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Background CD146 is a tight junction-associated molecule involved in maintaining endothelial barrier, and balancing immune–inflammation response, in cardiovascular disease. Notably, peripheral CD146+ cells significantly upsurge under vessel dyshomeostasis such as acute myocardial injury (AMI), appearing to be a promising therapeutic target. In this study, with a new view of gene correlation, we aim at deciphering the complex underlying mechanism of CD146+ cells’ impact in the development of AMI. Methods Transcription dataset GSE 66,360 of CD146+ blood cells from clinical subjects was downloaded from NCBI. Pearson networks were constructed and the clustering coefficients were calculated to disclose the differential connectivity genes (DCGs). Analysis of gene connectivity and gene expression were performed to reveal the hub genes and hub gene clusters followed by gene enrichment analysis. Results and conclusions Among the total 23,520 genes, 27 genes out of 126 differential expression genes were identified as DCGs. These DCGs were found in the periphery of the networks under normal condition, but transferred to the functional center after AMI. Moreover, it was revealed that DCGs spontaneously crowded together into two functional models, CCL20 cluster and NR4A3 cluster, influencing the CD146-mediated signaling pathways during the pathology of AMI for the first time.

Secondary injury following cortical stroke includes delayed gliosis and eventual neuronal loss in the thalamus. However, the effects of aging and the potential to ameliorate this gliosis with NMDA receptor (NMDAR) antagonism are not established. We used the permanent distal middle cerebral artery stroke model (pdMCAO) to examine secondary thalamic injury in young and aged mice. At 3 days post-stroke (PSD3), slight microgliosis (IBA-1) and astrogliosis (GFAP) was evident in thalamus, but no infarct. Gliosis increased dramatically through PSD14, at which point degenerating neurons were detected. Flow cytometry demonstrated a significant increase in CD11b+/CD45int microglia (MG) in the ipsilateral thalamus at PSD14. CCR2-RFP reporter mouse further demonstrated that influx of peripheral monocytes contributed to the MG/Mϕ population. Aged mice demonstrated reduced microgliosis and astrogliosis compared with young mice. Interestingly, astrogliosis demonstrated glial scar-like characteristics at two years post-stroke, but not by 6 weeks. Lastly, treatment with memantine (NMDAR antagonist) at 4 and 24 h after stroke significantly reduced gliosis at PSD14. These findings expand our understanding of gliosis in the thalamus following cortical stroke and demonstrate age-dependency of this secondary injury. Additionally, these findings indicate that delayed treatment with memantine (an FDA approved drug) provides significant reduction in thalamic gliosis.

Vascular contributions to cognitive impairment and dementia (VCID) includes multiple disorders that are identified by cognitive deficits secondary to cerebrovascular pathology. The risk of VCID is higher in people after the age of 70, and, currently, there is no effective treatment. Vascular endothelial cells (VEC) are critical components of the brain vasculature and neurovascular unit and their health is vital to the capacity of the brain vasculature to respond to stressors. However, aged VEC may enter an irreversible replicative-arrest state (senescence), which has been associated with dementia. E2F transcription factor 1 (E2F1) regulates cell cycle progression and DNA damage repair. Importantly, E2F1 deficiency is associated with cell senescence. We hypothesized that E2F1 downregulation contributes to senescence in the cerebral endothelium during aging. We used cultured primary VEC from young (4-months old, mo) and aged (18-mo) male and female mice for RNA sequencing, plasmid-based gene delivery, high-resolution microscopy, and (4-, 12-, and 18-mo) mice of the bilateral carotid artery stenosis (BCAS) model, which produces chronic cerebral hypoperfusion and recapitulates some of the features seen in patients with VCID. We found that overexpression of E2F1 reduced the levels of senescence-associated phenotypes in cultured VEC from young mice that were exposed to oxygen and glucose deprivation (pE2f1 was reduced (~40%) in cultured primary VEC from aged mouse brains compared with young cells (pIn conclusion , our study identifies E2F1 as a potential regulator of endothelial senescence in mice and highlights the contribution of aging as an important factor in losing endothelial resilience.

Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. However, how the transcriptional signature of these cells is altered in aging and stroke remains poorly defined. Here, we compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq). Our unbiased approach identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG with aging or ischemic stroke. Further analyses suggested that this gene may be a novel regulator of MG inflammation in aging and stroke. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke and sham controls. We used qRT-PCR to validate our scRNAseq findings and compare regional transcriptional changes. Human microglial cells (HMC3; subjected to OGD) and human autopsy brain sections (stroke versus non-stroke) were used to examine IFI27l2 protein expression. Results: Using comprehensive scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes. In contrast, a negative correlation was shown with genes involved in promoting the phagocytic MG phenotype (e.g. Spp1, Cst7, Lpl, and Itgax). Our qRT-PCR results validated the central scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in thalamus of aged brains (p = 0.0412) and trending in cortex (p = 0.23) (n=4 young/aged). Following stroke, Ifi27l2a and other markers of MG activation were significantly increased in both brain regions (n= 4-6). In human tissues, we observed an increase in IFI27l2 protein in HMC3 cells with OGD and in brain of stroke patients. Conclusions: We identified Ifi272la as a highly up-regulated gene in MG in aging and following stroke. Our findings suggest that Ifi27l2a may be a novel regulator of neuroinflammation in MG, and thus provide the basis for a new therapeutic target to reduce inflammation in aging and stroke.

Introduction: The leptomeningeal collaterals (LMCs) are arterial anastomoses that interconnect the distal branches of the major cerebral arteries. They distribute blood flow from adequately to poorly perfused vascular territories when needed. In normal conditions, the LMCs experience little net flow. However, during ischemic stroke, the LMCs are recruited to provide rescue flow to the ischemic territory. During prolonged or permanent stroke, the LMCs can rapidly remodel to provide increased perfusion capacity. Von Willebrand factor (VWF) is a secreted protein expressed in cerebral endothelium and has recently been implicated in angiogenesis. We sought to determine if VWF expression was increased in remodeling LMCs in the post-stroke brain. Methods: C57BL6 mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). At 3 days after pdMCAO, mice were perfused with fluorescent tomato lectin to label vascular endothelium. The cortical surface was “planed off” by vibratome to provide largely contiguous cortical surface vessel preparations containing distalmost arterioles (DMAs) and the connected LMCs. VWF immunofluorescence intensity was measured in LMCs and the connecting DMAs of hemispheres ipsilateral and contralateral to the stroke. Inner diameter of the LMCs and DMAs were measured with Image J. Results: At 3 days post-stroke, LMCs of the stroke hemisphere demonstrated significant outward remodeling. Mean inner diameter of the ipsilateral versus contralateral LMCs were 44.5 ± 5.9 um versus 28.2 ± 1.2 um, respectively (p Conclusions: We demonstrate that VWF is highly expressed in the remodeling LMCs (but not connecting DMAs) of the post-stroke brain. These findings suggest a novel and selective role for endothelial VWF in the LMC response to prolonged recruitment during ischemic stroke.

