Effects of Angiotensin II Infusion and Inhibition of Nitric Oxide Synthase on the Rat Aorta
In previous studies, we showed that in vivo infusion of angiotensin II (Ang II) to adult rats induced vascular changes in gene expression, and this effect did not depend solely on blood pressure elevation. To determine whether nitric oxide can influence the effects of Ang II on the vessel wall, we administered to rats Ang II separately or in combination with the arginine analogue Nω-nitro-l-arginine methyl ester, which inhibits nitric oxide synthase chronically when given in vivo. We measured changes in aortic medial thickness, the association of macrophages with the endothelial surface of the aorta, the presence of proliferating cell nuclear antigen in the intima and adventitia as an index of aortic cell cycle changes, and the expression of immunodetectable fibronectin as an index of changes in the extracellular matrix. After 18 days of nitric oxide inhibition, the major changes were increased medial thickness and a 3.5-fold increase in the number of adherent macrophages. Rats treated with two different doses of Ang II for 3 days had a fivefold and threefold increase in the number of proliferating cells from the intimal and adventitial regions, respectively. Combined treatment resulted in increased medial thickness, intimal and adventitial cell proliferation, and macrophage adherence. An increased and altered pattern of fibronectin distribution was found in all treatment groups. Losartan administration prevented the effects of Ang II but not of nitric oxide inhibition, whereas administration of l-arginine prevented both intimal macrophage adherence and increased adventitial proliferation in rats given combined treatment. The data suggest that nitric oxide selectively influences macrophage association with the arterial wall, whereas Ang II and nitric oxide may have opposing effects on arterial cell proliferation.
The cellular events associated with the response of large blood vessels to experimental hypertension, which include vascular hypertrophy and hyperplasia, functional changes in the endothelium, and remodeling processes that involve increased extracellular matrix deposition, are thought to be involved in the pathogenesis of vascular disease.1 2 Ang II is a potent vasoconstrictor that has direct effects on vascular smooth muscle and endothelial cells and is thought to modulate the vascular changes that occur in different forms of experimental hypertension.3 4 5 NO is an important vasodilator implicated as a causative factor in hypertension and has been shown recently to influence the proliferation of vascular cells.6 7 Thus, Ang II and NO have opposing effects on vascular tone and may have analogous opposing roles in the vascular changes accompanying hypertension.
The direct infusion of Ang II via osmotic minipumps to otherwise untreated rats has been used for study of the role of Ang II in the regulation of different tissue responses to hypertension; this experimental model has been recently reviewed.8 We have shown that increased aortic fibronectin expression is a rapid and sensitive index of vascular remodeling that occurs in response to Ang II infusion9 10 and have also reported that cardiac fibrosis occurs as a response to pressor doses of Ang II.11 Recently, we have shown12 that the effects of Ang II infusion on the heart were markedly modified by chronic inhibition of NO synthase activity, which was accomplished by administration of the arginine analogue L-NAME, to inhibit NO production. In that study,12 we found that the development of cardiac fibrosis, characterized biochemically by increased fibronectin expression, and an accompanying proliferative and inflammatory response occurred when subpressor doses of Ang II and L-NAME were given concurrently but not when either agent was given alone, implying an important regulatory role for NO in modulating Ang II–induced cardiac fibrosis.
In the present study, to assess the role of Ang II and NO on vascular tissue in vivo, we compared the effects of infused Ang II, NO inhibition, and the combination of both treatments on aortic tissue using morphological criteria to measure cellular changes. We found characteristic responses to both treatments, including medial hypertrophy and fibronectin deposition. In addition, Ang II promoted both adventitial and endothelial cell proliferation, and NO inhibition selectively caused adherence of monocytes and macrophages to the vascular endothelium.
Human Ang II (acetate salt), l-arginine, and L-NAME were purchased from Sigma Chemical Co, and sodium pentobarbital (Nembutal) was from Abbott Laboratories. Losartan was generously provided by DuPont-Merck.
