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Background: Ang II (angiotensin II) type 1 (AT 1 ) receptors play a critical role in cardiovascular diseases such as hypertension. Rodents have 2 types of AT 1 receptor (AT 1A and AT 1B ) of which knock-in Tagln -mediated smooth muscle AT 1A silencing attenuated Ang II–induced hypertension. Although vascular remodeling, a significant contributor to organ damage, occurs concurrently with hypertension in Ang II–infused mice, the contribution of smooth muscle AT 1A in this process remains unexplored. Accordingly, it is hypothesized that smooth muscle AT 1A receptors exclusively contribute to both medial thickening and adventitial fibrosis regardless of the presence of hypertension. Methods: About 1 µg/kg per minute Ang II was infused for 2 weeks in 2 distinct AT 1A receptor silenced mice, knock-in Tagln -mediated constitutive smooth muscle AT 1A receptor silenced mice, and Myh11 -mediated inducible smooth muscle AT 1A together with global AT 1B silenced mice for evaluation of hypertensive cardiovascular remodeling. Results: Medial thickness, adventitial collagen deposition, and immune cell infiltration in aorta were increased in control mice but not in both smooth muscle AT 1A receptor silenced mice. Coronary arterial perivascular fibrosis in response to Ang II infusion was also attenuated in both AT 1A receptor silenced mice. Ang II–induced cardiac hypertrophy was attenuated in constitutive smooth muscle AT 1A receptor silenced mice. However, Ang II–induced cardiac hypertrophy and hypertension were not altered in inducible smooth muscle AT 1A receptor silenced mice. Conclusions: Smooth muscle AT 1A receptors mediate Ang II–induced vascular remodeling including medial hypertrophy and inflammatory perivascular fibrosis regardless of the presence of hypertension. Our data suggest an independent etiology of blood pressure elevation and hypertensive vascular remodeling in response to Ang II.

The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [63, 155]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, olmesartan, telmisartan, etc. are clinically used AT1 receptor blockers.

Table 1 lists a number of putative GPCRs identified by NC-IUPHAR [161], for which preliminary evidence for an endogenous ligand has been published, or for which there exists a potential link to a disease, or disorder. These GPCRs have recently been reviewed in detail [121]. The GPCRs in Table 1 are all Class A, rhodopsin-like GPCRs. Class A orphan GPCRs not listed in Table 1 are putative GPCRs with as-yet unidentified endogenous ligands.Table 1: Class A orphan GPCRs with putative endogenous ligands GPR3GPR4GPR6GPR12GPR15GPR17GPR20 GPR22GPR26GPR31GPR34GPR35GPR37GPR39 GPR50GPR63GPR65GPR68GPR75GPR84GPR87 GPR88GPR132GPR149GPR161GPR183LGR4LGR5 LGR6MAS1MRGPRDMRGPRX1MRGPRX2P2RY10TAAR2 In addition the orphan receptors GPR18, GPR55 and GPR119 which are reported to respond to endogenous agents analogous to the endogenous cannabinoid ligands have been grouped together (GPR18, GPR55 and GPR119).

Background: Clinical studies consistently show a strong association between aging and increased risk of intracranial aneurysmal rupture. Aging causes the accumulation of senescent cells that promote chronic inflammation through the senescence-associated secretory phenotype. Since inflammation is emerging as a vital component of the pathophysiology of intracranial aneurysms, the accumulation of senescent cells in the aneurysm wall may play a critical role in the rupture of intracranial aneurysm. We hypothesized that the pharmacological clearance of senescent cells reduces the risk of aneurysmal rupture. We tested this hypothesis using a well-established mouse model with a senescent cell-clearing senolytic drug, ABT263. Methods: We used male C57BL/6J mice and induced intracranial aneurysms by combining an elastase injection and hypertension. We treated mice with either ABT263 (50 mg/kg/day, oral gavage) or a vehicle from one day before aneurysm induction and continued for three weeks. The development of aneurysmal rupture was used as the primary endpoint. We also assessed the effects of ABT263 or vehicle on mRNA expression levels of senescent cell markers (p16 and p21) in cerebral arteries. Results: The pharmacological clearance of senescent cells with ABT263 significantly decreased the rupture rate in mice (P Conclusion: We demonstrated that pharmacological clearance of senescent cells decreased intracranial aneurysmal rupture. The reduction of rupture rate was associated with the reduction of senescence markers. These findings suggest a link between cellular senescence and aneurysmal rupture. Cellular senescence may be responsible for the age-dependent increase in aneurysm rupture. Senolytic drugs may serve as a novel therapy for preventing aneurysm rupture.

Introduction: Aging is a known risk factor for intracranial aneurysm rupture in human clinical studies. However, clinical studies only showed the association, but not the causality of aging and aneurysm rupture. Using our well-established mouse model, we directly tested the hypothesis that aging increases the rupture rate of aneurysms. Methods: We used both male and female C57BL/6J mice at 10 weeks and 18 months of age. We induced intracranial aneurysms by a combination of elastase injection and DOCA-salt hypertension. We compared the formation and rupture rates of intracranial aneurysms and the survival curve between young mice and aged mice of each sex. Results: Aged female mice had a higher rupture rate (86.7% vs 38.9%; P Conclusions: Aging promotes both the formation and rupture of intracranial aneurysms in both male and female mice. These data agree with clinical observations and will serve as a basis for further investigation of the roles of the aging-related biological changes in the development of aneurysm rupture.

Table 1 lists a number of putative GPCRs identified by NC-IUPHAR [161], for which preliminary evidence for an endogenous ligand has been published, or for which there exists a potential link to a disease, or disorder. These GPCRs have recently been reviewed in detail [121]. The GPCRs in Table 1 are all Class A, rhodopsin-like GPCRs. Class A orphan GPCRs not listed in Table 1 are putative GPCRs with as-yet unidentified endogenous ligands.Table 1: Class A orphan GPCRs with putative endogenous ligands GPR3GPR4GPR6GPR12GPR15GPR17GPR20 GPR22GPR26GPR31GPR34GPR35GPR37GPR39 GPR50GPR63GPR65GPR68GPR75GPR84GPR87 GPR88GPR132GPR149GPR161GPR183LGR4LGR5 LGR6MAS1MRGPRDMRGPRX1MRGPRX2P2RY10TAAR2 In addition the orphan receptors GPR18, GPR55 and GPR119 which are reported to respond to endogenous agents analogous to the endogenous cannabinoid ligands have been grouped together (GPR18, GPR55 and GPR119).

Introduction: Hypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients have a 50% greater cardiovascular risk than untreated normotensive subjects, presenting a need for additional targets. Vascular smooth muscle cells (VSMC) play a critical role in vascular contractility and regulation of blood pressure. Fragile X-related protein (FXR1) is a muscle-enhanced RNA binding protein. In addition to containing mRNA binding domains, FXR1 has an agenet-like domain for protein-protein interactions and four WH2 motif domains to mediate actin dynamics. The structure of FXR1 supports its role in post-transcriptional regulation and though largely ignored, cytoskeletal dynamics via potential protein-protein interactions. This study will test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions. Results: RNA immuno-precipitation sequencing (RIPseq) analysis in human VSMC identified that FXR1 binds to mRNA that participate in VSMC contractility and cytoskeletal reorganization, and FXR1 depletion decreases mRNA abundance and stability of these transcripts. Mass-spectrometry and co-immunoprecipitation identified that FXR1 interacts with members of the WAVE complex, a five-subunit protein complex involved in the formation of the actin cytoskeleton, including CYFIP1 (Cytoplasmic FMR1-interacting protein 1) and Actin Related Protein 2/3 complex (ARP2). The WAVE complex is activated by an ARP2-mediated interaction with Rac1, promoting actin remodeling and cytoskeletal reorganization in a GTP-dependent manner. FXR1 depletion decreases small GTPases RAC1 and CDC42 activation in VSMC. Additionally, FXR1 depletion decreases VSMC lamellipodia formation, adhesion, migration and collagen gel contraction. Novel, VSMC-specific FXR1 conditional knock out mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls. Conclusion: These data are the first to suggest that FXR1 regulates mRNA stability of contractile proteins and interacts with members of the WAVE complex. Deletion of FXR1 abrogates VSMC cytoskeletal reorganization, resulting in a hypotensive phenotype in VSMC-specific conditional knockout mice.

