Cerebral Parenchymal Arterioles Research Articles

Thrombin in Pregnancy and Preeclampsia: Expression, Localization and Vasoactivity in Brain and Microvessels from Rats.

Thrombin is a coagulation factor increased in pregnancy and further increased in preeclampsia (PE), a hypertensive disorder. Thrombin is also expressed in brain and may have a nonhemostatic role. We characterized thrombin expression and vasoactivity in brain cerebral parenchymal arterioles (PAs) in rat models of pregnancy and PE. PAs were isolated and pressurized from nonpregnant (NP), late-pregnant (LP) rats and rats with experimental preeclampsia (ePE). Reactivity to thrombin (1-50 U/mL) was measured in the absence and presence of inhibition of cyclooxygenase (COX) and nitric oxide synthase (NOS). Plasma levels of prothrombin, thrombin-antithrombin (TAT), tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) and cerebrospinal fluid (CSF) levels of TAT were compared via ELISA. Expression of protease-activated receptor (PAR) types 1 and 2 in PAs were measured by Western blot and immunohistochemistry. Neuronal thrombin expression was quantified in brains from all groups by immunohistochemistry. Prothrombin and TAT were elevated in ePE plasma compared to NP and LP. TAT was detected in CSF from all groups and significantly elevated in LP (NP: 0.137±0.014 ng/mL, LP: 0.241±0.015 ng/mL, ePE: 0.192±0.028 ng/mL; p<0.05). Thrombin caused modest vasoconstriction in PAs from all groups regardless of COX or NOS inhibition. PAR1 and PAR2 were found in PAs from all groups co-localized to smooth muscle. Thrombin expression in central neurons was decreased in both LP and ePE groups compared to NP. These findings suggest a role for thrombin and other hemostatic changes during pregnancy and PE beyond coagulation.

Neurovascular Uncoupling in TgF344‐AD Rats is Associated with Reduced Cerebral Capillary Endothelial Kir2.1 Expression

AbstractBackgroundAlzheimer’s disease (AD) is an emerging global health care crisis. Although reduced cerebral blood flow (CBF) is an early and persistent symptom in developing cognitive deficits, the underlying molecular and cellular mechanisms remain unclear. Recent studies suggested that restoration of capillary endothelial cells (ECs) inward‐rectifier potassium channels (Kir2.1) currents reversed cerebral hypoperfusion and enhanced neurovascular coupling in AD mice. The present study aims to investigate whether capillary ECs Kir2.1 expression is reduced in TgF344‐AD rats and whether it contributes to neurovascular uncoupling and cognitive deficits.MethodThree to six months old TgF‐344 AD rats expressing mutant human APP and PS1 and their age‐matched wildtype (WT) rats were studied. An eight‐arm water maze was used to test spatial learning and short and long‐term memory. Brain perfusion was mapped with a laser speckle imaging system. Whisker stimulation‐induced functional hyperemia was measured using laser Doppler flowmetry in vivo. Brain Kir2.1 expression was compared using western blot. Cerebral parenchymal arterioles (PAs) with attached capillaries were freshly isolated from AD and WT rats and mounted in a pressure myography. PAs response to capillaries focally injected KCL (10 mM) and Kir2.1 inhibitor (ML133) was compared.ResultAD rats exhibited learning and memory dysfunction at 6‐month of age compared with WT controls. However, cerebral hypoperfusion was observed in AD rats starting from 4‐month of age. Similarly, impaired functional hyperemia in AD rats started at 4‐month of age, which was exacerbated in 6‐month old AD rats. The expression of Kir2.1 in 6‐month AD brain tissue was significantly lower than in WT rats. AD PAs displayed less vasodilation (110.96 ± 3.92 % vs. 122.93 ± 1.97 %) and vasoconstriction (92.16 ± 3.47 % vs. 78.04 ± 2.96 %) in response to capillary administered KCL and ML133, respectively, compared to WT vessels.ConclusionReduced brain hypoperfusion precedes cognitive impairment in AD rats, associated with attenuated capillary EC Kir2.1 expression and neurovascular uncoupling.

Cerebral arteriolar and neurovascular dysfunction after chemically induced menopause in mice.

Cognitive decline is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of dementia, possibly because of reduced functional dilation in parenchymal arterioles. These vessels are bottlenecks of the cerebral microcirculation, and dysfunction can limit functional hyperemia in the brain. Large-conductance Ca2+-activated K+ channels (BKCa) are the final effectors of several pathways responsible for functional hyperemia, and their expression is modulated by estrogen. However, it remains unknown whether BKCa function is altered in cerebral parenchymal arterioles after menopause. Using a chemically induced model of menopause, the 4-vinylcyclohexene diepoxide (VCD) model, which depletes follicles while maintaining intact ovaries, we hypothesized that menopause would be associated with reduced functional vasodilatory responses in cerebral parenchymal arterioles of wild-type mice via reduced BKCa function. Using pressure myography of isolated parenchymal arterioles, we observed that menopause (Meno) induced a significant increase in spontaneous myogenic tone. Endothelial function, assessed as nitric oxide production and dilation after cholinergic stimulation or endothelium-dependent hyperpolarization pathways, was unaffected by Meno. BKCa function was significantly impaired in Meno compared with control, without changes in voltage-gated K+ channel activity. Cerebral functional hyperemia, measured by laser-speckle contrast imaging during whisker stimulation, was significantly blunted in Meno mice, without detectable changes in basal perfusion. However, behavioral testing identified no change in cognition. These findings suggest that menopause induces cerebral microvascular and neurovascular deficits.NEW & NOTEWORTHY Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BKCa channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.

