AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H1989-H1996, 2005. First published October 21, 2004; doi:10.1152/ajpheart.00839.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/H1989    most recent
00839.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forder, J. P.
Right arrow Articles by Greene, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forder, J. P.
Right arrow Articles by Greene, A. S.

Angiogenic protection from focal ischemia with angiotensin II type 1 receptor blockade in the rat

Joan P. Forder, Diane H. Munzenmaier, and Andrew S. Greene

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 18 August 2004 ; accepted in final form 18 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.

cerebrovascular regulation; rat model; losartan; stroke

STROKE IS the number three killer in the United States and the leading cause of severe, long-term disability (28). The American Stroke Association estimated that ~700,000 Americans would experience a stroke in 2004 (28). Stroke results in a core area of cell death (infarct) due to interrupted circulation, with a larger surrounding area of compromised cell function resulting from reduced perfusion (penumbra) (28). The extent of the infarct that develops after alterations in perfusion depends on the ability of the affected tissue to compensate through metabolic adjustments or by restoration of blood flow (31). Without sufficient blood flow, cells must reduce metabolic activity to protect against apoptosis or face irreversible damage (31). A greater degree of vascularization may increase the viability of affected tissue and act to limit the extent of infarct.

One known angiogenic pathway involves ANG II. ANG II has been shown to work through both type 1 (AT1) and type 2 (AT2) receptors, which display opposing vasomotor and angiogenic actions (21). Losartan is an AT1 receptor antagonist that is used clinically to lower blood pressure (7) but has also been shown to decrease the incidence of stroke (3). Many studies have implied that blockade of the AT1 receptor allows for a more active angiogenic and neuroprotective role of the AT2 receptor in ischemic stroke (1, 26). However, direct evidence of this is currently lacking. Recent studies have shown that chronic, but not acute, pretreatment with an AT1 receptor antagonist significantly improves neurological outcome and reduces infarct size after temporary middle cerebral artery (MCA) occlusion followed by reperfusion in normotensive rats (15, 18, 19).

Previous work in our laboratory (22) showed that chronic oral administration of losartan in adult rats initiates a significant angiogenic response in the brain. To further explore the angiogenic protective effects of AT1 receptor blockade against ischemia in normotensive rats, we hypothesized that administration of losartan in the drinking water for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct by an increase in cerebral vascularization mediated by angiogenesis. Compared with untreated animals that received the same surgical intervention, chronic losartan pretreatment resulted in increased vessel density, increased average vessel size, increased cerebral surface vascularization, maintained vascular delivery, and reduced infarct size.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animal procedures were conducted in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Male Sprague-Dawley rats (12 wk of age; n = 6 in each group) weighing 350–400 g were used for this study. All animals had free access to standard laboratory chow and were fasted overnight before surgery. Animals in the experimental groups were provided with losartan (50 mg/day; generously donated by Merck) in the drinking water for 2 wk before surgery and 3 days after surgery. The daily intake of drug was closely monitored and adjusted as follows. A stock solution of losartan was created at a concentration of 1 mg/ml drinking water. Each rat was housed individually and had access to its own drinking bottle. The amount of water consumed was measured daily, and the concentration of the drug was adjusted according to the previous day’s measurement. Records were kept on each individual rat and then averaged. The average losartan consumption in rats was 50.07 ± 0.54 mg/day.

Permanent focal occlusion was induced over the whisker barrel cortex as previously described (7) and as modified from Wei et al. (29). Briefly, rats were anesthetized with a 0.01 ml/kg intramuscular injection of ketamine (100 mg/kg), acepromazine (2 mg/kg), and xylazine (50 mg/kg) and maintained at 37°C. The right external carotid artery (ECA) was cannulated for dye injections. After placement in a stereotaxic apparatus, a 6- to 8-mm cranial window was created over the right somatosensory cortex (2 mm caudal and 5 mm lateral to bregma). Small boluses (10–20 µl) of the vital dye Patent Blue Violet (10 mmol/l; Sigma) were injected into the right ECA to demonstrate transits through surface cortical vessels. A video recording was obtained at this time. For all rats, three critical arteriolar branches of the MCA around the barrel cortex were selected and electrically cauterized (Promhovet). The bolus injections of dye were repeated, and another video recording was made. For the untreated group, dye transits displayed complete lack of flow into surface vessels. For the three-vessel losartan (3V) group, surgery was halted after three arteriolar branches were cauterized. However, for the 3V group, dye transits indicated that retrograde flow occurred into the three cauterized vessels. For the remaining rats [all-vessel losartan (AV) group], cauterization continued until dye transits displayed complete lack of flow into surface vessels. A video recording was obtained at this time point for this group of rats. The window was replaced, and the scalp was sutured. The catheter was removed, and the anterior neck was sutured. All rats were allowed to recover and resume normal activities for 72 h after surgery. No evidence of gross neurological deficits was seen in any animal. To visualize the extent of hypoxia after focal ischemia surgery, six rats in each of three groups (untreated, 1 wk losartan treated, and 2 wk losartan treated) were infused with 0.50 ml of Hypoxyprobe-1 (30 mg/ml iv; Chemicon International) over a 30-s time period through the left femoral vein 1 h before cauterization of surface vessels.

