Thereza Christina Barja-Fidalgo

High fat diet induces central obesity, insulin resistance and microvascular dysfunction in hamsters

Microvascular dysfunction is an early finding in obesity possibly related to co-morbidities like diabetes and hypertension. Therefore we have investigated changes on microvascular function, body composition, glucose and insulin tolerance tests (GTT and ITT) on male hamsters fed either with high fat (HFD, n=20) or standard (Control, n=21) diet during 16 weeks. Total body fat and protein content were determined by carcass analysis, aorta eNOS and iNOS expression by immunoblotting assay and mean blood pressure (MAP) and heart rate (HR) by an arterial catheter. Microvascular reactivity in response to acetylcholine and sodium nitroprusside, functional capillary density (FCD), capillary recruitment induced by a hyperinsulinemic status and macromolecular permeability after 30 min ischemia was assessed on either cheek pouch or cremaster muscle preparations. Compared to Control, HFD animals have shown increased visceral fat (6.0 ± 0.8 vs. 13.8 ± 0.6g/100g BW), impaired endothelial dependent vasodilatation, decreased FCD (11.3 ± 1.3 vs. 6.8 ± 1.2/field) and capillary recruitment during hyperinsulinemia and increased macromolecular permeability after ischemia/reperfusion (86.4 ± 5.2 vs.105.2 ± 5.1 leaks/cm(2)), iNOS expression and insulin resistance. MAP, HR, endothelial independent vasodilatation and eNOS expression were not different between groups. Our results have shown that HFD elicits an increase on visceral fat deposition, microvascular dysfunction and insulin resistance in hamsters.

Chronic aerobic exercise associated to dietary modification improve endothelial function and eNOS expression in high fat fed hamsters.

Obesity is epidemic in the western world and central adipose tissue deposition points to increased cardiovascular morbidity and mortality, independently of any association between obesity and other cardiovascular risk factors. Physical exercise has been used as non-pharmacological treatment to significantly reverse/attenuate obesity comorbidities. In this study we have investigated effects of exercise and/or dietary modification on microcirculatory function, body composition, serum glucose, iNOS and eNOS expression on 120 male hamsters treated for 12 weeks with high fat chow (HF, n = 30) starting on the 21st day of birth. From week 12 to 20, animals were randomly separated in HF (no treatment change), return to standard chow (HFSC, n = 30), high fat chow associated to an aerobic exercise training program (AET) (HFEX, n = 30) and return to standard chow+AET (HFSCEX, n = 30). Microvascular reactivity in response to acetylcholine and sodium nitroprusside and macromolecular permeability increase induced by 30 minutes ischemia followed by reperfusion were assessed on the cheek pouch preparation. Total body fat and aorta eNOS and iNOS expression by immunoblotting assay were evaluated on the experimental day. Compared to HFSC and HFSCEX groups, HF and HFEX ones presented increased visceral fat [(mean±SEM) (HF)4.9±1.5 g and (HFEX)4.7±0.9 g vs. (HFSC)*3.0±0.7 g and (HFSCEX)*1.9±0.4 g/100 g BW]; impaired endothelial-dependent vasodilatation [Ach 10−8 M (HF)87.9±2.7%; (HFSC)*116.7±5.9%; (HFEX)*109.1±4.6%; (HFSCEX)*105±2.8%; Ach10−6 M (HF)95.3±3.1%; (HFSC)*126±6.2%; (HFEX)*122.5±2.8%; (HFSCEX)*118.1±4.3% and Ach10−4 M (HF)109.5±4.8%; (HFSC)*149.6±6.6%; (HFEX)*143.5±5.4% and (HFSCEX)*139.4±5.2%], macromolecular permeability increase after ischemia/reperfusion [(HF)40.5±4.2; (HFSC)*19.0±1.6; (HFEX)*18.6±2.1 and (HFSCEX)* 21.5±3.7 leaks/cm2), decreased eNOS expression, increased leptin and glycaemic levels. Endothelial-independent microvascular reactivity was similar between groups, suggesting that only endothelial damage had occurred. Our results indicate that an aerobic routine and/or dietary modification may cause significant improvements to high fat fed animals, diminishing visceral depots, increasing eNOS expression and reducing microcirculatory dysfunction.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Effect of obesity on biodistribution of nanoparticles

ABSTRACT Nanoparticles have specific features (lipophilicity, surface charge, composition and size). Studies regarding the biological behavior of nanoparticles in diseases such diabetics and obesity are scarce. Here, we evaluated two nanoparticles: magnetic core mesoporous silica (MSN) (58nm) and polycaprolactone (PCL) nanoparticle (280nm) in obese mice. Changes in the biodistribution were observed, especially considering the mononuclear phagocyte system (MPS), and the visceral fat tissue. Nonetheless, our data corroborates the influence of size in the biodistribution in obese animals, supporting that smaller nanoparticles, may show a higher tissue deposition at spleen, due the associated splenomegaly and the complications arising from this state. Finally, our study demonstrated that, in obesity, probably due the low‐grade inflammatory state associated with metabolic syndrome a difference in accumulation of nanoparticles was found, with profound impact in the tissue deposition of nanoparticles.