Mineralocorticoid receptor antagonism improves transient receptor potential vanilloid 4-dependent dilation of cerebral parenchymal arterioles and cognition in a genetic model of hypertension.

In a model of secondary hypertension, mineralocorticoid receptor (MR) antagonism during the development of hypertension prevents the impairment of transient receptor potential vanilloid 4 (TRPV4) activation in parenchymal arterioles (PAs) and cognitive impairment. However, it is unknown whether MR antagonism can improve these impairments when treatment begins after the onset of essential hypertension. We tested the hypothesis that MR activation in stroke-prone spontaneously hypertensive rats (SHRSP) leads to impaired TRPV4-mediated dilation in PAs that is associated with cognitive dysfunction and neuroinflammation. 20-22-week-old male SHRSP ± eplerenone (EPL; 100 mg/kg daily for 4 weeks) were compared to normotensive Sprague-Dawley (SD) rats. Pressure myography was used to assess PA function. Cognition was tested using Y-maze. Neuroinflammation was assessed using immunofluorescence and qRT-PCR. Carbachol-mediated endothelium-dependent dilation was impaired in SHRSP, and MR antagonism improved this without affecting myogenic tone. Dilation to TRPV4 agonist GSK1016790A was impaired in SHRSP, and ELP treatment restored this. Intermediate conductance potassium channel (IKCa)/small conductance potassium channel (SKCa)-mediated dilation was impaired by hypertension and unaffected by EPL treatment. TRPV4 and IKCa/SKCa channel mRNA expression were reduced in PAs from hypertensive rats, and EPL did not improve this. Impairments in PA dilation in SHRSP were associated with cognitive decline, microglial activation, reactive astrogliosis, and neuroinflammation; cognitive and inflammatory changes were improved with MR blockade. These data advance our understanding of the effects of hypertension on cerebral arterioles using a clinically relevant model and treatment paradigm. Our studies suggest TRPV4 and the MR are potential therapeutic targets to improve cerebrovascular function and cognition during hypertension.

Hypertensive Female Mice Are Protected from Impaired Parenchymal Arteriolar TRPV4‐Mediated Dilation and Reactive Astrogliosis

Hypertension is a leading risk factor for cerebral small vessel disease. Our lab has previously demonstrated that cerebral parenchymal arterioles (PAs) are dependent on transient receptor potential vanilloid 4 (TRPV4) function for endothelium‐dependent dilation, and that this dilation is impaired in angiotensin II (AngII)‐hypertensive male mice. This PA dilation impairment is associated with cognitive deficits and increased inflammation. However, sex differences in hypertension‐associated dementia development are not well understood. To better understand the effect of female sex hormones during hypertension, we tested the hypothesis that young, cycling hypertensive female mice will be protected from impaired TRPV4‐mediated PA dilation and reactive astrogliosis observed in male mice. AngII‐filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16‐18‐week‐old male C56BL/6 mice. Age‐matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail‐cuff plethysmography. Pressure myography was used to assess TRPV4‐mediated dilation in PAs. An anti‐glial fibrillary acidic protein (GFAP) antibody was used in paraformaldehyde‐fixed 40μm coronal brain sections to assess the quantity and morphology of astrocytes. Data are presented as means ± SEM (n=5‐10). Male data were compared by Student’s t‐test and female data were compared by one‐way ANOVA unless otherwise stated. Systolic blood pressure was elevated in AngII‐treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p=0.005) and in 1200ng AngII‐treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179 ± 7mmHg; p=0.0482) vs sham. PA dilation in response to the TRPV4 agonist GSK1016790A (10‐9‐10‐5M) was not impaired in AngII‐treated female mice (sham: 85 ± 5, 800ng AngII: 73 ± 7, 1200ng AngII: 77 ± 8% maximum dilation; p=0.4381), contrasting with what we have previously reported in male mice. There was an increase in astrocytes in the corpus callosum of hypertensive male mice (sham: 65 ± 2; AngII: 77 ± 5; p=0.039). Interestingly, sham female mice had more astrocytes than sham male mice, and AngII infusion reduced the quantity (sham: 81 ± 3, 800ng AngII: 71 ± 3, 1200ng AngII: 69 ± 3; p=0.011). Sholl analysis was used to assess astrocyte morphology. Astrocytes from hypertensive male mice had more arborization further from the soma compared to sham (p=0.0348 at 14‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice receiving the 1200ng dose AngII, however, had fewer arborizations away from the soma compared to sham (p=0.0263 at 13‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice are resistant to AngII‐induced hypertension and protected from the associated deleterious cerebrovascular effects, supporting our hypothesis.

