Aortic Rings Research Articles

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

Angiogenesis is a crucial process in the progression of various pathologies, like solid tumors, wet age-related macular degeneration, and chronic inflammation. Current anti-angiogenic treatments still have major drawbacks like limited efficacy in diseases that also rely on inflammation. Therefore, new anti-angiogenic approaches are sorely needed, and simultaneous inhibition of angiogenesis and inflammation is desirable. Here, we show that 2-desaza-annomontine (C81), a derivative of the plant alkaloid annomontine previously shown to inhibit endothelial inflammation, impedes angiogenesis by inhibiting CDC2-like kinases (CLKs) and WNT/β-catenin signaling. C81 reduced choroidal neovascularization in a laser-induced murine in vivo model, inhibited sprouting from vascular endothelial growth factor A (VEGF-A)-activated murine aortic rings ex vivo, and reduced angiogenesis-related activities of endothelial cells in multiple functional assays. This was largely phenocopied by CLK inhibitors and knockdowns, but not by inhibitors of the other known targets of C81. Mechanistically, CLK inhibition reduced VEGF receptor 2 (VEGFR2) mRNA and protein expression as well as downstream signaling. This was partly caused by a reduction of WNT/β-catenin pathway activity, as activating the pathway induced, while β-catenin knockdown impeded VEGFR2 expression. Surprisingly, alternative splicing of VEGFR2 was not detected. In summary, C81 and other CLK inhibitors could be promising compounds in the treatment of diseases that depend on angiogenesis and inflammation due to their impairment of both processes.Graphical abstract

Chemical Characterization of Phoenix dactylifera L. Seeds and their Beneficial Effects on the Vascular Response in Hypertensive Rats.

Although Phoenix dactylifera dates are traditionally consumed for their health benefits, no research has been done on the vascular response in hypertensive animals. This study evaluated the vascular relaxation of hydroalcoholic extracts from seeds of three varieties of P. dactylifera; Sukkari seed (SS), Ajwa seed (AS), and Mabroom seed (MS) on L-NAME-induced hypertension and spontaneously hypertensive rats (SHR). Results showed that all extracts (10µg/mL) caused relaxations higher than 60% in the aortic rings precontracted with 10- 6 M phenylephrine in normotensive rats, the SS extract was the most potent. Endothelial nitric oxide (NO) pathway is involved as significantly reduced vascular relaxation in denuded-endothelium rat aorta and with an inhibitor (10- 4 M L-Nω-Nitro arginine methyl ester; L-NAME) of endothelial nitric oxide synthase (eNOS). Confocal microscopy confirmed that 10µg/mL SS extract increases NO generation as detected by DAF-FM fluorescence in intact aortic rings. Consistent with these findings, vascular relaxation in intact aortic rings at 10µg/mL SS extract was significantly decreased in L-NAME-induced hypertensive rats (endothelial dysfunction model), but not in SHR. In both hypertensive models, the denuded endothelium blunted the vascular relaxation. In conclusion, the hydroalcoholic extract of the seed of P. dactylifera (Sukkari, Ajwa and Mabroom varieties) presents a potent endothelium-dependent vascular relaxation, via NO, in normotensive rats as well as in two different models of hypertension. This effect could be mediated by the presence of phenolic compounds identified by UHPLC-ESI-MS/MS, such as protocatechuic acid, and caftaric acid.

Effects of the Heterodimeric Neurotoxic Phospholipase A2 from the Venom of Vipera nikolskii on the Contractility of Rat Papillary Muscles and Thoracic Aortas.

Phospholipases A2 (PLA2s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA2s (svPLA2s) are extracellular proteins with a molecular mass of 13-14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA2 targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA2s were inconsistent, while the data on the dimeric svPLA2 crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA2 HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA2s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 μM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA2s. These are the first indications of the cardiac and vascular effects of true vipers' heterodimeric svPLA2s.