Secondary injury in the thalamus has been observed following cortical stroke in rodents and humans and is associated with worsened recovery. Interruption of this progressive injury reflects an important therapeutic goal. However, the mechanisms whereby primary cortical infarction leads to remote injury in distant regions of brain are not well defined. We used a mouse model of cortical stroke (which demonstrates delayed thalamic injury) to define the time course of thalamic gliosis and neuronal injury and then test the potential of delayed memantine treatment (an NMDA receptor antagonist) to attenuate this secondary injury. Methods: Cortical infarction was induced by permanent occlusion of the distal middle cerebral artery (pdMCAO) in male C57BL/6J mice (young and aged) and CCR2-RFP mice. Brain infarct, cell-specific injury, and gliosis were measured by cresyl violet, Fluoro-jade C (FJC), TTC, FACS, and immunofluorescence. In young mice, memantine was injected at 4 and 24 hours post-stroke (100 and 50 mg/kg, ip). Brains were evaluated at post-stroke day 3 and 14 (PSD3 and PSD14). Results: At PSD3, the primary infarct was restricted to the cortex of the MCA territory, with no infarct detected in the thalamus of young mice. However, by PSD 14, neurons in the ipsilateral thalamus exhibited significant injury (FJC positive, condensed pyknotic nuclei). Gliosis was first detectable in the ipsilateral thalamus at PSD3 and progressively increased to PSD14 (anti-GFAP and Iba1). Infiltration of peripheral-derived monocytes was determined to be one source of the activated microglia in the thalamus (CCR2-RFP reporter mice, n=3). Interestingly, pdMCAO mice allowed to recover for two years demonstrated persistent astrogliosis (cortex and thalamus), though microgliosis was no longer evident (n=2). Aged mice subjected to pdMCAO also demonstrated gliosis in thalamus at PSD14, albeit to a lesser extent than young mice (n=5 each age). Finally, delayed treatment with memantine resulted in significantly attenuated gliosis and neuronal loss in the thalamus at PSD14 (young mice, n=9 each). Conclusions: These results further define gliosis in the mechanism of secondary injury and importantly demonstrate attenuation of secondary injury by delayed NMDA receptor antagonism.

Remote secondary injury in the thalamus has been observed following cortical infarct, however the mechanisms are not well understood. We used the distal MCAO stroke model (pdMCAO) to explore the cellular and temporal gliosis response in secondary thalamic injury in mice. At 3 days post-stroke (PSD3), primary infarct was limited to the cortex, with no infarct in the thalamus. However, at 2 weeks after stroke (PSD14), the ipsilateral thalamus demonstrated degenerating and severely damaged neurons. Staining for GFAP (astrogliosis) or IBA-1 (microgliosis) was first apparent in the ipsilateral thalamus by PSD3, and showed a progressive increase through PSD14. The number of activated microglia was increased within the thalamus at PSD14, reflecting proliferation of resident microglia as well as infiltration of peripheral monocytes. Interestingly, astrogliosis within the thalamus was enduring, as it was still evident at two years post-stroke. Furthermore, the astrogliosis at two years (but not at 6 weeks) demonstrated glial scar-like characteristics. Lastly, we demonstrated that post-stroke treatment with an NMDA receptor antagonist (memantine) reduces gliosis in the thalamus at PSD14. These findings highlight the development of lasting secondary injury in the thalamus following cortical stroke and support the value of memantine treatment in the mitigation of this injury.

Experimental guidelines have been proposed to improve the rigor and reproducibility of experimental stroke studies in rodents. As brain temperature is a strong determinant of ischemic injury, tight management of brain or body temperature (Tcore) during the experimental protocol is highly recommended. However, little guidance is provided regarding how or for how long temperature support should be provided. We compared a commonly used heat support method (cage on heating pad) with a low-cost custom built warm ambient air cage (WAAC) system. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra-ischemic period (60 min) and post-ischemic period (3 hrs). Neurologic deficit score showed significantly less variance at post-stroke day 1 (PSD1) in WAAC system mice. Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54% lower in the WAAC system group. In summary, we provide a simple low-cost heat support system that provides superior Tcore management in mice during the intra-ischemic and post-ischemic periods, which results in reduced variability of experimental outcomes.HighlightsWe describe the fabrication of a low-cost mouse cage warming system (warmed ambient air cage; WAAC system) that can be assembled and applied in any stroke laboratory.The WAAC system provides more precise control of post-stroke mouse body temperature compared with traditional heating pad warming system.The more precise control of post-stroke core temperature reduces variability in some experimental measures in more severely injured mice.

Background: CD146 is a tight junction associated molecule involved in maintaining endothelial barrier and balancing immune-inflammation response in cardiovascular disease. Notably, the peripheral CD146+ cells significantly upsurge under vessel dyshomeostasis like acute myocardial injury (AMI), appearing to be promising therapeutic targets. In this study, in a new view of gene correlation, we aim at deciphering the underlying complex mechanism of CD146+ cells in the development of AMI. Methods: Transcription dataset GSE 66360 of CD146+ blood cells from clinical subjects were downloaded from NCBI. Pearson networks were constructed and the clustering coefficients were calculated to disclose the differential connectivity genes (DCGs). Analysis of gene connectivity and gene expression was performed to reveal the hub genes and hub genes clusters followed by gene enrichment analysis. Results and Conclusions: Among the total 23520 genes, 27 genes out of 126 differential expression genes are identified as DCGs. Those DCGs normally stay in the peripheral of networks while transfer to the functional central position under AMI situation. Moreover, it is revealed that DCGs spontaneously crowd together into two functional models, CCL20 cluster and NR4A3 cluster, influencing the CD146-mediated signaling pathways during the pathology of AMI for the first time.

Background and Purpose: Delayed neurological deficits are a devastating consequence of subarachnoid hemorrhage (SAH), which affects about 30% of surviving patients. Although a very serious concern, delayed deficits are understudied in experimental SAH models; it is not known whether rodents recapitulate the delayed clinical decline seen in SAH patients. We hypothesized that mice with SAH develop delayed functional deficits and that microthrombi and infarction correlate with delayed decline. Methods: Adult C57BL/6J mice of both sexes were subjected to endovascular perforation to induce SAH. Mice were allowed to survive for up to 1 week post-ictus and behavioral performance was assessed daily. Postmortem microthrombi, large artery diameters (to assess vasospasm), and infarct volume were measured. These measures were analyzed for differences between SAH mice that developed delayed deficits and SAH mice that did not get delayed deficits. Correlation analyses were performed to identify which measures correlated with delayed neurological deficits, sex, and infarction. Results: Twenty-three percent of males and 47% of females developed delayed deficits 3 to 6 days post-SAH. Female mice subjected to SAH had a significantly higher incidence of delayed deficits than male mice with SAH. Mice that developed delayed deficits had significantly more microthrombi and larger infarct volumes than SAH mice that did not get delayed deficits. Microthrombi positively correlated with infarct volume, and both microthrombi and infarction correlated with delayed functional deficits. Vasospasm did not correlate with either infarction delayed functional deficits. Conclusions: We discovered that delayed functional deficits occur in mice following SAH. Sex differences were seen in the prevalence of delayed deficits. The mechanism by which microthrombi cause delayed deficits may be via formation of infarcts.

