Cerebral Vascular Smooth Muscle Cells Research Articles

Inhibition of Soluble Epoxide Hydrolase Ameliorates Cerebral Blood Flow Autoregulation and Cognition in Alzheimer's Disease and Diabetes-Related Dementia Rat Models.

Alzheimer's Disease and Alzheimer's Disease-related dementias (AD/ADRD) pose major global healthcare challenges, with diabetes mellitus (DM) being a key risk factor. Both AD and DM-related ADRD are characterized by reduced cerebral blood flow, although the exact mechanisms remain unclear. We previously identified compromised cerebral hemodynamics as early signs in TgF344-AD and type 2 DM-ADRD (T2DN) rat models. Genome-wide studies have linked AD/ADRD to SNPs in soluble epoxide hydrolase (sEH). This study explored the effects of sEH inhibition with TPPU on cerebral vascular function and cognition in AD and DM-ADRD models. Chronic TPPU treatment improved cognition in both AD and DM-ADRD rats without affecting body weight. In DM-ADRD rats, TPPU reduced plasma glucose and HbA1C levels. Transcriptomic analysis of primary cerebral vascular smooth muscle cells from AD rats treated with TPPU revealed enhanced pathways related to cell contraction, alongside decreased oxidative stress and inflammation. Both AD and DM-ADRD rats exhibited impaired myogenic responses and autoregulation in the cerebral circulation, which were normalized with chronic sEH inhibition. Additionally, TPPU improved acetylcholine-induced vasodilation in the middle cerebral arteries (MCA) of DM-ADRD rats. Acute TPPU administration unexpectedly caused vasoconstriction in the MCA of DM-ADRD rats at lower doses. In contrast, higher doses or longer durations were required to induce effective vasodilation at physiological perfusion pressure in both control and ADRD rats. Additionally, TPPU decreased reactive oxygen species production in cerebral vessels of AD and DM-ADRD rats. These findings provide novel evidence that chronic sEH inhibition can reverse cerebrovascular dysfunction and cognitive impairments in AD/ADRD, offering a promising avenue for therapeutic development.

CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small- and medium-sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid β, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and varying cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody, and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in a total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. The overexpression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reductions in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

Amyloid Beta Oligomers Activate Death Receptors and Mitochondria-Mediated Apoptotic Pathways in Cerebral Vascular Smooth Muscle Cells; Protective Effects of Carbonic Anhydrase Inhibitors.

Amyloid beta (Aβ) deposition within the brain vasculature is an early hallmark of Alzheimer's disease (AD), which triggers loss of brain vascular smooth muscle cells (BVSMCs) in cerebral arteries, via poorly understood mechanisms, altering cerebral blood flow, brain waste clearance, and promoting cognitive impairment. We have previously shown that, in brain endothelial cells (ECs), vasculotropic Aβ species induce apoptosis through death receptors (DRs) DR4 and DR5 and mitochondria-mediated mechanisms, while FDA-approved carbonic anhydrase inhibitors (CAIs) prevent mitochondria-mediated EC apoptosis in vitro and in vivo. In this study, we analyzed Aβ-induced extrinsic and intrinsic (DR- and mitochondria-mediated) apoptotic pathways in BVSMC, aiming to unveil new therapeutic targets to prevent BVSMC stress and death. We show that both apoptotic pathways are activated in BVSMCs by oligomeric Aβ42 and Aβ40-Q22 (AβQ22) and mitochondrial respiration is severely impaired. Importantly, the CAIs methazolamide (MTZ) and acetazolamide (ATZ) prevent the pro-apoptotic effects in BVSMCs, while reducing caspase 3 activation and Aβ deposition in the arterial walls of TgSwDI animals, a murine model of cerebral amyloid angiopathy (CAA). This study reveals new molecular targets and a promising therapeutic strategy against BVSMC dysfunction in AD, CAA, and ARIA (amyloid-related imaging abnormalities) complications of recently FDA-approved anti-Aβ antibodies.