3,3′-O-dimethylquercetin: A bi-functional vasodilator isolated from green propolis of the Caatinga Mimosa tenuiflora

In the search for novel, bi-functional compounds acting as CaV1.2 channel blockers and K+ channel stimulators, which represent an effective therapy for hypertension, 3,3′-O-dimethylquercetin was isolated for the first time from Brazilian Caatinga green propolis. Its effects were investigated through electrophysiological, functional, and computational approaches. In rat tail artery myocytes, 3,3′-O-dimethylquercetin blocked Ba2+ currents through CaV1.2 channels (IBa1.2) in a concentration-dependent manner, with the inhibition being reversed upon washout. The compound also shifted the voltage dependence of the steady-state inactivation curve to more negative potentials without affecting the slope of the inactivation and activation curves. Furthermore, the flavonoid stimulated KCa1.1 channel currents (IKCa1.1). In silico simulations provided additional evidence for the binding of 3,3′-O-dimethylquercetin to KCa1.1 and CaV1.2 channels and elucidated its mechanism of action. In depolarized rat tail artery rings, the flavonoid induced a concentration-dependent relaxation. Moreover, in rat aorta rings its antispasmodic effect was inversely related to the transmembrane K+ gradient. In conclusion, 3,3′-O-dimethylquercetin demonstrates effective in vitro vasodilatory properties, encouraging the exploration of its scaffold to develop novel derivatives for potential use in the treatment of hypertension.

Comparison effect of high-intensity interval training and moderate intensity continuous training on vascular function in a mouse model of lower extremity peripheral artery disease

Introduction
 Lower extremity peripheral artery disease (PAD), due to narrowing of the arteries supplying the legs, represents a major cause of physical disability. Exercise training is an important part of PAD treatment, improving functional capacity. Recent research suggests that exercise training-induced endothelial function improvement is involved in the benefits of exercise in PAD (Lee et al., 2023); however, the underlying molecular mechanisms remain unknown. In addition, the potential vascular benefits of high intensity training in PAD remain unexplored. Therefore, the aim of the study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on endurance capacity, vascular endothelial function and expression of genes related to nitric oxide (NO) bioavailability in a mouse model of PAD.
 Methods
 PAD disease model was generated in male C57BL/6 mice by ligation of the right iliac artery. Mice with PAD were divided into 3 groups: sedentary control group (SED), MICT (40 min running at 70% of maximal aerobic speed) and HIIT (8 intervals of 2.5 minutes running at 90% of maximal aerobic speed combined with 2.5 minutes of running at 50% of maximal aerobic speed). Mice were exercised three times per week for 8 weeks. Maximal running time was determined by an incremental test until exhaustion. Vascular reactivity was assessed by measuring vasoconstrictor responses to phenylephrine as well as endothelium-dependent and -independent vasodilator responses to acetylcholine and DEA-NONOate, respectively, in isolated aortic rings. Gene expression of antioxidant enzymes (catalase and superoxide dismutase isoforms), prooxidant NADPH oxidase subunits (p22phox and p47phox), enzyme involved in NO production (endothelial NO synthase), vascular smooth muscle cell relaxation (soluble guanylate cyclase) and receptors involved in vascular tone regulation (a1 and b2 adrenergic receptors) was determined in aortic tissue by qPCR.
 Results
 Maximal running time was improved in both MICT and HIIT as compared to SED at the end of the study, but with no significant difference between the two exercise groups. Phenylephrine-induced vasoconstriction as well as vasodilator responses to acetylcholine and DEA-NONOate were significantly improved in MICT compared to SED while no significant differences were reported between HIIT and SED. There was no significant difference in gene expression among the three groups.
 Discussion/Conclusion
 MICT and HIIT were equally effective at improving endurance capacity in mice with PAD. However, only MICT improved vascular reactivity.
 References
 Lee, J., Zarezadehmehrizi, A., LaVoy, E., Markofski, M., & Park, Y. (2023). Exercise training improves brachial artery endothelial function, but does not alter inflammatory biomarkers in patients with peripheral artery disease: A systematic review and meta-analysis. Journal of Cardiovascular Translational Research. Advance online publication. https://doi.org/10.1007/s12265-023-10451-0

Activation of ATP-sensitive potassium channels prevents doxorubicin-induced mitochondrial dysfunction in the heart and impaired vascular responses in rats