Delta-like 4 (Dll4) is a Notch receptor ligand classically thought to be expressed exclusively in endothelial cells. However, little is known about the cellular expression or functional role of Dll4 following ischemic stroke. Here, we present novel findings that Dll4 expression is shifted from endothelial cells to glial cells in the sub-acute stroke phase. Method: The permanent distal middle cerebral artery occlusion (dMCAO) model was performed to induce stroke. To investigate expression patterns of Dll4 protein and mRNA, immunostaining as well as single-cell RNA sequencing and single-cell fluorescent in situ hybridization were performed. To verify the role of endothelial Dll4 , tamoxifen-inducible, endothelial-specific Dll4 KO mice (Dll4iecKO) and Dll4 fl/fl (“floxed-only”) female mice (3 mos) were injected with tamoxifen (n=4) at 24 and 48 hrs post-stroke. To determine the effect of Notch inhibition at different time points, DAPT (100 mg/kg) or vehicle was administered at 24 or 72 hours post-stroke (n=4-5). Behavioral tests (foot fault) were conducted at Post Stroke Day (PSD) 1, 3, 7, and 14 and functional angiogenesis was assessed at PSD 14. Results: Elevated expression of Dll4 protein was observed in the microvasculature of the peri-infarct cortex (piCtx) as early as PSD 1. By PSD 3, however, we discovered Dll4 expression in microglia within the piCtx, with robust expression by PSD 14. Post-stroke deletion of endothelial Dll4 resulted in significant improvement in neurological function and increased capillary density in piCtx[l1] . DAPT given at 24 hours post-stroke improved outcome and capillary density; however, DAPT given at 72 hours (when microglia Dll4 expression is evident) appeared to worsen outcome. Conclusions: After stroke, expression of Dll4 shifts from exclusively endothelial (PSD 1) to mixed endothelial/microglial (PSD 3) to primarily microglial (PSD 14). Notch inhibition beginning at 24 hours post-stroke or endothelial-only deletion of Dll4 leads to increased angiogenesis and improved functional recovery. In contrast, Notch inhibition initiated at 72 hours post-stroke leads to worse outcome[l2] . These findings suggest a potential novel role of microglia in supporting reparative angiogenesis through Dll4/Notch signaling.

Background and Purpose— VWF (von Willebrand factor) strings mediate spontaneous platelet adhesion in the vascular lumen, which may lead to microthrombi formation and contribute to stroke pathology. However, the mechanism of VWF string attachment at the endothelial surface is unknown. We tested the novel hypothesis that VWF strings are tethered to the endothelial surface through an interaction between extracellular vimentin and the A2 domain of VWF. We further explored the translational value of blocking this interaction in a model of ischemic stroke. Methods— Human endothelial cells and pressurized cerebral arteries were stimulated with histamine to elicit VWF string formation. Recombinant proteins and antibodies were used to block VWF string formation. Mice underwent transient middle cerebral artery occlusion with reperfusion. Just before recanalization, mice were given either vehicle or A2 protein (recombinant VWF A2 domain) to disrupt the vimentin/VWF interaction. Laser speckle contrast imaging was used to monitor cortical perfusion. Results— Pressurized cerebral arteries produced VWF strings following histamine stimulation, which were reduced in arteries from Vim KO (vimentin knockout) mice. VWF string formation was significantly reduced in endothelial cells incubated with A2 protein or antivimentin antibodies. Lastly, A2 protein treatment significantly improved cortical reperfusion after middle cerebral artery occlusion. Conclusions— We provide the first direct evidence of cerebral VWF strings and demonstrate that extracellular vimentin significantly contributes to VWF string formation via A2 domain binding. Lastly, we show that pharmacologically targeting the vimentin/VWF interaction through the A2 domain can promote improved reperfusion after ischemic stroke. Together, these studies demonstrate the critical role of VWF strings in stroke pathology and offer new therapeutic targets for treatment of ischemic stroke.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Neonatal hypoxia&ndash, ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase &beta, (CaMKK &beta, ) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK &beta, in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK &beta, knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK &beta, KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood&ndash, brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK &beta, inhibition. In conclusion, inhibition of CaMKK &beta, exacerbated neonatal hypoxia&ndash, ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic&ndash, ischemic brain injury.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Fil: Tomilin, Viktor N. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Mamenko, Mykola. Augusta University. Medical College of Georgia. Department of Physiology; Estados Unidos Fil: Zaika, Oleg. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Ren, Guohui. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Marrelli, Sean P. University of Texas. Health Science Center at Houston. Department of Neurology; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Pochynyuk, Oleh. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Abstract: It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.

The conducted vasomotor response reflects electrical communication in the arterial wall and the distance signals spread is regulated by three factors including resident ion channels. This study defined the role of inward-rectifying K+channels (KIR) in governing electrical communication along hamster cerebral arteries. Focal KCl application induced a vasoconstriction that conducted robustly, indicative of electrical communication among cells. Inhibiting dominant K+conductances had no attenuating effect, the exception being Ba2+blockade of KIR. Electrophysiology and Q-PCR analysis of smooth muscle cells revealed a Ba2+-sensitive KIRcurrent comprised of KIR2.1/2.2 subunits. This current was surprisingly small and when incorporated into a model, failed to account for the observed changes in conduction. We theorized a second population of KIRchannels exist and consistent with this idea, a robust Ba2+-sensitive KIR2.1/2.2 current was observed in endothelial cells. When both KIRcurrents were incorporated into, and then inhibited in our model, conduction decay was substantive, aligning with experiments. Enhanced decay was ascribed to the rightward shift in membrane potential and the increased feedback arising from voltage-dependent-K+channels. In summary, this study shows that two KIRpopulations work collaboratively to govern electrical communication and the spread of vasomotor responses along cerebral arteries.

