Acetylcholine-induced Vasorelaxation Research Articles

Soluble guanylyl cyclase, the NO-receptor, regulates endothelium-dependent vascular relaxation via its transnitrosation activity.

We previously demonstrated that the NO-receptor soluble guanylyl cyclase (GC1) has the ability to transnitrosate other proteins in a reaction that involves, in some cases, oxidized Thioredoxin 1 (oTrx1). This transnitrosation cascade was established in vitro and we identified by mass spectrometry and mutational analysis Cys 610 (C610) of GC1 α-subunit as a major donor of S-nitrosothiols (SNO). To assay the relevance of GC1 transnitrosation under physiological conditions and in oxidative pathologies, we studied a knock-in mouse in which C610 was replaced with a serine (KI αC 610S ) under basal or angiotensin II (Ang II)-treated conditions. Despite similar GC1 expression and NO-stimulated cGMP-forming activity, the Ang II-treated KI mice displayed exacerbated oxidative pathologies including higher mean arterial pressure and more severe cardiac dysfunctions compared to the Ang II-treated WT. These phenotypes were associated with a drastic decrease in global S-nitrosation and in levels of SNO-Trx1 and SNO-RhoA in the KI mice. To investigate the mechanism underlying the dysregulation of blood pressure despite an intact NO-cGMP axis, pressure myography and in vivo intravital microscopy were conducted to analyze the vascular resistance tone. Both approaches indicated that, even in the absence of oxidative stress, the single mutation C610S led to a significant deficiency in acetylcholine-induced vasorelaxation while smooth muscle relaxation in response to NO remained unchanged. These findings indicate that the C610S mutation uncoupled the two NO signaling pathways involved in the endothelium and smooth muscle vasorelaxation and suggest that GC1-dependent S-nitrosation is a key player in endothelium-derived hyperpolarization.

Decreased Extracellular Vesicle Vasorin in Severe Preeclampsia Plasma Mediates Endothelial Dysfunction.

Preeclampsia (PE) is a serious pregnancy complication affecting 5-8% of pregnancies globally. It is a leading cause of maternal and neonatal morbidity and mortality. Despite its prevalence, the underlying mechanisms of PE remain unclear. This study aimed to determine the potential role of vasorin (VASN) in PE pathogenesis by investigating its levels in extracellular vesicles (EV) and its effects on vascular function. We conducted unbiased proteomics on urine-derived EV from severe PE (sPE) and normotensive pregnant women (NTP), identifying differential protein abundances. Out of one hundred and twenty proteins with ≥ ±1.5-fold regulation at P<0.05 between sPE and NTP, we focused on Vasorin (VASN), which is downregulated in sPE in urinary EV, in plasma EV and in the placenta and is a known regulator of vascular function. We generated EV with high VASN content from both human and murine placenta explants (Plex EV), which recapitulated disease-state-dependent effects on vascular function observed when treating murine aorta rings (MAR) or human aortic endothelial cells (HAEC) with murine or human plasma-derived EV. In normal murine pregnancy, VASN increases with gestational age (GA), and VASN is decreased in plasma EV, in placenta tissue and in Plex EV after intravenous administration of adenovirus encoding short FMS-like tyrosine kinase 1 (sFLT-1), a murine model of PE (murine-PE). VASN is decreased in plasma EV, in placenta tissue and in EV isolated from conditioned media collected from placenta explants (Plex EV) in patients with sPE as compared to NTP. Human sPE and murine-PE plasma EV and Plex EV impair migration, tube formation, and induces apoptosis in human aortic endothelial cells (HAEC) and inhibit acetylcholine-induced vasorelaxation in murine vascular rings (MAR). VASN over-expression counteracts the effects of sPE EV treatment in HAEC and MAR. RNA sequencing revealed that over-expression or knock down of VASN in HAEC results in contrasting effects on transcript levels of hundreds of genes associated with vasculogenesis, endothelial cell proliferation, migration and apoptosis. The data suggest that VASN, delivered to the endothelium via EV, regulates vascular function and that the loss of EV VASN may be one of the mechanistic drivers of PE. What is NewVASN in circulating plasma EV in sPE is reduced compared with VASN content in plasma EV of gestational age-matched pregnant women.VASN is encapsulated and transported in EV and plays a pro-angiogenic role during pregnancy.VASN should be explored both for its pro-angiogenic mechanistic role and as a novel biomarker and potential predictive diagnostic marker for the onset and severity of PE.What Are the Clinical Implications?VASN plays a role in maintaining vascular health and the normal adaptive cardiovascular response in pregnancy. A decrease of VASN is observed in sPE patients contributing to cardiovascular maladaptation.Strategies to boost diminished VASN levels and/or to pharmacologically manipulate mechanisms downstream of VASN may be explored for potential therapeutic benefit in PE.The decrease in EV-associated VASN could potentially be used as a (predictive) biomarker for PE.