At 72 h after surgery, all rats were anesthetized with 1 ml of pentobarbital sodium (50 mg/ml ip) and the brain was quickly dissected and placed at –20°C for 20 min. Each brain was coronally sliced (2 mm) and incubated for 15 min in 2% triphenyltetrazolium chloride (TTC) to visualize mitochondrial dehydrogenase activity. Each 2-mm slice was photographed at x0.63 magnification. Both hemispheric regions and the infarct region from each photographed image were traced and recorded (Metamorph version 4.6; Universal Imaging, Downingtown, PA) (Fig. 1A). From the 2-mm slices, the area was outlined from each region (infarct region, right and left hemispheres) and the percent volume of infarct was calculated.



View larger version (61K):
[in this window]
[in a new window]
  		Fig. 1. Immunohistochemical flowchart outlining visualization of cerebral microvessels. A: triphenyltetrazolium chloride (TTC)-stained 2-mm coronal section of the rat brain to visualize infarct area (white area). Right hemisphere (yellow outline) and infarct area (green outline) have been outlined and quantitated for area measurements. White bar, 2 mm. B: TTC-stained sections were cryosectioned at 4 µm and then stained immunohistochemically for CD31 (platelet endothelial cell adhesion molecule 1), a known vessel endothelial cell marker. Regions outlined in A were transposed onto x1 pictures of immunohistochemically stained sections for localization of infarct. This "map" was then used to determine infarct, penumbra, and control regions for x20 pictures of vessels. C: x20 pictures were obtained from within the infarct, penumbra, and control regions. Quantification of vessel density (no. of vessels/0.26 mm2), average vessel size (µm2), and total vessel area (vessel density x average vessel size) were obtained from these pictures as described in MATERIALS AND METHODS. Black bar, 0.2 µm.

 
The 2-mm slices were embedded in optimal cutting temperature compound (OCT; Tissue Tek) and frozen in 2-methylbutane maintained at –70°C by the addition of dry ice. Each frozen section was then used to produce 4-µm slices. These slices were immunohistochemically stained for either CD31 (platelet endothelial cell adhesion molecule 1) to visualize vessels or Hypoxyprobe-1 to visualize the extent of hypoxia. For both vessel visualization and visualization of hypoxia, slides were fixed in cold acetone at –20°C for 10 min, with endogenous peroxidase removed with 1:1 hydrogen peroxide-methanol for 10 min, and then blocked in 1% horse serum at room temperature for 30 min. Slides were incubated for 1 h in either monoclonal primary anti-rat CD31 antibody (BD Pharmingen, San Diego, CA) diluted in 3% horse serum (1:100) for visualization of vessels or monoclonal primary anti-rat Hypoxyprobe antibody (Chemicon International) diluted in 3% horse serum (1:500) for visualization of hypoxia. Slides were rinsed with Tris-buffered saline (TBS) and incubated for 30 min in biotinylated anti-mouse IgG, rat absorbed secondary antibody (Alexa Fluor 488, Molecular Probes, Eugene, OR) diluted in 3% horse serum (1:100). Slides were rinsed in TBS, incubated in the Vectastain Elite ABC system (Vector Laboratories, Burlingame, CA) for 30 min, and then quickly rinsed in distilled water before being exposed to diaminobenzidine (Vector) for 4 min. Slides were then rinsed in distilled water, air-dried, and coverslipped with the water-soluble mountant gelvatol. For vessel visualization, each 4-µm slice was photographed at x1 magnification and previously outlined regions from the 2-mm pictures were overlaid onto the x1 pictures to visualize the infarct region (Fig. 1B).

For vessel quantification, this overlaid image was used as a guide and x20 magnification pictures were obtained from within the infarct, penumbra, and control regions (Fig. 1C). The control regions consisted of regions within the right hemisphere but far removed from the infarct and identical (symmetrical) regions from the left (control) hemisphere. With Metamorph software, each x20 picture was analyzed for vessel density [the number of vessels in the field of view (FOV)], average vessel size of all vessels in the FOV, and total vessel area (product of vessel size x vessel density).

For hypoxia quantification, the x1 photographed images were analyzed with Metamorph software to determine the extent of hypoxia as a percentage of the affected hemisphere. Sham-treated animals were also analyzed at each time point to ensure that visualized hypoxia was due to focal occlusion. More specifically, each image was thresholded to eliminate any background effects. Measurements of the area of visualized Hypoxyprobe antibody as well as the total hemispheric area were obtained, and a calculation of the percent area covered by the displayed Hypoxyprobe antibody was made. It is important to note that no attempt was made to determine the intensity of the antibody staining but merely to look at the percentage of the area of the hemisphere that was displayed as taking up the Hypoxyprobe antibody.

