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Aims: Hypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction., Main Methods: Male Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain., Key Findings: SBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it., Significance: doxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction., Competing Interests: Declaration of competing interest We declare that there are no conflicts of interest associated with this publication., (Copyright © 2024. Published by Elsevier B.V.)

Background & Aims: The treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO., Methods: Male Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit., Results: NONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NO • ) scavengers (PTIO; 100 μM and hydroxocobalamin; 30 μM) and nitroxyl anion (NO - ) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 μM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 μM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K + channels (K ir ), voltage-operated K + channels (K V ), and large conductance Ca 2+ -activated K + channels (BK Ca ). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 μM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production., Conclusions: NONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO • and NO - . Its vasorelaxant effect involves sGC, PKG, K + channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Increasing evidence shows that cardiovascular diseases (CVDs) are associated with an increased risk of cognitive impairment and Alzheimer's diseases (AD). It is unknown whether systemic vascular dysfunction occurs prior to the development of AD, if this occurs in a sex-dependent manner, and whether endothelial cells play a role in the deposition of amyloid beta (Aβ) peptides. We hypothesized that vascular dysfunction occurs prior to the onset of amyloid pathology, thus escalating its progression. Furthermore, endothelial cells from female mice will present with an exacerbated formation of Aβ peptides due to an exacerbated pressure pulsatility. To test this hypothesis, we used a double transgenic mouse model of early-onset AD (APPswe/PSEN1dE9). We evaluated hippocampus-dependent recognition memory and the cardiovascular function by echocardiography and direct measurements of blood pressure through carotid artery catheterization. Vascular function was evaluated in resistance arteries, morphometric parameters in the aortas, and immunofluorescence in the hippocampus and aortas. We observed that endothelial dysfunction occurred prior to the onset of amyloid pathology irrespective of sex. However, during the onset of amyloid pathology, only female APP/PS1 mice had vascular stiffness in the aorta. There was elevated Aβ deposition which colocalized with endothelial cells in the hippocampus from female APP/PS1 mice. Overall, these data showed that vascular abnormalities may be an early marker, and potential mediator of AD, but exacerbated aortic stiffness and pressure pulsatility after the onset of amyloid pathology may be associated with a greater burden of Aβ formation in hippocampal endothelial cells from female but not male APP/PS1 mice., (© 2024. The Author(s).)

Aims: Our aim was to evaluate whether the hydrogen sulfide (H 2 S) donor, 4-carboxyphenyl-isothiocyanate (4-CPI), exerts cardioprotective effect in the two kidney- one clip (2K-1C) rats through oxidative stress and MMP-2 activity attenuation and compare it with the classical H 2 S donor, Sodium Hydrosulfide (NaHS)., Materials and Methods: Renovascular hypertension (two kidneys-one clip; 2K-1C) was surgically induced in male Wistar rats. After two weeks, normotensive (2K) and hypertensive rats were intraperitoneally treated with vehicle (0.6 % dimethyl sulfoxide), NaHS (0.24 mg/Kg/day) or with 4-CPI (0.24 mg/Kg/day), for more 4 weeks. Systolic blood pressure (SBP) was evaluated weekly by tail-cuff plethysmography. Heart function was assessed by using the Millar catheter. Cardiac hypertrophy and fibrosis were evaluated by hematoxylin and eosin, and Picrosirius Red staining, respectively. The H 2 S was analyzed using WSP-1 fluorimetry and the cardiac oxidative stress was measured by lucigenin chemiluminescence and Amplex Red. MMP-2 activity was measured by in-gel gelatin or in situ zymography assays. Nox1, gp91 phox , MMP-2 and the phospho-p65 subunit (Serine 279) nuclear factor kappa B (NF-κB) levels were evaluated by Western blotting., Key Findings: 4-CPI reduced blood pressure in hypertensive rats, decreased cardiac remodeling and promoted cardioprotection through the enhancement of cardiac H 2 S levels. An attenuation of oxidative stress, with inactivation of the p65-NF-κB/MMP-2 axis was similarly observed after NaHS or 4-CPI treatment in 2K-1C hypertension., Significance: H 2 S is a mediator that promotes cardioprotective effects and decreases blood pressure, and 4-CPI seems to be a good candidate to reverse the maladaptive remodeling and cardiac dysfunction in renovascular hypertension., Competing Interests: Declaration of competing interest We declare that there are no conflicts of interest associated with this publication., (Copyright © 2024. Published by Elsevier Inc.)

O-Linked attachment of β-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that impacts the function, activity, subcellular localization, and stability of target proteins. Physiologically, acute O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. To date, thousands of proteins have been revealed to be O-GlcNAcylated and this number continues to grow as the technology for the detection of O-GlcNAc improves. The attachment of a single O-GlcNAc is catalyzed by the enzyme O-GlcNAc transferase (OGT), and their removal is catalyzed by O-GlcNAcase (OGA). O-GlcNAcylation is regulated by the metabolism of glucose via the hexosamine biosynthesis pathway, and the metabolic abnormalities associated with pathophysiological conditions are all associated with increased flux through this pathway and elevate O-GlcNAc levels. While chronic O-GlcNAcylation is well associated with cardiovascular dysfunction, only until recently, and with genetically modified animals, has O-GlcNAcylation as a contributing mechanism of cardiovascular disease emerged. This review will address and critically evaluate the current literature on the role of O-GlcNAcylation in vascular physiology, with a view that this pathway can offer novel targets for the treatment and prevention of cardiovascular diseases., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Introduction: Increased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension., Methods: Male Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK., Results: 2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect., Conclusions: Increase in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension., Competing Interests: Declaration of Competing Interest We declare that there are no conflicts of interest associated with this publication., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The major pathophysiological characteristic of hypertension is the occurrence of small artery remodeling and endothelial dysfunction. There is also solid evidence showing that microcirculation abnormalities occur prior to the onset of hypertension. However, the mechanism(s) that trigger these changes prior to the elevation of blood pressure are unknown, and this may limit our ability to identify the cause of this disease and effectively treat it. In hypertension, as with aging, the vasculature becomes less susceptible to repair. One of the reasons is because endothelial cells start to deteriorate and present with exacerbated endothelial-to-mesenchymal transition (EndMT). Likewise, vascular smooth muscle cells (VSMC) also dedifferentiate into a synthetic phenotype, whereby they start to produce and secrete extracellular vesicles with a high migration and proliferation capacity for repairing vascular injury. Uncontrolled EndMT and/or VSMC phenotype switching contributes to vascular diseases, but the initial trigger for these conditions is unidentified. Importantly, EndMT and synthetic VSMC exhibit plasticity and can return to adopt an endothelial cell-like fate and present contractile phenotype again, respectively. Therefore, in this hypothesis we will take advantage of this plasticity, and we propose to manipulate this fate by inducing partial cellular reprogramming without passing through the pluripotent state. Specifically, we suggest that activation of the three master transcription factors, Oct-4, Sox-2, and Klf-4 (collectively termed OSK) will reprogram endothelial cells and prevent and reduce EndMT and VSMC synthetic phenotype. It was recently shown that activation of OSK was able to restore lost vision in old mice, and cancer risk was reduced by excluding c-Myc. Therefore, OSK treatment could provide new possibilities for vascular rejuvenation and treatment of hypertension., Competing Interests: Conflict of Interest None.

