A multi-disciplinary commentary on preclinical research to investigate vascular contributions to dementia.

Although dementia research has been dominated by Alzheimer's disease (AD), most dementia in older people is now recognised to be due to mixed pathologies, usually combining vascular and AD brain pathology. Vascular cognitive impairment (VCI), which encompasses vascular dementia (VaD) is the second most common type of dementia. Models of VCI have been delayed by limited understanding of the underlying aetiology and pathogenesis. This review by a multidisciplinary, diverse (in terms of sex, geography and career stage), cross-institute team provides a perspective on limitations to current VCI models and recommendations for improving translation and reproducibility. We discuss reproducibility, clinical features of VCI and corresponding assessments in models, human pathology, bioinformatics approaches, and data sharing. We offer recommendations for future research, particularly focusing on small vessel disease as a main underpinning disorder.

Acute, pro-contractile effects of prorenin on rat mesenteric arteries.

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin-angiotensin-aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca2+ ]i and force were recorded in arteries loaded with Fura-2AM. Spontaneously transient outward currents were recorded via perforated whole-cell patch-clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V-ATPase, caveolin-1, ryanodine receptors, and large conductance Ca2+ -activated K+ channels (BKCa ) in VSMCs. [Ca2+ ]i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1-adrenoreceptor-mediated contraction, associated with an increased number of Ca2+ waves, independent of voltage-gated Ca2+ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BKCa -mediated relaxation. Inhibition of the V-ATPase with 100 nM bafilomycin prevented prorenin-mediated inhibition of BKCa -derived relaxation. Renin (1 nM) had no effect on BKCa -mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BKCa and increasing intracellular Ca2+ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V-ATPase.

Uncoupling of Ca2+ sparks from BK channels in cerebral arteries underlies hypoperfusion in hypertension-induced vascular dementia.

The deficit in cerebral blood flow (CBF) seen in patients with hypertension-induced vascular dementia is increasingly viewed as a therapeutic target for disease-modifying therapy. Progress is limited, however, due to uncertainty surrounding the mechanisms through which elevated blood pressure reduces CBF. To investigate this, we used the BPH/2 mouse, a polygenic model of hypertension. At 8 mo of age, hypertensive mice exhibited reduced CBF and cognitive impairment, mimicking the human presentation of vascular dementia. Small cerebral resistance arteries that run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to diminished activity of large-conductance Ca2+-activated K+ (BK) channels-key vasodilatory ion channels of cerebral vascular smooth muscle cells. Activation of BK channels by transient intracellular Ca2+ signals from the sarcoplasmic reticulum (SR), termed Ca2+ sparks, leads to hyperpolarization and vasodilation. Combining patch-clamp electrophysiology, high-speed confocal imaging, and proximity ligation assays, we demonstrated that this vasodilatory mechanism is uncoupled in hypertensive mice, an effect attributable to physical separation of the plasma membrane from the SR rather than altered properties of BK channels or Ca2+ sparks, which remained intact. This pathogenic mechanism is responsible for the observed increase in constriction and can now be targeted as a possible avenue for restoring healthy CBF in vascular dementia.

Functionally linked potassium channel activity in cerebral endothelial and smooth muscle cells is compromised in Alzheimer's disease.

The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer's disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer's disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-ß similar to those in patients with Alzheimer's disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events ("Ca2+ sparks") in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-ß 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer's disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.

Cardiovascular comorbidities, inflammation, and cerebral small vessel disease.

Cerebral small vessel disease (cSVD) is the most common cause of vascular cognitive impairment and affects all levels of the brain's vasculature. Features include diverse structural and functional changes affecting small arteries and capillaries that lead to a decline in cerebral perfusion. Due to an ageing population, incidence of cSVD is continually rising. Despite its prevalence and its ability to cause multiple debilitating illnesses, such as stroke and dementia, there are currently no therapeutic strategies for the treatment of cSVD. In the healthy brain, interactions between neuronal, vascular, and inflammatory cells are required for normal functioning. When these interactions are disturbed, chronic pathological inflammation can ensue. The interplay between cSVD and inflammation has attracted much recent interest, and this review discusses chronic cardiovascular diseases, particularly hypertension, and explores how the associated inflammation may impact on the structure and function of the small arteries of the brain in cSVD. Molecular approaches in animal studies are linked to clinical outcomes in patients, and novel hypotheses regarding inflammation and cSVD are proposed that will hopefully stimulate further discussion and study in this important area.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

[Figure: see text].