Time-restricted feeding improves aortic endothelial relaxation by enhancing mitochondrial function and attenuating oxidative stress in aged mice

Age-related endothelial dysfunction is a pivotal factor in the development of cardiovascular diseases, stemming, at least in part, from mitochondrial dysfunction and a consequential increase in oxidative stress. These alterations are central to the decline in vascular health seen with aging, underscoring the urgent need for interventions capable of restoring endothelial function for preventing cardiovascular diseases. Dietary interventions, notably time-restricted feeding (TRF), have been identified for their anti-aging effects on mitochondria, offering protection against age-associated declines in skeletal muscle and other organs. Motivated by these findings, our study aimed to investigate whether TRF could similarly exert protective effects on endothelial health in the vasculature, enhancing mitochondrial function and reducing oxidative stress. To explore this, 12-month-old C57BL/6 mice were placed on a TRF diet, with food access limited to a 6-h window daily for 12 months. For comparison, we included groups of young mice and age-matched controls with unrestricted feeding. We evaluated the impact of TRF on endothelial function by measuring acetylcholine-induced vasorelaxation of the aorta. Mitochondrial health was assessed using fluororespirometry, and vascular reactive oxygen species (ROS) production was quantified with the redox-sensitive dye dihydroethidium. We also quantified 4-hydroxynonenal (4-HNE) levels, a stable marker of lipid peroxidation, in the aorta using ELISA. Our findings demonstrated that aged mice on a standard diet exhibited significant impairments in aortic endothelial relaxation and mitochondrial function, associated with elevated vascular oxidative stress. Remarkably, the TRF regimen led to substantial improvements in these parameters, indicating enhanced endothelial vasorelaxation, better mitochondrial function, and reduced oxidative stress in the aortas of aged mice. This investigation establishes a vital foundation, paving the way for subsequent clinical research aimed at exploring the cardiovascular protective benefits of intermittent fasting.

Kaempferol protects against cardiovascular abnormalities induced by nitric oxide deficiency in rats by suppressing the TNF-α pathway

Kaempferol is a natural flavonoid compound that exhibits various pharmacological actions. However, there are few reports regarding the role of kaempferol in cardiovascular abnormalities. This study aimed to assess whether kaempferol could prevent cardiovascular malfunction and hypertrophy provoked by chronic inhibition of nitric oxide (NO) formation in rats. Rats (180–200 g) were treated daily with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (40 mg/kg, in drinking water) for five weeks concomitant with kaempferol (oral administration) at a dose of 20 mg/kg or 40 mg/kg or lisinopril (5 mg/kg). Kaempferol partially prevented the progression of hypertension provoked by NO inhibition (p < 0.05). Left ventricular malfunction and hypertrophy present in hypertensive rats were alleviated by concurrent administration of kaempferol (p < 0.05). Furthermore, L-NAME rats had increased sympathetic nerve-mediated vasoconstriction and decreased acetylcholine-induced vasorelaxation and aortic wall thickening, which were resolved by kaempferol treatment (p < 0.05). Kaempferol restored tissue superoxide formation, malondialdehyde, catalase activity, plasma nitric oxide metabolites, tumor necrosis factor-alpha (TNF-α) and interleukin-6 in L-NAME rats (p < 0.05). Overexpression of tumor necrosis factor receptor 2 (TNFR2), phosphatidylinositol 3-kinases (PI3K), AKT serine/threonine kinase 1 (Akt1) and smad2/3 in heart tissue and upregulation of tumor necrosis factor receptor 1 (TNFR1), phosphorylated nuclear factor-kappaB (p–NF–κB) and transforming growth factor beta 1 (TGF-β1) in vascular tissue were suppressed by kaempferol (p < 0.05). In conclusion, kaempferol exerts antihypertensive, cardioprotective, antioxidant, and anti-inflammatory effects in NO-dependent hypertensive rats. The underlying mechanisms of kaempferol in preventing cardiovascular changes induced by L-NAME were due to the suppression of the TNF-α pathway.

Effects of prenatal nicotine exposure on vascular endothelial function and structure in offspring rats