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

The prevalence of cardiovascular disease has increased among middle-aged women in the United States, yet has declined in middle-aged men. In experimental stroke, middle-aged females have larger strokes and greater inflammation than age-matched males or younger females. The mechanism underlying this shift from an “ischemia-protected” to an “ischemia-sensitive” phenotype in aging females is unknown. One potential factor is an age-related increase in systemic factors that induce inflammation. Increased abdominal fat deposition is seen in women during middle age. Adipose tissue plays a key role in obesity-induced systemic inflammation, including increased pro-inflammatory cytokines. We hypothesized that age and sex differences in adipose immune cells promote an augmented pro-inflammatory milieu in middle-aged females driven by a balance shift between pro-inflammatory and anti-inflammatory T cells. Abdominal adipose tissue immune cells from young (3–4 months) and middle-aged (15–16 months) male and female C57BL/6J mice were analyzed by flow cytometry. Plasma triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined with colorimetric assays. Middle-aged mice had higher adipose tissue mass compared to young mice. Lipid profiling showed no sex differences in TG and LDL, but middle-aged females had lower HDL (0.84 ± 0.07 μg/μl) than middle-aged males (1.35 ± 0.06 μg/μl). Flow cytometry data demonstrated an age-associated increase in adipose tissue CD8+ T cells that was augmented by female sex, with middle-aged females having a higher percentage of CD8+ cells (34.4 ± 3.2% of CD3+ T cells) than middle-aged males (24.4 ± 2.2%). This increase in CD8+ T-cell proportion was adipose tissue-specific, as this change was not observed in blood. Middle-aged females had higher numbers of activated (CD69+) CD8+ T cells than males. In addition, female CD8+ T cells produced higher levels of IFN-γ, TNF-α, and granzyme B ex vivo, and females had higher adipose levels of IFN-γ, RANTES and MIP-1β than middle-aged males. In parallel, females had lower levels of regulatory T cells (Tregs), an anti-inflammatory T-cell subtype, compared to age-matched males. In conclusion, middle-aged females have a detrimental combination of elevated pro-inflammatory T cells and decreased anti-inflammatory Tregs in adipose tissue, which may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.

Remote secondary injury in the thalamus has been observed following rodent and human stroke and is associated with poorer recovery. However the mechanisms and resultant molecular changes whereby cortical infarction leads to remote injury in distant regions of brain are not well defined. The present studies utilize a mouse model of permanent distal MCAO (pdMCAO) with delayed secondary thalamic injury. We have measured time dependent molecular and cellular events, focusing on glial activation and neuronal death in the thalamus. These studies provide an important foundation for future studies aimed at reducing secondary brain injury. Methods: Cortical infarction by pdMCAO was induced by microcauterization of the distal MCA in male C57BL/6J mice (11-13 wks). Cresyl violet (CV), fluoro-jade C (FJC) and immunochemical staining were performed at 24 hours, 3 days, 1 week, and 2 weeks after stroke (n=5, each time point) and in shams (n=3). Results: First, we confirmed with TTC staining that the primary infarction induced by pdMCAO was restricted to the cortex of the MCA territory at 72 hours, and that no infarct was seen in the thalamus. Microglia and astrocyte activation (IBA-1 and GFAP, respectively) was first detectable in the ipsilateral ventral posteromedial nucleus (VPN) of the thalamus at 3 days after stroke, and progressively increased at 1 and 2 weeks. However, thalamic neuronal injury (by FJC staining) was not evident until 1 week after stroke. At 2 weeks following stroke, the number of FJC positive neurons further increased and significant morphological changes were evident by CV staining, including shrunken cytoplasm and condensed pyknotic nuclei. Conclusions: We present a mouse stroke model for studying the mechanisms of secondary thalamic injury. In this model, primary cortical injury results in delayed cellular events in the ipsilateral thalamus, including an initial activation of microglia and astrocytes with subsequent neuronal injury and death. These studies provide necessary foundation for designing future interventional strategies aimed at reducing secondary injury following stroke.

Hypothermia shows promise for stroke neuroprotection, but current cooling strategies cause undesirable side effects that limit their clinical applications. Increasing efforts have focused on pharmacological hypothermia as a treatment option for stroke. Previously, we showed that activation of a thermoregulatory ion channel, transient receptor potential vanilloid 1 (TRPV1), by dihydrocapsaicin (DHC) produces reliable hypothermia. In this study, we investigate the effects of TRPV1-mediated hypothermia by DHC on long-term ischemic stroke injury and functional outcome. Hypothermia initiated at 3.5 hours after stroke significantly reduced primary cortical injury. Interestingly, hypothermia by DHC also significantly reduced secondary thalamic injury, as DHC-treated stroke mice exhibited 53% smaller thalamic lesion size. DHC-treated stroke mice further demonstrated decreased neuronal loss and astrogliosis in the thalamus and less thalamic fiber loss by diffusion tensor imaging (DTI). Importantly, a single 8 hour treatment of hypothermia by DHC after stroke provided long-term improvement in functional outcome, as DHC-treated mice exhibited improved behavioral recovery at one month post-stroke. These findings indicate that TRPV1-mediated hypothermia is effective in reducing both primary cortical injury and remote secondary thalamic injury, and a single treatment can produce persistent effects on functional recovery. These data highlight the therapeutic potential for TRPV1 agonism for stroke treatment.

Introduction: The TRPV1 channel is a non-selective ion channel that is activated by capsaicinoids, temperature (>43 °C), and multiple factors produced during inflammation or injury. At this time, there is very little reported regarding a role of this channel in regulation of cerebral blood flow. Here we provide new evidence for TRPV1 in promoting microvascular reperfusion following stroke. Hypothesis: Endothelial TRPV1 channels contribute to cerebral reperfusion following ischemic stroke. Methods: Experiments were performed in male WT and TRPV1 knockout mice of C57BL/6 background. Immunofluorescence was performed in brain sections with TRPV1 and Pecam-1 antibodies. Pressurized artery preparations were performed with isolated superior cerebellar arteries. Relative cerebral blood flow measurements were performed by laser Doppler and laser speckle flowmetry in mice before, during, and after MCAO (20 or 30 min). Reperfusion was monitored continuously for 2-3 hours and again at 24 hours. Results: TRPV1 expression was positively detected by immunofluorescence in the cerebral microvasculature, as assessed by Pecam-1 co-localization. TRPV1 KO mice were used as a negative control. Pressurized cerebral arteries demonstrated modest vasodilation to TRPV1 agonist (capsaicin) that was significantly potentiated with H 2 O 2 pre-treatment. The potentiated component of vasodilation was lost with removal of the endothelium, demonstrating the endothelial source of the response. Following brief MCA occlusion (20 or 30 min), WT mice demonstrated an initial return of cerebral perfusion followed by a transient drop in perfusion (~25% drop). This drop in perfusion started at 20-30 minutes into the reperfusion phase and lasted approximately one hour, at which point perfusion was restored to pre-stroke baseline value. In TRPV1 KO mice, the drop in perfusion was more profound (~65% drop) and persisted through 3 hours. By 24 hours, flow was restored to baseline in both groups. Conclusions: These data demonstrate a functional role for TRPV1 channels during reperfusion. We speculate that endothelial TRPV1 channels are functionally potentiated during injury and contribute to the vasodilatory capacity of the cerebral microvasculature following stroke.

