Middle Cerebral Artery Remodeling Research Articles

RNF34 ablation promotes cerebrovascular remodeling and hypertension by increasing NADPH-derived ROS generation

Cerebrovascular remodeling is the most common cause of hypertension and stroke. Ubiquitin E3 ligase RING finger protein 34 (RNF34) is suggested to be associated with the development of multiple neurological diseases. However, the importance of RNF34 in cerebrovascular remodeling and hypertension is poorly understood. Herein, we used mice with a global RNF34 knockout as well as RNF34 floxed mice to delete RNF34 in endothelial cells and smooth muscle cells (SMCs). Our results showed that global RNF34 knockout mice substantially promoted angiotensin II (AngII)-induced middle cerebral artery (MCA) remodeling, hypertension, and neurological dysfunction. Endothelial cell RNF34 did not regulate the development of hypertension. Rather, SMC RNF34 expression is a critical regulator of hypertension and MCA remodeling. Loss of RNF34 enhanced AngII-induced mouse brain vascular SMCs (MBVSMCs) proliferation, migration and invasion. Furthermore, MCA and MBVSMCs from SMC RNF34-deficient mice showed increased superoxide anion and reactive oxygen species (ROS) generation as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, but exhibited no marked effect on mitochondria-derived ROS. Knockout of RNF34 promoted p22phox expression, leading to increased binding of p22phox/p47phox and p22phox/NOX2, and eventually NADPH oxidase complex formation. Immunoprecipitation assay identified that RNF34 interacted with p22phox. RNF34 deletion increased p22phox protein stability by inhibiting ubiquitin-mediated degradation. Blockade of NADPH oxidase activity or knockdown of p22phox significantly abolished the effects of RNF34 deletion on cerebrovascular remodeling and hypertension. Collectively, our study demonstrates that SMC RNF34 deficiency promotes cerebrovascular SMC hyperplasia and remodeling by increased NADPH-derived ROS generation via reducing p22phox ubiquitin-dependent degradation.

Smooth muscle cell-specific knockout of FBW7 exacerbates intracranial atherosclerotic stenosis

Intracranial atherosclerotic stenosis (ICAS), the most common cause of stroke worldwide, is associated with high risk of recurrent ischemic stroke. F-box and WD repeat domain containing protein 7 (FBW7), an ubiquitin E3 ligase, is recently suggested to be involved in atherogenesis. However, whether FBW7 affects cerebrovascular remodeling during ICAS remains unknowns. We found that the expression of FBW7 was decreased in mouse brain microvessels from high-fat diet (HFD)-fed atherosclerotic mice. The reduced FBW7 expression was negatively associated with the remodeling of middle cerebral artery (MCA). Specific loss of FBW7 in smooth muscle cells (SMCs) markedly potentiated brain vascular SMC (VSMC) proliferation, migration and subsequent MCA remodeling in atherosclerotic mice. The increase of total reactive oxygen species (ROS) generation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in brain microvessels and VSMCs were enhanced after knockout of FBW7, while the mitochondria-derived ROS was unchanged. Analysis of several key subunits of NADPH oxidase revealed that FBW7 deficiency augmented HFD-induced the increase of Nox1 expression, but had no effect on p47phox and p67phox phosphorylation as well as p22phox expression. Both NADPH oxidase specific inhibitor and Nox1 downregulation abrogated the effects of FBW7 deficiency on MCA remodeling. Immunoprecipitation assay identified that FBW7 interacted with Nox1. FBW7 knockout increased Nox1 protein stability by inhibiting ubiquitin-mediated degradation. Collectively, our study demonstrates that SMC-specific deficiency of FBW7 exacerbates ICAS by facilitating Nox1-derived ROS generation, VSMC proliferation and cerebrovascular remodeling.

Mineralocorticoid receptor antagonism prevents obesity-induced cerebral artery remodeling and reduces white matter injury in rats.