Angiotensin II Type 1a Receptor (AT1a) has been shown to play a critical role in cardiovascular diseases. We have demonstrated that ADAM17 mediates cardiovascular remodeling induced by angiotensin II (AngII) by using an SM22α Cre reporter, which expresses in smooth muscle cells (SMC) and cardiomyocytes. It is conceivable that AT1 signaling in SMC specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of SMC AT1a receptors via endogenous SM22α promoter knock-in (KISMKO) results in diminished hypertension and cardiac hypertrophy induced by AngII. However, we have limited understanding whether SMC AT1a receptors affect vascular fibrosis and cardiac hypertrophy. Thus, this study was designed to elucidate the roles of the SMC AT1a receptor in cardiovascular remodeling using two distinct SMC AT1a receptor deficient mice. One line is the KISMKO, and another line is Tamoxifen inducible SMMHC-Cre AT1aflox/flox mouse (SMMHCKO) silencing in SMC but not cardiac myocytes. 8 to 12-week-old male mice were infused with AngII (1 μg/kg/min) for 2 weeks. Cardiac hypertrophy was assessed by heart-to-body weight ratios and found to be reduced in KISMKO mice compared with control mice (6.04 mg/g versus 4.89 mg/g, p=0.032), whereas ratios were not altered in SMMHCKO mice (5.31 mg/g versus 5.47 mg/g, p=0.941). Histological assessment demonstrated that perivascular fibrosis in coronary arteries was increased in control mice and attenuated in both KISMKO and SMMHCKO mice (1.27 vs. 0.22, p

A fruit of the Prunus mume is a traditional and familiar food in Asian countries and is most often pickled or used to make wine and juice concentrate. Recently, Bainiku-ekisu, an infused juice concentrate of Prunus mume is attracting attention as a potential health promoting food. We previously reported that Bainiku-ekisu (BE) incubation attenuates the activation of growth-promoting signaling cascade induced by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMC). However, whether BE has any effect on an animal model of cardiovascular disease remains unknown. Therefore, this study was designed to elucidate whether BE could attenuate cardiovascular pathology induced by Ang II infusion in mice, representing a model of human hypertension. 8- to 10-week-old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump or sham-operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement. Cardiac hypertrophy was assessed by heart-to-body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular hypertrophy and perivascular fibrosis. Increase in heart-to-body weight ratios were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 μm vs. 71 μm, respectively, p

Background and Purpose: The incidences of intracranial aneurysm and aneurysmal subarachnoid hemorrhage are high in postmenopausal women. Although population-based studies suggest that hormone replacement therapy is beneficial for postmenopausal women with intracranial aneurysms, estrogen replacement may no longer be recommended for the prevention of chronic diseases given its association with adverse outcomes, such as cancer and ischemic stroke. The isoflavone daidzein and its intestinal metabolite equol are bioactive phytoestrogens and potent agonists of estrogen receptors. Given their estrogenic properties, we investigated whether the isoflavones daidzein and equol are protective against the formation and rupture of intracranial aneurysms in a mouse model of the postmenopausal state. Methods: We induced intracranial aneurysms in ovariectomized adult female mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. We fed the mice with an isoflavone-free diet with/without daidzein supplementation, or in a combination of intraperitoneal equol, or oral vancomycin treatment. We also used estrogen receptor beta knockout mice. Results: Both dietary daidzein and supplementation with its metabolite, equol, were protective against aneurysm formation in ovariectomized mice. The protective effects of daidzein and equol required estrogen receptor-β. The disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein’s protective effect against aneurysm formation. Mice treated with equol had lower inflammatory cytokines in the cerebral arteries, suggesting that phytoestrogens modulate inflammatory processes important to intracranial aneurysm pathogenesis. Conclusions: Our study establishes that both dietary daidzein and its metabolite, equol, protect against aneurysm formation in ovariectomized female mice through the activation of estrogen receptor-β and subsequent suppression of inflammation. Dietary daidzein’s protective effect required the intestinal conversion to equol. Our results indicate the potential therapeutic value of dietary daidzein and its metabolite, equol, for the prevention of the formation of intracranial aneurysms and related subarachnoid hemorrhage.