Oxidative stress and MMP activity are found in the hearts and arteries in hypertension and contribute to the resulting hypertrophy and dysfunction. Quercetin is a flavonoid that reduces MMP-2 activity and ameliorates hypertrophic vascular remodeling of hypertension. The hypothesis is that treatment of hypertensive rats with quercetin ameliorates coronary maladaptive remodeling and decreases hypertrophic cardiac dysfunction by decreasing oxidative stress and MMP activity. Male Sprague-Dawley two-kidney, one-clip (2K1C) and Sham rats were treated with quercetin (10 mg/kg/day) or its vehicle for 8 weeks by gavage. Rats were analyzed at 10 weeks of hypertension. Systolic blood pressure (SBP) was examined by tail-cuff plethysmography. Cardiac left ventricles were used to determine MMP activity by in situ zymography and oxidative stress by dihydroethidium. Immunofluorescence was performed to detect transforming growth factor (TGF)-β and nuclear factor kappa B (NFkB). Morphological analyses of heart and coronary arteries were done by H&E and picrosirius red, and cardiac function was measured by Langendorff. SBP was increased in 2K1C rats, and quercetin did not reduce it. However, quercetin decreased both oxidative stress and TGF-β in the left ventricles of 2K1C rats. Quercetin also decreased the accentuated MMP activity in left ventricles and coronary arteries of 2K1C rats. Quercetin ameliorated hypertension-induced coronary arterial hypertrophic remodeling, although it did not reduce cardiac hypertrophic remodeling and dysfunction. Quercetin decreases cardiac oxidative stress and TGF-β and MMP activity in addition to improving coronary remodeling, yet does not ameliorate cardiac dysfunction in 2K1C rats., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hypertension is the most important risk factor for the development of terminal cardiovascular diseases, such as heart failure, chronic kidney disease, and atherosclerosis. Lifestyle interventions to lower blood pressure are generally desirable prior to initiating pharmaceutical drug treatments, which may have undesirable side effects. Ketogenic interventions are popular but the scientific literature supporting their efficacy is specific to certain interventions and outcomes in animal models and patient populations. For example, although caloric restriction has its own inherent difficulties (e.g. it requires high levels of motivation and adherence is difficult), it has unequivocally been associated with lowering blood pressure in hypertensive patients. On the other hand, the antihypertensive efficacy of ketogenic diets is inconclusive, and this is surprising, given that these diets have been largely helpful in mitigating metabolic syndrome and promoting longevity. It is possible that side effects associated with ketogenic diets (e.g. dyslipidemia) aggravate the hypertensive phenotype. However, given the recent data from our group, and others, reporting that the most abundant ketone body, β-hydroxybutyrate, can have positive effects on endothelial and vascular health, there is hope that ketone bodies can be harnessed as a therapeutic strategy to combat hypertension. Therefore, we conclude this review with a summary of the type and efficacy of ketone supplements. We propose that ketone supplements warrant investigation as low-dose antihypertensive therapy that decreases total peripheral resistance with minimal adverse side effects., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Studies have shown that strength training (ST) with blood flow restriction (BFR) in which low load is used (20-50% of 1 maximum voluntary contraction - MVC) can produce positive adaptations similar to ST with loads equal to or greater than 70% 1 MVC. Furthermore, recent studies have investigated the effects of STBFR on muscle adaptations, but few studies investigated the effects of STBFR on vascular function. This study aimed to evaluate the effects of the STBFR program on the vascular reactivity of the abdominal aorta of Wistar rats with femoral arteriovenous blood flow restriction. Male rats were divided into four groups: sedentary sham (S/S), sedentary with blood flow restriction (S/BFR), trained sham (T/S), and trained with blood flow restriction (T/BFR). The animals in the S/BFR and T/BFR groups underwent surgery to BFR in the femoral artery and vein. After one week, the trained groups started the ST which consisted of climbing ladder, six sets of 10 repetitions with 50% of 1 MVC assessed by maximum loaded weight (MLW) carried out for four weeks. Concentration-response curves to Acetylcholine (ACh: 10 nM - 100 μM) and Phenylephrine (PHE: 1 nM - 30 μM) were performed in aortic rings with intact endothelium. The production of nitric oxide (NO) and reactive oxygen species (ROS) in situ and the vascular remodeling marker (MMP-2) were also measured. The ST increased the strength of the T/S and T/BFR groups in MLW tests. The S/BFR group showed a 22% reduction in relaxation to acetylcholine, but exercise prevented this reduction in the T/BFR group. In animals without BFR, ST did not alter the response to acetylcholine. An increase in NO production was seen in T/S and T/BFR showed a reduction in ROS production (62% and 40%, respectively). In conclusion low load ST with BFR promotes similar vascular function responses to ST without BFR. Low load ST with and without BFR is interventions that can improve performance with similar magnitudes. Both training methods could have some benefits for vascular health due to NO production in the aorta increased in the T/S group and decreased production of reactive oxygen species in the T/BFR group., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Cardiac transition from concentric (C-LVH) to eccentric left ventricle hypertrophy (E-LVH) is a maladaptive response of hypertension. Matrix metalloproteinases (MMPs), in particular MMP-2, may contribute to tissue remodeling by proteolyzing extra- and intracellular proteins. Troponin I and dystrophin are two potential targets of MMP-2 examined in this study and their proteolysis would impair cardiac contractile function. We hypothesized that MMP-2 contributes to the decrease in troponin I and dystrophin in the hypertensive heart and thereby controls the transition from C-LVH to E-LVH and cardiac dysfunction., Methods: Male Wistar rats were divided into sham or two kidney-1 clip (2K-1C) hypertensive groups and treated with water (vehicle) or doxycycline (MMP inhibitor, 15 mg/kg/day) by gavage from the tenth to the sixteenth week post-surgery. Tail-cuff plethysmography, echocardiography, gelatin zymography, confocal microscopy, western blot, mass spectrometry, in silico protein analysis and immunofluorescence were performed., Results: 6 out of 23 2K-1C rats (26%) had E-LVH followed by reduced ejection fraction. The remaining had C-LVH with preserved cardiac function. Doxycycline prevented the transition from C-LVH to E-LVH. MMP activity is increased in C-LVH and E-LVH hearts which was inhibited by doxycycline. This effect was associated with an increase in troponin I cleavage products and a decline in dystrophin in the left ventricle of E-LVH rats, which was prevented by doxycycline., Conclusion: Hypertension causes increased cardiac MMP-2 activity which proteolyzes troponin I and dystrophin, contributing to the transition from C-LVH to E-LVH and cardiac dysfunction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: Hypertension underlies endothelial dysfunction, and activation of vasorelaxation signaling with low dependence on nitric oxide (NO) represents a good alternative for vascular modulation. C-type natriuretic peptide (CNP) causes relaxation by increasing cyclic guanosine 3',5'-monophosphate (cGMP) or Gi-protein activation through its natriuretic peptide receptor-B or -C, respectively. We have hypothesized that CNP could exerts its effects and could overcome endothelial dysfunction in two kidney-one clip (2K-1C) hypertensive rat aorta. Here, we investigate the intracellular signaling involved in CNP effects in hypertension., Materials and Methods: The 2K-1C hypertension was induced in male Wistar rats (200 g). CNP-induced vascular relaxation and cGMP production were investigated in rat thoracic aortas. The natriuretic peptide receptor-B and -C localization was evaluated by immunofluorescence. Calcium mobilization was assessed in endothelial cells from rat aortas., Key Findings: CNP induced similar relaxation in normotensive and 2K-1C hypertensive rat aortas, which increased after endothelium removal. CNP-induced relaxation involved natriuretic peptide receptor-B and -C activation in 2K-1C rats. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) counter-regulated CNP-particulate GC (pGC) activation in aortas. CNP reduced endothelial calcium and increased cGMP production, which was lower in 2K-1C. CNP-induced cGMP-dependent protein kinase (PKG) and sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) activation was impaired in 2K-1C rat aorta., Significance: Our results indicated CNP triggered relaxation through its natriuretic peptide receptor-B and -C in 2K-1C rat aortas, and that CNP-induced relaxation overcomes endothelial dysfunction in hypertension. In addition, NOS and sGC activities counter-regulate CNP-pGC activation to induce vascular relaxation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This work presents a new procedure to synthesize ruthenium-phthalocyanine complexes and uses diverse spectroscopic techniques to characterize trans- [RuCl(Pc)DMSO] (I) (Pc = phthalocyanine) and trans -[Ru(Pc)(4-ampy) 2 ] (II) (4-ampy = 4-aminopyridine). The triplet excited-state lifetimes of (I) measured by nanosecond transient absorption showed that two processes occurred, one around 15 ns and the other around 3.8 μs. Axial ligands seemed to affect the singlet oxygen quantum yield. Yields of 0.62 and 0.14 were achieved for (I) and (II) , respectively. The lower value obtained for (II) probably resulted from secondary reactions of singlet oxygen in the presence of the ruthenium complex. We also investigate how axial ligands in the ruthenium-phthalocyanine complexes affect their photo-bioactivity in B16F10 murine melanoma cells. In the case of (I) at 1 μmol/L, photosensitization with 5.95 J/cm 2 provided B16F10 cell viability of 6%, showing that (I) was more active than (II) at the same concentration. Furthermore, (II) was detected intracellularly in B16F10 cell extracts. The behavior of the evaluated ruthenium-phthalocyanine complexes point to the potential use of (I) as a metal-based drug in clinical therapy. Changes in axial ligands can modulate the photosensitizer activity of the ruthenium phthalocyanine complexes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FQ declared a shared affiliation with several of the authors, RS, TM, LN, and LP, to the handling editor at time of review., (Copyright © 2021 Martins, Negri, Pernomian, Faial, Xue, Akhimie,Hamblin, Turro and da Silva.)

