Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.

Modified Qing' e Pills exerts anti-osteoporosis effects and prevents bone loss by enhancing type H blood vessel formation.

Objective: To explore whether the modified Qing' e Pills (MQEP) exerts anti-osteoporotic effects and prevents bone loss by enhancing angiogenesis. Methods: Network pharmacology was used to assess whether MQEP has a pro-angiogenic capacity and to predict its potential targets. Human umbilical vein endothelial cells were treated with glucocorticoids and MQEP to assess cell viability. The expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor, and angiotensin converting enzyme, which are associated with the activation of the renin-angiotensin-aldosterone system, and the expression of vascular endothelial growth factor and hypoxia-inducible factor 1 alpha, which are associated with the formation of type H blood vessels, were examined by western blot and RT-qPCR. Thereafter, the glucocorticoid-induced osteoporosis model was established and intervened with MQEP. Femur scanning was performed with micro-computed tomography; trabecular spacing, trabecular thickness, and trabecular number were observed and calculated; the expression of nuclear factor-kappa B ligand and osteoprotegerin was detected by ELISA, and the ratio was calculated to evaluate the degree of bone resorption. Finally, type H blood vessels that were highly coupled to osteogenic cells were identified by immunohistochemistry staining and flow cytometry. Results: This is the first study to reveal and confirm that MQEP could prevent bone loss in glucocorticoid-induced osteoporosis by promoting the expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor, which are highly associated with type H blood vessel formation. In vitro experiments confirmed that MQEP could effectively promote the proliferation of vascular endothelial cells and alleviate glucocorticoids-induced activation of the renin-angiotensin-aldosterone system, thereby reducing vascular injury. Conclusion: MQEP exerts anti-osteoporosis effects and prevents bone loss by alleviating vascular injury caused by renin-angiotensin-aldosterone system activation and promoting type H blood vessel formation.

C21 preserves endothelial function in the thoracic aorta from DIO mice: role for AT2, Mas and B2 receptors.

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

In silico Nigellidine (N. sativa) bind to viral spike/active-sites of ACE1/2, AT1/2 to prevent COVID-19 induced vaso-tumult/vascular-damage/comorbidity.

COVID-19, a global-pandemic binds human-lung-ACE2. ACE2 causes vasodilatation. ACE2 works in balance with ACE1. The vaso-status maintains blood-pressure/vascular-health which is demolished in Covid-19 manifesting aldosterone/salt-deregulations/inflammations/endothelial-dysfunctions/hyper-hypo- tension, sepsis/hypovolemic-shock and vessel-thrombosis/coagulations. Here, nigellidine, an indazole-alkaloid was analyzed by molecular-docking for binding to different Angiotensin-binding-proteins (enzymes, ACE1(6en5)/ACE2(4aph)/receptors, AT1(6os1)/AT2(5xjm)) and COVID-19 spike-glycoprotein(6vsb). Nigellidine strongly binds to the spike-protein at the hinge-region/active-site-opening which may hamper proper-binding of nCoV2-ACE2 surface. Nigellidine effectively binds in the Angiotensin- II binding-site/entry-pocket (-7.54 kcal/mol, -211.76, Atomic-Contact-Energy; ACE-value) of ACE2 (Ki 8.68 and 8.3 µmol) in comparison to known-binder EGCG (-4.53) and Theaflavin-di-gallate (-2.85). Nigellidine showed strong-binding (Ki, 50.93 µmol/binding-energy -5.48 kcal/mol) to mono/multi-meric ACE1. Moreover, it binds Angiotensin-receptors, AT1/AT2 (Ki, 42.79/14.22 µmol, binding-energy, -5.96/-6.61 kcal/mol) at active-sites, respectively. This article reports the novel binding of nigellidine and subsequent blockage of angiotensin-binding proteins. The ACEs-blocking could restore Angiotensin-level, restrict vaso-turbulence in Covid patients and receptor-blocking might stop inflammatory/vascular impairment. Nigellidine may slowdown the vaso-fluctuations due to Angiotensin-deregulations in Covid patients. Angiotensin II-ACE2 binding (ACE-value -294.81) is more favorable than nigellidine-ACE2. Conversely, nigellidine-ACE1 binding-energy/Ki is lower than nigellidine-ACE2 values indicating a balanced-state between constriction-dilatation. Moreover, nigellidine binds to the viral-spike, closer-proximity to its ACE2 binding-domain. Taken together, Covid patients/elderly-patients, comorbid-patients (with hypertensive/diabetic/cardiac/renal-impairment, counting >80% of non-survivors) could be greatly benefited.

