Carotid dysfunction in senescent female mice is mediated by increased α1A-adrenoceptor activity and COX-derived vasoconstrictor prostanoids.

α-Adrenergic receptors are crucial regulators of vascular hemodynamics and essential pharmacological targets for cardiovascular diseases. With aging, there is an increase in sympathetic activation, which could contribute to the progression of aging-associated cardiovascular dysfunction, including stroke. Nevertheless, there is little information directly associating adrenergic receptor dysfunction in the blood vessels of aged females. This study determined the role of a-adrenergic receptors in carotid dysfunction of senescent female mice (accelerated-senescence prone, SAMP8), compared with a nonsenescent (accelerated-senescence prone, SAMR1). Vasoconstriction to phenylephrine (Phe) was markedly increased in common carotid artery of SAMP8 [area under the curve (AUC), 527 ± 53] compared with SAMR1 (AUC, 334 ± 30, P = 0.006). There were no changes in vascular responses to the vasoconstrictor agent U46619 or the vasodilators acetylcholine (ACh) and sodium nitroprusside (NPS). Hyperactivity to Phe in female SAMP8 was reduced by cyclooxygenase-1 and cyclooxygenase-2 inhibition and associated with augmented ratio of TXA2/PGI2 release (SAMR1, 1.1 ± 0.1 vs. SAMP8, 2.1 ± 0.3, P = 0.007). However, no changes in cyclooxygenase expression were seen in SAMP8 carotids. Selective α1A-receptor antagonism markedly reduced maximal contraction, whereas α1D antagonism induced a minor shift in Phe contraction in SAMP8 carotids. Ligand binding analysis revealed a threefold increase of α-adrenergic receptor density in smooth muscle cells (VSMCs) of SAMP8 vs. SAMR1. Phe rapidly increased intracellular calcium (Cai2+) in VSMCs via the α1A-receptor, with a higher peak in VSMCs from SAMP8. In conclusion, senescence intensifies vasoconstriction mediated by α1A-adrenergic signaling in the carotid of female mice by mechanisms involving increased Cai2+ and release of cyclooxygenase-derived prostanoids.NEW & NOTEWORTHY The present study provides evidence that senescence induces hyperreactivity of α1-adrenoceptor-mediated contraction of the common carotid. Impairment of α1-adrenoceptor responses is linked to increased Ca2+ influx and release of COX-derived vasoconstrictor prostanoids, contributing to carotid dysfunction in the murine model of female senescence (SAMP8). Increased reactivity of the common carotid artery during senescence may lead to morphological and functional changes in arteries of the cerebral microcirculation and contribute to cognitive decline in females. Because the elderly population is growing, elucidating the mechanisms of aging- and sex-associated vascular dysfunction is critical to better direct pharmacological and lifestyle interventions to prevent cardiovascular risk in both sexes.

Novel pharmacological approaches in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a severe vascular disease and a major public health issue with an unmet medical need for therapy. This disease is featured by a progressive dilation of the abdominal aorta, boosted by atherosclerosis, ageing, and smoking as major risk factors. Aneurysm growth increases the risk of aortic rupture, a life-threatening emergency with high mortality rates. Despite the increasing progress in our knowledge about the etiopathology of AAA, an effective pharmacological treatment against this disorder remains elusive and surgical repair is still the unique available therapeutic approach for high-risk patients. Meanwhile, there is no medical alternative for patients with small aneurysms but close surveillance. Clinical trials assessing the efficacy of antihypertensive agents, statins, doxycycline, or anti-platelet drugs, among others, failed to demonstrate a clear benefit limiting AAA growth, while data from ongoing clinical trials addressing the benefit of metformin on aneurysm progression are eagerly awaited. Recent preclinical studies have postulated new therapeutic targets and pharmacological strategies paving the way for the implementation of future clinical studies exploring these novel therapeutic strategies. This review summarises some of the most relevant clinical and preclinical studies in search of new therapeutic approaches for AAA.