Background: Several metal-based molecules that display cytotoxicity against multiple cell lines have been pursued in an attempt to fight against cancer and to overcome the typical side effects of drugs like cisplatin. In this scenario, ruthenium complexes have been extensively studied due to their activity in both in vitro and in vivo biological systems, including various cancer cell strains., Objective: We aimed to develop a method to synthesize novel [Ru(NO)(bpy) 2 L 2 ] 2+ complexes containing amino acid ligands by using an alternative Click Chemistry approach, namely the copper azide-alkyne cycloaddition reaction (CuAAC reaction), to construct nitrosyl/nitrite complexes bearing a modified lysine residue., Methods: We synthesized a new ligand by Click Chemistry approach and new compounds bearing the unprecedented ligand. Cytotoxicity was assessed by the classical MTT colorimetric assay. MCF-7 and MDAMB- 231 cells were used as breast cancer cell models. MCF-10 was used as a model of healthy cells., Results: Amino acid ligands related to N 3 -Lys(Fmoc) and the new pyLys were successfully synthesized by the diazotransfer reaction and the CuAAC reaction, respectively. The latter reaction involves coupling between N 3 -Lys(Fmoc) and 3ethynylpyridine. Both N 3 -Lys(Fmoc) and the new pyLys were introduced into the ruthenium bipyridine complex I, or cis-[Ru II (NO)(NO 2 )(bpy) 2 ] 2+ , to generate the common nitro-based complex III, which was further converted to the final complex IV. Results of the MTT assay proved the cytotoxic effect of cis- [Ru II (NO)(pyLysO-)(bpy) 2 ](PF 6 ) 2 against the mammalian breast cancer cells MCF-7 and MDA-MB231., Conclusion: The viability assays revealed that complex IV, bearing a NO group and a modified lysine residue, was able to release NO and cross tumor cell membranes. In this work, Complex IV was observed to be the most active ruthenium bipyridine complex against the mammalian breast cancer cells MCF-7 and MDA-MB231: it was approximately twice as active as cisplatin, whilst complexes I-III proved to be less cytotoxic than complex IV. Additional tests using healthy MCF 10A cells showed that complexes II-IV were three- to sixfold less toxic than cisplatin, which suggested that complex IV was selective against cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The aryl hydrocarbon receptor (AHR) is widely expressed in immune and non-immune cells of the gut and its activation has been correlated to the outcome of inflammatory bowel diseases (IBD). In ulcerative colitis and Crohn's disease, there is an excessive chronic inflammation with massive accumulation of leukocytes in the gut, in an attempt to constrain the invasion of pathogenic microorganisms on the damaged organ. Accordingly, it is known that dietary components, xenobiotics, and some chemicals or metabolites can activate AHR and induce the modulation of inflammatory responses. In fact, the AHR triggering by specific ligands during inflammatory conditions results in decreased IFNγ, IL-6, IL-12, TNF, IL-7, and IL-17, along with reduced microbial translocation and fibrosis in the gut. Moreover, upon AHR activation, there are increased regulatory mechanisms such as IL-10, IL-22, prostaglandin E 2 , and Foxp3, besides the production of anti-microbial peptides and epithelial repair. Most interestingly, commensal bacteria or their metabolites may also activate this receptor, thus contributing to the restoration of gut normobiosis and homeostasis. In line with that, Lactobacillus reuteri, Lactobacillus bulgaricus, or microbial products such as tryptophan metabolites, indole-3-pyruvic acid, urolithin A, short-chain fatty acids, dihydroxyquinoline, and others may regulate the inflammation by mechanisms dependent on AHR activation. Hence, here we discussed the potential modulatory role of AHR on intestinal inflammation, focused on the reestablishment of homeostasis through the receptor triggering by microbial metabolites. Finally, the development of AHR-based therapies derived from bacteria products could represent an important future alternative for controlling IBD.

