Mesenteric Bed Research Articles

Potent Vasodilator and Cellular Antioxidant Activity of Endemic Patagonian Calafate Berries (Berberis microphylla) with Nutraceutical Potential.

Hydroalcoholic extracts of Patagonian Calafate berry (Berberis microphylla) contain mono or disaccharide conjugated anthocyanins and flavonols. The Liquid Chromatography-Mass Spectrometry (LC-MS) chemical extract profile identified glycosylated anthocyanidins such as delphinidin-, petunidin- and malvidin-3-glucoside as the major constituents. The predominant flavonols were 3-O substituents quercetin-rutinoside or -rhamnoside. Anthocyanins doubled flavonols in mass (13.1 vs. 6 mg/g extract). Polyphenols vascular actions were examined in the rat arterial mesenteric bed bioassay; extract perfusion elicited concentration-dependent vasodilatation mimicked by conjugated anthocyanins standards. Vascular responses of main glycosylated anthocyanins were endothelium-dependent (p < 0.001) and mediated by NO production (p < 0.05). The anthocyanins antioxidant activity determined in isolated endothelial cells (CAA) showed a reduced redox potential as compared to the extract or quercetin. While in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, the anthocyanins showed an equivalent quercetin potency, the extract was 15-fold less active, proposing that the anthocyanin-induced vasodilation is not due to an antioxidant mechanism. The extract shows promising commercial nutraceutical potential.

"See fine world" -copy experience and thinking of membrane anatomy in laparoscopic radical gastrectomy (D2+CME)

Primary lesion removal and lymph node dissection are the main constituents of radical gastrectomy. However, the high recurrence rate after D2 radical gastrectomy for advanced gastric cancer has not improved. Recently, studies have found that discrete tumor deposits in the mesogastrium may be an important factor affecting the prognosis of gastric cancer after surgery. With the development of laparoscopic equipment, the ever-expanding "submicroscopic vision" makes it possible to completely remove the mesogastrium. Professor Gong Jianping advocated "membrane anatomy" to optimize the concept of radical gastrectomy: D2- based complete mesenteric resection (CME), namely D2+CME procedure. To prevent the leakage of tumor cells into the surgical field, as histological barrier, the intact mesogastrium should be located. The essential difference between D2+CME and previous D2/D2+systematic mesogastrium excision (SME), en-bloc mesogastric excision (EME) is as follow: double-factor guiding (lymph nodes and discrete tumor deposits) vs. single factor guiding (lymph nodes only). After practicing dozens of radical gastrectomy (D2+CME) authors believe that its conceptual connotation (double factor guiding) and operational extension (above mesentery bed) cover D2. In D2+CME surgery, depending on the anatomical identification under the magnified field of view, the conformal space between gastric mesentery and mesenteric beds is unique operational plane with repeatability. These findings and considerations address one problem: where is the precise boundary of en bloc principle in radical gastrectomy? In author's opinion, with laparoscopy and "sub-microsurgery" progression and detection of discrete tumor deposit metastasis, survival benefit from definition of en bloc boundary in radical gastrectomy will be widely recognized. Meanwhile, D2+CME procedure is an appropriate way for study. Although the development of the "membrane anatomy" concept for gastric cancer still requires many further clinical and basic researches, it is reasonable to foresee that D2+CME surgery will guide a concept-optimized era for gastric cancer surgery.

Apocynin Alters Redox Signaling in Blood Vessels of Spontaneously Hypertensive Rats

Apocynin reduces blood pressure and prevents development of endothelial dysfunction in SHR. Mechanisms underlying the effects of apocynin in SHR remain unclear. Angiotensin (Ang) II regulates NOX, the major source of ROS in cardiovascular system. We hypothesized that apocynin, acting as NOX inhibitor, may alter pressor and vasoconstrictor effects of Ang II in SHR. We analyzed the effect of Ang II on blood pressure, reactivity on aorta and mesenteric resistance arteries, plasmatic antioxidant capacity, NOX and NOS expression and activity, AT1 and AT2 receptors expression in blood vessels of SHR treated with apocynin SHR treated with apocynin (30 mg/Kg/day, p.o.). In SHR, apocynin reduced pressor effect of Ang II, increased plasmatic antioxidant capacity, ablated aortic and mesenteric NOX‐dependent ROS production, NOX2 and p47phox overexpression demonstrating that apocynin acts as a NOX inhibitor in blood vessels of SHR. Moreover, apocynin increased aortic and plasmatic nitrate/nitrite levels, but did not alter mesenteric NOS activity or eNOS expression, diminished AT1 expression in aortas and mesenteric bed, and increased AT2 expression in aortas from SHR. Apocynin increased endothelial modulation on Ang II vasoconstriction in resistance arteries from SHR. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. These integrated data could help us to understand the promising effect of apocynin as an antihypertensive drug different from those currently used clinically.Support or Funding InformationApproved by local ethics committee CEUA FOA 450/2015.Financial support: CNPq, CAPES and FAPESP (2016/22180‐9).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beneficial Changes Promoted by L‐arginine and Aliskiren in Resistance Vessels in 2K1C hypertensive rats