Introduction: VWF strings can form within the vascular lumen following endothelial activation and have been implicated in platelet adhesion and worsened outcome following stroke. However, the molecular interactions facilitating VWF string anchorage to the endothelial surface are currently unknown. Here we examined the novel role of endothelial vimentin in mediating the anchorage of VWF strings within the cerebrovasculature. We hypothesize that VWF released from activated endothelium remains anchored at the luminal surface (i.e. VWF strings) through direct interaction with extracellular vimentin. Methods: Cultured endothelial cell (EC) experiments were performed with human umbilical vein EC (HUVECs) and human brain microvascular EC (HBMVECs). EC were stimulated with histamine (10 uM) under flow conditions. Specific protein interactions were probed with recombinant vimentin and A2 domain of VWF. Mouse middle cerebral and superior cerebellar arteries from WT and vimentin KO mice were set up in a pressurized artery chamber, stimulated with histamine, and then processed for VWF immunofluorescence to quantify VWF strings. VWF string formation was further evaluated by cranial window preparation using labeled platelets to detect real-time in vivo string formation. Results: In cultured EC, histamine stimulation promoted the production of long VWF strings that were significantly attenuated in the presence of recombinant vimentin or A2 domain of VWF. In pressurized cerebral arteries, histamine stimulation promoted VWF strings that aligned along the luminal endothelial surface. VWF string formation was reduced 2.9 fold in arteries from vimentin knockout mice (P=0.02). Histamine stimulation in the cranial window produced platelet-adherent strings in the pial microvasculature. Conclusions: These studies provide in vitro and in vivo evidence for a novel interaction between vimentin and the A2 domain of VWF at the endothelial surface which contributes to the anchorage of VWF strings in the microvasculature. This vimentin/VWF interaction critically regulates VWF-mediated platelet adhesion at the surface of activated endothelium and could thus provide a novel therapeutic target in the treatment of acute ischemic stroke.

Mild decrease in core temperature (therapeutic hypothermia) provides lasting neuroprotection following cardiac arrest or cerebral ischemia. However, current methods for producing therapeutic hypothermia trigger a cold-defense response that must be countered by sedatives, muscle paralytics, and mechanical ventilation. We aimed to determine methods for producing hypothermia in the conscious mouse by targeting two transient receptor potential channels involved in thermoregulation, two transient receptor potential (TRP) channels involved in thermoregulation, TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8).Controlled prospective animal study.Research laboratory at academic medical center.Conscious unrestrained young and aged male mice.Mice were treated with the TRPV1 agonist dihydrocapsaicin, a TRPM8 inhibitor ("compound 5"), or their combination and the effects on core temperature (Tcore) were measured by implanted thermocouples and wireless transponders.TRPV1 agonist dihydrocapsaicin produced a dose-dependent (2-4 mg/kg s.c.) drop in Tcore. A loading dose followed by continuous infusion of dihydrocapsaicin produced a rapid and prolonged (6 hr) drop of Tcore within the therapeutic range (32-34°C). The hypothermic effect of dihydrocapsaicin was augmented in aged mice and was not desensitized with repeated administration. TRPM8 inhibitor "compound 5" (20 mg/kg s.c.) augmented the drop in core temperature during cold exposure (8°C). When "compound 5" (30 mg/kg) was combined with dihydrocapsaicin (1.25-2.5 mg/kg), the drop in Tcore was amplified and prolonged.Activating warm receptors (TRPV1) produced rapid and lasting hypothermia in young and old mice. Furthermore, hypothermia induced by TRPV1 agonists was potentiated and prolonged by simultaneous inhibition of TRPM8.

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ∼30% in MCAs from rats undergoing apneas compared with those from a sham control group ( P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

We tested the hypothesis that TREK-1, a two-pore domain K channel, is involved with dilations in arteries. Because there are no selective activators or inhibitors of TREK-1, we generated a mouse line deficient in TREK-1. Endothelium-mediated dilations were not different in arteries from wild-type (WT) and TREK-1 knockout (KO) mice. This includes dilations of the middle cerebral artery to ATP, dilations of the basilar artery to ACh, and relaxations of the aorta to carbachol, a cholinergic agonist. The nitric oxide (NO) and endothelium-dependent hyperpolarizing factor components of ATP dilations were identical in the middle cerebral arteries of WT and TREK-1 KO mice. Furthermore, the NO and cyclooxygenase-dependent components were identical in the basilar arteries of the different genotypes. Dilations of the basilar artery to α-linolenic acid, an activator of TREK-1, were not affected by the absence of TREK-1. Whole cell currents recorded using patch-clamp techniques were similar in cerebrovascular smooth muscle cells (CVSMCs) from WT and TREK-1 KO mice. α-linolenic acid or arachidonic acid increased whole cell currents in CVSMCs from both WT and TREK-1 KO mice. The selective blockers of large-conductance Ca-activated K channels, penitrem A and iberiotoxin, blocked the increased currents elicited by either α-linolenic or arachidonic acid. In summary, dilations were similar in arteries from WT and TREK-1 KO mice. There was no sign of TREK-1-like currents in CVSMCs from WT mice, and there were no major differences in currents between the genotypes. We conclude that regulation of arterial diameter is not altered in mice lacking TREK-1.

The neuroprotective effects of exogenous erythropoietin (EPO) in animals and humans after brain injury may be afforded, in part, by the influence of EPO on cerebral arteries. We tested (1) if EPO itself is vasoactive and (2) if EPO enhances endothelium-mediated dilations, specifically those mediated by endothelium-derived hyperpolarizing factor (EDHF). Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect EPO receptor. Rat middle cerebral arteries (MCAs) were isolated, pressurized, and perfused in vitro. EPO was directly applied to MCAs to test its vasoactivity. Endothelium-mediated dilations were elicited by UTP, whereas EDHF-mediated dilations were elicited by UTP after inhibition of endothelial nitric oxide synthase and cyclooxygenase. mRNA and protein for EPO receptor was found in rat MCA. Abluminal application of 0.001-10 U/mL EPO, which is selective for vascular smooth muscle, did not alter vessel diameter. In contrast, luminal application of EPO, which is selective for endothelium, resulted in concentration-dependent dilations of up to 39 +/- 16% at 10 U/mL (p = 0.0018), though responses were variable. A single dose of EPO (1,000 U/kg) administered to rats 24 h prior to examining vascular function potentiated dilations to UTP 2.6-fold (p0.0001). EDHF-mediated dilations were potentiated 2.1-fold following in vivo EPO treatment (p = 0.0034). This study demonstrates that EPO can directly dilate rat MCAs via the endothelium, though not all vessels are responsive. Additionally, pre-treatment with EPO for 24 h in vivo potentiates endothelium-mediated dilations, specifically those mediated by EDHF. Thus, enhanced endothelium-mediated dilations may partially underlie the neuroprotective effects of EPO after brain injury.

