Mônica V. Marquezini

Glycosaminoglycan Distribution in Atherosclerotic Saphenous Vein Grafts

Glycosaminoglycan composition of normal saphenous veins and atherosclerotic saphenous vein grafts is reported. Dermatan sulfate is the main glycosaminoglycan present in both normal saphenous veins and saphenous vein grafts. These tissues also contain chondroitin sulfate and heparan sulfate. Although the total amount of glycosaminoglycans decreased in the grafts (compared with normal saphenous veins), the grafts showed an increase in the relative amounts of dermatan sulfate and chondroitin sulfate. Heparan sulfate was decreased, compared with normal controls. These findings suggest the involvement of blood vessel glycosaminoglycans (not only the arterial glycosaminoglycans) in the process of atherosclerosis.

Changes in cardiac heparan sulfate proteoglycan expression and streptozotocin-induced diastolic dysfunction in rats

BackgroundChanges in the proteoglycans glypican and syndecan-4 have been reported in several pathological conditions, but little is known about their expression in the heart during diabetes. The aim of this study was to investigate in vivo heart function changes and alterations in mRNA expression and protein levels of glypican-1 and syndecan-4 in cardiac and skeletal muscles during streptozotocin (STZ)-induced diabetes.MethodsDiabetes was induced in male Wistar rats by STZ administration. The rats were assigned to one of the following groups: control (sham injection), after 24 hours, 10 days, or 30 days of STZ administration. Echocardiography was performed in the control and STZ 10-day groups. Western and Northern blots were used to quantify protein and mRNA levels in all groups. Immunohistochemistry was performed in the control and 30-day groups to correlate the observed mRNA changes to the protein expression.ResultsIn vivo cardiac functional analysis performed using echocardiography in the 10-day group showed diastolic dysfunction with alterations in the peak velocity of early (E) diastolic filling and isovolumic relaxation time (IVRT) indices. These functional alterations observed in the STZ 10-day group correlated with the concomitant increase in syndecan-4 and glypican-1 protein expression. Cardiac glypican-1 mRNA and skeletal syndecan-4 mRNA and protein levels increased in the STZ 30-day group. On the other hand, the amount of glypican in skeletal muscle was lower than that in the control group. The same results were obtained from immunohistochemistry analysis.ConclusionOur data suggest that membrane proteoglycans participate in the sequence of events triggered by diabetes and inflicted on cardiac and skeletal muscles.

Influence of ACTN3 R577X polymorphism on ventilatory thresholds related to endurance performance

Abstract The purpose of this study was to verify the association between ACTN3 polymorphism and physiological parameters related to endurance performance. A total of 150 healthy male volunteers performed a maximal incremental running test to determine the speeds corresponding to ventilatory threshold (VT) and respiratory compensation point (RCP). Participants were genotyped and divided into terciles based on the analysed variables. Genotype frequencies were compared through χ2 test between lower and higher terciles, with the lowest or highest values of each analysed variable. ACTN3 XX genotype was over-represented in higher tercile for VT and RCP. Odds ratio also showed significantly higher chances of XX individuals to be in higher tercile compared to RR (7.3) and RR + RX (3.5) for VT and compared to RR genotype (8.1) and RR + RX (3.4) for RCP. Thus, XX individuals could attain the VT and RCP at higher speeds, suggesting that they are able to sustain higher running speeds in lower exercise intensity domains. It could result in higher lipid acids oxidation, saving muscle glycogen and delaying the fatigue during prolonged exercises, which could be the advantage mechanism of this genotype to endurance performance.

Lacrimal Cytokines Assessment in Subjects Exposed to Different Levels of Ambient Air Pollution in a Large Metropolitan Area

Background Air pollution is one of the most environmental health concerns in the world and has serious impact on human health, particularly in the mucous membranes of the respiratory tract and eyes. However, ocular hazardous effects to air pollutants are scarcely found in the literature. Design Panel study to evaluate the effect of different levels of ambient air pollution on lacrimal film cytokine levels of outdoor workers from a large metropolitan area. Methods Thirty healthy male workers, among them nineteen professionals who work on streets (taxi drivers and traffic controllers, high pollutants exposure, Group 1) and eleven workers of a Forest Institute (Group 2, lower pollutants exposure compared to group 1) were evaluated twice, 15 days apart. Exposure to ambient PM2.5 (particulate matter equal or smaller than 2.5 μm) was 24 hour individually collected and the collection of tears was performed to measure interleukins (IL) 2, 4, 5 and 10 and interferon gamma (IFN-γ) levels. Data from both groups were compared using Student’s t test or Mann- Whitney test for cytokines. Individual PM2.5 levels were categorized in tertiles (lower, middle and upper) and compared using one-way ANOVA. Relationship between PM2.5 and cytokine levels was evaluated using generalized estimating equations (GEE). Results PM2.5 levels in the three categories differed significantly (lower: ≤22 μg/m3; middle: 23–37.5 μg/m3; upper: >37.5 μg/m3; p<0.001). The subjects from the two groups were distributed unevenly in the lower category (Group 1 = 8%; Group 2 = 92%), the middle category (Group 1 = 89%; Group 2 = 11%) and the upper category (Group 1 = 100%). A significant relationship was found between IL-5 and IL-10 and PM2.5 levels of the group 1, with an average decrease of 1.65 pg/mL of IL-5 level and of 0.78 pg/mL of IL-10 level in tear samples for each increment of 50 μg/m3 of PM2.5 (p = 0.01 and p = 0.003, respectively). Conclusion High levels of PM2.5 exposure is associated with decrease of IL-5 and IL-10 levels suggesting a possible modulatory action of ambient air pollution on ocular surface immune response.