One of the side effects of the anticancer drug doxorubicin is its mitotoxicity. At the same time, a sufficient number of functioning mitochondria is required for normal energy supply to the heart. The system of ATP-sensitive potassium channels (KATP -channels) of cytoplasmic and mitochondrial membranes is considered to be the central metabolic sensor of energy supply, and their opening triggers mechanisms of protection against cell damage and death under the influence of pathological factors. The aim of our study was to investigate the effect of KATP-channel opener flocalin on doxorubicin-induced mitochondrial dysfunction in the heart, impaired vascular contraction-relaxation function, and oxidative stress. Acute cardiotoxicity was modelled by short-term intraperitoneal injection of doxorubicin in a total dose of 15 mg/kg. To prevent damage, animals were administered flocalin at a dose of 2.5 mg/kg for 5 consecutive days. It was found that the rate of formation of superoxide anion (•O2 - ) and hydroxyl radical (•OH) in the heart mitochondria significantly increased after administration of doxorubicin by 10.5 and 3.4 times, respectively, and the level of H2O2 increased by 5.3 times. When rats were administered flocalin against the background of doxorubicin, oxidative stress indicators were significantly reduced, namely, the rate of •O2 - and •ON generation was 4 and 1.6 times lower, respectively, and the H2O2 levels were 4.6 times lower. Under conditions of impaired redox status in the rat heart after doxorubicin administration, mitochondrial permeability transition pore opening was activated: the amplitude of spontaneous swelling doubled, and Ca2+-induced swelling increased 1.5-fold. The use of flocalin reduced the amplitude of mitochondrial swelling in calcium-free medium by 84.6%, and under the conditions of action inducer of mPTP opening calcium, this index was restored to control values. Endothelium-dependent relaxation of aorta preparations of doxorubicin-treated animals to acetylcholine (0.1 μmol/l) was 47% less than in the control group. Contractions of aortic rings in these animals under the influence of norepinephrine (10 μmol/l) were reduced by 59% compared to control rats. When flocalin, a KATP-channel opener, was injected into rats, the contractile responses of isolated rat aortic rings were restored almost to the values of control animals, while the endothelium-dependent vasodilator effects of acetylcholine (0.1 μmol/l) under the influence of flocalin were restored by 69% compared with animals injected with doxorubicin. Thus, the opening of KATP-channels by flocalin prevents doxorubicininduced mitochondrial dysfunction in the heart, reduces oxidative stress and prevents vascular contractionrelaxation disorders.

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry's disease.

Fabry's disease is an X-linked lysosomal storage disorder caused by reduced activity of α-galactosidase A (GAL), leading to premature death on account of renal, cardiac, and vascular organ failure. Accumulation of the GAL substrate globotriaosylceramide (Gb3) in endothelial and smooth muscle cells is associated with early vascular cell damage, suggesting endothelial dysfunction as a driver of cardiorenal organ failure. Here, we studied the vascular expression of the key angiogenic factors, VEGFα and its antagonist angiostatin, in Fabry α-GAL-Tg/KO mice and determined circulating VEGFα and angiostatin serum levels in patients with Fabry's disease and healthy controls. Cryopreserved aortic vessels from six α-GAL-Tg/KO and six wild-type (WT) mice were obtained and VEGFα and angiostatin levels were determined by performing Western blot analysis. VEGFα expression was visualized by an immunohistochemical staining of paraffin aortic rings. In addition, VEGFα and angiostatin serum levels were measured by using an enzyme-linked immunosorbent assay in 48 patients with genetically verified Fabry's disease (50% male) and 22 healthy controls and correlated with disease severity markers such as lyso-Gb3, albuminuria, NTproBNP, high-sensitive troponin T (hsTNT), and myocardial wall thickness. It was found that there was a significant increase in VEGFα protein expression (1.66 ± 0.35 vs. 0.62 ± 0.16, p = 0.0009) and a decrease in angiostatin expression (0.024 ± 0.007 vs. 0.053 ± 0.02, p = 0.038) in aortic lysates from α-GAL-Tg/KO compared with that from WT mice. Immunohistochemical staining revealed an adventitial VEGFα signal in α-GAL-Tg/KO mice, whereas no VEGFα signal could be detected in WT mice aortas. No differences in aortic angiostatin expression between α-GAL-Tg/KO- and WT mice could be visualized. The serum levels of VEGFα were significantly upregulated in patients with Fabry's disease compared with that in healthy controls (708.5 ± 426.3 vs. 458.5 ± 181.5 pg/ml, p = 0.048) and positively associated with albuminuria (r = 0.82, p < 0.0001) and elevated NTproBNP (r = 0.87, p < 0.0001) and hsTNT values (r = 0.41, p = 0.048) in male patients with Fabry's disease. For angiostatin, no significant difference was found between patients with Fabry's disease and healthy controls (747.6 ± 390.3 vs. 858.8 ± 599.3 pg/ml). In conclusion, an overexpression of VEGFα and downregulation of its counter player angiostatin in aortic tissue of α-GAL-Tg/KO mice support the hypothesis of an underlying vasculopathy in Fabry's disease. Elevated VEGFα serum levels were also observed in patients with Fabry's disease and were positively associated with elevated markers of organ manifestation in males. These findings suggest that angiogenetic markers, such as VEGFα, may be potentially useful biomarkers for the detection of endothelial dysfunction in classical Fabry's disease.