During surgery, observation of the surface vessels indicated a possible increase in the extent of vascular branching visible through the cranial window of losartan-treated animals. To quantitate this observation, a precauterization image was captured for each rat and each image was converted to gray scale (Microsoft Photoshop version 7.0). With Metamorph software, automated measurement of the percentage of cortex area within a predetermined rectangular area (3 mm x 2 mm) over the whisker barrel cortex-constituting vessels was obtained.

To quantify microvascular delivery of blood within the whisker barrel cortex, a dye transit event was captured from each of the pre- and post-three-vessel cauterization videos from all rats in all groups (untreated, 3V, and AV groups) as well as the post-all-vessels cauterization videos from the AV group. This method involved using Adobe Premier software, by which 5 frames/s were captured starting 1 s before visible identification of dye in the arterioles and lasting until all visible dye had left the venules. For each event, each frame was converted to gray scale (Microsoft Photoshop version 7.0) and each frame of each event was stacked in the computer’s frame buffer to allow for automated analysis of dye intensity changes over the event period. A standardized circular area of 0.5 mm2 was positioned in an area that was determined to be devoid of large visible vessels ("avascular" region), and dye intensity data were collected for all frames in each event. The same avascular region was used in all videos obtained for each rat.

Data analysis. A two-way analysis of variance was performed for all measurements. All data are expressed as means ± SE. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Infarct size. Cauterization of vessels feeding the whisker barrel cortex of control animals resulted in a reproducible focal lesion confined to the whisker barrel cortex (Fig. 2A, left). TTC staining of the 2-mm-thick slices demonstrated a sharp delineation of the infarct region that was confined to the cortex. Only one of six rats in the 3V group displayed any infarct (Fig. 2A, right). When a bolus injection of dye was repeated after the three main arterioles were cauterized, all rats in this losartan group displayed backfilling of dye into the major surface arterioles, whereas untreated animals did not. For the remaining six losartan-pretreated animals (AV group), cauterization was continued beyond the three original vessels until a bolus injection of dye revealed no backfilling into the major surface arterioles. This cauterization resulted in 8–14 occlusion points (Fig. 2A, middle). For the AV group, the average infarct size was significantly smaller (0.40% ± 0.16 of right hemisphere) than in untreated rats (2.91% ± 0.54 of right hemisphere) but was significantly larger than the average infarct size of the 3V group (0.031% ± 0.03 of right hemisphere; Fig. 2B).



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2. Comparison of infarct size between untreated and losartan-pretreated rats (50 mg/day, oral administration for 2 wk before focal ischemia surgery). A: visual representation of treatment groups. Losartan-pretreated animals subjected to the same surgical protocol as untreated animals displayed no visible infarct (losartan 3-vessel group). Losartan-pretreated animals that received between 8 and 12 cauterizations displayed a significantly decreased infarct size compared with untreated animals (losartan all-vessels group). White bar, 2 mm. B: quantification of infarct size between treatment groups. Data represent the infarct size as % of the right hemisphere. Results are presented as means ± SE; n = 6 in each group. *Significantly different from untreated group; {ddagger}significantly different from losartan all-vessels group (P < 0.05).