Experimental Animal Models
Male Wistar rats weighing 200 to 225 g were purchased from Charles River Breeding Laboratories, Inc (Wilmington, Mass) and allowed 1 week to adjust to the facilities before all experimental protocols. Under standardized conditions, rats were given L-NAME in their drinking water at a dosage of 40 mg/kg per day. Control rats were given only water. Routine monitoring showed that the rats consumed approximately 50 mL of drinking water each day whether or not L-NAME was included, and the drinking patterns did not change throughout all treatment protocols. After 15 days, control rats or those given L-NAME were anesthetized, and osmotic minipumps (Alzet, Alza Corp) containing Ang II dissolved in 0.15 mol/L NaCl and 1 mmol/L acetic acid were implanted to deliver the drug at a dosage of 0.225 or 0.720 mg/kg per day. Losartan was given at a dosage of 20 mg/kg per day ad libitum via the drinking water with treatment initiated 1 day before pump implantation. l-Arginine was given in the drinking water 7 days before pump implantation at a dosage of 4 g/kg per day. Systolic pressures were obtained by tail-cuff plethysmography. Blood pressure levels were determined before Ang II treatment and 4 to 6 hours before death. In all experiments, the reported value at each time point represented an average of multiple recordings. Sodium pentobarbital was used as surgical anesthesia (50 mg/kg) and for overdosing (0.5 g/kg) rats.
Tissue Preparation and Immunohistochemistry
Rat thoracic aorta was removed, rinsed with phosphate-buffered saline, and carefully cleaned in buffer to remove adherent adventitial fat; care was taken not to damage either the endothelium or the adventitial material closest to the medial layer. The tissue was then fixed with 10% formalin for 24 hours and subsequently embedded in paraffin. Serial sections were cut at a thickness of 4 μm and routinely stained with hematoxylin and eosin.
For immunodetection of PCNA, sections were treated with 3% hydrogen peroxide for 5 minutes and then incubated for 1 hour with an antibody to PCNA (PC-10, Dako Corp) that was coupled to horseradish peroxidase. With the EPOS system of the supplier, detection was accomplished by reaction with 3,3′-diaminobenzidine, including nickel ion (Vector Laboratories).
For immunodetection of macrophages, sections were initially treated with 0.1% protease type XXIV (Sigma) for 5 minutes at room temperature, then incubated for 5 minutes with 3% hydrogen peroxide, and subsequently treated for 1 hour with an antibody directed against rat monocytes/macrophages (ED-1, mouse anti-rat monocyte and macrophage antibody, Biosource International) used at a dilution of 1:100. The sections were then treated with biotinylated anti-mouse IgG antibody for 30 minutes at a dilution of 1:200 and reacted with an avidin-biotin complex (Vectastain Elite ABC kit, Vector). Detection was performed with 3,3′-diaminobenzidine and counterstained with Gill No 2 hematoxylin (Sigma).
Fibronectin immunodetection involved preliminary digestion with protease type XXIV and hydrogen peroxide as described above, followed by incubation for 1 hour with a rabbit anti-rat fibronectin antibody (Calbiochem) used at a dilution of 1:2000. Biotinylated anti-rabbit IgG was used as second antibody at a dilution of 1:200, and detection was accomplished with an avidin-biotin complex and 3,3′-diaminobenzidine as described above.
The circumferential length of the internal elastic lamina and the medial thickness were measured with OPTIMAS 4.02 image-analysis software (Bioscan, Inc) in conjunction with an Olympus microscope, video camera, and on-line computer. Four randomly selected aortic ring sections were used to obtain length and thickness measurements. Medial thickness data for each rat were based on the average of four separate determinations for each of the four sections. Cells containing either PCNA or monocyte/macrophage antigen were counted manually with a Nikon microscope at a final magnification of either ×200 or ×400. When cells were counted in the adventitia, only those positively stained cells located within 40 μm of the external elastic lamina were included.
