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Am J Physiol Heart Circ Physiol 288: H805-H812, 2005; doi:10.1152/ajpheart.00696.2004
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Control of oxidative stress in microcirculation of spontaneously hypertensive rats

F. A. DeLano, R. Balete, and G. W. Schmid-Schönbein

Microcirculation Laboratory, Department of Bioengineering and The Whitaker Institute of Biomedical Engineering, University of California San Diego, La Jolla, California

Submitted 14 July 2004 ; accepted in final form 28 September 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
One mechanism for organ damage in individuals with arterial hypertension may be due to oxygen free radical production. This study was designed to localize free radicals in a microvascular network of mature spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. Because glucocorticoids play a role in pressure elevation of SHRs, we investigated their role in microvascular free radical formation. Oxygen radical production in mesentery was detected by tetranitroblue tetrazolium reduction to formazan aided by digital light-absorption measurements. Formazan deposits were observed in the endothelial cells and lumens of all microvessels and in lymphatic endothelia but were fewer in tissue parenchyma. The formazan distribution in younger (14–16 wk old) WKY rats and SHRs was heterogeneous with low values in capillaries and small arterioles/venules (<30 µm) but enhanced deposits in larger venules. Adrenalectomy served to reduce the formazan density in SHRs to the level of WKY rats, whereas dexamethasone supplementation of the adrenalectomized rats caused elevation in the larger venules of SHRs. In older (40 wk old) SHRs, formazan levels were elevated in all hierarchies of microvessels. After pressure reduction was employed with chronic hydralazine treatment, the formazan deposits were reduced in all locations of the microcirculation in both WKY rats and SHRs. Elevated formazan deposits were also found in lymphatic endothelium. These results suggest that oxygen free radical production is elevated in both high- and low-pressure regions of SHR microcirculation via a process that is controlled by glucocorticoids. Older SHRs have higher formazan levels than younger SHRs in all microvessels. Chronic hydralazine treatment, which serves to reduce arterial blood pressure, attenuates tetranitroblue tetrazolium reduction in WKY rats and SHRs even in venules of the microcirculation, which has no micropressure elevation. Free radical production may be a more global condition in SHRs and may not be limited to arteries and arterioles.

mesentery; arteriole; capillary; venule; superoxide; nitroblue tetrazolium; dexamethasone; Wistar-Kyoto; adrenalectomy; hydralazine

ELEVATION OF ARTERIAL blood pressure is accompanied by a significant risk factor for cardiovascular complications such as stroke, myocardial infarction, atherosclerosis, or renal failure. But the mechanisms that lead to such vascular complications in hypertension are still uncertain. A number of studies indicate that elevated oxygen free radical production may play a role in experimental models of hypertension (12, 23, 25, 26, 29, 36, 44, 47) and in human hypertensive individuals (21, 31). Oxygen free radicals such as superoxide, hydrogen peroxide, hydroxyl radical, or peroxynitrite may cause local vascular damage while at the same time producing the hallmarks of hypertension, i.e., increased vascular tone, reduced fluid shear stress response, and arterial pressure elevation (8, 9, 19, 53).

Free radicals in hypertension may be derived from a variety of cells in vivo (35, 38, 44) and even from plasma (22). It is important to determine the cellular sources at the level of the microcirculation. For this purpose, we utilize here the microcirculation of the spontaneously hypertensive rat (SHR) model of hypertension with an approach that serves to display the degree of free radical formation in all cells in the microcirculation.

Hypertension in SHRs strongly depends on adrenal glucocorticoids. Adrenalectomy serves to normalize blood pressure in SHRs to the level of their normotensive Wistar-Kyoto (WKY) controls, whereas supplementation of adrenalectomized SHRs leads to significantly elevated blood pressure compared with WKY rats (1, 24, 30, 45). There are still few studies that serve to explore to what degree glucocorticoids influence free radical formation in SHRs (44). We hypothesize that oxygen free radical production in SHRs may be enhanced by glucocorticoids.

Thus the objective of this study was to examine in the microcirculation of SHRs and their normotensive WKY controls the level and localization of oxygen free radical production by means of tetranitroblue tetrazolium (TNBT) reduction to formazan. To explore the role of glucocorticoids, the two rat strains were studied before and after adrenalectomy and supplementation with a synthetic glucocorticoid. Because glucocorticoids directly affect blood pressure, we also examined TNBT reduction in a set of SHRs treated with an alternative technique, hydralazine treatment, to reduce blood pressure. The study was carried out on the mesentery to facilitate detailed delineation of the reaction with digital microscopy in multiple segments of the microcirculation at different blood pressures. We also examined TNBT reduction in lymphatic endothelium with substantially lower fluid pressures.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animal procedures were reviewed and approved by the University of California San Diego Animal Subjects Committee.

