Abstract 052: Rho Kinase and Endoplasmic Reticulum Stress Mediate Peripheral Vascular Dysfunction in a Model of Small Vessel Disease of the Brain

Hypertension (HT) and CADASIL are clinical conditions of small vessel disease. Vascular dementia is a major feature in CADASIL, and a serious consequence of HT. CADASIL is a monogenic condition due to mutations in NOTCH3 , which is expressed almost exclusively in VSMCs. We hypothesised that altered NOTCH3 signalling in CADASIL and HT are associated with small vessel disease. Small arteries from gluteal biopsies from CADASIL patients (n=14), HT patients (n=3) and healthy controls (n=10) were investigated. Vascular function was assessed by myography. Cultured VSMCs were used to assess signaling through NOTCH3, NO, ER stress (gene array) and Rho kinase (ELISA). CADASIL and HT patients exhibited endothelial dysfunction (Max response: CADASIL 41.7±3%, HT 54.1±2% vs Control 98.2±4%). Pre-incubation with N-acetyl-cysteine ameliorated vasorelaxation. Only CADASIL displayed impaired endothelium-independent relaxation (Max response: CADASIL 53±1.9% vs Control 93±8.9%) and contraction (Max response: CADASIL 78±1.3% vs control 102±5%) (p<0.05). AngII-induced contraction was elevated in HT (98%), yet reduced in CADASIL (28%) (vs control 64% max contraction: p<0.05), despite VSMCs from both conditions displaying increased AT 1 R mRNA expression (HT: 5.1; CADASIL: 3.8; fold vs control; p<0.05). VSMCs from CADASIL and HT have decreased expression of CAMK1, SIRT2 and VEGFA; important in NO signalling (0.5 fold; p<0.05 vs control). VSMC levels of NOTCH3 and NOTCH ligand, JAG1, were increased in CADASIL (3.5, 2.5 fold) and HT (3.0, 2.6 fold, p<0.05). Downstream targets, HEY1 and HEYL, were elevated in CADASIL (3.8, 4.2 fold) and HT (1.9, 2.6 fold) (p<0.05). CADASIL but not HT VSMCs exhibited increased expression of ER stress markers. Rho kinase activity was increased in VSMCs from CADASIL (2.5 fold) and HT (2 fold) vs control (p<0.05). These data demonstrate that in CADASIL and HT, vascular dysfunction, is associated with aberrant NOTCH3 and Rho kinase signalling. In CADASIL, but not HT, endothelium-independent relaxation and ER stress were increased. Our results demonstrate a putative role for NOTCH3 -Rho kinase in vascular dysfunction in conditions of small vessel disease and suggest that ER stress and oxidative stress may be important in vascular injury in CADASIL.

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Abstract MP31: Peripheral Vascular Dysfunction In A Model Of Small Vessel Disease Of The Brain (CADASIL) Involves Impaired Redox-sensitive Cyclic GMP/PKG Signaling

Hypertension ◽

10.1161/hyp.76.suppl_1.mp31 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Karla B Neves ◽

Hannah Morris ◽

Rheure Alves-lopes ◽

Augusto C Montezano ◽

Rhian M Touyz

Keyword(s):

Soluble Guanylate Cyclase ◽

Small Vessel Disease ◽

Vascular Dysfunction ◽

Plasma Lipid ◽

Fold Increase ◽

Mesenteric Arteries ◽

Small Vessel ◽

Resistance Arteries ◽

Vessel Disease ◽

The Brain

CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3 R169C ) and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of Notch3 (WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p<0.01), suggesting increased Notch3 activation. CADASIL mice exhibited endothelial-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent dysfunction (pD 2 7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr 495 ) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (p<0.05). Plasma lipid peroxidation (0.8±0.1 vs. 2.0±0.3; p<0.05) and vascular reactive oxygen species (ROS) production (7.2±1.9 vs. 75.4±35.0; p<0.05) were increased in TgNotch3 R169C mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H 2 O 2 levels were decreased in TgNotch3 R169C mice (1.9±0.2 vs. 1.1±1.9; p<0.05), which was associated with reduced activation of protein kinase G (PKG). Observations in TgNotch3 R169C mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p<0.05) and sGC oxidation were also increased. Our findings demonstrate that the vasculopathy associated with a CADASIL Notch3 gain-of-function mutation in peripheral small vessels involves reduction in eNOS activation and redox-sensitive processes leading to impaired sGC/cGMP signalling pathway. We identify a potential new therapeutic target in CADASIL, for which there are no disease-specific treatments.

