Isolated Mouse Aorta Research Articles

Portulaca Oleracea L. (Purslane) Extract Protects Endothelial Function by Reducing Endoplasmic Reticulum Stress and Oxidative Stress through AMPK Activation in Diabetic Obese Mice.

Portulaca oleracea L. (purslane) is a food and a traditional drug worldwide. It exhibits anti-inflammatory, anti-oxidative, anti-tumor, and anti-diabetic bioactivities; but its activity on diabetic-associated endothelial dysfunction is unknown. This study aimed to investigate the effect of purslane on endothelial function and the underlying mechanisms. Male C57BL/6 mice had 14-week ad libitum access to a high-fat rodent diet containing 60% kcal% fat to induce obesity and diabetes whereas purslane extract (200 mg/kg/day) was administered during the last 4 weeks via intragastric gavage. Primary rat aortic endothelial cells and isolated mouse aortas were cultured with a risk factor, high glucose or tunicamycin, together with purslane extract. By ESI-QTOF-MS/MS, flavonoids and their glycoside products were identified in the purslane extract. Exposure to high glucose or tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations in aortas and induced endoplasmic reticulum (ER) stress and oxidative stress with the downregulation of 5' AMP-activated protein kinase (AMPK)/ endothelial nitric oxide synthase (eNOS) signaling. Co-incubation with purslane significantly ameliorated these impairments. The effects of purslane were abolished by Compound C (AMPK inhibitor). Four-week purslane treatment ameliorated aortic relaxations, ER stress, and oxidative stress in diabetic obese mice. This study supported that purslane protected endothelial function, and inhibited ER stress and oxidative stress in vasculature through AMPK/eNOS activation, revealing its therapeutic potential against vascular complications in diabetes.

Characterisation of pomegranate juice polyphenols identifies oenothein B as an endothelium-dependent vasodilator and regulator of endothelial function that enhances actions of oligomeric procyanidins

Abstract Background Cardiovascular disease mortality is reduced in individuals with high daily consumption of dietary polyphenols. Optimal daily intakes, choice of polyphenol, and mechanism of action have yet to be fully defined. Pomegranate juice (PJ) reduces blood pressure and improves endothelial function. These effects are attributed to the high levels of polyphenols, primarily ellagitannins. Purpose The aims of these investigations were (1) to identify PJ polyphenol(s) that altered endothelial function and could potentially lower blood pressure, and (2) to compare these endothelial actions to oligomeric procyanidins (OPC) purified from apple polyphenol extracts. Methods PJ polyphenols were purified by Sephadex LH-20 chromatography and RP-HPLC, and identified by LC-MS/MS and NMR. Fractions were screened for biological activity using cultured bovine aortic endothelial cells by measuring the degree of inhibition of endothelin-1 synthesis. Endothelial actions were further assessed by measuring changes in intracellular Ca2+ using FLIPR Ca2+-6 assay, qRT-PCR (EDN1, eNOS and KLF2 mRNAs), and NO synthesis (nitrite). Results Six PJ polyphenols were isolated. These were identified as punicalin, four punicalagin isomers, and oenothein B (OTB, a cyclic dimeric ellagitannin; LC-MS [M-H]-= 1567.1). At concentrations ≤5 µg/ml only OTB inhibited ET-1 synthesis (IC50 1.5 µg/ml). The identity of OTB was confirmed by NMR, and by comparison with OTB purified from Epilobium species. Further characterisation of the actions of OTB on endothelial cells showed pre-incubation with 1.25, 2.5 and 5 µg/ml OTB increased binding of biotinylated-OPC by 1.7, 2.3 and 3.8 fold. Subsequent studies of the interactions between OTB and OPC showed synergistic effects for decreases in ET-1 synthesis, EDN1 mRNA, and increases in KLF2 mRNA, eNOS mRNA and NO release. The effect of combining OTB (1 µg/ml) and OPC (1 µg/ml) lasted for over 24 h (relative to control: ET-1 -57%, EDN1 mRNA -63%, eNOS mRNA +69%, KLF2 mRNA +356%), when individually these polyphenols no longer had any significant effect. Intracellular Ca2+ responses also showed synergistic interactions, however the profile of [Ca2+] changes for OTB and OPC alone differed, suggesting separate signalling pathways initiating these endothelial responses. Endothelium-dependent vasodilator effects of OTB were confirmed on isolated mouse aorta (EC50 0.27 µg/ml). Conclusions These effects of OTB may underlie previously reported decreases in blood pressure and improvements in endothelial function after pomegranate juice consumption. Detailed understanding of the mechanism of action of OTB, and its interactions with OPC could lead to new therapeutic modalities for treating endothelial dysfunction in a broad spectrum of people at risk of cardiovascular disease.