To clarify the effect of prenatal nicotine exposure (PNE) on vascular endothelial function (VEF) in offspring rats, and to explore whether these effects are long-lasting and sex-dependent. Pregnant Sprague-Dawley rats were randomized into two groups and exposed to either 102 mg/mL nicotine (experimental group) or normal saline (control group) through a subcutaneously implanted osmotic micropump. Vascular rings from the thoracic aorta were collected from offspring rats at 1, 4, and 12 months of age. The specimens were used to determine VEF using functional tests and to observe morphologic and pathologic changes of blood vessels in hematoxylin and eosin-stained samples with light microscopy. The percentage of acetylcholine-induced endothelium-dependent vasorelaxation (EDV) of the thoracic aorta was significantly higher in the experimental group than in the control group in 1-, 4-, and 12-month-old female offspring rats (18.68±2.32 vs. 13.85±6.10, 17.44±3.91 vs. 10.77±5.77, and 30.96±22.26 vs. 18.94±13.61, respectively; P &lt;0.05). The percentage of acetylcholine-induced EDV was significantly lower in the experimental group than in the control group in 1-, 4-, and 12-month-old male offspring rats (10.31±4.05 vs. 16.05±14.80, 5.57±2.81 vs. 12.12±5.62, and 11.98±7.24 vs. 58.87±32.43, respectively; P &lt;0.05). The hematoxylin &amp; eosin-stained vascular rings of the offspring rats displayed an uneven lumen, scattered intimal thickening, partial shedding and vacuolar degeneration of endothelial cells, and inflammatory cell infiltration and phagocytosis in the experimental group. In addition, overt smooth muscle atrophy in tunica media, disordered cell arrangement, and unclear structure of the elastic fiber layer was observed. None of these histopathologic changes were found in the control group. In adult offspring rats, PNE not only affected the VEF of the thoracic aorta but also led to pathologic changes in the vascular structure. Additionally, the effects of PNE on VEF were sex-specific, manifesting primarily as significantly improved VEF in female offspring rats and significantly impaired VEF in male offspring rats, lasting into adulthood.

Mesenteric Artery Reactivity in the Development of Metabolic Syndrome in Rats Fed on a High-Fat Diet

Higt fat diet can lead to the development of metabolic syndrome (MS). However, the question of the mechanisms of pathophysiological processes in MS has not been studied enough. The aim of the work was to study the effect of a high-fat diet (HFD) on the reactivity of the mesenteric arteries of Wistar rats in vivo, as well as to evaluate the change in the mechanisms of endothelium-dependent arterial dilatation in HFD. The HFD-group of rats (n = 25) received HFD containing 50% animal fat for 10 weeks, the control group (n = 25) received a standard diet. The effect of HFD on endothelium-dependent and endothelium-independent responses of the mesenteric arteries under the action of agonists in the absence and with the use of blockers of NO-synthase (L-NAME), cyclooxygenase (indomethacin), and K+-channels (tetraethylammonium) was assessed using photomicrography and video recording of mesenteric artery diameter in vivo. HFD in rats led to the development of MS, including dyslipidemia, hyperglycemia and insulin resistance, and an increase in blood pressure. MS was accompanied by impaired functional state of the mesenteric arteries. In rats of the HFD group, compared with the control group, there was an increase in the constrictor reaction to phenylephrine by 29%, as well as a decrease in the reactivity of vessels previously contracted by phenylephrine under the action of acetylcholine by 36%. Pre-incubation of vessels with blockers reduced the amplitude of relaxation under the action of acetylcholine, compared with the initial acetylcholine-induced vasorelaxation, in HFD-group rats: with L-NAME – by 47%, L-NAME and indomethacin – by 50%, L-NAME, indomethacin and tetraethylammonium – by 65%; in the control group – by 69, 72 and 83%, respectively. HFD had no significant effect on the amplitude of vasodilation under the action of sodium nitroprusside. Thus, endothelial dysfunction in HFD-treated rats was mediated both by impairment of NO-dependent mechanisms of vasodilation, in particular, by a decrease in NO production by the endothelium, and by a decrease in the effectiveness of ВКСа. The decrease in NO bioavailability in HFD was partially compensated by the activation of endothelial hyperpolarization mechanisms (mediated by IKCa and SKCa activities) in acetylcholine-induced vasodilation.

Role of Perivascular Adipose Tissue in Aorta Reactivity from Obese and Hyperglycemic CD-1 Mice: New Insights into Perivascular Adipose Tissue.

Background: Perivascular adipose tissue (PVAT) plays an essential role in cardiovascular homeostasis. However, during obesity and diabetes, its role in vascular tone regulation is unclear. This study aimed to evaluate the function of the PVAT on aorta reactivity in the lean and cafeteria (CAF) diet-induced obese-hyperglycemic mice model. Methods: Aorta reactivity to phenylephrine, KCl, and acetylcholine was analyzed in lean (n = 6) and obese mice (n = 6). Also, nitric oxide (NO-) and cyclooxygenase participation, in the presence (n = 6) and absence (n = 6) of PVAT, were examined in the aortas. Results: After a CAF diet for 19 weeks, obese mice showed increased body weight, glucose intolerance, and hypercholesterolemia concerning lean mice. Vascular reactivity to phenylephrine was reduced significantly in the aorta of obese mice. In contrast, the contraction produced by KCl (80 mM) was increased in the aorta of obese mice independent of PVAT. Acetylcholine-induced vasorelaxation diminished in the aortas of obese mice in the presence of PVAT. Nonselective inhibition of cyclooxygenases likely shows that PVAT and endothelium release vasorelaxant prostanoids. Conclusions: The results suggest that PVAT modulates aorta reactivity by releasing NO-, decreasing the α1-adrenergic response to phenylephrine, and probably releasing vasorelaxant prostanoids. The data suggest that PVAT regulates the vascular smooth muscle and endothelial function in a CAF diet-induced obese-hyperglycemic mice model.