Midlife obesity is a risk factor for dementia development. Obesity has also been linked to hyperaldosteronism, and this can be modeled in rats by high fat (HF) feeding from weaning. Aldosterone, or activation of the mineralocorticoid receptor (MR) causes cerebrovascular injury in lean hypertensive rats. We hypothesized that rats fed a HF diet would show inward middle cerebral artery (MCA) remodeling that could be prevented by MR antagonism. We further proposed that the cerebral artery remodeling would be associated with white mater injury. Three-week-old male Sprague-Dawley rats were fed a HF diet±the MR antagonist canrenoic acid (Canr) for 17weeks. Control rats received normal chow (control NC). MCA structure was assessed by pressure myography. The MCAs from HF fed rats had smaller lumens and thicker walls when compared to arteries from control NC rats; Canr prevented the MCA remodeling associated with HF feeding. HF feeding increased the mRNA expression of markers of cell proliferation and vascular inflammation in cerebral arteries and Canr treatment prevented this. White mater injury was increased in the rats fed the HF diet and this was reduced by Canr treatment. The expression of doublecortin, a marker of new and immature neurons was reduced in HF fed rats, and MR antagonism normalized this. These data suggest that HF feeding leads to MR dependent remodeling of the MCA and this is associated with markers of dementia development.

Angiotensin-(1–7) counteracts the effects of Ang II on vascular smooth muscle cells, vascular remodeling and hemorrhagic stroke: Role of the NFкB inflammatory pathway

Angiotensin (Ang)-(1–7) is a potential vasoprotective peptide. In the present study, we investigated its counteractive effects to Ang II on vascular smooth muscle cells (VSMCs) and intracerebral hemorrhagic stroke (ICH) through inflammatory mechanism. In in vitro experiments, human brain VSMCs (HBVSMCs) were treated with vehicle, Ang II, Ang II+Ang-(1–7), Ang II+A-779 or Ang II+Ang-(1–7)+A-779 (Mas receptor antagonist). HBVSMC proliferation, migration and apoptosis were determined by methyl thiazolyltetrazolium, wound healing assay and flow cytometry, respectively. In in vivo experiments, C57BL/6 mice were divided into vehicle, Ang II, Ang II+Ang-(1–7), Ang II+A-779 or Ang II+Ang-(1–7)+A-779 groups before they were subjected to collagenase-induced ICH or sham surgery. Hemorrhage volume and middle cerebral artery (MCA) remodeling were determined by histological analyses. Levels of NFκB, inhibitor of κBα (IκBα), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were measured by western blot or ELISA. We found that 1) Ang II increased HBVSMC migration, proliferation and apoptosis, and increased the blood pressure (BP), neurological deficit score, MCA remodeling and hemorrhage volume in ICH mice. 2) Ang-(1–7) counteracted these effects of Ang II, which was independent of BP, with the down-regulation of NFκB, up-regulation of IκBα, and decreased levels of TNF-α, MCP-1 and IL-8. 3) The beneficial effects of Ang-(1–7) could be abolished by A-779. In conclusion, Ang-(1–7) counteracts the effects of Ang II on ICH via modulating NFκB inflammation pathway in HBVSMCs and cerebral microvessels.

Middle cerebral artery remodeling following transient brain ischemia is linked to early postischemic hyperemia: a target of uric acid treatment.

Ischemia impairs blood supply to the brain, and reperfusion is important to restore cerebral blood flow (CBF) and rescue neurons from cell death. However, reperfusion can induce CBF values exceeding the basal values before ischemia. This hyperemic effect has been associated with a worse ischemic brain damage, albeit the mechanisms that contribute to infarct expansion are not clear. In this study, we investigated the influence of early postischemic hyperemia on brain damage and middle cerebral artery (MCA) properties and the effect of treatment with the endogenous antioxidant uric acid (UA). The MCA was occluded for 90 min followed by 24 h reperfusion in adult male Sprague-Dawley rats. Cortical CBF increases at reperfusion beyond 20% of basal values were taken as indicative of hyperemia. UA (16 mg/kg) or vehicle (Locke's buffer) was administered intravenously 135 min after MCA occlusion. Hyperemic compared with nonhyperemic rats showed MCA wall thickening (sham: 22.4 ± 0.8 μm; nonhyperemic: 23.1 ± 1.2 μm; hyperemic: 27.8 ± 0.9 at 60 mmHg; P < 0.001, hyperemic vs. sham) involving adventitial cell proliferation, increased oxidative stress, and interleukin-18, and more severe brain damage. Thus MCA remodeling after ischemia-reperfusion takes place under vascular oxidative and inflammatory stress conditions linked to hyperemia. UA administration attenuated MCA wall thickening, induced passive lumen expansion, and reduced brain damage in hyperemic rats, although it did not increase brain UA concentration. We conclude that hyperemia at reperfusion following brain ischemia induces vascular damage that can be attenuated by administration of the endogenous antioxidant UA.