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Antioxidant and Vasorelaxant Properties of Phaseolus vulgaris Linn (Fabaceae) Immature Pods Extract on the Thoracic Aorta of NMRI Mice

Introduction: Oxidative stress, through the increased bioavailability of reactive oxygen species (ROS), is a major cause of hypertension. The resulting endothelial remodeling promotes the production of vasoconstrictor substances leading to an increase in blood pressure. This study aimed to evaluate the antioxidant and vasorelaxant properties of the decoction (PAD), ethyl acetate (EAP), and residual aqueous (ARP) fractions of immature Phaseolus vulgaris pods on NMRI mice thoracic aorta rings. Methods: Phytochemical screening was performed by high-performance thin-layer chromatography. Folin-Ciocalteu and aluminum trichloride colorimetric methods were used to quantify total polyphenol compounds (TPC) and total flavonoids (TFC), respectively. Antioxidant activities of the extracts were determined by 2,29-azinobis-3-ethylbenzothiazoline-6-sulfonic (ABTS●), 2,2-Diphenyl-1-picrylhydrazyl (DPPH●), and ferric ion (FRAP) radical reduction methods. The DMT 620M ADInstruments myograph technique was used to evaluate the ex-vivo vasodilatory effects of Phaseolus vulgaris extracts on aortic rings. Results: Chromatographic fingerprints showed the presence of flavonoids, coumarins, tannins, steroids, triterpenes, and saponins in the extracts studied. The TPC (61.07±0.04 mgGAE/g) and TFC (6.16±0.03 mgQE/g) of EAP were statistically significant compared to that of PAD and ARP (p<0.001). The antioxidant power of Trolox was statistically significant compared to all studied extracts (p<0.05). It should be noted that among these extracts, EAP showed the better antiradical capacities for ABTS (IC50=71.87±0.30 µg/mL) and DPPH (IC50=9.93±0.00 µg/mL). However, for FRAP activity, it was the PAD extract (T=170.68±0.11 µgAAE/g) that obtained the best score. In terms of vasorelaxant activity, all extracts induced concentration-dependent relaxation of aortic rings precontracted with U46619. Pharmacodynamic parameters were significantly in favor of EAP [EAP(E+)=(Emax=100.06±0.00%; pD2=1.24±0.01) and EAP(E-)=(Emax=101.01±0.00%; pD2=0.84±0.02)] followed by PAD and then ARP. Conclusion: Phaseolus vulgaris immature pod extracts possess concentration-dependent vasorelaxant effects on isolated mouse aorta. These preliminaries results were scientific evidence to support the use of this plant in traditional and complementary medicine for the treatment of hypertension.

Abstract 15395: Caveolin-1 is a Primary Determinant of Endothelial Stiffening Associated With Dyslipidemia, Disturbed Flow and Ageing

Objective: Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Approach and Results: Elastic moduli of cultured endothelial cells and of endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy. We found that a loss of Cav-1 abrogates the uptake of oxLDL (n=5-8) and oxLDL-induced endothelial stiffening (n=4-5) but has no effect on endothelial stiffness in the absence of oxLDL (n=4). A loss of Cav-1 also abrogated endothelial stiffening induced by disturbed flow, which in turn is oxLDL dependent (n=6). Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor (n=4) but not for endothelial stiffening induced by a direct application of 7ketocholesterol (n=8). Genetic deletion of Cav-1 also led to profound changes in endothelial stiffness in vivo : it abrogated endothelial stiffening observed in the disturbed flow region of the aortic arch (n=5-6), abrogated endothelial stiffening induced by feeding the mice high fat diet (4-6 weeks, n=5) and significantly blunted endothelial stiffening that develops with advanced age (n=5-6). This effect was independent of stiffening of the sub-endothelium layer (n=5-8). Conclusions: Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo . Mechanistically, Cav-1 regulates endothelial stiffening via CD36-mediated uptake of oxLDL, whereas exposure to 7ketocholesterol circumvents this step.

