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Accumulating evidence indicates that dietary phenolic compounds can prevent obesity-related disorders. We investigated whether the consumption of polyphenol-rich jabuticaba peel and seed powder (JPSP) could ameliorate the progression of diet-induced obesity in mice. Male mice were fed a control diet or a high-fat (HF) diet for 9 weeks. After this period, mice were fed control, HF or HF diets supplemented with 5 % (HF-J5), 10 % (HF-J10) or 15 % (HF-J15) of JPSP, for 4 additional weeks. Supplementation with JPSP not only attenuated HF-induced weight gain and fat accumulation but also ameliorated the pro-inflammatory response associated with obesity, as evidenced by the absence of mast cells in the visceral depot accompanied by lower IL-6 and TNF-α at the tissue and circulating levels. JPSP-supplemented mice also exhibited smaller-sized adipocytes, reduced levels of leptin and higher levels of adiponectin, concomitant with improved glucose metabolism and insulin sensitivity. The magnitude of the observed effects was dependent on JPSP concentration with HF-J10- and HF-J15-fed mice showing metabolic profiles similar to control. This study reveals that the consumption of JPSP protects against the dysfunction of the adipose tissue and metabolic disturbances in obese mice. Thus, these findings indicate the therapeutic potential of the phenolic-rich JPSP in preventing obesity-related disorders.

Adipose tissue depots are invested with an extensive capillary network that is closely associated with maintenance of adipose functions and enables healthy tissue expansion. The capillary network displays a high level of plasticity, demonstrating either growth (angiogenesis) or regression (rarefaction) under various physiological/pathological conditions, which has significant consequences for cardiometabolic health. Thus, the visualization and quantification of adipose vascular networks is an important aspect of studying factors that regulate adipose tissue health. This chapter provides an overview of several methods to quantify adipose vascularization. In-depth protocols are provided for the visualization of vascular structures by staining and imaging of whole-mount adipose tissues or paraffin-embedded adipose tissue sections, together with the quantitative analysis of vascularization from these images.

Throughout the global coronavirus pandemic, we have seen an unprecedented volume of COVID-19 researchpublications. This vast body of evidence continues to grow, making it difficult for research users to keep up with the pace of evolving research findings. To enable the synthesis of this evidence for timely use by researchers, policymakers, and other stakeholders, we developed an automated workflow to collect, categorise, and visualise the evidence from primary COVID-19 research studies. We trained a crowd of volunteer reviewers to annotate studies by relevance to COVID-19, study objectives, and methodological approaches. Using these human decisions, we are training machine learning classifiers and applying text-mining tools to continually categorise the findings and evaluate the quality of COVID-19 evidence.

ObjectiveTo assess the effectiveness and safety of different preparations and doses of non-steroidal anti-inflammatory drugs (NSAIDs), opioids, and paracetamol for knee and hip osteoarthritis pain and physical function to enable effective and safe use of these drugs at their lowest possible dose.DesignSystematic review and network meta-analysis of randomised trials.Data sourcesCochrane Central Register of Controlled Trials (CENTRAL), Medline, Embase, regulatory agency websites, and ClinicalTrials.gov from inception to 28 June 2021.Eligibility criteria for selecting studiesRandomised trials published in English with ≥100 patients per group that evaluated NSAIDs, opioids, or paracetamol (acetaminophen) to treat osteoarthritis.Outcomes and measuresThe prespecified primary outcome was pain. Physical function and safety outcomes were also assessed.Review methodsTwo reviewers independently extracted outcomes data and evaluated the risk of bias of included trials. Bayesian random effects models were used for network meta-analysis of all analyses. Effect estimates are comparisons between active treatments and oral placebo.Results192 trials comprising 102 829 participants examined 90 different active preparations or doses (68 for NSAIDs, 19 for opioids, and three for paracetamol). Five oral preparations (diclofenac 150 mg/day, etoricoxib 60 and 90 mg/day, and rofecoxib 25 and 50 mg/day) had ≥99% probability of more pronounced treatment effects than the minimal clinically relevant reduction in pain. Topical diclofenac (70-81 and 140-160 mg/day) had ≥92.3% probability, and all opioids had ≤53% probability of more pronounced treatment effects than the minimal clinically relevant reduction in pain. 18.5%, 0%, and 83.3% of the oral NSAIDs, topical NSAIDs, and opioids, respectively, had an increased risk of dropouts due to adverse events. 29.8%, 0%, and 89.5% of oral NSAIDs, topical NSAIDs, and opioids, respectively, had an increased risk of any adverse event. Oxymorphone 80 mg/day had the highest risk of dropouts due to adverse events (51%) and any adverse event (88%).ConclusionsEtoricoxib 60 mg/day and diclofenac 150 mg/day seem to be the most effective oral NSAIDs for pain and function in patients with osteoarthritis. However, these treatments are probably not appropriate for patients with comorbidities or for long term use because of the slight increase in the risk of adverse events. Additionally, an increased risk of dropping out due to adverse events was found for diclofenac 150 mg/day. Topical diclofenac 70-81 mg/day seems to be effective and generally safer because of reduced systemic exposure and lower dose, and should be considered as first line pharmacological treatment for knee osteoarthritis. The clinical benefit of opioid treatment, regardless of preparation or dose, does not outweigh the harm it might cause in patients with osteoarthritis.Systematic review registrationPROSPERO number CRD42020213656