Catecholamines participate in angiogenesis, an important tumor development process. However, the way catecholamines interact with their receptors has not been completely elucidated, and doubts still remain as to whether these interactions occur between catechol and/or amine sites and particular amino acid residues on the catecholamine receptors. To evaluate how catechol and amine groups contribute to angiogenesis, we immobilized the catechol site through ruthenium ion (Ru) coordination, to obtain species with the general formula [Ru(NH 3 ) 4 (catecholamine-R)]Cl. We then assessed the angiogenic activity of the complexes in a chorioallantoic membrane model (CAM) and examined vascular reactivity and calcium mobilization in rat aortas and vascular cells. [Ru(NH 3 ) 4 (catecholamine-R)]Cl acted as partial agonists and/or antagonists of their respective receptors and induced calcium mobilization. [Ru(NH 3 ) 4 (isoproterenol)] + [Ru(NH 3 ) 4 (noradrenaline)] + , and [Ru(NH 3 ) 4 (adrenaline)] + behaved as antiangiogenic complexes, whereas [Ru(NH 3 ) 4 (dopamine)] + proved to be a proangiogenic complex. In conclusion, catecholamines and [Ru(NH 3 ) 4 (catecholamine-R)]Cl can modulate angiogenesis, and catechol group availability can modify the way these complexes impact the vascular tone, suggesting that catecholamines and their receptors interact differently after catecholamine coordination to ruthenium., (This journal is © The Royal Society of Chemistry 2020.)

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Acute or chronic exposure to diabetes-related stressors triggers a specific psychological and behavior stress syndrome called diabetes distress, which underlies depressive symptoms in most diabetic patients. Distressed and/or depressive diabetic adults exhibit higher rates of cardiovascular mortality and morbidity, which have been correlated to macrovascular complications evoked by diabetic behavior stress. Recent experimental findings clearly point out that oxidative stress accounts for the vascular dysfunction initiated by the exposure to life stressors in diabetic conditions. Moreover, oxidative stress has been described as the main autocrine and paracrine mechanism of cardiovascular damage induced by endothelial microparticles (anuclear ectosomal microvesicles released from injured endothelial cells) in diabetic subjects. Such robust relationship between oxidative stress and cardiovascular diseases strongly suggests a critical role for endothelial microparticles as the primer messengers of the redox-dependent vascular dysfunction underlying diabetes distress. Here, we provide novel perspectives opened in the view of endothelial microparticles as promising diagnostic and pharmacotherapeutic biomarkers of cardiovascular risk in distressed diabetic patients.

Compared with age-matched men, premenopausal women are largely protected from coronary artery disease, a difference that is lost after menopause. The effects of oestrogens are mediated by the activation of nuclear receptors (ERα and ERβ) and by the G protein-coupled oestrogen receptor (GPER). This study aims to evaluate the potential role of GPER in coronary circulation in female and male rats. The baseline coronary perfusion pressure (CPP) and the concentration-response curve with a GPER agonist (G-1) were evaluated in isolated hearts before and after the blockade of GPER. GPER, superoxide dismutase (SOD-2), catalase and gp91phox protein expression were assessed by Western blotting. Superoxide production was evaluated ' in situ ' via dihydroethidium fluorescence (DHE). GPER blockade significantly increased the CPP in both groups, demonstrating the modulation of coronary tone by GPER. G-1 causes relaxation of the coronary bed in a concentration-dependent manner and was significantly higher in female rats. No differences were detected in GPER, SOD-2 and catalase protein expression. However, gp91phox expression and DHE fluorescence were higher in male rats, indicating elevated superoxide production. Therefore, GPER plays an important role in modulating coronary tone and reactivity in female and male rats. The observed differences in vascular reactivity may be related to the higher superoxide production in male rats. These findings help to elucidate the role of GPER-modulating coronary circulation, providing new information to develop a potential therapeutic target for the treatment of coronary heart disease., (© 2017 Society for Endocrinology.)

Objectives: To investigate the mechanisms underlying the effects of chronic restraint stress on the vascular contractile response induced by angiotensin (Ang) II in rat carotid., Methods: Concentration-response curves for AngII were obtained in endothelium-intact or endothelium-denuded carotid rings, in the absence or presence of SC-560 (COX-1 inhibitor), SC-236 (COX-2 inhibitor), wortmannin (PI 3 K-Akt inhibitor), ML171 (NOX-1 inhibitor), VAS2870 (NOX-4 inhibitor), tiron (O2- scavenger) or PEG-catalase (H 2 O 2 scavenger). 6-ketoPGF 1α , TXB 2 , O2- or H 2 O 2 levels and superoxide dismutase and catalase activity or expression were also measured in rat carotid., Key Findings: Stress increased AngII potency in rat carotid. Muscular COX-1 or COX-2-derived metabolites negatively modulated AngII-induced contraction in control rat carotid. Endothelial COX-1 or COX-2-derived metabolites positively modulated AngII-induced contraction in stressed rat carotid. PI 3 K-Akt, NOX-1, NOX-4, O2- and H 2 O 2 positively modulated AngII-induced contraction in stressed rat carotid. Stress increased 6-ketoPGF 1α or H 2 O 2 generation and reduced catalase activity in rat carotid. Protein expression of COX-1, NOX-4 or p-Akt was increased in stressed rat carotid., Conclusions: Stress increases AngII potency in rat carotid by a mechanism that involves the increased generation of PGI 2 and H 2 O 2 and the activation of Akt pathway. Such mechanism could play a pathophysiological role in cardiovascular diseases correlated with stress., (© 2016 Royal Pharmaceutical Society.)