 
Angiogenesis. Only one of the six 2-wk losartan-pretreated animals displayed any visual infarct. Therefore, in the animals that did not display any visible infarct, an area of the whisker barrel cortex equivalent to the infarct size and location of the animal that did show a visible infarct was used for analysis and was termed the "area at risk." The decrease in vessel density seen in the infarct region of untreated animals in response to focal ischemia was attenuated in 1-wk losartan-pretreated animals and increased even further in 2-wk losartan-treated animals (Fig. 3A). The increase in vessel density seen in control regions of untreated animals in response to focal ischemia was not seen in 1-wk or 2-wk losartan-pretreated animals. The increase in vessel density seen in the penumbra region of untreated animals in response to focal ischemia was also present in 1-wk, but not in 2-wk, losartan-pretreated animals. Comparing sham-surgery animals, a significant increase in vessel density was seen in all cortical areas of 2-wk losartan-pretreated animals (102.5 vessels/0.26 mm2) compared with untreated animals (76.5 vessels/0.26 mm2) (P < 0.001). Analysis of all sham-surgery groups indicated no significant differences throughout the cortex as follows: untreated sham-surgery group: control region = 77.75 ± 4.93, area at risk = 75.42 ± 4.26, penumbra region = 76.29 ± 5.48 [not significant (NS)]; 1-wk losartan-pretreated sham-surgery group: control region = 78.09 ± 4.89, area at risk = 82.02 ± 5.73, penumbra region = 82.84 ± 5.79 (NS); 2-wk losartan-pretreated sham-surgery group: control region = 101.74 ± 6.61, area at risk = 103.31 ± 5.24, penumbra region = 102.44 ± 4.17 (NS). Therefore, all three cortical regions were combined for each treatment group (Fig. 3A). The vessel density was significantly increased in 2-wk losartan-pretreated sham-surgery animals (102.5 vessels/0.26 mm2) compared with untreated sham-surgery animals (76.5 vessels/0.26 mm2). Vessel density in 1-wk pretreated sham-surgery animals was not significantly different from untreated sham-surgery animals (Fig. 3A).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3. Comparison of vessel density, average vessel size, and total vessel area between treatment groups. A: vessel density within the region at risk (dark gray bars) was protected in losartan-pretreated animals. Vessel density increased in the penumbra region (open bars) of the untreated and 1-wk losartan-pretreated animals but not in the 2-wk losartan-pretreated animals. Vessel density within control regions (light gray bars) increased in untreated animals but not in the 1-wk or 2-wk losartan-treated animals. Filled bars, sham-surgery animals. B: remodeling of the microvasculature was seen as an average vessel size increase from sham-surgery animals in each treatment group. C: total vessel area decreased in the region at risk in untreated animals but was protected in losartan-pretreated animals. Results are presented as means ± SE; n = 6 in each group. *Significantly different from sham-surgery animals; {ddagger}significantly different from untreated group (P < 0.05).

 
The average vessel size in control and penumbra regions of untreated focal ischemia animals was significantly increased from that of sham-surgery animals (Fig. 3B) and remained increased in both 1-wk and 2-wk losartan-pretreated animals. Although the average vessel size within the area at risk of the untreated animals was not significantly different from sham-surgery animals, it was significantly increased in both 1-wk and 2-wk losartan-pretreated animals (P < 0.001). Comparing sham-surgery animals, no significant difference in average vessel size was seen between treatment groups. Furthermore, no significant difference was seen in all cortical regions within each sham-surgery group (control, area at risk, or penumbra). Therefore, all three cortical regions were combined for each treatment group.

Total vessel area is a product of vessel density and average vessel size. A significant decrease was seen in the total vessel area in the infarct region in untreated animals in response to focal ischemia (3,765.8 µm2/0.26 mm2) compared with total vessel area in a comparable region in untreated sham-surgery animals (4,810.6 µm2/0.26 mm2) (P < 0.01; Fig. 3C). The total vessel area in the infarct area of 1-wk losartan-pretreated animals was significantly increased (6,894.9 µm2/0.26 mm2) compared with a comparable region in 1-wk losartan-pretreated sham-surgery animals (4,319.2 µm2/0.26 mm2) (P < 0.001). The total vessel area was significantly increased in the area at risk in 2-wk losartan-pretreated animals (10,674.3 µm2/0.26 mm2) compared with 2-wk losartan-pretreated sham-surgery animals (7,859.5 µm2/0.26 mm2) (P < 0.001; Fig. 3C). The increase in total vessel area seen in control regions of untreated animals in response to focal ischemia was also seen in 1-wk losartan-pretreated animals but was not seen in 2-wk losartan-pretreated animals (Fig. 3C). The increase in total vessel area seen in the penumbra region of untreated animals in response to focal ischemia was also present in 1-wk and 2-wk losartan-pretreated animals (Fig. 3C). Comparing sham-surgery animals, no significant difference was seen in all cortical regions within each sham-surgery group (control, area at risk, or penumbra). Therefore, all three cortical regions were combined for each treatment group (Fig. 3C). A significant increase in total vessel area was seen in all cortical areas of 2-wk losartan-pretreated sham-surgery animals (7,859.5 µm2/0.26 mm2) compared with untreated sham-surgery animals (4,810.6 µm2/0.26 mm2) (P < 0.001). A trend toward significance was seen in 1-wk losartan-pretreated sham-surgery animals compared with untreated sham-surgery animals. Therefore, the changes seen within each region appear to increase in a time-dependent manner.

Extent of hypoxia. Immunohistochemistry of the hypoxic region with Hypoxyprobe-1 antibody revealed a significantly smaller visible hypoxic region within the hemisphere that received focal occlusion in 2-wk losartan-pretreated animals (3.9%) compared with untreated animals (13.8%) (P < 0.001; Fig. 4). The extent of the hypoxic area was not significantly different between untreated and 1-wk losartan-pretreated animals.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4. Area of hypoxia comparison between treatment groups. Losartan pretreatment reduced the size of the hypoxic area after focal ischemia. Results are presented as means ± SE; n = 6 in each group. *Significantly different from untreated group (P < 0.05).