Renovascular hypertension is characterized by increased angiotensin II and oxidative stress and by endothelial dysfunction. By acting on multiple targets and promoting diverse actions, angiotensin II (Ang II) plays a pivotal role in vascular dysfunction. The purpose of this study was to test whether the administration of aliskiren (ALSK) and L‐arginine (L‐arg) would restore endothelial dysfunction and reduce inflammation and oxidative stress in a rat renovascular hypertension model. Hypertension was induced by clipping the right renal artery, and the following five groups were divided: SHAM operated; 2‐kidney, 1‐clip (2K1C); 2K1C plus ALSK; 2K1C plus L‐ARG; and 2K1C plus ALSK+ L‐ARG. The vascular reactivity to norepinephrine (NE; 10(−9) to 2 × 10(−3) mol/L) and acetylcholine (ACh; 10(−10) to 10(−3) mol/L) was evaluated in the mesenteric arteriolar bed through concentration‐effect curves. The structure was assessed by histology and biomolecular analyzes were done via western blot, lipid peroxidation, DHE and DAF. For 4 weeks, blood pressure (BP) was monitored and endothelium‐dependent vasoconstriction and relaxation were assessed in mesenteric arteriolar. ALSK+L‐arg reduced BP and the contractile response to norepinephrine and improved acetylcholine relaxation. Incubation with N‐nitro‐L‐arginine methyl ester (L‐NAME) and L‐NAME+ Indometacin increased the response to norepinephrine in the 2K1C group and the effect was greater and partially improved, respectively, with the treatments. However, It was improved acetylcholine relaxation with the both of the treatments. The bioavailability of nitric oxide increased with the treatments, and the inflammation and oxidative stress was reduced with the treatments. L‐arginine and aliskiren attenuates the morphofunctional deleterious effects of Ang II on resistance vessels. The benefits of L‐arginine and aliskiren seem to occur through restoring the balance of ROS/NO. Therefore, this study opened new avenues for further clinical targeting of the treatment of cardiovascular diseases related to activation of the renin‐angiotensin system. In conclusion, combined ALSK+L‐arg treatment normalizes BP and prevents endothelial dysfunction in 2K1C hypertensive rats.Support or Funding InformationThis research was supported by a CNPq research grants. This study was funded by Fundação de Amparo a Pesquisa do Espirito Santo (FAPES‐Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq‐Brazil).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitric Oxide‐Mediated Nanomolar Vasodilator Potencies of Glycosylated Anthocyanins do not Correlate with Redox Potential

Calafate is an endemic Patagonian shrub (Berberis Microphylia); its berries are rich in polyphenols, including 3‐glycosylated anthocyanins (GA, delphinidin, petunidin or malvidin) or mono or disaccharide conjugated quercetin (Q). The chemical profile of Calafate berries is well characterized and indicates its high redox potential; pharmacological properties are virtually unknown. From ethno‐pharmaceutical reports we hypothesized that Calafate polyphenols are vasodilators and ascertained whether this effect is related to their cell antioxidant activity (CAA).Vascular responses of GA and Q were assessed perfusing the intact rat arterial mesenteric bed or following endothelium denudation. The role of NO was assessed in mesenteries perfused with an eNOS inhibitor (L‐NNA). Luminally accessible NO was determined by chemiluminescence; intracellular NO was quantified by DAF fluorescence in isolated mesentery endothelial cells. Antioxidant capacity was quantified either by 1,1‐Diphenyl‐2‐picryl‐hydrazyl antioxidant assay (DPPH) or by CAA assessment. Extracellular ATP was assessed by chloroacetaldehyde derivatization to yield fluorescent purines; the nucleotide is related to NO production in endothelial cells.GA proved potent vasodilators with EC50s of 84.1 ± 0.3, 65.8 ± 0.4 and 17.7 ± 0.1 nM, respectively; aglycones exhibited similar EC50s. The maximal effect of GA did not exceed 56 ± 3.7% efficacy compared to acetylcholine (EC50 was 43.4 ± 0.1 nM with 100% efficacy) while the Q EC50 was 12.8 ± 0.1 μM, with 99 ± 4.1%. The GA‐induced vasodilatation was totally endothelium‐dependent; while the Q response showed partial endothelium‐dependence. The degree of GA vasodilatation correlated with luminal NO (r2 = 0.83), confirming NO as the final intracellular signal. 30 μM Q increased both extracellular ATP secretion from endothelial cells plus intracellular DAF‐fluorescence (P&lt;0.05), an indication that part of the Q response involves NO as the final effector. While Q exhibited the highest antioxidant effect in both the DPPH or CAA (p&lt;0.01), the GA showed a 60‐fold lower potential than Q in the CAA test, indicating that the CAA is not related to its vasodilator profile, which apparently relies on eNOS allosteric modulation.In sum, GA are novel and potent endothelium‐dependent vasodilators, linked to modulation of NO production.Support or Funding InformationFunded by FONDECYT 117‐0842 and 21141226 to CC and CEDENNA FB0807This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Superimposing a high-fat diet on Schistosoma mansoni infection affects renin-angiotensin system components in the mouse kidney.

