Distinct potassium channel types in brain capillary pericytes.

Capillaries, composed of electrically coupled endothelial cells and overlying pericytes, constitute the vast majority of blood vessels in the brain. The most arteriole-proximate three to four branches of the capillary bed are covered by α-actin-expressing, contractile pericytes. These mural cells have a distinctive morphology and express different markers compared with their smooth muscle cell (SMC) cousins but share similar excitation-coupling contraction machinery. Despite this similarity, pericytes are considerably more depolarized than SMCs at low intravascular pressures. We have recently shown that pericytes, such as SMCs, possess functional voltage-dependent Ca2+ channels and ATP-sensitive K+ channels. Here, we further investigate the complement of pericyte ion channels, focusing on members of the K+ channel superfamily. Using NG2-DsRed-transgenic mice and diverse configurations of the patch-clamp technique, we demonstrate that pericytes display robust inward-rectifier K+ currents that are primarily mediated by the Kir2 family, based on their unique biophysical characteristics and sensitivity to micromolar concentrations of Ba2+. Moreover, multiple lines of evidence, including characteristic kinetics, sensitivity to specific blockers, biophysical attributes, and distinctive single-channel properties, established the functional expression of two voltage-dependent K+ channels: KV1 and BKCa. Although these three types of channels are also present in SMCs, they exhibit distinctive current density and kinetics profiles in pericytes. Collectively, these findings underscore differences in the operation of shared molecular features between pericytes and SMCs and highlight the potential contribution of these three K+ ion channels in setting pericyte membrane potential, modulating capillary hemodynamics, and regulating cerebral blood flow.

A new model for evaluating pressure-induced vascular tone in small cerebral arteries.

The capacity of small cerebral arteries (SCAs) to adapt to pressure fluctuations has a fundamental physiological role and appears to be relevant in different pathological conditions. Here, we present a new computational model for quantifying the link, and its contributors, between luminal pressure and vascular tone generation in SCAs. This is assembled by combining a chemical sub-model, representing pressure-induced smooth muscle cell (SMC) signalling, with a mechanical sub-model for the tone generation and its transduction at tissue level. The devised model can accurately reproduce the impact of luminal pressure on different cytoplasmic components involved in myogenic signalling, both in the control case and when combined with some specific pharmacological interventions. Furthermore, the model is also able to capture and predict experimentally recorded pressure-outer diameter relationships obtained for vessels under control conditions, both in a Ca 2 + -free bath and under drug inhibition. The modularity of the proposed framework allows the integration of new components for the study of a broad range of processes involved in the vascular function.

Mechanosensing by Vascular Endothelium.

Mechanical forces influence different cell types in our bodies. Among the earliest forces experienced in mammals is blood movement in the vascular system. Blood flow starts at the embryonic stage and ceases when the heart stops. Blood flow exposes endothelial cells (ECs) that line all blood vessels to hemodynamic forces. ECs detect these mechanical forces (mechanosensing) through mechanosensors, thus triggering physiological responses such as changes in vascular diameter. In this review, we focus on endothelial mechanosensing and on how different ion channels, receptors, and membrane structures detect forces and mediate intricate mechanotransduction responses. We further highlight that these responses often reflect collaborative efforts involving several mechanosensors and mechanotransducers. We close with a consideration of current knowledge regarding the dysregulation of endothelial mechanosensing during disease. Because hemodynamic disruptions are hallmarks of cardiovascular disease, studying endothelial mechanosensing holds great promise for advancing our understanding of vascular physiology and pathophysiology.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries.

Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca2+ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca2+ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca2+ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca2+ signals and could, therefore, have a profound impact on central nervous system blood flow control.

Vascular calcium signalling and ageing.

Changes in cellular Ca2+ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca2+ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing per se on vascular Ca2+ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca2+ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca2+ channels, Ca2+ -activated K+ channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca2+ handling was suppressed in ageing mainly via reduced expression of intracellular Ca2+ release channels, and Ca2+ reuptake or efflux pumps. An increase in mitochondrial Ca2+ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca2+ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca2+ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world's ageing population, future studies should be directed at solving specific ageing-induced Ca2+ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases.

Traumatic Brain Injury Impairs Systemic Vascular Function Through Disruption of Inward-Rectifier Potassium Channels.

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2-induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.

Differential restoration of functional hyperemia by antihypertensive drug classes in hypertension-related cerebral small vessel disease.

Dementia resulting from small vessel diseases (SVDs) of the brain is an emerging epidemic for which there is no treatment. Hypertension is the major risk factor for SVDs, but how hypertension damages the brain microcirculation is unclear. Here, we show that chronic hypertension in a mouse model progressively disrupts on-demand delivery of blood to metabolically active areas of the brain (functional hyperemia) through diminished activity of the capillary endothelial cell inward-rectifier potassium channel, Kir2.1. Despite similar efficacy in reducing blood pressure, amlodipine, a voltage-dependent calcium-channel blocker, prevented hypertension-related damage to functional hyperemia whereas losartan, an angiotensin II type 1 receptor blocker, did not. We attribute this drug class effect to losartan-induced aldosterone breakthrough, a phenomenon triggered by pharmacological interruption of the renin-angiotensin pathway leading to elevated plasma aldosterone levels. This hypothesis is supported by the finding that combining losartan with the aldosterone receptor antagonist eplerenone prevented the hypertension-related decline in functional hyperemia. Collectively, these data suggest Kir2.1 as a possible therapeutic target in vascular dementia and indicate that concurrent mineralocorticoid aldosterone receptor blockade may aid in protecting against late-life cognitive decline in hypertensive patients treated with angiotensin II type 1 receptor blockers.