Aim Critical limb ischaemia (CLI) is characterized by inadequate angiogenesis, arteriolar remodelling and chronic myopathy, which are most severe in type 2 diabetic patients. Hypertriglyceridaemia, commonly observed in these patients, compromises macrovascular function. However, the effects of high-fat diet-induced increases in circulating lipids on microvascular remodelling are not established. Here, we investigated if high-fat diet would mimic the detrimental effect of type 2 diabetes on post-ischaemia vascular remodelling and muscle regeneration, using a mouse model of hindlimb ischaemia. Methods Male C57Bl6/J mice were fed with normal or high-fat diets for 8 weeks prior to unilateral femoral artery ligation. Laser doppler imaging was used to assess limb perfusion recovery. Vascular recovery, inflammation, myofibre regeneration and fibrosis were assessed at 4 or 14 days post-ligation by histology and RNA analyses. Capillary-level haemodynamics were assessed by intravital microscopy of control and regenerating muscles 14 days post-ligation. Results High-fat diet increased muscle succinate dehydrogenase activity and capillary-level oxygen supply. At 4 days post-ligation, no diet differences were detected in muscle damage, inflammatory infiltration or capillary activation. At 14 days post-ligation, high fat-fed mice displayed accelerated limb blood flow recovery, elevated capillary and arteriole densities as well as greater red blood cell supply rates and capillary-level oxygen supply. Regenerating muscles from high fat-fed mice displayed lower interstitial fat and collagen deposition. Conclusion The muscle-level adaptations to high-fat diet improved multiple aspects of muscle recovery in response to ischaemia and did not recapitulate the worse outcomes seen in diabetic CLI patients.

Impaired angiogenesis is a hallmark of metabolically dysfunctional adipose tissue in obesity. However, the underlying mechanisms restricting angiogenesis within this context remain ill-defined. Here, we demonstrate that induced endothelial-specific depletion of the transcription factor Forkhead Box O1 (FoxO1) in male mice led to increased vascular density in adipose tissue. Upon high-fat diet feeding, endothelial cell FoxO1-deficient mice exhibited even greater vascular remodeling in the visceral adipose depot, which was paralleled with a healthier adipose tissue expansion, higher glucose tolerance and lower fasting glycemia concomitant with enhanced lactate levels. Mechanistically, FoxO1 depletion increased endothelial proliferative and glycolytic capacities by upregulating the expression of glycolytic markers, which may account for the improvements at the tissue level ultimately impacting whole-body glucose metabolism. Altogether, these findings reveal the pivotal role of FoxO1 in controlling endothelial metabolic and angiogenic adaptations in response to high-fat diet and a contribution of the endothelium to whole-body energy homeostasis., eLife digest In the body, thread-like blood vessels called capillaries weave their way through our tissues to deliver oxygen and nutrients to every cell. When a tissue becomes bigger, existing vessels remodel to create new capillaries that can reach far away cells. However, in obesity, this process does not happen the way it should: when fat tissues expand, new blood vessels do not always grow to match. The starved fat cells can start to dysfunction, which causes a range of issues, from inflammation and scarring of the tissues to problems with how the body processes sugar and even diabetes. Yet, it is still unclear why exactly new capillaries fail to form in obesity. What we know is that a protein called FoxO (short for Forkhead box O) is present in the cells that line the inside of blood vessels, and that it can stop the development of new capillaries. FoxO controls how cells spend their energy, and it can force them to go into a resting state. During obesity, the levels of FoxO actually increase in capillary cells. Therefore, it may be possible that FoxO prevents new blood vessels from growing in the fat tissues of obese individuals. To find out, Rudnicki et al. created mice that lack the FoxO protein in the cells lining the capillaries, and then fed the animals a high-fat diet. These mutant mice had more blood vessels in their fat tissue, and their fat cells looked healthier. They also stored less fat than normal mice on the same diet, and their blood sugar levels were normal. This was because the FoxO-deprived cells inside capillaries were burning more energy, which they may have obtained by pulling sugar from the blood. These results show that targeting the cells that line capillaries helps new blood vessels to grow, and that this could mitigate the health problems that arise with obesity, such as high levels of sugar (diabetes) and fat in the blood. However, more work is needed to confirm that the same cellular processes can be targeted to obtain positive health outcomes in humans.