Hyperuricaemia Does Not Interfere with Aortopathy in a Murine Model of Marfan Syndrome.

Redox stress is involved in the aortic aneurysm pathogenesis in Marfan syndrome (MFS). We recently reported that allopurinol, a xanthine oxidoreductase inhibitor, blocked aortopathy in a MFS mouse model acting as an antioxidant without altering uric acid (UA) plasma levels. Hyperuricaemia is ambiguously associated with cardiovascular injuries as UA, having antioxidant or pro-oxidant properties depending on the concentration and accumulation site. We aimed to evaluate whether hyperuricaemia causes harm or relief in MFS aortopathy pathogenesis. Two-month-old male wild-type (WT) and MFS mice (Fbn1C1041G/+) were injected intraperitoneally for several weeks with potassium oxonate (PO), an inhibitor of uricase (an enzyme that catabolises UA to allantoin). Plasma UA and allantoin levels were measured via several techniques, aortic root diameter and cardiac parameters by ultrasonography, aortic wall structure by histopathology, and pNRF2 and 3-NT levels by immunofluorescence. PO induced a significant increase in UA in blood plasma both in WT and MFS mice, reaching a peak at three and four months of age but decaying at six months. Hyperuricaemic MFS mice showed no change in the characteristic aortic aneurysm progression or aortic wall disarray evidenced by large elastic laminae ruptures. There were no changes in cardiac parameters or the redox stress-induced nuclear translocation of pNRF2 in the aortic tunica media. Altogether, the results suggest that hyperuricaemia interferes neither with aortopathy nor cardiopathy in MFS mice.

Anti-TGFß (Transforming Growth Factor ß) Therapy With Betaglycan-Derived P144 Peptide Gene Delivery Prevents the Formation of Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

Objective: We investigated the effect of a potent TGFß (transforming growth factor ß) inhibitor peptide (P144) from the betaglycan/TGFß receptor III on aortic aneurysm development in a Marfan syndrome mouse model. Approach and Results: We used a chimeric gene encoding the P144 peptide linked to apolipoprotein A-I via a flexible linker expressed by a hepatotropic adeno-associated vector. Two experimental approaches were performed: (1) a preventive treatment where the vector was injected before the onset of the aortic aneurysm (aged 4 weeks) and followed-up for 4 and 20 weeks and (2) a palliative treatment where the vector was injected once the aneurysm was formed (8 weeks old) and followed-up for 16 weeks. We evaluated the aortic root diameter by echocardiography, the aortic wall architecture and TGFß signaling downstream effector expression of pSMAD2 and pERK1/2 by immunohistomorphometry, and Tgfß1 and Tgfß2 mRNA expression levels by real-time polymerase chain reaction. Marfan syndrome mice subjected to the preventive approach showed no aortic dilation in contrast to untreated Marfan syndrome mice, which at the same end point age already presented the aneurysm. In contrast, the palliative treatment with P144 did not halt aneurysm progression. In all cases, P144 improved elastic fiber morphology and normalized pERK1/2-mediated TGFß signaling. Unlike the palliative treatment, the preventive treatment reduced Tgfß1 and Tgfß2 mRNA levels. Conclusions: P144 prevents the onset of aortic aneurysm but not its progression. Results indicate the importance of reducing the excess of active TGFß signaling during the early stages of aortic disease progression.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Treatment with Standard and Low Dose of Conjugated Equine Estrogen Differentially Modulates Estrogen Receptor Expression and Response to Angiotensin II in Mesenteric Venular Bed of Surgically Postmenopausal Hypertensive Rats.