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK 1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ET A antagonist), BQ-788 (ET B antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK 1 -mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ET B -mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Protective effects of MAS activation have spurred clinical interests in developing MAS agonists. However, current bases that drive this process preclude that physiological concentrations of peptide MAS agonists induce an atypical signaling that does not reach the metabotropic efficacy of constitutive activation. Canonical activation of MAS-coupled G proteins is only achieved by supraphysiological concentrations of peptide MAS agonists or physiological concentrations of chemically modified analogues. These pleiotropic differences are because of two overlapped binding domains: one non-metabotropic site that recognizes peptide agonists and one metabotropic domain that recognizes modified analogues., Objective: It is feasible that supraphysiological concentrations of peptide MAS agonists undergo to chemical modifications required for binding to metabotropic domain. Receptor oligomerization enhances pharmacological parameters coupled to metabotropic signaling. The formation of receptor-signalosome complex makes the transduction of agonists more adaptive. Considering the recent identification of MAS-signalosome, we aimed to postulate the reverse induced fit hypothesis in which MAS-signalosome would trigger chemical modifications required for agonists bind to MAS metabotropic domain., Methods: Here we cover rational perspectives for developing novel metabotropic MAS agonists in the view of the reverse induced-fit hypothesis., Results: Predicting a 3D model of MAS metabotropic domain may guide the screening of chemical modifications required for metabotropic efficacy. Pharmacophore-based virtual screening would select potential metabotropic MAS agonists from virtual libraries from human proteome., Conclusions: Rational perspectives that consider reverse induced fit hypothesis during MAS activation for developing metabotropic MAS agonists represents the best approach in providing MAS ligands with constitutive efficacy at physiological concentrations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Diabetes mellitus is associated with reactive oxygen and nitrogen species accumulation. Behavioral stress increases nitric oxide production, which may trigger a massive impact on vascular cells and accelerate cardiovascular complications under oxidative stress conditions such as Diabetes. For this study, type-1 Diabetes mellitus was induced in Wistar rats by intraperitoneal injection of streptozotocin. After 28 days, cumulative concentration-response curves for angiotensin II were obtained in endothelium-intact carotid rings from diabetic rats that underwent to acute restraint stress for 3h. The contractile response evoked by angiotensin II was increased in carotid arteries from diabetic rats. Acute restraint stress did not alter angiotensin II-induced contraction in carotid arteries from normoglycaemic rats. However acute stress combined with Diabetes increased angiotensin II-induced contraction in carotid rings. Western blot experiments and the inhibition of nitric oxide synthases in functional assays showed that neuronal, endothelial and inducible nitric oxide synthase isoforms contribute to the increased formation of peroxynitrite and contractile hyperreactivity to angiotensin II in carotid rings from stressed diabetic rats. In summary, these findings suggest that the increased superoxide anion generation in carotid arteries from diabetic rats associated to the increased local nitric oxide synthases expression and activity induced by acute restrain stress were responsible for exacerbating the local formation of peroxynitrite and the contraction induced by angiotensin II., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: To evaluate the role of nitric oxide, reactive oxygen species (ROS), and natriuretic peptide receptor-B activation in C-type natriuretic peptide-anti-contractile effect on Phenylephrine-induced contraction in aorta isolated from septic rats., Methods and Results: Cecal ligation and puncture (CLP) surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery (RMA). Measurement of survival rate, mean arterial pressure (MAP), plasma nitric oxide, specific protein expression, and localization were evaluated. Septic rats had a survival rate about 37% at 4 h after the surgery, and these rats presented hypotension compared to control-operated (Sham) rats. Phenylephrine-induced contraction was decreased in sepsis. C-type natriuretic peptide (CNP) induced anti-contractile effect in aortas. Plasma nitric oxide was increased in sepsis. Nitric oxide-synthase but not natriuretic peptide receptor-B expression was increased in septic rat aortas. C-type natriuretic peptide-anti-contractile effect was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA, and basal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were lower in septic rats. Phenylephrine and CNP enhanced ROS production. However, stimulated ROS production was low in sepsis., Conclusion: CNP induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation.

Emerging data point the crosstalk between dyslipidemia and renin-angiotensin system (RAS). Advanced dyslipidemia is described to induce RAS activation in the vasculature. However, the interplay between early dyslipidemia and the RAS remains unexplored. Knowing that hamsters and humans have a similar lipid profile, we investigated the effects of early and advanced dyslipidemia on angiotensin II-induced contraction. Cumulative concentration-response curves for angiotensin II (1.0pmol/l to 1.0µmol/l) were obtained in the hamster thoracic aorta. We also investigated the modulatory action of NAD(P)H oxidase on angiotensin II-induced contraction using ML171 (Nox-1 inhibitor, 0.5µmol/l) and VAS2870 (Nox-4 inhibitor, 5µmol/l). Early dyslipidemia was detected in hamsters treated with a cholesterol-rich diet for 15 days. Early dyslipidemia decreased the contraction induced by angiotensin II and the concentration of Nox-4-derived hydrogen peroxide. Advanced dyslipidemia, observed in hamsters treated with cholesterol-rich diet for 30 days, restored the contractile response induced by angiotensin II by compensatory mechanism that involves Nox-4-mediated oxidative stress. The hyporresponsiveness to angiotensin II may be an auto-inhibitory regulation of the angiotensinergic function during early dyslipidemia in an attempt to reduce the effects of the upregulation of the vascular RAS during the advanced stages of atherogenesis. The recovery of vascular angiotensin II functionality during the advanced phases of dyslipidemia is the result of the upregulation of redox-pro-inflammatory pathway that might be most likely involved in atherogenesis progression rather than in the recovery of vascular function. Taken together, our findings show the early phase of dyslipidemia may be the most favorable moment for effective atheroprotective therapeutic interventions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: Our main objective was to investigate the mechanisms underlying the effects of hyperhomocysteinaemia (HHcy) on contractile response mediated by α1-adrenoceptors in the rat corpus cavernosum., Methods: Concentration-response curves for phenylephrine (PE) were obtained in strips of corpus cavernosum, in absence or after incubation with tiron, tempol or polyethylene glycol (PEG)-catalase combined or not with tempol. We also measured the superoxide anion (O2(-)) and hydrogen peroxide (H2O2) generation, superoxide dismutase (SOD) and catalase activity and α-actin expression in rat corpus cavernosum from both groups., Key Findings: HHcy increased PE-induced contraction in cavernosal strips. Tiron, PEG-catalase or tempol increased PE-induced contraction in strips from control rats, but it was not altered by tiron or PEG-catalase in HHcy rats, whereas tempol reduced this response. The combination of PEG-catalase and tempol did not alter the contractile response to PE in both groups. HHcy increased O2(-) generation and SOD activity, whereas H2O2 concentration was reduced. Finally, HHcy did not alter catalase activity or expression of α-actin., Conclusions: The major new finding from this study is that HHcy induced a marked increase in PE-induced contraction in rat corpus cavernosum by a mechanism that involves increased O2(-) generation and it could play a role in the pathogenesis of erectile dysfunction associated with HHcy., (© 2015 Royal Pharmaceutical Society.)