Animal groups and blood pressure. Age (14–16 wk)- and weight (300–350 g)-matched SHRs and normotensive WKY rats (n = 5 male rats/group; Charles River Laboratory; Wilmington, MA) and a cohort of bilaterally adrenalectomized SHRs and WKY rats (n = 4 rats/group) were enrolled in the study. One group of adrenalectomized WKY rats and SHRs was administered replacement therapy with dexamethasone (Sigma; St. Louis, MO) at a rate of 0.5 mg·kg–1·day–1 sc for 5 days (n = 4 rats/group). The adrenalectomized rats were fed standard rat diet and 0.9% (wt/vol) NaCl in their drinking water ad libitum and were maintained in a light-controlled holding facility for 1 wk. Completeness of the adrenalectomy was confirmed by postmortem examination of the suprarenal region.

To examine the role of blood pressure in oxygen free radical formation, groups of male WKY rats and SHRs (n = 3 rats/group) were treated at the age of 14 wk for 10–14 days as well as at 26 wk with hydralazine hydrochloride (200 mg/l of drinking water ad libitum; average consumption, 2.7–3 mg·kg–1·day–1). Control rats were age and gender matched on regular drinking water without hydralazine.

The rats were given local anesthesia (4% xylocaine im; Astra; Westborough, MA) for placement of a polyethylene-50 femoral artery catheter (Clay Adams; Parsippany, NJ) and measurement of systemic blood pressures. After rats recovered from the surgical procedure, blood pressure values were continuously monitored in conscious, free-roaming animals via a transducer (Statham; Madison, WI) for 30 min (52, 56). Thereafter, 50 mg/kg iv pentobarbital sodium (Abbot Laboratories; North Chicago, IL) was given as general anesthesia.

Mesentery preparation. The animals were placed on a water-heated (37°C) stage. The mesentery was gently exposed through a short abdominal midline incision and was continuously superfused with Krebs-Henseleit bicarbonate-buffered solution saturated with a 95% nitrogen-5% carbon dioxide gas mixture. For this purpose, only selected segments of the small intestine were manipulated with cotton wicks soaked in saline, and no contact was made with the mesentery tissue per se. The mesentery sectors were immediately treated to minimize formation of oxygen free radicals due to tissue exposure.

Free radical detection. TNBT (Vector Laboratories; Burlingame, CA) reduction to formazan crystals in the presence of superoxide was used to detect superoxide radicals in vivo as previously described (48). No glucose, however, was used as a substrate, because it yielded a labeling pattern that was too intense for quantitative image analysis. Fresh TNBT (prepared approximately every 10 min from a fresh solution) was topically applied by constant drip for 1 h on selected mesenteric sectors. At the end of this period, Krebs-Henseleit (36.5°C; pH 7.4) was used to wash TNBT from the specimen for 15 min. The mesentery tissue was fixed a topical application of 10% formalin (Sigma) for 15 min, excised, and then stored in 10% formalin. The formazan deposits are stable for several weeks under these conditions, so a detailed analysis of the staining pattern in all segments of the microcirculation can be carried out after completion of the experiment. This approach avoids the need to record the labeling pattern in a living tissue under a time constraint as is the case during use of other free radical detection techniques (44), and it also permits analysis in all segments of the microvascular network. Special care was taken to standardize the anesthesia, tissue handling, and TNBT labeling among groups.

Measurement techniques. The mesentery microcirculation is located at the distal segments of the blood supply from the superior mesenteric artery. In this study, only those regions of the mesentery without adipose tissue were examined, because the presence of adipose cells surrounding individual microvessels may limit the penetration of the TNBT solution and thereby reduce formazan crystal formation. Larger mesenteric arterioles are generally embedded in adipose tissue, which cannot be readily penetrated by the TNBT solution. Therefore, the largest arterioles included in the analysis were ~25 to 30 µm in lumen diameter in the younger animals and ≤60 µm in the older group. Venules had diameters ≤60 µm. Lymphatics were mostly of the contractile type with spontaneous peristalsis and lymphatic intraluminal valves.

The TNBT reaction could be blocked in large part on single leukocytes in suspension by superoxide dismutase (3) and was abolished in the presence of the hydroxyl radical scavenger dimethylthiourea (2 mM; Aldrich Chemical; Milwaukee, WI; results not shown). The reaction could also be blocked in the plasma by intravenously administered superoxide dismutase conjugated with polyethylene glycol (2 mg/kg; Sigma). But the formation of formazan crystals was not completely blocked in endothelial cells in vivo, because superoxide dismutase after intravascular administration does not readily penetrate the endothelial cell, pericytes, or smooth muscle cells where most of the reaction product may be located even if conjugated with heparin.

Image analysis. To detect formazan crystal density in the microvasculature, bright-field images of individual microvessels were recorded through a Leica intravital microscope. Our analysis was limited to microvessels located outside adipose tissue. Tissue overviews were recorded with x10 and x20 objectives and, for the purpose of optical density measurements, with a x60 objective (numerical aperture, 0.9). The images were captured with a color charge-couple device camera (DEI-470; Optronics Engineering; Goleta, CA), digitized (512 x 512, 8-bit gray-level resolution) on a Power Mac laboratory computer (Apple Computer; Cupertino, CA), and analyzed in digital format (NIH Image 1.61 public domain software).

Average light intensities over the microvessel (Iv) and over the adjacent tissue (It), which contained no significant formazan deposits (Fig. 1, top), were recorded, and the light absorption (A) was computed as A = ln(Iv/It).