Bevacizumab reduces neurocan content and gene expression in newborn rat retina in vitro.

PURPOSE Extracellular matrix (ECM) and cellular membrane proteoglycans (PGs) play important roles in neural differentiation and cell adhesion. Vascular endothelial growth factor, an important signal protein in vascular and retinal neural cell development, is retained in the ECM due to its high affinity for PG. Bevacizumab, an anti-VEGF agent, has been extensively used for treating retinal diseases in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on neurocan, phosphacan, and syndecan-3 PG levels in newborn rat retina. METHODS Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 48 hours. Immunohistochemical staining was assessed against neurocan, phosphacan, and syndecan-3. Proteoglycan content was quantified based on the intensity of immunohistochemical labeling. Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. The results from the treatment and control groups were compared. RESULTS No significant difference in the staining intensity and mRNA expression of phosphacan and syndecan-3 was observed between the groups. However, a significant decrease in neurocan content and mRNA expression was observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Is the COL5A1 rs12722 Gene Polymorphism Associated with Running Economy

The COL5A1 rs12722 polymorphism is considered to be a novel genetic marker for endurance running performance. It has been postulated that COL5A1 rs12722 may influence the elasticity of tendons and the energetic cost of running. To date, there are no experimental data in the literature supporting the relationship between range of motion, running economy, and the COL5A1 rs12722 gene polymorphism. Therefore, the main purpose of the current study was to analyze the influence of the COL5A1rs12722 polymorphism on running economy and range of motion. One hundred and fifty (n = 150) physically active young men performed the following tests: a) a maximal incremental treadmill test, b) two constant-speed running tests (10 km•h−1 and 12 km•h−1) to determine the running economy, and c) a sit-and-reach test to determine the range of motion. All of the subjects were genotyped for the COL5A1 rs12722 single-nucleotide polymorphism. The genotype frequencies were TT = 27.9%, CT = 55.8%, and CC = 16.3%. There were no significant differences between COL5A1 genotypes for running economy measured at 10 km•h−1 (p = 0.232) and 12 km•h−1 (p = 0.259). Similarly, there were no significant differences between COL5A1 genotypes for range of motion (p = 0.337). These findings suggest that the previous relationship reported between COL5A1 rs12722 genotypes and running endurance performance might not be mediated by the energetic cost of running.

Traffic-Related Air Pollution Effect on Fast Glycemia of Aged Obese Type 2 Diabetic Mice

Recent experimental data have provided associations between ambient PM2.5 (fine particulate matter; diameter ≤ 2.5 μm) and propensity to inflammation and chronic diseases especially among susceptible groups, such as elderly people. There is cumulative evidence that type-2 diabetes mellitus is a chronic inflammatory state aggravated by factors that promote endothelium inflammation. Accordingly our hypothesis that the exposure of aged obese population to PM2.5 might aggravate type-2 diabetes, we used a model of aged, diet-induced obese mice. C57BL6 male mice were fed with regular chow (n=30; RC) or high-fat chow (n=36; HF) during one-year and randomly assigned to filtered (FA-RC, n=16; FA-HF, n=19) or PM2.5 concentrated air (600 μg.m-3) (EXP-RC, n=14; EXPHF, n=17) chambers to have a daily 1 hour exposition during consecutive 30- days. Fast glycemia was measured before the animals were euthanized. The Institution’s Ethics Committee approved all experimental procedures. Heart mRNA content of selected migration, signalization and adhesion proteins were measured by SYBR Green fluorescence Real Time RT-PCR protocol using appropriate primers. There were no difference between RC-EXP and RC-FA nor between HF-EXP and HF-FA body weight. Regarding fast glycemia, both, RC and HF groups, were diabetic, but only the HF group was affected by acute exposure to PM2.5 (mean ± SD, EXP-HF vs FA- HF, 172.8 ± 23.4 vs 156.7 ± 17.6, p <0.05; EXP-RC vs FA-RC, 149.8 ± 19.2, 139.7 ± 15.3, ns; ANOVA). The gene expression profile of E-selectin, IL-6, VCAM-1, ICAM-1 and MMP-9, was differently affected by PM2.5 in heart and lung. Proteins activated by inflammatory stimuli involved in the inhibition of insulin signaling are being investigated.