Integration of single-nucleus RNA sequencing and network disturbance to elucidate crosstalk between multicomponent drugs and trigeminal ganglia cells in migraine

Ethnopharmacological relevanceMigraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) plays an important role. TG is composed of multiple neuronal and non-neuronal cell types, which is related to “neuro-inflammation-vascular” disorder in migraine. Tou Tong Ning capsule (TTNC), a CFDA-approved traditional Chinese medicine for treating migraine, has the characteristics of “multicomponents, multitargets, multipathways". Aim of the studyTo clarify the mechanism of TTNC and elucidate crosstalk between multicomponent drugs and neuronal and non-neuronal functions and cells in migraine. Materials and methodsWe integrated single-nucleus RNA sequencing and a quantitative evaluation algorithm of the disturbance of multitarget drugs on the disease network and explored the specific pathology of migraine and corresponding compounds. A cerebrovascular smooth muscle spasmolytic activity experiment was carried out to verify the results of the bioinformatics analysis. ResultsTTNC exhibited its regulation activities in neuronal and non-neuronal aspects based on drugs attack to four subnetworks and cell specific networks, which explored the MoA of TTNC in comprehensive and refined perspectives. Compared to neuronal regulation, TTNC showed more significant attack score on non-neuronal biological function (smooth muscle and vessel). And TTNC compound clusters C1, C6 and C7, targeting non-neuronal function and cells, had larger group area than C10, C4 and C6 for neuronal function and cell, which implied that TTNC may mainly regulate the non-neuronal function, e.g., vessel smooth muscle contraction. Contraction of cerebrovascular smooth muscle of mice ex vivo confirmed the vasodilation activity of TTNC and active compounds from C1, C6, C9 (Emodin, Luteolin and Levistilide A). Literature mining confirmed the vasospasmodolytic activity and neuroprotective effect of TTNC. ConclusionsThe study found that TTNC may primarily alleviate non-neuronal functional disorders in migraine by relaxing cerebral vascular smooth muscle cell spasm to alleviate migraine. Integrating single-nucleus RNA sequencing data and network disturbance tools provides a new strategy for the pharmacological mechanism of multicomponent drugs through cell subtyping.

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.

An Engineered Adeno-Associated Virus Capsid Mediates Efficient Transduction of Pericytes and Smooth Muscle Cells of the Brain Vasculature.

Neurodegeneration and cerebrovascular disease share an underlying microvascular dysfunction that may be remedied by selective transgene delivery. To date, limited options exist in which cellular components of the brain vasculature can be effectively targeted by viral vector therapeutics. In this study, we characterize the first engineered adeno-associated virus (AAV) capsid mediating high transduction of cerebral vascular pericytes and smooth muscle cells (SMCs). We performed two rounds of in vivo selection with an AAV capsid scaffold displaying a heptamer peptide library to isolate capsids that traffic to the brain after intravenous delivery. One identified capsid, termed AAV-PR, demonstrated high transduction of the brain vasculature, in contrast to the parental capsid, AAV9, which transduces mainly neurons and astrocytes. Further analysis using tissue clearing, volumetric rendering, and colocalization revealed that AAV-PR enabled high transduction of cerebral pericytes located on small-caliber vessels and SMCs in the larger arterioles and penetrating pial arteries. Analysis of tissues in the periphery indicated that AAV-PR also transduced SMCs in large vessels associated with the systemic vasculature. AAV-PR was also able to transduce primary human brain pericytes with higher efficiency than AAV9. Compared with previously published AAV capsids tropisms, AAV-PR represents the first capsid to allow for effective transduction of brain pericytes and SMCs and offers the possibility of genetically modulating these cell types in the context of neurodegeneration and other neurological diseases.

MicroRNA-130b May Induce Cerebral Vasospasm after Subarachnoid Hemorrhage via Modulating Kruppel-like Factor 4.

