Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell Ca2+ signals require an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature.

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary inositol 1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell IP3R-mediated Ca2+ signals requires an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial IP3-evoked Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo within 120 seconds. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

The post-arteriole transitional zone: a specialized capillary region that regulates blood flow within the CNS microvasculature.

The brain is an energy hog, consuming available energy supplies at a rate out of all proportion to its relatively small size. This outsized demand, largely reflecting the unique computational activity of the brain, is met by an ensemble of neurovascular coupling mechanisms that link neuronal activity with local increases in blood delivery. This just-in-time replenishment strategy, made necessary by the limited energy-storage capacity of neurons, complicates the nutrient-delivery task of the cerebral vasculature, layering on a temporo-spatial requirement that invites - and challenges - mechanistic interpretation. The centre of gravity of research efforts to disentangle these mechanisms has shifted from an initial emphasis on astrocyte-arteriole-level processes to mechanisms that operate on the capillary level, a shift that has brought into sharp focus questions regarding the fine control of blood distribution to active neurons. As these investigations have drilled down into finer reaches of the microvasculature, they have revealed an arteriole-proximate subregion of CNS capillary networks that serves a regulatory function in directing blood flow into and within downstream capillaries. They have also illuminated differences in researchers' perspectives on the vascular structures and identity of mural cells in this region that impart the vasomodulatory effects that control blood distribution. In this review, we highlight the regulatory role of a variably named region of the microvasculature, referred to here as the post-arteriole transition zone, in channeling blood flow within CNS capillary networks, and underscore the contribution of dynamically contractile perivascular mural cell - generally, but not universally, recognized as pericytes - to this function.

Retrospective study of medical student scholarship and career trajectory following a mentored preclinical cardiovascular summer research fellowship.

OBJECTIVES: Developing a preclinical training infrastructure for cardiovascular clinician-scientists is an academic workforce priority. The Cardiovascular Research Institute of Vermont developed a cardiovascular summer research fellowship (SRF), wherein medical student awardees were selected by merit-based application and completed mentored research between the first and second years. We aimed to study the impact of the SRF on medical student scholarship and career planning. DESIGN: Retrospective survey study. SETTING: Single academic medical centre. PARTICIPANTS: All SRF participants from 2015 to 2020. INTERVENTIONS: Not applicable. PRIMARY AND SECONDARY OUTCOME MEASURES: Prior SRF participants were surveyed to ascertain current position, research engagement and perspectives regarding SRF experience. Comparisons to American Association of Medical Colleges Graduation Questionnaire data from equivalent years were made using χ2 tests. RESULTS: Survey response rate was 87% (20/23), 55% were women. Median time from SRF completion was 2 years (IQR 0.75-2.25), with 75% still enrolled in medical school and 25% in residency. As a result of the first-year summer programme, 45% published a peer-reviewed abstract or manuscript, which was equivalent to the national rate for graduating students (53%, p=0.4). Most respondents (80%) were active in additional research projects during school separate from the SRF, 90% anticipated a career involving research (vs 53% nationally, p<0.001) and 75% planned to pursue a career in cardiovascular medicine. CONCLUSION: Medical students completing a mentored cardiovascular SRF after their first year have a high rate of academic scholarship, with publication rate already equivalent to national peer graduates. Preclinical SRF students strongly anticipate cardiovascular medicine and research careers.

Adenosine signaling activates ATP-sensitive K+ channels in endothelial cells and pericytes in CNS capillaries.

The dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuron- and glial-derived compounds that enhance regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the physiological bridge that translates brain activity into vascular function. In other beds, the ATP-sensitive K+ (KATP) channel regulates VM in vascular smooth muscle, which is absent in the capillary network. Here, with transgenic mice that expressed a dominant-negative mutant of the pore-forming Kir6.1 subunit specifically in brain cECs or pericytes, we demonstrated that KATP channels were present in both cell types and robustly controlled VM. We further showed that the signaling nucleotide adenosine acted through A2A receptors and the Gαs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo increased cerebral blood flow (CBF), an effect that was blunted by expression of the dominant-negative Kir6.1 mutant in either capillary cell type. These findings establish an important role for KATP channels in cECs and pericytes in the regulation of CBF.

Impact of an institutional grant award on early career investigator applicants and peer reviewers.