The levels of the full-length form of the (pro)renin receptor (PRR), a component of the renin-angiotensin system (RAS), may be reduced in the membranes of kidneys in renal diseases. This study aimed to investigate the RAS components in the kidneys of mice submitted to a combination of a high-fat diet and Schistosoma mansoni infection. Female BALB/c mice were maintained on a control or high-fat diet from 3 weeks of age. After 10 weeks on the designated diets, half the mice in each group were infected with S. mansoni cercariae. The blood and kidneys were harvested 8 weeks after infection. The high-fat diet increased the number of eggs in the feces and the number of adult worms in the mesenteric bed. Schistosoma mansoni infection reduced the plasma levels of glucose, triglycerides, and HDL cholesterol in the control and high-fat diet groups. In mice on the control diet, S. mansoni infection resulted in increased expression of IL-6 in the kidneys; however, in mice on the high-fat diet, the levels of IL-6 were reduced and those of superoxide anions were increased. The RAS components evaluated were ACE2, renin, PRR, AT1R, and AT2R, and the levels of PRR were found to be reduced in the kidneys of infected mice on the high-fat diet. The finding regarding PRR is not yet clear. However, combining a high-fat diet and S. mansoni infection resulted in increased oxidative stress in the kidney that can aggravate hypertension as well as its associated complications.

AdipoRon, adiponectin receptor agonist improves vascular function in the mesenteric arteries of type 2 diabetic mice

AdipoRon, adiponectin receptor agonist improves vascular function in the mesenteric arteries of type 2 diabetic mice

Apocynin alters redox signaling in conductance and resistance vessels of spontaneously hypertensive rats

Chronic treatment with apocynin reduces blood pressure and prevents endothelial dysfunction development in spontaneously hypertensive rats (SHR). Mechanisms underlying apocynin effects on SHR remain unclear. Compared to diapocynin and other drugs, apocynin is a weak antioxidant, which suggests that its effects on SHR are associated with other mechanisms besides its antioxidant capacity. Angiotensin (Ang) II regulates NOX, the major reactive oxygen species (ROS) source in the cardiovascular system. We hypothesized that, by inhibiting NOX, apocynin could alter Ang II pressor and vasoconstrictor effects on SHR. We analyzed how Ang II affects blood pressure and vascular reactivity in aorta and mesenteric resistance arteries and evaluated plasma antioxidant capacity, NOX isoforms and subunits, NOS isoforms, AT1 and AT2 receptors expression, ROS production, and NOS activity in apocynin-treated SHR blood vessels (30 mg/Kg/day, p.o.). In SHR, apocynin reduced Ang II pressor effects, increased plasmatic antioxidant capacity, and blunted aortic and mesenteric NOX-dependent oxidants production and NOX2 and p47phox overexpression, which demonstrated that apocynin inhibits NOX in SHR blood vessels. Moreover, apocynin raised plasmatic and aortic nitrate/nitrite levels, maintained NOS activity and eNOS, p-eNOS, nNOS, iNOS, sGC-α, and sGC-β expression in mesenteric bed, diminished AT1 expression in aorta and mesenteric bed, and elevated AT2 expression in SHR aorta. Apocynin increased Ang II vasoconstriction endothelial modulation in SHR resistance arteries. All these results showed that in vivo treatment with apocynin alters several mechanisms that reduce Ang II pressor and vasoconstrictor effects on SHR. Such apocynin effects involve other mechanisms besides vascular ROS modulation, which improves NO availability in SHR vascular cells. These integrated data could help us to understand the promising apocynin activity as an antihypertensive drug that acts differently from the drugs that are currently being used in the clinical setting.

Effect of oral magnesium sulfate administration on lectin-like oxidized low-density lipoprotein receptor-1 gene expression to prevent atherosclerosis in diabetic rat vessels.