Abstract 091: Does Smooth Muscle Cullin-3 Regulate The Renin-angiotensin System?

Cullin-3 (CUL3) is a scaffold protein involved in the formation and function of the CUL3-RING E3 ubiquitin ligase complex. Loss of smooth muscle CUL3 promotes vascular dysfunction, arterial stiffness, and severe hypertension (HTN). We hypothesized that conditional deletion of CUL3 in smooth muscle cells causes severe HTN through a renin-angiotensin system (RAS)-dependent mechanism. Mice carrying a conditional allele of CUL3 were bred to mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). Tamoxifen was administered to generate smooth muscle CUL3 knockout (S-CUL3KO). Three weeks after tamoxifen, mean arterial pressure (MAP) was 140±1 vs. 108±1 mmHg (S-CUL3KO vs. ISM-CRE). Aorta from S-CUL3KO mice exhibited impaired acetylcholine-induced vasorelaxation (ACh, max relaxation: 19±11% vs. 84±5%, p&lt;0.05). S-CUL3KO mice also manifested impaired vasorelaxation to sodium nitroprusside (SNP, max relaxation: 15±12% vs. 96±2%, p&lt;0.05). Captopril administration (7 days) markedly reduced MAP to 84±0.3 mmHg and improved vasorelaxation in response to both ACh (72±6%) and SNP (87±4%) in S-CUL3KO mice, suggesting RAS-dependency of the HTN. Q-PCR analyses and RNAScope showed no difference in renin expression in the kidney between the S-CUL3KO and control mice suggesting a blunting of the baroreceptor mechanism regulating renin. In preliminary studies, lamin A/C, a crucial component of the nuclear mechanotransducer controlling expression of renin, displayed a trend to increased protein expression in S-CUL3KO animals (6±2 vs. 2±1 normalized RFU, p=0.12). Similarly, expression of connexin 40, a component responsible renin signal transduction in the renin cells, was significantly higher in S-CUL3KO mice (8±1 vs. 3±1 normalized RFU, p&lt;0.05). Our data suggest that CUL3 may play a vital role in baroreceptor mechanism in the renin cells via regulation of components of the baroreflex sensor (connexin 40) and transducer (lamin A/C) which are essential for responding to changes in perfusion pressure. Hence, understanding CUL3 target proteins and the mechanism of the impaired baroreceptor mechanism is beneficial in developing new treatments of hypertension.

Actions of Esomeprazole on the Maternal Vasculature in Lean and Obese Pregnant Mice with Impaired Nitric Oxide Synthesis: A Model of Preeclampsia.

Preeclampsia is a devastating, multisystem disorder of pregnancy. It has no cure except delivery, which if premature can impart significant neonatal morbidity. Efforts to repurpose pregnancy-safe therapeutics for the treatment of preeclampsia have led to the assessment of the proton pump inhibitor, esomeprazole. Preclinically, esomeprazole reduced placental secretion of anti-angiogenic sFlt-1, improved endothelial dysfunction, promoted vasorelaxation, and reduced maternal hypertension in a mouse model. Our understanding of the precise mechanisms through which esomeprazole works to reduce endothelial dysfunction and enhance vasoreactivity is limited. Evidence from earlier studies suggested esomeprazole might work via the nitric oxide pathway, upregulating endothelial nitric oxide synthase (eNOS). Here, we investigated the effect of esomeprazole in a mouse model of L-NAME-induced hypertension (decreased eNOS activity). We further antagonised the model by addition of diet-induced obesity, which is relevant to both preeclampsia and the nitric oxide pathway. Esomeprazole did not decrease blood pressure in this model, nor were there any alterations in vasoreactivity or changes in foetal outcomes in lean mice. We observed similar findings in the obese mouse cohort, except esomeprazole treatment enhanced ex vivo acetylcholine-induced vasorelaxation. As acetylcholine induces nitric oxide production, these findings hint at a function for esomeprazole in the nitric oxide pathway.

Proteomic Profiling and Pathway Analysis of Acid Stress-Induced Vasorelaxation of Mesenteric Arteries In Vitro.

Although metabolic acidosis is associated with numerous pathophysiological conditions and its vasorelaxation effects have been well described in different animal and culture models, the molecular mechanisms of acidosis-induced vasorelaxation are not fully understood. Mesenteric artery models have been used extensively to examine the vascular response to various pathophysiological conditions. Our previous studies and several other reports have suggested the vascular responses of goat mesenteric arteries and human arteries to various stimuli, including acidic stress, are highly similar. In this study, to further identify the signaling molecules responsible for altered vasoreactivity in response to acidic pH, we examined the proteomic profile of acid stress-induced vasorelaxation using a goat mesenteric artery model. The vascular proteomes under acidic pH were compared using 2D-GE with 7 cm IPG strips and mini gels, LC-MS/MS, and MALDI TOF MS. The unique proteins identified by mass spectroscopy were actin, transgelin, WD repeat-containing protein 1, desmin, tropomyosin, ATP synthase β, Hsp27, aldehyde dehydrogenase, pyruvate kinase, and vitamin K epoxide reductase complex subunit 1-like protein. Out of five protein spots identified as actin, three were upregulated > 2-fold. ATP synthase β was also upregulated (2.14-fold) under acid stress. Other actin-associated proteins upregulated were transgelin, desmin, and WD repeat-containing protein 1. Isometric contraction studies revealed that both receptor-mediated (histamine) and non-receptor-mediated (KCl) vasocontraction were attenuated, whereas acetylcholine-induced vasorelaxation was augmented under acidosis. Overall, the altered vasoreactivity under acidosis observed in the functional studies could possibly be attributed to the increase in expression of actin and ATP synthase β.