Lactic acid fermented Elaeagnus multiflora Thunb. fruit: suppressive effect of its extracts on angiogenesis

BackgroundAngiogenesis has been considered as one of the hallmarks of tumor progression and cancer malignancy. Meanwhile, the fruit of Elaeagnus multiflora Thunb. or cherry silverberry (CS), was found to have strong biological activities including anti-cancer and anti-angiogenesis. In this study, the influence of the extracts of E. multiflora Thunb. fruits fermented by mono- and co-culture of L. plantarum and L. casei on the new blood vessel formation in human umbilical vascular endothelial cell (HUVEC) stimulated by vascular endothelial growth factor (VEGF) as well as the underlying mechanism was elucidated.ResultsSulforhodamine B, scratch wound-healing, Transwell migration, capillary-like tube formation, rat aortic ring assays, and morphological analysis were performed to determine the effect of the fermented fruit extracts on the VEGF-induced angiogenic events (motility, migration, invasion, formation of capillary-like tube, blood vessel sprouting ex vivo). The findings showed that at concentration of 25–50 µg/ml, the extracts of CS fermented by mixed cultures is the most effective in inhibiting angiogenesis in HUVECs. Moreover, analysis of the biomarker proteins related to angiogenesis through Western blot technique showed that the fermented extracts exert their anti-angiogenic activity by regulating the VEGFR2 signaling pathway and its possible downstream targets namely Erk ½ and FAK.ConclusionTaken together, the results suggest that the extracts of CS fruits fermented by co-culture of L. plantarum and L. casei has the potential to be utilized in the development of functional food and ingredient with anti-angiogenic properties.Graphical

Absorbed Bioactive Compounds Replicate Guanxin II-Induced Endothelium-Associated in/ex vivo Vasodilation.

To develop an interference-free and rapid method to elucidate Guanxin II (GX II)'s representative vasodilator absorbed bioactive compounds (ABCs) among enormous phytochemicals. The contents of ferulic acid, tanshinol, and hydroxysafflor yellow A (FTA) in GX II/rat serum after the oral administration of GX II (30 g/kg) were detected using ultra-performance liquid chromatography-mass spectrometry. Totally 18 rats were randomly assigned to the control group (0.9% normal saline), GX II (30 g/kg) and FTA (5, 28 and 77 mg/kg) by random number table method. Diastolic coronary flow velocity-time integral (VTI), i.e., coronary flow or coronary flow-mediated dilation (CFMD), and endothelium-intact vascular tension of isolated aortic rings were measured. After 12 h of exposure to blank medium or 0.5 mmol/L H2O2, endothelial cells (ECs) were treated with post-dose GX II of supernatant from deproteinized serum (PGSDS, 300 µL PGSDS per 1 mL of culture medium) or FTA (237, 1539, and 1510 mg/mL) for 10 min as control, H2O2, PGSDS and FTA groups. Nitric oxide (NO), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), superoxide dismutase (SOD), malondialdehyde (MDA) and phosphorylated phosphoinositide 3 kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed. PGSDS was developed as a GX II proxy of ex vivo herbal crude extracts. PGSDS effectively eliminates false responses caused by crude GX II preparations. When doses equaled the contents in GX II/its post-dose serum, FTA accounted for 98.17% of GX II -added CFMD and 92.99% of PGSDS-reduced vascular tension. In ECs, FTA/PGSDS was found to have significant antioxidant (lower MDA and higher SOD, P<0.01) and endothelial function-protective (lower VEGF, ET-1, P<0.01) effects. The increases in aortic relaxation, endothelial NO levels and phosphorylated PI3K/Akt/eNOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester (L-NA, eNOS inhibitor) and wortmannin (PI3K/AKT inhibitor), respectively, indicating an endothelium-dependent vasodilation via the PI3K/AKT-eNOS pathway (P<0.01). This study provides a strategy for rapidly and precisely elucidating GX II's representative in/ex vivo cardioprotective absorbed bioactive compounds (ABCs)-FTA, suggesting its potential in advancing precision ethnomedicine.