Standard hormone therapy for menopausal women [conjugated equine estrogen (CEE) 0.625 mg] has been associated with increased risk of venous thrombosis. Regimens containing a lower CEE dose (0.30 mg) have been used clinically to decrease side effects of supraphysiologic doses of estrogen. In this study, we determined the effects of standard (SD) and low dose (LD) of CEE on venular function in ovariectomized (OVX) spontaneously hypertensive rats (SHR). Contractions by angiotensin-II (Ang-II 10 µM) in perfused mesenteric venular bed were markedly increased in OVX (21.5 ± 1.3 mmHg) compared with Sham (14.7 ± 1.1 mm Hg, P < 0.05). CEE-SD did not modify Ang-II responses in OVX, whereas CEE-LD restored Ang-II contraction to Sham levels. Endothelial nitric oxide synthase (eNOS) inhibition by L-NAME increased Ang-II contractions in Sham and CEE-LD and was without effect in venules of OVX SHR and CEE-SD. In OVX there was decreased NO generation in association with diminished eNOS phosphorylation and increased O2- generation in the venular wall. CEE-LD reverted the deleterious effects of ovariectomy. Although CEE-SD augmented eNOS phosphorylation in OVX, it was unable to increase NO levels, probably owing to its inability to reduce O2- Distinct effects by CEE-SD and CEE-LD parallel the differential modulation of Ang-II and estrogen receptors. Compared with Sham, CEE-LD increases Ang II receptor type 2, whereas CEE-SD modified ERß expression in the venous bed. Interestingly, both CEE doses increased G protein-coupled estrogen receptor in OVX. Our data suggest that estrogen dose is an important factor for venous function. Although CEE-LD reversed deleterious effects of OVX, CEE-SD showed null effects despite its ability to increase eNOS activity.

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome.

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-ß signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-ß, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 µm(2); Marfan Nox4(-/-): 8,795 ± 824 µm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-ß expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS.

Middle cerebral artery remodeling following transient brain ischemia is linked to early postischemic hyperemia: a target of uric acid treatment.

Ischemia impairs blood supply to the brain, and reperfusion is important to restore cerebral blood flow (CBF) and rescue neurons from cell death. However, reperfusion can induce CBF values exceeding the basal values before ischemia. This hyperemic effect has been associated with a worse ischemic brain damage, albeit the mechanisms that contribute to infarct expansion are not clear. In this study, we investigated the influence of early postischemic hyperemia on brain damage and middle cerebral artery (MCA) properties and the effect of treatment with the endogenous antioxidant uric acid (UA). The MCA was occluded for 90 min followed by 24 h reperfusion in adult male Sprague-Dawley rats. Cortical CBF increases at reperfusion beyond 20% of basal values were taken as indicative of hyperemia. UA (16 mg/kg) or vehicle (Locke's buffer) was administered intravenously 135 min after MCA occlusion. Hyperemic compared with nonhyperemic rats showed MCA wall thickening (sham: 22.4 ± 0.8 µm; nonhyperemic: 23.1 ± 1.2 µm; hyperemic: 27.8 ± 0.9 at 60 mmHg; P < 0.001, hyperemic vs. sham) involving adventitial cell proliferation, increased oxidative stress, and interleukin-18, and more severe brain damage. Thus MCA remodeling after ischemia-reperfusion takes place under vascular oxidative and inflammatory stress conditions linked to hyperemia. UA administration attenuated MCA wall thickening, induced passive lumen expansion, and reduced brain damage in hyperemic rats, although it did not increase brain UA concentration. We conclude that hyperemia at reperfusion following brain ischemia induces vascular damage that can be attenuated by administration of the endogenous antioxidant UA.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid during reperfusion promotes multifaceted brain and vascular protection in spontaneously hypertensive rats with transient ischaemic stroke.