Effects of local cryotherapy on systemic endothelial activation, dysfunction, and vascular inflammation in adjuvant-induced arthritis (AIA) rats

AimThis study explored the systemic vascular effects of local cryotherapy with a focus on endothelial changes and arterial inflammation in the model of rat adjuvant-induced arthritis (AIA).MethodsCryotherapy was applied twice a day on hind paws of AIA rats from the onset of arthritis to the acute inflammatory phase. Endothelial activation was studied in the aorta by measuring the mRNA levels of chemokines (CXCL-1, MCP-1 (CCL-2), MIP-1α (CCL-3)) and adhesion molecules (ICAM-1, VCAM-1) by qRT-PCR. Endothelial dysfunction was measured in isolated aortic and mesenteric rings. Aortic inflammation was evaluated via the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6) by qRT-PCR and leucocyte infiltration analysis (flow cytometry). Plasma levels of TNF-α, IL-6, IL-1β, IL-17A, and osteoprotegerin (OPG) were measured using Multiplex/ELISA.ResultsAIA was associated with an increased aortic expression of CXCL-1 and ICAM-1 as well as an infiltration of leucocytes and increased mRNA expression of IL-6, IL-1β, and TNF-α. Local cryotherapy, which decreased arthritis score and structural damages, reduced aortic mRNA expression of CXCL-1, IL-6, IL-1β, and TNF-α, as well as aortic infiltration of leucocytes (T lymphocytes, monocytes/macrophages, neutrophils) and improved acetylcholine-induced vasorelaxation in the aorta and mesenteric arteries. Plasma levels of IL-17A and OPG were significantly reduced by cryotherapy, while the number of circulating leucocytes was not. IL-17A levels positively correlated with endothelial activation and dysfunction.ConclusionIn the AIA model, local cryotherapy reduced systemic endothelial activation, immune cell infiltration, and endothelial dysfunction. Mechanistically, the reduction of circulating levels of IL-17A appears as the possible link between joint cooling and the remote vascular effects.

Alterations in aortic vasorelaxation in rats with epilepsy induced by the electrical amygdala kindling model

Cardiovascular alterations are frequently related to epilepsy in both clinical and experimental models, and have been hypothesized as a potential contributor to sudden unexpected death in epilepsy (SUDEP). Further, the frequency of generalized tonic-clonic seizures (GTCS) is a primary risk factor for SUDEP. Therefore, we aimed to evaluate the vascular response of rats subjected to the electrical amygdala kindling model of epilepsy. Male Wistar rats were randomly distributed into the following groups: without seizures (sham, n = 8), 5 GTCS (5 S, n = 5), and 10 GTCS (10 S, n = 6). One day after the last seizure, the rats were euthanized, and the thoracic aorta rings with (E+) and without (E-) endothelium were used to evaluate vascular reactivity ex vivo using the organ bath system. The maximum response to acetylcholine-induced vasorelaxation in the E+ aortic ring was lower in the 5 S group than in the sham and 10 S groups. A reduced concentration of sodium nitroprusside was required to induce vasorelaxation in the E- aortic rings. These results suggest an impairment in endothelial function and alterations in the nitric oxide (NO) pathway. In conclusion, epilepsy altered the vasorelaxation of the aortic rings and the number of seizures influenced these alterations; therefore, an analysis of endothelial function in patients with a high risk of SUDEP may be beneficial.

Vascular Endothelial Dysfunction in the Thoracic Aorta of Rats with Ischemic Acute Kidney Injury: Contribution of Indoxyl Sulfate