Ifanosine: Olea europaea L. and Hyphaene thebaica L. combination, from traditional utilization to rational formulation: Preclinical and clinical efficacy on hypertensives patients

Ethnopharmacological relevanceOlea europaea L. and Hyphaene thebaica L. are commonly employed by traditional healers in Africa for treating and preventing hypertension, either individually or in a polyherbal preparation (Ifanosine). Aim of the studyThe primary aim was to assess the antihypertensive effects of Olea europaea L. leaves aqueous extract (OEL), Hyphaene thebaica L. mesocarp extract (HT), and the Ifanosine on isolated rat aorta rings. The secondary objective was to evaluate the clinical benefits of a new oral formulation of Ifanosine. Materials and methodsIn vitro studies using an isometric transducer examined the antihypertensive effects of HT, OEL, and Ifanosine on rat aorta. Ussing chambers technic were employed to measure mucosal to serosal fluxes and total transepithelial electrical conductance (Gt) to assess the intestinal bioavailability of HT, OEL, and Ifanosine. HPLC was utilized to determine the phytochemical composition of OEL and HT extracts. Subchronic toxicity investigations involved two groups of rats, treated with either water (control) or Ifanosine at 5 g/kg for 28 days. Clinical benefits of the new Ifanosine formulation were evaluated in an observational study with 32 hypertensive patients receiving a fixed oral dose of 3.5 mg three times a day for 30 days. ResultsAqueous extracts induced dose-dependent relaxation of rat aorta rings, with HT and OEL having higher IC50 values than Ifanosine (IC50 = 44.76 ± 1.35 ng/mL, 58.67 ± 1.02 ng/mL, and 29.46 ± 0.26 ng/mL, respectively). The pA2 values of OEL and HT were 1 and 0.6, respectively, while Ifanosine was 0.06. Intestinal bioavailability studies revealed better Prazosin bioavailability than plant extracts. Toxicological studies demonstrated the safety of Ifanosine, supported by histological examinations and biochemical parameters in rat blood. Biochemical analyses indicated flavonoids and phenolic acids as dominant active constituents. Clinical benefits in humans included reduced SBP, DBP, LDL-c, VLDL-c, and TAG, and increased HDL-c without overt adverse effects. ConclusionThis study validates the traditional use of OEL and HT for hypertension and advocates for alternative and combinatorial polyphytotherapy (ACP) to enhance traditional remedies.

N-phenyl and N-benzyl substituted succinimides: Preclinical evaluation for their antihypertensive effect and underlying mechanism