Until now, renin-angiotensin system (RAS) hyperactivity was largely thought to result from angiotensin II (Ang II) dependent stimulation of the Ang II type 1 receptor (AT1R). Here we assessed the role of soluble (pro)renin receptor (sPRR), a product of site-1 protease-mediated cleavage of (pro)renin receptor (PRR), as a possible ligand of the AT1R in mediating: (i) endothelial cell dysfunction in vitro; and (ii) arterial dysfunction in mice with diet-induced obesity. Primary human umbilical vein endothelial cells (HUVECs) treated with a recombinant histidine-tagged sPRR (sPRR-His) exhibited IκBα degradation concurrent with NF-κB p65 activation. These responses were secondary to sPRR-His evoked elevations in Nox4-derived H(2)O(2) production that resulted in inflammation, apoptosis and reduced NO production. Each of these sPRR-His-evoked responses was attenuated by AT1R inhibition using Losartan (Los) but not ACE inhibition using captopril (Cap). Further mechanistic exploration revealed that sPRR-His activated AT1R downstream Gq signaling pathway. Immunoprecipitation coupled with autoradiography experiments and radioactive ligand competitive binding assays indicate sPRR directly interacts with AT1R via Lysine(199) and Asparagine(295). Important translational relevance was provided by findings from obese C57/BL6 mice that sPRR-His evoked endothelial dysfunction was sensitive to Los. Besides, sPRR-His elevated blood pressure in obese C57/BL6 mice, an effect that was reversed by concurrent treatment with Los but not Cap. Collectively, we provide solid evidence that the AT1R mediates the functions of sPRR during obesity-related hypertension. Inhibiting sPRR signaling should be considered further as a potential therapeutic intervention in the treatment and prevention of cardiovascular disorders involving elevated blood pressure.

Potential roles for neutrophils in the pathophysiology of intracranial aneurysm have long been suggested by clinical observations. The presence of neutrophil enzymes in the aneurysm wall has been associated with significant increases in rupture risk. However, the mechanisms by which neutrophils may promote aneurysm rupture are not well understood. Neutrophil extracellular traps (NETs) were implicated in many diseases that involve inflammation and tissue remodeling, including atherosclerosis, vasculitis, and abdominal aortic aneurysm. Therefore, we hypothesized that NETs may promote the rupture of intracranial aneurysm, and that removal of NETs can reduce the rate of rupture. We employed both pharmacological and genetic approaches for the disruption of NETs and used a mouse model of intracranial aneurysm to investigate the roles of NETs in the development of intracranial aneurysm rupture. Here, we showed that NETs are detected in human intracranial aneurysms. Both global and granulocyte-specific knockout of peptidyl arginine deiminase 4 (an enzyme essential for NET formation) reduced the rate of aneurysm rupture. Pharmacological blockade of the NET formation by Cl-amidine also reduced the rate of aneurysm rupture. In addition, the resolution of already formed NETs by deoxyribonuclease was effective against aneurysm rupture. Inhibition of NETs formation with Cl-amidine decreased mRNA expression of proinflammatory cytokines (intercellular adhesion molecule 1 [ICAM-1], interleukin 1 beta [IL-1β], monocyte chemoattractant protein-1 [MCP-1], and tumor necrosis factor alpha [TNF-α]) in cerebral arteries. These data suggest that NETs promote the rupture of intracranial aneurysm. Pharmacological removal of NETs, by inhibition of peptidyl arginine deiminase 4 or resolution of already-formed NETs, may represent a potential therapeutic strategy for preventing aneurysmal rupture.