Lípides nitrados (NO2-FA) são apontados como uma nova classe de mediadores lipídicos, podendo atuar como reservatórios endógenos de óxido nítrico (&#8226NO) bem como moduladores pluripotentes de sinalização celular. Recentemente, tem sido sugerido que os doadores de &#8226NO estariam envolvidos na regulação da angiogênese. Evidências contundentes indicam ainda que o processo de neovascularização poderia contribuir para a patogênese de uma serie de condições clínicas, entre elas a aterosclerose. Contudo, apesar de diversos estudos terem explorado os efeitos biológicos dos NO2-FA, os efeitos destes compostos sobre o processo de angiogênese não haviam sido descritos. Dessa maneira, o presente trabalho investigou os efeitos dos NO2-FA (derivados da nitração do ácido linoléico e oléico) noprocesso de angiogênese. Demonstrou-se que os NO2-FA podem atuar como mediadores pró-angiogênicos. Este efeito foi caracterizado em células endoteliais humanas, assim como, em modelos ex vivo e in vivo. Nas células endoteliais, observou-se que os No2-FA não influenciaram a proliferação ou a viabilidade celular, ao passo que estimularam a migração. Demonstrou-se também que os NO2-FA podem modular o brotamento ex vivo de novos vasos, em cultura de anéis de aorta de rato, bem como o processo angiogênico in vivo observado na membrana corioalantóica de embrião de galinha. Adicionalmente, os NO2-FA induziram a expressão do fator de crescimento endotelial vascular (VEGF), que é o principal mediador do processo de angiogênese. Em relação ao mecanismo de ação, os achados sugerem que os efeitos demonstrados seriam via mecanismos dependentes de &#8226NO, uma vez que foram abolidos na presença de um seqüestrador de &#8226NO, enquanto concentrações equivalentes dos lípides precursores não demonstraram qualquer influência nas condições experimentais utilizadas neste estudo. Por fim, os efeitos pró-angiogênicos dos NO2-FA foram mediados pela estabilização da proteína do fator induzível por hipóxia -1α (HIF-1α), uma vez que estes compostos promoveram acúmulo desta proteína e falharam em demonstrar efeitos indutores em células knockdown para o gene HIF-1α. Em conjunto, estes resultados indicam que os NO2-FA podem modular a migração de células endoteliais e estimular o processo de angiogênese resultante da ativação de HIF-1a via mecanismo dependente de &#8226NO. Nitrated lipids (NO2-FA) are described as a new class of Iipid mediators that are able to act as endogenously nitric oxide (&#8226NO) reservoirs as well as pluripotent cell signaling modulators. Furthermore, recent findings suggest that &#8226NO donors could be involved in the regulation of angiogenesis. Compelling evidence also indicate that the neovascularization process might contribute to the pathogenesis of many clinical conditions, such as atherosclerosis. However, although several studies have explored the NO2-FA biological properties, the effects of these compounds on the angiogenic process remain unknown. Hence, the present study investigated the effects of the NO2-FA (derivates from the nitration of Iinoleic and oleic acids at physiological concentrations) on angiogenesis processo It is demonstrated that the No2-FA could act as pro-angiogenic mediators. This effect was observed not only in human endothelial cells but also in ex vivo and in vivo models. Using endothelial cells, it is showed that NO2-FA failed to affect cell proliferation ar influence cellular viability, but significantly stimulated cell migration. It was also found that the NO2-FA might modulate the ex vivo sprouting of new vessels as well as the in vivo angiogenic process, while inducing the expression of the vascular endothelial growth factor, the main mediator of angiogenesis. The data are consistent with the hypothesis that the observed effects mediated by NO-dependent mechanisms, since the presence of a &#8226NO scavenger abrogated the induced effects, whereas equimolar concentrations of its precursors, showed no effect on angiogenesis under our experimental conditions. Finally, the pro-angiogenic effects of NOrFA were mediated by the stabilization of the hypoxia inducible factor-1α (HIF-1α) protein, because these compounds increased the protein amount and failed to show inductive effects in HIF-1α knockdown cells. Taken together, these findings indicated that NO2-FA might modulate the endothelial cell migration and stimulate the process of angiogenesis by the HIF-1α induction through a &#8226NO-dependent mechanism.

Background: Impaired capillary growth (angiogenesis) in skeletal muscle and adipose tissue contributes to the development of metabolic disorders in obese males. This association remains unexplored in females, despite mounting evidence that endothelial cells have sex-specific transcriptional profiles. Therefore, herein we assessed whether males and females show distinct angiogenic capacities in response to diet-induced obesity. Methods: Age-matched male and female mice were fed normal chow or high-fat obesogenic diets for 16 weeks. At the end of diet period, systemic glucose disposal was assessed as well as insulin sensitivity of skeletal muscle and visceral adipose tissue. Capillary content and the expression of angiogenic regulators were also evaluated in these tissues. Results: When placed on a high-fat diet, female mice gained less weight than males and showed a metabolic phenotype similar to NC-fed mice, contrasting with the impaired whole-body glucose metabolism observed in high-fat-fed males. However, high-fat-feeding elevated serum lipid levels similarly in male and female mice. Although skeletal muscle of high-fat-fed female mice had higher insulin sensitivity than male counterparts, no sex difference was detected in muscle capillarization. Metabolic functions of perigonadal white adipose tissue (pgWAT) were retained in high-fat-fed females, as evidenced by smaller adipocytes with preserved insulin sensitivity, greater responsiveness to isoproterenol, higher expression of Adiponectin and a lower ratio of Leptin:Adiponectin mRNA. An enhanced browning phenotype was detected in HF-fed female adipocytes with upregulation of Ucp1 expression. PgWAT from high-fat-fed females also showed augmented capillary number and expression of endothelial cell markers, which was associated with elevated mRNA levels of pro-angiogenic mediators, including vascular endothelial growth factor A (Vegfa) and its receptor (Vegfr2), the Notch ligand Jagged-1 (Jag1) and Angiopoietin-2 (Angpt2). Conclusion: Taken together, our findings provide novel evidence that visceral adipose tissue of female mice display greater levels of pro-angiogenic factors and vascularity than males in response to high-fat diet. This phenotype is associated with preserved metabolic homeostasis at both tissue and systemic levels. Our study discloses that a thus-far-unappreciated sex-specific difference in the regulation of adipose angiogenesis may contribute to an individual's susceptibility to developing adipose dysfunction and obesity-related metabolic disturbances.

