Mackerel and Seaweed Burger as a Functional Product for Brain and Cognitive Aging Prevention.

Most world countries are experiencing a remarkable aging process. Meanwhile, 50 million people are affected by Alzheimer's disease (AD) and related dementia and there is an increasing trend in the incidence of these major health problems. In order to address these, the increasing evidence suggesting the protective effect of dietary interventions against cognitive decline during aging may suggest a response to this challenge. There are nutrients with a neuroprotective effect. However, Western diets are poor in healthy n-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA), iodine (I), and other nutrients that may protect against cognitive aging. Given DHA richness in chub mackerel (Scomber colias), high vitamin B9 levels in quinoa (Chenopodium quinoa), and I abundance in the seaweed Saccorhiza polyschides, a functional hamburger rich in these nutrients by using these ingredients was developed and its formulation was optimized in preliminary testing. The effects of culinary treatment (steaming, roasting, and grilling vs. raw) and digestion on bioaccessibility were evaluated. The hamburgers had high levels of n-3 PUFAs in the range of 42.0-46.4% and low levels of n-6 PUFAs (6.6-6.9%), resulting in high n-3/n-6 ratios (>6). Bioaccessibility studies showed that the hamburgers could provide the daily requirements of eicosapentaenoic acid (EPA) + DHA with 19.6 g raw, 18.6 g steamed, 18.9 g roasted, or 15.1 g grilled hamburgers. Polyphenol enrichment by the seaweed and antioxidant activity were limited. The hamburgers contained high levels of Se and I at 48-61 µg/100 g ww and 221-255 µg/100 g ww, respectively. Selenium (Se) and I bioaccessibility levels were 70-85% and 57-70%, respectively, which can be considered high levels. Nonetheless, for reaching dietary requirements, considering the influence of culinary treatment and bioaccessibility, 152.2-184.2 g would be necessary to ensure daily Se requirements and 92.0-118.1 g for I needs.

Fatty acids, selenium, and vitamin B12 in chub mackerel (Scomber colias) as nourishment considering seasonality and bioaccessibility as factors.

Chub mackerel (Scomber colias) is an underutilised fish abundant in the North Atlantic and a nutritious food with high docosahexaenoic acid (DHA), selenium (Se), and vitamin B12 contents, affected by seasonality. Hence, the bioaccessibility of fatty acid (FA), Se, and vitamin B12 in chub mackerel was studied. Daily consumptions of 51 g, 35 g, and 14 g of March, June, and October chub mackerel, respectively, would ensure EPA + DHA requirements. Vitamin B12 requirements would be met by daily consumptions of 15-18 g of chub mackerel. For Se, larger amounts would be needed, 126-133 g/day. For FA, bioaccessibility often exceeded 80 %. Additionally, Se and vitamin B12 bioaccessibilities were 91-95 % and 77-83 %, respectively. Thus, bioaccessibility-based consumption frequencies for meeting dietary requirements are similar to those above, being the highest consumption frequencies associated to Se requirements, 137-146 g/day. Chub mackerel proved to be an excellent source of bioaccessible essential nutrients.

Corrigendum: Patterns of street food purchase in cities from Central Asia.

[This corrects the article DOI: 10.3389/fnut.2022.925771.].

Patterns of Street Food Purchase in Cities From Central Asia.

Street food makes a significant contribution to the diet of many dwellers in low- and middle-income countries and its trade is a well-developed activity in the central Asian region. However, data on its purchase and nutritional value is still scarce. This study aimed to describe street food purchasing patterns in central Asia, according to time and place of purchase. A multicentre cross-sectional study was conducted in 2016/2017 in the main urban areas of four central Asian countries: Dushanbe (Tajikistan), Bishkek (Kyrgyzstan), Ashgabat (Turkmenistan) and Almaty (Kazakhstan). Street food markets (n = 34) and vending sites (n = 390) were selected by random and systematic sampling procedures. Data on the purchased foods and beverages were collected by direct observation. Time and geographic location of the purchases was registered, and their nutritional composition was estimated. A total of 714 customers, who bought 852 foods, were observed. Customers' influx, buying rate and purchase of industrial food were higher in city centers compared to the outskirts (median: 4.0 vs. 2.0 customers/10 min, p < 0.001; 5.0 vs. 2.0 food items/10 min, p < 0.001; 36.2 vs. 28.7%, p = 0.004). Tea, coffee, bread and savory pastries were most frequently purchased in the early morning, bread, main dishes and savory pastries during lunchtime, and industrial products in the mid-morning and mid-afternoon periods. Energy and macronutrient density was highest at 11:00-12:00 and lowest at 09:00-10:00. Purchases were smaller but more energy-dense in city centers, and higher in saturated and trans-fat in the peripheries. This work provides an overview of the street food buying habits in these cities, which in turn reflect local food culture. These findings from the main urban areas of four low- and middle-income countries which are currently under nutrition transition can be useful when designing public health interventions customized to the specificities of these food environments and their customers.

Nutritional Characterization of Street Food in Urban Turkmenistan, Central Asia.