Biochemical and cytochemical characterization of extracellular proteoglycans in the inner circular smooth muscle layer of dog small intestine.

Current literature concerning smooth muscle blood vessels has shown versican as the main proteoglycan (PG) component of the matrix. To show whether smooth muscle matrix has the same PG distribution when present in organs, other than the blood vessels, the inner circular smooth muscle layer of the small intestine was obtained by dissection as a highly purified tissue and analyzed by biochemical and cytochemical methods. The smooth muscle layer PGs were extracted from dog small intestine with 4 M guanidine‐HCl in the presence of proteinase inhibitors, purified by charge equilibrium, isolated by equilibrium CsCl density gradients, and analyzed in terms of anion exchange, size, and glycosaminoglycan (GAG) distribution. Proteoheparan sulfate itself represented 91.5% of the PGs present in this tissue. The remainder was proteodermatan sulfate. Cytochemical analyses using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinases ABC and heparitinase III showed the arrangement and identification of PGs in basal lamina and intramuscular connective tissues. The PGs in the basal lamina were proteoheparan sulfate, and those associated with collagen fibrils in the endomysium and perimysium were rich in dermatan sulfate. In contrast to the blood vessels, inner circular muscle smooth tissue in intestine has, as the main PG, perlecan.

The genetics of human running: ACTN3 polymorphism as an evolutionary tool improving the energy economy during locomotion

Abstract Background: Covering long distances was an important trait to human evolution and continues to be highlighted for health and athletic status. This ability is benefitted by a low cost of locomotion (CoL), meaning that the individuals who are able to expend less energy would be able to cover longer distances. The CoL has been shown to be influenced by distinct and even ‘opposite’ factors, such as physiological and muscular characteristics, which are genetically inherited. In this way, DNA alterations could be important determinants of the characteristics associated with the CoL. A polymorphism in the ACTN3 gene (R577X) has been related to physical performance, associating the X allele with endurance and the R allele with strength/power abilities. Aim: To investigate the influence of ACTN3 genotypes on the CoL. Subjects and methods: One hundred and fifty healthy male individuals performed two constant speed tests (at 10 and 12 km/h) to determine the CoL. Results: Interestingly, the results showed that heterozygous individuals (RX genotype) presented significantly lower CoL compared to RR and XX individuals. Conclusions: It is argued that RX genotype might generate an intermediate strength-to-endurance phenotype, leading to a better phenotypic profile for energy economy during running and, consequently, for long-term locomotion.

Cellular stress response in human Müller cells (MIO-M1) after bevacizumab treatment

&NA; Bevacizumab, an anti‐vascular endothelial growth factor (VEGF) agent, is widely used in the treatment of retinal vascular diseases. However, due to the essential role Müller cell derived‐VEGF plays in the maintenance of retinal neurons and glial cells, cell viability is likely to be affected by VEGF inhibition. We therefore evaluated the effect of bevacizumab‐induced VEGF inhibition on Müller cells (MIO‐M1) in vitro. MIO‐M1 cells were cultured for 12 or 24 h in media containing bevacizumab at 0.25 or 0.5 mg/mL. Controls were cultured in medium only. Cell viability was determined with the trypan blue exclusion test and MTT assay. Caspase‐3, beclin‐1, glial fibrillary acidic protein (GFAP) and vimentin content were quantified by immunohistochemistry. Gene expression was evaluated by real‐time quantitative PCR. Treatment with bevacizumab did not reduce MIO‐M1 cell viability, but increased metabolic activity at 24 h (0.5 mg/mL) and induced apoptosis and autophagy, as shown by the increased caspase‐3 levels at 12 h (0.25 and 0.5 mg/mL) and the increased beclin levels at 24 h (0.5 mg/mL). Caspase‐3 mRNA was upregulated at 12 h and downregulated at 24 h in cells treated with bevacizumab at 0.25 mg/mL. Bevacizumab treatment was also associated with structural protein abnormalities, with decreased GFAP and vimentin content and upregulated GFAP and vimentin mRNA expression. Although bevacizumab did not significantly affect MIO‐M1 cell viability, it led to metabolic and molecular changes (apoptosis, autophagy and structural abnormalities) suggestive of significant cellular toxicity. HighlightsAnti‐VEGF drugs are used as treatment of vascular retinopathies but its affect on retinal glial cells survival is unknown.Anti‐VEGF bevacizumab did not reduce Müller cell viability but caused increased metabolic activity, apoptosis and autophagy.Bevacizumab treatment was also associated with structural protein abnormalities, including GFAP and vimentin.We conclude that bevacizumab led to metabolic and molecular changes suggestive of cellular toxicity to Müller cells.Exposure to bevacizumab seems to triggers an adaptive mechanism in Müller cells capable of reducing cellular damage.