Biologically active angiotensin fragments, including the hexapeptide 3-8 (AngIV) and Ang-(1-7), are formed in the rat circulation from angiotensin precursors, and may contribute to the vascular actions of the RAS (64). Stimulation of receptors distinct from AT1 and AT2 by AngIV increases the expression of plasminogen activator inhibitor 1 (PAI-1) mRNA and protein production in cultured bovine endothelial cells (65). We recently identified a binding site for Ang-(1-7) in bovine aortic endothelial cells (66) and showed that Ang-(1-7) inhibited vascular smooth-muscle cell (VSMC) proliferation in vitro (67) and inhibited in vivo neointima formation in carotid artery-injured rats (68). In this study, losartan administration increased plasma Ang-(1-7) levels and reduced monocyte adherence only in mRen2 rats. Additional studies will be required to determine whether the reduction in monocyte activation and adhesion in losartan-treated rats might be due in part to stimulation of non-AT1 receptors or the action of angiotensin peptides other than Ang II.
The ultimate importance of Ang II-induced monocyte activation, recruitment, and infiltration into the vascular wall remains to be determined. The suggestion that blood pressures and RAS activation differentially affect endothelial function and monocyte adherence is supported by studies in rats (68) indicating that tissue damage associated with leukocyte adhesion and infiltration is not necessarily preceded by endothelial cell injury. DeLano et al. (69) demonstrated that activated leukocytes adhere and migrate to cause subendothelial injury. Monocytic clusters like those observed in this study in hypertensive mRen2 rats may release cytokines like tumor necrosis factor-α and interleukins that promote coagulant properties of the endothelium and circulating blood elements (70-73). These findings may relate experimental studies to clinical studies in which RAS activity, leukocyte activation, and Ang II plasma levels correlated with the incidence of ischemic cardiovascular events in humans with essential hypertension. The recently completed Evaluation of Losartan in the Elderly (ELITE) study suggested a decrease in mortality in patients with congestive heart failure treated with losartan compared with those treated with captopril (74). Studies in humans with combined angiotensin-converting enzyme (ACE) inhibition and AT1-receptor antagonism indicated that humans maximally treated with ACE inhibitors still had significant residual AT1-receptor stimulation (75). Results from these in vivo and in vitro studies support the hypothesis that pathways regulated by AT1-receptor stimulation may be integral to the maladaptive changes in hypertensive and atherosclerotic arteries responsible for the events resulting in myocardial ischemia.
In summary, we normalized the blood pressure in transgenic rats with prolonged Ang II-dependent hypertension by using either the AT1-receptor antagonist losartan or the nonspecific vasodilator hydralazine. Both strategies were effective in reducing the extent of endothelial injury, but only AT1-receptor antagonism significantly reduced the activation of circulating monocytes and their adhesion to the endothelium. Our results suggest a direct humoral effect of angiotensin peptides on monocyte adhesion-promoting mechanisms, and support a role for the RAS in monocyte adherence to the endothelium of mRen2 rats that is independent of the injurious effects of increased blood pressure.
Acknowledgment: Losartan was kindly provided by the Merck Human Health Division, West Point, PA. These studies were supported in part by a Medical School Grant from Merck, Inc., and by a grant from the National Institutes of Health, HL-51952.
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