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for invitro experimentation. In vitro and invivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 (KLF4), oe-KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH. Elevated miR-130b and reduced KLF4 were found in SAH patients and rat models of SAH. KLF4 was the target gene of miR-130b. miR-130b promoted the proliferation and migration of cVSMCs through the Inhibition of KLF4. Besides, KLF4 inhibited the proliferation and migration of cVSMCs through blockage of the p38/MAPK pathway. Furthermore, invivo assay confirmed the inhibitory effect of decreased miR-130b in CVS following SAH. In conclusion, miR-130b may activate the p38/MAPK signaling pathway through targeted inhibition of KLF4, thereby contributing to some extent to the development of cerebral vasospasm after SAH.

S-18-2: HYPERTENSION AS A DRIVER OF CEREBRAL SMALL VESSEL DISEASE AND DEMENTIA

Hypertension is the principal cause of vascular dementia. Unfortunately however, lowering blood pressure after the onset of cognitive decline does not slow disease progression and currently, vascular dementia is a disease with no available treatment. A principal feature of hypertension-related vascular dementia is a reduction in cerebral blood flow. Precisely how elevated blood pressure reduces cerebral blood flow is unknown and to investigate this further we used a polygenic model of hypertension, the BPH/2 mouse. Aged 8 months, BPH/2 mice exhibited cognitive impairment mimicking the human presentation of vascular dementia and in addition, cerebral blood flow was reduced. Small cerebral resistance arteries which run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to lack of activity of large conductance Ca2+ activated K+ channels (BK channels), key vasodilatory ion channels of cerebral vascular smooth muscle cells. BK channels are activated by transient intracellular signals from the sarcoplasmic reticulum called Ca2+ sparks, leading to hyperpolarisation and vasodilation. Combining patch clamp electrophyiology, high speed confocal imaging and proximity ligation assays we demonstrated that while the biophysical properties of BK channels and Ca2+ sparks remained intact, physical separation of the plasma membrane from the sarcoplasmic reticulum uncoupled the vasodilatory mechanism. This novel pathogenic mechanism is responsible for the increased constriction and can be now targeted as a possible avenue to restore healthy cerebral blood flow in vascular dementia.

Amyloid‐mediated TRAIL DRs activation leads to progressive degeneration of cerebral vascular smooth muscle cells

AbstractBackgroundVascular accumulation of amyloid beta (Ab) leads to loss of vascular smooth muscle cells (VSMC) in cerebral arteries via unknown mechanisms, promoting cerebral blood flow alterations and contributing to cognitive impairment. Ab species with different lengths or mutated familial variants may have differential effects on VSMC. Ab1‐42, the main pathogenic form of amyloid in the AD brain with the highest propensity to form aggregates, increased apoptosis in brain vascular cells whereas Ab1‐40 was found to be less toxic. By comparison, the familial Ab variant E22Q, induced the most cell death of all the amyloid variants tested. However, there has been little progress in defining the pathways leading to VSMC degeneration in AD and related disorders. The aim of this study was to identify Ab‐mediated cell death mechanisms in VSMC.MethodHuman cerebral VSMC were treated with pre‐oligomerized variants of Ab for 2‐8hrs (acute treatment) and 24‐72hrs (chronic treatment). Analysis of fragmented nucleosomes and visualization of cytochrome c (Cyt. C) expression by immunocytochemistry were used to measure apoptosis. Activation of intrinsic and extrinsic apoptotic pathways were examined by western blot and caspase‐glo assays. The effects of Ab on mitochondrial function were examined using a Seahorse XF96 Analyzer and TMRM fluorescent dye in live cells.ResultsAcute Ab treatment in VSMC up‐regulated tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) death receptors DR4 and DR5 with activation of caspase 8 and transient activation of Nuclear Factor k B (NF‐kB). DR4 and DR5 expression remained high with chronic Ab treatment and lead to caspase activation followed by Cyt. C release and apoptosis. 24hrs of Ab treatment also decreased mitochondrial membrane potential and significantly impaired mitochondrial respiration.ConclusionAcute exposure of VSMC to Ab causes up‐regulation and activation of DR4/DR5, that, over time, results in mitochondrial dysfunction, caspase activation and apoptosis. Our data suggests that an early and transient activation of NF‐kB may also contribute to the toxic effects of Ab, possibly by inducing pro‐inflammatory pathways in the neurovascular unit.

Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity.

Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor α (ERα)-NRF1-OMI-mitophagy signaling was involved in the dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) under simulated microgravity. Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.

VEGFR2 in vascular smooth muscle cells mediates H2S-induced dilation of the rat cerebral basilar artery

The aim of present study was to study whether the vascular endothelial growth factor receptor 2 (VEGFR2) mediates hydrogen sulfide (H2S)-induced relaxation of the rat cerebral vasculature. Relaxation of cerebral basilar artery (CBA) and vascular smooth muscle cells (VSMCs) was measured by using a pressure myograph system and image analysis system, respectively. The intracellular calcium concentration ([Ca2+]i) in VSMCs was detected using fluorescence imaging analysis. We found that H2S donor NaHS induced significant relaxation of VSMCs from the CBA of wild type rat, but in VEGFR2 knockdown VSMCs, NaHS-induced relaxation reduced markedly. In addition, NaHS-induced vasodilation of rat CBA also attenuated obviously when the expression of VEGFR2 was knocked down in vivo. In addition, pretreatment with the VEGFR2 blocker SU5416 likewise lowered the NaHS-induced relaxation of rat CBA. Nevertheless, the VEGFR2 agonist, vascular endothelial growth factor 164 (VEGF164), induced a concentration-dependent relaxation of CBA, which is similar to the effect of NaHS. Furthermore, we found that both NaHS and VEGF164 significantly inhibited the U46619-induced increase of [Ca2+]i fluorescence intensity in the VSMCs. However, the inhibitory effect of NaHS on the [Ca2+]i fluorescence intensity in VSMCs was markedly inhibited by pretreatment with SU5416 or VEGFR2 knockdown. These findings indicated that H2S-induced CBA dilation and reduction of [Ca2+]i in VSMCs occur by acting on VEGFR2.

Molecular Mechanism of Electroacupuncture Regulating Cerebral Arterial Contractile Protein in Rats with Cerebral Infarction Based on MLCK Pathway.

To explore the effect of electroacupuncture (EA) intervention on the vasoconstriction of cerebral artery smooth muscle cells after cerebral infarction. Male Wistar rats were randomly divided into 3 groups by a random number table: the model group (n=24), the EA group (n=24), and the normal group (n=6). The model and the EA groups were divided into different time subgroups at 0.5, 1, 3, and 6 h after middle cerebral artery occlusion (MCAO), with 6 rats in each subgroup. MCAO model was established using intraluminal suture occlusion method. The EA group was given EA treatment at acupoint Shuigou (GV 26) instantly after MCAO for 20 min. The contents of cerebrovascular smooth muscle MLCK, the 3 subunits of myosin light chain phosphatase (MLCP) MYPT1, PP1c-δ and M20, as well as myosin-ATPase activity were detected using immunohistochemistry and Western blotting. The overall expression level of the MYPT1 and PP1c-δ in the model group was significantly higher (P<0.01). After EA intervention, the 0.5 h group expression level was close to that of the normal group (P>0.05), and the other subgroups were still significantly higher than the normal group (P<0.01). After EA intervention, the expression level of each subgroup was significantly lower than the corresponding model group. There was a significant difference between the 0.5 and 1 h subgroups (P<0.01), while a difference was also observed between the 3 and 6 h subgroups (P<0.05). The dynamic change rule gradually increased with the prolongation of infarction time within 6 h after infarction. EA intervention can inhibit contraction of cerebral vascular smooth muscle cells and regulate smooth muscle relaxation by regulating MLCK pathway.

Peroxisome proliferator‑activated receptor β/δ regulates cerebral vasospasm after subarachnoid hemorrhage via modulating vascular smooth muscle cells phenotypic conversion.