Post‐menopausal Cerebral Microvascular Alterations in a Mouse Model of Alzheimer’s Disease

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p&lt;0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p&lt;0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p&lt;0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p&lt;0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.

Endothelial KIR channel dysfunction in aged cerebral parenchymal arterioles

Aging is associated with cognitive decline via mechanisms that are not fully understood. Endothelial dysfunction is known to occur in parenchymal arterioles, the bottlenecks of the cerebral microcirculation. Dilation of these arterioles is highly dependent on changes in extracellular K+ ([K+]E), caused by activity of inwardly rectifying K+ channels (KIR2) or by opening of Ca2+‐activated small‐ and intermediate‐conductance K+ channels (KCa2.3 / KCa3.1, respectively). The effects of aging on activity of these channels in the endothelium of cerebral parenchymal arterioles remain undetermined, as well as their impact on endothelium‐dependent dilation. Thus, we hypothesized that advanced age will impair both KIR2 and KCa2.3 / KCa3.1 function in parenchymal arterioles of Aged mice (&gt;24 months) when compared to Young (4‐6 months). Data are means ± SEM, Young vs. Aged males and females. No sex differences were observed, thus the data were combined. Using electrophysiology, we observed that freshly‐isolated endothelial cell tubes from Aged mice showed a similar hyperpolarization to the purinergic receptor agonist 2‐methyl‐S‐ATP (1 µM), which purportedly activates the downstream effectors KIR2 and KCa2.3 / KCa3.1, than those from Young mice (Δmembrane potential (Vm): ‐21.0 ± 1.7 vs ‐23.9 ± 5.1 mV, n = 5‐7), although vasodilatory responses of pressurized parenchymal arterioles were smaller (%Vasodilation: 22.4 ± 3.8 vs. 12.7 ± 3.2%, p&lt;0.05, Student’s t‐test, n = 7‐6). Activity of KCa2.3 / KCa3.1 after activation by NS‐309 (1 µM) was unchanged by aging, as evidenced by similar hyperpolarization (ΔVm: ‐36.1 ± 2.4 vs. ‐32.4 ± 2.2 mV, n = 13‐14) and vasodilation (%Vasodilation: 16.5 ± 2.0 vs. 21.3 ± 3.2%, n = 8‐6). Activation of KIR2 by 15 mM [K+]E induced a smaller hyperpolarization in Aged than Young (ΔVm: ‐11.0 ± 1.1 vs ‐6.9 ± 0.7 mV, p&lt;0.05, Student’s t‐test, n = 13‐13), with a paradoxical larger vasodilation that was unchanged by endothelium removal (%Vasodilation: 10.7 ± 2.3 vs. 25.0 ± 3.6% vs. 27.3 ± 4.5, one‐way ANOVA, Young endothelium intact vs. Aged endothelium intact vs Aged endothelium removed, n = 7‐13‐6), suggesting a compensation by smooth muscle KIR2. Lastly, spontaneous myogenic tone was higher in parenchymal arterioles from Aged mice, and endothelium removal did not further enhance myogenic tone (%myogenic tone: 25.4 ± 1.7 vs. 34.3 ± 2.3 vs 36.4 ± 3.2%, p&lt;0.05, one‐way ANOVA, Young endothelium intact vs. Aged endothelium intact vs Aged endothelium removed, n = 22‐23‐6). In conclusion, aging impairs endothelial KIR2 channels and increases resting myogenic tone, which may underlie vascular dysfunction and exacerbate cognitive decline.

Luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, reverses cerebrovascular dysfunction and cognitive impairments in 18-mo-old diabetic animals.

Diabetes mellitus (DM) is a leading risk factor for age-related dementia, but the mechanisms involved are not well understood. We previously discovered that hyperglycemia induced impaired myogenic response (MR) and cerebral blood flow (CBF) autoregulation in 18-mo-old DM rats associated with blood-brain barrier (BBB) leakage, impaired neurovascular coupling, and cognitive impairment. In the present study, we examined whether reducing plasma glucose with a sodium-glucose cotransporter-2 inhibitor (SGLT2i) luseogliflozin can ameliorate cerebral vascular and cognitive function in diabetic rats. Plasma glucose and HbA1c levels of 18-mo-old DM rats were reduced, and blood pressure was not altered after treatment with luseogliflozin. SGLT2i treatment restored the impaired MR of middle cerebral arteries (MCAs) and parenchymal arterioles and surface and deep cortical CBF autoregulation in DM rats. Luseogliflozin treatment also rescued neurovascular uncoupling, reduced BBB leakage and cognitive deficits in DM rats. However, SGLT2i did not have direct constrictive effects on vascular smooth muscle cells and MCAs isolated from normal rats, although it decreased reactive oxygen species production in cerebral vessels of DM rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.NEW & NOTEWORTHY This study demonstrates that luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, improved CBF autoregulation in association with reduced vascular oxidative stress and AGEs production in the cerebrovasculature of 18-mo-old DM rats. SGLT2i also prevented BBB leakage, impaired functional hyperemia, neurodegeneration, and cognitive impairment seen in DM rats. Luseogliflozin did not have direct constrictive effects on VSMCs and MCAs isolated from normal rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.