Abstract 224: Chronic Infusion Of Angiotensin-(1-7) Alleviates Cerebral Hemorrhagic Injury Compromised By Angiotensin II

Angiotensin-(1-7) [Ang-(1-7)] is a potential vascular protective peptide which counteracts the effects of Ang II. In this study, we investigated the role of Ang-(1-7) in hemorrhagic stroke. Adult male C57BL/6 mice implanted with telemetric probe for recording arterial blood pressure (BP) were divided into four minipump treatment groups: saline, Ang II, Ang II + Ang-(1-7), Ang II + Ang-(1-7) + A-779. After 2 weeks of treatment, mice were subjected to induction of hemorrhagic stroke by microinjection of collagenase (type VII; 0.075 U in 0.5 μL) in striatum. Mice were sacrificed at twenty-four hours after ICH induction. Brains were dissected and cut into coronal slices for histological analysis of middle cerebral artery (MCA)remodeling and hemorrhagic size. The plasma levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were determined by ELISA and the levels of NFκB and inhibitor of kappa B (IκB) in cerebral microvasculature were determined by western blot. We found (Figure): 1) Ang II increased BP, MCA remodeling and hemorrhagic size. 2) Ang-(1-7) significantly decreased the effects of Ang II on MCA and hemorrhage without affecting the BP, which were abolished by A-779. 3) Ang-(1-7) induced a 26-32% reduction in plasma level of TNF-α, MCP-1 and IL-8 increased by Ang II. 4) Ang-(1-7) also counter-regulated Ang II-induced NFƙB up-regulation and IƙB down-regulation. In conclusion, Ang-(1-7) counteracts Ang II on vascular remodeling and hemorrhagic injury by alleviating NFκB-related inflammation.

Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats.

Hypertension causes vascular inflammation evidenced by an increase in perivascular macrophages and proinflammatory cytokines in the arterial wall. Perivascular macrophage depletion reduced tumor necrosis factor (TNF)-α expression in cerebral arteries of hypertensive rats and attenuated inward remodeling, suggesting that TNF-α might play a role in the remodeling process. We hypothesized that TNF-α inhibition would improve middle cerebral artery (MCA) structure and reduce damage after cerebral ischemia in hypertensive rats. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with the TNF-α inhibitor etanercept (ETN; 1.25 mg·kg(-1)·day(-1) ip daily) or PBS (equivolume) for 6 wk. The myogenic tone generation, postischemic dilation, and passive structure of MCAs were assessed by pressure myography. Cerebral ischemia was induced by MCA occlusion (MCAO). Myogenic tone was unchanged, but MCAs from SHRSP + ETN had larger passive lumen diameter and reduced wall thickness and wall-to-lumen ratio. Cerebral infarct size was increased in SHRSP + ETN after transient MCAO, despite an improvement in dilation of nonischemic MCA. The increase in infarct size was linked to a reduction in the number of microglia in the infarct core and upregulation of markers of classical macrophage/microglia polarization. There was no difference in infarct size after permanent MCAO or when untreated SHRSP subjected to transient MCAO were given ETN at reperfusion. Our data suggests that TNF-α inhibition attenuates hypertensive MCA remodeling but exacerbates cerebral damage following ischemia/reperfusion injury likely due to inhibition of the innate immune response of the brain.