Caveolin-1 is a primary determinant of endothelial stiffening associated with dyslipidemia, disturbed flow, and ageing

Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells (EC) exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Elastic moduli of cultured ECs and of the endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy (AFM). We found that a loss of Cav-1 abrogates the uptake of oxLDL and oxLDL-induced endothelial stiffening, as well as endothelial stiffening induced by disturbed flow (DF), which was also oxLDL dependent. Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor. Genetic deletion of Cav-1 abrogated endothelial stiffening observed in the DF region of the aortic arch, and induced by a high fat diet (4–6 weeks) and significantly blunted endothelial stiffening that develops with advanced age. This effect was independent of stiffening of the sub-endothelium layer. Additionally, Cav-1 expression significantly increased with age. No differences in elastic modulus were observed between the sexes in advanced aged wild type and Cav-1 knockout mice. Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo.

Measurement of Endothelium-dependent Vasorelaxation in the Mouse Thoracic Aorta using Tensometric Small Volume Chamber Myography.

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

Identification of Piezo1 channels in perivascular adipose tissue (PVAT) and their potential role in vascular function

The vasculature constantly experiences distension/pressure exerted by blood flow and responds to maintain homeostasis. We hypothesized that activation of the stretch sensitive, non-selective cation channel Piezo1 would directly increase vascular contraction in a way that might be modified by perivascular adipose tissue (PVAT). The presence and function of Piezo1 was investigated by RT-PCR, immunohistochemistry, and isolated tissue bath contractility. Superior and mesenteric resistance arteries, aortae, and their PVATs from male Sprague Dawley rats were used. Piezo1 mRNA was detected in aortic vessels, aortic PVAT, mesenteric vessels, and mesenteric PVAT. Both adipocytes and stromal vascular fraction of mesenteric PVAT expressed Piezo1 mRNA. In PVAT, expression of Piezo1 mRNA was greater in magnitude than that of Piezo2, transient receptor potential cation channel, subfamily V, member 4 (TRPV4), anoctamin 1, calcium activated chloride channel (TMEM16), and Pannexin1 (Panx1). Piezo1 protein was present in endothelium and PVAT of rat aortic and in PVAT of mesenteric artery. The Piezo1 agonists Yoda1 and Jedi2 (1 nM - 10 µM) did not stimulate aortic contraction [max < 10% phenylephrine (PE) 10 µM contraction] or relaxation in tissues + or -PVAT. Depolarizing the aorta by modestly elevated extracellular K+ did not unmask aortic contraction to Yoda1 (max <10% PE 10 µM contraction). Finally, the Piezo1 antagonist Dooku1 did not modify PE-induced aorta contraction + or -PVAT. Surprisingly, Dooku1 directly caused aortic contraction in the absence (Dooku1 =26 ± 11; Vehicle = 11 ± 11%PE contraction) but not in the presence of PVAT (Dooku1 = 2 ± 1; Vehicle = 8 ± 5% PE contraction). Thus, Piezo1 is present and functional in the isolated rat aorta but does not serve direct vascular contraction with or without PVAT. We reaffirmed the isolated mouse aorta relaxation to Yoda1, indicating a species difference in Piezo1 activity between mouse and rat.

Alpha-mangostin improves endothelial dysfunction in db/db mice through inhibition of aSMase/ceramide pathway.

Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha‐mangostin has been reported to have anti‐diabetic capacity in recent years. Here, we investigated the protective function of alpha‐mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha‐mangostin improved impaired endothelium‐dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up‐regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha‐mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha‐mangostin increased phosphorylation of eNOS and NO production in high glucose‐treated aortas. Alpha‐mangostin normalized high glucose‐induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha‐mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation.

Pharmacological and Genetic Evidence of Dopamine Receptor 3-Mediated Vasoconstriction in Isolated Mouse Aorta.