Chronic kidney disease (CKD) and cardiovascular disease are known to be linked, and the involvement of indoxyl sulfate (IS), a type of uremic toxin, has been suggested as one of the causes. It is known that IS induces vascular dysfunction through overproduction of reactive oxygen species (ROS). On the other hand, the involvement of IS in the vascular dysfunction associated with acute kidney injury (AKI) is not fully understood. Therefore, we investigated this issue using the thoracic aorta of rats with ischemic AKI. Ischemic AKI was induced by occlusion of the left renal artery and vein for 45 min, followed by reperfusion 2 weeks after contralateral nephrectomy. One day after reperfusion, there was marked deterioration in renal function evidenced by an increase in plasma creatinine. Furthermore, blood IS levels increased markedly due to worsening renal function. Seven days and 28 days after reperfusion, blood IS levels decreased with the improvement in renal function. Of note, acetylcholine-induced vasorelaxation deteriorated over time after reperfusion, contradicting the recovery of renal function. In addition, 28 days after reperfusion, we observed a significant increase in ROS production in the vascular tissue. Next, we administered AST-120, a spherical adsorbent charcoal, after reperfusion to assess whether the vascular endothelial dysfunction associated with the ischemic AKI was due to a temporary increase in blood IS levels. AST-120 reduced the temporary increase in blood IS levels after reperfusion without influencing renal function, but did not restore the impaired vascular reactivity. Thus, in ischemic AKI, we confirmed that the vascular endothelial function of the thoracic aorta is impaired even after the recovery of kidney injury, probably with excessive ROS production. IS, which increases from ischemia to early after reperfusion, may not be a major contributor to the vascular dysfunction associated with ischemic AKI.

Endothelial BBSome is essential for vascular, metabolic, and retinal functions

ObjectivesEndothelial cells that line the entire vascular system play a pivotal role in the control of various physiological processes, including metabolism. Additionally, endothelial dysfunction is associated with many pathological conditions, including obesity. Here, we assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins in endothelial cells. MethodsWe studied the effects of BBSome disruption in endothelial cells on vascular function, body weight, glucose homeostasis, and the liver and retina. For this, we generated mice with selective BBSome disruption in endothelial cells through Bbs1 gene deletion. ResultsWe found that endothelial cell–specific BBSome disruption causes endothelial dysfunction, as indicated by the impaired acetylcholine-induced vasorelaxation in both the aorta and mesenteric artery. This was associated with an increase in the contractile response to thromboxane A2 receptor agonist (U46619) in the mesenteric artery. Mechanistically, we demonstrated that mice lacking the Bbs1 gene in endothelial cells show elevated vascular angiotensinogen gene expression, implicating renin-angiotensin system activation in the vascular changes evoked by endothelial BBSome deficiency. Strikingly, our data indicate that endothelial BBSome deficiency increases body weight and fat mass and causes hepatosteatosis along with alterations in hepatic expression of lipid metabolism–related genes and metabolomics profile. In addition, electroretinogram and optical coherence tomography analyses revealed functional and structural abnormalities in the retina, evoked by absence of the endothelial BBSome. ConclusionsOur findings demonstrate that the BBSome in endothelial cells is required for the regulation of vascular function, adiposity, hepatic lipid metabolism, and retinal function.

Renal Denervation Improves Exercise Performance in the Setting of Severe Cardiometabolic HFpEF

Background Metabolic comorbidities associated with heart failure with preserved ejection fraction (HFpEF) have complicated the treatment of this debilitating syndrome. Systemic metabolic dysregulation, cardiac dysfunction, and elevated oxidative stress in cardiometabolic HFpEF manifest in severe exercise intolerance, diminished quality of life, and repeated hospitalizations. Renal denervation (RDN) is a device-based therapy designed for treatment of resistant hypertension. RDN has recently been shown to attenuate the severity of metabolic syndrome in preclinical models. To date, RDN has not been evaluated in the setting of severe HFpEF. Objective To investigate the effects of RDN on exercise tolerance, vascular reactivity, and skeletal muscle mitochondrial function in a clinically relevant cardiometabolic model of HFpEF. Methods 20-week-old male ZSF1 obese rats (n=13-14 per group) were randomized to undergo either Sham-RDN or radiofrequency (RF)-RDN treatment on week 4 of a 10-week HFpEF protocol. At baseline, 4-, and 8-weeks of the study protocol, rats were subject to a treadmill exercise protocol. At 10 weeks, thoracic aorta and gastrocnemius muscle samples were collected for vascular reactivity and mitochondrial respiration studies, respectively. Results Compared to the sham-RDN group, RDN-treated ZSF1 obese rats demonstrated significant improvement in exercise capacity at 4 weeks following treatment (Figure A). Acetylcholine-induced vasorelaxation was significantly (p < 0.01 vs. Sham-RDN) following RDN treatment (Figure B). Furthermore, we observed significantly (p < 0.01) improved respiration (State 3) in skeletal muscle mitochondria isolated from RDN-treated animals compared to sham-RDN (Figure C). Conclusion Our data indicate that RDN improves exercise tolerance in the setting of severe HFpEF. Ablation of the renal sympathetic nerves enhanced vascular function which is critical for exercise performance. The improvement in skeletal muscle mitochondrial respiration that we observed likely contributes to the improved exercise tolerance following RDN. These provocative findings suggest that inhibition of renal sympathetic nerve activity by RDN improves peripheral vascular and skeletal muscle function to improve exercise capacity. Furthermore, these findings strongly support RDN as a viable therapy to treat patients that suffer from cardiometabolic HFpEF.

Liver circadian clock disruption alters perivascular adipose tissue gene expression and aortic function in mice.