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.

Nanoscale coupling of junctophilin-2 and ryanodine receptors regulates vascular smooth muscle cell contractility.

Junctophilin proteins maintain close contacts between the endoplasmic/sarcoplasmic reticulum (ER/SR) and the plasma membrane in many types of cells, as typified by junctophilin-2 (JPH2), which is necessary for the formation of the cardiac dyad. Here, we report that JPH2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) isolated from cerebral arteries and that acute knockdown diminishes the area of sites of interaction between the SR and plasma membrane. Superresolution microscopy revealed nanometer-scale colocalization of JPH2 clusters with type 2 ryanodine receptor (RyR2) clusters near the cell surface. Knockdown of JPH2 had no effect on the frequency, amplitude, or kinetics of spontaneous Ca2+ sparks generated by transient release of Ca2+ from the SR through RyR2s, but it did nearly abolish Ca2+ spark-activated, large-conductance, Ca2+-activated K+ (BK) channel currents. We also found that JPH2 knockdown was associated with hypercontractility of intact cerebral arteries. We conclude that JPH2 maintains functional coupling between RyR2s and BK channels and is critically important for cerebral arterial function.

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

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

Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility.

Junctional membrane complexes facilitate excitation-contraction coupling in skeletal and cardiac muscle cells by forming subcellular invaginations that maintain close (≤20 nm) proximity of ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) with voltage-dependent Ca2+ channels in the plasma membrane. In fully differentiated smooth muscle cells, junctional membrane complexes occur as distributed sites of peripheral coupling. We investigated the role of the cytoskeleton in maintaining peripheral coupling and associated Ca2+ signaling networks within native smooth muscle cells of mouse and rat cerebral arteries. Using live-cell confocal and superresolution microscopy, we found that the tight interactions between the SR and the plasma membrane in these cells relied on arching microtubule structures present at the periphery of smooth muscle cells and were independent of the actin cytoskeleton. Loss of peripheral coupling associated with microtubule depolymerization altered the spatiotemporal properties of localized Ca2+ sparks generated by the release of Ca2+ through type 2 RyRs (RyR2s) on the SR and decreased the number of sites of colocalization between RyR2s and large-conductance Ca2+-activated K+ (BK) channels. The reduced BK channel activity associated with the loss of SR-plasma membrane interactions was accompanied by increased pressure-induced constriction of cerebral resistance arteries. We conclude that microtubule structures maintain peripheral coupling in contractile smooth muscle cells, which is crucial for the regulation of contractility and cerebral vascular tone.

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

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

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

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

Molecular and functional significance of Ca(2+)-activated Cl(-) channels in pulmonary arterial smooth muscle.

Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca(2+) levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K(+) channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca(2+) channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl(-) and is activated by a rise in intracellular Ca(2+) concentration (Ca(2+)-activated Cl(-) channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca(2+) activating CaCCs, which include stimulation by mobilization from intracellular Ca(2+) stores and Ca(2+) entry through voltage-dependent and voltage-independent Ca(2+) channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH.

Potent vasorelaxant activity of the TMEM16A inhibitor T16A(inh) -A01.

BACKGROUND AND PURPOSE: T16A(inh) -A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A(inh) -A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone. EXPERIMENTAL APPROACH: Single channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery. KEY RESULTS: In rabbit pulmonary artery myocytes T16A(inh) -A01 (1-30 µM) inhibited single calcium (Ca(2+) )-activated chloride (Cl(-) ) channels and whole cell currents activated by 500 nM free Ca(2+) . Similar effects were observed for single Ca(2+) -activated Cl(-) channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, F(act) (10 µM), increased single channel and whole cell Ca(2+) -activated Cl(-) currents in rabbit pulmonary arteries. In isometric tension studies, T16A(inh) -A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC(50) of 1.6 µM and suppressed the methoxamine concentration-effect curve. T16A(inh) -A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 µM T16A(inh) -A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K(+) ) channel blockers. T16A(inh) -A01 (10 µM) also relaxed human visceral adipose arteries by 88 ± 3%. CONCLUSIONS AND IMPLICATIONS: T16A(inh) -A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels.