Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Dec 3.
Published in final edited form as: Neurochem Int. 2022 Jun 7;158:105375. doi: 10.1016/j.neuint.2022.105375

Contralesional angiotensin type 2 receptor activation contributes to recovery in experimental stroke

Abdelrahman Y Fouda a,b,*, Heba A Ahmed c, Bindu Pillai d,e, Anna Kozak d,e, Trevor Hardigan f, Adviye Ergul g,h, Susan C Fagan d,e, Tauheed Ishrat c,i,**
PMCID: PMC9719365  NIHMSID: NIHMS1844106  PMID: 35688299

Abstract

We and others have previously shown that angiotensin II receptor type 2 receptor (AT2R) is upregulated in the contralesional hemisphere after stroke in normoglycemic Wistar rats. In this study, we examined the expression of AT2R in type 2 diabetic Goto-Kakizaki (GK) rats and control Wistars after stroke. We also tested the contribution of the contralesional AT2R in recovery after stroke through a specific knockdown of the AT2R in this hemisphere only. Two experiments were conducted. In the first experiment, GK rats were subjected to middle cerebral artery occlusion (MCAO) and treated with the angiotensin II receptor type 1 receptor (AT1R) blocker candesartan or saline at reperfusion. Stroke outcomes, as well as AT2R expression, were examined and compared to control Wistars at 24 h. In the second experiment, localized AT2R knockdown was achieved through intrastriatal injection of short hairpin RNA (shRNA) lentiviral particles or non-targeting control into the left-brain hemisphere of Wistar rats. After 14 days, rats were subjected to right MCAO and treated with the AT2R agonist, Compound 21 (C21), or saline for 7 days. Behavioral outcomes were assessed for up to 10 days. In the first experiment, stroke reduced the expression of AT2R in GK rats. Candesartan treatment failed to improve the neurobehavioral outcomes, preserve vascular integrity or reduce oxidative/nitrative stress or apoptotic markers at 24 h post stroke in these animals. In the second experiment, contralesional AT2R knockdown reduced the C21-mediated functional recovery after stroke. In conclusion, contralesional AT2R upregulation after stroke is blunted in diabetic rats which show reduced sensitivity to post-stroke candesartan treatment. Contralesional AT2R could be involved in C21-mediated functional recovery after stroke.

Keywords: Stroke, Angiotensin receptors, Diabetes, Candesartan, Compound 21, contralesional hemisphere

1. Introduction

Angiotensin II activates two main types of receptors, the angiotensin II type 1 and type 2 receptors (AT1R and AT2R). Literature from the past decade has unequivocally proven the protective effect of AT1R blockade after stroke (Saavedra, 2012; Villapol and Saavedra, 2015). These protective effects are at least partially attributed to the indirect stimulation of the AT2R through increased availability of its endogenous ligand, angiotensin II (Alhusban et al., 2013; Iwai et al., 2004; Li et al., 2005). Studies have shown the protective role of pre-and post-stroke AT2R stimulation (Joseph et al., 2014; McCarthy et al., 2009, 2012, 2014a; Min et al., 2014). We previously found that the AT2R agonist compound 21 (C21) provided sustained functional recovery after ischemic stroke. This was associated with upregulation of the AT2R and the neurotrophin brain-derived neurotrophic factor (BDNF) in the contralesional hemisphere (Alhusban et al., 2015). Similarly, a previous study has documented the contralesional upregulation of AT2R using autoradiography after an experimental stroke (Kagiyama et al., 2003). However, the role of this contralesional receptor upregulation remains unclear.

Reports from human and animal studies have shown functional and structural cortical reorganization in both hemispheres after stroke (Dodd et al., 2017; Huang et al., 2020). Furthermore, modulation of the contralesional reorganization is under investigation to achieve optimal post-stroke recovery (Buetefisch, 2015; Volz et al., 2017). Published literature from our lab and others has reported biochemical contralesional changes after experimental stroke as well (Alhusban et al., 2015; Guan et al., 2011; Herz et al., 2012; Madinier et al., 2013; Reitmeir et al., 2011; Sist et al., 2014). However, few studies have examined the possible role of these changes in post-stroke recovery. Many studies use the contralesional hemisphere as control and thus report markers measured in the ischemic hemisphere as fold changes from the contralesional side. Such way of reporting misses the opportunity to detect changes in the contralesional side and may lead to erroneous interpretations with respect to changes in the stroked hemisphere.

Diabetes is a major risk factor for ischemic stroke and accounts, for ~26% of ischemic strokes cases (Kissela et al., 2005). Moreover, stroke outcome is usually worse in diabetic/hyperglycemic patients (Bruno et al., 2008). Experimental studies attributed this worsened outcome to increased oxidative stress, as well as changes in cerebrovascular structure and myogenic tone (Ergul et al., 2009). Diabetes and hyperglycemia have been shown to activate the local tissue renin-angiotensin systems (RAS). Diabetes activates renal RAS, an effect that has been implicated in the development of diabetic nephropathy (Peti-Peterdi et al., 2008). Similarly, high glucose levels are known to activate the RAS in different cell types, including vascular smooth muscle cells (Lavrentyev et al., 2007) and cardiomyocytes (Singh et al., 2007). Therefore, acute RAS modulation after stroke may yield different outcomes in diabetic versus non-diabetic animals. Pretreatment with AT1R blockers for 14 days improved stroke outcome in streptozotocin (STZ)-induced type 1 diabetic rats (Kusaka et al., 2004) and type 2 diabetic mice (Iwanami et al., 2010). We have recently shown that AT2R stimulation with C21 improved survival and preserved cognitive and sensorimotor functions in diabetic rats after stroke (Jackson-Cowan et al., 2021; Jackson et al., 2020). To our knowledge, post-stroke treatment with AT1R blockers has not been tested in experimental models of diabetes.