Abstract 35: Gamma Adducin Dysfunction Leads To Cerebrovascular Distention, Blood Brain Barrier Leakage, And Cognitive Deficits In The Fawn-hooded Hypertensive Rats

We have previously identified an inactivating mutation of ADD3 in FHH rats which is associated with impaired myogenic reactivity of renal arterioles and podocyte function, and contributes to the development of CKD. We have found that SNPs in human ADD1 or ADD3 in the same region as Add3 in FHH rats are linked to reductions in brain volumes and impaired performance on cognitive tests in 4,286 elderly patients (67-90 years old) in the Atherosclerosis Risk in Communities Neurocognitive Study (ARIC-NCS), but the mechanisms of these pathologies are unclear. The present study examined cerebral hemodynamics and cognitive function in FHH versus FHH.1BN and FHH. Add3 rats that express the WT Add3 gene. The myogenic responses of the middle cerebral artery (MCA) and parenchymal arterioles (PA) were impaired in FHH rats. MCA diameter decreased by 15-20% in FHH.1BN (n = 27) and FHH. Add3 (n = 10) transgenic rats, but increased by 9 ± 3% in FHH rats (n = 15) when perfusion pressure was increased from 40 to 160 mmHg. PA diameter increased by 3.16 ± 2.79% in FHH (n = 5) rats versus a 19 ± 3% and 13 ± 2% decrease FHH. Add3 (n = 4) and FHH.1BN (n = 6), respectively, when pressure was increased from 10 to 40 mmHg. Autoregulation of surface and deep cortical blood flow was impaired in FHH rats and rose by 48 ± 3% (n = 22) and 41 ± 3% (n = 12), respectively, versus 32 ± 3% (n = 7) and 16 ± 5% (n = 6) in FHH. Add3 rats when MAP was increased from 100 to 160 mmHg. By using a fluorescent microscope to examine 60 μm brain sections, it was revealed that the outer diameters of PAs were distended in FHH in comparison to FHH.1BN and FHH. Add3 transgenic rats when systemic pressure was increased to 160 mmHg. Blood brain barrier leakage was also greater in FHH rats than in FHH.1BN and FHH. Add3 rats after acute elevations in pressure. FHH (n=16) rats took 40- 50% longer to navigate an eight-arm water maze than FHH. Add3 (n=11) and FHH.1BN (n=7) rats. These results indicate that variants that alter Add3 function promote cognitive dysfunction in FHH rats by altering cerebral hemodynamics and may play a similar role in cognitive deficits in elderly patients in the ARIC-NCS study. This study suggests that blood pressure should be strictly controlled in hypertensive patients identified with ADD3 variants to prevent dementia.

Activation of the Central Renin-Angiotensin System Causes Local Cerebrovascular Dysfunction.

Hypertension is a leading risk factor for cerebrovascular disease and loss of brain health. While the brain renin-angiotensin system (RAS) contributes to hypertension, its potential impact on the local vasculature is unclear. We tested the hypothesis that activation of the brain RAS would alter the local vasculature using a modified deoxycorticosterone acetate (DOCA) model. C57BL/6 mice treated with DOCA (50 mg SQ; or shams) were given tap H2O and H2O with 0.9% NaCl for 1 to 3 weeks. In isolated cerebral arteries and parenchymal arterioles from DOCA-treated male mice, endothelium- and nitric oxide-dependent dilation was progressively impaired, while mesenteric arteries were unaffected. In contrast, cerebral endothelial function was not significantly affected in female mice treated with DOCA. In males, mRNA expression of renal Ren1 was markedly reduced while RAS components (eg, Agt and Ace) were increased in both brain and cerebral arteries with central RAS activation. In NZ44 reporter mice expressing GFP (green fluorescent protein) driven by the angiotensin II type 1A receptor (Agtr1a) promoter, DOCA increased GFP expression ≈3-fold in cerebral arteries. Impaired endothelial responses were restored to normal by losartan, an AT1R (angiotensin II type 1 receptor) antagonist. Last, DOCA treatment produced inward remodeling of parenchymal arterioles. These findings suggest activation of the central and cerebrovascular RAS impairs endothelial (nitric oxide dependent) signaling in brain through expression and activation of AT1R and sex-dependent effects. The central RAS may be a key contributor to vascular dysfunction in brain in a preclinical (low renin) model of hypertension. Because the brain RAS is also activated during aging and other diseases, a common mechanism may promote loss of endothelial and brain health despite diverse cause.