Hypertension is the most prevalent modifiable risk factor for stroke and is associated with worse functional outcomes. Pharmacological inhibition of histone deacetylases by suberoylanilide hydroxamic acid (SAHA) modulates gene expression and has emerged as a promising therapeutic approach to reduce ischaemic brain injury. Here, we have tested the therapeutic potential of SAHA administered during reperfusion in adult male spontaneously hypertensive (SHR) rats subjected to transient middle cerebral artery occlusion (tMCAO; 90 min occlusion/24 h reperfusion). Animals received a single dose of SAHA (50 mg/kg) or vehicle i.p. at 1, 4, or 6 h after reperfusion onset. The time-course of brain histone H3 acetylation was studied. After tMCAO, drug brain penetrance and beneficial effects on behavioural outcomes, infarct volume, oedema, angiogenesis, blood-brain barrier integrity, cerebral artery oxidative stress and remodelling, and brain and vascular inflammation were evaluated. SAHA increased brain histone H3 acetylation from 1 to 6 h after injection, reaching the ischaemic brain administered during reperfusion. Treatment given at 4 h after reperfusion onset improved neurological score, reduced infarct volume and oedema, attenuated microglial activation, prevented exacerbated MCA angiogenic sprouting and blood-brain barrier breakdown, normalised MCA oxidative stress and remodelling, and modulated brain and cerebrovascular cytokine expression. Overall, we demonstrate that SAHA administered during early reperfusion exerts robust brain and vascular protection after tMCAO in hypertensive rats. These findings are aligned with previous research in ischaemic normotensive mice and help pave the way to optimise the design of clinical trials assessing the effectiveness and safety of SAHA in ischaemic stroke.

Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta.

AIMS: Transient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. MAIN METHODS: Aorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. KEY FINDINGS: TRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SIGNIFICANCE: In conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold.

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

Targeting mitochondrial stress with Szeto-Schiller 31 prevents experimental abdominal aortic aneurysm: Crosstalk with endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction and inflammation contribute to a myriad of cardiovascular diseases. Deleterious crosstalk of mitochondria and persistent endoplasmic reticulum (ER) stress triggers oxidative stress, which is involved in the development of vascular diseases. This study determined if inhibition of mitochondrial stress reduces aneurysm development in angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE-/- ) mice and its effect on ER stress. EXPERIMENTAL APPROACH: The mitochondria-targeted tetrapeptide, Szeto-Schiller 31 (SS31), ameliorated mitochondrial dysfunction and the enhanced expression of ER stress markers triggered by Ang II in ApoE-/- mice, and limited plasmatic and vascular reactive oxygen species (ROS) levels. Interestingly, SS31 improved survival, reduced the incidence and severity of abdominal aortic aneurysm (AAA), and the Ang II-induced increase in aortic diameter as evaluated by ultrasonography, resembling the response triggered by the classic ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyrate (PBA). KEY RESULTS: Disorganization of the extracellular matrix, increased expression of metalloproteinases and pro-inflammatory markers and infiltration of immune cells induced by Ang II in the abdominal aorta were effectively reduced by SS31 and ER inhibitors. Further, C/EBP homologous protein (CHOP) deficiency in ApoE-/- mice attenuated Ang II-mediated increase in vascular diameter and incidence of AAA, suggesting its contribution to the favourable response induced by ER stress inhibition. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that inhibition of mitochondrial stress by SS31 limits AAA formation and increases survival through a reduction of vascular remodelling, inflammation and ROS, and support that attenuation of ER stress contributes to the favourable response elicited by SS31.

Electronegative LDL Is Associated with Plaque Vulnerability in Patients with Ischemic Stroke and Carotid Atherosclerosis.