Cerebral vasospasm (CVS) is a common complication of subarachnoid hemorrhage (SAH) with high deformity rates and cerebral vascular smooth muscle cells (VSMCs) phenotypic switch is considered to be involved in the regulation of CVS. However, to the best of the authors' knowledge, its underlying molecular mechanism remains to be elucidated. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been demonstrated to be involved in the modulation of vascular cells proliferation and maintains the autoregulation function of blood vessels. The present study investigated the potential effect of PPARβ/δ on CVS following SAH. A model of SAH was established by endovascular perforation on male adult Sprague-Dawley rats, and the adenovirus PPARβ/δ (Ad-PPARβ/δ) was injected via intracerebroventricular administration prior to SAH. The expression levels of phenotypic markers α-smooth muscle actin and embryonic smooth muscle myosin heavy chain were measured via western blotting or immunofluorescence staining. The basilar artery diameter and vessel wall thickness were evaluated under fluorescence microscopy. SAH grade, neurological scores, brain water content and brain swelling were measured to study the mechanisms of PPARβ/δ on vascular smooth muscle phenotypic transformation. It was revealed that the expression levels of synthetic proteins were upregulated in rats with SAH and this was accompanied by CVS. Activation of PPARβ/δ using Ad-PPARβ/δ markedly upregulated the contractile proteins elevation, restrained the synthetic proteins expression and attenuated SAH-induced CVS by regulating the phenotypic switch in VSMCs at 72 h following SAH. Furthermore, the preliminary study demonstrated that PPARβ/δ downregulated ERK activity and decreased the expression of phosphorylated (p-)ETS domain-containing protein Elk-1 and p-p90 ribosomal S6 kinase, which have been demonstrated to serve an important role in VSMC phenotypic change. Additionally, it was revealed that Ad-PPARβ/δ could positively improve CVS by ameliorating the diameter of the basilar artery and mitigating the thickness of the vascular wall. Furthermore, subsequent experiments demonstrated that Ad-PPARβ/δ markedly reduced the brain water content and brain swelling and improved the neurological outcome. Taken together, the present study identified PPARβ/δ as a useful regulator for the VSMCs phenotypic switch and attenuating CVS following SAH, thereby providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

Abstract 34: Diminished Contractile Capability In Cerebral Vascular Smooth Muscle Cells In The TgF344-AD Rat Model Of Alzheimer's Disease

We recently reported that cerebral vascular dysfunction leads to impaired autoregulation of cerebral blood flow (CBF), neurovascular coupling (NVC), and blood-brain barrier (BBB) leakage. The present study examined if cerebral vascular dysfunction precedes cognitive impairment in the TgF344-AD (AD) rat model of Alzheimer's disease. In the present study, we confirmed that the AD rats develop learning and memory deficits beginning at 24-week of age using an eight-arm water maze. AD rats (n = 11) took a longer time to escape and displayed more errors than age-matched wildtype (WT) rats (n = 6). We also completed a longitudinal comparison of the myogenic response (MR) of the middle cerebral artery (MCA) and found that the MR was similar in AD and WT rats at 8- to 12-week of age when perfusion pressure was increased from 40 to 180 mmHg. However, the MR was significantly reduced in 16-week old AD rats (n = 6) as the inner diameter of the MCA only decreased by 8.2 ± 2.4% when perfusion pressure was increased from 40 to 180 mmHg compared with 14.5% ± 2.0% in age-matched WT rats (n = 6). The impaired MR of the MCA was exacerbated in AD rats with aging. Autoregulation of CBF AD rats (n = 4) in vivo was impaired in the surface and deep cortex at 24-week of age compared to age-matched WT rats (n = 4). Furthermore, we found the contractile capability of the cerebral vascular smooth muscle cells (VSMCs) isolated from AD rats (n = 4) was significantly reduced compared with WT rats (n = 4), detected by the reduction in size of 15.7 ± 0.9% vs. 25.4 ± 1.0% using a collagen gel-based assay kit. These results provide evidence that cerebral VSMC dysfunction, impaired MR, and autoregulation of CBF precede the development of memory and learning deficits in the TgF344-AD rat model. However, the underlying mechanisms for the loss of VSMCs contractility in this AD model overexpressing mutant human amyloid precursor protein ( APPsw ) and presenilin 1 ( PS1ΔE9 ) genes remain to be determined. Nevertheless, these results provide novel insight into the vascular contribution to AD.