Sexual Dimorphisms in Hypertension‐Associated Cerebrovascular Damage

Hypertension is a major risk factor for cerebral small vessel disease (CVSD), which increases stroke and dementia risk. Hypertension alters artery structure, impairing blood flow regulation and preventing nutrient delivery to the brain parenchyma. We previously showed angiotensin II (AngII)-hypertension in male mice reduces overall pial blood flow measured by scanning laser Doppler and causes inward hypotrophic remodeling in cerebral parenchymal arterioles (PAs). Epidemiological evidence suggests sex differences exist in CVSD, with large vessel damage occurring more in males and small vessel damage occurring more in females. We hypothesized hypertensive male and female mice would have reductions in pial blood flow measured by laser speckle contrast and inward hypotrophic remodeling in PAs. AngII-filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16-18-week-old male C56BL/6 mice. Age-matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail-cuff plethysmography. Pial artery blood flow was measured in the frontal, parietal, and temporal areas of both brain hemispheres. After euthanasia, PAs were isolated and mounted on a pressure myograph for structure assessment by increasing intraluminal pressure from 3 to 120mmHg in 20mmHg increments. Data are presented as means ± SEM (n=6-9). PA data are reported at an intraluminal pressure of 40mmHg. Systolic blood pressure was elevated in AngII-treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p<0.05) and in 1200ng AngII-treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179* ± 7mmHg; p<0.05) vs sham. There was a sex difference in blood flow alterations during hypertension. Male AngII treated mice had reduced flow in the parietal region (sham: 361 ± 19, AngII: 301 ± 9.3A.U.; p<0.05) but not frontal (sham: 359 ± 19, AngII: 327 ± 11A.U.; p=0.19) or temporal regions (sham: 292 ± 13, AngII: 284 ± 15A.U.; p=0.68) vs sham. AngII treated female mice had reductions in pial flow in the parietal (sham: 403 ± 9, 800ng AngII: 336* ± 19, 1200ng AngII: 314* ± 33 A.U.; p<0.05), frontal (sham: 378 ± 7, 800ng AngII: 314* ± 13, 1200ng AngII: 279* ± 24 A.U.; p<0.05), and temporal (sham: 338 ± 8.4, 800ng AngII: 273* ± 11, 1200ng AngII: 278* ± 25 A.U.; p<0.05) regions vs sham. PAs from male AngII mice had reduced outer diameter (sham: 56 ± 3, AngII: 46 ± 2μm; p<0.05), lumen diameter (sham: 47 ± 1, AngII: 40 ± 2μm; p=0.05), and wall area (sham: 719 ± 115, AngII: 405 ± 24μm2; p<0.05) vs sham, reproducing previous findings. PAs from AngII treated female mice did not exhibit significant reductions in outer diameter (sham: 57 ± 3, 800ng AngII: 50 ± 5, 1200ng AngII: 47 ± 4μm; p=0.17) or lumen diameter (sham: 49 ± 2, 800ng AngII: 43 ± 5, 1200ng AngII: 41 ± 4μm; p=0.22, but showed a modest reduction in wall area (sham: 647 ± 64, 800ng AngII: 534 ± 78, 1200ng AngII: 439 ± 58μm2; p=0.09) vs sham. These data suggest the mechanism of hypertension-induced vascular damage differs between males and females. Understanding sex differences in CVSD is critical for dementia and stroke prevention.

Amyloid‐β(1‐42) Inhibits Ca2+ Transients Induced by N‐Methyl‐D‐Aspartate Receptor in The Cerebral Vasculature

Endothelium‐dependent dilation of cerebral arteries, a process dependent on transient increases in intracellular calcium, contributes to neurovascular coupling (NVC), which is altered in cerebral amyloid angiopathy (CAA).The N‐methyl‐D‐aspartate receptor (NMDAR), a nonselective cation channel with high Ca2+ permeability, has been shown to mediate endothelium‐dependent dilation in cerebral arteries. NMDAR activity is reduced by amyloid‐β, which accumulates around the cerebral vasculature during CAA. We hypothesized that amyloid‐β impairs NMDAR‐induced Ca2+ transients in endothelial cells of cerebral arteries, which impairs endothelium‐mediated dilation in mice. All animal experiments were approved by the University of Arizona IACUC. Data are means ± SEM, both in male and female mice (no sex differences were observed). Cerebral arteries isolated from mice expressing the genetically‐encoded calcium indicator GCaMP8 in endothelial cells (cdh5:Gcamp8) were prepared en face for time‐lapse imaging of endothelial Ca2+ transients induced by NMDAR activation. In fields of view that displayed Ca2+ transients, we found that the NMDAR agonist NMDA (1 μM) increased the frequency of endothelial Ca2+ transients compared to baseline (0.22 ± 0.06 vs 0.58 ± 0.15 Hz, baseline vs NMDA, n = 10 and 13 fields of view from at least 3 mice, p &lt; 0.05, one way ANOVA). Pre‐incubation (5 minutes) of preparations with the NMDAR antagonist D‐AP5 (10 μM) prevented NMDA induction of endothelial cell Ca2+ transients (frequency: 0.14 ± 0.05, n = 10 fields of view, p &lt; 0.05 vs NMDA, one‐way ANOVA). These data suggest that endothelial NMDAR Ca2+ transients can be stimulated in cerebral arteries via NMDA. We then tested whether the peptide amyloid‐β(1‐42), commonly found in CAA, blunted NMDAR‐elicited Ca2+ transients. Cerebral artery preparations were incubated for 30 minutes with 5 μM amyloid‐β(1‐42), then exposed to NMDA. Our preliminary data suggests that pre‐incubation of preparations with amyloid‐β blunts NMDA‐dependent induction of endothelial cell Ca2+ transients (0.21 ± 0.04, n = 10 fields of view, p &lt; 0.05 vs NMDA, one‐way ANOVA). In order to evaluate the effects of amyloid‐β on dilation of cerebral arterioles, we then performed ex vivo pressure myography experiments with cerebral parenchymal arterioles from a mouse model of familial Alzheimer’s disease without aging (5x‐FAD) or wildtype littermates. Our preliminary results suggest that NMDA‐elicited dilation of parenchymal arterioles may be impaired in 5x‐FAD mice (30 μM NMDA, vasodilation (%): 11.06 ± 0.78 vs 6.21 ± 2.09, wildtype vs 5x‐FAD, n = 3 arterioles from 3 mice, p = 0.067, two‐tailed Student’s t‐test). These preliminary data suggest that NMDA receptors in the cerebrovascular endothelium of wildtype mice mediate arteriolar dilation via an increase in Ca2+ transients. Further, amyloid‐β may impair the activity of endothelial NMDA receptors and thus contribute to neurovascular dysfunction via impaired arteriolar dilation in individuals with CAA.Support or Funding InformationNational Heart, Lung and Blood Institute (R00HL140106 to PWP). Supported in part by the Undergraduate Biology Research Program with funds from the University of Arizona Office of the Provost and the College of Medicine.