The mitochondria are highly plastic and dynamic organelles; mitochondrial dysfunction has been reported to play causative roles in diabetes, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). However, the relationship between mitochondrial fission and NAFLD pathogenesis remains unknown. We aimed to investigate whether alterations in mitochondrial fission could play a role in the progression of NAFLD.Mice were fed a standard diet or choline-deficient, L-amino acid-defined (CDAA) diet with vehicle or mitochondrial division inhibitor-1.Substantial enhancement of mitochondrial fission in hepatocytes was triggered by 4 weeks of feeding and was associated with changes reflecting the early stage of human nonalcoholic steatohepatitis (NASH), steatotic change with liver inflammation, and hepatocyte ballooning. Excessive mitochondrial fission inhibition in hepatocytes and lipid metabolism dysregulation in adipose tissue attenuated liver inflammation and fibrogenesis but not steatosis and the systemic pathological changes in the early and chronic fibrotic NASH stages (4- and 12-week CDAA feeding). These beneficial changes due to the suppression of mitochondrial fission against the liver and systemic injuries were associated with decreased autophagic responses and endoplasmic reticulum stress in hepatocytes. Injuries to other liver cells, such as endothelial cells, Kupffer cells, and hepatic stellate cells, were also attenuated by the inhibition of mitochondrial fission in hepatocytes.Taken together, these findings suggest that excessive mitochondrial fission in hepatocytes could play a causative role in NAFLD progression by liver inflammation and fibrogenesis through altered cell cross-talk. This study provides a potential therapeutic target for NAFLD.

Angiotensin II (Ang II) AT1 receptor has been shown to play a critical role in several cardiovascular diseases. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affect hypertensive fibrosis. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock-In, KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and control (AT1a flox/flox Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks. In control mice, Ang II infusion induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly decreased in AT1a SMKO mice compared with control mice (6.04 versus 4.89, p=0.032). Cardiac hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 μm vs. 56.4 μm, p

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism in cell culture. However, surveillance of vascular-related journals for the past 5 years demonstrated that less than 20% of articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in vascular cell types with particular attention paid to changes in glucose consumption upon angiotensin II (AngII) stimulation. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without 100 nM AngII stimulation. In all cell types after a 48-hour incubation in relatively low glucose media (1 g/L in 6 well dish with 1mL), medium glucose concentration was reduced to almost 0. Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48 hours in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngII was observed in AF. PDGF-BB also stimulated glucose consumption in AF which was associated with a trend of cell proliferation. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of GRP78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Introduction: Hypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients have a 50% greater cardiovascular risk than untreated normotensive subjects presenting a need for additional targets. Vascular smooth muscle cells (VSMC) are the fulcrum of vascular disease, particularly hypertension. VSMC play a critical role in vascular contractility and the regulation of blood pressure. Fragile X-related protein (FXR1) is a muscle-enhanced RNA binding protein and we previously found siRNA knock down of FXR1 increases inflammatory mRNA stability. Overexpression of FXR1 decreases inflammatory mRNA stability in VSMC. Little is known concerning FXR1 protein binding partners and its role in vascular disease. The specific aim of this study is to test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions. Results: Gene ontology of RNA immuno-precipitation sequencing analysis in human VSMC identified that FXR1 binds to mRNA that participate in VSMC contractility and regulation of blood pressure- related pathways. FXR1 depletion in human VSMC decreases mRNA abundance of contractile machinery such as RhoA, Cortactin, ARP2 and Dynactin. While considered an RNA stability protein, mass-spectrometry identified that FXR1 interacts with proteins related to contractile processes such as cell migration, adhesion and stress fiber formation. Additionally, siRNA knock down of FXR1 decreased VSMC adhesion, migration and collagen gel contraction. FXR1 depletion decreases CDC42 activation in VSMC. CDC42 is a small GTPase involved in cell morphology, adhesion and migration. In order to establish an in vivo role of FXR1 in vascular disease we generated a novel, VSMC-specific FXR1 conditional knock out mouse (FXR1 VSMC/VSMC ). Preliminary data indicates that these mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls. Conclusion: These data are the first to suggest FXR1 regulates blood pressure and vascular contractility potentially by two mechanisms: mRNA stability and functional activity by protein-protein interactions. These findings suggest FXR1 as target for therapeutic intervention to regulate blood pressure.