Iron chelation suppresses secondary bleeding after intracerebral hemorrhage in angiotensin II-infused mice.

AimsSecondary bleeding and further hematoma expansion (HE) aggravate brain injury after intracerebral hemorrhage (ICH). The majority of HE results from hypertensive ICH. Previous study reported higher iron content in the brains of hypertensive patients. Iron overload exacerbates the risk of hemorrhagic transformation in thromboembolic stroke mice. Whether iron overload during the process of hypertension participates in secondary bleeding of hypertensive ICH remains unclear.MethodsHypertension was induced by continuous infusion of angiotensin II (Ang II) with an osmotic pump into C57BL/6 mice. ICH was simulated by intrastriatal injection of the liquid polymer Onyx‐18. Iron chelation and iron overload was achieved by deferoxamine mesylate or iron dextran injection. Secondary bleeding was quantified by measuring the hemoglobin content in the ipsilateral brain hemisphere.ResultsAng II‐induced hypertensive mice showed increased iron accumulation in the brain and expanded secondary hemorrhage after ICH modeling. Moreover, iron chelation suppressed while iron overload aggravated secondary bleeding. Mechanistically, iron exacerbated the loss of contractile cerebral vascular smooth muscle cells (VSMCs), aggravated blood–brain barrier (BBB) leakage in Ang II‐induced hypertensive mice, and increased glial and MMP9 accumulation after ICH.ConclusionIron overload plays a key role in secondary bleeding after ICH in Ang II‐induced hypertensive mice. Iron chelation during the process of Ang II‐induced hypertension suppresses secondary bleeding after ICH.

Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity

ObjectiveExposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. MethodsThree-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca2+ distribution, inositol 1,4,5-trisphosphate receptor (IP3R) abundance, and the activities of voltage-gated K+ channels (KV) and Ca2+-activated K+ channels (BKCa) were examined in rat cerebral vascular smooth muscle cells (VSMCs). ResultsAn increase of cytoplasmic Ca2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP3 R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of KV and BKCa decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP3R expression levels and restored the activities of the KV and BKCa channels. MitoTEMPO restored the Ca2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. ConclusionThe present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

20-HETE-promoted cerebral blood flow autoregulation is associated with enhanced pericyte contractility

We previously reported that deficiency in 20-HETE or CYP4A impaired the myogenic response and autoregulation of cerebral blood flow (CBF) in rats. The present study demonstrated that CYP4A was coexpressed with alpha-smooth muscle actin (α-SMA) in vascular smooth muscle cells (VSMCs) and most pericytes along parenchymal arteries (PAs) isolated from SD rats. Cell contractile capabilities of cerebral VSMCs and pericytes were reduced with a 20-HETE synthesis inhibitor, HET0016, but restored with 20-HETE analog WIT003. Similarly, intact myogenic responses of the middle cerebral artery and PA of SD rats decreased with HET0016 and were rescued by WIT003. The myogenic response of the PA was abolished in SS and was restored in SS.BN5 and SS.Cyp4a1 rats. HET0016 enhanced CBF and impaired its autoregulation in the surface and deep cortex of SD rats. These results demonstrate that 20-HETE has a direct effect on cerebral mural cell contractility that may play an essential role in controlling cerebral vascular function.