The study was designed to investigate the antihypertensive potential of 2-(2, 5-dioxo-1-phenylpyrrolidin-3-yl)-3-(4-isopropylphenyl)-2-methylpropanal (Comp-1) and 2-(1-benzyl-2,5-dioxopyrrolidin-3-yl)-3-(4-isopropylphenyl)-2-methylpropanal (Succ-5) in rats. The study results showed that, just like nifedipine (the standard reference drug), the test compounds, Comp-1 (at doses of 15 and 20 mg/kg) and Succ-5 (at a dose of 20 mg/kg) had significant antihypertensive effect against deoxycorticosterone acetate-salted rats. The test compounds maintained the level of cardiac markers troponin I and creatinine kinase myocardial bands (CK-MB) in serum, and modulate the oxidative stress markers Glutathione s-transferase (GST) activity, reduced glutathione (GSH), catalase levels, and lipid peroxidation (LPO). These compounds also reduced the expression of inflammatory markers, including cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) in heart tissues. Furthermore, in the ex-vivo study, the test substances relaxed the contractions induced by phenylephrine (PE) and potassium (K+). Vasodilation was endothelium-independent because the test substances showed nearly the same effect in aortic rings with intact endothelium, denuded endothelium, and with L-NAME pretreatment. The test compounds shifted the calcium curve to the right, i.e., contraction was inhibited and decreased the maximal response. This study demonstrated the antihypertensive, anti-inflammatory, antioxidant, and vasodilate effects of the test compounds. In addition, the results supported the phenomenon of calcium channel blockades responsible for vasodilation.

Inhibitors of gastric acid secretion increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling.

The use of inhibitors of gastric acid secretion (IGAS), especially proton pump inhibitors (PPI), has been associated with increased cardiovascular risk. While the mechanisms involved are not known, there is evidence supporting increased oxidative stress, a major activator of matrix metalloproteinases (MMP), as an important player in such effect. However, there is no study showing whether other IGAS such as histamine H2-receptor blockers (H2RB) cause similar effects. This study aimed at examining whether treatment with the H2RB ranitidine promotes oxidative stress resulting in vascular MMP activation and corresponding functional and structural alterations in the vasculature, as compared with those found with the PPI omeprazole. Male Wistar rats were treated (4 weeks) with vehicle (2% tween 20), omeprazole (10 mg/Kg/day; i.p.) or ranitidine (100 mg/Kg/day; gavage). Then the aorta was collected to perform functional, biochemical, and morphometric analysis. Both ranitidine and omeprazole increased gastric pH and oxidative stress assessed in situ with the fluorescent dye dihydroethidium (DHE) and with lucigenin chemiluminescence assay. Both IGAS augmented vascular activated MMP-2. These findings were associated with aortic remodeling (increased media/lumen ratio and number of cells/μm2). Both IGAS also impaired the endothelium-dependent relaxation induced by acetylcholine (isolated aortic ring preparation). This study provides evidence that the H2RB ranitidine induces vascular dysfunction, redox alterations, and remodeling similar to those found with the PPI omeprazole. These findings strongly suggest that IGAS increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling, which helps to explain the increased cardiovascular risk associated with the use of those drugs.

Vasorelaxant effects of ellagitannins isolated from Cuphea carthagenensis.

Cuphea carthagenensis (Jacq.) J. F. Macbr. is a popular plant in Brazilian folk medicine owing to its hypotensive and central nervous system depressant effects. This study aimed to validate the hypotensive effect of the plant's aqueous extract (AE) in rats and examine the vascular actions of three hydrolyzable tannins, oenothein B, woodfordin C, and eucalbanin B, isolated from AE. Systolic blood pressure in unanesthetized rats was determined using the non-invasive tail-cuff method. Oral treatment of normotensive rats with 0.5 and 1.0 g/kg/day AE induced a dose-related hypotensive effect after 1 week. In rat aortic rings pre-contracted with noradrenaline, all ellagitannins (20 - 180 µM) induced a concentration-related vasorelaxation. This effect was blocked by either removing the endothelium or pre-incubating with NG-nitro-l-arginine methyl ester (10 µM), an inhibitor of nitric oxide (NO) synthase. In KCl-depolarized rat portal vein preparations, the investigated compounds did not affect significantly the maximal contractile responses and pD2 values of the concentration-response curves to CaCl2. Our results demonstrated the hypotensive effect of C. carthagenensis AE in unanesthetized rats. All isolated ellagitannins induced vasorelaxation in vitro via activating NO synthesis/NO release from endothelial cells, without altering the Ca2+ influx in vascular smooth muscle preparations. Considering the low oral bioavailability of ellagitannins, the determined in vitro actions of these compounds are unlikely to account for the hypotensive effect of AE in vivo. It remains to be determined the role of the bioactive ellagitannin-derived metabolites in the hypotensive effect observed after oral treatment of unanesthetized rats with the plant extract.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Gestational Hypoxia Impaired Endothelial Nitric Oxide Synthesis Via miR-155-5p/NADPH Oxidase/Reactive Oxygen Species Axis in Male Offspring Vessels.