The liver plays a central role that influences cardiovascular disease outcomes through regulation of glucose and lipid metabolism. It is recognized that the local liver molecular clock regulates some liver-derived metabolites. However, it is unknown whether the liver clock may impact cardiovascular function. Perivascular adipose tissue (PVAT) is a specialized type of adipose tissue surrounding blood vessels. Importantly, cross talk between the endothelium and PVAT via vasoactive factors is critical for vascular function. Therefore, we designed studies to test the hypothesis that cardiovascular function, including PVAT function, is impaired in mice with liver-specific circadian clock disruption. Bmal1 is a core circadian clock gene, thus studies were undertaken in male hepatocyte-specific Bmal1 knockout (HBK) mice and littermate controls (i.e., flox mice). HBK mice showed significantly elevated plasma levels of β-hydroxybutyrate, nonesterified fatty acids/free fatty acids, triglycerides, and insulin-like growth factor 1 compared with flox mice. Thoracic aorta PVAT in HBK mice had increased mRNA expression of several key regulatory and metabolic genes, Ppargc1a, Pparg, Adipoq, Lpl, and Ucp1, suggesting altered PVAT energy metabolism and thermogenesis. Sensitivity to acetylcholine-induced vasorelaxation was significantly decreased in the aortae of HBK mice with PVAT attached compared with aortae of HBK mice with PVAT removed, however, aortic vasorelaxation in flox mice showed no differences with or without attached PVAT. HBK mice had a significantly lower systolic blood pressure during the inactive period of the day. These new findings establish a novel role of the liver circadian clock in regulating PVAT metabolic gene expression and PVAT-mediated aortic vascular function.

Hyperuricemia impaired nitric oxide bioavailablity and deteriorated pulmonary arterial hypertension via a uric acid transporter, URATv1 in xanthine oxidoreductase (XOR)-independent manner

Abstract Background Hyperuricemia occurs in approximately 80% in patients with pulmonary arterial hypertension (PAH) and is positively correlated with pulmonary arterial pressure (PAP). It has been reported that uric acid (UA) reduced endothelium derived nitric oxide (NO) production in porcine pulmonary arterial endothelial cells (PAEC). However, the effects of UA and xanthine oxidoreductase (XOR), catalytic enzyme of UA, on the development of PAH have not been fully elucidated. Purpose We examined the followings; (1) the effects of hyperuricemia on the endothelial function and the development of PAH in rats (2) the therapeutic effects of UA transporter inhibitor on PAH in rats, and (3) the role of XOR in PAH in mice. Methods We used normal and 5-wk Sugen5416/Hypoxia/Normoxia-exposed (SU/Hx/Nx) rats. Gene expression levels of URATv1, a UA transporter, were measured by RT-PCR. We determined the isometric tension of PA rings isolated from normal rats. The study with the isolated perfused lung preparation was performed in SU/HX/Nx rats. To investigate the chronic effect of UA on the development of PAH, hyperuricemia was induced by the administration of 2% oxonic acid (OA) in diet for 6-wk. Benzbromarone (BBR, 10mg/kg/day, diet, from weeks 0 to 5), a URATv1 transporter inhibitor, was administered in the SU/Hx/Nx-rats with or without 2%OA. To examine the role of XOR in PAH, XOR+/− and wild type (WT) mice were exposed to 3-wk Nx or Hx (10% O2). Results The mRNA of URATv1 was detected in the normal lungs. Isometric tension study showed that UA (8 mg/dl) inhibited acetylcholine-induced vasorelaxation. In perfused lung preparations, UA acutely increased estimated PVR in a dose-dependent manner (1.6–16.0mg/dl) with reducing cGMP levels in the lungs. BBR significantly attenuated the pressor response to UA. UA levels in the plasma and the lung tissues were significantly elevated in SU/Hx/Nx-rats with 2%OA (normal vs. vehicle vs. 2%OA, plasma: 0.24±0.01 vs. 0.80±0.14 and 1.44±0.17 mg/dl; lung tissues: 68±3 vs. 142±3 and 377±46 pmol/g tissue). They exhibited further elevation of right ventricle systolic pressure (RVSP) (31±2 vs. 72±6 vs. 101±3 mmHg) and Ea (a marker of RV afterload) (0.24±0.04 vs. 0.97±0.15 vs. 2.36±0.49 mmHg/μL) with the exacerbation of occlusive lesions of PAs. BBR had no changes in the UA levels in the plasma (1.93±0.30 mg/dL), but significantly reduced the UA levels in the lung tissues (101±10 pmol/g tissue) and attenuated the increase in RVSP (53±8mmHg) and Ea (0.21±0.05 mmHg/mL) in the SU/Hx/Nx-rats with 2%OA. On the other hand, BBR had no effects on RVSP (76±7 mmHg) and Ea (0.91±0.15 mmHg/mL) in the SU/Hx/Nx-rats without 2%OA. There were no significant differences in RVSP between XOR+/− mice with Hx and WT with Hx (26±2 vs. 26±2 mmHg). Conclusions Hyperuricemia itself impairs endothelial function and deteriorates PAH via URATv1 in a XOR-independent manner. UA can be a novel therapeutic target for PAH. Funding Acknowledgement Type of funding source: None

