Endothelial KIR2 channel dysfunction in aged cerebral parenchymal arterioles.

Aging is associated with cognitive decline via incompletely understood mechanisms. Cerebral microvascular dysfunction occurs in aging, particularly impaired endothelium-mediated dilation. Parenchymal arterioles are bottlenecks of the cerebral microcirculation, and dysfunction causes a mismatch in nutrient demand and delivery, leaving neurons at risk. Extracellular nucleotides elicit parenchymal arteriole dilation by activating endothelial purinergic receptors (P2Y), leading to opening of K+ channels, including inwardly-rectifying K+ channels (KIR2). These channels amplify hyperpolarizing signals, resulting in dilation. However, it remains unknown if endothelial P2Y and KIR2 signaling are altered in brain parenchymal arterioles during aging. We hypothesized that aging impairs endothelial P2Y and KIR2 function in parenchymal arterioles. We observed reduced dilation to the purinergic agonist 2-methyl-S-ADP (1 µM) in arterioles from Aged (>24-month-old) mice when compared to Young (4-6 months of age) despite similar hyperpolarization in endothelial cells tubes. No differences were observed in vasodilation or endothelial cell hyperpolarization to activation of small- and intermediate-conductance Ca2+-activated K+ channels (KCa2.3 / KCa3.1) by NS309. Hyperpolarization to 15 mM [K+]E was smaller in Aged than Young mice, despite a paradoxical increased dilation in Aged arterioles to 15 mM [K+]E that was unchanged by endothelium removal. KIR2 Inhibition attenuated vasodilatory responses to 15 mM [K+]E and 1 µM 2-me-S-ADP in both Young and Aged arterioles. Further, we observed a significant increase in myogenic tone in Aged parenchymal arterioles, which was not enhanced by endothelium removal. We conclude that aging impairs endothelial KIR2 channel function in the cerebral microcirculation with possible compensation by smooth muscle cells.

Vascular Dysfunction in Polycystic Kidney Disease: A Mini-Review.

Polycystic kidney disease (PKD) is one of the most common hereditary kidney diseases, which is characterized by progressive cyst growth and secondary hypertension. In addition to cystogenesis and renal abnormalities, patients with PKD can develop vascular abnormalities and cardiovascular complications. Progressive cyst growth substantially alters renal structure and culminates into end-stage renal disease. There remains no cure beyond renal transplantation, and treatment options remain largely limited to chronic renal replacement therapy. In addition to end-stage renal disease, patients with PKD also present with hypertension and cardiovascular disease, yet the timing and interactions between the cardiovascular and renal effects of PKD progression are understudied. Here, we review the vascular dysfunction found in clinical and preclinical models of PKD, including the clinical manifestations and relationship to hypertension, stroke, and related cardiovascular diseases. Finally, our discussion also highlights the critical questions and emerging areas in vascular research in PKD.

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.

Nanoscale remodeling of ryanodine receptor cluster size underlies cerebral microvascular dysfunction in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) results from mutations in the gene encoding dystrophin which lead to impaired function of skeletal and cardiac muscle, but little is known about the effects of the disease on vascular smooth muscle cells (SMCs). Here we used the mdx mouse model to study the effects of mutant dystrophin on the regulation of cerebral artery and arteriole SMC contractility, focusing on an important Ca2+-signaling pathway composed of type 2 ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) and large-conductance Ca2+-activated K+ (BK) channels on the plasma membrane. Nanoscale superresolution image analysis revealed that RyR2 and BKα were organized into discrete clusters, and that the mean size of RyR2 clusters that colocalized with BKα was larger in SMCs from mdx mice (∼62 RyR2 monomers) than in controls (∼40 RyR2 monomers). We further found that the frequency and signal mass of spontaneous, transient Ca2+-release events through SR RyR2s ("Ca2+ sparks") were greater in SMCs from mdx mice. Patch-clamp electrophysiological recordings indicated a corresponding increase in Ca2+-dependent BK channel activity. Using pressure myography, we found that cerebral pial arteries and parenchymal arterioles from mdx mice failed to develop appreciable spontaneous myogenic tone. Inhibition of RyRs with tetracaine and blocking of BK channels with paxilline restored myogenic tone to control levels, demonstrating that enhanced RyR and BK channel activity is responsible for the diminished pressure-induced constriction of arteries and arterioles from mdx mice. We conclude that increased size of RyR2 protein clusters in SMCs from mdx mice increases Ca2+ spark and BK channel activity, resulting in cerebral microvascular dysfunction.