Dopamine receptors (DRs) are generally considered as mediators of vasomotor functions. However, when used in pharmacological studies, dopamine and/or DR agonists may not discriminate among different DR subtypes and may even stimulate alpha1 and beta-adrenoceptors. Here, we tested the hypothesis that D2R and/or D3R may specifically induce vasoconstriction in isolated mouse aorta. Aorta, isolated from wild-type (WT) and D3R−/− mice, was mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE), acetylcholine (ACh), and the D3R agonist 7-hydrxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), with or without the D2R antagonist L741,626 and the D3R antagonist SB-277011-A. The vasoconstriction to PE and the vasodilatation to ACh were not different in WT and D3R−/−; in contrast, the contractile responses to 7-OH-DPAT were significantly weaker in D3R−/−, though not abolished. L741,626 did not change the contractile response induced by 7-OH-DPAT in WT or in D3R−/−, whereas SB-277011-A significantly reduced it in WT but did not in D3R−/−. D3R mRNA (assessed by qPCR) was about 5-fold more abundant than D2R mRNA in aorta from WT and undetectable in aorta from D3R−/−. Following transduction with lentivirus (72-h incubation) delivering synthetic microRNAs to specifically inactivate D2R (LV-miR-D2) or D3R (LV-miR-D3), the contractile response to 7-OH-DPAT was unaffected by LV-miR-D2, while it was significantly reduced by LV-miR-D3. These data indicate that, at least in mouse aorta, D3R stimulation induces vasoconstriction, while D2R stimulation does not. This is consistent with the higher expression level of D3R. The residual vasoconstriction elicited by high concentration D3R agonist in D3R−/− and/or in the presence of D3R antagonist is likely to be unrelated to DRs.

Edible Bird's Nest Protects Against Hyperglycemia-Induced Oxidative Stress and Endothelial Dysfunction.

Increased oxidative stress by hyperglycemia is a major cause of vascular complications in diabetes. Bird’s nest, which is made from the saliva of swiftlets has both medicinal and nutritional values dated back to ancient China. However, its role in improving endothelial dysfunction due to diabetes is yet to be elucidated. The present study examined the protective effect and mechanism of action of the aqueous extract of hydrolyzed edible bird nest (HBN) on endothelium in models of diabetes, in vitro and in vivo. Male db/m+ and db/db mice were orally administered with or without HBN and glibenclamide for 28 days, followed by vascular reactivity studies in mouse aortas. Human umbilical vein endothelial cells (HUVECs) and isolated mouse aorta from C57BL/6J were treated with high glucose (HG), HBN, sialic acid (SA), glibenclamide, and apocynin, respectively. The effects of HBN on reactive oxygen species (ROS) production and nitric oxide (NO) bioavailability were assessed by Western blot, 2′,7′-dichlorofluorescin-diacetate (DCF-DA), and 4-amino-5-methylamino-2′,7′ difluorofluorescein (DAF-FM DA) in HUVECs, isolated mouse aorta, and db/db diabetic mice. HBN significantly reversed the endothelial dysfunction in diabetic mice and isolated mouse aorta. HBN normalized ROS over-production of NOX2 and nitrotyrosine, reversed the reduction of anti-oxidant marker, SOD-1 as well as restored NO bioavailability in both HUVECs challenged with HG and in db/db diabetic mice. Similarly, HG-induced elevation of oxidative stress in HUVECs were reversed by SA, glibenclamide, and apocynin. This attests that HBN restores endothelial function and protects endothelial cells against oxidative damage induced by HG in HUVECs, isolated mouse aorta, and db/db diabetic mice via modulating ROS mechanism, which subsequently increases NO bioavailability. This result demonstrates the potential role of HBN in preserving endothelial function and management of micro- or macrovascular complications in diabetes.

Omentin-1 protects against high glucose-induced endothelial dysfunction via the AMPK/PPARδ signaling pathway

High glucose-induced endothelial dysfunction is a critical initiating factor in the development of diabetic vascular complications. Omentin-1 has been regarded as a novel biomarker of endothelial function in subjects with type-2 diabetes (T2D); however, it is unclear whether omentin-1 has any direct effect in ameliorating high glucose-induced endothelial dysfunction. In the present study, we analyzed the effect of omentin-1 on high glucose-induced endothelial dysfunction in isolated mouse aortas and mouse aortic endothelial cells (MAECs). Vascular reactivity in aortas was measured using wire myography. The expression levels of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor δ (PPARδ), Akt, endothelial nitric-oxide synthase (eNOS), and endoplasmic reticulum (ER)-stress markers in MAECs were determined by Western blotting. The production of reactive oxygen species (ROS) and nitric oxide (NO) was assessed by diluted fluoroprobe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM DA), respectively. We found that ex vivo treatment with omentin-1 reversed impaired endothelial-dependent relaxations (EDR) in mouse aortas after high-glucose insult. Elevated ER-stress markers, oxidative stress, and reduction of NO production induced by high glucose in MAECs were reversed by omentin-1 treatment. Omentin-1 also effectively reversed tunicamycin-induced ER stress responses in MAECs, as well as ameliorated impairment of endothelial-dependent relaxation in mouse aortas. Moreover, omentin-1 increased AMPK phosphorylation with a subsequent increase in PPARδ expression, while also restoring the decreased phosphorylation of Akt and eNOS. The effects of omentin-1 were abolished by cotreatment of compound C (AMPK inhibitor) and GSK0660 (PPARδ antagonist). These data indicate that omentin-1 protects against high glucose-induced vascular-endothelial dysfunction through inhibiting ER stress and oxidative stress and increasing NO production via activation of AMPK/PPARδ pathway.