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Abstract P242: Microvascular Changes in Brain During Salt-sensitive Hypertension: Molecular Mechanisms and Implications for Small Vessel Disease

Hypertension ◽

10.1161/hyp.68.suppl_1.p242 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Travice M De Silva ◽

Justin Grobe ◽

Frank Faraci

Keyword(s):

Molecular Mechanisms ◽

Small Vessel Disease ◽

Rho Kinase ◽

Renin Angiotensin System ◽

Microvascular Dysfunction ◽

Small Vessel ◽

Vessel Disease ◽

Microvascular Changes ◽

Pial Arterioles ◽

The Brain

Hypertension is a major risk factor for small vessel disease (SVD), a leading contributor to stroke and dementias. Mechanisms that underlie SVD in brain are poorly defined, with no specific therapy at present. Because parenchymal and pial arterioles are targets of the SVD process, we examined microvascular changes in a model using deoxycorticosterone-salt (DOCA) to activate the brain renin-angiotensin system (RAS), with resulting salt-sensitive (sodium- and fluid-dependent) hypertension. Male C57Bl/6 mice were treated with DOCA and given the choice of drinking H 2 O or H 2 O with 0.9% NaCl for 3 wks. Along with a modest elevation in mean arterial pressure (79±2 vs 95±3 mmHg, P<0.05), DOCA impaired endothelium-dependent dilation of both isolated parenchymal (baseline diameter of 15±1 μm) and pial arterioles (37±1 μm) in a pathway specific manner. Endothelium-dependent hyperpolarization was intact while eNOS-mediated vasodilation was markedly impaired along with reductions in phosphorylation in AKT (an upstream activator of eNOS). Local inhibition of angiotensin II type 1 (AT1-R) or mineralocorticoid receptors (MR) or Rho kinase (including ROCK2), restored endothelial function in DOCA-treated mice. Inner diameter of maximally dilated parenchymal arterioles was reduced approximately 20% by DOCA (P<0.05 vs sham). DOCA increased mRNA expression of RAS components (eg, AGT, ACE) in both brain and cerebral vessels. In NZ44 reporter mice that express GFP driven by the AT1A-R promoter, DOCA increased cerebrovascular GFP protein expression about 3-fold (P<0.05). Thus, DOCA activates both the brain and the cerebrovascular RAS, impairs select pathways affecting parenchymal and pial arteriolar function, while producing inward microvascular remodeling. AT1R, MR and ROCK2 are key contributors to cerebral microvascular dysfunction in this clinically relevant model of SVD.

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Small Blood Vessel Disease in the Brain Theme Issue Overlapping protein accumulation profiles of CADASIL and CAA: Is there a common mechanism driving cerebral small vessel disease?

American Journal Of Pathology ◽

10.1016/j.ajpath.2020.11.015 ◽

2020 ◽

Author(s):

Kelly Z. Young ◽

Gang Xu ◽

Simon G. Keep ◽

Jimo Borjigin ◽

Michael M. Wang

Keyword(s):

Blood Vessel ◽

Small Vessel Disease ◽

Cerebral Small Vessel Disease ◽

Small Vessel ◽

Common Mechanism ◽

Protein Accumulation ◽

Theme Issue ◽

Small Blood Vessel ◽

Vessel Disease ◽

The Brain

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Role of Purinergic Signalling in Endothelial Dysfunction and Thrombo-Inflammation in Ischaemic Stroke and Cerebral Small Vessel Disease