Captopril treatment improves endothelium dependent dilation and middle cerebral artery remodeling in hypertensive rats (841.8)

Hypertension is associated with cognitive deficits. Angiotensin converting enzyme inhibitors (ACEI) improve cognitive decline in hypertensive patients. We hypothesized that centrally active ACEI captopril (CTP) would alleviate cognitive function by improving endothelium dependent dilation and middle cerebral artery (MCA) remodeling from stroke prone spontaneously hypertensive rats (SHRSP). Data are shown as mean±SEM, SHRSP vs CTP and are analyzed by Student’s t‐test. 12 week old male SHRSP received CTP (50 mg/kg/day) for 6 weeks. Cognitive function, evaluated by Morris water maze, showed no difference between groups. CTP reduced systolic blood pressure (178±8 vs 131±6 mmHg, p&lt;0.05). MCA reactivity and passive structure was assessed by pressure myography (data shown at 80 mmHg). CTP did not increase lumen diameter (232.6±8.1 vs 249.1±8.1). CTP reduced wall thickness (21.9±0.1 vs 17.3±1.0 µm, p&lt;0.05) and wall/lumen ratio (0.1±0.01 vs 0.07±0.01, p&lt;0.05). CTP improved endothelial function as evidenced by enhanced dilation to 10 µM adenosine diphosphate (change in diameter from baseline at: 36.2±1.0 vs 54.2± 5.3 µm, p&lt;0.05). CTP increased resting pial artery perfusion (673±32 vs 750±201 laser Doppler units, p&lt;0.05). These data suggests that while CTP does not affect cognitive abilities, it may contribute to increased baseline perfusion by enhancing dilation and reversing remodeling of MCAs.

Smooth Muscle Cells Specific Mineralocorticoid Deletion Does Not Alter Middle Cerebral Artery Structure in Mice

We demonstrated that remodeling of the middle cerebral artery (MCA) in rats is in part dependent on the mineralocorticoid receptor (MR). MR mediated MCA remodeling leads to reduction in lumen diameter and increase in wall thickness. These changes can increase stroke risk and damage after cerebral ischemia. Recent studies demonstrate that MR in smooth muscle cells (SMC) directly regulates gene expression and function. We hypothesized that MR deletion in SMC play a role in MCA remodeling. SMC‐specific MR deletion (SMC‐MR−/−, n=8) in mice was achieved through expression of CRE‐recombinase driven by a SMC‐specific promoter and loxP sequences flanking exons 4 and 5 of the MR gene. MR floxed, Cre negative (WT, n=7) littermates were controls. MCAs from 9 months‐old mice were mounted on a pressure myograph to assess passive structure. Data are mean ± SEM, SMC‐MR−/− vs WT, at 80mmHg intralumenal pressure. SMC‐MR−/− did not alter MCA lumen diameter (151.6±2.8 vs 155.7±2.6μm), outer diameter (169.1±2.8 vs 171.7±2.8μm), wall thickness (8.75±0.2 vs 8.0±0.3μm) and the wall‐to‐lumen ratio (0.06±0.01 vs 0.05±0.01). MR deletion did not alter extracellular parameters in the MCA, such as stress (696±24 vs 785.8±33), stiffness (β‐coefficient: 5.58±0.4 vs 5.56±0.3), and distensibility (68.4±5.6 vs 71.5±5.2). This data suggest that under normal physiological conditions SMC‐MR does not have deleterious effects in the MCA in mice.

Etanercept, a tumor‐necrosis factor (TNF‐α) inhibitor, improves endothelial function of contralateral middle cerebral artery after cerebral ischemia in hypertensive rats

Middle cerebral artery (MCA) occlusion (MCAO) causes endothelial dysfunction in the ischemic MCA (Marrelli et al., AJP 276:H33‐H41), and in small mesenteric arteries (Martinez‐Revelles et al., JPET 325: 363–9). The effect of MCAO on the contralateral MCA (CL MCA) however is unknown. In hypertensive rats MCA remodeling impairs artery function, and etanercept (ETN) attenuates this. We hypothesized that CL MCA dilation is impaired after MCAO and that ETN will improve this. 12 week old stroke prone spontaneously hypertensive rats, ETN‐treated from 6 weeks of age (ETN IR) or untreated (IR), underwent 1 hour of MCAO and 47 hours reperfusion. SHAM controls were generated. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry. CL MCAs were mounted in a pressure myograph under physiological conditions. Endothelial function was assessed by intraluminal perfusion of ADP. Data are presented as mean ± SEM. Tone generation was not different between the groups. ADP mediated dilation was impaired in the IR group and this was ameliorated by ETN treatment (p&lt;0.05). This caused a small, but insignificant (p=0.12), increase in post‐MCAO CBF in the ETN IR group. Our data suggest that ETN improves CL MCA endothelial function and may improve post‐MCAO CBF in hypertensive rats. (AHA0840122N) SHAM (n=2) ETN IR (n=3) IR (n=4) Tone (%) 32.6±5.4 33.2±7.1 36.9±4.7 Maximum increase in lumen diameter with ADP (μm) 62.5±12.5 60.6±7.5 28.2±7.8 Flow (% of pre‐MCAO) 94.5±10.9 86.6±7.0 62.6±10.1