Phosphorylation and activation of endothelial nitric oxide synthase by red fruit (Pandanus conoideus Lam) oil and its fractions.

Red fruit (Pandanus conoideus Lam) oil (RFO) is utilized by inhabitants of the Papua Island to treat diseases such as infections, cancer, and cardiovascular disease, but the mechanism of action is unknown. We have recently shown that RFO stimulates nitric oxide (NO) production in endothelial cells. The present study was conducted to investigate the molecular mechanism of endothelial NO synthase (eNOS) activation by RFO. NO production by endothelial cells was determined with electron paramagnetic resonance. The vascular function of isolated mouse aorta was examined using a wire myograph system. Phosphorylation of eNOS was studied with Western blot analyses. RFO induced concentration-dependent vasodilation in isolated mouse aorta. The vasodilator effect of RFO was lost in endothelium-denuded aorta and in aorta from mice deficient in eNOS. Treatment of human EA.hy 926 endothelial cells with RFO led to an enhancement of eNOS phosphorylation at serine 1177 and NO production. The RFO-induced eNOS phosphorylation and NO production were reduced by inhibitors of Akt or AMPK, but not by an inhibitor of CaMKII. The effects of RFO were decreased by pharmacological inhibition of PI3K, indicating an involvement of the PI3K-Akt pathway. Moreover, acetone-soluble fractions and oily fractions of RFO showed higher efficacies than the RFO polar fraction in activating eNOS. RFO contains highly active compounds that enhance NO production through Akt- or AMPK-mediated eNOS phosphorylation. The increase in endothelial NO production is likely to represent one of the molecular mechanisms responsible for the therapeutic effects of RFO.

SILDENAFIL-MIL-89 FORMULATION AS POTENTIAL DRUG DELIVERY SYSTEM IN PULMONARY ARTERIAL HYPERTENSION

Pulmonary Arterial Hypertension (PAH) is an aggressive disease with poor prognosis, no available cure, and low survival rates. Currently, there are several classes of vasodilator drugs that are commonly used as treatment strategies for PAH including sildenafil, a phosphodiesterase type 5-inhibitor. Despite their clinical benefits, these therapies are hindered by their systemic side effects. This limitation could be overcome using controlled drug release with the potential for targeted drug delivery using a nanomedicine approach. In the current study, we have evaluated the use of a highly porous nano-sized preparation of iron-based metal-organic framework (MOF) commonly referred to as MIL-89 for loading with sildenafil. We have previously shown that MIL-89 is relatively non-toxic in a range of human cell types and well tolerated in vivo. Here, we report the use of a nano-formulation of MIL-89 (nanoMIL-89) as drug delivery for the phosphodiesterase type 5-inhibitor sildenafil. nanoMIL-89 was first prepared and then successfully loaded with sildenafil ([email protected]). [email protected] was shown to release sildenafil in a biphasic manner with an initial rapid release over 6 hours followed by a more sustained release over 72 hours. Finally, the vasodilator effect of [email protected] was measured over 8 hours using isolated mouse aorta. Consistent with drug release kinetics, Sil-nanoMIL-89 induced vasodilation after a lag phase of 3 hours. [email protected] is a promising nano-formulation prototype of sildenafil displaying delayed drug release and vasodilator activity. Further pharmacological assessment of [email protected] in animal models for PAH is now required and constitutes the subject of an ongoing investigation.

Pharmacological inhibition of the F1 -ATPase/P2Y1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation.