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

Aims Angiotensin II (AngII) is a potential contributor to the development of abdominal aortic aneurysm (AAA). In aortic vascular smooth muscle cells (VSMCs), exposure to AngII induces mitochondrial fission via dynamin-related protein 1 (Drp1). However, pathophysiological relevance of mitochondrial morphology in AngII-associated AAA remains unexplored. Here, we tested the hypothesis that mitochondrial fission is involved in the development of AAA. Methods and results Immunohistochemistry was performed on human AAA samples and revealed enhanced expression of Drp1. In C57BL6 mice treated with AngII plus β-aminopropionitrile, AAA tissue also showed an increase in Drp1 expression. A mitochondrial fission inhibitor, mdivi1, attenuated AAA size, associated aortic pathology, Drp1 protein induction, and mitochondrial fission but not hypertension in these mice. Moreover, western-blot analysis showed that induction of matrix metalloproteinase-2, which precedes the development of AAA, was blocked by mdivi1. Mdivi1 also reduced the development of AAA in apolipoprotein E-deficient mice infused with AngII. As with mdivi1, Drp1+/− mice treated with AngII plus β-aminopropionitrile showed a decrease in AAA compared to control Drp1+/+ mice. In abdominal aortic VSMCs, AngII induced phosphorylation of Drp1 and mitochondrial fission, the latter of which was attenuated with Drp1 silencing as well as mdivi1. AngII also induced vascular cell adhesion molecule-1 expression and enhanced leucocyte adhesion and mitochondrial oxygen consumption in smooth muscle cells, which were attenuated with mdivi1. Conclusion These data indicate that Drp1 and mitochondrial fission play salient roles in AAA development, which likely involves mitochondrial dysfunction and inflammatory activation of VSMCs.

Chronic inflammation of the arterial wall has been implicated in the development of abdominal aortic aneurysm (AAA). However, the detailed molecular mechanism(s) by which inflammatory cells contributes to AAA pathogenesis remains largely unclear. In their article in Clinical Science, Krishna et al. have reported that depletion of CD11c+ dendritic cells inhibited experimental AAA formation in mice. The authors also demonstrated a decrease in CD4 and CD8 positive T cells in the circulation, lower plasma neutrophil elastase activity, and aortic matrix remodeling. These novel findings will help clarify the underlying mechanisms of AAA progression and may provide a new target for future therapeutic research in AAA formation.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Table 1 lists a number of putative GPCRs identified by NC-IUPHAR [161], for which preliminary evidence for an endogenous ligand has been published, or for which there exists a potential link to a disease, or disorder. These GPCRs have recently been reviewed in detail [121]. The GPCRs in Table 1 are all Class A, rhodopsin-like GPCRs. Class A orphan GPCRs not listed in Table 1 are putative GPCRs with as-yet unidentified endogenous ligands.Table 1: Class A orphan GPCRs with putative endogenous ligands GPR3GPR4GPR6GPR12GPR15GPR17GPR20 GPR22GPR26GPR31GPR34GPR35GPR37GPR39 GPR50GPR63GRP65GPR68GPR75GPR84GPR87 GPR88GPR132GPR149GPR161GPR183LGR4LGR5 LGR6MAS1MRGPRDMRGPRX1MRGPRX2P2RY10TAAR2 In addition the orphan receptors GPR18, GPR55 and GPR119 which are reported to respond to endogenous agents analogous to the endogenous cannabinoid ligands have been grouped together (GPR18, GPR55 and GPR119).

