Impaired intracellular Ca2+ signaling contributes to age-related cerebral small vessel disease in Col4a1 mutant mice.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca2+ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1+/G1344D mice.

ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves.

PI3K block restores age-dependent neurovascular coupling defects associated with cerebral small vessel disease.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.

PI3K block restores age-dependent neurovascular coupling defects associated with cerebral small vessel disease.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP 2 ) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K + (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP 2 by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD. One-sentence summary: PI3K inhibition rescues neurovascular coupling defects in cerebral small vessel disease.

Endothelial cell TRPA1 activity exacerbates cerebral hemorrhage during severe hypertension.

Introduction: Hypoxia-induced dilation of cerebral arteries orchestrated by Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) cation channels on endothelial cells is neuroprotective during ischemic stroke, but it is unknown if the channel has a similar impact during hemorrhagic stroke. TRPA1 channels are endogenously activated by lipid peroxide metabolites generated by reactive oxygen species (ROS). Uncontrolled hypertension, a primary risk factor for the development of hemorrhagic stroke, is associated with increased ROS production and oxidative stress. Therefore, we hypothesized that TRPA1 channel activity is increased during hemorrhagic stroke. Methods: Severe, chronic hypertension was induced in control (Trpa1 fl/fl) and endothelial cell-specific TRPA1 knockout (Trpa1-ecKO) mice using a combination of chronic angiotensin II administration, a high-salt diet, and the addition of a nitric oxide synthase inhibitor to drinking water. Blood pressure was measured in awake, freely-moving mice using surgically placed radiotelemetry transmitters. TRPA1-dependent cerebral artery dilation was evaluated with pressure myography, and expression of TRPA1 and NADPH oxidase (NOX) isoforms in arteries from both groups was determined using PCR and Western blotting techniques. In addition, ROS generation capacity was evaluated using a lucigenin assay. Histology was performed to examine intracerebral hemorrhage lesion size and location. Results: All animals became hypertensive, and a majority developed intracerebral hemorrhages or died of unknown causes. Baseline blood pressure and responses to the hypertensive stimulus did not differ between groups. Expression of TRPA1 in cerebral arteries from control mice was not altered after 28 days of treatment, but expression of three NOX isoforms and the capacity for ROS generation was increased in hypertensive animals. NOX-dependent activation of TRPA1 channels dilated cerebral arteries from hypertensive animals to a greater extent compared with controls. The number of intracerebral hemorrhage lesions in hypertensive animals did not differ between control and Trpa1-ecKO animals but were significantly smaller in Trpa1-ecKO mice. Morbidity and mortality did not differ between groups. Discussion: We conclude that endothelial cell TRPA1 channel activity increases cerebral blood flow during hypertension resulting in increased extravasation of blood during intracerebral hemorrhage events; however, this effect does not impact overall survival. Our data suggest that blocking TRPA1 channels may not be helpful for treating hypertension-associated hemorrhagic stroke in a clinical setting.

Vascular mechanotransduction.

This review aims to survey the current state of mechanotransduction in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), including their sensing of mechanical stimuli and transduction of mechanical signals that result in the acute functional modulation and longer-term transcriptomic and epigenetic regulation of blood vessels. The mechanosensors discussed include ion channels, plasma membrane-associated structures and receptors, and junction proteins. The mechanosignaling pathways presented include the cytoskeleton, integrins, extracellular matrix, and intracellular signaling molecules. These are followed by discussions on mechanical regulation of transcriptome and epigenetics, relevance of mechanotransduction to health and disease, and interactions between VSMCs and ECs. Throughout this review, we offer suggestions for specific topics that require further understanding. In the closing section on conclusions and perspectives, we summarize what is known and point out the need to treat the vasculature as a system, including not only VSMCs and ECs but also the extracellular matrix and other types of cells such as resident macrophages and pericytes, so that we can fully understand the physiology and pathophysiology of the blood vessel as a whole, thus enhancing the comprehension, diagnosis, treatment, and prevention of vascular diseases.

Faulty TRPM4 channels underlie age-dependent cerebral vascular dysfunction in Gould syndrome.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is necessary for TRPM4 activity. Dialyzing SMCs with PIP2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-ß) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-ß signaling pathways stimulates PI3K to deplete PIP2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP2 depletion, revealing an age-dependent mechanism of cSVD.