Owing to the high risk of recurrence, identifying indicators of carotid plaque vulnerability in atherothrombotic ischemic stroke is essential. In this study, we aimed to identify modified LDLs and antioxidant enzymes associated with plaque vulnerability in plasma from patients with a recent ischemic stroke and carotid atherosclerosis. Patients underwent an ultrasound, a CT-angiography, and an 18F-FDG PET. A blood sample was obtained from patients (n = 64, 57.8% with stenosis ≥50%) and healthy controls (n = 24). Compared to the controls, patients showed lower levels of total cholesterol, LDL cholesterol, HDL cholesterol, apolipoprotein B (apoB), apoA-I, apoA-II, and apoE, and higher levels of apoJ. Patients showed lower platelet-activating factor acetylhydrolase (PAF-AH) and paraoxonase-1 (PON-1) enzymatic activities in HDL, and higher plasma levels of oxidized LDL (oxLDL) and electronegative LDL (LDL(-)). The only difference between patients with stenosis ≥50% and <50% was the proportion of LDL(-). In a multivariable logistic regression analysis, the levels of LDL(-), but not of oxLDL, were independently associated with the degree of carotid stenosis (OR: 5.40, CI: 1.15-25.44, p < 0.033), the presence of hypoechoic plaque (OR: 7.52, CI: 1.26-44.83, p < 0.027), and of diffuse neovessels (OR: 10.77, CI: 1.21-95.93, p < 0.033), indicating that an increased proportion of LDL(-) is associated with vulnerable atherosclerotic plaque.

Molecular Mechanisms Underlying the Effects of Olive Oil Triterpenic Acids in Obesity and Related Diseases.

Dietary components exert protective effects against obesity and related metabolic and cardiovascular disturbances by interfering with the molecular pathways leading to these pathologies. Dietary biomolecules are currently promising strategies to help in the management of obesity and metabolic syndrome, which are still unmet medical issues. Olive oil, a key component of the Mediterranean diet, provides an exceptional lipid matrix highly rich in bioactive molecules. Among them, the pentacyclic triterpenic acids (i.e., oleanolic acid) have gained clinical relevance in the last decade due to their wide range of biological actions, particularly in terms of vascular function, obesity and insulin resistance. Considering the promising effects of these triterpenic compounds as nutraceuticals and components of functional foods against obesity and associated complications, the aim of our review is to decipher and discuss the main molecular mechanisms underlying these effects driven by olive oil triterpenes, in particular by oleanolic acid. Special attention is paid to their signaling and targets related to glucose and insulin homeostasis, lipid metabolism, adiposity and cardiovascular dysfunction in obesity. Our study is aimed at providing a better understanding of the impact of dietary components of olive oil in the long-term management of obesity and metabolic syndrome in humans.

An Imidazoline 2 Receptor Ligand Relaxes Mouse Aorta via Off-Target Mechanisms Resistant to Aging.

Imidazoline receptors (IR) are classified into three receptor subtypes (I1R, I2R, and I3R) and previous studies showed that regulation of I2R signaling has neuroprotective potential. In order to know if I2R has a role in modulating vascular tone in health and disease, we evaluated the putative vasoactive effects of two recently synthesized I2R ligands, diethyl (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole -1-phosphonate (B06) and diethyl [(1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H-imidazol-4-yl]phosphonate] (MCR5). Thoracic aortas from Oncins France 1 (3- to 4-months-old) and C57BL/6 (3- to 4- and 16- to 17-months-old mice) were mounted in tissue baths to measure isometric tension. In young mice of both strains, MCR5 induced greater relaxations than either B06 or the high-affinity I2R selective ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), which evoked marginal responses. MCR5 relaxations were independent of I2R, as IR ligands did not significantly affect them, involved activation of smooth muscle KATP channels and inhibition of L-type voltage-gated Ca2+ channels, and were only slightly modulated by endothelium-derived nitric oxide (negatively) and prostacyclin (positively). Notably, despite the presence of endothelial dysfunction in old mice, MCR5 relaxations were preserved. In conclusion, the present study provides evidence against a functional contribution of I2R in the modulation of vascular tone in the mouse aorta. Moreover, the I2R ligand MCR5 is an endothelium-independent vasodilator that acts largely via I2R-independent pathways and is resistant to aging. We propose MCR5 as a candidate drug for the management of vascular disease in the elderly.

Serum from Stroke Patients with High-Grade Carotid Stenosis Promotes Cyclooxygenase-Dependent Endothelial Dysfunction in Non-ischemic Mice Carotid Arteries.