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  		Fig. 1. Micrograph of a mesentery microcirculation in a 40-wk-old Wistar-Kyoto (WKY) rat (top) and age-matched spontaneously hypertensive rat (SHR; bottom) with dark purple formazan deposits due to tetranitroblue tetrazolium (TNBT) reduction. Deposit pattern in an arteriole (A), capillary (C), and venule (V) is shown at higher magnifications (right). Window positioned over the WKY blood vessel (V) indicates a typical area over which light intensity (Iv) was measured. Windows A and C above and below the venule show the area used to measure background light intensity (It). Other than in some tissue mast cells, there were no measurable formazan deposits in mesentery tissue. This was further confirmed by comparison with mesentery that was prepared in an identical fashion but without TNBT incubation. Note the higher density of formazan in the larger venules of SHRs.

 
All measurements were carried out by computer-aided analysis thus minimizing operator error. The investigator who performed the image analysis was unaware with regard to animal group until the absorption measurements were completed and entered into a data spreadsheet. Mesentery specimens that were fixed without application of TNBT solution served as controls. Such specimens had some degree of light absorption due only to hemoglobin, which did not differ between strains.

Statistics. Measurements are presented as means ± SD. Comparisons of mean values among groups were carried out by unpaired Student's t-test. P < 0.05 was considered significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Blood pressure. The femoral arterial blood pressures were significantly elevated in SHRs (14–16 wk of age) compared with their age-matched WKY controls. Blood pressure values of SHRs were reduced to normotensive levels after adrenalectomy and were significantly elevated after dexamethasone treatment (Table 1, P < 0.05).


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  		Table 1. Femoral arterial blood pressure for rats at 14–16 wk of age

 
The older SHRs (40 wk of age, untreated control group) also had significantly elevated blood pressure compared with their normotensive WKY controls (Table 2). After 26 wk of hydralazine treatment, the mean blood pressure for SHRs was normalized.


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  		Table 2. Femoral arterial blood pressure of rats at 40 wk of age

 
We also examined a group of SHRs and WKY rats that were treated at age 14 wk for 11–14 days with hydralazine, and no reduction of blood pressure was observed (results not shown).

Tissue pattern of TNBT reduction. In all groups, formazan deposits in the mesentery were largely limited to locations close to microvessels with some deposits in mast cells but with almost undetectable deposits in many regions of the interstitial space (see Fig. 1). There was no preferred localization of labeled mast cells regarding adjacency to arterioles, capillaries, or venules.

Formazan deposits could be detected in every hierarchy of microvessels but with a markedly nonuniform distribution along the microvascular network. Capillaries and smaller arterioles and venules in general had on average lower levels of formazan crystals than larger arterioles and venules (see Fig. 1, insets). These deposits were detectable in nonuniform formazan clusters located in close proximity to or within the endothelium. The deposition of formazan crystals was without a particular preference for vascular branch points or other locations along individual microvessels. Frequently, we saw arterioles or venules with high levels of formazan deposits giving rise to smaller side branches with low levels of deposits. The major change in formazan density occurred at branch points. Between branch points, we found a more uniform density of formazan deposits (see Fig. 1). Although mast cells in WKY rat mesentery had undetectable deposits, SHR mesentery showed evidence for localized formazan in the immediate vicinity of some but not all mast cells.

In addition to the formazan deposition in vascular endothelium, we also found significant TNBT reduction in the contractile lymphatics of the mesentery (Fig. 2) with enhancement at the (secondary) intralymphatic valves (Fig. 3) (34). The formazan deposits were uniform over the lymphatics (see Fig. 2) despite the highly attenuated endothelial morphology and were positioned frequently in immediate proximity to the strong formazan deposits in venules. Free radical production was also prominent on the leaflets of the lymphatic valves (see Fig. 3).



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  		Fig. 2. Micrographs of spontaneous TNBT reduction in lymphatics of the mesentery of a 40-wk-old WKY rat (top) and an age-matched SHR (bottom). Purple-black crystalline formazan deposits delineate free radical production along the endothelium of the lymphatic channels (Lym).

 


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  		Fig. 3. Micrographs of spontaneous TNBT reduction at lymphatic valves of a 40-wk-old WKY rat (top) and an age-matched SHR (bottom). Valves consist of bileaflets (Leaflet) positioned at a location in the lymphatic channel with enlargement of the lymphatic diameter and prominent formazan deposits.

 
Adrenal control of microvascular TNBT reduction. In the 14- to 16-wk-old SHRs, larger venules (>30 µm) had higher levels of formazan deposits as determined by light absorption compared with the corresponding venules of age-matched WKY rats (Fig. 4, top). In contrast, in capillaries and smaller arterioles or venules (<30 µm), we found no significant density differences for the formazan deposits between WKY rats and SHRs in any part of the mesentery microcirculation.



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  		Fig. 4. Means ± SD of light absorption values (see METHODS) in 14- to 16-wk-old WKY rats and SHRs under control conditions (top), with adrenalectomy (middle), and with adrenalectomy and dexamethasone supplementation (bottom). Number of vessels in each group is derived from 3 rats and 10–12 vessels in each vessel category per rat. *P < 0.05 compared with WKY rats.