The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hyperglycemia increases the generation of reactive oxygen species and affects systems that regulate the vascular tone including renin-angiotensin system. Stress could exacerbate intracellular oxidative stress during Diabetes upon the activation of angiotensin AT1/NADPH oxidase pathway, which contributes to the development of diabetic cardiovascular complications. For this study, type-I Diabetes was induced in Wistar rats by intraperitoneal injection of streptozotocin. 28 days after streptozotocin injection, the animals underwent to acute restraint stress for 3 h. Cumulative concentration-response curves for angiotensin II were obtained in carotid rings pre-treated or not with Nox or cyclooxygenase inhibitors. Nox1 or Nox4 expression and activity were assessed by Western blotting and lucigenin chemiluminescence, respectively. The role of Nox1 and Nox4 on reactive oxygen species generation was evaluated by flow cytometry and Amplex Red assays. Cyclooxygenases expression was assessed by real-time polymerase chain reaction. The contractile response evoked by angiotensin II was increased in diabetic rat carotid. Acute restraint stress increased this response in this vessel by mechanisms mediated by Nox4, whose local expression and activity in generating hydrogen peroxide are increased. The contractile hyperreactivity to angiotensin II in stressed diabetic rat carotid is also mediated by metabolites derived from cyclooxygenase-2, whose local expression is increased. Taken together, our findings suggest that acute restraint stress exacerbates the contractile hyperreactivity to angiotensin II in diabetic rat carotid by enhancing Nox4-driven generation of hydrogen peroxide, which evokes contractile tone by cyclooxygenases-dependent mechanisms. Finally, these findings highlight the harmful role played by acute stress in modulating diabetic vascular complications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The important role played by aryl hydrocarbon receptor activation in the pathophysiology of atherosclerosis induced by cigarette smoke exposure has spurred the clinical interest in the development of aryl hydrocarbon receptor antagonists with atheroprotective efficacy. A few aryl hydrocarbon receptor antagonists were developed but the lack of structural information regarding the receptor ligand binding domain resulted in several limitations in the pharmacological properties of these compounds including partial agonism, allosterism, non-selectivity, cytotoxicity and susceptibility to bioactivation. These limitations make the progress of preclinical and clinical assays with the available aryl hydrocarbon receptor antagonists difficult. There is a great interest in developing pure, competitive, selective, nontoxic and resistant to bioactivation aryl hydrocarbon receptor antagonists. Current technology permits the development of pharmacologically ideal antagonists based on the chemical features of the aryl hydrocarbon receptor ligand binding domain. According to these characteristics, chlorinated derivatives of trans-stilbene meta-substituted with electrophilic aromatic directing groups would be effective prototypes for pure, competitive, selective, nontoxic and resistant to bioactivation antagonists for such receptor., (Copyright © 2015 Elsevier B.V. All rights reserved.)

AT1 antagonists effectively prevent atherosclerosis since AT1 upregulation and angiotensin II-induced proinflammatory actions are critical to atherogenesis. Despite the classic mechanisms underlying the vasoprotective and atheroprotective actions of AT1 antagonists, the cross-talk between angiotensin-converting enzyme-angiotensin II-AT1 and angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axes suggests other mechanisms beyond AT1 blockage in such effects. For instance, angiotensin-converting enzyme 2 activity is inhibited by reactive oxygen species derived from AT1-mediated proinflammatory signaling. Since angiotensin-(1-7) promotes antiatherogenic effects, we hypothesized that the vasoprotective and atheroprotective effects of AT1 antagonists could result from their inhibitory effects on the AT1-mediated negative modulation of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Interestingly, our results showed that early atherosclerosis triggered in thoracic aorta from high cholesterol fed-Apolipoprotein E-deficient mice impairs angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality by a proinflammatory-redox AT1-mediated pathway. In such mechanism, AT1 activation leads to the aortic release of tumor necrosis factor-α, which stimulates NAD(P)H oxidase/Nox1-driven generation of superoxide and hydrogen peroxide. While hydrogen peroxide inhibits angiotensin-converting enzyme 2 activity, superoxide impairs MAS functionality. Candesartan treatment restored the functionality of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis by inhibiting the proinflammatory-redox AT1-mediated mechanism. Candesartan also promoted vasoprotective and atheroprotective effects that were mediated by MAS since A779 (MAS antagonist) co-treatment inhibited them. The role of MAS receptors as the final mediators of the vasoprotective and atheroprotective effects of candesartan was supported by the vascular actions of angiotensin-(1-7) upon the recovery of the functionality of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Balloon injury in diabetic rats triggers a sensory neurocompensatory response that restores the blood flow in contralateral carotid. These vasoprotective effects result from H2O2-mediated relaxation that counteracts AT1-dependent contractile hyperreactivity. The most important mechanism from the renin-angiotensin-system in counteracting AT1-mediated effects is that one is mediated by Mas receptors. Thus, we hypothesized that the vasoprotective effects of balloon neurocompensation in diabetic rats could result from the improvement of Mas signaling by H2O2-mediated sensory mechanisms. NK1 receptors are sensory components whose activation could lead to H2O2 generation upon TGFβ1 release and ALK5-mediated Nox4 upregulation. Based on this, we aimed to investigate: (1) the role of the TGFβ1/ALK5-Nox4-H2O2 pathway on modulating Mas signaling in diabetic rat contralateral carotid; and (2) the contribution of Mas signaling in the control of local blood flow. Our results showed that balloon neurocompensation restored diabetic rat contralateral carotid flow by improving Mas signaling through NK1-mediated TGFβ1 release. TGFβ1/ALK5 activation enhanced Nox4 expression and Nox4-driven generation of H2O2. In turn, H2O2 enhanced the local Mas-mediated relaxation. Since restenosis impairs diabetic rat ipsilateral carotid flow, the restoration of diabetic rat contralateral carotid flow may prevent further damages in cerebral irrigation by carotid pathways after angioplasty during diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Renin-Angiotensin system plays an important role in the regulation of systemic blood pressure as well as in fluid and electrolyte balance. It is divided into two described axes, the ACE - Ang II - AT1 receptor, with Ang II as the main mediator, and the ACE2 - Ang-(1-7) - Mas receptor, with Ang-(1-7) responsible for the main effects. The main vascular effect induced by Ang II is contraction, while Ang-(1-7) includes relaxation in several vascular beds. Ang II also activates several cytokines that are important in the genesis of vascular inflammation and hypertrophy. In this context, Ang-(1-7) seems to have a protective role. Both AT1 and Mas receptors modulate, in different ways, the generation of, which are involved in the control of vascular tone and the genesis of vascular dysfunction triggered by several diseases, including diabetes mellitus, arterial hypertension and atherosclerosis. Thereby, this review presents an overview of the modulation played by the whole Renin-Angiotensin system on the reactive oxygen species-mediated control of vascular tone and the oxidative stress-elicited vascular dysfunction.