Tadalafil ameliorates memory deficits, oxidative stress, endothelial dysfunction and neuropathological changes in rat model of hyperhomocysteinemia induced vascular dementia

Aim The present study investigates the potential of Tadalafil, a phosphodiesterase-5 inhibitor, in a rat model of hyperhomocysteinemia induced vascular dementia. Methods Hyperhomocysteinemia induced vascular dementia in Wistar rats was produced by administering l-Methionine (1.7 g/kg/day; p.o.×8 weeks). Learning and memory was assessed by employing Morris water maze (MWM) test. Endothelial dysfunction was assessed through acetylcholine-induced endothelial-dependent vasorelaxation and serum nitrite levels. Various other biochemical and histopathological estimations were also performed. Results l-Methionine produced significant impairment in acetylcholine-induced endothelium-dependent vasorelaxation and a decrease in serum nitrite levels indicating endothelial dysfunction. Further, these animals performed poorly on Morris water maze, depicting impairment of learning and memory. There was a significant rise in brain oxidative stress level (indicated by an increase in brain thiobarbituric acid reactive species and a decrease in reduced glutathione levels). Increase in brain acetylcholinesterase activity; brain myeloperoxidase activity and brain neutrophil infiltration (a marker of inflammation) were also observed. Tadalafil (5 and 10 mg/kg, p.o.)/Donepezil (0.5 mg/kg, i.p., serving as standard) treatment ameliorated l-Methionine induced endothelial dysfunction; memory deficits; biochemical and histopathological changes in a significant manner. Conclusions It may be concluded that tadalafil has shown efficacy in the rat model of l-Methionine induced vascular dementia and that phosphodiesterase-5 can be considered as an important therapeutic target for the treatment of vascular dementia.

Arachidonic Acid Metabolites of CYP450 Enzymes and HIF-1α Modulate Endothelium-Dependent Vasorelaxation in Sprague-Dawley Rats under Acute and Intermittent Hyperbaric Oxygenation.

Acetylcholine-induced vasorelaxation (AChIR) and responses to reduced pO2 (hypoxia-induced relaxation (HIR), 0% O2) were assessed in vitro in aortic rings of healthy male Sprague-Dawley rats (N = 252) under hyperbaric (HBO2) protocols. The studied groups consisted of the CTRL group (untreated); the A-HBO2 group (single HBO2; 120 min of 100% O2 at 2.0 bars); the 24H-HBO2 group (examined 24 h after single exposure) and the 4D-HBO2 group (four consecutive days of single HBO2). AChIR, sensitivity to ACh and iNOS expression were decreased in the A-HBO2 group. HIR was prostanoid- and epoxyeicosatrienoic acid (EET)-mediated. HIF-1α expression was increased in the 24H-HBO2 and 4D-HBO2 groups. LW6 (HIF-1α inhibitor) decreased HIR in the 24H-HBO2 group. HBO2 affected the expression of COX-1 and COX-2. CYP2c11 expression was elevated in the 24H-HBO2 and 4D-HBO2 groups. Concentrations of arachidonic acid (AA) metabolites 14(15)-DiHET, 11(12)-DiHET and 8(9)-DiHET were increased in A-HBO2 and 24H-HBO2. An increased concentration of 8(9)-EET was observed in the A-HBO2 and 24h-HBO2 groups vs. the CTRL and 4D-HBO2 groups, and an increased concentration of 5(6)-DiHET was observed in the 24H-HBO2 group vs. the 4D-HBO2 group. The 20-HETE concentration was increased in the A-HBO2 group. All were determined by LC-MS/MS of the aorta. The results show that AChIR in all groups is mostly NO-dependent. HIR is undoubtedly mediated by the CYP450 enzymes’ metabolites of AA, whereas HIF-1α contributes to restored HIR. Vasoconstrictor metabolites of CYP450 enzymes contribute to attenuated AChIR and HIR in A-HBO2.

Sex differences in cardio-metabolic and cognitive parameters in rats with high-fat diet-induced metabolic dysfunction.

Excessive dietary fat intake plays important roles in the process of metabolic dysfunction and increases susceptibilities to chronic diseases such as hypertension. Few previous studies, however, have accurately reflected real-world medical conditions. In addition, studies performed to date have not examined detailed sex-differences in cardio-metabolic and cognitive parameters, precluding the development of sex-tailored interventions for patients with metabolic dysfunction who are susceptible to hypertension and cognitive impairment. In this study, using rats with HFD-induced metabolic dysfunction that made them susceptible to hypertension and cognitive impairment, we demonstrate that male rats show greater impairment of acetylcholine-induced vasorelaxation of the carotid artery and systolic blood pressure compared to female rats. These findings may provide a basis for the early detection of carotid artery dysfunction and systolic blood pressure increase, especially in males.