All values are expressed as mean±SE. Measurements were compared with one-way ANOVA. Subsequent comparisons were performed with two-tailed, unpaired Student's t test.
The Table⇓ summarizes changes in systolic pressure and aortic medial thickness that occurred after long-term treatment of the rats with either L-NAME for 18 days, Ang II administered for 3 days at either a low or high dose, or a combination of L-NAME and the low dose of Ang II. Also included are data from rats given the combined treatment plus either losartan (20 mg/kg per day) or L-arginine (4 g/kg per day) as described in “Methods.” L-NAME treatment increased blood pressure to hypertensive levels after only 7 days of treatment (mean systolic pressure=155 mm Hg). After 2 weeks, the rats were clearly hypertensive. The relatively low dose of Ang II caused a slight but statistically significant increase in blood pressure after 3 days of treatment, yet when administered to rats pretreated with L-NAME, there was no additive effect on systolic pressure. Effects on medial thickness were found in all treatment groups and were most marked in rats given both L-NAME and Ang II. Of particular interest was the effect of the low dose of Ang II, which increased medial thickness within 3 days. With the higher dose of Ang II, the effects on medial thickness appeared to be greater, but the measured values did not approach statistical significance when both doses of Ang II were compared. When either losartan or l-arginine was given to rats also receiving both L-NAME and Ang II, neither substance appreciably modulated the hypertensive response.
Fig 1⇓ compares representative photomicrographs of aortic tissue from untreated rats and those given a combination of both L-NAME and Ang II. The hematoxylin and eosin–stained sections (Fig 1A and 1B⇓⇓) showed a marked increase in medial thickness at least partly due to increased cellular mass. In rats given L-NAME, leukocyte adhesion to the intima was a common occurrence. To assess this change, we used an antibody specific for rat monocytes/macrophages. As noted in Fig 1C and 1D⇓⇓, in rats given both L-NAME and Ang II, there was a striking increase in positively stained cells on the intimal surface and in the subintimal space. Rats receiving only Ang II did not differ significantly from the untreated rats with respect to macrophage accumulation, whereas rats given only L-NAME for 18 days did have macrophages associated with the vascular endothelium, similar to the section shown in Fig 1D⇓.
Morphometric analysis of the different treatment groups is summarized in Fig 2⇓. The clear increase in adherent macrophages found in rats given L-NAME with or without Ang II reflected cells found both on the intimal surface and with a subintimal location. The cells found in aortas from untreated rats were localized almost exclusively on the endothelial surface. Rats given only Ang II at either a low or high dose had no significant change in the number of adherent cells compared with the control group. When l-arginine was given during the last 10 days of the 18-day treatment with L-NAME, macrophage adherence to the aorta was almost completely suppressed. In separate studies (data not shown), we found that if L-NAME was given for only 5 days to rats, macrophage adherence did not increase, indicating that long-term treatment with L-NAME was necessary to influence endothelial cell–leukocyte interactions.
To assess the effects of Ang II and L-NAME on cell proliferation, we used PCNA as a marker for cell cycle activity. Fig 3⇓ contrasts the number of PCNA-positive cells found in the aortic intima and adventitia of control rats and those given only Ang II at a low dose. In untreated rats, the number of PCNA-positive cells was relatively low, with most of the cells localized within the adventitia. When Ang II was given, there was a clear increase in PCNA-positive cells both associated with endothelial cells and within the adventitia. PCNA-positive cells in the intima, although infrequent, almost always had the appearance of endothelial cells and were not found in a subendothelial location. Although PCNA-positive cells within the aortic media also were occasionally found, they were less numerous than in the intima or adventitia and showed no obvious change between treatment groups.