Transforming Growth Factor-ß and the Renin-Angiotensin System in Syndromic Thoracic Aortic Aneurysms: Implications for Treatment.

Thoracic aortic aneurysms (TAAs) are permanent pathological dilatations of the thoracic aorta, which can lead to life-threatening complications, such as aortic dissection and rupture. TAAs frequently occur in a syndromic form in individuals with an underlying genetic predisposition, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). Increasing evidence supports an important role for transforming growth factor-ß (TGF-ß) and the renin-angiotensin system (RAS) in TAA pathology. Eventually, most patients with syndromic TAAs require surgical intervention, as the ability of present medical treatment to attenuate aneurysm growth is limited. Therefore, more effective medical treatment options are urgently needed. Numerous clinical trials investigated the therapeutic potential of angiotensin receptor blockers (ARBs) and ß-blockers in patients suffering from syndromic TAAs. This review highlights the contribution of TGF-ß signaling, RAS, and impaired mechanosensing abilities of aortic VSMCs in TAA formation. Furthermore, it critically discusses the most recent clinical evidence regarding the possible therapeutic benefit of ARBs and ß-blockers in syndromic TAA patients and provides future research perspectives and therapeutic implications.

The protective role of melatonin against the effects of different doses of caffeine on the fetus.

Skeletal system and some organs development changes in rat fetuses with 30 and 60 mg/kg caffeine and melatonin's (10 mg/kg) protective role against rat fetuses were investigated. Groups (n = 4) were formed as Control, LDC, HDC, LDC+melatonin, HDC+melatonin and melatonin. Fetuses were taken by cesarean section and stained using dual skeletal staining method and FESEM. TRAP and AP immune-reactivity concentrations were calculated.  Oxidative stress and inflammatory markers were also measured by liver, bone and placenta samples.  TNF-α, IL-1ß, IL-6, VEGF-A, SOST and Fetuin-A levels were measured in tissue by using ELISA. TBARS, SOD, GSH, GSSG, TOS, TAS, measured by spectrophotometric assay method.  The mRNA levels of Agtr2 gene expressed in placental tissues of control rats and in placental tissues of rats exposed to HDC, LDC, MEL, HDC+MEL, LDC+MEL were analyzed by Real-time PCR. The gene expressions of Agtr2 were significantly upregulated in the placentas exposed to HDC, MEL, HDC+MEL and LDC+MEL (P<0,001). No significant difference in samples of the LDC group (P>0,05). According to these data, caffeine used during pregnancy delayed ossification; melatonin, a powerful antioxidant, was found to eliminate this effect. Besides, changes in angiotensin receptor expression observed in response to a caffeine and melatonin exposure result from high dose and join effect.

TMAO, a seafood-derived molecule, produces diuresis and reduces mortality in heart failure rats.

Trimethylamine-oxide (TMAO) is present in seafood which is considered to be beneficial for health. Deep-water animals accumulate TMAO to protect proteins, such as lactate dehydrogenase (LDH), against hydrostatic pressure stress (HPS). We hypothesized that TMAO exerts beneficial effects on the circulatory system and protects cardiac LDH exposed to HPS produced by the contracting heart. Male, Sprague-Dawley and Spontaneously-Hypertensive-Heart-Failure (SHHF) rats were treated orally with either water (control) or TMAO. In vitro, LDH with or without TMAO was exposed to HPS and was evaluated using fluorescence correlation spectroscopy. TMAO-treated rats showed higher diuresis and natriuresis, lower arterial pressure and plasma NT-proBNP. Survival in SHHF-control was 66% vs 100% in SHHF-TMAO. In vitro, exposure of LDH to HPS with or without TMAO did not affect protein structure. In conclusion, TMAO reduced mortality in SHHF, which was associated with diuretic, natriuretic and hypotensive effects. HPS and TMAO did not affect LDH protein structure.