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

OBJECTIVE: 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. METHODS: Three-week-old male Sprague-Dawley rats were fed a HF diet ± the MR antagonist canrenoic acid (Canr) for 17 weeks. Control rats received normal chow (control NC). MCA structure was assessed by pressure myography. RESULTS: 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. CONCLUSIONS: These data suggest that HF feeding leads to MR dependent remodeling of the MCA and this is associated with markers of dementia development.

The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

KEY POINTS: The angiotensin II receptor type 1b (AT1 Rb ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT1 Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT1 Ra or AT1 Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT1 Rb mRNA is ∼30-fold higher than AT1 Ra in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT1 Rb for pressure-induced vasoconstriction. ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT1 R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1 R - AT1 Ra and AT1 Rb - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1 Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1 Ra knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT1 R blocker losartan (1 µm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT1 R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1 Rb mRNA was ∼30-fold greater than AT1 Ra in cerebral arteries, and knockdown of AT1 Rb selectively diminished myogenic constriction. We conclude that AT1 Rb , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

DOCA-salt hypertension impairs artery function in rat middle cerebral artery and parenchymal arterioles.

OBJECTIVE: Chronic hypertension induces detrimental changes in the structure and function of surface cerebral arteries. Very little is known about PAs, which perfuse distinct neuronal populations in the cortex and may play a role in cerebrovascular disorders. We investigated the effect of DOCA-salt induced hypertension on endothelial function and artery structure in PAs and MCAs. METHODS: Uninephrectomized male Sprague-Dawley rats were implanted with a subcutaneous pellet containing DOCA (150 mg/kg b.w.) and drank salt water (1% NaCl and 0.2% KCl) for 4 weeks. Sham rats were uninephrectomized and drank tap water. Vasoreactivity and passive structure in the MCAs and the PAs were assessed by pressure myography. RESULTS: Both MCAs and PAs from DOCA-salt rats exhibited impaired endothelium-dependent dilation (P<.05). In the PAs, addition of NO and COX inhibitors enhanced dilation in DOCA-salt rats (P<.05), suggesting that dysfunctional NO and COX-dependent signaling could contribute to impaired endothelium-mediated dilation. MCAs from DOCA-salt rats exhibited inward remodeling (P<.05). CONCLUSIONS: Hypertension-induced MCA remodeling coupled with impaired endothelium-dependent dilation in both the MCAs and PAs may exacerbate the risk of cerebrovascular accidents and the associated morbidity and mortality.

Regulation of myogenic tone and structure of parenchymal arterioles by hypertension and the mineralocorticoid receptor.

Proper perfusion is vital for maintenance of neuronal homeostasis and brain function. Changes in the function and structure of cerebral parenchymal arterioles (PAs) could impair blood flow regulation and increase the risk of cerebrovascular diseases, including dementia and stroke. Hypertension alters the structure and function of large cerebral arteries, but its effects on PAs remain unknown. We hypothesized that hypertension increases myogenic tone and induces inward remodeling in PAs; we further proposed that antihypertensive therapy or mineralocorticoid receptor (MR) blockade would reverse the effects of hypertension. PAs from 18-wk-old stroke-prone spontaneously hypertensive rats (SHRSP) were isolated and cannulated in a pressure myograph. At 50-mmHg intraluminal pressure, PAs from SHRSP showed higher myogenic tone (%tone: 39.1 ± 1.9 vs. 28.7 ± 2.5%, P < 0.01) and smaller resting luminal diameter (34.7 ± 1.9 vs. 46.2 ± 2.4 µm, P < 0.01) than those from normotensive Wistar-Kyoto rats, through a mechanism that seems to require Ca(2+) influx through L-type voltage-gated Ca(2+) channels. PAs from SHRSP showed inward remodeling (luminal diameter at 60 mmHg: 55.2 ± 1.4 vs. 75.7 ± 5.1 µm, P < 0.01) and a paradoxical increase in distensibility and compliance. Treatment of SHRSP for 6 wk with antihypertensive therapy reduced PAs' myogenic tone, increased their resting luminal diameter, and prevented inward remodeling. In contrast, treatment of SHRSP for 6 wk with an MR antagonist did not reduce blood pressure or myogenic tone, but prevented inward remodeling. Thus, while hypertensive remodeling of PAs may involve the MR, myogenic tone seems to be independent of MR activity.

Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles.

Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.

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.

The effects of hypertension on the cerebral circulation.

Maintenance of brain function depends on a constant blood supply. Deficits in cerebral blood flow are linked to cognitive decline, and they have detrimental effects on the outcome of ischemia. Hypertension causes alterations in cerebral artery structure and function that can impair blood flow, particularly during an ischemic insult or during periods of low arterial pressure. This review will focus on the historical discoveries, novel developments, and knowledge gaps in 1) hypertensive cerebral artery remodeling, 2) vascular function with emphasis on myogenic reactivity and endothelium-dependent dilation, and 3) blood-brain barrier function. Hypertensive artery remodeling results in reduction in the lumen diameter and an increase in the wall-to-lumen ratio in most cerebral arteries; this is linked to reduced blood flow postischemia and increased ischemic damage. Many factors that are increased in hypertension stimulate remodeling; these include the renin-angiotensin-aldosterone system and reactive oxygen species levels. Endothelial function, vital for endothelium-mediated dilation and regulation of myogenic reactivity, is impaired in hypertension. This is a consequence of alterations in vasodilator mechanisms involving nitric oxide, epoxyeicosatrienoic acids, and ion channels, including calcium-activated potassium channels and transient receptor potential vanilloid channel 4. Hypertension causes blood-brain barrier breakdown by mechanisms involving inflammation, oxidative stress, and vasoactive circulating molecules. This exposes neurons to cytotoxic molecules, leading to neuronal loss, cognitive decline, and impaired recovery from ischemia. As the population ages and the incidence of hypertension, stroke, and dementia increases, it is imperative that we gain a better understanding of the control of cerebral artery function in health and disease.

Improvement in middle cerebral artery structure and endothelial function in stroke-prone spontaneously hypertensive rats after macrophage depletion.

BACKGROUND: Inflammation is involved in the pathogenesis of hypertension. Hypertensive animals have an increased number of perivascular macrophages in cerebral arteries. Macrophages might be involved in remodeling of the cerebral vasculature. We hypothesized that peripheral macrophage depletion would improve MCA structure and function in hypertensive rats. METHODS: For macrophage depletion, six-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were treated with CLOD, 10 mL/kg every three or four days, i.p., or vehicle (PBS lipo). MCA structure and function were analyzed by pressure and wire myography. RESULTS: Blood pressure was not affected by CLOD. The number of perivascular CD163-positive cells per microscopic field was reduced in the brain of SHRSP+CLOD. CLOD treatment caused an improvement in endothelium-dependent dilation after intralumenal perfusion of ADP and incubation with Ach. Inhibition of NO production blunted the Ach response, and endothelium-independent dilation was not altered. At an intralumenal pressure of 80 mmHg, MCA from SHRSP+CLOD showed increased lumen diameter, decreased wall thickness, and wall-to-lumen ratio. Cross-sectional area of pial arterioles from SHRSP+CLOD was higher than PBS lipo. CONCLUSIONS: These results suggest that macrophage depletion attenuates MCA remodeling and improves MCA endothelial function in SHRSP.

Ultrasound, photoacoustic, and magnetic resonance imaging to study hyperacute pathophysiology of traumatic and vascular brain injury.