 
After adrenalectomy, the SHRs had lower light-absorption values without significant differences between the WKY rats and SHRs (Fig. 4, middle). In contrast, after supplementation of the adrenalectomized WKY rats and SHRs with dexamethasone, the light absorption in venules was significantly raised without a significant effect on the population of small (<30 µm) arterioles. The light absorption in smaller venules (10–30 µm) of the SHR mesentery (Fig. 4, bottom) was reduced below the value in the WKY group after dexamethasone supplementation.

Microvascular TNBT reduction after chronic hydralazine treatment. In the 40-wk-old WKY rats and SHRs (controls without treatment), average TNBT reduction was significantly elevated compared with the younger, untreated counterparts (14–16 wk old) listed above. This increase with age was observed in all microvessels (P < 0.05). In addition, the formazan deposits in older SHRs were elevated in all groups of microvessels compared with WKY rats at the same ages (Fig. 5, top). Even capillaries, which in the younger group exhibited few formazan deposits and no difference between strains, had higher levels of light absorption in the older SHRs compared with the age-matched WKY rats.



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  		Fig. 5. Means ± SD of light absorption values in 40-wk-old WKY rats and SHRs without treatment (top) and after hydralazine treatment for 26 wk (bottom). Number of vessels in each group is derived from 3 rats and 10–12 vessels in each vessel category per rat. *P < 0.05 compared with WKY rats; **P < 0.05 compared with no hydralazine treatment.

 
When the blood pressure of the SHRs was reduced by 26-wk hydralazine treatment, we saw a significant reduction in formazan deposits (Fig. 5, bottom). This reduction was detected across all hierarchies of microvessels and was visible in both rat strains, i.e., was not specific to SHRs.

Free radical production in interstitium and microlymphatics. The average level of formazan deposits in interstitial cells was elevated in SHRs (40 wk of age; Fig. 6). Hydralazine treatment served to reduce the formazan levels in both WKY rats and SHRs but failed to reduce the difference between SHRs and WKY rats. A similar elevation of the light absorption in SHRs was detected in lymphatics on both the wall and the intraluminal leaflets (Fig. 7).



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  		Fig. 6. Means ± SD of light absorption values in mesentery interstitium of 40-wk-old WKY rats and SHRs without treatment (control) and after hydralazine treatment for 26 wk. Number of measurements in each group is derived from 3 rats per group and 20–30 interstitial measurements per rat. *P < 0.05 compared with WKY rats.

 


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  		Fig. 7. Means ± SD of light absorption values in mesentery lymphatics of 40-wk-old WKY rats and SHRs without treatment (control) and after hydralazine treatment for 26 wk. Number of measurements in the lymphatic wall is n = 10, derived from 10 lymphatics of 10 rats, and n = 8 valves from 8 rats for both WKY rats and SHRs. *P < 0.05 compared with WKY rats.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The localization of oxygen free radical production may be an important lead in deciphering the mechanisms for organ injury in the microcirculation of hypertensive individuals. The present experimental approach yields a pattern of formazan deposit that is quite specialized. We found low levels in the tissue parenchyma and the highest levels in the endothelial lining of the microvessels. In younger SHRs, larger venules tended to exhibit higher levels of TNBT reduction to formazan, whereas capillaries and small arterioles or venules showed lower levels. In older SHRs, on average, all microvessels exhibited elevated levels of formazan. The formazan deposits suggest that endothelial cells, circulating cells, and plasma may be important sources of oxygen free radicals.

A key feature brought to light by display of the microvascular formazan distribution is that the production of reactive oxygen species is not limited to the arteriolar side. Formazan is equally distributed on the arterial and on the venous side but appears at lower levels in capillaries. This pattern of reactive oxygen production has been encountered in all forms of hypertension investigated to date with a variety of experimental approaches (23, 44, 47, 48). Enhanced oxygen free radical production in SHRs or hypertensive Dahl rats can be documented in circulating leukocytes and in plasma that passes through both high- and low-pressure regions (38, 39). Enhancement of free radical production is also prominent in SHR lymphatics, which are vessels that serve to drain the interstitium with only connective-tissue connections to the microvessels in the mesentery.

These observations suggest that the mechanisms that mediate free radical production in hypertension may not be limited to the arterial and/or arteriolar side of the circulation (17), but instead may represent a more general vascular condition that affects both high- and low-pressure regions of the circulation. One such mechanism may be due to glucocorticoids. Glucocorticoids have the ability to directly influence the formation of oxygen free radicals and blood pressure (28, 52). Depletion of glucocorticoids by adrenalectomy and supplementation with dexamethasone affect the blood pressure and TNBT reduction. The blood pressure of SHRs is more affected by supplementation with glucocorticoids than the blood pressure of WKY rats. In the mesentery microvessels, glucocorticoids affect free radical formation mostly on the venous side without pressure elevation (56), and they directly influence enzymes that are involved in oxygen free radical formation (see below). The present observations of free radical formation in endothelium of arterioles and venules are in agreement with previous measurements using hydroethidine as an alternative technique for detecting superoxide (44).