Aims/IntroductionThe purpose of the present study was to investigate the possible effect of oral magnesium sulfate (MgSO 4) in the reduction of atherosclerosis plaques through inhibition of lectin‐like low‐density lipoprotein receptor‐1 (LOX‐1) gene expression in diabetic vessels.Materials and methodsA total of 50 rats were divided into five groups, including non‐diabetic control, Mg‐treated non‐diabetic control, chronic diabetic, Mg‐treated chronic diabetic and insulin‐treated chronic diabetic. The induction of diabetes was carried out by streptozotocin. The Mg‐treated chronic diabetic and Mg‐treated non‐diabetic control groups were treated with 10 g/L of MgSO 4 added to their drinking water. The insulin‐treated chronic diabetic group received 2.5 U/kg of insulin twice per day. The fasting blood glucose level and bodyweight were determined weekly. Blood pressure measurement and the intraperitoneal glucose tolerance test were carried out after 16 weeks, and the plasma levels of Mg, lipid profile and oxidized low‐density lipoprotein cholesterol (oxLDL) were determined. The mesenteric bed was isolated and perfused according to the McGregor method. The aorta was isolated for LOX‐1 genes and proteins expression, and pathological investigation.ResultsMgSO 4 administration improved blood pressure, sensitivity to phenylephrine, intraperitoneal glucose tolerance test, lipid profile and plasma ox‐LDL level, and also lowered the blood glucose level to the normal range, and decreased LOX‐1 gene and protein expressions. Insulin decreased blood pressure, sensitivity to phenylephrine, blood glucose, lipid profiles and plasma oxLDL level, but it did not decrease LOX‐1 gene and protein expressions.ConclusionsThe present findings suggested that MgSO 4 improves blood pressure and vessel structure through decreasing oxLDL, and LOX‐1 gene and protein expressions; however, insulin did not repair vessel structure, and LOX‐1 gene and protein expressions.

Abstract 17311: Gut Microbiota Derived Metabolites Impact Lymphatic Inflammatory Mechanisms

Diet induced changes in gut microbial composition and dysregulated immune responses are key players in the progression of metabolic syndrome (MetSyn) and obesity.The lymphatics that are directly exposed to the gut microbiota in the mesenteric bed are central players in inflammatory and immune responses and are impaired during MetSyn. However, the mechanisms remain unknown.Endogenous tryptophan derived gut microbiota metabolites such as indole, significantly limits inflammation and immune response. We hypothesize that microbiota influence the lymphatic mesenteric microenvironment via such discrete metabolites to impact metabolic disease. We evaluated the effects of indole, tryptamine and indole-3-acetate (I-3A) on inflammatory mechanisms in vitro and in vivo using cultured models of lymphatic endothelium (HDLECs) and rat models of high fructose diet induced MetSyn and LPS induced acute inflammation. HDLECs were treated with varying doses of indole, tryptamine and I-3A (100uM-500uM) in presence or absence of LPS (20ng/ml) and its effects on inflammatory cytokines were evaluated. Rats were injected with LPS (10mg/kg, 24hrs) with or without indole (20mg/kg) to assess for inflammation. Further, rats were also subjected to a 7-week high fructose (60%) diet regimen and gavaged with indole for 4 weeks prior to completion of the diet regimen and metabolic parameters were measured. Our results show that indole decreases levels of inflammatory cytokines as cfos, cjun, IL1β, MCP1, JAK1, SOCS3 and SOCS1 induced by LPS in the HDLECs and in the mesenteric arcades and lymph nodes. Similar effects are also observed with tryptamine and I-3A. Flow cytometric analysis of tissues show that indole significantly suppresses the population of inflammatory MHCII + /cd45 + cd11b + cells (p&lt;0.02) and augments Foxp3+/cd4+ cells (p&lt;0.003) in the mesenteric arcades, lymph nodes and spleen that are induced in LPS treated animals. In MetSyn animals, indole reduces levels of glucose, triglyceride and epididymal fat content that is also associated with concomitant decrease in expression of inflammatory cytokines. Taken together, we show that indole and other tryptophan metabolites limit lymphatic inflammatory pathways and also decreases metabolic parameters associated with MetSyn and thus can be a viable therapeutic intervention for metabolic disease.

64 - O-GlcNAcylation is decreased in aorta and mesenteric artery in normotensive and hypertensive pregnant rats

O-glycosylation by N-acetyl-glucosamine (O-GlcNAc) decreases nitric oxide synthase phosphorylation leading to a lower nitric oxide (NO) bioavailability. We performed the analysis of O-GlcNAc, OGT and OGA expression by Western Blot and OGA activity by biochemical analysis in aortic and mesenteric bed homogenates from non-pregnant (NP) and pregnant (P) Wistar and spontaneously hypertensive rats (SHR) with 12 weeks old (n= 6). Aorta rings or mesenteric artery rings (n= 6), with and without endothelium, or with endothelium in the presence of L-NAME (100 µM) were incubated in the absence or presence of PugNAc (100 μM) and stimulated with cumulative concentrations of PE. The results were compared among groups (Student`s t test or ANOVA, p