Heart failure is a common cause of death in industrialized countries with aging populations. Japan, however, has lower rates of heart failure and fewer deaths linked to this disease than the United States or Europe, despite having the highest proportion of elderly people in the world. Dietary differences between these regions may explain the lower rate of heart failure in Japan. The Japanese diet is rich in seafood, which contains nutrients that promote heart health, such as omega-3 fatty acids. Seafood also contains other compounds, including trimethylamine oxide (TMAO). Fish that live in deep waters undergo high pressures, which can damage their proteins, but TMAO seems to protect the proteins from harm. In humans, eating seafood increases TMAO levels in the blood and urine, but it is unclear what effects this has on heart health. Increased levels of TMAO in the blood are associated with cardiovascular diseases, but scientists are not sure whether TMAO itself harms the heart. A toxic byproduct of gut bacteria called TMA is converted in TMAO in the body, so it is possible that TMA rather than TMAO is to blame. To assess the effects of dietary TMAO on heart failure, Gawrys-Kopczynska et al. fed the compound to healthy rats and rats with heart failure for one year. TMAO had no effects on the healthy rats. Of the rats with heart failure that were fed TMAO, all of them survived the year, while one third of rats with heart failure that were not fed TMAO died. TMAO-treated rats with heart failure had lower blood pressure and urinated more than untreated rats with the condition. The experiments suggest that dietary TMAO may mimic the effects of heart failure treatments, which remove excess water and salt and lower pressure on the heart. More studies are needed to confirm whether TMAO has this same effect on humans.

AT2 receptor stimulation inhibits phosphate-induced vascular calcification.

Vascular calcification is a common finding in atherosclerosis and in patients with chronic kidney disease. The renin-angiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the hypothesis that angiotensin II type 2 receptor (AT2) stimulation has inhibitory effects on phosphate-induced vascular calcification. In vivo, calcification of the thoracic aorta induced by an adenine and high-phosphate diet was markedly attenuated in smooth muscle cell-specific AT2-overexpressing mice (smAT2-Tg) compared with wild-type and AT2-knockout mice (AT2KO). Similarly, mRNA levels of relevant osteogenic and vascular smooth muscle cell marker genes were unchanged in smAT2-Tg mice, while their expression was significantly altered in wild-type mice in response to high dietary phosphate. Ex vivo, sections of thoracic aorta were cultured in media supplemented with inorganic phosphate. Aortic rings from smAT2-Tg mice showed less vascular calcification compared with those from wild-type mice. In vitro, calcium deposition induced by high-phosphate media was markedly attenuated in primary vascular smooth muscle cells derived from smAT2-Tg mice compared with the two other mouse groups. To assess the underlying mechanism, we investigated the effect of PPAR-γ, which we previously reported as one of the possible downstream effectors of AT2 stimulation. Treatment with a PPAR-γ antagonist attenuated the inhibitory effects on vascular calcification observed in smAT2-Tg mice fed an adenine and high-phosphate diet. Our results suggest that AT2 activation represents an endogenous protective pathway against vascular calcification. Its stimulation may efficiently reduce adverse cardiovascular events in patients with chronic kidney disease.

Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system.

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

Qishenyiqi Dropping Pill attenuates myocardial fibrosis in rats by inhibiting RAAS-mediated arachidonic acid inflammation.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: In China, Qishenyiqi Dropping Pill (QSDP), a Chinese medicine formula containing Astragalus membranaceus (Fisch.) Bunge, Salvia miltiorrhiza Bunge, Panax notoginseng (Burkill) F.H.Chen and Dalbergia odorifera T.C.Chen, has been used frequently in traditional folk medicine for treatment of coronary heart diseases (CHD) and heart failure (HF). AIM OF STUDY: Previous study has shown that QSDP has definite therapeutic effects on promoting the heart function on CHD patients. The present study was designed to study the anti-fibrosis effects of QSDP on HF rats and to explore the underlying molecular mechanisms. MATERIALS AND METHODS: HF rat model was induced by left anterior descending (LAD) coronary artery ligation. Two-dimensional (2D) echocardiography was adopted to evaluate heart functions. Immunohistochemical (IHC) method and Western-blot were used to detect expression of critical proteins in renin-angiotensin-aldosterone system (RAAS) or arachidonic acid (AA) metabolic pathway. RESULTS: Heart functions were seriously injured in the model group. Expressions of fibrotic markers, such as collagen Ⅰ, collagen Ⅲ, matrix metallopeptidase 2 (MMP2) and MMP9 were elevated in the model group. RAAS pathway was activated. Interestingly, AA pathway was also up-regulated in the model group and it was down-regulated by angiotensin converting enzyme inhibitors (ACEIs) drug Captopril. Expressions of the important signal-transuding proteins, including NF-κB, JAK1/STAT3 and Akt, all increased remarkably in the model group. Treatment with QSDP could attenuate myocardial fibrosis by inhibiting RAAS-activated pathway, as indicated by decreased angiotensin type 1 receptor (AT1) and increased AT2 expression. Expressions of phospholipase A2 (PLA2), cyclooxygenase 1 (COX1) and COX2 were also down-regulated in the QSDP-treated group. In addition, "therapeutic" QSDP administration seemed to down-regulate expressions of NF-κB, JAK1/ STAT3 and Akt which may play important roles in myocardial fibrosis. CONCLUSION: QSDP can exert anti-fibrosis effect by down-regulating RAAS pathway, and subsequently inhibiting expressions of proteins in AA pathway.