Nitric oxide (NO) is the most important vasodilator secreted by vascular endothelial cells, and its abnormal synthesis is involved in the development of cardiovascular disease. The prenatal period is a critical time for development and largely determines lifelong vascular health in offspring. Given the high incidence and severity of gestational hypoxia in mid-late pregnancy, it is urgent to further explore whether it affects the long-term synthesis of NO in offspring vascular endothelial cells. Pregnant Sprague-Dawley rats were housed in a normoxic or hypoxic (10.5% O2) chamber from gestation days 10 to 20. The thoracic aortas of fetal and adult male offspring were isolated for experiments. Gestational hypoxia significantly reduces the NO-dependent vasodilation mediated by acetylcholine in both the fetal and adult offspring thoracic aorta rings. Meanwhile, acetylcholine-induced NO synthesis is impaired in vascular endothelial cells from hypoxic offspring thoracic aortas. We demonstrate that gestational hypoxic offspring exhibit a reduced endothelial NO synthesis capacity, primarily due to increased expression of NADPH oxidase 2 and enhanced reactive oxygen species. Additionally, gestational hypoxic offspring show elevated levels of miR-155-5p in vascular endothelial cells, which is associated with increased expression of NADPH oxidase 2 and reactive oxygen species generation, as well as impaired NO synthesis. The present study is the first to demonstrate that gestational hypoxia impairs endothelial NO synthesis via the miR-155-5p/NADPH oxidase 2/reactive oxygen species axis in offspring vessels. These novel findings indicate that the detrimental effects of gestational hypoxia on fetal vascular function can persist into adulthood, providing new insights into the development of vascular diseases.

A 3D-Printed Externally Adjustable Symmetrically Extensible (EASE) Aortic Annuloplasty Ring for Root Repair and Aortic Valve Regurgitation.

The valve-sparing aortic root replacement (VSARR) procedure was developed to preserve the aortic valve apparatus to replace aneurysmal aortic roots with synthetic grafts and to eliminate associated aortic regurgitation (AR). However, residual post-repair AR is not uncommon and has been found to be associated with recurrent AR and future reoperation. We designed and manufactured a 3D-printed, external adjustable symmetrically extensible (EASE) aortic annuloplasty ring that can symmetrically reduce the aortic annulus diameter via a radial constriction, compliant mechanism. An ex vivo porcine VSARR model with annular dilation and AR was developed (n = 4) and used for hemodynamic, echocardiography, and high-speed videography data collection. After ring annuloplasty repair using the EASE aortic ring, the regurgitant fraction decreased from 23.6 ± 6.9% from the VSARR model to 7.4 ± 5.6% (p = 0.05), which was similar to that measured from baseline with a regurgitant fraction of 10.2 ± 3.9% (p = 0.34). The leaflet coaptation height after annuloplasty repair also significantly increased from that measured in VSARR model (0.4 ± 0.1cm) to 0.9 ± 0.1cm (p = 0.0004), a level similar to that measured in baseline (1.1 ± 0.1cm, p = 0.28). Using an ex vivo VSARR model, the EASE ring successfully reduced AR by reducing the annular diameter and improving leaflet coaptation. With its broad applicability and ease of use, this device has the potential to have a significant impact on patients suffering worldwide from AR due to root aneurysms.

Angiogenesis is promoted by hypoxic cervical carcinoma-derived extracellular vesicles depending on the endothelial cell environment