The fact that free radical production in SHRs is not limited to microvascular regions with elevated blood pressure is further highlighted by the present observations of elevated TNBT reduction in mesentery lymphatics. In a variety of species under normal conditions, these vessels typically operate at pressures ≤10 mmHg (33). There are presently, however, no measurements of lymphatic pressures in SHRs.

The question then arises, is blood pressure elevation the cause for free radical production, or vice versa, is it possible that free radical production may cause pressure elevation? Perhaps both events occur more or less simultaneously. Acute elevation of blood pressure per se in vitro serves to raise oxygen free radical production (14). But to date, no chronic in vivo experimental model is available that serves to examine the role of blood pressure elevation on free radical production. Most models either have an effect on fluid shear stress or rely on administration of an agent such as glucocorticoid or angiotensin that has its own effect on oxygen radical formation, be it at high or low pressures. We examined here the effect of an alternative method of blood pressure reduction with chronic hydralazine treatment (for 26 wk). We found that blood pressure and oxygen free radical production reduction go hand in hand, albeit on both the arteriolar and venular sides of the microcirculation, and in some microvessels in both WKY rats and SHRs (see Fig. 5). In our pilot study with a shorter period of hydralazine treatment, we saw no blood pressure reduction as well as no reduction in formazan deposits in any segment of the microcirculation of either strain (results not shown). The hydralazine treatment per se may be inconsequential for free radical production unless it is accompanied by a blood pressure reduction. In addition, we found that the level of TNBT reduction increased with age in all microvessels, a reaction that was not accompanied by significant elevation of the mean arterial blood pressure or venular pressure.

Furthermore, in the past, we were able to obtain evidence in human hypertensive subjects to indicate that there may already be elevated hydrogen peroxide production in normotensive individuals at genetic risk for hypertension years before their blood pressure is elevated (22). Thus there is a lack of a spatial correlation between blood pressure and free radical production in the microcirculation, while at the same time, several treatments to reduce blood pressure are closely correlated with reduction of free radical production in both high and low fluid-pressure regions. This evidence, combined with the fact that prehypertensive individuals may already be producing enhanced free radical production before blood pressure elevation, suggests that there may be systemic mechanisms that control free radical production in addition to just pressure elevation. The fact that SHRs have an enhanced level of glucocorticoid and mineralocorticoid receptors (7) and after adrenalectomy their blood pressure responds significantly more strongly to glucocorticoids (46) is in line with this hypothesis. Comparing adrenalectomy and hydralazine treatment in SHRs, it is noteworthy that both serve to reduce TNBT reduction and blood pressure, but hydralazine is not specific to SHRs and instead affects free radical production in the normotensive WKY rats as well on both the arteriolar and venular sides.

Increased sensitivity to adrenal glucocorticoids has also been observed in human essential hypertensive subjects in the form of a skin-blanching test (50). This sensitivity may be one of the underlying processes in hypertension. Blood pressures of SHRs may also be hyperresponsive to mineralocorticoid and its metabolites (10, 13), a trend that is not uniformly confirmed (46). Blood pressure is significantly elevated by aldosterone supplementation in adrenalectomized SHRs (10, 37). However, the effect of mineralocorticoids on oxygen free radical formation in microvessels as players in blood pressure regulation and organ injury remains to be examined.

The plasma of essential hypertensive individuals has the ability to produce oxygen free radicals at a level that correlates with their blood pressure (22). Individuals with a family history of hypertension but still without elevated blood pressure already have levels of free radical production above those of normotensive controls. This evidence suggests that the free radical production may precede the blood pressure elevation. The level of free radical production is a heritable trait in humans and may be attributed to genetic factors (21).

Among a number of different enzymes that may be involved in free radical production in SHRs are NAD(P)H oxidase (55) and xanthine oxidase (41). The gender dimorphism with male SHRs presenting higher superoxide anion concentration under basal conditions than female SHRs may act via a mechanism that depends on NAD(P)H oxidase components (6). There could also be depletion of antioxidant enzymes (16, 26, 32, 40, 54). These enzyme systems can be modulated by renal and adrenal hormones, a situation that may be closely linked to the specific complications encountered in SHRs. The level of xanthine oxidase may be influenced by adrenal hormones (20, 52) and in SHRs can be inhibited by a glucocorticoid receptor inhibitor (44). The enzyme is involved in the production of oxygen free radicals of SHRs, Dahl hypertensive model rats, and dexamethasone-induced hypertensive model animals (41, 47, 52). Renal transplantation with kidneys from normotensive donor rats (4) as well as adrenalectomy serve to reduce blood pressure in SHRs (30, 42, 44). Furthermore, the evidence points to the fact that oxygen free radical production is controlled by the renin-angiotensin system (40, 55) as well as by an adrenal pathway (28). Scavenging of superoxide by nitric oxide may be attenuated by reduced NO activity (5, 18, 27) possibly via glucocorticoids (42, 51).