Angiotensin AT2 receptor stimulation is anti-inflammatory in lipopolysaccharide-activated THP-1 macrophages via increased interleukin-10 production.

Macrophages have an important role in the pathogenesis of hypertension and associated end-organ damage via the activation of the Toll-like receptors, such as Toll-like receptor-4 (TLR4). Accumulating evidence suggests that the angiotensin AT2 receptor (AT2R) has a protective role in pathological conditions involving inflammation and tissue injury. We have recently shown that AT(2)R stimulation is renoprotective, which occurs in part via increased levels of anti-inflammatory interleukin-10 (IL-10) production in renal epithelial cells; however, the role of AT(2)R in the inflammatory activity of macrophages is not known. The present study was designed to investigate whether AT(2)R activation stimulates an anti-inflammatory response in TLR4-induced inflammation. The effects of the anti-inflammatory mechanisms that occurred following pre-treatment with the AT(2)R agonist Compound 21 (C21) (1 µmol ml(-1)) on the cytokine profiles of THP-1 macrophages after activation by lipopolysaccharide (LPS) (1 µg ml(-1)) were studied. The AT(2)R agonist dose-dependently attenuated LPS-induced tumor necrosis factor-α (TNF-α) and IL-6 production but increased IL-10 production. IL-10 was critical for the anti-inflammatory effects of AT(2)R stimulation because the IL-10-neutralizing antibody dose-dependently abolished the AT(2)R-mediated decrease in TNF-α levels. Further, enhanced IL-10 levels were associated with a sustained, selective increase in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) but not p38 mitogen-activated protein kinase (MAPK). Blocking the activation of ERK1/2 before C21 pre-treatment completely abrogated this increased IL-10 production in response to the AT(2)R agonist C21, while there was a partial reduction in IL-10 levels following the inhibition of p38. We conclude that AT(2)R stimulation exerts a novel anti-inflammatory response in THP-1 macrophages via enhanced IL-10 production as a result of sustained, selective ERK1/2 phosphorylation, which may have protective roles in hypertension and associated tissue injury.

Venthamarai chooranam, a polyherbal Siddha medicine, alleviates hypertension via AT1R and eNOS signaling pathway in 2K1C hypertensive rats.

The present study was aimed to scientifically demonstrate the anti-hypertensive action of Venthamarai chooranam (VMC) in renal hypertensive rats. Two Kidney One Clip (2K1C) Goldblatt model was adopted to induce hypertension in rats. Male Sprague Dawley rats (270-320 g) were randomized into sham (n = 6), vehicle-treated 2K1C (n = 9) and VMC-treated 2K1C (400 mg/kg, p.o; n = 8) and monitored for nine weeks. Systolic blood pressure (SBP), plasma nitrate/nitrite, carotid endothelial nitric oxide synthetase (eNOS), renal angiotensin type 1 receptor (AT1R), angiotensin type 2 receptor (AT2R), TNFα, IL-6, thioredoxin 1 (TRX1), and thioredoxin reductase 1 (TRXR1) mRNA expressions were studied. VMC upregulated eNOS expression which in turn improved plasma nitric oxide and decreased SBP in hypertensive rats. It down-regulated AT1R and simultaneously upregulated AT2R expression in comparison to vehicle-treated 2K1C rats. Further, renal TNFα and IL-6 expressions were down-regulated while TRX1 and TRXR1 were upregulated by VMC. VMC potentially interacts with renin-angiotensin components and endothelial functions, and thereby exerts its antihypertensive action. This is the first study to demonstrate the mechanism of anti-hypertensive action of VMC in an animal model of renovascular hypertension.

Synthesis and evaluation of isoleucine derived angiotensin II AT(2) receptor ligands.

Sixteen new C-terminally modified analogues of 2, a previously described potent and selective AT2R ligand, were designed, synthesized and evaluated for their affinity to the AT2R receptor. The introduction of large, hydrophobic substituents was shown to be beneficial and the most active compound (17, Ki=8.5 µM) was over 12-times more potent than the lead compound 2.