In this study, we hypothesized that 1) AT1R blockade with candesartan would improve early functional outcomes in diabetic animals, 2) the angiotensin II receptors in the nonischemic hemisphere are modulated differently in control and diabetic animals, and 3) silencing of the contralesional AT2R would block the C21-mediated functional recovery after stroke.

2. Materials and methods

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Veterans Affairs Medical Center.

2.1. Animals and experimental cerebral ischemia

Two major experiments were conducted as follows:

2.2. Experiment 1

This experiment was conducted to study the effect of AT1R blockade on stroke injury, AT1R and AT2R expression, and acute outcomes in diabetic Goto-Kakizaki (GK) rats.

Adult male Wistar, and type 2 diabetic GK rats (Taconic), weighing between 280 and 300 g, were used. Diabetes was confirmed by measuring blood glucose levels daily for 8–12 days before middle cerebral artery occlusion (MCAO). GK rats had an average blood glucose level of 162.6 ± 15.9 mg/dL confirming the presence of persistent hyperglycemia as we described previously (Abdelsaid et al., 2017).

Animals were either sham-operated or subjected to 3 h MCAO using an intraluminal suture model of stroke as described previously (Guan et al., 2011). Animals were randomized to receive 1 mg/kg of candesartan (AT1R blocker, a gift from AstraZeneca) or saline at reperfusion via tail vein injection (Fig. 1A). At 24 h, animals were deeply anesthetized with ketamine/xylazine intramuscular and transcardially perfused with 240 mL of ice-cold phosphate-buffer saline (PBS). Brains were harvested and the two hemispheres were snap-frozen separately as described previously (Guan et al., 2011). The duration of ischemia/reperfusion and dose of 1 mg/kg of candesartan were selected to compare the response of GK rats to our previous studies in the control Wistars using the same experimental conditions with the same surgeon performing the strokes (Fagan et al., 2006; Kozak et al., 2009). Sham animals were subjected to the same surgical procedure without actual MCAO occlusion. For inclusion/exclusion criteria, we used the modified neurological deficit score; animals with no apparent deficits obtained 0; signs of forelimb flexion, 1; reduced resistance to push, 2 and with circling 3. For consistent MCAO completion, only animals with a score of ~3 at reperfusion were included in further analysis and treatment groups.

Fig. 1. GK rats show reduced response to AT1R blockade after stroke.

Fig. 1.

Open in a new tab

A) Schematic diagram of experiment 1 study design and endpoints. GK rats were subjected to 3 h MCAO then treated with IV 1 mg/kg of the AT1R blocker, candesartan, at reperfusion. Animals were sacrificed at 24 h. B) Continuous blood pressure monitoring by telemetry showed an average basal blood pressure of 109 ± 2 mm Hg. MCAO increased blood pressure abruptly to 147 ± 4 mm Hg. Treatment with a single dose of candesartan reduced blood pressure to an average of 120 ± 3 mm Hg during the first 24 h, versus 135 ± 3 mm Hg for saline-treated animals. X-axis represents the time of day in hours. Animals were stroked at the same time (9:00) every day to control for any diurnal variation in blood pressure. Light is turned off in animal facility from 18:00 to 6:00 as represented by black line on X-axis. MAP: mean arterial pressure. C) Neurobehavioral testing was conducted using the three-point Bederson score. Animals scored 3 and 2.7 on average in the saline and candesartan-treated groups, respectively. D) Assessment of hemoglobin content in brain tissue homogenate was conducted using a specific ELISA kit. Hemoglobin content decreased from 80.9 ± 25 to 64 ± 19 μg/mg protein with treatment. The change, however, did not reach significance. E, F) Analysis of NY and 4-HNE levels in both saline and candesartan-treated groups was conducted using slot blot. No change was observed in the oxidative or nitrative stress markers with treatment at 24 h. G) Quantification of cleaved caspase-3 at the same time point showed a trend towards decreasing the cell death marker (18% decrease) with the treatment, that did not reach significance (n = 6 and 8 in saline and candesartan-treated groups, respectively), ns: no statistical significance, * = p < 0.05 vs corresponding contralesional side.

2.2.1. Blood pressure telemetry

Blood pressure (BP) transmitters (Data Sciences) were implanted into animals’ abdominal cavity according to the manufacturer’s protocol and as described previously (Fagan et al., 2006). Blood pressure was measured continuously and recorded for 2 days before and 1 day after MCAO.

2.2.2. Hemoglobin content (vascular integrity) measurement

Brain hemorrhage was quantified by measuring tissue hemoglobin content using a colorimetric hemoglobin detection assay (QuantiChrom, BioAssay Systems) according to the manufacturer’s protocol (Ishrat et al., 2013).

2.2.3. Slot blot

Detection of 4-hydorxynonenal (4-HNE), a marker of lipid peroxidation, and nitrotyrosine (NY), a marker of nitrosative stress, was done using slot blot technique (Alhusban et al., 2015). Membranes were incubated with primary anti-nitrotyrosine (Millipore) or anti-4 hydroxynonenal (Alpha Diagnostic) antibodies, followed by peroxidase-labeled goat anti-mouse IgG. Densitometric measurements were done using Image-J software.

2.2.4. Determination of basilar artery reactivity

Isometric tension exerted by the vessels was recorded via a force transducer using the wire-myograph technique (Danish Myo Technologies, Denmark). The myograph chambers were filled with Krebs buffer (NaCl 118.3, NaHCO3 25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.5 and Dextrose 11.1 mM), gassed with 95% O2 and 5% CO2 and maintained at 37 °C. Basilar arteries were isolated from GK and Wistar rats and vessel segments mounted in the chamber using 40 μm-thin wires and adjusted to a baseline tension of 0.4 g. Viability was tested by measuring the vasorelaxation in response to 70 mM KCl. Vessels were then preconstricted to 60% of the baseline tension with 100 nM serotonin (5-HT) and cumulative dose-response curves to C21 (0.1 nM - 1 μM) were generated. The relaxation response was expressed a % change of 5HT response. In additional experiments, vessels were equilibrated and then incubated with 100 nM C21 ± 1 μM PD123319 (AT2R antagonist) for 30 min. Vessels were then preconstricted with 5HT and endothelium-dependent relaxation to acetylcholine (Ach, 1 nM - 1 μM) was assessed. Sensitivity (half maximal effective concentration - EC50) and area under the curve (AUC) values were calculated from the respective dose-response equations. C21 was provided as a kind gift from Vicore Pharma (Göteborg, Sweden) and PD123319 was purchased from Sigma- Aldrich.