Fig 4⇓ summarizes the morphometric analysis of each of the treatment groups with respect to intimal and adventitial changes in PCNA-positive cells. Ang II treatment was the major factor responsible for the increased number of cells in the aorta. L-NAME treatment alone, despite the increased blood pressure, did not lead to changes in PCNA reactivity, whereas Ang II, even when given at a relatively low dose, caused a marked change in the number of PCNA-positive cells. The relative change in cell number was actually greater within the intima than the adventitia, but the number of PCNA-positive cells was appreciably greater in the adventitial region. Losartan treatment given to rats receiving both Ang II and L-NAME attenuated the increase in Ang II–induced PCNA-positive cells, and the effect appeared to be more prominent in the adventitia than in the intima. Interestingly, l-arginine also caused a moderate reduction of PCNA-stained cells in the adventitia. When the PCNA-positive cells were examined closely in the groups given Ang II alone, adventitial fibroblasts and endothelial cells appeared to be the predominant cell types labeled, whereas when L-NAME was included in the treatment, evidence for both intimal and adventitial macrophages containing PCNA was found, representing between 20% and 30% of the total positive cells in each region. In experiments not summarized in Fig 4⇓, rats given L-NAME for only 8 days showed no change over control levels in any aortic region.
We performed experiments to correlate the data obtained using PCNA as a marker of cell proliferation with that of tritiated thymidine. We used an autoradiographic procedure in which tritiated thymidine was given to rats intraperitoneally at 8-hour intervals beginning 24 hours before death.11 In those studies, we found that within the first day after Ang II infusion at the higher dose, essentially no incorporation of labeled thymidine into aortic cells occurred. Incorporation was obvious during the 2nd and 3rd days and occurred predominantly in the aortic adventitia. Visual observation and quantitative estimation of labeled thymidine incorporation during the 2nd and 3rd days of Ang II treatment gave results that were similar to those reported in Fig 4⇑. Thus, PCNA immunodetection appears to be a reliable index of cell proliferation in response to Ang II treatment.
We had shown previously that increased aortic fibronectin expression was a characteristic change accompanying several forms of experimental hypertension, including Ang II infusion. To determine whether NO influences aortic fibronectin, we used immunohistochemical procedures in the different treatment groups. Fig 5⇓ shows that in control tissue, fibronectin was present throughout the intima, media, and adventitia but was most prevalent in the extracellular regions surrounding the medial smooth muscle cells closest to the intima. After treatment with L-NAME and Ang II, the immunodetectable material was more abundant throughout the media and was particularly concentrated in the region separating the intima and media, as evidenced by the narrow band of intensely stained fibronectin indicated by the arrow in Fig 5D⇓. This pattern was found in all rats examined that were given either Ang II alone, L-NAME alone, or combined treatment.
We have found that phenotypic changes in aortic endothelial cells, smooth muscle cells, and adventitial fibroblasts occurred after in vivo treatments with L-NAME and Ang II. These changes included increased medial thickness, cell cycle changes for several vascular cell types, increased extracellular matrix deposition, and inflammation. We were able to dissociate the effects of NO and Ang II on at least two important cellular responses: the adhesion of leukocytes to vascular endothelial cells, which occurred only when NO production was inhibited, and the proliferation of adventitial fibroblasts, which solely depended on Ang II administration and did not occur when NO production was inhibited, despite the associated increase in blood pressure. These selective effects of Ang II and NO inhibition on aortic responses are in striking contrast to the synergistic response we recently reported in the heart,12 in which cardiac fibrosis was rapidly induced in rats given both Ang II and NO blockade but was absent when either treatment was given alone.
The increased adhesion of monocytes/macrophages to the aorta after inhibition of NO production was a marked and specific response to L-NAME treatment. l-Arginine administration during the latter half of the 18-day treatment prevented the increased adhesion of leukocytes yet did not reduce the hypertension that characterized long-term L-NAME treatment, suggesting that hypertension alone was not the causative factor. The induction of adhesion molecules on the surface of endothelial cells to facilitate leukocyte adhesion and recruitment has been documented in association with endothelial dysfunction and foam cell lesion development.13 14 With respect to experimental hypertension, monocytes have been identified on the endothelial cell surface of aorta and coronary arteries from spontaneously hypertensive rats but not the corresponding controls with the use of an antibody specific for rat monocytes/macrophages.15 Similar findings were reported in rats made hypertensive by mineralocorticoid and salt treatment.16 More recently, changes in leukocyte adhesion between the spontaneously hypertensive rat and control rats were found in cerebral blood vessels17 and mesenteric vessels,18 and a large increase in intimal mononuclear cells was found after only 14 days of treatment in the two-kidney, one clip hypertensive rat.19 NO is a possible paracrine mediator regulating adhesion of leukocytes to the vascular endothelium.20 Recent studies have indicated that in hypertension, vascular NO levels may be decreased in part because of decomposition by interaction with superoxide anion, which increases in hypertension.21 22 Our findings clearly indicate an important role for NO in modulating monocyte adhesion to the macrovasculature and suggest that this effect is not mediated through hemodynamic mechanisms.
The change in the amount of PCNA-positive cells was perhaps the most obvious effect of Ang II treatment and was most apparent in the adventitia. Many studies show that Ang II can influence cell proliferation of cultured vascular smooth muscle cells, but much less information is available regarding the effects of Ang II on adventitial fibroblasts. Aortic ligation induced increased DNA synthesis of adventitial fibroblasts, and it was suggested that the adventitia participates in the development of vascular hypertrophy and arterial disease produced by aortic ligation.23 During remodeling processes that occur in pulmonary hypertension, changes in adventitial connective tissue metabolism were induced by a factor derived from vascular smooth muscle.24 Interestingly, a recent report indicates that the adventitia is a barrier to NO in the pulmonary artery.25 A few studies have used aortic adventitial fibroblasts in tissue culture and shown that when fibroblasts are taken from spontaneously hypertensive rats, proliferative activity26 and expression of cyclin27 differ from comparable cells obtained from Wistar-Kyoto controls. Although a plethora of studies implicate paracrine relationships between endothelial and vascular smooth muscle cells, the role of the adventitial cell population in the modulation of smooth muscle cell function has not been examined intensively. Our findings of major alterations in the adventitia in response to Ang II infusion suggest that the adventitia may participate in and contribute to the changes observed in the remainder of the vessel wall.
An increase in medial thickness was produced by all treatment protocols and was associated with hypertension in all cases, except perhaps for administration of the low dose of Ang II, during which systolic pressure was elevated above control levels, but did not exceed 160 mm Hg, and persisted for a relatively short time of less than 72 hours. The changes in medial thickness that occur in experimental hypertension are most commonly due to hypertrophy of smooth muscle cells and increased extracellular matrix deposition. Fibronectin distribution, which we measured as an index of vascular changes in extracellular matrix, was localized throughout the aorta but was somewhat focal, with “patchy” accumulation present throughout the medial layer. After treatment with either L-NAME or Ang II, the increases were obvious in the media, particularly near the intimal-medial boundary, where a thick band of immunodetectable fibronectin was a consistent finding. This increase in aortic fibronectin expression is consistent with biochemical data we published previously showing that several models of experimental hypertension, including Ang II infusion and deoxycorticosterone-salt hypertension, led to increased steady-state levels of fibronectin mRNA.9 However, the effect does not depend solely on increased blood pressure, as we have shown previously that Ang II–induced aortic fibronectin expression was not attenuated simply by lowering of blood pressure.10 In the current study, we found that the increased fibronectin deposition was as marked in rats given a low dose of Ang II as when blood pressure was raised significantly higher by long-term L-NAME treatment (see Fig 5B⇑ versus 5D). If the effects of L-NAME on fibronectin expression are not solely due to hemodynamic effects related to blood pressure, then NO could play an important regulatory role in determining the deposition of fibronectin and other extracellular matrix components that occurs in response to vascular injury or remodeling processes.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|PCNA||=||proliferating cell nuclear antigen|
This work was supported by National Institutes of Health grant HL-55001. Susan Hope provided excellent technical support for the care and treatment of the animals.
- Received January 3, 1996.
- Revision received March 15, 1996.
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