[Background]: Obesity and insulin resistance/diabetes are important risk factors for cardiovascular diseases and demand safe and efficacious therapeutics., [Objective]: To assess the effects of a new thiazolidine compound—GQ-11—on obesity and insulin resistance induced by a diabetogenic diet in LDL receptor-deficient (LDLr−/−) mice, [Methods]: Molecular docking simulations of GQ-11, PPARα and PPARγ structures were performed. Male C57BL/6J LDLr−/− mice fed a diabetogenic diet for 24 weeks were treated with vehicle, GQ-11 or pioglitazone or (20 mg/kg/day) for 28 days by oral gavage. Glucose tolerance test, insulin, HOMA-IR, adipokines (leptin, adiponectin) and the lipid profile were assessed after treatment. Adipose tissue was analysed by X-ray analysis and morphometry; gene and protein expression were evaluated by real-time PCR and western blot, respectively., [Results]: GQ-11 showed partial agonism to PPARγ and PPARα. In vivo, treatment with GQ-11 ameliorated insulin sensitivity and did not modify subcutaneous adipose tissue and body weight gain. In addition, GQ-11 restored adipokine imbalance induced by a diabetogenic diet and enhanced Glut-4 expression in the adipose tissue. Improved insulin sensitivity was also associated with lower levels of MCP-1 and higher levels of IL-10. Furthermore, GQ-11 reduced triglycerides and VLDL cholesterol and increased HDL-cholesterol by upregulation of Apoa1 and Abca1 gene expression in the liver., [Conclusion]: GQ-11 is a partial/dual PPARα/γ agonist that demonstrates anti-diabetic effects. Additionally, it improves the lipid profile and ameliorates chronic inflammation associated with obesity in atherosclerosis-prone mice.

Background Atherosclerosis is a complex disorder with a multifactorial pathogenesis. We previously indicated that the new TZD LPSF/GQ-02 inhibits hepatic steatosis and inflammation, which are reported as risk factors for atherosclerosis development. Here, we explored the effects of LPSF/GQ-02 on atherosclerosis in LDLr−/− mice comparing two treatment periods. Methods and results LDLr −/− mice were fed a high-fat diet for 10 and 12 weeks and received oral treatment with LPSF/GQ-02 (30 mg/kg/day) or pioglitazone (20 mg/kg/day) for 15 and 30 days, respectively. Both treatment protocols with LPSF/GQ-02 resulted in lower collagen density in the atherosclerotic lesions. In addition, the treatment for 15 days also decreased mRNA levels of CD40, MCP-1, ABCG1 and upregulated PPARα, whereas the 30-days treatment reduced the protein levels of LOX-1, p-IκBα and p-NFκB. Conclusion This study provides evidence that LPSF/GQ-02 affects the composition and growth of atherosclerotic lesions in LDLr −/− mice. Moreover, our data also support previous findings showing anti-inflammatory properties of LPSF/GQ-02 and reinforce the therapeutic potential of this TZD for treating atherosclerosis and inflammation-related disorders.

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.

Angiotensinogen (AGT) gene polymorphisms have been linked to increased risk of hypertension, but the data remain controversial. In this study we review the most commonly investigated polymorphisms at the AGT locus (other than M235T) and provide summary estimates regarding their association with essential hypertension, while addressing heterogeneity, as well as publication biases. Data on 26 818 subjects from 46 studies for the 4 most-studied AGT variants (T174M in exon 2 and 3 promoter variants: A-6G, A-20C, and G-217A) were meta-analyzed. Statistically significant associations with hypertension were identified for the T174M (odds ratio [OR]: 1.19; 95% CI: 1.07 to 1.33; P =0.002) and G-217A (OR: 1.37; 95% CI: 1.17 to 1.59; P =0.00006) polymorphisms. A dual but consistent effect was observed for the −20C allele, which was associated with a decreased risk of hypertension in populations of mixed and European ancestries (OR: 0.64; 95% CI: 0.44 to 0.92; P =0.02 and OR: 0.77; 95% CI: 0.65 to 0.91; P =0.003, respectively), but with a 24% increase in the odds of hypertension in Asian subjects (OR: 1.24; 95% CI: 1.04 to 1.48; P =0.02). No association of the A-6G variant with hypertension was detected. Current studies support the notion that single variants at the AGT might modulate the risk of hypertension but indicate caution in interpreting these results because of the putative presence of publication bias and gene-environment interactions.

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with 111InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates multiple pathways involved in the pathogenesis of obesity and atherosclerosis. Here, we evaluated the therapeutic potential of GQ-177, a new thiazolidinedione, on diet-induced obesity and atherosclerosis. The intermolecular interaction between PPARγ and GQ-177 was examined by virtual docking and PPAR activation was determined by reporter gene assay identifying GQ-177 as a partial and selective PPARγ agonist. For the evaluation of biological activity of GQ-177, low-density lipoprotein receptor-deficient (LDLr) C57/BL6 mice were fed either a high fat diabetogenic diet (diet-induced obesity), or a high fat atherogenic diet, and treated with vehicle, GQ-177 (20 mg/kg/day), pioglitazone (20 mg/kg/day, diet-induced obesity model) or rosiglitazone (15 mg/kg/day, atherosclerosis model) for 28 days. In diet-induced obesity mice, GQ-177 improved insulin sensitivity and lipid profile, increased plasma adiponectin and GLUT4 mRNA in adipose tissue, without affecting body weight, food consumption, fat accumulation and bone density. Moreover, GQ-177 enhanced hepatic mRNA levels of proteins involved in lipid metabolism. In the atherosclerosis mice, GQ-177 inhibited atherosclerotic lesion progression, increased plasma HDL and mRNA levels of PPARγ and ATP-binding cassette A1 in atherosclerotic lesions. GQ-177 acts as a partial PPARγ agonist that improves obesity-associated insulin resistance and dyslipidemia with atheroprotective effects in LDLr mice., This study was supported by São Paulo Research Foundation (FAPESP grant 2012/51316-5 to DSPA) and National Council for Scientific and Technological Development (National Institute of Science and Technology for Pharmaceutical Innovation (INCT_if/CNPq) grant 573663/2008-4 to IRP and CNPq/MICCIN grant BFU2011-2476 to DSPA, LBG and BLH). FAC was supported by CNPq fellowship (201602/2011-03), JCS was supported by FAPESP fellowship (2012/14360-6) and MR was supported by the FAPESP (2009/53072-3) and the CNPq (151568/2013-8) fellowships.