2.3. Experiment 2

This experiment was conducted to study the effect of in vivo unilateral AT2R knockdown (KD) on stroke outcomes in non-diabetic rats treated with either vehicle or AT2R agonist (C21).

Male Wistar rats (200–220 g) received two intrastriatal microinjections of short hairpin RNA (shRNA) lentiviral particles against AT2R (SMARTchoice lentiviral rat Agtr2 hCMV-turboGFP shRNA, 1 × 108 TU/mL, Dharmacon, # SH-090219-01-10), or non-targeting control vector (NTC) into the left brain hemisphere to achieve localized AT2R KD. Intrastriatal microinjections were conducted in a stereotaxic frame under isoflurane anesthesia using the following coordinates: 0.5 mm anteroposterior (AP), 3 mm lateral relative to bregma. The needle was advanced 5 mm dorsoventral, 2 μL injected slowly over 2 min followed by a wait time of 1 min. The needle was then retracted up 1 mm then another 2 μL injected over 2 min followed by a wait of 1 min then slow retraction of the needle. Animals were allowed to recover for 14 days to allow for lentiviral particles integration in the genome, shRNA expression, and AT2R KD. After 14 days, one set of animals was sacrificed to confirm the KD, while the other set was subjected to 90 min right MCAO. These rats were randomized to receive either C21 0.03 mg/kg or saline intraperitoneal (IP) at reperfusion and daily for 7 days then were sacrificed at day 10 (Fig. 3A).

Fig. 3. Effect of contralesional AT2R knockdown and C21 treatment on functional recovery after stroke in Wistars.

Fig. 3.

Open in a new tab

A) Schematic diagram of experiment 2 study design and endpoints. Rats received two intrastriatal injections of short hairpin RNA (shRNA) lentiviral particles against AT2R, or non-targeting control vector (NTC) into the left brain hemisphere to achieve localized AT2R knockdown. Rats were subjected to 90 min middle cerebral artery occlusion (MCAO) on the right hemisphere and received C21 0.03 mg/kg or saline intraperitoneal (i.p.) at reperfusion and daily for 7 days. Animals from 4 groups (C21/NTC, C21/shRNA, saline/NTC, and saline/shRNA) were followed for 10 days using a battery of behavioral tests, then euthanized at day 10. B) Diagram of intrastriatal injection of lentiviral particles. C) Western blotting confirmed the successful knockdown of the AT2R in the left side (contralesional hemisphere) of brain collected 14 days after the intrastriatal injection, n = 2 per group. D-F) All groups showed worsened outcome on days 1–3 then later recovered on days 7–10. C21/NTC group showed better functional outcome on Bederson score, as well as a trend towards more weight gain and better performance on rotarod test. Saline/shRNA group was associated with the least functional recovery, n = 7–8 per group * = p < 0.05 vs other groups.

2.3.1. Behavioral outcome analysis

Rats were assessed for sensorimotor deficits in a blinded fashion using modified Bederson and rotarod tests. Modified Bederson test was scored on 0–3 scale in which animals were assessed for forelimb flexion, decreased resistance to lateral push and circling movement (one point for each). For rotarod test, the maximum running time a rat can achieve on an accelerating rod was measured as described previously (Alhusban et al., 2015).

2.3.2. Western blot

Protein expression was measured by western blotting as described previously (Guan et al., 2011). Membranes were probed using antibodies against cleaved caspase-3 (Cell Signaling), AT1R (Abcam) and AT2R (Abcam), were used. GAPDH (Cell Signaling) and β-actin (Sigma Aldrich) were used as loading controls. Band optical densities were quantified using ImageJ software (NIH), and divided by the respective loading control (GAPDH, Cell Signaling) and (β-actin, Sigma Aldrich), then normalized to the control group.

2.4. Statistical analysis

Results were expressed as mean ± standard error of the mean (±SEM) for all figures except Bederson score which is presented as median/range. Data was statistically analyzed using two-sample unpaired Student’s t-test, Mann-Whitney test or two-way ANOVA test followed by Tukey post hoc analysis. Two-way repeated measure ANOVA or Friedmann’s test were conducted for the behavioral data analysis using SAS 9.4. Post hoc pair-wise comparisons between groups over time were performed using a Bonferroni adjustment to the overall alpha level. Results were considered statistically significant at p < 0.05.

3. Results

3.1. The beneficial effect of AT1R blockade on blood pressure and acute stroke outcomes is blunted in diabetic GK rats

GK rats recorded a mean baseline arterial pressure of 109 ± 2 mm Hg, about 10 mm Hg higher than Wistars, consistent with the previously published data by other groups (Cheng et al., 2001). Middle cerebral artery occlusion resulted in ~30 mm Hg increase in blood pressure. Treatment with candesartan (1 mg/kg) at reperfusion reduced blood pressure but failed to bring it down to pre-stroke levels suggesting an impaired sensitivity to angiotensin blockade after stroke (Fig. 1B). This came in contrast to our previous studies, in which the same candesartan dose administered at reperfusion successfully reduced the elevation in blood pressure in normotensive normoglycemic Wistars (Fagan et al., 2006) and in hypertensive rats (Kozak et al., 2008).