Differential expression of angiotensin II type 1 receptor subtypes within the cerebral microvasculature.

Arteries and arterioles constrict in response to intraluminal pressure to generate myogenic tone, but the molecular nature of the vascular force-sensing mechanism is not fully characterized. Here, we investigated the role of angiotensin II type 1 receptors (AT1Rs) on vascular smooth muscle cells in the development of myogenic tone in cerebral parenchymal arterioles from mice. We found that pretreatment with the AT1R blocker losartan inhibited the development of myogenic tone in these vessels but did not alter the luminal diameter of arterioles with preestablished tone. Rodents express two AT1R isotypes: AT1Ra and AT1Rb. We previously demonstrated that AT1Rb is expressed at much higher levels compared with AT1Ra in cerebral pial arteries and is required for myogenic contractility in these vessels, whereas AT1Ra is unnecessary for this function. Here, we found that AT1Ra and AT1Rb are expressed at similar levels in parenchymal arterioles and that genetic knockout of AT1Ra blunted the ability of these vessels to generate myogenic tone. We also found that AT1Rb and total AT1R expression levels are much lower in parenchymal arterioles compared with pial arteries and that parenchymal arterioles are less sensitive to the vasoconstrictive effects of the endogenous AT1R ligand angiotensin II (ANG II). We conclude that 1) AT1Rs are critical for the initiation, but not the maintenance, of myogenic tone in parenchymal arterioles, and 2) lower levels of AT1Rb and total AT1R in parenchymal arterioles compared with pial arteries result in differences in myogenic and ANG II-induced vasoconstriction between these vascular segments.NEW & NOTEWORTHY Myogenic tone is critical for appropriate regulation of cerebral blood flow, but the mechanisms used by vascular smooth muscle cells to detect changes in intraluminal pressure are not fully characterized. Here, we demonstrate angiotensin II receptor type 1 (AT1R) is indispensable to initiation, but not maintenance, of myogenic tone in cerebral parenchymal arterioles. Furthermore, we demonstrate differences in AT1R expression levels lead to critical differences in contractile regulation between parenchymal arterioles and cerebral pial arteries.

Prostaglandin E2, a postulated mediator of neurovascular coupling, at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance-sized human cerebral parenchymal arterioles.To achieve this goal PGE2-induced isotonic vasomotor responses were assessed in parenchymal arterioles isolated from fronto-temporo-parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined.In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10−8 to 10-6 mol/l) caused significant, endothelium-independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1-dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA and protein expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles.PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human patients.

Prostaglandin E2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles: implications for neurovascular coupling responses

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance‐sized human cerebral parenchymal arterioles.To achieve this goal PGE2‐induced isotonic responses were assessed in parenchymal arterioles isolated from fronto‐temporo‐parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined.In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10−8 to 10−6 mol/l) caused significant, endothelium‐independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1‐dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles. In contrast, rat vessels expressed predominantly EP1 receptors.PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human subjects.Support or Funding InformationThis work was supported by grants from the National Research, Development and Innovation Office to PT (NKFI‐FK123798) and AK (K108444), the Hungarian Academy of Sciences Bolyai Research Scholarship BO/00634/15 to PT, the PTE AOK‐KA 3/2016 04.01/F to PT, the ÚNKP‐18‐4‐PTE‐6 New National Excellence Program of the Ministry of Human Capacities, Higher Education Institutional Excellence Programme – Grant No. 20765‐3/2018/FEKUTSTRAT, EFOP‐3.6.2.‐16‐2017‐00008, GINOP‐2.3.2‐15‐2016‐00048 and GINOP‐2.3.3‐15‐2016‐00032 to PT and AB, the Hungarian National Brain Research Program Grant No. 2017‐1.2.1‐NKP‐2017‐00002 to AB; the Marie Curie Actions SMARTER 7th Framework Program of the European Union 606998 to NSz and AK, National Institute of Health R01‐AG055395, R01‐NS100782, R01‐AT006526 to ZU.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mineralocorticoid receptor antagonism improves parenchymal arteriole dilation via a TRPV4-dependent mechanism and prevents cognitive dysfunction in hypertension.