Apocynin treatment attenuated middle cerebral artery remodeling in life‐long obesity in Sprague‐Dawley rats

Metabolic syndrome is a disorder that includes abdominal obesity and hypertension, and these increase stroke risk. Diet‐induced obesity and hypertension lead to remodeling of the middle cerebral artery (MCA) through a mechanism dependent on superoxide anion (O2•−). In the vasculature, most O2•− is generated by the enzyme NADPH‐oxidase (Nox). We hypothesized that Nox inhibition with apocynin (Apo) would attenuate obesity‐induced MCA remodeling in rats. Three‐week old male Sprague‐Dawley rats fed a high‐fat diet (HF) for 17 weeks were treated with Apo (HF+Apo, n=7) or vehicle (HF, n=12). Rats fed normal chow were controls (NC, n=11). MCAs were mounted on a pressure myograph to assess passive structure. Data are mean ± SEM, NC vs HF vs HF+Apo. Rats fed HF had higher visceral fat:body weight (1.42±0.13, 3.92±0.19, 4.27±0.45, p&lt;0.05). Apo prevented the HF‐induced increase in mean arterial pressure (126±3, 146±6, 125±3mmHg, p&lt;0.01). At 80mmHg intralumenal pressure, Apo increased MCA lumen diameter (266±6.6, 245±8.3, 280±8.3μm, p&lt;0.05), decreased wall thickness (20.2±1.5, 33.0±2.8, 18.0±1.2μm, p&lt;0.05) and wall‐to‐lumen ratio (0.08±0.01, 0.14±0.01, 0.07±0.01, p&lt;0.05). HF±Apo had no effect on distensibility (56.9±5.6, 48.8±4.6, 50.3±4.3). These data suggest that Apo prevented the increase in blood pressure in rats fed HF. This might be responsible for the beneficial effects of Apo in the MCA structure.

Abstract 3713: Possible Role Of Circulating Cellular Microparticles In Progressive Hypertension And Middle Cerebral Artery Remodeling

Circulating cellular microparticles (MPs) are potential biomarkers for cardiovascular diseases and are also suggested to be involved in the pathogenesis of hypertension and stroke. We assessed the hypothesis that circulating MPs could have implications in influencing mean arterial pressure (MAP) and middle cerebral artery (MCA) remodeling via changes in their levels and contents. Male C57BL/6 mice were fed with normal diet (control) or induced to progressive hypertension by infusion with angiotensin II (ANG II, 42µg/kg/h, osmotic minipump) and fed with high-salt diet (HSD, 8% NaCl) plus L-NAME (120 mg/kg/day, in drinking water) for 2 (for MPs donor) and 4 weeks (for MP infusion study). Circulating MPs isolated from control (cMPs) or hypertensive (hMPs) mice were isolated as we previously described (Chen, et al, AJP, 2011). The levels of circulating endothelial MPs (EMPs) and endothelial progenitor cells-MPs (EPC-MPs) were analyzed using flow cytometry by staining with CD144 (EMPs), CD34 and VEGFR2 (EPC-MPs), respectively. The mRNA levels of CXCR4, endothelial nitric oxide synthase (eNOS), NOX2 and tumor necrosis factor-alpha (TNF-a) in circulating MPs were determined by quantitative real-time RT-PCR. In separate experiment, control or hypertensive mice were treated with infusion of cMPs or hMPs (1x10 5 /100µl) via tail vein once per week for two continuous weeks, and sacrificed at the end of experiment (week four). MAP was measured using radiotelemetric probe. The lumen/wall in the MCA was determined by Hematoxylin and Eosin staining. Results showed ( table ): 1) Mice infused with ANG II and fed with HSD and L-NAME showed progressive hypertension and MCA remodeling. 2) The levels of EMP and EPC-MPs were significantly increased in hypertensive mice. 3) hMPs carried less CXCR4 and eNOS, and higher NOX2 and TNF-a mRNAs when compared with cMPs. 4) Infusion of hMPs increased MAP and MCA remodeling in hypertensive mice, but only induced MCA remodeling in control mice. 5) cMPs decreased MAP and MCA in hypertensive mice, but had no effect on MCA remodeling and MAP in control mice. In conclusion, circulating MPs could be served as biomarkers and used for developing therapeutic avenues for progressive hypertensive and MCA remodeling.