BACKGROUND AND PURPOSE: Cerebrovascular dynamics and pathomechanisms that evolve in the minutes and hours following traumatic vascular injury in the brain remain largely unknown. We investigated the pathophysiology evolution in mice within the first 3 hours after closed-head traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH), two significant traumatic vascular injuries. METHODS: We took a multimodal imaging approach using photoacoustic imaging, color Doppler ultrasound, and MRI to track injury outcomes using a variety of metrics. RESULTS: Brain oxygenation and velocity-weighted volume of blood flow (VVF) values significantly decreased from baseline to 15 minutes after both TBI and SAH. TBI resulted in 19.2% and 41.0% ipsilateral oxygenation and VVF reductions 15 minutes postinjury, while SAH resulted in 43.9% and 85.0% ipsilateral oxygenation and VVF reduction (p < .001). We found partial recovery of oxygenation from 15 minutes to 3 hours after injury for TBI but not SAH. Hemorrhage, edema, reduced perfusion, and altered diffusivity were evident from MRI scans acquired 90-150 minutes after injury in both injury models, although the spatial distribution was mostly focal for TBI and diffuse for SAH. CONCLUSIONS: The results reveal that the cerebral oxygenation deficits immediately following injuries are reversible for TBI and irreversible for SAH. Our findings can inform future studies on mitigating these early responses to improve long-term recovery.

BK Ca nitrosylation is associated with cerebral microvascular dysfunction in female 5x-FAD mice.

Cerebral microvascular dysfunction and nitro-oxidative stress are present in patients with Alzheimer's disease (AD) and may contribute to disease progression and severity. Large conductance Ca 2+ -activated K + channels (BK Ca ) play an essential role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. BK Ca can be modified in a pro-nitro-oxidative environment, resulting in decreased activity and vascular hyper-contractility, which can compromise cerebral blood flow regulation. We hypothesized that reductions in BK Ca function in cerebral arteries, as a consequence of nitro-oxidative stress, are associated with blunted neurovascular responses in the 5x-FAD model of AD. Using pressure myography, we observed that posterior communicating arteries (PComA) from 5 months-old female 5x-FAD mice showed higher spontaneous myogenic tone than wild-type (WT) littermates. Constriction to the BK Ca blocker iberiotoxin (30 nM) was smaller in 5x-FAD than WT, suggesting lower basal BK Ca activity, which was independent of alterations in intracellular Ca 2+ transients or BK Ca mRNA expression. These vascular changes were associated with higher levels of oxidative stress in female 5x-FAD and a higher level of S-nitrosylation in the BK Ca α-subunit. In females, pre-incubation of PComA from 5x-FAD with the reducing agent DTT (10 µM) rescued iberiotoxin-induced contraction. Female 5x-FAD mice showed increased expression of iNOS mRNA, lower resting cortical perfusion atop the frontal cortex, and impaired neurovascular coupling responses. No significant differences between male 5x-FAD and WT were observed for all parameters above. These data suggest that the exacerbation in BK Ca S-nitrosylation contributes to cerebrovascular and neurovascular impairments in female 5x-FAD mice. Significance Statement: Cerebral vascular dysfunction is increasingly recognized as a hallmark of Alzheimer's disease and other dementias. Impaired microvascular regulation can lead to deficits in blood flow to the brain. An intrinsic property of the resistance vasculature is to constrict when pressurized (myogenic tone), generating a vasodilatory reserve. Detrimental over-constriction is prevented by vascular feedback mechanisms, including the opening of large-conductance Ca 2+ -activated K + channels (BK Ca ). Here, using a combination of molecular biology tools with ex vivo and in vivo vascular assessments, we show a novel mechanism associated with BK Ca dysfunction in the cerebral microvasculature of female 5x-FAD mice. We report increased BK Ca S-nitrosylation linked to reduced activity and, consequently, higher basal myogenic tone. These changes were associated with lower perfusion of the frontal cortex and impaired neurovascular reactivity, suggesting that nitro-oxidative stress is an important mechanism of vascular dysfunction in Alzheimer's disease.

PNA5, A Novel Mas Receptor Agonist, Improves Neurovascular and Blood-Brain-Barrier Function in a Mouse Model of Vascular Cognitive Impairment and Dementia.