In postcapillary venules of the younger group (see Fig. 4), we saw no difference in formazan deposits between WKY rats and SHRs. The formazan deposits in postcapillary venules (<30 µm) may have even been on average higher in WKY rats than in SHRs (not significant). The trend was observed in the control WKY rat and SHR groups before and after adrenalectomy and in the adrenalectomized groups after supplementation with dexamethasone, and it may be influenced by leukocyte-endothelial interaction. The postcapillary venules are subject to regular interaction with circulating leukocytes. The rolling is especially prominent in smaller postcapillary venules and may serve as a signal-transduction mechanism that stimulates endothelial cells and their free radical production. Leukocyte rolling on or adhesion to endothelium in postcapillaries is greatly inhibited in SHRs (2). Adrenalectomy serves to increase the leukocyte-endothelium interaction in SHRs, whereas dexamethasone is well recognized as inhibiting the interaction (43). Thus an increased leukocyte-endothelial interaction in WKY rat postcapillary venules may, in part, account for the trend in oxygen free radical formation observed in this particular class of microvessels.

What may be the consequences of enhanced free radical formation in SHRs? In addition to a role in signaling arteriolar tone due to NO annihilation (11, 18, 23) they may also cause frank cell death (15). It is interesting to note that the pattern of formazan deposits with preference for microvascular endothelium is closely mirrored by markers for endothelial apoptosis in the mesentery microcirculation of SHRs (24). Adrenalectomy serves to reduce endothelial apoptosis to levels that are indistinguishable between WKY rats and SHRs. Glucocorticoids also enhance apoptosis in SHR microcirculation (24).