Previous findings from our laboratory have shown that dilations to endothelium-derived hyperpolarizing factor (EDHF) in rat middle cerebral artery (MCA) are less in females compared to males. Myoendothelial gap junctions (MEGJs) appear to mediate the transfer of hyperpolarization between endothelium and smooth muscle in males. In the present study, we hypothesized that MEGJs are the site along the EDHF pathway which is compromised in female rat MCA. Membrane potential in endothelium was measured using the voltage-sensitive dye di-8-ANEPPS and in smooth muscle using intracellular glass microelectrodes in the presence of l-NAME (3x10(-5 )M) and indomethacin (10(-5 )M). Electron microscopy was used to assess MEGJ characteristics. In endothelial cells, the di-8-ANEPPS fluorescence ratio change to 10(-5 )M UTP was similar in males (-2.9+/-0.5%) and females (-3.2+/-0.2%), indicating comparable degrees of endothelial cell hyperpolarization. However, smooth muscle cell hyperpolarization to 10(-5 )M UTP was significantly attenuated in females (0 mV hyperpolarization; -31+/-1.5 mV resting) compared to males (8 mV hyperpolarization; -28+/-1.7 mV resting). Ultrastructural evidence suggested that MEGJ frequency and area of contact were comparable between males and females. Taken together, our data suggest that in rat MCA, MEGJ frequency does not account for the reduced EDHF responses observed in females compared to males. We conclude that reduced myoendothelial coupling and/or homocellular coupling within the media may account for these differences.

There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.

The present study evaluated the role of endothelial intermediate conductance calcium-sensitive potassium channels (IKCa) in the mechanism of endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in pressurized cerebral arteries. Male rat middle cerebral arteries (MCA) were mounted in an isolated vessel chamber, pressurized (85 mmHg), and luminally perfused (100 μl/min). Artery diameter was measured simultaneously with either endothelial intracellular Ca2+concentration ([Ca2+]i; fura-2) or changes in endothelial membrane potential [4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS)]. Nitric oxide synthase and cyclooxygenase inhibitors were present throughout. Luminal application of UTP produced EDHF-mediated dilations that correlated with significant endothelial hyperpolarization. The dilation and endothelial hyperpolarization were virtually abolished by inhibitors of IKCachannels but not by selective inhibitors of small or large conductance KCachannels (apamin and iberiotoxin, respectively). Additionally, direct stimulation of endothelial IKCachannels with 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial hyperpolarization and vasodilatation that were blocked by inhibitors of IKCachannels. 1-EBIO hyperpolarized the endothelium but did not affect endothelial [Ca2+]i. We conclude that the mechanism of EDHF-mediated dilations in cerebral arteries requires stimulation of endothelial IKCachannels to promote endothelial hyperpolarization and subsequent vasodilatation.

Background and Purpose— The contribution of endothelium-derived hyperpolarizing factor (EDHF) to ATP-mediated dilations is significantly attenuated in the rat middle cerebral artery of intact and estrogen-treated ovariectomized (OVX) females compared with males and vehicle-treated OVX females. Since an increase in endothelial calcium appears to be a critical prerequisite in the EDHF response, we tested the hypothesis that endothelial cell intracellular calcium ([Ca 2+ ] i ) fails to reach sufficient levels to elicit robust EDHF-mediated dilations in females and that this effect is mediated by estrogen. Methods— Vascular diameter and [Ca 2+ ] i were measured concomitantly in perfused middle cerebral artery segments with the use of videomicroscopy and fura 2 fluorescence, respectively. Results— In the presence of N G -nitro- l -arginine methyl ester and indomethacin, the dilation to 10 −5 mol/L ATP was significantly reduced ( P 2+ ] i increased to comparable levels in intact females (461±116 nmol/L), estrogen-treated OVX females (417±50 nmol/L), intact males (421±77 nmol/L), and vehicle-treated OVX females (530±92 nmol/L). In response to luminal ATP (10 −5 mol/L), smooth muscle cell [Ca 2+ ] i decreased to a greater degree in males (37±4%; n=8) compared with females (21±5%; n=7) and in vehicle-treated OVX females (18±7%; n=7) compared with estrogen-treated OVX females (3±5%; n=9). Conclusions— Our data suggest that loss of a factor coupling EDHF to reduction of ionized smooth muscle cell [Ca 2+ ] i accounts for the attenuated EDHF-mediated dilations in the female middle cerebral artery.

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF)–mediated dilations are potentiated after several pathologies, including ischemia/reperfusion (I/R). However, no study to date has addressed the mechanism by which this potentiation occurs. This study tested the hypothesis that potentiated EDHF-mediated dilations are due to altered endothelial Ca 2+ handling after I/R. Methods— Rat middle cerebral arteries (MCAs) were isolated after 2 hours of MCA occlusion and 24 hours of reperfusion (or sham surgery). This model has been previously demonstrated to produce potentiated EDHF-mediated dilations. MCAs were studied in a pressurized/perfused vessel chamber equipped for the simultaneous measurement of endothelial Ca 2+ (with fura 2) and artery diameter. Measures were made after luminal administration of UTP (P2Y 2 purinoceptor agonist), 2 MeS-ATP (P2Y 1 purinoceptor agonist), and Br-A23187 (receptor-independent Ca 2+ ionophore) for sham and I/R MCAs. Results— I/R resulted in significantly potentiated UTP-mediated dilations (through a P2Y 2 purinoceptor) and endothelial Ca 2+ responses in the presence of N G -nitro- l -arginine methyl ester (L-NAME) and indomethacin. Endothelial Ca 2+ and diameter responses were also significantly potentiated with 2 MeS-ATP (through a P2Y 1 purinoceptor) when L-NAME and indomethacin were absent. Br-A23187, a receptor-independent Ca 2+ ionophore, produced significantly potentiated endothelial Ca 2+ responses after I/R in the presence of L-NAME/indomethacin. Evaluation of artery diameter as a function of endothelial Ca 2+ demonstrated no differences between sham and I/R groups. Conclusions— These findings demonstrate that I/R results in augmented endothelial Ca 2+ responses that appear to be downstream of the receptor level. Moreover, these data suggest that this augmented Ca 2+ response contributes to the potentiated EDHF-mediated dilations after I/R.