Ume ( Prunus mume ), is a traditional and familiar food in Asian countries, and is most often pickled or used to make wine and juice concentrate. Recently, Ume is attracting attention as a health food. We previously reported that Ume attenuates the activation of EGFR-ERK cascade and ROS production induced by angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) of thoracic aorta. However, how Ume works on vascular inflammation and ER stress still remains unknown. AngII may enhance inflamm-aging via ER stress contributing to arterial stiffness and hypertension.We used primary culture of rat VSMC for experiment. We obtained 5 fractions (methanol, hexane, dichloromethane, ethyl acetate, and water) extracted from Ume juice. VSMC were pretreated with each fraction for 30 minutes followed by Ang II or tumor necrosis factor (TNF)-α stimulation. Stimulation period was 24 and 48 hours for Ang II, 6 hours for TNF-α. As the preliminary screening, we tested the cell response and solubility of each fraction (n=1~2). Pretreatment with 500μg/mL hexane fraction (PMF-H) showed the tendency to decrease VCAM1 expression under Ang II condition. PMF-H was completely soluble in the media at the concentration of 150 μg/mL. Based on this result, we focused on 150μg/mL PMF-H. DMSO was used as vehicle. In Western blot analysis, PMF-H attenuates ATF6 expression under both AII (p=0.0351, n=5) and TNF-α (p=0.0199, n=5) condition compared to vehicle. VCAM1 expression was significantly enhanced by Ang II stimulation (p=0.0471, n=5), and attenuated by PMF-H. PMF-H also reduced VCAM1 expression under TNF-α stimulation compared to vehicle (p=0.0377, n=5). GRP78 and Crystallin AB did not show any significant change. ATF6 and VCAM1 are key modulators of ER stress and vascular inflammation that may lead to cellular aging. Our results indicate PMF-H attenuates ATF6 and VCAM1 induced by Ang II or TNF-α stimulation. Thus, identification of the active ingredient in hexane fraction of Ume is desired as it may constitute a good candidate for a supplement or drug for anti-inflamm-aging therapy.

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the sm22α promoter (KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and Controls (AT1a flox/flox KIsm22α-Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump. In Control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly increased in Control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in Controls after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKOs. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis, whereas these phenotypic changes were attenuated in SMKO mice (n=4). In conclusion, AT1a receptors from VSMC could mediate Ang II-induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined, the data contrast to the past manuscript showing a protective effect in AT1a flox/flox S100A4-Cre+/- mice (fibroblast silencing) with Ang II infusion.

Hypertension is a complex disorder and risk factor for cardiovascular disease, ultimately contributing to premature death. The societal burden of hypertension is enormous, therefore better understanding of molecular mechanisms underlying hypertension and associated pathological vascular remodeling is needed for mechanism-targeted therapy development. Endoplasmic reticulum (ER) stress occurs in cells under increased protein synthesis, Ca 2+ flux, or ROS generating environments- all of which are induced by Angiotensin II signaling in vascular cells. ER stress leads to the accumulation of misfolded proteins, and mediates cell death, fibrotic, and hypertrophic responses. The aim of this study was to target ER stress via chemical chaperone 3-hydroxy-2-naphthoic acid (3HNA) or genetic overexpression of chaperone glucose-regulated protein 78 (GRP78) to reduce protein aggregation and pathological response in VSMCs. Rat primary VSMCs were treated with 500 μM 3HNA or 30 MOI lacZ-GRP78 prior to stimulation with Ang II (100 nM). Protein synthesis assessed via puromycin incorporation revealed ER stress inhibition blocked Ang II associated protein synthesis in VSMCs, fold change relative to control of 1.7 (saline + Ang II) to 1.2 (3-HNA + Ang II) treated cells (P

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism. However, there is a lack of attention paid to the supply and utilization of glucose in many cultured experiments. Surveillance of vascular related journals for the past 5 years demonstrated that less than 20% of published articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in various cell types with particular attention paid to changes in glucose consumption. By using distinct glucose concentrations in Dulbecco's Modified Eagle's medium (DMEM), we identified ideal glucose media for stimulation experiments. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II (AII) stimulation. In all cell types after a 48h incubation in relatively low glucose media (1 g/L), medium glucose concentration was reduced to almost 0 (EC 0.01±0.01 g/L, VSMC 0.13±0.05 g/L, AF 0.02±0.01 g/L). Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48h in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AII treatment enhanced glucose consumption in AF and low passage VSMC but not in EC. In AF, AII induction of target proteins varied depending on the glucose concentration used. In low glucose media induction of Grp78 or hexokinase II was highest, whereas induction of VCAM1 was lowest. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AII-induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.