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by National Heart, Lung, and Blood Institute Grant HL-10881.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. W. Schmid-Schönbein, Dept. of Bioengineering, Whitaker Institute for Biomedical Engineering, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0412 (E-mail: gwss{at}bioeng.ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Aoki K, Tankaw H, and Fujinami T. Pathological studies on the endocrine organs of the spontaneously hypertensive rat. Jpn Heart J 4: 426–442, 1963.
  2. Arndt H, Smith CW, and Granger DN. Leukocyte-endothelial cell adhesion in spontaneously hypertensive and normotensive rats. Hypertension 21: 667–673, 1993.[Abstract/Free Full Text]
  3. Barroso-Aranda J, Chavez-Chavez RH, Mathison JC, Suematsu M, and Schmid-Schönbein GW. Circulating neutrophil kinetics during tolerance in hemorrhagic shock using bacterial lipopolysaccharide. Am J Physiol Heart Circ Physiol 266: H415–H421, 1994.[Abstract/Free Full Text]
  4. Bianchi G, Fox U, Di Francesco GF, Giovanetti AM, and Pagetti D. Blood pressure changes produced by kidney cross-transplantation between spontaneously hypertensive rats and normotensive rats. Clin Sci Mol Med 47: 435–448, 1974.[ISI][Medline]
  5. Boegehold MA. Flow-dependent arteriolar dilation in normotensive rats fed low- or high salt diets. Am J Physiol Heart Circ Physiol 269: H1407–H1414, 1995.[Abstract/Free Full Text]
  6. Dantas AP, Franco Mdo C, Silva-Antonialli MM, Tostes RC, Fortes ZB, Nigro D, and Carvalho MH. Gender differences in superoxide generation in microvessels of hypertensive rats: role of NAD(P)H-oxidase. Cardiovasc Res 61: 22–29, 2004.[Abstract/Free Full Text]
  7. Delano FA and Schmid-Schönbein GW. The glucocorticoid and mineralocorticoid receptor distribution in a microvascular network of the spontaneously hypertensive rat. Microcirculation 11: 69–78, 2004.[CrossRef][ISI][Medline]
  8. Frisbee JC, Roman RJ, Falck JR, Linderman JR, and Lombard JH. Impairment of flow-induced dilation of skeletal muscle arterioles with elevated oxygen in normotensive and hypertensive rats. Microvasc Res 60: 37–48, 2000.[CrossRef][ISI][Medline]
  9. Fukuda S, Yasu T, Kobayashi N, Ikeda N, and Schmid-Schönbein GW. Contribution of fluid shear response in leukocytes to hemodynamic resistance in the spontaneously hypertensive rat. Circ Res 95: 100–108, 2004.[Abstract/Free Full Text]
  10. Gorsline J and Morris DJ. The hypertensinogenic activity of 19-nor-deoxycorticosterone in the adrenalectomized spontaneously hypertensive rat. J Steroid Biochem 23: 535–536, 1985.[CrossRef][ISI][Medline]
  11. Gryglewski RJ, Palmer RMJ, and Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454–456, 1986.[CrossRef][Medline]
  12. Hibino M, Okumura K, Iwama Y, Mokuno S, Osanai H, Matsui H, Toki Y, and Ito T. Oxygen-derived free radical-induced vasoconstriction by thromboxane A2 in aorta of the spontaneously hypertensive rat. J Cardiovasc Pharmacol 33: 605–610, 1999.[CrossRef][ISI][Medline]
  13. Horiuchi M, Nishiyama H, Hama J, Takenaka T, Kondo H, Kino H, Nagata S, Sugimura K, and Katori R. Characterization of renal aldosterone receptors in genetically hypertensive rats. Am J Physiol Renal Fluid Electrolyte Physiol 264: F286–F291, 1993.[Abstract/Free Full Text]
  14. Huang A, Sun D, Kaley G, and Koller A. Superoxide released to high intra-arteriolar pressure reduces nitric oxide-mediated shear stress- and agonist-induced dilations. Circ Res 83: 960–965, 1998.[Abstract/Free Full Text]
  15. Ito H, Torii M, and Suzuki T. Comparative study on free radical injury in the endothelium of SHR and WKY aorta. Clin Exp Pharmacol Physiol Suppl 22: S157–159, 1995.[Medline]
  16. Ito H, Torii M, and Suzuki T. Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens 17: 803–816, 1995.[ISI][Medline]
  17. Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, and Hamilton CA. Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium. Hypertension 33: 1353–1358, 1999.[Abstract/Free Full Text]
  18. Koller A and Huang A. Impaired nitric oxide-mediated flow-induced dilatation in arterioles of spontaneously hypertensive rats. Circ Res 74: 416–421, 1994.[Abstract/Free Full Text]
  19. Koller A and Huang A. Shear stress-induced dilation is attenuated in skeletal muscle arterioles of hypertensive rats. Hypertension 25: 758–763, 1995.[Abstract/Free Full Text]
  20. Kurosaki M, Zanotta S, Li Calzi M, Garattini E, and Terao M. Expression of xanthine oxidoreductase in mouse mammary epithelium during pregnancy and lactation: regulation of gene expression by glucocorticoids and prolactin. Biochem J 319: 801–810, 1996.[Medline]
  21. Lacy F, Kailasam MT, O'Connor DT, Schmid-Schönbein GW, and Parmer RJ. Circulating oxygen radicals in human essential hypertension: role of heredity, gender, and ethnicity. Hypertension 36: 878–884, 2000.[Abstract/Free Full Text]
  22. Lacy F, O'Connor DT, and Schmid-Schönbein GW. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J Hypertens 16: 291–303, 1998.[CrossRef][ISI][Medline]
  23. Lenda DM, Sauls BA, and Boegehold MA. Reactive oxygen species may contribute to reduced endothelium-dependent dilation in rats fed high salt. Am J Physiol Heart Circ Physiol 279: H7–H14, 2000.[Abstract/Free Full Text]
  24. Lim HH, DeLano FA, and Schmid-Schönbein GW. Life and death labeling in the microcirculation of the spontaneously hypertensive rat. J Vasc Res 38: 228–236, 2001.[CrossRef][ISI][Medline]
  25. Miyazaki H, Shoji H, and Lee MC. Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy. Redox Rep 7: 260–265, 2002.[CrossRef][ISI][Medline]
  26. Nakazono K, Watanabe N, Matsuno K, Sasaki J, Sato T, and Inoue M. Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci USA 88: 10045–10048, 1991.[Abstract/Free Full Text]
  27. Nurkiewicz TR and Boegehold MA. Limitation of arteriolar myogenic activity by local nitric oxide: segment-specific effect of dietary salt. Am J Physiol Heart Circ Physiol 277: H1946–H1955, 1999.[Abstract/Free Full Text]
