Mitochondrial Ca2+-coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund’s adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human post-mortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ipsilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions

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

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-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β 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

Video1_Endothelial cell TRPA1 activity exacerbates cerebral hemorrhage during severe hypertension.MP4

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 (Trpa1fl/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.</p