We have previously shown neuroprotection and functional recovery post-stroke with candesartan treatment in Wistars (Fagan et al., 2006; Kozak et al., 2009). Under the same experimental setup, candesartan administration did not result in a significant improvement in the neurobehavioral outcome of GK rats at 24 h. Bederson score decreased only modestly with the treatment (Fig. 1C). Similarly, candesartan treatment did not significantly preserve vascular integrity in GK rats as assessed by tissue hemoglobin content (Fig. 1D), although there was a trend towards vascular preservation.

On the molecular level, oxidative stress markers, nitrotyrosine (NY), and 4-hydroxynonenal (4-HNE), were comparable in saline- and candesartan-treated animals (Fig. 1E, 1F). There was a trend of decreasing the pro-apoptotic cleaved caspase-3 with treatment but this did not reach significance (Fig. 1G). Taken together, these results show that candesartan treatment exhibited no benefit post-stroke in diabetic GK rats.

3.2. GK rats have reduced AT2R expression after stroke and show impaired response to endothelium-dependent relaxation with AT2R stimulation

Since the post-stroke protective effects of AT1R blockade have been attributed to the indirect stimulation of the AT2R (Alhusban et al., 2013; Iwai et al., 2004; Li et al., 2005), we examined the AT1R and AT2R expression in brains of GK rats and their Wistar controls in which we have previously shown profound neurovascular protection and recovery with candesartan. Sham-operated Wistar and GK rats showed no significant difference in AT1R or AT2R expression (Fig. 2A, 2B). AT1R showed as two bands at 45 and 60 kDa, due to differential protein glycosylation and were quantified separately. Both bands showed a similar trend, with stronger upregulation of the 45-kDa band in the stroked hemisphere after MCAO in Wistars and GK rats. Candesartan treatment decreased the AT1R expression in Wistars but failed to decrease it in GK rats (Fig. 2C, 2D).

Fig. 2. GK rats show reduced AT2R expression after stroke as compared to Wistars.

Fig. 2.

Open in a new tab

A, B) Western blotting showed no difference in baseline angiotensin receptors expression between sham Wistars and GK rats. C, D) Quantification of AT1R expression showed a significant upregulation in the ischemic hemispheres of Wistars and GKs at 24 h. Candesartan decreased the receptor expression in Wistars with no change with treatment in GKs. E) Quantification of AT2R expression showed a significant upregulation in contralateral hemisphere of Wistars. Candesartan treatment further increased the receptor expression. F) GK rats showed a significant decrease in AT2R expression that was not rescued by candesartan treatment at 24 h, n = 3–4, 6, 6 for sham, saline and candesartan groups, respectively, * = p < 0.05. G) Preincubation with C21 enhanced relaxation to Ach in control animals (AUC: vehicle 146.7 + 3.9 vs C21 pretreatment 229.6 + 11.6) which was abolished by the blockade of AT2R (176.9 + 25.5, p = 0.007). Similarly, C21 improved sensitivity in control animals (EC50: vehicle 110.5 + 32 vs C21 pretreatment 11.9 + 2) which was abolished in the presence of PD129319 (93.8 + 31, p = 0.04), *C21 vs other groups. H) Basilar artery relaxation was impaired in diabetic GK rats as compared to controls and C21 had no effect.

AT2R expression was upregulated at 24 h after 3 h-MCAO in the contralesional hemisphere of Wistars as compared to shams. Candesartan treatment further augmented the MCAO-induced AT2R upregulation (Fig. 2E). GK animals, on the other hand, showed reduced AT2R expression after stroke and candesartan treatment failed to enhance the receptor expression (Fig. 2F).

To further examine the differential response to AT2R stimulation in GK and Wistar rats, basilar artery reactivity in response to increasing doses of the AT2R agonist, C21 (0.1 nM - 1 μM), was examined using wire-myography. Basilar arteries of Wistar but not GK rats showed a small (7–10%) relaxation to C21 starting at 10−7 M (data not shown). Since the response was small, in the next set of experiments, the ability of C21 to enhance endothelium-dependent relaxation was measured. Pre-incubation with C21 (100 nM) enhanced relaxation to Ach in control rats (AUC, vehicle 146.7 ± 3.9 vs C21 pretreatment 229.6 ± 11.6), which was abolished by the blockade of AT2R (176.9 ± 25.5, p = 0.007) (Fig. 2G). Similarly, C21 improved sensitivity (EC50) in control rats as indicated by a leftward shift in the dose-response curve (vehicle 110.5 ± 32 nM vs C21 pretreatment 11.9 ± 2 nM), which was abolished in the presence of PD123319 (93.8 ± 31 nM, p = 0.04) suggesting that these effects were AT2R mediated (Fig. 2H). Basilar artery relaxation was impaired in diabetic GK rats as we have shown previously (Sachidanandam et al., 2006) and C21 had no effect (Fig. 2H).

3.3. Effect of contralesional AT2R knockdown and C21 treatment on functional recovery after stroke in Wistars

To study the possible protective effect of contralesional AT2R upregulation in Wistars, we performed a unilateral knock down of the receptor expression in the contralesional hemisphere using intrastriatal injections of short hairpin RNA (shRNA). To further augment the differences between the control and knockdown animals, we used the AT2R agonist, C21. This resulted in a 2X2 study design with four groups as follows: C21/NTC, C21/shRNA, Sal/NTC, and Sal/shRNA (Fig. 3A and B). AT2R lentiviral shRNA successfully knocked down the receptor expression in the left hemisphere as confirmed by Western blotting (Fig. 3C). Animals were then subjected to right 90 min-MCAO occlusion and followed up for 10 days. Rats received C21 0.03 mg/kg or saline IP at reperfusion and daily for 7 days post-stroke as shown in (Fig. 3A).

All groups showed significant deficits on days 1–3 but later recovered on days 7–10. C21/NTC group showed better functional outcome on Bederson score at day 10 (Fig. 3D). C21/NTC also showed a trend towards better recovery on rotarod test (Fig. 3E), as well as weight gain at days 7–10 (Fig. 3F).