  28. Rajashree S and Puvanakrishnan R. Dexamethasone induced alterations in enzymatic and nonenzymatic antioxidant status in heart and kidney of rats. Mol Cell Biochem 181: 77–85, 1998.[CrossRef][ISI][Medline]
  29. Rathaus M and Bernheim J. Oxygen species in the microvascular environment: regulation of vascular tone and the development of hypertension. Nephrol Dial Transplant 17: 216–221, 2002.[Abstract/Free Full Text]
  30. Ruch W, Baumann JB, Häusler A, Otten UH, Siegl H, and Girard J. Importance of the adrenal cortex for the development and maintenance of hypertension in spontaneously hypertensive rats. Acta Endocrinol 105: 417–424, 1984.[Medline]
  31. Sagar S, Kallo IJ, Kaul N, Ganguly NK, and Sharma BK. Oxygen free radicals in essential hypertension. Mol Cell Biochem 111: 103–108, 1992.[ISI][Medline]
  32. Sato M, Yanagisawa H, Nojima Y, Tamura J, and Wada O. Zn deficiency aggravates hypertension in spontaneously hypertensive rats: possible role of Cu/Zn-superoxide dismutase. Clin Exp Hypertens 24: 355–370, 2002.[CrossRef][ISI][Medline]
  33. Schmid-Schonbein GW. Microlymphatics and lymph flow. Physiol Rev 70: 987–1028, 1990.[Abstract/Free Full Text]
  34. Schmid-Schönbein GW. The second valve system in lymphatics. Lymph Res Biol 1: 25–31, 2003.[CrossRef][Medline]
  35. Schmid-Schönbein GW, Seiffge D, DeLano FA, Shen K, and Zweifach BW. Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension 17: 323–330, 1991.[Abstract/Free Full Text]
  36. Schnackenberg CG, Welch WJ, and Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role of nitric oxide. Hypertension 32: 59–64, 1998.[Abstract/Free Full Text]
  37. Semafuko WE and Morris DJ. Effect of high calcium diet on the development of high blood pressure in intact spontaneously hypertensive rats and in adrenalectomized spontaneously hypertensive rats treated with aldosterone. Steroids 56: 131–135, 1991.[CrossRef][ISI][Medline]
  38. Shen K, DeLano FA, Zweifach BW, and Schmid-Schönbein GW. Circulating leukocyte counts, activation, and degranulation in Dahl hypertensive rats. Circ Res 76: 276–283, 1995.[Abstract/Free Full Text]
  39. Shen K, Sung KL, Whittemore DE, DeLano FA, Zweifach BW, and Schmid-Schönbein GW. Properties of circulating leukocytes in spontaneously hypertensive rats. Biochem Cell Biol 73: 491–500, 1995.[ISI][Medline]
  40. Shou I, Wang LN, Suzuki S, Fukui M, and Tomino Y. Effects of antihypertensive drugs on antioxidant enzyme activities and renal function in stroke-prone spontaneously hypertensive rats. Am J Med Sci 314: 377–384, 1997.[ISI][Medline]
  41. Suzuki H, DeLano FA, Parks DA, Jamshidi N, Granger DN, Ishii H, Suematsu M, Zweifach BW, and Schmid-Schönbein GW. Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats. Proc Natl Acad Sci USA 95: 4754–4759, 1998.[Abstract/Free Full Text]
  42. Suzuki H, Miura S, Mori M, Akiba Y, Nagahashi S, Zweifach BW, Ishii H, and Schmid-Schönbein GW. Protective role of adrenal glucocorticoids for gastric mucosa in spontaneously hypertensive rats. J Gastroenterol Hepatol 14: 364–371, 1999.[CrossRef][ISI][Medline]
  43. Suzuki H, Schmid-Schönbein GW, Suematsu M, DeLano FA, Forrest MJ, Miyasaka M, and Zweifach BW. Impaired leukocyte-endothelial cell interaction in spontaneously hypertensive rats. Hypertension 24: 719–727, 1994.[Abstract/Free Full Text]
  44. Suzuki H, Swei A, Zweifach BW, and Schmid-Schönbein GW. In vivo evidence for microvascular oxidative stress in spontaneously hypertensive rats. Hydroethidine microfluorography. Hypertension 25: 1083–1089, 1995.[Abstract/Free Full Text]
  45. Suzuki H, Zweifach BW, Forrest MJ, and Schmid-Schönbein GW. Modification of leukocyte adhesion in spontaneously hypertensive rats by adrenal corticosteroids. J Leukoc Biol 57: 20–26, 1995.[Abstract]
  46. Suzuki H, Zweifach BW, and Schmid-Schönbein GW. Dependance of elevated mesenteric arteriolar tone on glucocorticoids in spontaneously hypertensive rats. Int J Microcirc Clin Exp 15: 309–315, 1995.[ISI][Medline]
  47. Swei A, Lacy F, Delano FA, Parks DA, and Schmid-Schönbein GW. A mechanism of oxygen free radical production in the Dahl hypertensive rat. Microcirculation 6: 179–187, 1999.[CrossRef][ISI][Medline]
  48. Swei A, Lacy F, DeLano FA, and Schmid-Schönbein GW. Oxidative stress in the Dahl hypertensive rat. Hypertension 30: 1628–1633, 1997.[Abstract/Free Full Text]
  49. Walker BR, Best R, Shackleton CH, Padfield PL, and Edwards CR. Increased vasoconstrictor sensitivity to glucocorticoids in essential hypertension. Hypertension 27: 190–196, 1996.[Abstract/Free Full Text]
  50. Wallerath T, Witte K, Schäfer SC, Schwarz PM, Prellwitz W, Wohlfart P, Kleinert H, Lehr HA, Lemmer B, and Förstermann U. Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension. Proc Natl Acad Sci USA 96: 13357–13362, 1999.[Abstract/Free Full Text]
  51. Wallwork CJ, Parks DA, and Schmid-Schönbein GW. Xanthine oxidase activity in the dexamethasone-induced hypertensive rat. Microvasc Res 66: 30–37, 2003.[CrossRef][ISI][Medline]
  52. Yang D, Feletou M, Boulanger CM, Wu HF, Levens N, Zhang JN, and Vanhoutte PM. Oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats. Br J Pharmacol 136: 104–110, 2002.[CrossRef][ISI][Medline]
  53. Yuan YV, Kitts DD, and Godin DV. Heart and red blood cell antioxidant status and plasma lipid levels in the spontaneously hypertensive and normotensive Wistar-Kyoto rat. Can J Physiol Pharmacol 74: 290–297, 1996.[CrossRef][ISI][Medline]
  54. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, and Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension 32: 488–495, 1998.[Abstract/Free Full Text]
  55. Zweifach BW, Kovalcheck S, DeLano FA, and Chen P. Micropressure-flow relationship in a skeletal muscle of spontaneously hypertensive rats. Hypertension 3: 601–614, 1981.[Free Full Text]



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