BACKGROUND: Obtaining research funding support is integral to a successful career in science. Training and practice in grant writing, as well as engagement in peer review of grant applications may help lead to successful research funding. However, there is little evidence on the impact of institutional programs on the career development of early career investigators (ECIs). OBJECTIVES: Understand the impact of participation in an institutional research award program on the career development of ECIs. METHODS: The Cardiovascular Research Institute of Vermont established an Early Career Research (ECR) award program in 2018. ECIs who participated as applicants or reviewers in the first 3 years of the program (2018-2020) were surveyed to understand the impact of the ECR award program on their grant writing and professional development. RESULTS: Ninety-four percent of 17 applicants and 90% of 19 reviewers completed the survey. Ninety-two percent of funded and 75% of unfunded applicants, and 87% of reviewers reported that the program was beneficial to their professional development. Similarly, 85% of funded applicants, 75% of unfunded applicants, and 80% of reviewers reported improvement in their grant-writing skills. All respondents reported they would recommend the ECR award program to their peers. CONCLUSIONS: This single-institution ECR award program had a positive impact on ECI's professional development and grant-writing skills and may lead to further extramural funding opportunities.

Local IP3 receptor-mediated Ca2+ signals compound to direct blood flow in brain capillaries.

Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP3 receptor-mediated Ca2+ events, ranging from small, subsecond protoevents, reflecting Ca2+ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca2+ signals are driven by neuronal activity, which engages Gq protein-coupled receptor signaling, and are enhanced by Ca2+ entry through TRPV4 channels. The resulting Ca2+-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.

Apelin Does Not Impair Coronary Artery Relaxation Mediated by Nitric Oxide-Induced Activation of BKCa Channels.

Apelin-APJ receptor signaling regulates vascular tone in cerebral and peripheral arteries. We recently reported that apelin inhibits BKCa channel function in cerebral arteries, resulting in impaired endothelium-dependent relaxations. In contrast, apelin causes endothelium-dependent relaxation of coronary arteries. However, the effects of apelin on BKCa channel function in coronary arterial myocytes have not yet been explored. We hypothesized that apelin-APJ receptor signaling does not have an inhibitory effect on coronary arterial BKCa channels and hence does not alter nitric oxide (NO)-dependent relaxation of coronary arteries. Patch clamp recording was used to measure whole cell K+ currents in freshly isolated coronary smooth muscle cells. Apelin had no effect on the increases in current density in response to membrane depolarization or to NS1619 (a BKCa channel opener). Moreover, apelin did not inhibit NO/cGMP-dependent relaxations that required activation of BKCa channels in isolated coronary arteries. Apelin-APJ receptor signaling caused a marked increase in intracellular Ca2+ levels in coronary arterial smooth muscle cells, but failed to activate PI3-kinase to increase phosphorylation of Akt protein. Collectively, these data provide mechanistic evidence that apelin has no inhibitory effects on BKCa channel function in coronary arteries. The lack of inhibitory effect on BKCa channels makes it unlikely that activation of APJ receptors in coronary arteries would adversely affect coronary flow by creating a vasoconstrictive environment. It can be expected that apelin or other APJ receptor agonists in development will not interfere with the vasodilator effects of endogenous BKCa channel openers.

PIP2 Improves Cerebral Blood Flow in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a leading cause of dementia and a substantial healthcare burden. Despite this, few treatment options are available for controlling AD symptoms. Notably, neuronal activity-dependent increases in cortical cerebral blood flow (CBF; functional hyperemia) are attenuated in AD patients, but the associated pathological mechanisms are not fully understood at the molecular level. A fundamental mechanism underlying functional hyperemia is activation of capillary endothelial inward-rectifying K+ (Kir2.1) channels by neuronally derived potassium (K+), which evokes a retrograde capillary-to-arteriole electrical signal that dilates upstream arterioles, increasing blood delivery to downstream active regions. Here, using a mouse model of familial AD (5xFAD), we tested whether this impairment in functional hyperemia is attributable to reduced activity of capillary Kir2.1 channels. In vivo CBF measurements revealed significant reductions in whisker stimulation (WS)-induced and K+-induced hyperemic responses in 5xFAD mice compared with age-matched controls. Notably, measurements of whole-cell currents in freshly isolated 5xFAD capillary endothelial cells showed that Kir2.1 current density was profoundly reduced, suggesting a defect in Kir2.1 function. Because Kir2.1 activity absolutely depends on binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the channel, we hypothesized that capillary Kir2.1 channel impairment could be corrected by exogenously supplying PIP2. As predicted, a PIP2 analog restored Kir2.1 current density to control levels. More importantly, systemic administration of PIP2 restored K+-induced CBF increases and WS-induced functional hyperemic responses in 5xFAD mice. Collectively, these data provide evidence that PIP2-mediated restoration of capillary endothelial Kir2.1 function improves neurovascular coupling and CBF in the setting of AD.