Background Under the hypothesis that the G-308A polymorphism in the promoter region of the tumor necrosis factor α gene might increase tumor necrosis factor α expression, several investigations have been performed to examine the influence of the -308A allele on the risk of cardiovascular events. The results of these studies, however, have been conflicting. To provide a more robust estimate of the putative effect of the G-308A polymorphism on the risk of cerebrocardiovascular events, we did 2 meta-analyses that examined the role of the -308A variant in both ischemic heart disease (IHD) and ischemic stroke. Methods We applied both fixed- and random-effects models to combine odds ratios (OR) and 95% CIs, and publication bias and heterogeneity were explored. Results Data of 17030 subjects from 23 studies were used. Overall, in populations predominantly of European ancestry, no association between the G-308A polymorphism and IHD under a dominant model (AA + GA vs GG) was observed: OR, 1.07; 95% CI, 0.94-1.21; P = .32. Similarly, the -308A allele was not associated with ischemic stroke considering all studies: OR, 0.99; 95% CI, 0.70-1.41, P = .96. However, analysis by ancestry revealed that Asian subjects harboring the -308A variant were approximately 40% less likely to have stroke compared to the GG genotype: OR, 0.62; 95% CI, 0.44-0.86; P = .004. Conclusions These results suggest that the G-308A polymorphism is unlikely to be associated with the development of IHD, whereas it might be a protective factor for ischemic stroke in Asians only.

The leaves of Passiflora alata Dryander and Passiflora edulis Sims, traditionally used in American countries to treat both anxiety and nervousness by folk medicine, are rich in polyphenols, which have been reported as natural antioxidants. In this study, the antioxidant activities of P. edulis and P. alata hydroalcoholic leaf extracts were verified in in vitro and ex vivo assays. P. alata showed a higher total reactive antioxidant potential than did P. edulis. The antioxidant activities of both extracts were significantly correlated with polyphenol contents. In addition, both extracts attenuated ex vivo iron-induced cell death, quantified by lactate dehydrogenase leakage, and effectively protected against protein damage induced by iron and glucose. These findings demonstrate that the P. alata and P. edulis leaf extracts have potent in vitro and ex vivo antioxidant properties and might be considered as possible new sources of natural antioxidants.

Propolis has been shown to modulate the angiogenesis in bothin vitroandin vivomodels. Thus, we aimed to evaluate the antiangiogenic properties of an ethanolic extract of Chilean propolis (EEP) and Pinocembrin (Pn). Migration, formation of capillary-like structures of endothelial cells, and sprouting from rat aortic rings were used to assess the antiangiogenic properties of EEP or Pn. In addition, microRNAs and VEGFA mRNA expression were studied by qPCR. ERK1/2 phosphorylation and HIF1αstabilization were assessed by western blot. EEP or Pn attenuated the migration, the capillary-like tube formation, and the sprouting in thein vitroassays. In addition, the activation of HIF1αand ERK1/2 and the VEGFA mRNA expression was significantly inhibited in a dose-dependent manner. In summary, these results suggest that HIF1αand ERK1/2 phosphorylation could be involved in the antiangiogenic effect of Chilean propolis, but more studies are needed to corroborate these findings.

et al., Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that improve insulin-mediated glucose uptake and possess beneficial vasculoprotective actions. However, because undesirable side effects are associated with these drugs, novel TZDs are under development. In this study, we evaluated the biological activity of LYSO-7, a new indole-thiazolidine, on PPAR activation, inflammation and atherogenesis using a gene reporter assay, lipopolysaccharide (LPS)-activated RAW 264.7 cell culture, and a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. LYSO-7 shows low cytotoxicity in RAW 264.7 cells and at 2.5 μmol/L induces PPARα and PPARγ transactivation as well as inhibits LPS-induced nitrite production and the mRNA gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). In addition, treatment with LYSO-7 reduces the development of atherosclerosis in LDLr−/− mice, improves the lipid profile, blood glucose levels, and downregulates CD40 and CD40L expression without affecting the body weight of the animals. Altogether, our data show that LYSO-7 possesses anti-inflammatory properties and that treatment with this TZD attenuates atherosclerosis progression in LDLr−/− mice by modulating lipid metabolism and inflammation. Thus, LYSO-7 shows potential as a new drug candidate for the treatment of atherosclerosis., This study was supported by FAPESP grant 2012/51316-5 (to DSPA), INCT_if/CNPq grant 573663/2008-4 (to IRP), and CNPq/MICCIN grant BFU2011-2476 (to DSPA, LBG and BLH). FAC was supported by CNPq scholarship (201602/2011-03), and MR was supported by a postdoctoral fellowship from FAPESP (2009/53072-3).