Atherosclerosis is responsible for 20% of ischemic strokes, and severe carotid stenosis is associated with a higher incidence of first-ever and recurrent strokes. The release of pro-inflammatory mediators into the blood in severe atherosclerosis may aggravate endothelial dysfunction after stroke contributing to impair disease outcomes. We hypothesize that environments of severe carotid atherosclerotic disease worsen endothelial dysfunction in stroke linked to enhanced risk of further cerebrovascular events. We mounted nonischemic common carotid arteries from 2- to 4-month-old male Oncins France 1 mice in tissue baths for isometric contraction force measurements and exposed them to serum from men with a recent ischemic stroke and different degrees of carotid stenosis: low- or moderate-grade stenosis (LMGS; < 70%) and high-grade stenosis (HGS; ≥ 70%). The results show that serum from stroke patients induced an impairment of acetylcholine relaxations in mice carotid arteries indicative of endothelium dysfunction. This effect was more pronounced after incubation with serum from patients with a recurrent stroke or vascular death within 1 year of follow-up. When patients were stratified according to the degree of stenosis, serum from HGS patients induced more pronounced carotid artery endothelial dysfunction, an effect that was associated with enhanced circulating levels of IL-1ß. Mechanistically, endothelial dysfunction was prevented by both nonselective and selective COX blockade. Altogether, the present findings add knowledge on the understanding of the mechanisms involved in the increased risk of stroke in atherosclerosis and suggest that targeting COX in the carotid artery wall may represent a potential novel therapeutic strategy for secondary stroke prevention.

Allopurinol blocks aortic aneurysm in a mouse model of Marfan syndrome via reducing aortic oxidative stress.

BACKGROUND: Increasing evidence indicates that redox stress participates in MFS aortopathy, though its mechanistic contribution is little known. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mouse aortae. Here we address the contribution of xanthine oxidoreductase (XOR), which catabolizes purines into uric acid and ROS in MFS aortopathy. METHODS AND RESULTS: In aortic samples from MFS patients, XOR protein expression, revealed by immunohistochemistry, increased in both the tunicae intima and media of the dilated zone. In MFS mice (Fbn1C1041G/+), aortic XOR mRNA transcripts and enzymatic activity of the oxidase form (XO) were augmented in the aorta of 3-month-old mice but not in older animals. The administration of the XOR inhibitor allopurinol (ALO) halted the progression of aortic root aneurysm in MFS mice. ALO administrated before the onset of the aneurysm prevented its subsequent development. ALO also inhibited MFS-associated endothelial dysfunction as well as elastic fiber fragmentation, nuclear translocation of pNRF2 and increased 3'-nitrotyrosine levels, and collagen maturation remodeling, all occurring in the tunica media. ALO reduced the MFS-associated large aortic production of H2O2, and NOX4 and MMP2 transcriptional overexpression. CONCLUSIONS: Allopurinol interferes in aortic aneurysm progression acting as a potent antioxidant. This study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and warrants the evaluation of ALO therapy in MFS patients.

Arachnoid membrane as a source of sphingosine-1-phosphate that regulates mouse middle cerebral artery tone.

Growing evidence indicates that perivascular tissue is critical to modulate vessel function. We hypothesized that the arachnoid membrane surrounding middle cerebral artery (MCA) regulates its function via sphingosine-1-phosphate (S1P)-induced vasoconstriction. The MCA from 3- to 9-month-old male and female wild-type (Oncine France 1 and C57BL/6) mice and sphingosine kinase 2 knockout (SphK2-/-) mice in the C57BL/6 background was mounted in pressure myographs with and without arachnoid membrane. Raman microspectroscopy and imaging were used for in situ detection of S1P. The presence of arachnoid tissue was associated with reduced external and lumen MCA diameters, and with an increase in basal tone regardless of sex and strain background. Strong S1P-positive signals were detected in the arachnoid surrounding the MCA wall in both mice models, as well as in a human post-mortem specimen. Selective S1P receptor 3 antagonist TY 52156 markedly reduced both MCA vasoconstriction induced by exogenous S1P and arachnoid-dependent basal tone increase. Compared to 3-month-old mice, the arachnoid-mediated contractile influence persisted in 9-month-old mice despite a decline in arachnoid S1P deposits. Genetic deletion of SphK2 decreased arachnoid S1P content and vasoconstriction. This is the first experimental evidence that arachnoid membrane regulates the MCA tone mediated by S1P.