4. Discussion

Collectively, our results showed an upregulation of AT2R in the nonischemic hemisphere after stroke in Wistars but not in GK rats, and that GK rats show an impaired response to angiotensin system modulation suggesting a possible protective role of the contralesional hemisphere and this receptor in ischemic injury. Our finding that AT2R KD in the contralesional hemisphere leads to impaired functional recovery with C21 provides further evidence to support this concept.

In the current study, there was no benefit of post-stroke candesartan treatment in type 2 diabetic GK rats, as opposed to our previous findings in Wistars (Fagan et al., 2006; Kozak et al., 2009). This was correlated with: 1. Post-stroke downregulation of AT2R in GKs versus its upregulation in Wistars, 2. Upregulation of AT2R with candesartan treatment in Wistars but not GKs, and 3. Enhanced cerebrovascular relaxation in response to AT2R stimulation in Wistar but not GKs. In our previous studies, candesartan improved neurovascular stroke outcome in Wistar rats after 3 h MCAO when given I.V. at reperfusion at a dose of 1 mg/kg (Fagan et al., 2006; Guan et al., 2011; Kozak et al., 2009). This effect was associated with the amelioration of oxidative stress and BP reduction to baseline values. Subjecting diabetic GK rats to the same experimental conditions showed no improvement with candesartan treatment. While this is the first report to our knowledge to examine post-stroke treatment with AT1R blocker in a diabetic model, other groups have demonstrated that chronic pretreatment with AT1R blocker successfully reduces stroke severity in diabetic rodents (Iwanami et al., 2010; Kusaka et al., 2004). This beneficial role of chronic pretreatment with AT1R blocker on stroke outcome was attributed to the amelioration of oxidative and inflammatory milieu of diabetes. Our results show that such an effect cannot be achieved acutely with a single dose after stroke when oxidative and inflammatory effectors peak.

Clinically, chronic candesartan treatment is associated with a reduction in stroke incidence (Lithell et al., 2003). Moreover, preadmission use of AT1R blockers is associated with reduced 30-day mortality after stroke (Sundboll et al., 2014). Interestingly, in a meta-analysis of 4 clinical trials, AT1R blockers provided less protection against stroke incidence in diabetic as compared to non-diabetic patients, demonstrating a relatively reduced efficacy of AT1R blockers pretreatment as well in the diabetic setting (Turnbull et al., 2005).

While AT1R plays a detrimental role after stroke, AT2R activation improves stroke outcome (Sumners et al., 2013). Furthermore, previous studies suggest a cross-talk between the two receptors. AT1R activation inhibits the expression of AT2R mRNA while its blockade increases the AT2R expression (De Paolis et al., 1999). Likewise, overexpression of AT2R downregulates AT1R expression (Jin et al., 2002) and its stimulation promotes positive self-feedback (Shibata et al., 1997). A report by Kagiyama et al. has shown increased contralesional AT2R using quantitative autoradiography in Sprague-Dawley rats after transient MCAO (Kagiyama et al., 2003). In accord, we found a contralesional AT2R upregulation after stroke in Wistar rats, which was further augmented by candesartan treatment. On the other hand, GK rats showed AT2R downregulation after stroke that was not rescued by candesartan. The reason behind this downregulation remains unknown and will be explored in future studies.

C21 has been reported to elicit vasorelaxation in basilar arteries from spontaneously hypertensive rats (SHRs) (McCarthy et al., 2014b). Using the same C21 concentration range, we did not see significant vasorelaxation in basilar arteries from Wistars or GK rats. However, preincubation with C21 enhanced the endothelium-mediated relaxation in Wistars but not in GK rats. These findings suggest an impaired AT2R sensitivity in GK animals at baseline compared to Wistars. This reduced sensitivity and further AT2R downregulation after stroke could be the reasons behind reduced candesartan efficacy in these animals compared to Wistar controls. Further studies are warranted, however, to prove the causative relationship between diabetes and the dysregulation of AT2 receptor expression and sensitivity.

To examine the role of contralesional AT2R upregulation after stroke, we achieved localized AT2R KD in the contralesional striatum of Wistars. On the behavioral level, AT2R KD abrogated C21 mediated functional recovery. We have previously shown C21 mediated functional recovery after stroke is associated with upregulation of the AT2R and the neurotrophin BDNF in the contralesional hemisphere (Alhusban et al., 2015). Our results indicate that the contralesional AT2R upregulation is involved in the C21 mediated functional recovery. It is likely that contralesional AT2R stimulation promotes recovery by enhancing BDNF expression. A previous study showed that infusion of a BDNF antisense oligonucleotide to the contralesional hemisphere negated motor improvement with rehabilitation therapy post-stroke (Ploughman et al., 2009). Future studies are needed to examine the mechanisms underlying the contralesional AT2R contribution to C21 protective effects.

Due to its pleiotropic effects, it is likely that the AT2R activation acts by combining intranuclear and membrane mechanisms (Ahmed and Ishrat, 2020). C21 functions by selective stimulation of the AT2R, which promotes vasodilation improves cerebral blood flow (CBF) and reduces oxidative stress (Ahmed et al., 2022). It also effectively reduces inflammation by decreasing the expression of pro-inflammatory cytokines and increasing the expression of anti-inflammatory cytokine, IL-10. In the CNS, IL-10 inhibits the release of pro-inflammatory cytokines from microglia, stimulates other anti-inflammatory responses, and increases cell survival (Jackson et al., 2018). Several pre-clinical studies, including our own, have attributed the benefits of C21 in cerebrovascular conditions like stroke and vascular cognitive impairment to the reduction of inflammation and oxidative stress, partly by enhancing the expression and overall activity of PPARγ and IL-10, in addition to increasing levels of neurotrophic factors like VEGF and BDNF (Eldahshan et al., 2019; Jackson et al., 2018; Peluso et al., 2018). This study aims to obtain preclinical information to address whether contralesional angiotensin type 2 receptor activation contributes to recovery in experimental stroke. Being a preliminary study, a few limitations need to be addressed in future research. The interpretations are made from a limited number of endpoints following MCAO. However, we demonstrated that delayed administration of C21, 3 days post-stroke, reduced mortality and improved sensorimotor and cognitive deficits in male and female high-fat diet (HFD)-induced diabetic rats (Jackson-Cowan et al., 2021; Jackson et al., 2020). The longer-term studies to evaluate the minimal efficacious dose, window of opportunity, and effect of sex on CBF, neuronal survival, vascular inflammatory markers, infarct volume, and functional cognitive outcome for AT2R stimulation are needed to assess the actual usage and impact of C21, a promising AT2R agonist in GK rats.