Brain endothelial cell TRPA1 channels initiate neurovascular coupling.

Cerebral blood flow is dynamically regulated by neurovascular coupling to meet the dynamic metabolic demands of the brain. We hypothesized that TRPA1 channels in capillary endothelial cells are stimulated by neuronal activity and instigate a propagating retrograde signal that dilates upstream parenchymal arterioles to initiate functional hyperemia. We find that activation of TRPA1 in capillary beds and post-arteriole transitional segments with mural cell coverage initiates retrograde signals that dilate upstream arterioles. These signals exhibit a unique mode of biphasic propagation. Slow, short-range intercellular Ca2+ signals in the capillary network are converted to rapid electrical signals in transitional segments that propagate to and dilate upstream arterioles. We further demonstrate that TRPA1 is necessary for functional hyperemia and neurovascular coupling within the somatosensory cortex of mice in vivo. These data establish endothelial cell TRPA1 channels as neuronal activity sensors that initiate microvascular vasodilatory responses to redirect blood to regions of metabolic demand.

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury.

Traumatic brain injury (TBI) acutely impairs dynamic regulation of local cerebral blood flow, but long-term (>72 h) effects on functional hyperemia are unknown. Functional hyperemia depends on capillary endothelial cell inward rectifier potassium channels (Kir2.1) responding to potassium (K+) released during neuronal activity to produce a regenerative, hyperpolarizing electrical signal that propagates from capillaries to dilate upstream penetrating arterioles. We hypothesized that TBI causes widespread disruption of electrical signaling from capillaries-to-arterioles through impairment of Kir2.1 channel function. We randomized mice to TBI or control groups and allowed them to recover for 4 to 7 days post-injury. We measured in vivo cerebral hemodynamics and arteriolar responses to local stimulation of capillaries with 10 mM K+ using multiphoton laser scanning microscopy through a cranial window under urethane and α-chloralose anesthesia. Capillary angio-architecture was not significantly affected following injury. However, K+-induced hyperemia was significantly impaired. Electrophysiology recordings in freshly isolated capillary endothelial cells revealed diminished Ba2+-sensitive Kir2.1 currents, consistent with a reduction in channel function. In pressurized cerebral arteries isolated from TBI mice, K+ failed to elicit the vasodilation seen in controls. We conclude that disruption of endothelial Kir2.1 channel function impairs capillary-to-arteriole electrical signaling, contributing to altered cerebral hemodynamics after TBI.

Apelin inhibits an endothelium-derived hyperpolarizing factor-like pathway in rat cerebral arteries.

Apelin has complex vasomotor actions inasmuch as the peptide may cause either vasodilation or vasoconstriction depending on the vascular bed and experimental conditions. In cerebral arteries, apelin inhibits endothelium-dependent relaxations mediated by nitric oxide (NO); however, its effects on relaxation to other endothelium-derived substances (e.g. prostacyclin, endothelium-derived hyperpolarizing factors(s) (EDHF)) are unknown. The present study was designed to determine effects of apelin on endothelium-dependent relaxations that are independent of NO in rat cerebral arteries. In arterial rings contracted with 5-HT, A23187 caused endothelium-dependent relaxation that was unaffected by inhibitors of eNOS, guanylyl cyclase or cyclooxygenase, but was attenuated by MS-PPOH, a selective inhibitor of cytochrome P450 catalyzed synthesis of epoxyeicosatrienoic acids (EETs) and by 14,15-EE(Z)E, an EET-receptor antagonist. Apelin inhibited A23187-induced relaxation, as well as relaxations evoked by exogenous 11,12- and 14,15-EET. These effects of apelin were mimicked by the selective BKCa channel blocker, iberiotoxin. The APJ receptor antagonist, F13A abolished the effects of apelin on A23187-induced relaxations. Both 11,12- and 14,15-EET also increased BKCa channel current density in isolated cerebral artery smooth muscle cells, effects that were inhibited in a similar manner by apelin and iberiotoxin. These findings provide evidence that apelin impairs endothelium-dependent relaxation of cerebral arteries by inhibiting an NO-independent pathway (i.e. "EDHF-like") involving activation of smooth muscle cell BKCa channels by endothelium-derived EETs. Inhibition of such pathway may create an environment favoring vasoconstriction in cerebral arteries.

Vascular effects of apelin: Mechanisms and therapeutic potential.