Plasma sICAM-1 as a Biomarker of Carotid Plaque Inflammation in Patients with a Recent Ischemic Stroke.

18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) identifies carotid plaque inflammation and predicts stroke recurrence in patients with atherothrombotic stroke. The aim of the study was to identify plasma inflammatory biomarkers associated with plaque inflammation according to 18F-FDG uptake. We conducted a prospective study of consecutive adult patients with a recent (< 7 days) anterior circulation ischemic stroke and at least one atherosclerotic plaque in the ipsilateral internal carotid artery. We included 64 patients, 57.8% of whom showed a carotid stenosis ≥ 50%. All patients underwent an early (< 15 days from inclusion) 18F-FDG PET, and a blood sample was obtained at days 7 ± 1 from the stroke. The plasma concentration of 16 inflammation-related molecules was analyzed in a Luminex using xMAP technology. Multivariable linear regression was used to assess the association between plasma biomarkers and the standardized uptake value (SUV) of 18F-FDG uptake. Soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), and fractalkine (FKN) were independently associated with plaque inflammation (ß = 0.121, 95% CI 0.061-0.181, p < 0.001; ß = 0.144, 95% CI 0.012-0.276, p = 0.033; ß = 0.136, 95% CI 0.037-0.235, p = 0.008). In a multivariable logistic regression analysis, sICAM-1 was associated with SUVmax ≥ 2.85 (OR = 1.02, 95% CI 1.00-1.03, p = 0.020). Multivariable Cox regression was used to assess the association between biomarkers and stroke recurrence. sICAM-1 was associated with stroke recurrence (HR = 1.03, 95% CI 1.00-1.05, p = 0.002). In summary, elevated concentrations of sICAM-1 were associated with carotid plaque inflammation and an increased risk of stroke recurrence in patients with recent ischemic stroke and carotid atherosclerosis.

Sex-Dependent End-of-Life Mental and Vascular Scenarios for Compensatory Mechanisms in Mice with Normal and AD-Neurodegenerative Aging.

Life expectancy decreases with aging, with cardiovascular, mental health, and neurodegenerative disorders strongly contributing to the total disability-adjusted life years. Interestingly, the morbidity/mortality paradox points to females having a worse healthy life expectancy. Since bidirectional interactions between cardiovascular and Alzheimer's diseases (AD) have been reported, the study of this emerging field is promising. In the present work, we further explored the cardiovascular-brain interactions in mice survivors of two cohorts of non-transgenic and 3xTg-AD mice, including both sexes, to investigate the frailty/survival through their life span. Survival, monitored from birth, showed exceptionally worse mortality rates in females than males, independently of the genotype. This mortality selection provided a "survivors" cohort that could unveil brain-cardiovascular interaction mechanisms relevant for normal and neurodegenerative aging processes restricted to long-lived animals. The results show sex-dependent distinct physical (worse in 3xTg-AD males), neuropsychiatric-like and cognitive phenotypes (worse in 3xTg-AD females), and hypothalamic-pituitary-adrenal (HPA) axis activation (higher in females), with higher cerebral blood flow and improved cardiovascular phenotype in 3xTg-AD female mice survivors. The present study provides an experimental scenario to study the suggested potential compensatory hemodynamic mechanisms in end-of-life dementia, which is sex-dependent and can be a target for pharmacological and non-pharmacological interventions.