Mesenchymal stem cells (MSC) prevented the progression of renovascular hypertension, improved renal function and architecture.

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×10(5) cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1ß, TNF-α angiotensinogen, ACE, and Ang II receptor AT1 were elevated, whereas AT2 levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant-induced arthritis.

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant-induced arthritis (AIA) by losartan. Adjuvant-induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor-α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin-1ß in vitro, the effects of the AT2R agonist CGP42112 (10(-8) -10(-5)  M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of CGP42112 (5, 10 and 20 µg/kg) were evaluated in vivo by intra-articular injection in AIA rats. After treatment with losartan, the down-regulation of AT1R expression and up-regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up-regulation of AT2R expression. Intra-articular injection with CGP42112 (10 and 20 µg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up-regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.

Central insulin-angiotensin II interaction in blood pressure regulation in fructose overloaded rats.

The aim of the present study was to determine if insulin is able to modulate the pressor response to intracerebroventricularly administered angiotensin II in insulin resistant fructose overloaded rats. Male Sprague-Dawley rats were divided into two groups: 1) Control group (C) with tap water to drink for 6 weeks (n=36); and 2) fructose treated (F), with fructose solution (10% w/v) to drink for 6 weeks (n=36). On the day of the experiment, anesthetized male C and F rats were intracerebroventricularly infused with insulin (12 mU/h, n=15) or Ringer's solution as vehicle (n=15) for 2h. Immediately, changes in mean arterial pressure (MAP) in response to an intracerebroventricular subpressor dose of angiotensin II (5 pmol, n=10) or vehicle (n=5) were measured for 10 min. Then, hypothalami were removed and Akt and ERK1/2 phosphorylation levels were determined. In a subset of C (n=10) and F (n=20) animals, PD98059 (p44/42 MAPK inhibitor) or vehicle was administered intracerebroventricularly at a flow rate of 5 µl/min for 1 min. Ten minutes later, insulin (12 mU/h, n=5 for each group) or vehicle (Ringer's solution, only in the F group, n=5) was perfused for 2h at a flow rate of 4 µl/h, and cardiovascular parameters were measured every 15 min. Immediately, changes in MAP and HR in response to a subpressor dose of Ang II (5 pmol/2 µl) were evaluated for 10 min (n=5 for each group). In other subset of animals (n=6 for each group), AT1 and AT2 hypothalamic receptor levels were measured by Western blotting. Intracerebroventricular insulin pre-treatment increased the pressor response to angiotensin II in C rats. In F rats (with or without insulin pretreatment), the pressor response to angiotensin II was higher than that in vehicle pre-treated C animals, but similar to that observed in C after insulin infusion. In C rats phospho-ERK 1/2 hypothalamic levels significantly increased after angiotensin II injection in insulin pretreated animals compared to vehicle pre-treated rats, suggesting that MAPK activation might be involved in insulin potentiation of blood pressure response to angiotensin II in the brain. Phospho-ERK 1/2 hypothalamic levels were significantly increased in vehicle treated F rats compared to C, suggesting that basal MAPK activation might play a role in the enhanced response to angiotensin II observed in these animals. Finally, in F rats, either after vehicle or insulin infusion, angiotensin II injection was associated with a similar increase in phospho-ERK 1/2 hypothalamic levels, comparable to that observed after angiotensin II injection in insulin pre-treated C animals. ERK 1/2 blockade significantly reduced MAP in F rats compared to C. Moreover, ERK 1/2 inhibition completely abolished the Ang II pressor response in F rats and in insulin pre-treated C animals. All these findings suggest that insulin-angiotensin II interaction at hypothalamic level might be involved in the increase in blood pressure observed in the insulin resistant state.

Early changes of hypothalamic angiotensin II receptors expression in gestational protein-restricted offspring: effect on water intake, blood pressure and renal sodium handling.

The current study examines changes in the postnatal hypothalamic angiotensin receptors by maternal protein restriction (LP), and its impact on in uteri programming of hypertension in adult life. The data show that LP male pup body weight was significantly reduced when compared to that of control (NP) pups. Also, immunoblotting analysis demonstrated a significantly decreased expression of type 1 AngII receptors (AT1R) in the entire hypothalamic tissue extract of LP rats at 12 days of age compared to age-matched NP offspring. Conversely, the expression of the type 2 AngII (AT2R) receptors in 12-day- and 16-week-old LP hypothalamus was significantly increased. The current data show the influence of central AngII administration on water consumption in a concentration-dependent fashion, but also demonstrate that the water intake response to AngII was strikingly attenuated in 16-week-old LP. These results may be related to decreased brain arginine vasopressin (AVP) expression appearing in maternal protein-restricted offspring. The present investigation shows an early decrease in fractional urinary sodium excretion in maternal protein-restricted offspring. The decreased fractional sodium excretion was accompanied by a fall in proximal sodium excretion and occurred despite unchanged creatinine clearance. These effects were associated with a significant enhancement in arterial blood pressure in the LP group, but the precise mechanism of these phenomena remains unknown.