The endothelial nitric oxide synthase (eNOS) plays an important role in the control of the vascular tone. This work aimed to evaluate the role of an α1-adrenoceptor agonist phenylephrine (PE) on eNOS activity and downstream signaling pathway activation in normotensive (2K) and renal hypertensive (2K-1C) intact-endothelium rat aortas. Concentration-effect curves were performed for PE in intact-endothelium aortas from 2K and 2K-1C rats, in the absence of or in the presence of NOS or soluble guanylyl cyclase (sGC) inhibitor. Intact endothelium aortas were stimulated with PE in organ chambers and eNOS Ser(1177)/Thr(495) phosphorylation expression was evaluated by western blot. Nitric Oxide (NO) production was evaluated in isolated endothelial cells from 2K and 2K-1C rat aortas by flow-cytometry using NO selective fluorescent probe, DAF-2DA. The sGC activity/expression was also evaluated. PE-induced contractile response is lower in 2K-1C than in 2K intact-endothelium rat aorta. This is due to higher eNOS Ser(1177) phosphorylation in 2K-1C, which induces the eNOS overactivation. It was abolished by NOS or sGC inhibition. Phenylephrine reduces NO production in 2K as compared to the basal level, but it is not modified in 2K-1C. In PE-stimulated endothelial cells, the NO production is higher in 2K-1C than in 2K. Phenylephrine induces higher cGMP production in 2K-1C than in 2K, despite the lower expression of sGC in 2K-1C. Our results suggest that alpha1-adrenoceptor activation contributes to the increased activity of the enzyme eNOS by Ser(1177) phosphorylation in 2K-1C intact-endothelium aorta, which consequently decreases PE-induced contractile response., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We investigated the involvement of nitric oxide (NO) and reactive oxygen species (ROS) on autonomic cardiovascular parameters, vascular reactivity, and endothelial cells isolated from aorta of monosodium glutamate (MSG) obese rats. Obesity was induced by administration of 4 mg/g body wt of MSG or equimolar saline [control (CTR)] to newborn rats. At the 60th day, the treatment was started with N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg) or 0.9% saline. At the 90th day, after artery catheterization, mean arterial pressure (MAP) and heart rate were recorded. Plasma was collected to assess lipid peroxidation. Endothelial cells isolated from aorta were evaluated by flow cytometry and fluorescence intensity (FI) emitted by NO-sensitive dye [4,5-diaminofluoresceindiacetate (DAF-2DA)] and by ROS-sensitive dye [dihydroethidium (DHE)]. Vascular reactivity was made by concentration-response curves of acetylcholine. MSG showed hypertension compared with CTR. Treatment with L-NAME increased MAP only in CTR. The MSG induced an increase in the low-frequency (LF) band and a decrease in the high-frequency band of pulse interval. L-NAME treatment increased the LF band of systolic arterial pressure only in CTR without changes in MSG. Lipid peroxidation levels were higher in MSG and were attenuated after L-NAME. In endothelial cells, basal FI to DAF was higher in CTR than in MSG. In both groups, acetylcholine increased FI for DAF from basal. The FI baseline to DHE was higher in MSG than in CTR. Acetylcholine increased FI to DHE in the CTR group, but decreased in MSG animals. We suggest that reduced NO production and increased production of ROS may contribute to hypertension in obese MSG animals., (Copyright © 2014 the American Physiological Society.)

We investigated the mechanisms underlying the vasorelaxant and hypotensive actions of the labdane-type diterpene ent-3-acetoxy-labda-8(17),13-dien-15-oic acid (labda-15-oic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats. cAMP and cGMP were measured by enzyme immunoassay (EIA) whereas nitrate measurement was performed by chemiluminescence. Nitric oxide (NO) concentration ([NO]c) was measured in endothelial cells by flow cytometry. The cytosolic calcium concentration ([Ca2+]c) in vascular smooth muscle cells (VSMC) was measured by confocal microscopy. Blood pressure measurements were performed in conscious rats. Labda-15-oic acid inhibited the contraction induced by phenylephrine and serotonin in either endothelium-intact or endothelium-denuded rat aortic rings. The labdane significantly reduced CaCl2-induced contraction in a Ca2+-free solution containing KCl or phenylephrine. Labda-15-oic acid (0.1–300 μmol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl. In endothelium-intact rings, the relaxation induced by labda-15-oic acid was affected by L-NAME, 7-nitroindazole, ODQ, hemoglobin, Rp-8-Br-Pet-cGMPS and thapsigargin. Blockade of K+ channels with 4-aminopyridine, apamin, charybdotoxin and glibenclamide affected the relaxation induced by labda-15-oic acid. The labdane increased cGMP and nitrate levels but did not affect cAMP levels in endothelium-intact aortas. Labda-15-oic acid increased [NO]c in endothelial cells and decreased [Ca2+]c in VSMC. The hypotension induced by intravenous administration of labda-15-oic acid (0.3–3 mg/kg) was partially reduced by L-NAME. In conclusion, the mechanisms underlying the cardiovascular actions of the labdane involve the activation of the endothelial NO-cGMP pathway, the opening of K+ channels and the alteration on Ca2+ mobilization.

There are many evidences indicating a compensatory mechanism in contralateral carotids following balloon injury. Previously it was observed α1-adrenoceptor-mediated hyper-reactivity and impairment of calcium influx in contralateral carotids 4 days after injury. At a later stage, α1-adrenoceptor-mediated contraction is similar to the control and we hypothesized that downstream signaling was normal. In the present study, we aimed to evaluate α1-adrenoceptor-mediated calcium influx in contralateral carotids 15 days after balloon injury. Concentration-response curves for CaCl2 in presence of the α1-adrenoceptor agonist (phenylephrine), measurement of the intracellular calcium transient and the levels of reactive oxygen species using fluorescent dyes were performed in control and contralateral carotids. Phenylephrine-induced intracellular calcium mobilization in contralateral carotids was not altered, while phenylephrine-induced calcium influx was reduced in the contralateral artery. Nitric oxide synthase inhibitors, L-NAME or L-NNA, restored this response, but nitrite and nitrate levels were decreased in contralateral carotids. Additionally, a rise in oxygen free radicals was observed in contralateral carotids. Furthermore, Tiron, a superoxide anion scavenger, restored α1-adrenoceptor-mediated calcium influx in contralateral carotids to the control level. Similar results were observed with the selective potassium channels blockers 4-aminopyridine and charybdotoxin. In conclusion, data showed that balloon catheter injury resulted in increased superoxide anions levels, activation of potassium channels (Kv and BKCa), inhibition of calcium channels (Cav) and preservation of α1-adrenoceptor-mediated contraction at a later stage after injury., (© 2013 Published by Elsevier B.V.)

We hypothesized that endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species during type I-diabetes impairs carotid ACE2-angiotensin-(1-7)-Mas axis functionality, which accounts for the impaired carotid flow in diabetic rats. We also hypothesized that angiotensin-(1-7) chronic treatment of diabetic rats restores carotid ACE2-angiotensin-(1-7)-Mas axis functionality and carotid flow. Relaxant curves for angiotensin II or angiotensin-(1-7) were obtained in carotid from streptozotocin-induced diabetic rats. Superoxide or hydrogen peroxide levels were measured by flow cytometry in carotid endothelial cells. Carotid flow was also determined. We found that endothelial AT1-activated NAD(P)H oxidase-driven generation of superoxide and hydrogen peroxide in diabetic rat carotid impairs ACE2-angiotensin-(1-7)-Mas axis functionality, which reduces carotid flow. In this mechanism, hydrogen peroxide derived from superoxide dismutation inhibits ACE2 activity in generating angiotensin-(1-7) seemingly by activating I(Cl,SWELL0, while superoxide inhibits the nitrergic Mas-mediated vasorelaxation evoked by angiotensin-(1-7). Angiotensin-(1-7) treatment of diabetic rats restored carotid ACE2-angiotensin-(1-7)-Mas axis functionality by triggering a positive feedback played by endothelial Mas receptors, that blunts endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species. Mas-mediated antioxidant effects also restored diabetic rat carotid flow, pointing to the contribution of ACE2-angiotensin-(1-7)-Mas axis in maintaining carotid flow.

Endothelium-derived factors play an important role in vascular tone control. This study aimed to evaluate how endothelium and reactive oxygen species (ROS) contribute to phenylephrine (PE)-induced contraction in renovascular hypertensive (2K-1C) and normotensive (2K) rats aortas. The effects of the superoxide scavenger Tiron (0.1mM and 1mM) or catalase (30 U/ml, 90 U/ml, 150 U/ml and 300 U/ml) on the PE-induced contraction were evaluated in both intact endothelium (E+) and denuded (E-) aortas. Endothelium removal increased the PE-induced contractions. The maximum contractile response decreased only in 2K-1C rat E+ aorta, and catalase (30 U/ml, 90 U/ml, 150 U/ml) partially reversed this effect. Endothelium increased the basal hydrogen peroxide (H2O2) production in 2K and 2K-1C rats aortas. PE-stimulated H2O2 production was higher in 2K-1C (E+/E-) than in 2K (E+/E-). Inhibition of the enzymes cyclooxygenase, NADPH-oxidase, xanthine-oxidase, and superoxide dismutase reduced the PE-stimulated H2O2 production in 2K-1C rat aorta. The decreased contraction to PE in 2K-1C rat aorta is partially due to endothelial H2O2 production; however, in denuded aorta, it contributes to maintaining the contractile response. Superoxide plays an important role on the PE-induced contraction in 2K rat denuded aorta, whereas in 2K-1C rat aorta, it is H2O2 that plays an important role in this effect., (© 2013 Elsevier B.V. All rights reserved.)

Objectives: Our main objectives were to investigate the affinity properties of endothelial and muscular α1D -adrenoceptors and to characterize the cross-talk between endothelial α1D -adrenoceptors and β2 -adrenoceptors in rat carotid., Methods: Relaxation and contraction concentration-response curves for phenylephrine (α1 -adrenergic agonist) were obtained in carotid rings in absence or presence of increasing concentrations of BMY7378 (α1D -adrenergic antagonist), combined or not with increasing concentration of ICI-118,551 (β2 -adrenergic antagonist). Schild analysis was used to estimate the affinity constant from pA2 values of BMY7378., Key Findings: BMY7378 produced an unsurmountable antagonism on phenylephrine-induced relaxation but a surmountable antagonism on phenylephrine-induced contraction. BMY7378 potency was higher in inhibiting the relaxation than the contraction induced by phenylephrine because the rightward shifts induced by BMY7378 were greater in the relaxation. The apparent pA2 value for BMY7378 in phenylephrine-induced relaxation was greater than in contraction. When combined with ICI-118,551, BMY7378 yielded a surmountable antagonism on phenylephrine-induced relaxation and presented a pA2 value similar to that obtained in phenylephrine-induced contraction., Conclusions: Endothelial α1D -adrenoceptors, which mediates rat carotid relaxation, present high ligand affinity because of the cross-talk with β2 -adrenoceptors, which explains the higher potency of phenylephrine in inducing relaxation than contraction and the atypical unsurmountable antagonism produced by BMY7378 on phenylephrine-induced relaxation., (© 2013 Royal Pharmaceutical Society.)

The purpose from this study was to investigate the consequences of sensory neurocompensation to carotid balloon injury in diabetic rats on angiotensin II-induced contraction and basal blood flow in contralateral carotid. Concentration-response curves for angiotensin II and blood flow were obtained in contralateral carotid from non-treated or capsaicin-treated streptozotocin-induced diabetic rats that underwent carotid balloon injury. Diabetes increased angiotensin II-induced contraction and impaired the blood flow in non-operated rat carotid. In diabetic rats, balloon injury led to neointima formation, which reduced the blood flow in ipsilateral carotid. Carotid balloon injury in diabetic rats reduced angiotensin II-induced contraction and restored the blood flow in contralateral carotid when compared to diabetic non-operated rat carotid. Capsaicin inhibited the effects evoked by carotid balloon injury on diabetic rat contralateral carotid. Endothelium removal, PEG-catalase (hydrogen peroxide scavenger) or l-NPA (neuronal nitric oxide synthase, nNOS, inhibitor) increased angiotensin II-induced contraction in contralateral carotid from diabetic operated rats to the levels observed in diabetic non-operated rat carotid. Our findings suggest that carotid balloon injury in diabetic rats elicits a neurocompensation that attenuates the diabetic hyperreactivity to angiotensin II in contralateral carotid by a sensory nerves-dependent mechanism mediated by hydrogen peroxide derived from endothelial nNOS. This sensory mechanism also restored the blood flow in this vessel, compensating the impaired blood flow in diabetic rat ipsilateral carotid. Thus, our major conclusions are that Diabetes confers a vasoprotective significance to the neurocompensation to carotid balloon injury in preventing further damage at carotid cerebral irrigation after angioplasty in diabetic subjects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Endothelial dysfunction is the hallmark of hypertension, which is a multifactorial disorder. In the cardiovascular system reactive oxygen species play a pivotal role in controlling the endothelial function and vascular tone. Physiologically, the endothelium-derived relaxing factors (EDRFs) and endothelium-derived contractile factors (EDCFs) that have functions on the vascular smooth muscle cells. The relaxation induced by the EDRFs nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarization factor (EDHF) could be impaired in hypertension. The impaired ability of endothelial cells to release NO along with enhanced EDCFs production has been described to contribute to the endothelium dysfunction, which appears to lead to several cardiovascular diseases. The present review discusses the role of oxidative stress, vascular endothelium, and vascular tone control by EDRFs, mainly NO, and EDCFs in different models of experimental hypertension.

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P)H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT(1)-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT(1)-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P)H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT(1)-receptor expression and the reduced angiotensin II potency., (Copyright © 2011 Elsevier B.V. All rights reserved.)