In conclusion, this study demonstrates the effect of underlying disease conditions on the response to AT1R blocker treatment after stroke. Although candesartan treatment improved stroke outcomes in Wistar rats, it failed to achieve such improvement in diabetic GK rats, under the same experimental conditions. Differential expression and activity of the AT2R may account for this impaired response in GKs. In addition, the contralesional AT2R may be involved in C21 mediated functional recovery after stroke. Taken together, our results suggest that the contralesional hemisphere plays an important role in brain healing after stroke.

Acknowledgements

This is study was supported by Veterans Affairs (VA) Merit Review (BX000347), VA Senior Research Career Scientist Award (IK6 BX004471), National Institute of Health (NIH) R01 NS083559 and R01 NS104573 (multi-PI, Susan C. Fagan as co-PI) to Adviye Ergul; K99/R00 award 4 R00 EY029373-03 and UAMS Hornick award to Abdelrahman Y. Fouda; and NIH R01-NS097800 to T. Ishrat.

The authors thank Vicore Pharma (Göteborg, Sweden) for the supply of C21 and AstraZeneca for the supply of candesartan.

Footnotes

Declaration of competing interest

The authors declare that they have no conflict of interest.

Compliance with ethical standards

This study was approved by the Institutional Review Board, and was performed in accordance with standard ethical procedures.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The data generated and analyzed in this study will be made available from the corresponding author on reasonable request.