The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

Red fruit (Pandanus conoideus Lam) oil stimulates nitric oxide production and reduces oxidative stress in endothelial cells

Red fruit oil (RFO) is used in traditional medicine for the treatment of a number of diseases. However, evidence for the biological effects and action mechanisms is still lacking. In the present study, we show for the first time that RFO stimulated the phosphorylation of the endothelial nitric oxide synthase (eNOS) and enhanced the NO production in human endothelial cells. In isolated mouse aorta, RFO induced a vasodilation, with a significant effect evident at a concentration as low as 1:100,000 dilution. The RFO-induced vasodilation could be completely prevented by eNOS inhibition, indicating that RFO contains highly potent substances stimulating eNOS activity. In addition, RFO reduced oxidative stress in endothelial cells, as demonstrated with three different methods. In the comet assay, RFO significantly decreased oxidative DNA damage induced by genotoxicants. These results suggest that enhanced endothelial NO production and reduced oxidative stress may represent part of the molecular mechanisms responsible for the therapeutic effects of RFO.

5,6-DiHETE attenuates vascular hyperpermeability by inhibiting Ca2+ elevation in endothelial cells

Although more than 100 lipid metabolites have been identified, their bioactivities remain unknown. In a previous study, we discovered that the production of several lipid metabolites in the intestines dramatically changed in colitis. Of these metabolites, 5,6-dihydroxyeicosatetraenoic acid (DiHETE) possesses novel anti-inflammatory activity in the vasculature. In this study, we used mouse and human umbilical vein endothelial cell (HUVEC) models to elucidate the mechanisms underlying the vascular activity of lipid metabolites, particularly those related to the release of histamine, a major proinflammatory mediator that stimulates endothelial cells to produce NO, a mediator of vascular relaxation and hyperpermeability, by activating intracellular Ca2+ concentration-dependent signaling. In a mouse ear, the administration of 5,6-DiHETE did not induce inflammatory reactions, and pretreatment with 5,6-DiHETE inhibited histamine-induced inflammation, specifically vascular dilation and hyperpermeability. In an isolated mouse aorta, 5,6-DiHETE treatment did not influence vascular contraction but attenuated acetylcholine-induced vascular relaxation. In HUVECs, treatment with 5,6-DiHETE inhibited histamine-induced endothelial barrier disruption and inhibited the production of NO. Most notably, 5,6-DiHETE inhibited histamine-induced increases in intracellular Ca2+ concentrations in HUVECs. Our findings suggest that 5,6-DiHETE attenuates vascular hyperpermeability during inflammation by inhibiting endothelial Ca2+ elevation, which might lead to a novel pharmacological strategy against inflammatory diseases.

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

Comparative analysis of endothelial cell and sub-endothelial cell elastic moduli in young and aged mice: Role of CD36

ObjectiveTo perform comparative analysis of the role of scavenger receptor CD36 on endothelial vs. sub-endothelial elastic modulus (stiffness) in the aortas of young and aged mice. Approaches and ResultsElastic moduli of endothelial and sub-endothelial layers of freshly isolated mouse aortas were quantified using atomic force microscopy. In young mice (4–6 months old), we found that while endothelial stiffness is markedly reduced in aortas of CD36−/−mice, as compared to WT controls, no difference between CD36−/− and WT aortas is observed in the stiffness of the sub-endothelial layer in denuded arteries. Additionally, inhibition of myosin phosphorylation also decreases the elastic modulus in the EC, but not the sub-EC layer in WT mice. Moreover, inhibiting CD36 mediated uptake of oxLDL in intact WT aortas abrogated oxLDL-induced endothelial stiffening. Further analysis of aged mice (22–25 months) revealed that aging resulted not only in significant stiffening of the denuded arteries, as was previously known, but also a comparable increase in the elastic modulus of the endothelial layer. Most significantly, this stiffening in the EC layer is dependent on CD36, whereas the denuded layer is not affected. ConclusionsOur results show that the role CD36 in stiffening of cellular components of intact aortas is endothelial-specific and that genetic deficiency of CD36 protects against endothelial stiffening in aged mice. Moreover, these data suggest that endothelial stiffness in intact mouse aortas depends more on the expression of CD36 than on the stiffness of the sub-endothelial layer.

Paeonol Attenuates LPS-Induced Endothelial Dysfunction and Apoptosis by Inhibiting BMP4 and TLR4 Signaling Simultaneously but Independently.

Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.