 
Surface vascularization. Quantification of the percent area covered by surface vessels revealed that 2-wk losartan-pretreated animals displayed a significantly higher surface vascularization (31.8% ± 0.92 of surface area) compared with animals given normal drinking water (untreated animals; 16.7% ± 0.84 of surface area) (Fig. 5). Surface vascularization was not significantly different between untreated and 1-wk losartan-pretreated animals.



View larger version (54K):
[in this window]
[in a new window]
  		Fig. 5. Surface vessel area comparison between groups. Animals pretreated with 50 mg/day losartan in the drinking water for 2 wk before infarct surgery displayed a significantly larger vessel surface area compared with untreated animals. Results are presented as means ± SE; n = 6 in each group. *Significantly different from untreated group (P < 0.05). Blue bar, 2 mm.

 
Vascular delivery. Small bolus injections of dye were made before and after cauterization for each rat. Precauterization dye intensity changes in the avascular area of the cranial window for all animals indicated a rapid initial increase in intensity with a slower emptying (Fig. 6). Cauterization of control animals resulted in cessation of vascular delivery into the area. However, for losartan-pretreated animals, the same cauterization resulted in only a delayed vascular delivery. Cauterization of an additional 8–14 points in the losartan-pretreated animals resulted in complete cessation of vascular delivery into the area. One-week losartan-pretreated animals displayed an attenuated, but still present, vascular delivery after the standard three-vessel occlusion of surface vessels (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Vascular delivery comparison between groups both before and after cauterization. Precauterization dye transit time for all groups ({bullet}) indicates a rapid vascular delivery that dissipates within 2–3 s from onset. Dye transit time after 3-vessel cauterization ({square}) in untreated animals (A) indicates occluded vascular delivery, whereas in the losartan-pretreated animals (50 mg/day, B) the same cauterizations displayed vascular delivery that was delayed but equivalent to that of precauterization. For losartan-pretreated animals, vascular delivery was eliminated only after 8–14 cauterizations ({blacktriangleup}, C). Results are presented as means ± SE; n = 6 in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides the first quantitative description of a vascular angiogenic protective role for the AT1-specific antagonist losartan against cerebral ischemia. This protection was characterized by decreased infarct size, increased microvascular measures of vessel density, average vessel size, and total vessel area, reduced resultant hypoxia, increased surface vascularization, and sustained vascular delivery in losartan-pretreated animals in a time-dependent manner. These findings provide strong evidence for increased collateralization in the cerebral vascular network resulting in little or no visible infarct. The increase in average vessel size after focal ischemia provides additional evidence for a remodeling response in support of previously postulated theory (28). Remodeling provides greater blood flow, whereas the increase in collateralization would provide alternate routes for blood flow into tissue that has been subjected to ischemic stroke. This combination could provide adequate tissue perfusion and assist in maintaining the viability of the tissue. The increased vessel density seen in the penumbra of infarcted rats that had not been given losartan implies a compensatory adaptation 72 h after focal ischemia. Even when all surface vessels were cauterized in losartan-pretreated animals, the infarct size was still significantly smaller than in untreated animals, suggesting possible cerebral collateralization from deeper vessels and remodeling of the existing vascular bed. As indicated in the 1-wk losartan-pretreated animals, in which remodeling but not increased vessel density was seen, this remodeling may precede the growth of new vessels and provide a degree of vascular protection.

Previous studies demonstrated a decrease in infarct size in various animal models after chronic losartan pretreatment (5, 19). However, the length of pretreatment, the mode of administration, and the animal used varies from study to study. For example, a single bolus pretreatment dose of losartan (10 mg/kg) significantly increased the survival rate of gerbils subjected to gradual occlusion of the carotid artery while increasing blood flow in the ipsilateral hemisphere (5). Such an acute effect of losartan may involve immediate opening of ipsilateral circulation, which would be crucial in the gerbil as this species has an incomplete circle of Willis and therefore cannot obtain increased blood flow from the contralateral hemisphere to the same extent as other species.

In a recent study using spontaneously hypertensive rats (SHR), the ANG I-converting enzyme inhibitor captopril, the Ca2+ channel blocker nicardipine, or the AT1 receptor blocker candesartan was given to rats for 3 (acute) or 28 (chronic) days (19). The size of infarct and cerebral blood flow (CBF) were measured after permanent MCA occlusion (19). Acute administration did not affect either infarct size or CBF, which is in contrast to the effect seen in gerbil studies and therefore does not support the vasodilation theory of protection from infarct. Rats, having a complete circle of Willis, would be more capable of collateral recruitment than gerbils. Thus, as outlined by Ito et al. (19), if increased blood flow due to vasodilation of ipsilateral vessels was the mechanism responsible for the decreased infarct size in gerbils, then rats should also see an increased blood flow and decreased infarct size with acute administration of the AT1 blocker. However, this was not the case (19). We did not measure the effect of acute losartan pretreatment on blood flow in our normotensive rat strain in the current study but plan to do so in the future.