In cerebral vessels, there is at least one factor or mechanism not involving nitric oxide/endothelium-derived relaxing factor (NO/EDRF) or prostacyclin that is responsible for dilations when endothelial receptors are stimulated. This factor or mechanism is termed endothelium-derived hyperpolarizing factor (EDHF). In rat middle cerebral arteries, branches of the middle cerebral arteries and penetrating arterioles, ATP or UTP elicits the release of EDHF through stimulation of P2Y 2 receptors on the endothelium. The dilations are characterized by the involvement of calcium-activated potassium channels (K Ca ) and hyperpolarizations in vascular smooth muscle. In smaller arteries and in arterioles, EDHF appears to be more important than in larger cerebral arteries. Finally, the EDHF response is upregulated 24 h following mild traumatic brain injury (TBI). EDHF may play an important role in the regulation of cerebral blood flow during normal physiological conditions and an even greater role following pathological conditions such as TBI.

Background Transient receptor potential C3 ( TRPC 3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell ( EC ) hyperpolarization and endothelium‐dependent hyperpolarization–mediated vasodilation. However, the mechanism by which TRPC 3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC 3 trafficking to the plasma membrane, where it provides the source of Ca 2+ influx for small conductance calcium‐activated K + (SK Ca ) channel activation and sustained EC hyperpolarization. Methods and Results Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC 3 blocker (Pyr3) produced significant attenuation of endothelium‐dependent hyperpolarization–mediated vasodilation and endothelial Ca 2+ response ( EC ‐specific Ca 2+ biosensor) to intraluminal ATP . Pyr3 treatment also resulted in a reduced ATP ‐stimulated global Ca 2+ and Ca 2+ influx in primary cultures of cerebral endothelial cells. Patch‐clamp studies with freshly isolated cerebral EC s demonstrated 2 components of EC hyperpolarization and K + current activation in response to ATP . The early phase was dependent on intermediate conductance calcium‐activated K + channel activation, whereas the later sustained phase relied on SK C a channel activation. The SK C a channel–dependent phase was completely blocked with TRPC 3 channel inhibition or in ECs of TRPC 3 knockout mice and correlated with increased trafficking of TRPC 3 (but not SK C a channel) to the plasma membrane. Conclusions We propose that TRPC 3 dynamically regulates SK C a channel activation through receptor‐dependent trafficking to the plasma membrane, where it provides the source of Ca 2+ influx for sustained SK C a channel activation, EC hyperpolarization, and endothelium‐dependent hyperpolarization–mediated vasodilation.

The present study was designed to evaluate the role of endothelial intracellular Ca2+concentration ([Ca2+]i) in the difference between P2Y1- and P2Y2-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca2+indicator, for simultaneous measurement of endothelial [Ca2+]iand diameter. Luminal administration of 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP), an endothelial P2Y1agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca2+]iincreases up to ∼300 nM (resting [Ca2+]i= 145 nM). UTP, an endothelial P2Y2agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca2+]iincreases to >400 nM. In the presence of NG-nitro-l-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca2+]ithreshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y1and P2Y2purinoceptors result in part from differential [Ca2+]iresponses. Consistent with this finding, these studies also demonstrate a higher [Ca2+]ithreshold for EDHF-dependent responses compared with NO.

The effect of luminal shear stress was studied in cerebral arteries and arterioles. Middle cerebral arteries (MCA) and penetrating arterioles (PA) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized. After the development of spontaneous tone, inside diameters were 186 ± 5 μm ( n = 28) for MCA and 65 ± 3 μm ( n = 37) for PA. MCA and PA constricted ∼20% with increasing flow. Flow-induced constriction persisted in MCA and PA after removal of the endothelium. After removal of the endothelium, the luminal application of a polypeptide containing the Arg-Gly-Asp amino acid sequence (inhibitor of integrin attachment) abolished the flow-induced constriction. Similarly, an antibody specific for the β3-chain of the integrin complex significantly inhibited the flow-induced constriction. The shear stress-induced constriction was accompanied by an increase in vascular smooth muscle Ca2+. For example, a shear stress of 20 dyn/cm2constricted MCA 8% ( n = 5) and increased Ca2+from 209 ± 17 to 262 ± 29 nM ( n = 5). We conclude that isolated cerebral arteries and arterioles from the rat constrict to increased shear stress. Because the endothelium is not necessary for the response, the shear forces must be transmitted across the endothelium, presumably by the cytoskeletal matrix, to elicit constriction. Integrins containing the β3-chain are involved with the shear stress-induced constrictions.

Despite a critical role for calcium in endothelial regulation of cerebrovascular tone, endothelial intracellular calcium ([Ca 2+ ] i ) has never been measured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth muscle [Ca 2+ ] i and diameter in a pressurized/perfused cerebral vessel. In a pressurized rat middle cerebral artery, fura-2 AM was administered selectively to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluorescence before and after removal of the endothelium. Removal of the endothelium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioadenosine triphosphate (2MeS-ATP, a selective endothelial P2 receptor agonist) was used to infer the selectivity of fura-2 loading. It was reasoned that 2MeS-ATP should produce a decrease in smooth muscle [Ca 2+ ] i and an increase in endothelial [Ca 2+ ] i in selectively loaded vessels, consistent with its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca 2+ ] i went from 252±8 to 82±9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca 2+ ] i went from 137±11 to 271±20 nM. Thus, a method is provided which allows for selective measurement of endothelial or smooth muscle [Ca 2+ ] i with simultaneous measurement of diameter in a pressurized cerebral vessel.

The effects of stimulating P2Y1 or P2Y2 purinoceptors on the endothelium of isolated middle cerebral arteries (MCAs), third-order branches of the MCA (bMCAs), and penetrating arterioles (PAs) of the rat were studied. After pressurization and development of spontaneous tone (25% contraction), resting diameters for MCAs, bMCAs, and PAs were 203 ± 5 ( n = 50), 99 ± 2 ( n = 42), and 87 ± 2 μm ( n = 53), respectively. Luminal application of the P2Y1-selective agonist 2-methylthioadenosine 5′-triphosphate elicited dose-dependent dilations (or loss of intrinsic tone) in MCAs but not in bMCAs or PAs. The dilation in MCAs was completely blocked by removal of the endothelium or by nitro-l-arginine methyl ester (10−5 M), an inhibitor of NO synthase. Luminal application of the P2Y2-selective agonist ATP elicited dilations in MCAs, bMCAs, and PAs. Removal of the endothelium abolished the dilations in all vessel groups. Dilations in MCAs have been shown to involve both NO and endothelium-derived hyperpolarizing factor (EDHF). The dilations in bMCAs and PAs had a minor NO component and prominent EDHF component; that is, 1) the dilations to ATP were not diminished by the combined inhibition of NO synthase and cyclooxygenase, 2) the dilations were accompanied by significant hyperpolarizations of the vascular smooth muscle (∼15 mV), and 3) the dilations were completely abolished by the calcium-activated potassium channel blocker charybdotoxin. We concluded that the role of NO in purinoceptor-induced dilations diminishes along the cerebrovascular tree in the rat, whereas the role of EDHF becomes more prominent.