277 - Encapsulation of tDodSNO generates a nitric oxide releasing nanoparticle

The development of drugs for the controlled delivery of therapeutic nitric oxide (NO) is an ongoing challenge. Current options include organonitrates and nitroprusside which have severe limitations to their use, with organonitrates inducing tolerance and nitroprusside releasing toxic cyanide ions. An alternative is the s-nitrosothiol drug class which breaks down to release NO; however, this NO release is normally too rapid to be therapeutically useful. The molecule tDodSNO has recently been developed as a stable s-nitrosothiol which is resistant to metabolic and thermal degradation. Its key property is that it can be selectively degraded by light, with photoactivation causing NO release. To render this s-nitrosothiol suitable for in vivo applications we report encapsulating tDodSNO within a nanoparticle consisting of a co-polymer of styrene and maleic acid (SMA). The resulting SMA-tDodSNO was fully water soluble, and the nanoparticle also further protected tDodSNO from degradation by glutathione, with a half-life increase of 24 hours. Encapsulation did not affect tDodSNO’s NO release profile, with similar NO levels (0-2 µM) recorded over a range of photoactivation levels for both tDodSNO and SMA-tDodSNO. Moving into cardiovascular activity assays, photoactivation of SMA-tDodSNO allowed controllable vasodilation of rat aortic rings, with vascular tone proportional to photoactivation. Additionally, in rat mesenteric beds, photoactivated SMA-tDodSNO induced selective hyperpermeability to macromolecules. In comparison to nitroprusside and the s-nitrosothiols GSNO and SNAP, without photoactivation SMA-tDodSNO induced vasodilation at a 40 fold higher EC50. However, upon photoactivation, SMA-tDodSNO was the only molecule to display enhanced NO release, with its EC50 decreasing to GSNO levels. This EC50 shift demonstrated that SMA-tDodSNO can be used to selectively induce NO dependent signalling by photoactivation. Future development of SMA-tDodSNO is therefore predicted to find applications for treating cardiovascular disease, as well as for enhancing the delivery of macromolecules, including antibodies and nanoconstructs, to target tissue.

19 - Apocynin reduces pressor and vasoconstrictor effects of Ang II in SHR

NAD(P)H oxidase (NOX) activity and expression can be activated by angiotensin (Ang) II. We have previously observed that apocynin, a NOX inhibitor, prevented endotelial dysfunction and reduced blood pressure by increasing nitric oxide (NO) and reducing ROS concentrations in endothelial cells. We evaluated the effect of apocynin on the contractile responses to Ang II in resistance arteries of SHR and the mechanisms involved on these effects. SHR were treated from the 4th to the 10th week of life with apocynin (30 mg/Kg). Wistar rats were used as normotensive control. Using mesenteric arteries, we performed concentration-response curves to Ang II and determined eNOS and NOX isoforms and subunits expressions, lucigenin chemiluminescence and nitrate/nitrite levels. Data were expressed as mean ± SEM. Apocynin increased endothelium modulation and/or NOS activity on Ang II vasoconstrictor responses in mesenteric arteries of SHR. Treatment with apocynin did not alter NO synthase activity, and eNOS, NOX1, NOXO1, and NOX4 expressions, however, it decreased NOX2 and p47phox expressions in mesenteric beds of SHR treated. Moreover, treatment apocynin was able to decrease ROS production in these vessels. The lower reactivity of Ang II in resistance arteries would lead to lower peripheral vascular resistance and consequently the reduction of mean arterial pressure and Ang II pressor effect in SHR treated with apocynin, as previously observed. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. Ethics Comittee CEUA FOA 450/2015. Financial Support FAPESP 2016/22180-9, CAPES and CNPq.

Encapsulation of tDodSNO generates a photoactivated nitric oxide releasing nanoparticle for localized control of vasodilation and vascular hyperpermeability

We report the synthesis and characterization of a photoactive nitric oxide (NO) releasing nanoparticle (NP) by encapsulation of the NO donor tert-dodecane S-nitrosothiol (tDodSNO) into a co-polymer of styrene and maleic anhydride (SMA) to afford SMA-tDodSNO. Encapsulation did not affect tDodSNO's stability or NO release profile, but imparted water solubility and protection from degradation reactions with glutathione. Under photoactivation the NP acted as a potent NO donor, with photoactivation acting as a switch to induce localized vasodilation in aortic rings (EC50* 660 nM at 2700 W/m2) and cause vascular hyperpermeability in mesenteric beds (8-fold increase in dye uptake at 1 µM SMA-tDodSNO with 460 W/m2 photoactivation). The NP was markedly superior as a photoactive NO donor in comparison to the S-nitrosothiols GSNO and SNAP, which are commonly used in experimental studies, as well as sodium nitroprusside, a clinically used vasodilator. Future development of this NP may find wide ranging therapeutic applications for treating cardiovascular disease and other disorders related to NO signaling, as well as enhancing macromolecular drug delivery to target organs through selective hyperpermeability. Supporting information describing the biophysical characterization of SMA-tDodSNO is supplied in an accompanying Data in Brief article (Alimoradi et al., doi: 10.1016/j.dib.2018.10.149).