The ethanolic extract of Symphyopappus casarettoi syn. Eupatorium casarettoi was partionated in hexanic, chloroformic and methanolic fractions. Extract and fractions were tested for their antioxidant activity in vitro and ex vivo assays. The methanolic fraction showed a higher antioxidant potential compared to the others fractions, which was correlated with its total phenol content. In addition, the ethanolic extract and the methanolic fraction attenuated ex vivo iron-induced cell death, quantified by lactate dehydrogenase leakage, and effectively protected against lipid damage induced by iron. These findings suggest that the ethanolic extract of S. casarettoi inflorescence and its methanolic fraction have in vitro and ex vivo antioxidant properties.

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.

Objective— Nitro-fatty acids (NO 2 -FAs) are emerging as a new class of cell signaling mediators. Because NO 2 -FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO 2 -FAs in angiogenesis. Methods and Results— The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 μmol/L, both NO 2 -FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO 2 -FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO 2 -FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA–mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO 2 -FAs. Conclusion— Our data disclose a novel physiological role for NO 2 -FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

Thiazolidinediones (TZDs) are a class of drugs used for treatment of type 2 diabetes. However, the therapy with currently available TDZs (e.g. rosiglitazone) is associated with important side effects, such as edema and weight gain, suggesting that the investigation of alternative TZDs with better pharmacological properties is warranted. In this study, we investigated both anti-inflammatory and antioxidant properties of a new chemically modified TZD, the arylidene-thiazolidinedione 5-(4-methanesulfonyl-benzylidene)-3-(4-nitrobenzyl)-thiazolidine-2,4-dione (SF23), and compared the results to those obtained with rosiglitazone. We found that our SF23 displays a weaker affinity for PPAR¿, up-regulating in a lower magnitude the expression of both PPAR¿ and CD36 compared to rosiglitazone. In lipopolysaccharide (LPS)-stimulated macrophages, SF23 decreased nitrite production and attenuated the mRNA expression of both iNOS and COX-2. These anti-inflammatory effects were comparable to those obtained with rosiglitazone. Interestingly, SF23, but not rosiglitazone, prevented LPS-induced mitochondrial membrane hyperpolarization, apoptosis, reactive oxygen species (ROS) generation, and the expression of NADPH oxidase subunits, Nox1 and Nox2. In addition, in macrophages from Nrf2¿/¿ mice, SF23 protected against LPSinduced cellular death and ROS production, whereas rosiglitazone was only able to protect normal Nrf2¿/¿ cells against oxidative injury, suggesting that, unlike rosiglitazone, the antioxidant activity of SF23 might be Nrf2-independent. Finally, in macrophages exposed to high concentrations of glucose, SF23 induced significant increases in the mRNA expression of glucose transporters, insulin receptor substrate and mitoNEET. Altogether, our data indicate that our new chemically modified TDZ displays similar anti-inflammatory properties, but superior antioxidant effects on the LPS-stimulated macrophages compared to rosiglitazone.

The vascular effects of nitrolinoleate (LNO2), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO2 was capable to deliver free radical nitric oxide (·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO2 for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO2 were observed in vivo by intravital microscopy assays. LNO2 decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO2 reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO2 on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO2 involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO2 inhibited adhesion molecules expression and promoted ·NO inactivation of the CD40-CD40L system, both important processes of the inflammatory response. © 2010 Elsevier Inc. All rights reserved., This study was supported by Fundação de Apoio à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico/Instituto do Milênio-Redoxoma.

Studies on the relationship between endothelial nitric oxide (NOS3) gene variants and hypertension have been conflicting. To explore this hypothesis further, we performed a meta-analysis and re-evaluated the relationship between the three most widely studied NOS3 polymorphisms and hypertension status and blood pressure levels.Data on 40,413 subjects from 53 studies were combined in five distinct meta-analyses, and heterogeneity and publication bias were explored.Heterogeneity was observed in all meta-analyses. By a random-effects model, carriers of the four 27-basepair repeat variable number of tandem repeats in intron 4 were associated with a 28% increase in the risk of hypertension compared with those homozygous for the 5 repeat: odds ratio (OR) 1.28, 95% confidence interval (CI) 1.11-1.47, P=0.001. In Asian individuals, Asp allele carriers displayed a similar association: OR 1.28, 95% CI 1.06-1.54, P=0.01, as well as a 2 mmHg increase in both systolic (P=0.04) and diastolic (P=0.009) blood pressure levels. Furthermore, meta-regression analysis indicated that the effect of the Glu298Asp genotype on the risk of hypertension might be dependent on total cholesterol status. No effect of the T-786C variant on hypertension was detected. There was evidence that such findings might be a result of selectively reporting/publishing positive reports.Our results suggest that current data on the relationship between NOS3 variants and hypertension are subject not only to important heterogeneity but also to publication bias. Future research should preferentially focus on gene-environment interactions as well as haplotype analyses.