IntroductionCancer needs perfusion for its growth and metastasis. Cancer cell-derived extracellular vesicles (CA-EVs) alter the tumor microenvironment (TME), potentially promoting angiogenesis. We hypothesize that conditions in the tumor, e.g., hypoxia, and in the target cells of the TME, e.g., nutrient deprivation or extracellular matrix, can affect the angiogenic potential of CA-EVs, which would contribute to explaining the regulation of tumor vascularization and its influence on cancer growth and metastasis. MethodsCA-EVs were isolated and characterized from cervical carcinoma cell lines HeLa and SiHa cultured under normoxia and hypoxia, and their angiogenic potential was evaluated in vitro in three endothelial cells (ECs) lines and aortic rings, cultured in basal (growth factor-reduced) or complete medium. ResultsHypoxia increased EV production 10–100 times and protein content 2–4 times compared to normoxic CA-EVs. HeLa-EVs contained six times more RNA than SiHa-EVs, and this concentration was not affected by hypoxia. Treatment with CA-EVs increased tube formation and sprouting in ECs and aortic rings cultured in basal medium and long-term stabilized the stablished vascular networks formed by ECs cultured in complete medium. ConclusionHypoxia differentially affects CA-EVs in a cell line-dependent manner. The cellular environment (nutrient availability and extracellular matrix scaffold) influences the effect of CA-EV on the angiogenic potential of ECs.

Association of Segment-Specific Pulse Wave Velocity With Vascular Calcification: The ARIC (Atherosclerosis Risk in Communities) Study.

Pulse wave velocity (PWV) is a noninvasive measure of arterial stiffness and predictor of cardiovascular disease. However, the association between PWV and vascular calcification across different vascular beds has not been fully investigated. This study aimed to quantify the association between PWV and multiterritory calcification and to explore whether PWV can identify individuals with vascular calcification beyond traditional risk factors. Among 1351 older adults (mean age, 79.2 years [SD, 4.1]) from the ARIC (Atherosclerosis Risk in Communities) study, we measured segment-specific PWVs: heart-carotid, heart-femoral, carotid-femoral, heart-ankle, brachial-ankle, and femoral-ankle. Dependent variables were high calcium score (≥75th percentile of Agatston score) across different vascular beds: coronary arteries, aortic valve ring, aortic valve, mitral valve, ascending aorta, and descending aorta. Quartiles of carotid-femoral, heart-femoral, heart-ankle, and brachial-ankle PWV were significantly associated with coronary artery calcium (eg, adjusted odds ratio [OR] for the highest versus lowest quartile of carotid-femoral PWV, 1.84 [95% CI, 1.24-2.74]). Overall, PWVs were most strongly associated with descending aorta calcification, with significant results for carotid-femoral, heart-femoral, heart-ankle, and brachial-ankle PWV (eg, adjusted OR for the highest versus lowest quartile of carotid-femoral PWV, 3.99 [95% CI, 2.61-6.17]). In contrast, femoral-ankle PWV was inversely associated with descending aorta calcification. Some PWVs improved the discrimination of coronary artery calcium and descending aorta calcification beyond traditional risk factors. The associations of PWV with vascular calcification varied substantially across segments, with descending aorta calcification most closely linked to PWVs. Our study suggests that some PWVs, especially carotid-femoral PWV, are helpful to identify individuals with coronary artery calcium and descending aorta calcification.

Abnormal aortic origin of coronary arteries

The anomalous aortic origin of the coronary arteries has an estimated prevalence of 0.02-5.7%. It can be associated with sudden death when it has an interarterial or intramural pathway or be damaged during interventions on the mitral, pulmonary and/or aortic annulus or percutaneous closure of an interatrial septal defect. To identify these patients by imaging techniques such as transthoracic color Doppler echocardiography (TTE), computed tomography (CT) multislice angiography or coronary angiography. The imaging techniques used for the detection of coronary anomalies were TTE, multislice coronary angiography or coronary angiography according to what is generally accepted. Fifteen patients were identified; in 12 of them the suspicion was due to TTE and in the remaining 3, CT multislice angiography was diagnostic. The circumflex artery was the coronary artery most involved, associated or not with another coronary anomaly (12/15 patients) and in the other three cases, the anomalous coronary artery had an interarterial course, with the right coronary arteries and the anterior descending coronary arteries being involved. The under diagnosis by TTE of coronary artery abnormalities may be due to the difficulty of visualization that is accentuated with age. Their detection is crucial because they can both, lead to sudden death associated with an intramural and/or interarterial pathway and complicate an interventional procedure on the interatrial septum or within the mitral, pulmonary and/or aortic rings.

FS145, the first flea-derived disintegrin, inhibits angiogenesis through specifically binding integrin αvβ3

FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αββ structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72–81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvβ3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvβ3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.