Abstract P276: Hydrogen Sulfide Regulates Local Blood Flow in Conscious, Free-Moving Rats

Inhibiting cystathionine gamma-lyase (CSE) to prevent H 2 S synthesis augments constriction in isolated mesenteric and renal arteries, but H 2 S regulation of resistance in these vascular beds in vivo is unknown. This study evaluated regulation of blood flow and resistance in the mesenteric and renal vascular beds in chronically instrumented rats. Based on studies in isolated arteries, we hypothesized that endogenous production of H 2 S by CSE decreases vascular resistance more in mesenteric than in renal arteries. Rats were anesthetized with inhaled isofluorane (2%) and instrumented with femoral vein catheters, a telemeter blood pressure device and a pulsed Doppler flow probe (Crystal Biotech) on either the superior mesenteric artery or the renal artery (after denervation of the renal artery by removing all visible nerves and painting the surface of the artery with 10% phenol). After five to seven days of recovery from surgery, arterial pressure (AP), heart rate (HR) and relative blood flow (Q) was recorded. Rats then received a daily injection of the irreversible CSE inhibitor, propargylglycine (PAG, 50 mg/kg/day). After five injections, AP was significantly increased in rats with mesenteric flow probes (110±3 start to 122±4 mmHg post PAG, p&lt;0.05 n=7 rats per group). Relative mesenteric resistance (AP/Q) normalized to starting values was increased in PAG rats (150±7% starting value) compared to no change in rats receiving saline (98±3% starting value) (p&lt;0.05, n=7 rats per group). In the rats instrumented with renal flow probes, AP was also increased in the PAG but not in the saline rats (115±6 start to 143±5 mmHg post PAG, p = 0.0014) and calculated renal resistance increased (198% ± 14% versus 103 ± 5% starting value, p=0.0003, n = 5 rats). Heart rate was not changed by any of the treatments. Our results suggest that endogenous H 2 S is an important regulator of vascular resistance in both the mesenteric and renal circulations and that the contribution may be larger in the renal bed than in the mesenteric bed.

Influence of Luehea divaricata Mart. extracts on peripheral vascular resistance and the role of nitric oxide and both Ca+2-sensitive and Kir6.1 ATP-sensitive K+ channels in the vasodilatory effects of isovitexin on isolated perfused mesenteric beds

BackgroundLuehea divaricata Mart. (Malvaceae) is an important medicinal species widely used by indigenous and riverside populations of the Brazilian Pantanal region. It has been shown that the several extracts obtained from leaves of this species have important cardioprotective effects. Nevertheless, the secondary metabolites responsible for this activity, as well as the molecular mechanisms responsible for their pharmacological effects remain unknown. PurposeTo carry out a biomonitoring study to identify possible active metabolites present in different ESLD fractions and evaluate the mechanisms responsible for the vasodilatory effects on isolated perfused mesenteric beds. MethodsFirst, ESLD was obtained from L. divaricata leaves and a liquid-liquid fractionation was performed. The resulting fractions were analyzed by liquid chromatography-mass spectrometry. Then, the possible vasodilatory effects of ESLD, chloroform, ethyl acetate, n-butanolic and aqueous fractions on perfused arterial mesenteric vascular beds were evaluated. Finally, the molecular mechanisms involved in vasodilator responses of the aqueous fraction and its chemical component, isovitexin, on the mesenteric arteriolar tone were also investigated. ResultsIn preparations with functional endothelium ESLD, n-butanolic, aqueous fraction and isovitexin dose-dependently reduced the perfusion pressure in mesenteric vascular beds. Endothelium removal or inhibition of nitric oxide synthase enzymes by L-NAME reduced the vasodilatory effects induced by aqueous fraction and isovitexin. Perfusion with nutritive solution containing 40 mM KCl abolished the vasodilatory effect of all aqueous fractions and Isovitexin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channels blocker, tetraethylammonium, a non-selective KCa (calcium-activated) potassium channels blocker, or apamin, a potent blocker of small conductance Ca2+-activated (SK KCa) potassium channels reduced by around 70% vasodilation induced by all aqueous fractions and isovitexin doses. In addition, association of tetraethylammonium and glibenclamide, or L-NAME and glibenclamide, fully inhibited aqueous fraction and Isovitexin -induced vasodilation. ConclusionThis study showed that AqueFr obtained from Luehea divaricata and its metabolite - isovitexin - has important vasodilatory effects on MVBs. Apparently, these effects are dependent on endothelium-NO release and both SK KCa K+ channels and Kir6.1 ATP-sensitive K+ channels activation in the vascular smooth muscle.