The Ito et al. (19) study found that, in contrast to acute pretreatment, chronic pretreatment of either captopril or candesartan, but not nicardipine, reduced infarct size. The reduced infarct size cannot be accounted for by an increased CBF because nicardipine and captopril both significantly decreased CBF in the peripheral area of ischemia whereas chronic pretreatment with candesartan in SHR resulted in only a slight decrease in CBF (19). Chronic pretreatment with candesartan did result in a concurrent increased MCA external diameter and a reduced media thickness of the MCA (19). Because all three drugs decreased blood pressure equally but only captopril and candesartan reduced infarct size, Ito et al. (19) concluded that the protection against ischemia is dependent on ANG II system inhibition. The present study used a normotensive rat strain and indicates an angiogenic component of ischemic protection that supports the findings of Ito et al. (19). Although we have found a strong effect with losartan, recent studies indicate that other AT1 receptor antagonists may cross the blood-brain barrier more effectively (12, 13).

It is important to note that although the MCA is a resistance vessel, the downstream arterioles also play a major role in local blood flow regulation and must be examined for possible increased compliance and sensitivity to ischemic injury. Furthermore, AT1 blockade has been shown to increase plasma ANG II levels (15), thus providing for an increased stimulation of other ANG II receptor subtypes. Either of these possibilities would further support the explanation put forward by Ito et al. (19) in that the protection against ischemia is dependent on ANG II system inhibition.

In Wistar rats, chronic pretreatment with candesartan resulted in significant improvement in neurological outcome and reduced infarct size and edema of the hemisphere with focal ischemia (15). Although both that study and the present study used normotensive rats and obtained similar findings concerning reduced infarct size, some significant differences are found. We administered losartan orally for 14 days as opposed to subcutaneously for 5 days. Previous studies have found that 7–14 days of pretreatment are necessary with AT1 receptor blockade before normalization of the cerebrovascular regulation can occur (23). Our focal ischemic model was restricted to the somatosensory whisker barrel cortex of the rat, whereas the Groth et al. study (15) involved a more extensive complete MCA occlusion, thus allowing for more extensive neurological damage. Groth et al. (15) measured neurological behavioral outcome of the occluded rats, whereas we used quantitative vessel area and vascular delivery measures to assess the protective effect. With the use of a 14-day pretreatment period, a focal occlusion model, and quantitative measures, the present study has defined a possible angiogenic component to ischemic protection after stroke.

The decrease in infarct size following chronic administration of an AT1 receptor antagonist such as losartan could be due to a number of different mechanisms: NO-dependent or bradykinin-dependent vasodilation of existing vessels, neuroprotection, or angiogenesis. Although strong evidence has been provided for the role of ANG II in limiting the decrease in blood flow after ischemia through the rapid recruitment of preexisting collateral circulation in noncerebral tissues (2, 23), direct evidence of this effect after cerebral focal ischemia has been lacking. Such a mechanism has been postulated in SHR (19) without direct experimental evidence. However, in a direct comparison between SHR and Wistar-Kyoto rats, only SHR displayed NO-dependent vasodilation after AT2 stimulation (24). Other studies have reported a vasodilatory protective role of losartan initiated through a kinin-dependent mechanism in the aorta of stroke-prone SHR (11). However, this response could not be repeated in normotensive animals (11). Other studies have indicated that in SHR it is not the increase in CBF that is responsible for decreases in focal ischemic infarct size (30). Together, these studies indicate that vasodilation, through an NO- or kinin-dependent mechanism, cannot fully account for the protective role of losartan in reducing infarct size in normotensive animals after focal ischemia.

A neuroprotective action of AT1 antagonist pretreatment has been reported in the rat brain (4). Such a protective action has been shown to significantly improve the neurological outcome of focal cerebral ischemia with markedly reduced expression of c-Fos and c-Jun proteins in the cortex in the ipsilateral hemisphere (4). However, the presence of these proteins indicates cellular stimulation in response to stressors such as ischemia (16) but is not necessarily indicative of increased cellular death. Thus there may not be a direct neuroprotective role of AT1 antagonist pretreatment but instead a decreased ischemic stressor, as indicated by the reduced expression of these proteins. It has also been suggested that ischemia induces an overexpression of AT2 receptors in neuronal cell cultures, thus providing a cellular adaptation to hypoxia (26). Chronic administration of AT1 antagonist has been shown to remove the negative feedback to renin release and thus results in an increase in circulating plasma ANG II. This increased ANG II is believed to increase AT2 receptor stimulation without the opposing actions of AT1 receptor activation. However, direct evidence for this has not been reported to date. It is also possible that increased ANG II levels may result in increased metabolites such as ANG III or ANG IV and that the specific receptors to these metabolites may be involved in this phenomenon.