Apelin is a vasoactive peptide and is an endogenous ligand for APJ receptors, which are widely expressed in blood vessels, heart, and cardiovascular regulatory regions of the brain. A growing body of evidence now demonstrates a regulatory role for the apelin/APJ receptor system in cardiovascular physiology and pathophysiology, thus making it a potential target for cardiovascular drug discovery and development. Indeed, ongoing studies are investigating the potential benefits of apelin and apelin-mimetics for disorders such as heart failure and pulmonary arterial hypertension. Apelin causes relaxation of isolated arteries, and systemic administration of apelin typically results in a reduction in systolic and diastolic blood pressure and an increase in blood flow. Nonetheless, vasopressor responses and contraction of vascular smooth muscle in response to apelin have also been observed under certain conditions. The goal of the current review is to summarize major findings regarding the apelin/APJ receptor system in blood vessels, with an emphasis on regulation of vascular tone, and to identify areas of investigation that may provide guidance for the development of novel therapeutic agents that target this system.

Activation of Large Conductance, Calcium-Activated Potassium Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Isolated Rat Coronary Arteries.

Apelin increases coronary blood flow, cardiac contractility, and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and polymerase chain reaction analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in 4,5-diaminofluorescein fluorescence that was abolished by nitro-l-arginine (NLA) and F13A (H-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala-OH), an APJ receptor antagonist, consistent with increased nitric oxide (NO) production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch-clamp studies in isolated smooth muscle cells demonstrated that the NO donors, diethyl amine NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated potassium channel (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. These results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.

Apelin Reduces Nitric Oxide-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large-Conductance, Calcium-Activated K Channels.

Activation of the apelin/APJ receptor signaling system causes endothelium-dependent and nitric oxide (NO)-dependent relaxation in several peripheral arteries. The effects of apelin in cerebral arteries are unknown; however, apelin inhibits voltage-dependent increases in large-conductance, calcium-activated K channel (BKCa) currents in cerebral artery smooth muscle cells. Because NO-induced relaxation of cerebral arteries is mediated, in part, by activation of BKCa channels, the goals of this study were to determine the net effect of apelin in cerebral arteries, as well as test the hypothesis that the actions of apelin in cerebral arteries are secondary to stimulation of APJ receptors. Immunoblot and quantitative reverse transcription polymerase chain reaction analyses detected APJ receptors in cerebral arteries of male Sprague-Dawley rats, and immunofluorescence studies using confocal microscopy confirmed APJ receptor localization in smooth muscle cells. In myograph studies, apelin itself had no direct vasomotor effect but inhibited relaxations to the NO-donor, diethylamine NONOate, and to the endothelium-dependent vasodilator, bradykinin. These effects of apelin were mimicked by the selective BKCa-channel blocker, iberiotoxin, and suppressed by the APJ receptor antagonist, F13A. Apelin also inhibited relaxations evoked by the BKCa-channel openers, NS1619 and BMS 191011, but had no effect on relaxation to levcromakalim, a selective KATP-channel opener. Apelin had no effect on diethylamine NONOate-induced or bradykinin-induced increases in cyclic guanosine monophosphate levels. Patch clamp recordings demonstrated that apelin and iberiotoxin each suppressed the increase in BKCa currents induced by DEA and NS1619 in freshly isolated cerebral artery smooth muscle cells. The results demonstrate that apelin inhibits NO-induced relaxation of cerebral arteries through a mechanism involving activation of APJ receptors and inhibition of BKCa channels in cerebral arterial smooth muscle cells.

Effects of Thiazolidinediones on metabolism and cancer: Relative influence of PPARγ and IGF-1 signaling.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-gamma (PPARγ) agonists. TZDs are orally effective medicines for metabolic syndrome and type 2 diabetes. In addition to metabolic effects these molecules also possess anti-cancer effects. Data from diabetes clinical trials also support anti-cancer effects of TZDs. The anti-cancer effects of TZDs neither correlate well with their ability to activate PPARγ receptor, nor are affected by the presence of PPARγ receptor antagonists. Accumulating evidence suggests that TZDs act as selective inhibitors of insulin-like growth factor-1 (IGF-1) receptor signaling, and IGF-1 signaling is known to be aberrantly regulated in various cancers. Structural analysis of TZDs suggest that the presence of 5-exo C-C single bond of the thiazolidine-2,4-dione ring is important for the metabolic effects but not for anti-cancer effects, as inclusion of C=C double bond at this position promotes antagonistic properties to the PPARγ receptor without compromising its anti-proliferative effects. The objectives of this review includes summarization of the relative influence of TZDs on PPARγ and IGF-1 signaling in mediating pharmacological effects, and to discuss the possibility of multiple pharmacophores, and thereby independent regulation of PPARγ and IGF-1 signaling.