Biomonitoring the cardiorenal effects of Luehea divaricata Mart.: An ethnoguided approach

Ethnopharmacological relevanceLuehea divaricata Mart. (Malvaceae) is an important medicinal species that is widely used as a diuretic in the Brazilian Pantanal region. An ethanolic supernatant that was obtained from an infusion of leaves of this species (ESLD) was recently shown to exert hypotensive and diuretic activity. Nevertheless, the secondary metabolites that are responsible for this activity and the molecular mechanisms of pharmacological action remain unknown. AimWe performed a detailed study to identify possible active metabolites that are present in different ESLD fractions and investigated their effects on renal and peripheral arteriolar tone. We further evaluated their interrelations with sustained diuretic and hypotensive actions. Materials and methodsThe ESLD was first obtained from L. divaricata leaves, and liquid-liquid fractionation was performed. The fractions were analyzed by liquid chromatography-mass spectrometry. An ethyl acetate fraction (AceFr), n-butanolic fraction (ButFr), and aqueous fraction (AqueFr) were then orally administered in male Wistar rats in a single dose or daily for 7 days. The doses were previously defined based on the yield that was obtained from each fraction. Hydrochlorothiazide was used as a positive control. Blood pressure, heart rate, urinary volume, pH, density, and urinary sodium, potassium, chloride, and calcium levels were measured. Serum levels of nitrite, thiobarbituric acid reactive species, nitrotyrosine, aldosterone, vasopressin, and plasma angiotensin converting enzyme activity were also measured. Finally, the direct effects of the ButFr on renal and mesenteric arteriolar tone and the role of nitric oxide and prostaglandins in the renal and hemodynamic effects were investigated. ResultsOf the fractions that were tested, only the ButFr exerted significant diuretic and saluretic effects. The AceFr and ButFr also had acute hypotensive effects, but only the ButFr maintained its response after 7 days of treatment. Prolonged treatment with the ButFr increased serum nitrite levels and significantly reduced oxidative and nitrosative markers of stress. Additionally, the ButFr caused a vasodilatory response in the renal and mesenteric arteriolar beds through the release of nitric oxide and prostaglandins. Finally, the diuretic and hypotensive effects of the ButFr were completely blocked by pretreatment with Nω-nitro-L-arginine methyl ester and indomethacin, thus demonstrating the direct involvement of nitric oxide and prostaglandins in these effects. ConclusionThe ButFr that was obtained from Luehea divaricata exerted sustained diuretic and hypotensive effects. These effects were apparently attributable to the release of nitric oxide and prostaglandins, which reduce renal and peripheral arteriolar tone and lead to an increase in the glomerular filtration rate and a reduction of global peripheral resistance. These findings suggest that the ButFr may be a potential complementary therapy for several conditions in which diuretic and hypotensive effects are required.

Diuretic herb Gomphrena celosioides Mart. (Amaranthaceae) promotes sustained arterial pressure reduction and protection from cardiac remodeling on rats with renovascular hypertension

Ethnopharmacological relevanceGomphrena celosioides Mart., belonging to the Amaranthaceae family, is a weed known as “perpétua,” and its ethnopharmacological use is to treat of urinary tract disorders and kidney stones. Urinary tract disorders and kidney stones could include several pathological conditions such hypertension, diuretic and lithiasic problems. In the present work a model of renovascular hypertension was developed in vivo to investigate its usefulness as an antihypertensive drug. Aim of the studyEvaluate the effect of acute and 28 day oral administration of G. celosioides extract on systemic arterial pressure and diuresis of renovascular-hypertensive rats, as well as its effect on cardiac remodeling and vascular reactivity. Materials and methodsEthanolic extract of G. celosioides (EEGC) was used. To induce renovascular hypertension, adult male Wistar rats were submitted to Goldblatt 1K1C or 2K1C surgery. The mean arterial pressure (MAP) of 1K1C animals was directly assessed by cannulation of the carotid artery before and after intraduodenal acute administration of 30, 100 or 300 mg/kg of EEGC. For the 4-week assay, 2K1C animals received daily treatments with water (control group), 100 mg/kg EEGC or 15 mg/kg enalapril for 28 days. Diuresis and caudal blood pressure were assessed weekly, and at the 28th day of treatment, the MAP was directly quantified shortly before euthanasia. Internal organs were removed, weighed and routinely processed for histology and the left ventricle wall was measured. Blood was collected for biochemical analysis and mechanism investigation by quantification of angiotensin converting enzyme (ACE) activity and aldosterone, nitrite and thiobarbituric acid reactive substances (TBARS) concentration. The rats’ mesenteric beds were isolated and cannulated to have their pressure variation assessed after crescent doses of phenylephrine (Phe), acetylcholine (ACh) and sodium nitroprusside (SNP). ResultsEEGC acutely reduced MAP the dose of 100 mg/kg. In the 4-week assay, EEGC acted as diuretic after acute administration after 1, 2, 3 and 4 weeks of treatment. EEGC also acted as an antihypertensive and it showed significant difference already after 1 week (and after 3 and 4 weeks) compared to control, with its MAP close to pre-surgery values at the end of the experiment. It promoted ACE inhibition, which led to lower aldosterone levels. The lower TBARS and higher nitrite concentration found in the EEGC group suggest antioxidant activity and NO maintenance. Moreover, EEGC counteracted the impairment of vascular reactivity induced by renovascular hypertension. The extract group presented thinner left ventricle wall compared to the control, meaning reduced hypertension-induced cardiac remodeling. ConclusionsThe G. celosioides diuretic effect is maintained on renovascular hypertensive rats and can reduce the blood pressure after the first week of treatment by inhibiting ACE and these effects are longstanding and strong enough to promote protection against cardiac remodeling. Therefore, it shows potential as an antihypertensive drug.