STIM1 is the key that unlocks airway smooth muscle remodeling and hyperresponsiveness during asthma.

Airway smooth muscle remodeling and hyperresponsiveness are critical determinants of asthma severity, but the precise mechanisms regulating these disease processes remain elusive. In their latest study published in PNAS, Trebak and colleagues demonstrate that STIM1 (stromal-interacting molecule 1) expression is upregulated in airway smooth muscle cells during asthma and facilitates Ca2+ influx to drive airway remodeling and hyperresponsiveness.

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

Nitric Oxide Signals Through IRAG to Inhibit TRPM4 Channels and Dilate Cerebral Arteries.

Nitric oxide (NO) relaxes vascular smooth muscle cells (SMCs) and dilates blood vessels by increasing intracellular levels of cyclic guanosine monophosphate (cGMP), which stimulates the activity of cGMP-dependent protein kinase (PKG). However, the vasodilator mechanisms downstream of PKG remain incompletely understood. Here, we found that transient receptor potential melastatin 4 (TRPM4) cation channels, which are activated by Ca2+ released from the sarcoplasmic reticulum (SR) through inositol triphosphate receptors (IP3Rs) under native conditions, are essential for SMC membrane depolarization and vasoconstriction. We hypothesized that signaling via the NO/cGMP/PKG pathway causes vasodilation by inhibiting TRPM4. We found that TRPM4 currents activated by stretching the plasma membrane or directly activating IP3Rs were suppressed by exogenous NO or a membrane-permeable cGMP analog, the latter of which also impaired IP3R-mediated release of Ca2+ from the SR. The effects of NO on TRPM4 activity were blocked by inhibition of soluble guanylyl cyclase or PKG. Notably, upon phosphorylation by PKG, IRAG (IP3R-associated PKG substrate) inhibited IP3R-mediated Ca2+ release, and knockdown of IRAG expression diminished NO-mediated inhibition of TRPM4 activity and vasodilation. Using superresolution microscopy, we found that IRAG, PKG, and IP3Rs form a nanoscale signaling complex on the SR of SMCs. We conclude that NO/cGMP/PKG signaling through IRAG inhibits IP3R-dependent activation of TRPM4 channels in SMCs to dilate arteries. SIGNIFICANCE STATEMENT: Nitric oxide is a gaseous vasodilator produced by endothelial cells that is essential for cardiovascular function. Although NO-mediated signaling pathways have been intensively studied, the mechanisms by which they relax SMCs to dilate blood vessels remain incompletely understood. In this study, we show that NO causes vasodilation by inhibiting the activity of Ca2+-dependent TRPM4 cation channels. Probing further, we found that NO does not act directly on TRPM4 but instead initiates a signaling cascade that inhibits its activation by blocking the release of Ca2+ from the SR. Thus, our findings reveal the essential molecular pathways of NO-induced vasodilation-a fundamental unresolved concept in cardiovascular physiology.

Transient Receptor Potential Channel Ankyrin 1: A Unique Regulator of Vascular Function.

TRPA1 (transient receptor potential ankyrin 1), the lone member of the mammalian ankyrin TRP subfamily, is a Ca2+-permeable, non-selective cation channel. TRPA1 channels are localized to the plasma membranes of various cells types, including sensory neurons and vascular endothelial cells. The channel is endogenously activated by byproducts of reactive oxygen species, such as 4-hydroxy-2-noneal, as well as aromatic, dietary molecules including allyl isothiocyanate, a derivative of mustard oil. Several studies have implicated TRPA1 as a regulator of vascular tone that acts through distinct mechanisms. First, TRPA1 on adventitial sensory nerve fibers mediates neurogenic vasodilation by stimulating the release of the vasodilator, calcitonin gene-related peptide. Second, TRPA1 is expressed in the endothelium of the cerebral vasculature, but not in other vascular beds, and its activation results in localized Ca2+ signals that drive endothelium-dependent vasodilation. Finally, TRPA1 is functionally present on brain capillary endothelial cells, where its activation orchestrates a unique biphasic propagation mechanism that dilates upstream arterioles. This response is vital for neurovascular coupling and functional hyperemia in the brain. This review provides a brief overview of the biophysical and pharmacological properties of TRPA1 and discusses the importance of the channel in vascular control and pathophysiology.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

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.

The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.