References

  1. Abdelsaid M, Coucha M, Hafez S, Yasir A, Johnson MH, Ergul A, 2017. Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes. Diabetologia 60, 740–750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmed HA, Ishrat T, 2020. The brain AT2R-a potential target for therapy in alzheimer’s disease and vascular cognitive impairment: a comprehensive Review of clinical and experimental therapeutics. Mol. Neurobiol. 3458–3484. 10.1007/s12035-020-01964-. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahmed HA, Ismael S, Salman M, Devlin P, McDonald MP, Liao FF, Ishrat T, 2022. Direct AT2R stimulation slows post-stroke cognitive decline in the 5XFAD alzheimer’s disease mice. Mol. Neurobiol. 4124–4140. 10.1007/s12035-022-02839-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alhusban A, Fouda AY, Bindu P, Ishrat T, Soliman S, Fagan SC, 2015. Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke. J. Hypertens. 33, 170–180. [DOI] [PubMed] [Google Scholar]
  5. Alhusban A, Kozak A, Ergul A, Fagan SC, 2013. AT1 receptor antagonism is proangiogenic in the brain: BDNF a novel mediator. J. Pharmacol. Exp. Therapeut. 344, 348–359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bruno A, Kent TA, Coull BM, Shankar RR, Saha C, Becker KJ, Kissela BM, Williams LS, 2008. Treatment of hyperglycemia in ischemic stroke (THIS): a randomized pilot trial. Stroke 39, 384–389. [DOI] [PubMed] [Google Scholar]
  7. Buetefisch CM, 2015. Role of the contralesional hemisphere in post-stroke recovery of upper extremity motor function. Front. Neurol. 6, 214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng ZJ, Vaskonen T, Tikkanen I, Nurminen K, Ruskoaho H, Vapaatalo H, Muller D, Park JK, Luft FC, Mervaala EM, 2001. Endothelial dysfunction and salt-sensitive hypertension in spontaneously diabetic Goto-Kakizaki rats. Hypertension 37, 433–439. [DOI] [PubMed] [Google Scholar]
  9. De Paolis P, Porcellini A, Gigante B, Giliberti R, Lombardi A, Savoia C, Rubattu S, Volpe M, 1999. Modulation of the AT2 subtype receptor gene activation and expression by the AT1 receptor in endothelial cells. J. Hypertens. 17, 1873–1877. [DOI] [PubMed] [Google Scholar]
  10. Dodd KC, Nair VA, Prabhakaran V, 2017. Role of the contralesional vs. Ipsilesional hemisphere in stroke recovery. Front. Hum. Neurosci. 11, 469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eldahshan W, Ishrat T, Pillai B, Sayed M, Alwhaibi A, Fouda AY, Ergul A, Fagan SC, 2019. Angiotensin type 2 receptor stimulation with compound 21 improves neurological function after stroke in female rats: a pilot study. Am. J. Physiol. Heart Circ. Physiol. H1192–H1201. 10.1152/ajpheart.00446.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ergul A, Li W, Elgebaly MM, Bruno A, Fagan SC, 2009. Hyperglycemia, diabetes and stroke: focus on the cerebrovasculature. Vasc. Pharmacol. 51, 44–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fagan SC, Kozak A, Hill WD, Pollock DM, Xu L, Johnson MH, Ergul A, Hess DC, 2006. Hypertension after experimental cerebral ischemia: candesartan provides neurovascular protection. J. Hypertens. 24, 535–539. [DOI] [PubMed] [Google Scholar]
  14. Guan W, Somanath PR, Kozak A, Goc A, El-Remessy AB, Ergul A, Johnson MH, Alhusban A, Soliman S, Fagan SC, 2011. Vascular protection by angiotensin receptor antagonism involves differential VEGF expression in both hemispheres after experimental stroke. PLoS One 6, e24551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Herz J, Reitmeir R, Hagen SI, Reinboth BS, Guo Z, Zechariah A, ElAli A, Doeppner TR, Bacigaluppi M, Pluchino S, Kilic U, Kilic E, Hermann DM, 2012. Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions. Neurobiol. Dis. 45, 1077–1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang X, Wang X, Yang M, Pan X, Duan M, Wen X, Cai H, Jiang G, Chen L, 2020. Spontaneous neuronal plasticity in the contralateral motor cortex and corticospinal tract after focal cortical infarction in hypertensive rats. J. Stroke Cerebrovasc. Dis. 29, 105235. [DOI] [PubMed] [Google Scholar]
  17. Ishrat T, Pillai B, Ergul A, Hafez S, Fagan SC, 2013. Candesartan reduces the hemorrhage associated with delayed tissue plasminogen activator treatment in rat embolic stroke. Neurochem. Res. 38, 2668–2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T, Horiuchi M, 2004. Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation. Circulation 110, 843–848. [DOI] [PubMed] [Google Scholar]
  19. Iwanami J, Mogi M, Tsukuda K, Min LJ, Sakata A, Jing F, Iwai M, Horiuchi M, 2010. Low dose of telmisartan prevents ischemic brain damage with peroxisome proliferator-activated receptor-gamma activation in diabetic mice. J. Hypertens. 28, 1730–1737. [DOI] [PubMed] [Google Scholar]
  20. Jackson-Cowan L, Eldahshan W, Dumanli S, Dong G, Jamil S, Abdul Y, Althomali W, Baban B, Fagan SC, Ergul A, 2021. Delayed administration of angiotensin receptor (AT2R) agonist C21 improves survival and preserves sensorimotor outcomes in female diabetic rats post-stroke through modulation of microglial activation. Int. J. Mol. Sci. 22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jackson L, Dong G, Althomali W, Sayed MA, Eldahshan W, Baban B, Johnson MH, Filosa J, Fagan SC, Ergul A, 2020. Delayed administration of angiotensin II type 2 receptor (AT2R) agonist compound 21 prevents the development of post-stroke cognitive impairment in diabetes through the modulation of microglia polarization. Transl. Stroke Res. 11, 762–775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jackson L, Eldahshan W, Fagan SC, Ergul A, 2018. Within the brain: the renin angiotensin system. Int. J. Mol. Sci. 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jin XQ, Fukuda N, Su JZ, Lai YM, Suzuki R, Tahira Y, Takagi H, Ikeda Y, Kanmatsuse K, Miyazaki H, 2002. Angiotensin II type 2 receptor gene transfer downregulates angiotensin II type 1a receptor in vascular smooth muscle cells. Hypertension 39, 1021–1027. [DOI] [PubMed] [Google Scholar]
  24. Joseph JP, Mecca AP, Regenhardt RW, Bennion DM, Rodriguez V, Desland F, Patel NA, Pioquinto DJ, Unger T, Katovich MJ, Steckelings UM, Sumners C, 2014. The angiotensin type 2 receptor agonist Compound 21 elicits cerebroprotection in endothelin-1 induced ischemic stroke. Neuropharmacology 81, 134–141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kagiyama T, Kagiyama S, Phillips MI, 2003. Expression of angiotensin type 1 and 2 receptors in brain after transient middle cerebral artery occlusion in rats. Regul. Pept. 110, 241–247. [DOI] [PubMed] [Google Scholar]