Canrenoic Acid, a Mineralocorticoid Receptor Antagonist, Attenuates Resistance Artery Remodeling in Stroke‐Prone Spontaneous Hypertensive Rats

Chronic hypertension causes structural changes in the resistance vasculature through the process of remodeling. Middle cerebral artery (MCA) remodeling increases the risk for ischemic stroke. We have previously shown that the mineralocorticoid receptor (MR) is involved in MCA remodeling. Therefore, we hypothesized that MR antagonism with canrenoic acid (CANR) would attenuate the process. Six‐week old male stroke‐prone spontaneously hypertensive rats (SHRSP) were treated with CANR (20mg/kg/day, n=7) in the drinking water for six weeks. Untreated SHRSP were used as controls (n=12). MCA and mesenteric resistant artery (MRA) passive structure was analyzed by pressure myography. Data are shown as mean ± SEM, CANR vs control respectively. In the MCA, at 80mmHg, CANR treatment increased lumen diameter (277±6 vs 240±5μm, p&lt;0.05) and decreased wall thickness (17.1±1.1 vs 25.6±1.2μm, p&lt;0.05) and wall‐to‐lumen ratio (0.06±0.01 vs 0.11±0.01, p&lt;0.05), without changing vessel stiffness (β‐coefficient: 10.4±1.4 vs 8.16±1.6, p&lt;0.05). In the MRA, at 90 mmHg, CANR treatment reduced wall thickness (17.0±0.7 vs 27±1.7μm, p&lt;0.05) and wall‐to‐lumen ratio (0.16±0.01 vs 0.10±0.01, p&lt;0.05), without changing lumen diameter (274±8 vs 265±9μm) or vessel stiffness (β‐coefficient: 5.5±0.7 vs 5.5±0.5). These data suggest that MR antagonism attenuates cerebral and peripheral remodeling by different mechanisms.

Ischemia/Reperfusion Injury Causes an Outward Remodeling of the Middle Cerebral Artery.

Currently, the most effective form of treatment for acute ischemic stroke is to recannulate the blocked vessel to reintroduce blood flow to the brain. The long‐term effects of this ischemia/reperfusion injury (I/R) on cerebral vessel structure have not been investigated. To study this, 10‐week‐old Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion using the intralumenal suture occlusion technique. After 1 hour of ischemia, the monofilament was withdrawn to allow reperfusion of the brain tissue. Fourteen days later the rats were euthanized and the MCA was studied by pressure myography. I/R caused an increase in the MCA lumen (284.4±5.6 vs 263.7±7.5 μm at 80 mmHg, I/R vs control, p&lt;0.05) and outer diameter (316.6±5.0 vs 295.7±6.1 μm at 80 mmHg, I/R vs control, p&lt;0.05). This change in MCA structure occurred without a change in wall thickness (32.1±1.8 vs 32.0±3.3μm at 80 mmHg, I/R/vs control), wall/lumen ratio (0.11±0.008 vs 0.12±0.014 at 80 mmHg, I/R vs control), or vessel stiffness (β‐coefficient: 5.9±0.4 vs 6.3±0.3, I/R vs control). I/R injury had no effect on the ability of the vessel to generate tone or contract in response to serotonin 2 weeks after the insult. The mechanisms for the outward MCA remodeling remain to be elucidated, as does the effect of the change in structure on the outcome of future ischemic events.

Abstract 5081: Circulating Microparticles as Potential Biomarkers for Primary Intracerebral Hemorrhage Stroke

Introduction: Primary intracerebral hemorrhage (ICH) stroke is a serious complication of uncontrolled hypertension. Recent clinical studies have shown that circulating microparticles (MPs) from endothelia (EMPs), platelets (PMPs) and endothelial progenitor cells (EPC-MPs) are potential biomarkers for cardiovascular diseases. However, the role of circulating MPs in ICH stroke is unknown. Methods and Results: The human renin and angiotensinogen transgenic (R+A+) and the control (R−A−) mice (male, 8 –10 weeks) were fed with normal diet or high salt diet (8% NaCl, HSD) plus nitric oxide synthase inhibitor L-NAME (120 mg/kg/day) in drinking water for two weeks. Mean arterial pressure (MAP) was measured using telemetric probes. Circulating MPs were identified by membrane markers, CD62E (for EMPs), CD62P (for PMPs), and CD34 and VEGFR2 (for EPC-MPs) using flow cytometry. The severity of ICH was determined by histological analysis of total bleeding area. The lumen/wall in the middle cerebral artery (MCA) was histologically measured. Results (Table and Figure ) showed: The R+A+ mice were hypertensive and had higher basal level of circulating PMPs and EMPs with no difference in EPC-MPs; After fed with HSD and L-NAME, R+A+ mice had severe hypertension and 50% of them developed ISH stroke within two weeks; There was a further increase of EMPs and a decrease of EPC-MPs in R+A+ mice with ICH stroke; There were strong correlations of EMPs (r=0.44~0.61) and EPC-MPs (r=−0.52~0.78) with MAP, MCA lumen/wall and total area of bleeding sites. Conclusion: Circulating EPC-MPs and EMPs could serve as useful biomarkers for uncontrolled hypertension, MCA remodeling and ICH stroke. Table. Blood pressure, circulating MPs and MCA lumen/wall in R+A+ and R−A− mice fed with normal diet or HSD and L-NAME for two weeks Fig. Hemorrhages in the brain (A, D: brainstem: B, E: cerebellum; C,F: basal ganglia) and MCA remodeling, A–C; R+A+ mice fed with HSD+L-NAME; D–F: R−A− mice fed with HSD+L-NAME; G: R−A− mice fed with normal diet, H: R−A− mice fed with HSD+L-NAME; I: R+A+ mice fed with HSD+L-NAME

Antioxidant treatment with tempol prevents obesity induced remodeling of middle cerebral arteries in Sprague‐Dawley rats

Obesity is a risk factor for stroke, especially when associated with diabetes. Cerebral vessel remodeling may contribute to this increased risk of cerebrovascular accidents. We hypothesized that a diet rich in fat and carbohydrates would contribute to middle cerebral artery (MCA) remodeling, and that oxidative stress would play a key role in the process. Three week old male Sprague‐Dawley rats were fed a high‐fat (HF) diet containing 36% fat and 36% carbohydrate (n=21), or HF plus the super oxide dismutase mimetic, tempol (n=9) or regular chow (n= 18) for 18 weeks. MCA's were mounted in a pressure myograph under calcium‐free conditions to assess vascular structure. All data are shown as mean±SEM. Body weight was higher in the groups that received HF diet (442±6, 484±19, 374±9 grams, HF, HF+tempol and control); the same was true for visceral fat weight (16.2±0.9, 22.1±3.1, 5.7±0.5 grams, HF, HF+tempol, control). Blood glucose was increased in the HF group (197±10, 143±5 mg/dl, HF vs control) and treatment with tempol reversed this effect (115±10 mg/dl). Wall/lumen ratio was higher in the HF rats and reduced by tempol treatment (0.15±0.02, 0.08±0.01, 0.08±0.01, HF, HF+tempol, control, at 80mmHg p&lt;0.05), the same was true for wall thickness and wall area. These results suggest that a HF diet increases MCA remodeling and blood glucose, and tempol treatment reverses these processes. ADA‐7‐07‐RA28, AHA‐0840122N