Mitochondrial DNA Activates NLRP3 Inflammasome and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetic Mice

IntroductionNLRP3 inflammasome is a molecular platform of innate immune system that regulates the inflammatory response through the activation of caspase‐1 and IL‐1β. It can be activated by several ligands, for example, mitochondrial DNA (mDNA). Some studies have shown that circulating mDNA is increased in patients with diabetes and play a role in the development of the disease; however, its role in vascular changes is still unknown. In this sense, we tested the hypothesis that mDNA contributes to vascular inflammatory/oxidative processes associated to type 1 diabetes (T1D) via NLRP3 inflammasome activation.MethodsWild‐type and NLRP3‐deficient (NLRP3−/−) mice were treated with vehicle (Veh) or streptozotocin (40 mg/kg) (T1D) for 5 days. Vascular reactivity was determined in mesenteric arteries. Caspase‐1 and IL‐1β expression were evaluated by western blot. Pancreatic mDNA was extracted from control (cmDNA) and diabetic animals (dmDNA) for endothelial cells stimulation. ROS generation was determined by chemiluminescence. Hydrogen peroxide (H2O2) production and calcium influx were determined by fluorescence. Data are presented as mean ± standard error of the mean.ResultsDiabetes increased vascular caspase‐1 and IL‐1β activation [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1; 4.8±1.1 vs. 0.8±0.5 vs. the Veh, respectively, p &lt;0.05]. However, this activation was attenuated in T1D NLRP3−/−. Mesenteric arteries from T1D exhibited decreased ACh‐induced vasodilatation vs. Veh [(Emax), 46.6±4.0 vs. 91.5±2.8, n=4–5, p&lt;0.05], which was not observed in T1D NLRP3−/−. The NLRP3 inhibitor MCC950 acutely improved endothelium‐dependent vasodilation in T1D [(Emax), 62.5±3.8 vs. 85.6±3.7, n=4–5, p&lt;0.05]. ROS generation [(Relative luminescence unit, RLU), 1.5×107±2.3 vs. 48.9×103±1.1, n=4–5, p&lt;0.05] as well as H2O2 production [(Relative fluorescence unit, RFU), 1082±242.6 vs. 232.0±42.8, n=4–5, p&lt;0.05] were lower in mesenteric bed from T1D NLRP3−/− than T1D. Only incubation with dmDNA reduced ACh‐induced vasodilation [(Emax), 48.4 ± 4.1 vs. 90.7 ± 3.4, n=4–5, p&lt;0.05], which was attenuated by the presence of an antioxidant [(Emax), 48.4 ± 4.1 vs. 71.5 ± 3.1, n=4–5, p&lt;0.05]. Similarly, only LPS‐primed cells incubated with dmDNA had significant NLRP3 activation (caspase‐1 and IL‐1β activation, respectively) [(u.a.), 1.6 ± 0.2 vs. 0.9 ± 0.1; and 1.6 ± 0.2 vs. 1.0 ± 0.1, n=4–5, p&lt;0.05]. The dmDNA induced in a time‐dependent way both calcium influx and ROS generation in endothelial cells, being the last reversed by the presence of an antioxidant. Likewise, T1D patients had increased circulating mDNA vs. healthy volunteers and also higher NLRP3 serum expression, and caspase‐1/IL‐1β activation.ConclusionThe T1D increases mDNA release which in turn stimulates increased calcium influx, induces ROS generation, and triggers vascular NLRP3 inflammasome activation contributing to inflammatory response and endothelial dysfunction in diabetes.Support or Funding InformationFinancial Support: FAPESP and CAPES. Ethics Committee number (026/2015).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.