Hypertension and mineralocorticoid receptor activation cause cerebral parenchymal arteriole remodeling; this can limit cerebral perfusion and contribute to cognitive dysfunction. We used a mouse model of angiotensin II-induced hypertension to test the hypothesis that mineralocorticoid receptor activation impairs both transient receptor potential vanilloid (TRPV)4-mediated dilation of cerebral parenchymal arterioles and cognitive function. Mice (16-18 wk old, male, C57Bl/6) were treated with angiotensin II (800 ng·kg-1·min-1) with or without the mineralocorticoid receptor antagonist eplerenone (100 mg·kg-1·day-1) for 4 wk; sham mice served as controls. Data are presented as means ± SE; n = 5-14 mice/group. Eplerenone prevented the increased parenchymal arteriole myogenic tone and impaired carbachol-induced (10-9-10-5 mol/l) dilation observed during hypertension. The carbachol-induced dilation was endothelium-derived hyperpolarization mediated because it could not be blocked by N-nitro-l-arginine methyl ester (10-5 mol/l) and indomethacin (10-4 mol/l). We used GSK2193874 (10-7 mol/l) to confirm that in all groups this dilation was dependent on TRPV4 activation. Dilation in response to the TRPV4 agonist GSK1016790A (10-9-10-5 mol/l) was also reduced in hypertensive mice, and this defect was corrected by eplerenone. In hypertensive and eplerenone-treated animals, TRPV4 inhibition reduced myogenic tone, an effect that was not observed in arterioles from control animals. Eplerenone treatment also improved cognitive function and reduced microglia density in hypertensive mice. These data suggest that the mineralocorticoid receptor is a potential therapeutic target to improve cerebrovascular function and cognition during hypertension. NEW & NOTEWORTHY Vascular dementia is a growing public health issue that lacks effective treatments. Transient receptor potential vanilloid (TRPV)4 channels are important regulators of parenchymal arteriole dilation, and they modulate myogenic tone. The data presented here suggest that TRPV4 channel expression is regulated by the mineralocorticoid receptor (MR). MR blockade also improves cognitive function during hypertension. MR blockade might be a potential therapeutic approach to improve cerebrovascular function and cognition in patients with hypertension.

Changes in Cerebral Arteries and Parenchymal Arterioles With Aging: Role of Rho Kinase 2 and Impact of Genetic Background.

Vascular aging fundamentally contributes to large and small vessel disease. Despite the importance of such changes for brain function, mechanisms that mediate such changes are poorly defined. We explored mechanisms that underlie changes with age, testing the hypothesis that ROCK (Rho kinase) plays an important role. In C57BL/6 mice, baseline diameters of isolated pressurized parenchymal arterioles were similar in adult (4-5 month) and old mice (22±1 month; ≈15±1 µm). Endothelium-dependent dilation was impaired in old mice compared with adults in a pathway-specific manner. Vasodilation to NS-309 (which activates small- and intermediate-conductance Ca2+ activated K+ channels in endothelial cells) was intact while endothelial nitric oxide synthase-mediated vasodilation was reduced by ≥60%, depending on the concentration (P<0.05). A similar reduction was present in basilar arteries. Inhibiting both ROCK isoforms with Y-27632 restored the majority of endothelial function in old mice. Because genetic background is a determinant of vascular disease, we performed similar studies using FVB/N mice. Endothelial dysfunction was seen with aging in both FVB/N and C57BL/6 mice although the magnitude was increased ≈2-fold in the latter strain (P<0.05). In both strains of mice, age-induced endothelial dysfunction was reversed by inhibition of ROCK2 with SLX-2119. Thus, aging impairs endothelial function in both cerebral arteries and parenchymal arterioles, predominantly via effects on endothelial nitric oxide synthase-dependent regulation of vascular tone. The magnitude of these changes was influenced by genetic background and mediated by ROCK2.