  26. Kissela BM, Khoury J, Kleindorfer D, Woo D, Schneider A, Alwell K, Miller R, Ewing I, Moomaw CJ, Szaflarski JP, Gebel J, Shukla R, Broderick JP, 2005. Epidemiology of ischemic stroke in patients with diabetes: the greater Cincinnati/Northern Kentucky Stroke Study. Diabetes Care 28, 355–359. [DOI] [PubMed] [Google Scholar]
  27. Kozak A, Ergul A, El-Remessy AB, Johnson MH, Machado LS, Elewa HF, Abdelsaid M, Wiley DC, Fagan SC, 2009. Candesartan augments ischemia-induced proangiogenic state and results in sustained improvement after stroke. Stroke 40, 1870–1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kozak W, Kozak A, Johnson MH, Elewa HF, Fagan SC, 2008. Vascular protection with candesartan after experimental acute stroke in hypertensive rats: a dose-response study. J. Pharmacol. Exp. Therapeut. 326, 773–782. [DOI] [PubMed] [Google Scholar]
  29. Kusaka I, Kusaka G, Zhou C, Ishikawa M, Nanda A, Granger DN, Zhang JH, Tang J, 2004. Role of AT1 receptors and NAD(P)H oxidase in diabetes-aggravated ischemic brain injury. Am. J. Physiol. Heart Circ. Physiol. 286, H2442–H2451. [DOI] [PubMed] [Google Scholar]
  30. Lavrentyev EN, Estes AM, Malik KU, 2007. Mechanism of high glucose induced angiotensin II production in rat vascular smooth muscle cells. Circ. Res. 101, 455–464. [DOI] [PubMed] [Google Scholar]
  31. Li J, Culman J, Hortnagl H, Zhao Y, Gerova N, Timm M, Blume A, Zimmermann M, Seidel K, Dirnagl U, Unger T, 2005. Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury. Faseb. J. 19, 617–619. [DOI] [PubMed] [Google Scholar]
  32. Lithell H, Hansson L, Skoog I, Elmfeldt D, Hofman A, Olofsson B, Trenkwalder P, Zanchetti A, 2003. The Study on Cognition and Prognosis in the Elderly (SCOPE): principal results of a randomized double-blind intervention trial. J. Hypertens. 21, 875–886. [DOI] [PubMed] [Google Scholar]
  33. Madinier A, Bertrand N, Rodier M, Quirie A, Mossiat C, Prigent-Tessier A, Marie C, Garnier P, 2013. Ipsilateral versus contralateral spontaneous post-stroke neuroplastic changes: involvement of BDNF? Neuroscience 231, 169–181. [DOI] [PubMed] [Google Scholar]
  34. McCarthy CA, Facey LJ, Widdop RE, 2014a. The protective arms of the renin-angiontensin system in stroke. Curr. Hypertens. Rep. 16, 440. [DOI] [PubMed] [Google Scholar]
  35. McCarthy CA, Vinh A, Broughton BR, Sobey CG, Callaway JK, Widdop RE, 2012. Angiotensin II type 2 receptor stimulation initiated after stroke causes neuroprotection in conscious rats. Hypertension 60, 1531–1537. [DOI] [PubMed] [Google Scholar]
  36. McCarthy CA, Vinh A, Callaway JK, Widdop RE, 2009. Angiotensin AT2 receptor stimulation causes neuroprotection in a conscious rat model of stroke. Stroke 40, 1482–1489. [DOI] [PubMed] [Google Scholar]
  37. McCarthy CA, Vinh A, Miller AA, Hallberg A, Alterman M, Callaway JK, Widdop RE, 2014b. Direct angiotensin AT2 receptor stimulation using a novel AT2 receptor agonist, compound 21, evokes neuroprotection in conscious hypertensive rats. PLoS One 9, e95762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Min LJ, Mogi M, Tsukuda K, Jing F, Ohshima K, Nakaoka H, Kan-No H, Wang XL, Chisaka T, Bai HY, Iwanami J, Horiuchi M, 2014. Direct stimulation of angiotensin II type 2 receptor initiated after stroke ameliorates ischemic brain damage. Am. J. Hypertens. 1036–1044. 10.1093/ajh/hpu015. [DOI] [PubMed] [Google Scholar]
  39. Peluso AA, Bertelsen JB, Andersen K, Mortsensen TP, Hansen PB, Sumners C, Bader M, Santos RA, Steckelings UM, 2018. Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase. Clin. Sci. (Lond.) 132, 777–790. [DOI] [PubMed] [Google Scholar]
  40. Peti-Peterdi J, Kang JJ, Toma I, 2008. Activation of the renal renin-angiotensin system in diabetes–new concepts. Nephrol. Dial. Transplant. 23, 3047–3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ploughman M, Windle V, MacLellan CL, White N, Doré JJ, Corbett D, 2009. Brain-derived neurotrophic factor contributes to recovery of skilled reaching after focal ischemia in rats. Stroke 40, 1490–1495. [DOI] [PubMed] [Google Scholar]
  42. Reitmeir R, Kilic E, Kilic U, Bacigaluppi M, ElAli A, Salani G, Pluchino S, Gassmann M, Hermann DM, 2011. Post-acute delivery of erythropoietin induces stroke recovery by promoting perilesional tissue remodelling and contralesional pyramidal tract plasticity. Brain 134, 84–99. [DOI] [PubMed] [Google Scholar]
  43. Saavedra JM, 2012. Angiotensin II AT(1) receptor blockers as treatments for inflammatory brain disorders. Clin. Sci. (Lond.) 123, 567–590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sachidanandam K, Harris A, Hutchinson J, Ergul A, 2006. Microvascular versus macrovascular dysfunction in type 2 diabetes: differences in contractile responses to endothelin-1. Exp. Biol. Med. 231, 1016–1021. [PubMed] [Google Scholar]
  45. Shibata K, Makino I, Shibaguchi H, Niwa M, Katsuragi T, Furukawa T, 1997. Upregulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells. Biochem. Biophys. Res. Commun. 239, 633–637. [DOI] [PubMed] [Google Scholar]
  46. Singh VP, Le B, Bhat VB, Baker KM, Kumar R, 2007. High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol. 293, H939–H948. [DOI] [PubMed] [Google Scholar]
  47. Sist B, Fouad K, Winship IR, 2014. Plasticity beyond peri-infarct cortex: spinal up regulation of structural plasticity, neurotrophins, and inflammatory cytokines during recovery from cortical stroke. Exp. Neurol. 252, 47–56. [DOI] [PubMed] [Google Scholar]
  48. Sumners C, Horiuchi M, Widdop RE, McCarthy C, Unger T, Steckelings UM, 2013. Protective arms of the renin-angiotensin-system in neurological disease. Clin. Exp. Pharmacol. Physiol. 40, 580–588. [DOI] [PubMed] [Google Scholar]
  49. Sundboll J, Schmidt M, Horvath-Puho E, Christiansen C, Pedersen L, Botker H, Sorensen H, 2014. Preadmission use of ACE inhibitors or angiotensin receptor blockers and short-term mortality after stroke. J. Neurol. Neurosurg. Psychiatry. 10.1136/jnnp-2014-308948. [DOI] [PubMed] [Google Scholar]
  50. Turnbull F, Neal B, Algert C, Chalmers J, Chapman N, Cutler J, Woodward M, MacMahon S, 2005. Effects of different blood pressure-lowering regimens on major cardiovascular events in individuals with and without diabetes mellitus: results of prospectively designed overviews of randomized trials. Arch. Intern. Med. 165, 1410–1419. [DOI] [PubMed] [Google Scholar]
  51. Villapol S, Saavedra JM, 2015. Neuroprotective effects of angiotensin receptor blockers. Am. J. Hypertens. 28, 289–299. [DOI] [PubMed] [Google Scholar]
  52. Volz LJ, Vollmer M, Michely J, Fink GR, Rothwell JC, Grefkes C, 2017. Time-dependent functional role of the contralesional motor cortex after stroke. Neuroimage Clin. 16, 165–174. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data generated and analyzed in this study will be made available from the corresponding author on reasonable request.

RESOURCES