The remaining possible mechanism involves an angiogenic role of losartan. The angiogenic role of ANG II has been well documented in noncerebral organs such as skeletal muscle (14, 21) kidney (16, 17), rabbit cornea (6, 9), and various cell lines (25). These studies indicate systemically that it is the AT1 receptor that mediates angiogenesis and vasoconstriction, whereas the AT2 receptor opposes these actions. There have been recent indications that the roles of these two receptors may differ in the brain (22), with AT1 receptor blockade increasing cerebral microvessel density in rats. Thus pretreatment with losartan would block the angiogenic inhibition and favor microvessel proliferation. This increased angiogenesis would provide a more dense vascular bed. When a vessel is occluded, as is the case in stroke, the increased angiogenesis could allow for blood flow to the area from alternative routes. Evidence for this correlation between increased angiogenesis and more positive outcomes after stroke in humans showed that increased angiogenesis correlated strongly with longer survival rates after stroke when measured postmortem (20).

The present study provides direct evidence of increased angiogenesis, increased surface vascularization, and maintained microvascular delivery after chronic administration of losartan followed by permanent focal ischemia to small surface branches of the MCA over the whisker barrel cortex of normotensive rats. Because losartan is clinically administered to combat hypertension, it may also provide an inherent vascular protective role against cerebral ischemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-29587 and MH-51358.