There is evidence supporting a role for 5-10 methylenetetrahydrofolate reductase (MTHFR) gene variants in acute lymphoblastic leukemia (ALL). To provide a more robust estimate of the effect of MTHFR polymorphisms on the risk of ALL, we did a meta-analysis to reevaluate the association between the two most commonly studied MTHFR polymorphisms (C677T and A1298C) and ALL risk. All case-control studies investigating an association between the C677T or A1298C polymorphisms and risk of ALL were included. We applied both fixed-effects and random-effects models to combine odds ratio (OR) and 95% confidence intervals (95% CI). Q-statistic was used to evaluate the homogeneity and both Egger and Begg-Mazumdar tests were used to assess publication bias. The meta-analysis of the C677T polymorphism and risk of childhood ALL included 13 studies with a total of 4,894 individuals. Under a fixed-effects model, the TT genotype failed to be associated with a statistically significant reduction of childhood ALL risk (TT versus CT + CC: OR, 0.88; 95% CI, 0.73-1.06; P = 0.18). However, individuals homozygous for the 677T allele exhibited a 2.2-fold decrease in risk of adult ALL (TT versus CT + CC: OR, 0.45; 95% CI, 0.26-0.77; P = 0.004). In both cases, no evidence of heterogeneity was observed. No association between the A1298C variant and susceptibility to both adult and childhood ALL was disclosed. Our findings support the proposal that the common genetic C677T polymorphism in the MTHFR contributes to the risk of adult ALL, but not to the childhood ALL susceptibility. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1956–63)

The leaf extract of Passiflora alata Dryander (P. alata) has been demonstrated to possess antioxidant activity in vitro. The aim of this study was to investigate the effects of P. alata leaf extract pretreatment on carbon tetrachloride-treated rats. Male Wistar rats were randomly allocated into four groups: group 1 (control - vehicle), group 2 and 3 (P. alata extract - 1 and 5mg/kg, respectively) and group 4 (trolox - 0.18mg/kg). Rats received daily pretreatment by oral gavage for 30 days followed by a single dose of CCl(4) (3ml/kg i.p. in vegetable oil) on the 30th day and were killed after 6h. The pretreatment with the P. alata extract provided significant protection to liver, evidenced by lower degree of necrosis, decreased lipid peroxidation (TBARS) and higher catalase and superoxide dismutase activities. Additionally, pretreated-rats with P. alata (5mg/kg) showed significantly decreased cardiac TBARS levels. Our results indicate that a low oral dose of P. alata leaf extract has both hepato and cardioprotective effects on rats treated with CCl(4).

Background. Autosomal dominant polycystic kidney disease (ADPKD) is a renal disease characterized by an important variability in clinical course, which cannot be fully explained by the genetic heterogeneity of the disease. Although the role for the angiotensin I-converting enzyme (ACE) insertion/deletion (I/D) polymorphism as a modifier factor in ADPKD renal deterioration has been suggested, direct evidence from genetic association studies remain inconclusive. To provide a more robust estimate of the putative effect of the ACE I/D polymorphism on the renal progression in ADPKD, we performed a meta-analysis pooling data from all relevant studies in which the role of the ACE I/D variant in ADPKD clinical features was evaluated. Methods. We applied a random-effects model to combine odds ratio and 95% confidence intervals. Q-statistic was used to evaluate the homogeneity, and both Egger’s and Begg–Mazumdar tests were used to assess publication bias. Results. Altogether, three distinct meta-analyses were generated using data from 13 studies. Despite the absence of publication bias and the presence of homogeneity among study results, the DD genotype failed to show an influence on risk of end-stage renal disease (ESRD), mean age at ESRD or risk of hypertension in ADPKD patients when compared with I-allele carriers (DD vs ID þ II). Likewise, meta-analyses carried out separately for Caucasian and Asian studies showed no indication of an association between the DD genotype and a faster renal deterioration in ADPKD. Conclusion. These findings do not support the hypothesis that the enhanced ACE activity associated with the D allele might promote a significantly worse prognosis in patients with ADPKD.

Many biological properties have been attributed to ruthenium complexes including anti-tumor activity and the attenuation of reperfusion damage and infarct size. In this work, we characterize the antioxidant activity of trans-[RuCl2(nic)4] where nic is 3-pyridinecarboxylic acid and trans-[RuCl2(i-nic)4] where i-nic is 4-pyridinecarboxylic acid by (i) evaluation of total antioxidant potential (TRAP); (ii) prevention of DNA damage induced by hydrogen peroxide using the alkaline comet assay; and (iii) the prevention of lipid peroxidation and cell death induced by iron in liver slices. Our results suggest that nic has stronger antioxidant potential when compared to the i-nic. Higher doses (above 200 microM) of these compounds gave genotoxic effects, but the antioxidant potential could be obtained with the use lower doses (0.1-10 microM).

Meta-analysis has become an important statistical tool in genetic association studies, since it may provide more powerful and precise estimates. However, meta-analytic studies are prone to several potential biases not only because the preferential publication of ``positive'' studies but also due to difficulties in obtaining all relevant information during the study selection process. In this letter, we point out major problems in meta-analysis that may lead to biased conclusions, illustrating an empirical example of two recent meta-analyses on the relation between MTHFR polymorphisms and risk of acute lymphoblastic leukemia that, despite the similarity in statistical methods and period of study selection, provided partially conflicting results.