Biomolecules ◽

10.3390/biom11070994 ◽

2021 ◽

Vol 11 (7) ◽

pp. 994

Author(s):

Natasha Ting Lee ◽

Lin Kooi Ong ◽

Prajwal Gyawali ◽

Che Mohd Nasril Che Mohd Nassir ◽

Muzaimi Mustapha ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Dysfunction ◽

Inflammatory Response ◽

Small Vessel Disease ◽

Ischemia Reperfusion ◽

Cerebral Small Vessel Disease ◽

Small Vessel ◽

Purinergic Signalling ◽

Vessel Disease ◽

The Brain

The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood–brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain.

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Physiology and Clinical Relevance of Enlarged Perivascular Spaces in the Aging Brain

Neurology ◽

10.1212/wnl.0000000000013077 ◽

2021 ◽

pp. 10.1212/WNL.0000000000013077

Author(s):

Corey W Bown ◽

Roxana O Carare ◽

Matthew S Schrag ◽

Angela L Jefferson

Keyword(s):

Fluid Transport ◽

Small Vessel Disease ◽

Treatment Strategies ◽

Small Vessel ◽

Aging Brain ◽

Perivascular Spaces ◽

Vessel Disease ◽

Clinical Consequences ◽

The Brain

Perivascular spaces (PVS) are fluid filled compartments that are part of the cerebral blood vessel wall and represent the conduit for fluid transport in and out of the brain. PVS are considered pathologic when sufficiently enlarged to be visible on magnetic resonance imaging. Recent studies have demonstrated that enlarged PVS (ePVS) may have clinical consequences related to cognition. Emerging literature points to arterial stiffening and abnormal protein aggregation in vessel walls as two possible mechanisms that drive ePVS formation. In this review, we describe the clinical consequences, anatomy, fluid dynamics, physiology, risk factors, and in vivo quantification methods of ePVS. Given competing views of PVS physiology, we detail the two most prominent theoretical views and review ePVS associations with other common small vessel disease markers. As ePVS are a marker of small vessel disease and ePVS burden is higher in Alzheimer’s disease, a comprehensive understanding about ePVS is essential in developing prevention and treatment strategies.

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Response to "Carotid Flow Augmentation as a Risk for Small Vessel Disease of the Brain"

American Journal of Hypertension ◽

10.1038/ajh.2010.122 ◽

2010 ◽

Vol 23 (9) ◽

pp. 933-933

Author(s):

K. Kohara ◽

N. Ochi ◽

Y. Tabara ◽

T. Miki

Keyword(s):

Small Vessel Disease ◽

Small Vessel ◽

Vessel Disease ◽

The Brain ◽

Carotid Flow

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Risk factors for small-vessel disease revealed by magnetic resonance imaging of the brain.

Nosotchu ◽

10.3995/jstroke.18.10 ◽

1996 ◽

Vol 18 (1) ◽

pp. 10-18

Author(s):

Tatsuo Kohriyama ◽

Shinya Yamaguchi ◽

Eiji Tanaka ◽

Yasuhiro Yamamura ◽

Shigenobu Nakamura

Keyword(s):

Magnetic Resonance Imaging ◽

Risk Factors ◽

Magnetic Resonance ◽

Small Vessel Disease ◽

Small Vessel ◽

Resonance Imaging ◽

Vessel Disease ◽

The Brain

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Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420765112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E796-E805 ◽

Cited By ~ 40

Author(s):

Fabrice Dabertrand ◽

Christel Krøigaard ◽

Adrian D. Bonev ◽

Emmanuel Cognat ◽

Thomas Dalsgaard ◽

...

Keyword(s):

Smooth Muscle ◽

Genetic Model ◽

Small Vessel Disease ◽

Early Stage ◽

Mesenteric Arteries ◽

Small Vessel ◽

Pial Arteries ◽

Vessel Disease ◽

Myogenic Responses ◽

The Brain

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3R169C mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3R169C mice. This effect was associated with an ∼60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3R169C mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3R169C mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.

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