   ACKNOWLEDGMENTS
 
The authors thank Merck for the generous donation of losartan compound.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Greene, Dept. of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (E-mail: agreene{at}mcw.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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Achard JM, Fournier A, Mazouz H, Caride VJ, Penar PL, and Fernandez LA. Protection against ischemia: a physiological function of the renin-angiotensin system. Biochem Pharmacol 62: 261–271, 2001.[CrossRef][Web of Science][Medline]
  2. Campbell DJ. Endogenous angiotensin II levels and the mechanisms of action of angiotensin-converting enzyme inhibitors and angiotensin receptor type 1 antagonists. Clin Exp Pharmacol Physiol 3: S125–S131, 1996.
  3. Dahlof B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, deFaire U, Fyhrquist F, Ibsen H, Kristiansson K, Lederballe-Pedersen O, Lindholm LH, Nieminen MS, Omvik P, Oparil S, and Wedel H. Cardiovascular morbidity and mortality in the Losartan Intervention For Endpoint reduction in hypertension study (LIFE): a randomized trial against atenolol. Lancet 359: 995–1003, 2002.[CrossRef][Web of Science][Medline]
  4. Dai WJ, Funk A, Herdegen T, Unger T, and Culman J. Blockade of central angiotensin AT1 receptors improves neurological outcome and reduces expression of AP-1 transcription factors after focal brain ischemia in rats. Stroke 30: 2391–2398, 1999.[Abstract/Free Full Text]
  5. Fernandez LA, Spencer DD, and Kaczmar T. Angiotensin II decreases mortality rate in gerbils with unilateral carotid ligation. Stroke 17: 82–85, 1986.[Abstract/Free Full Text]
  6. Fernandez LA, Twickler J, and Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med 105: 141–145, 1985.[Web of Science][Medline]
  7. Ferrario C, Abdelhamed AI, and Moore M. AII antagonists in hypertension, heart failure, and diabetic nephropathy: focus on losartan. Curr Med Res Opin 20: 279–293, 2004.[CrossRef][Web of Science][Medline]
  8. Forder JP, Greene AS, and Munzenmaier DH. Focal ischemia model of stroke in the rat. Experimental Biology meeting abstracts. FASEB J 17: A553, 2003.
  9. Gimbrone MA, Cotran RS, Leapman SB, and Folkman J. Tumor growth and neovascularization: an experimental model using the rabbit cornea. J Natl Cancer Inst 52: 413–427, 1974.
  10. Gohlke P, Linz W, Scholkens BA, Wiemer G, and Unger T. Cardiac and vascular effects of long-term losartan treatment in stroke-prone spontaneously hypertensive rats. Hypertension 28: 397–402, 1996.[Abstract/Free Full Text]
  11. Gohlke P, Pees C, and Unger T. AT2 receptor stimulation increases aortic cyclic GMP in SHRSP by a kinin-dependent mechanism. Hypertension 31: 349–355, 1998.[Abstract/Free Full Text]
  12. Gohlke P, Von Kugelgen S, Jurgensen T, Kox T, Rascher W, Culman J, and Unger T. Effects of orally applied candesartan cilexetil on central responses to angiotensin II in conscious rats. J Hypertens 20: 909–918, 2002.[CrossRef][Web of Science][Medline]
  13. Gohlke P, Weiss S, Jansen A, Wienen W, Stangier J, Rascher W, Culman J, and Unger T. AT1 receptor antagonist telmisartan administered peripherally inhibits central responses to angiotensin II in conscious rats. J Pharmacol Exp Ther 298: 62–70, 2001.[Abstract/Free Full Text]
  14. Greene AS. Life and death in the microcirculation: a role for angiotensin II. Microcirculation 5: 101–107, 1998.[CrossRef][Web of Science][Medline]
  15. Groth W, Blume A, Gohlke P, Unger T, and Culman J. Chronic pretreatment with candesartan improves recovery from focal cerebral ischaemia in rats. J Hypertens 21: 2175–2182, 2003.[CrossRef][Web of Science][Medline]
  16. Hernandez I and Greene AS. Hemodynamic and microcirculatory changes during development of renal hypertension. Am J Physiol Heart Circ Physiol 268: H33–H38, 1995.[Abstract/Free Full Text]
  17. Ilich N, Hollenberg NK, Williams DH, and Abrams HL. Time course of increased collateral arterial and venous endothelial cell turnover after renal stenosis in the rat. Circ Res 45: 579–582, 1979.[Abstract/Free Full Text]
  18. Ito T, Nishimura Y, and Saavedra J. Pre-treatment with candesartan protects from cerebral ischaemia. J Renin Angiotensin Aldosterone Syst 2: 174–179, 2001.[Abstract/Free Full Text]
  19. Ito T, Yamakawa H, Bregonzio C, Terron JA, Falcon-Neri A, and Saavedra JM. Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin II AT1 antagonist. Stroke 33: 2297–2303, 2002.[Abstract/Free Full Text]
  20. Krupinski J, Kaluza J, Kumar P, Kumar S, and Wang JM. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25: 1794–1799, 1994.[Abstract]
  21. Munzenmaier DH and Greene AS. Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension 27: 760–765, 1996.[Abstract/Free Full Text]
  22. Munzenmaier DH and Greene AS. Angiotensin II receptor blockade increases cerebral microvessel density in rats (Abstract). J Cereb Blood Flow Metab 23: 490, 2003.[CrossRef][Web of Science][Medline]
  23. Nishimura Y, Ito T, and Saavedra JM. Angiotensin II AT1 blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats. Stroke 31: 2478–2486, 2000.[Abstract/Free Full Text]
  24. Pees C, Unger T, and Gohlke P. Effect of angiotensin AT2 receptor stimulation on vascular cyclic GMP production in normotensive Wistar Kyoto rats. Int J Biochem Cell Biol 35: 963–972, 2003.[CrossRef][Web of Science][Medline]
  25. Schelling P, Ganten D, Speck G, and Fischer H. Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells. J Cell Physiol 98: 503–513, 1979.[CrossRef][Web of Science][Medline]
  26. Shibata K, Makino I, Shibaguchi H, Niwa M, Ohgami Y, Fujiwara M, Furukawa T, and Katsuragi T. Expression of angiotensin type-2 receptors in rat brain during the cell injury. Nippon Yakurigaku Zasshi 112: 53P–57P, 1998.
  27. Von Lutterotti N, Camargo MJ, Campbell WG Jr, Mueller FB, Timmermans PB, Sealey JE, and Laragh JH. Angiotensin II receptor antagonist delays renal damage and stroke in salt-loaded Dahl salt-sensitive rats. J Hypertens 10: 949–957, 1992.[Web of Science][Medline]
  28. Wei G, Dawson VL, and Zweier JL. Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim Biophys Acta 1455: 23–34, 1999.[Medline]
  29. Wei L, Rovainen CM, and Woolsey TA. Ministrokes in rat barrel cortex. Stroke 26: 1459–1462, 1995.[Abstract/Free Full Text]
  30. Yamamoto S, Golanov EV, and Reis DJ. Reductions in focal ischemic infarctions elicited from cerebellar fastigial nucleus do not result from elevations in cerebral blood flow. J Cereb Blood Flow Metab 13: 1020–1024, 1993.[Web of Science][Medline]
  31. Zhang RL, Lu CZ, Ren HM, and Xiao BG. Metabolic changes of arachidonic acid after cerebral ischemia-reperfusion in diabetic rats. Exp Neurol 184: 746–752, 2003.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
W. Kozak, A. Kozak, M. H. Johnson, H. F. Elewa, and S. C. Fagan
Vascular Protection with Candesartan after Experimental Acute Stroke in Hypertensive Rats: A Dose-Response Study
J. Pharmacol. Exp. Ther., September 1, 2008; 326(3): 773 - 782.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
H.-S. Sun, T. A. Doucette, Y. Liu, Y. Fang, L. Teves, M. Aarts, C. L. Ryan, P. B. Bernard, A. Lau, J. P. Forder, et al.
Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat
Stroke, September 1, 2008; 39(9): 2544 - 2553.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. H. Munzenmaier and A. S. Greene
Chronic angiotensin II AT1 receptor blockade increases cerebral cortical microvessel density
Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H512 - H516.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/H1989    most recent
00839.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forder, J. P.
Right arrow Articles by Greene, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forder, J. P.
Right arrow Articles by Greene, A. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.