Oral administration of an angiotensin-converting enzyme 2 activator ameliorates diabetes-induced cardiac dysfunction.

We evaluated the hypothesis that activation of endogenous angiotensin-converting enzyme (ACE) 2 would improve cardiac dysfunction induced by diabetes. Ten days after diabetes induction (streptozotocin, 50 mg/kg, i.v.), male Wistar rats were treated with the ACE2 activator 1-[[2-(dimethylamino)ethyl]amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl)sulfonyl]oxy]-9H-xanthen-9-one (XNT, 1 mg/kg/day, gavage) or saline (control) for 30 days. Echocardiography was performed to analyze the cardiac function and kinetic fluorogenic assays were used to determine cardiac ACE and ACE2 activities. Cardiac ACE2, ACE, Mas receptor, AT(1) receptor, AT(2) receptor and collagen types I and III mRNA and ACE2, ACE, Mas, AT(1) receptor, AT(2) receptor, ERK1/2, Akt, AMPK-α and AMPK-ß(1) protein were measured by qRT-PCR and western blotting techniques, respectively. Histological sections of hearts were analyzed to evaluate the presence of hypertrophy and fibrosis. Diabetic animals presented hyperglycemia and diastolic dysfunction along with cardiac hypertrophy and fibrosis. XNT treatment prevented further increase in glycemia and improved the cardiac function, as well as the hypertrophy and fibrosis. These effects were associated with increases in cardiac ACE2/ACE ratios (activity: ~26%; mRNA: ~113%; and protein: ~188%) and with a decrease in AT(1) receptor expression. Additionally, XNT inhibited ERK1/2 phosphorylation and prevented changes in AMPK-α and AMPK-ß(1) expressions. XNT treatment did not induce any significant change in AT(2) receptor and Akt expression. These results indicate that activation of intrinsic cardiac ACE2 by oral XNT treatment protects the heart against diabetes-induced dysfunction through mechanisms involving ACE, ACE2, ERK1/2, AMPK-α and AMPK-ß(1) modulations.

Candesartan restored cardiac Hsp72 expression and tolerance against reperfusion injury in hereditary insulin-resistant rats.

AIMS: We tested the hypothesis that candesartan, an angiotensin II (AII) type 1 receptor antagonist, would restore the depressed phosphatidylinositol 3 (PI3) kinase-dependent Akt phosphorylation, an essential signal to induce heat-shock protein 72 (Hsp72) in response to hyperthermia, in Otsuka Long-Evans Tokushima fatty (OLETF) rats. METHODS AND RESULTS: At 14 weeks of age, male OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats were treated with candesartan (0.25 mg/kg/day) for 2 weeks. Thereafter, hyperthermia (43°C for 20 min) was applied. We observed the following: (i) Candesartan did not improve insulin sensitivity in OLETF rats. (ii) Candesartan restored depressed PI3 kinase-dependent Akt phosphorylation and Hsp72 expression in OLETF rat hearts. (iii) Cardiac ventricular tissue contents of AII were greater in OLETF rats, which were suppressed by candesartan. (iv) Cardiac levels of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphorylation were greater in OLETF rats, which were suppressed by candesartan. In cultured cardiomyocytes, application of AII induced PTEN phosphorylation, which was suppressed by candesartan. (v) In high-fat diet insulin-resistant rats, similar results were observed with respect to Hsp72 expression, Akt phosphorylation and PTEN phosphorylation. (vi) In isolated, perfused heart experiments, reperfusion-induced cardiac functional recovery was suppressed in OLETF rat hearts, which was improved by candesartan. CONCLUSION: Our results suggest that the depression of PI3 kinase-dependent Akt activation in response to hyperthermia in OLETF rats can be restored by candesartan. Substantial activation of the renin-angiotensin system, represented by increased myocardial AII content and subsequent PTEN phosphorylation, may underlie the pathogenesis which is ameliorated by candesartan.