Intact female stroke-prone hypertensive rats lack responsiveness to mineralocorticoid receptor antagonists

Data from the Framingham Heart Study suggest that women may be more sensitive to the deleterious cardiovascular remodeling effects of aldosterone. Previous studies from our laboratory have shown that chronic treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist, decreases ischemic cerebral infarct size and prevents remodeling of the middle cerebral artery (MCA) in male spontaneously hypertensive stroke-prone rats (SHRSP). Therefore, we hypothesized that MR antagonism would reduce ischemic infarct size and prevent MCA remodeling in female SHRSP. Six-week-old female SHRSP were treated for 6 wk with spironolactone (25 or 50 mg.kg(-1).day(-1)) or eplerenone (100 mg.kg(-1).day(-1)) and compared with untreated controls. At 12 wk, cerebral ischemia was induced for 18 h using the intraluminal suture occlusion technique, or the MCA was isolated for analysis of passive structure using a pressurized arteriograph. MR antagonism had no effect on infarct size or passive MCA structure in female SHRSP. To study the potential effects of estrogen, the above experiments were repeated in bilaterally ovariectomized (OVX) female SHRSP treated with spironolactone (25 mg.kg(-1).day(-1)). Infarct size and vessel structure in OVX SHRSP were not different from control SHRSP. Spironolactone had no effect on infarct size in OVX SHRSP. However, MCA lumen and outer diameters were increased in spironolactone-treated OVX SHRSP, suggesting an effect of estrogen. Cerebral artery MR expression, assessed by Western blotting, was increased in female, compared with male, SHRSP. These studies highlight an apparent sexual dimorphism of MR expression and activity in the cerebral vasculature from hypertensive rats.

Eplerenone Prevents Cerebral Vessel Remodeling in Male Hypertensive Rats

Aldosterone, a mineralocorticoid, promotes deleterious remodeling of the cardiovascular system. Spironolactone, a non-specific mineralocorticoid receptor (MR) antagonist, prevents remodeling of the middle cerebral artery (MCA) in male spontaneously hypertensive stroke-prone rats (SHRSP). We hypothesized that eplerenone (EPL), a specific MR antagonist, would also prevent remodeling of the MCA of male SHRSP. Six week old male SHRSP were treated with EPL (100 mg/kg/day) for 6 weeks. MCA structure was analyzed using a pressurized arteriograph under passive (calcium-free) conditions from 0 to 180 mmHg. Blood pressure (BP) was measured using telemetry or tail-cuff plethysmography. Lumen and outer diameters were increased (Table) and wall/lumen ratios were decreased (p<0.05, ANOVA) compared to untreated SHRSP; wall area was unchanged (p>0.05, ANOVA), indicating that EPL treatment prevented MCA remodeling. BP was not different between groups (Table), indicating a BP independent effect. In conclusion, the data indicate that effects seen previously with spironolactone are MR specific and highlight the involvement of MR activation in cerebral vessel remodeling.

Plaque Volume Measurement by Magnetic Resonance Imaging as an Index of Remodeling of Middle Cerebral Artery: Correlation with Transcranial Color Doppler and Magnetic Resonance Angiography

Background: The feasibility of a noninvasive evaluation of remodeling of the middle cerebral artery (MCA) by magnetic resonance imaging (MRI) was assessed. The results were correlated with magnetic resonance angiography (MRA) and transcranial color Doppler (TCD) findings. Methods: 26 patients (13 male and 13 female, age ranged from 46 to 82 years) who presented with symptoms of cerebrovascular accidents had TCD, MRA and MRI assessment of the MCA. The TCD and MRA findings of 40 MCAs accessible by TCD were correlated with the ratio of cross-sectional area of the vessel (VA), luminal area of the vessel (LA) and plaque load (PL). Results: The VA ratio and LA showed no correlation with TCD or MRA results. PL however was shown to be associated with both TCD and MRA. Conclusions: MRI could be used to assess remodeling in MCAs. PL was found to be associated with TCD and MRA findings.