Chronic Cerebral Hypoperfusion Impairs Transient Receptor Potential Channel Vanilloid 4 (TRPV4)‐Mediated Dilation in Cerebral Parenchymal Arterioles From Normotensive, but not Hypertensive Rats

Hypertension is a risk factor for cerebral small vessel disease and vascular cognitive impairment. Hypertension also increases the risk of other vascular injuries, such as carotid artery stenosis, that can place additional stress on the cerebral vasculature. To understand how hypertension affects the vascular response to reduced cerebral perfusion we studied normotensive Wistar Kyoto (WKY) rats and stroke prone spontaneously hypertensive rats (SHSRP). Rats underwent a bilateral carotid artery stenosis (BCAS) surgery or a sham surgery at 20 weeks of age, and they were studied 8 weeks later. In both cases the BCAS surgery impaired short‐term memory assessed by calculating the novel object exploration quotient (WKY: 0.6±0.04 vs 0.4±0.4, sham vs BCAS; n=11–13, p&lt;0.05; SHRSP: 0.6±0.05 vs 0.5±0.04 sham vs BCAS; n=16, p&lt;0.05). The effects of BCAS on the parenchymal arteriole (PA) function were assessed by pressure myography. BCAS had no effect of myogenic tone generation in either rat strain. In both WKY rats and SHRSP, BCAS caused a marked impairment in carbachol mediated endothelium‐dependent dilation (% dilation, WKY: 29.6±8.1 vs −10.2±7.4, sham vs BCAS; n=5–7, p&lt;0.05; SHRSP: 17.5±5.3 vs −0.22±2.9, sham vs BCAS; n=5–6 p&lt;0.05). Recent studies from our lab suggest that transient receptor potential channel vanilloid 4 (TRPV4) mediates endothelium‐dependent dilation in PAs. To assess the effects of BCAS on TRPV4‐dependent dilation, we used GSK1016790A, a potent and selective TRPV4 agonist. We found that TRPV4‐mediated dilation in the WKY rats was significantly impaired post‐BCAS (% dilation, 93.5±3.3 vs 57.7±13.3 Sham vs BCAS; n=3–4, p&lt;0.05); this effect was not observed in the SHRSP (% dilation, 57.2±1.6 vs 51.8±6.9 sham vs BCAS; n=5–6, p&lt;0.05). Interestingly TRPV4‐mediated dilation was impaired in the sham hypertensive rats compared to the sham WKY rats suggesting that hypertension impacts TRPV4 function. Together these data indicate that impaired TRPV4‐mediated dilation may contribute to the cognitive decline observed with BCAS in normotensive rats but not in rats with sustained hypertension.Support or Funding InformationSupported by 5P01HL070687‐12.

Does serelaxin treatment alter passive mechanical wall properties in small resistance arteries?

The peptide hormone relaxin is recognized for its connective tissue remodeling actions in the reproductive tract during pregnancy and parturition, but it also has vascular remodeling actions independent of pregnancy. Recombinant human relaxin (serelaxin) treatment in male and non-pregnant female rodents enhances passive arterial compliance in the renal vasculature. This review focuses on serelaxin's actions on passive mechanical wall properties in small arteries and highlights the diversity of responses to serelaxin treatment in rodents. Different experimental approaches (duration of serelaxin treatment, rat strain, age) and animal models of disease (obesity, hypertension) will be considered. Most studies in young rodents demonstrate that serelaxin treatment fails to alter passive compliance in resistance-size arteries (mesenteric and femoral arteries and cerebral parenchymal arterioles), suggesting that serelaxin's beneficial effects are minimal in healthy animals. Short-term serelaxin treatment (5d) in aged, obese, and spontaneously hypertensive rats (SHRs) is largely without effect on passive mechanical wall properties. However, a longer duration of serelaxin treatment in SHRs (14d) enhances passive compliance in large muscular arteries as well as resistance-size arteries. In conclusion, serelaxin is capable of vascular remodeling. Its actions are vascular bed-dependent, more prominent in disease, and likely requires a longer duration of treatment to be effective.

Rho kinase activity governs arteriolar myogenic depolarization

Cerebral arterioles contribute critically to regulation of local and global blood flow within the brain. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of fundamental control mechanisms at this level of circulation. Emerging evidence points to a key role of Rho-associated protein kinase in regulation of microvascular contractility. This study sought to decipher the mechanisms of Rho-associated protein kinase–mediated myogenic vasoconstriction in cerebral parenchymal arterioles. Here, we report that the Rho-associated protein kinase inhibitor H1152 strongly attenuated pressure-induced constriction, cytosolic [Ca2+] increases, and depolarization of isolated parenchymal arterioles. Further, the RhoA activator CN03 potentiated parenchymal arteriole myogenic constriction and depolarization, indicating important involvement of RhoA/Rho-associated protein kinase signaling in myogenic excitation-contraction mechanisms. Because of the well-established role of TRPM4 in pressure-induced depolarization, possible modulatory effects of Rho-associated protein kinase on TRPM4 currents were explored using patch clamp electrophysiology. TRPM4 currents were suppressed by H1152 and enhanced by CN03. Finally, H1152 elevated the apparent [Ca2+]-threshold for TRPM4 activation, suggesting that Rho-associated protein kinase activates TRPM4 by increasing its Ca2+-sensitivity. Our results support a novel mechanism whereby Rho-associated protein kinase–mediated myogenic vasoconstriction occurs primarily through activation of TRPM4 channels, smooth muscle depolarization, and cytosolic [Ca2+] increases in cerebral arterioles.