Effects of miR-7 on Hcy-induced rat cerebral arterial vascular smooth muscle cell proliferation, migration and inflammatory factor expression by targeting MMP-14 to regulate TLR4/NF-κB signaling pathway

The current research aimed to investigate the effect of miR-7 targeting matrix metalloproteinase 14 (MMP-14) on homocysteine (Hcy)-induced rat cerebral artery vascular smooth muscle cells (VSMCs) proliferation, migration and inflammatory factor expression and its possible mechanism. The expression of miR-7 and MMP-14 in Hcy-induced VSMCs were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot. Methyl Thiazolyl Tetrazolium (MTT) method, Transwell assays and enzyme-linked immunosorbent assay (ELISA) were performed to detect the effect of miR-7 and MMP-14 expression on the proliferation and migration, as well as interleukin 6 (IL-6) and tumor necrosis factor ɑ (TNF-ɑ) expression of Hcy-induced VSMCs. The interaction between miR-7 and MMP-14 was detected by dual-luciferase reporter gene assay. Western blot was applied to analyse the effects of miR-7 and MMP-14 expression on the Toll-like receptor (TLR4)/nuclear transcription factor-KB (NF-κB) signaling pathway. The results showed that after induced by Hcy, the expression of miR-7 in VSMCs was significantly reduced, the expression of MMP-14 was significantly increased, and the cell viability, the number of migrating cells, IL-6 and TNF-ɑ expression were significantly increased (P&lt;0.05). After overexpression of miR-7, the viability, migration cell numbers, IL-6 and TNF-ɑ expression of Hcy-induced VSMCs were significantly reduced (P&lt;0.05). miR-7 directly binds to MMP-14 and negatively regulates the expression of MMP-14. After overexpression of miR-7, the levels of TLR4 and p-NF-κB p65 in VSMCs were significantly reduced (P&lt;0.05); overexpression of MMP-14 could reduce the effect of miR-7 overexpression on TLR4 and p-NF-κB p65 expression in VSMCs (P&lt;0.05). Overexpression of MMP-14 and/or activation of the TLR4/NF-κB signaling pathway could reverse the effect of miR-7 overexpression on the proliferation, migration and IL-6 and TNF-ɑ expression of Hcy-induced VSMCs (P&lt;0.05). It is concluded that miR-7 can inhibit Hcy-induced rat cerebral artery VSMCs proliferation, migration, and inflammatory factor expression by targeting the regulation of MMP-14 expression and inhibiting the activation of the TLR4/NF-κB signaling pathway.

20-HETE Enzymes and Receptors in the Neurovascular Unit: Implications in Cerebrovascular Disease.

20-HETE is a potent vasoconstrictor that is implicated in the regulation of blood pressure, cerebral blood flow and neuronal death following ischemia. Numerous human genetic studies have shown that inactivating variants in the cytochrome P450 enzymes that produce 20-HETE are associated with hypertension, stroke and cerebrovascular disease. However, little is known about the expression and cellular distribution of the cytochrome P450A enzymes (CYP4A) that produce 20-HETE or the newly discovered 20-HETE receptor (GPR75) in the brain. The present study examined the cell types and regions in the rat forebrain that express CYP4A and GPR75. Brain tissue slices from Sprague Dawley (SD), Dahl Salt-Sensitive (SS) and CYP4A1 transgenic rat strains, as well as cultured human cerebral pericytes and cerebral vascular smooth muscle cells, were analyzed by fluorescent immunostaining. Tissue homogenates from these strains and cultured cells were examined by Western blot. In the cerebral vasculature, CYP4A and GPR75 were expressed in endothelial cells, vascular smooth muscle cells and the glial limiting membrane of pial arteries and penetrating arterioles but not in the endothelium of capillaries. CYP4A, but not GPR75, was expressed in astrocytes. CYP4A and GPR75 were both expressed in a subpopulation of pericytes on capillaries. The diameters of capillaries were significantly decreased at the sites of first and second-order pericytes that expressed CYP4A. Capillary diameters were unaffected at the sites of other pericytes that did not express CYP4A. These findings implicate 20-HETE as a paracrine mediator in various components of the neurovascular unit and are consistent with 20-HETE's emerging role in the regulation of cerebral blood flow, blood-brain barrier integrity, the pathogenesis of stroke and the vascular contributions to cognitive impairment and dementia. Moreover, this study highlights GPR75 as a potential therapeutic target for the treatment of these devastating conditions.