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.

Zinc drives vasorelaxation by acting in sensory nerves, endothelium and smooth muscle.

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity.

Cerebral small vessel diseases (SVDs) are a central link between stroke and dementia-two comorbidities without specific treatments. Despite the emerging consensus that SVDs are initiated in the endothelium, the early mechanisms remain largely unknown. Deficits in on-demand delivery of blood to active brain regions (functional hyperemia) are early manifestations of the underlying pathogenesis. The capillary endothelial cell strong inward-rectifier K+ channel Kir2.1, which senses neuronal activity and initiates a propagating electrical signal that dilates upstream arterioles, is a cornerstone of functional hyperemia. Here, using a genetic SVD mouse model, we show that impaired functional hyperemia is caused by diminished Kir2.1 channel activity. We link Kir2.1 deactivation to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid essential for Kir2.1 activity. Systemic injection of soluble PIP2 rapidly restored functional hyperemia in SVD mice, suggesting a possible strategy for rescuing functional hyperemia in brain disorders in which blood flow is disturbed.

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.

Aging, calcium channel signaling and vascular tone.

Calcium signaling in vascular smooth muscle is crucial for arterial tone regulation and vascular function. Several proteins, including Ca2+ channels, function in an orchestrated fashion so that blood vessels can sense and respond to physiological stimuli such as changes in intravascular pressure. Activation of the voltage-dependent Ca2+ channel, Cav1.2, leads to Ca2+ influx and consequently arterial tone development and vasoconstriction. Unique among Ca2+ channels, the vascular Cav3.2 T-type channel mediates feedback inhibition of arterial tone-and therefore causes vasodilation-of resistance arteries by virtue of functional association with hyperpolarizing ion channels. During aging, several signaling modalities are altered along with vascular remodeling. There is a growing appreciation of how calcium channel signaling alters with aging and how this may affect vascular function. Here, we discuss key determinants of arterial tone development and the crucial involvement of Ca2+ channels. We next provide an updated view of key changes in Ca2+ channel expression and function during aging and how these affect vascular function. Further, this article synthesizes new questions in light of recent developments. We hope that these questions will outline a roadmap for new research, which, undoubtedly, will unravel a more comprehensive picture of arterial tone dysfunction during aging.

PIP2: A critical regulator of vascular ion channels hiding in plain sight.

The phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), has long been established as a major contributor to intracellular signaling, primarily by virtue of its role as a substrate for phospholipase C (PLC). Signaling by Gq-protein-coupled receptors triggers PLC-mediated hydrolysis of PIP2 into inositol 1,4,5-trisphosphate and diacylglycerol, which are well known to modulate vascular ion channel activity. Often overlooked, however, is the role PIP2 itself plays in this regulation. Although numerous reports have demonstrated that PIP2 is critical for ion channel regulation, how it impacts vascular function has received scant attention. In this review, we focus on PIP2 as a regulator of ion channels in smooth muscle cells and endothelial cells-the two major classes of vascular cells. We further address the concerted effects of such regulation on vascular function and blood flow control. We close with a consideration of current knowledge regarding disruption of PIP2 regulation of vascular ion channels in disease.

The capillary Kir channel as sensor and amplifier of neuronal signals: Modeling insights on K+-mediated neurovascular communication.

Neuronal activity leads to an increase in local cerebral blood flow (CBF) to allow adequate supply of oxygen and nutrients to active neurons, a process termed neurovascular coupling (NVC). We have previously shown that capillary endothelial cell (cEC) inwardly rectifying K+ (Kir) channels can sense neuronally evoked increases in interstitial K+ and induce rapid and robust dilations of upstream parenchymal arterioles, suggesting a key role of cECs in NVC. The requirements of this signal conduction remain elusive. Here, we utilize mathematical modeling to investigate how small outward currents in stimulated cECs can elicit physiologically relevant spread of vasodilatory signals within the highly interconnected brain microvascular network to increase local CBF. Our model shows that the Kir channel can act as an "on-off" switch in cECs to hyperpolarize the cell membrane as extracellular K+ increases. A local hyperpolarization can be amplified by the voltage-dependent activation of Kir in neighboring cECs. Sufficient Kir density enables robust amplification of the hyperpolarizing stimulus and produces responses that resemble action potentials in excitable cells. This Kir-mediated excitability can remain localized in the stimulated region or regeneratively propagate over significant distances in the microvascular network, thus dramatically increasing the efficacy of K+ for eliciting local hyperemia. Modeling results show how changes in cEC transmembrane current densities and gap junctional resistances can affect K+-mediated NVC and suggest a key role for Kir as a sensor of neuronal activity and an amplifier of retrograde electrical signaling in the cerebral vasculature.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw.

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.