Endothelial-mediated dilations to selective P2Y1 and P2Y2 purinoceptor agonists [2-methylthioadenosine triphosphate (2MeS-ATP) and uridine 5′-triphosphate (UTP), respectively] were evaluated in middle cerebral arteries (MCAs) of rats after 2 h of ischemia followed by 24 h of reperfusion (I/R). MCAs were harvested, pressurized to 85 mmHg, and luminally perfused. 2MeS-ATP, which dilates by the synthesis and release of nitric oxide (NO), had significantly reduced maximum dilations following I/R. Reduced smooth muscle sensitivity to NO may explain the reduced dilation to 2MeS-ATP. In contrast, the dilations elicited by UTP were potentiated in that the concentration of agonist necessary to produce one-half of the maximum dilation was reduced by 75%. The potentiated dilation to UTP was the result of an endothelial factor having all the characteristics of the endothelium-derived hyperpolarizing factor (EDHF). That is, it was neither NO nor a cyclooxygenase metabolite, and its actions involved calcium-activated potassium channels and smooth muscle hyperpolarization. We conclude that the effect of I/R on endothelial-mediated dilations depends on the receptor system and the mechanism of dilation. Dilations elicited by 2MeS-ATP were attenuated, while dilations UTP were potentiated due to the upregulation of the EDHF mechanism.

The mechanisms associated with dysfunction of the cerebral vasculature following head trauma have not yet been fully elucidated. In an attempt to shed more light on the matter, we investigated the endothelial-mediated dilations in the rat middle cerebral artery (MCA) following severe traumatic brain injury (TBI). Rats were subjected to severe controlled cortical impact injury (CCI; 5 m/s, 130 ms duration, 3 mm deformation) over the right parietal cortex. At 24 h postinjury, ipsilateral segments of MCA and corresponding contralateral segments were isolated, mounted in a vessel chamber, and pressurized. The responses to 2 methylthio-ATP (2MeSATP), a selective agonist for the P2Y1 purinoceptors, N(omega)-nitro-L-arginine (L-NAME), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (SNAP), an exogenous NO donor, were determined. 2MeSATP elicited concentration dependent dilations in all MCAs studied. Ipsilateral MCAs harvested following TBI or sham-TBI, showed similar maximum dilations to 2MeSATP [70 +/- 4% (n = 17) and 72 +/- 6% (n = 13), respectively]. However, TBI reduced the concentration of 2MeSATP necessary to elicit one-half of the maximum dilation (EC50) from 15 to 9 nM (p0.05). Inhibition of NO synthase with 10(-5) M L-NAME abolished the dilation to 2MeSATP in both TBI and sham-TBI MCAs. The constriction to L-NAME was significantly reduced in TBI MCAs compared to sham vessels. Dilations to SNAP, an NO donor, were not altered by TBI indicating that the mechanisms of dilation involving NO in the vascular smooth muscle were not affected. Unlike other pathological conditions which often diminish endothelial-mediated responses, severe TBI enhanced the sensitivity to 2MeSATP without altering the maximum response.

Traditional methods of therapeutic hypothermia show promise for neuroprotection against cerebral ischemia-reperfusion (I/R), however, with limitations. We examined effectiveness and specificity of pharmacological hypothermia (PH) by transient receptor potential vanilloid 1 (TRPV1) channel agonism in the treatment of focal cerebral I/R. Core temperature (Tcore) was measured after subcutaneous infusion of TRPV1 agonist dihydrocapsaicin (DHC) in conscious C57BL/6 WT and TRPV1 knockout (KO) mice. Acute measurements of heart rate (HR), mean arterial pressure (MAP), and cerebral perfusion were measured before and after DHC treatment. Focal cerebral I/R (1 h ischemia + 24 h reperfusion) was induced by distal middle cerebral artery occlusion. Hypothermia (>8 h) was initiated 90 min after start of reperfusion by DHC infusion (osmotic pump). Neurofunction (behavioral testing) and infarct volume (TTC staining) were measured at 24 h. DHC (1.25 mg/kg) produced a stable drop in Tcore (33°C) in naive and I/R mouse models but not in TRPV1 KO mice. DHC (1.25 mg/kg) had no measurable effect on HR and cerebral perfusion but produced a slight transient drop in MAP (

Mild decrease of core temperature (32–34°C), also known as therapeutic hypothermia, is a highly effective strategy of neuroprotection from ischemia and holds significant promise in the treatment of stroke. However, induction of hypothermia in conscious stroke patients is complicated by cold-defensive responses, such as shivering and tachycardia. Although multiple thermoregulatory responses may be altered by modulators of thermosensitive ion channels, TRPM8 (transient receptor potential melastatin 8) and TRPV1 (TRP vanilloid 1), it is unknown whether these agents affect cold-induced shivering and tachycardia. The current study aimed to determine the effects of TRPM8 inhibition and TRPV1 activation on the shivering and tachycardic responses to external cooling. Conscious mice were treated with TRPM8 inhibitor compound 5 or TRPV1 agonist dihydrocapsaicin (DHC) and exposed to cooling at 10°C. Shivering was measured by electromyography using implanted electrodes in back muscles, tachycardic response by electrocardiography, and core temperature by wireless transmitters in the abdominal cavity. The role of TRPM8 was further determined using TRPM8 KO mice. TRPM8 ablation had no effect on total electromyographic muscle activity (vehicle: 24.0 ± 1.8; compound 5: 23.8 ± 2.0; TRPM8 KO: 19.7 ± 1.9 V·s/min), tachycardia (ΔHR = 124 ± 31; 121 ± 13; 121 ± 31 beats/min) and drop in core temperature (−3.6 ± 0.1; −3.4 ± 0.4; −3.6 ± 0.5°C) during cold exposure. TRPV1 activation substantially suppressed muscle activity (vehicle: 25.6 ± 3.0 vs. DHC: 5.1 ± 2.0 V·s/min), tachycardia (ΔHR = 204 ± 25 vs. 3 ± 35 beats/min) and produced a profound drop in core temperature (−2.2 ± 0.6 vs. −8.9 ± 0.6°C). In conclusion, external cooling-induced shivering and tachycardia are suppressed by TRPV1 activation, but not by TRPM8 inhibition. This suggests that TRPV1 agonists may be combined with external physical cooling to achieve more rapid and effective hypothermia.

Aims Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. Methods and results TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SKCa (apamin) or IKCa (TRAM-34) block, Pyr3 abolished the residual respective IKCa- and SKCa-dependent EDH-mediated vasodilation. Conclusion The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SKCa and IKCa activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.