It is well established that decreased brain blood flow, increased reactive oxygen species production (ROS), and pro-inflammatory mechanisms accelerate neurodegenerative disease progressions, including vascular cognitive impairment and dementia (VCID). Previous studies in our laboratory have shown that our novel glycosylated Angiotensin-(1-7) Mas receptor agonist PNA5 reverses cognitive deficits, decreases ROS production, and inhibits inflammatory cytokine production in our preclinical mouse model of VCID that is induced by chronic heart failure (VCID-HF). In the present study, the effects of VCID-HF and treatment with PNA5 on microglia activation, blood-brain-barrier (BBB) integrity, and neurovascular coupling were assessed in our mouse model of VCID-HF. Three-month-old male C57BL/6J mice were subjected to myocardial infarction (MI) to induce heart failure for four weeks and then treated with subcutaneous injections of extended-release PNA5. Microglia activation, BBB permeability, cerebral perfusion, and neurovascular coupling were assessed. Results show that in our VCID-HF model, there was an increase in microglial activation and recruitment within the CA1 and CA3 regions of the hippocampus, a disruption in BBB integrity, and a decrease in neurovascular coupling. Treatment with PNA5 reversed these neuropathological effects of VCID-HF, suggesting that PNA5 may be an effective disease-modifying therapy to treat and prevent VCID. This study identifies potential mechanisms by which heart failure may induce VCID and highlights the possible mechanisms by which treatment with our novel glycosylated Angiotensin-(1-7) Mas receptor agonist, PNA5, may protect cognitive function in our model of VCID.

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function-integral to the physiology and etiology of chronic degenerative diseases-requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia. In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial "tubes" (diameter, ~25 µm) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 °C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Amyloid-ß disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries.

Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid-ß(1-40) accumulates around blood vessels. In neurons, amyloid-ß impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid-ß(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR (NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid-ß(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid-ß(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid-ß(1-40), as well as in a mouse model of Alzheimer's disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid-ß(1-40) and impaired in 5x-FAD mice.

Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats.

Matrix metalloproteases (MMPs) are a family of zinc peptidases involved in extracellular matrix turnover. There is evidence that increased MMP activity is involved in remodeling of resistance vessels in chronic hypertension. Thus we hypothesized that inhibition of MMP activity with doxycycline (DOX) would attenuate vascular remodeling. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with DOX (50 mg·kg(-1)·day(-1) in the drinking water) for 6 wk. Untreated SHRSP were controls. Blood pressure was measured by telemetry during the last week. Middle cerebral artery (MCA) and mesenteric resistance artery (MRA) passive structures were assessed by pressure myography. MMP-2 expression in aortas was measured by Western blot. All results are means ± SE. DOX caused a small increase in mean arterial pressure (SHRSP, 154 ± 1; SHRSP + DOX, 159 ± 3 mmHg; P < 0.001). Active MMP-2 expression was reduced in aorta from SHRSP + DOX (0.21 ± 0.06 vs. 0.49 ± 0.13 arbitrary units; P < 0.05). In the MCA, at 80 mmHg, DOX treatment increased the lumen (273.2 ± 4.7 vs. 238.3 ± 6.3 µm; P < 0.05) and the outer diameter (321 ± 5.3 vs. 290 ± 7.6 µm; P < 0.05) and reduced the wall-to-lumen ratio (0.09 ± 0.002 vs. 0.11 ± 0.003; P < 0.05). Damage after transient cerebral ischemia (transient MCA occlusion) was reduced in SHRSP + DOX (20.7 ± 4 vs. 45.5 ± 5% of hemisphere infarcted; P < 0.05). In the MRA, at 90 mmHg DOX, reduced wall thickness (29 ± 1 vs. 22 ± 1 µm; P < 0.001) and wall-to-lumen ratio (0.08 ± 0.004 vs. 0.11 ± 0.008; P < 0.05) without changing lumen diameter. These results suggest that MMPs are involved in hypertensive vascular remodeling in both the peripheral and cerebral vasculature and that DOX reduced brain damage after cerebral ischemia.