Amblyomin-X é um inibidor de serinoprotease do tipo kunitz obtido de uma biblioteca de cDNA das glândulas salivares do carrapato Amblyomma cajannense. Dados preliminares mostraram que a injeção in vivo de Amblyomin-X reduziu a formação de massa tumoral induzida por células de melanoma B16F10 em camundongos. Com base neste dado e na ação de inibidores de serinoproteases sobre a angiogênese, este projeto foi delineado para caracterizar as ações do Amblyomin-X sobre a angiogênese in vivo e sobre funções da célula endotelial in vitro na vigência do fator de crescimento VEGF-A. Os efeitos do Amblyomin-X sobre a cinética de formação de novos vasos na rede microcirculatória in vivo foram investigados no modelo de câmara dorsal em camundongos e na membrana corioalantóica. Empregando cultura de células endoteliais de linhagem de microcirculação (t-End), foram avaliados os efeitos do Amblyomin-X sobre viabilidade e citoproteção celular (anexina e iodeto de propídio - PI), proliferação celular (incorporação do fluoróforo diacetato de carboxi-fluoresceína succinimidil éster) e ciclo celular, além da expressão das moléculas de adesão PECAM-1, ICAM-1, VCAM-1, β1 e β3 integrinas em ensaios de citometria de fluxo. A aderência celular em Matrigel foi quantificada por espectrofotometria, a formação de tubos foi mensurada por microscopia óptica e a expressão gênica das moléculas de adesão PECAM-1, ICAM-1 e VCAM-1 por RT-PCR. Os dados obtidos mostraram que a aplicação tópica do Amblyomin-X (100ng/10µL) reduziu a formação de novos vasos no tecido subcutâneo dorsal de camundongos, quando o tratamento foi iniciado anterior ou concomitantemente, mas não posteriormente ao tratamento ao VEGF (10ng/10µL) e, reduziu a formação de novos vasos na membrana corioalantóica, quando administrado concomitantemente com o VEGF (0,25ng/10µL). O Amblyomin-X não alterou a viabilidade de células t-End (1000ng/mL, 72 horas); inibiu a apoptose e apoptose tardia causada por meio de cultura carenciado (100ng/mL); inibiu a proliferação (10, 100 ou 1000ng/mL de Amblyomin-X, 48 e 72 horas); induziu parada nas fases G1/G0 do ciclo celular; diminuiu a aderência e a formação de tubos das células endoteliais; não alterou a expressão basal de PECAM-1, VCAM-1, ICAM-1, β1 e β3 integrinas, mas reduziu a expressão de PECAM-1, VCAM-1, ICAM-1 induzida pelo VEGF-A. A redução da expressão protéica da PECAM-1 e ICAM-1 parece não ser dependente de ação do Amblyomin-X na expressão gênica, já que os níveis de RNAm não foram afetados pela ação do Amblyomin-X. Em conjunto, os dados obtidos neste trabalho mostram que o Amblyomin-X inibe a formação de novos vasos in vivo, por interferir, possivelmente, em mecanismos relacionados a sinalização da célula endotelial induzida pelo VEGF-A, em especial com a proliferação, adesão , tubulogênese e expressão de moléculas de adesão da família das imunoglobulinas. Amblyomin-X is a type-kunitz serineprotease inhibitor obtained from a cDNA library of the Amblyomma cajannense salivary glands. Preliminary data showed that in vivo injection of the protein reduced the formation of tumor-induced B16F10 melanoma cells in mice. Based on this data and on actions of serinorpoteases inhibitors on angiogenesis, this project aimed to characterize the actions of Amblyomin-X on angiogenesis in vivo and on in vitro endothelial cells functions in the presence of the growth factor VEGF. The effects of Amblyomin-X on the in vivo formation of microcirculatory new vessels were investigated in the dorsal chamber model in mice and in the chorioallantoic membrane assay. Mice microvascular endothelial cell lineage (t-End) was employed to evaluate the effects of Amblyomin-X on cytoprotection and cell viability (Annexin-V and propidium iodide-PI), cell proliferation (incorporation of the fluorophore fluorescein diacetate carboxy-succinimidil ester), cell cycle, membrane expression of PECAM-1, ICAM-1, VCAM-1, β1 and β3 integrins adhesion molecules using flow cytometry. Cell adhesion was assessed in matrigel by spectrophotometry, the formation of tubes was measured by optical microscopy and adhesion molecules PECAM-1, ICAM-1 and VCAM-1 gene expression was evaluated by RT-PCR. Data obtained showed that topical application of Amblyomin-X (100ng/10µL) reduced the formation of new vessels, only when Amblyomin-X treatment started before or simultaneously to VEGF-A stimulation (10ng/10µL). Amblyomin-X (100ng/10µL) also reduced the formation of new vessels in the chorioallantoic membrane assay, when coadministered with VEGF (0.25ng/10µL). The Amblyomin-X did not alter the viability of t-End (1000ng/mL, 72 hours), inhibited apoptosis and late apoptosis caused by deprivated serum (100ng/mL), inhibited proliferation (10, 100 or 1000ng/mL, 48 and 72 hours), induced G1/G0 arrest in cell cycle phases, decreased the cell adhesion and tube formation (100ng/mL), did not alter the basal expression of PECAM-1, VCAM-1, ICAM-1, β1 or β3 integrin, but reduced the PECAM-1, VCAM-1, ICAM-1 induced by VEGF-A. Impaired PECAM-1 and ICAM-1 expression seems not be dependent on gene expression, as, mRNA levels were not affected by the action of Amblyomin-X. Together, the data obtained shows that the Amblyomin-X inhibits new vessel formation in vivo, by interfering, possibly, with mechanisms related to VEGF-A induced endothelial cell signaling, especially with the proliferation, adhesion, tubulogenesis and expression of adhesion molecules.