Objective: Describing the availability and nutritional composition of the most commonly available street foods in Ashgabat, Turkmenistan. Methods: One hundred sixty-one street food vending sites (six public markets) were assessed, through a collection of data on vending sites' characteristics and food availability, and samples of commonly available foods (21 homemade; 11 industrial), for chemical analysis. Results: Fruit, beverages, and food other than fruit were available in 6.8, 29.2, and 91.9% of all vending sites, respectively. Regarding the latter, 52.7% of the vending sites sold only homemade products (main dishes, snacks, cakes, biscuits and pastries, bread, ice-cream chocolate and confectionery, savory pastries and sandwiches), 37.2% only industrial (ice-cream, chocolate and confectionery, cakes, biscuits and pastries, snacks, bread and savory pastries) and 10.1% both. Homemade foods presented significantly higher total fat [homemade 11.6 g (range 6.6-19.4 g); industrial 6.2 g (range 4.0-8.6 g), p = 0.001], monounsaturated, polyunsaturated and trans-fat, and sodium and potassium content per serving. Industrial wafers presented the highest mean saturated (11.8 g/serving) and trans-fat (2.32 g/serving) content. Homemade hamburgers presented the highest mean sodium content (1889 mg/serving). Conclusions: Strategies to encourage the production and sales of healthier street foods, especially homemade, are needed to promote healthier urban food environments in urban Turkmenistan.

A Cross-Sectional Study of the Street Foods Purchased by Customers in Urban Areas of Central Asia.

This study aimed to describe street food purchases in cities from Central Asia, considering customers' characteristics and the nutritional composition of the foods and beverages. Cross-sectional studies were conducted in 2016/2017 in Dushanbe (Tajikistan), Bishkek (Kyrgyzstan), Ashgabat (Turkmenistan) and Almaty (Kazakhstan). Direct observation was used to collect data on the purchases made by street food customers, selected by random and systematic sampling. Nutritional composition was estimated using data from chemical analyses, food composition tables or food labels. A total of 714 customers (56.6% females, 55.5% aged ≥35 years, 23.3% overweight/obese) were observed, who bought 852 foods and beverages, the most frequent being savoury pastries/snacks (23.2%), main dishes (19.0%), sweet pastries/confectionery (17.9%), tea/coffee (11.3%) and soft drinks/juices (9.8%). Fruit was the least purchased food (1.1%). Nearly one-third of customers purchased industrial food items (31.9%). The median energy content of a street food purchase was 529 kcal/serving. Saturated and trans-fat median contents were 4.7 g/serving and 0.36 g/serving (21.4% and 16.5% of maximum daily intake recommendations, respectively). Median sodium and potassium contents were 745 mg/serving (37.3% of maximum recommendation) and 304 mg/serving (8.7% of minimum recommendation), respectively. In general, the purchases observed presented high contents of energy, saturated-fat, trans-fat and sodium, and low levels of potassium. Policies towards the improvement of these urban food environments should be encouraged.

The chemical composition and lipid profile of the chub mackerel (Scomber colias) show a strong seasonal dependence: Contribution to a nutritional evaluation.

The seasonal variation in chemical and lipid composition of chub mackerel (Scomber colias) was evaluated monthly over one year (proximate chemical composition and lipid profile: fatty acid (FA) and lipid classes distribution). Significant seasonal changes regarding fat content were noticed (1.3-10.3 g/100 g), with the lowest fat content obtained in February (during spawning period), and the highest in September. Regarding the FA profile, the main fluctuations were recorded in saturated (SFA) and polyunsaturated fatty acids (PUFA). The highest SFA content was registered between March and August (25.3-32.3%). PUFA (the most abundant group) reached its maximum percentual content between December and February (60.9 and 66.9%, respectively). In absolute terms, PUFA attained 5352.6 mg/100 g edible part in September, where 2473.8 mg/100 g of docosahexaenoic acid (DHA, C22:6n-3), representing 46.2% of total PUFA. DHA lowest level was 519.8 mg/100 g, registered in low-fat chub mackerel. Together DHA and EPA (eicosapentaenoic acid, C20:5n-3) represented 75% of the total PUFA and 84% of n-3 PUFA. Triacylglycerols (TAG) with 82.2-92.1% of total lipid content) and phospholipids (4.4-8.4%) were the main lipid classes. Polar lipid fraction (phospholipids), was predominantly constituted by PUFA (68.6-74.5%), mainly DHA (45.2-55.1%), with the highest percentage recorded in low-fat chub mackerel. High relative contents of PUFA (36.6-49.1%) were also found in TAG. Having into account the data obtained, chub mackerel is a privileged source of DHA even in a lean species whereby its consumption should be recommended as part a healthy dietary regime.

Collaborative study: Quantification of total folate in food using an efficient single-enzyme extraction combined with LC-MS/MS.

Quantification of the specific folate vitamers to estimate total folate in foods is not standardized. A collaborative study, including eight European laboratories, was conducted in order to determine the repeatability and reproducibility of the method for folate quantification in foods using the plant-origin γ-glutamyl hydrolase as part of the extraction procedure. The seven food samples analyzed represent the food groups; fruits, vegetables, dairy products, legumes, offal, fish, and fortified infant formula. The homogenization step was included, and six folate vitamers were analyzed using LC-MS/MS. Total folate content, expressed as folic acid equivalent, was 17-490 µg/100 g in all samples. Horwitz ratio values were within the acceptable range (0.60-1.94), except for fish. The results for fortified infant formula, a certified reference material (NIST 1869), confirmed the trueness of the method. The collaborative study is part of a standardization project within the Nordic Committee on Food Analysis (NMKL).

Effects of Industrial Boiling on the Nutritional Profile of Common Octopus (Octopus vulgaris).

Industrial cooking of common octopus (Octopus vulgaris) under well-established procedures is advantageous for current consumers, which demand healthy and convenient food. This work aimed to evaluate the effect of industrial water boiling, without the addition of salt, on the nutritional profile of common octopus. True retentions (TRs) were calculated for essential nutrients and toxic elements. After boiling, the moisture content decreased, resulting in a concentration of other constituents (protein, fat, fatty acids, majority of amino acids, phosphorus, zinc, and iodine). High TRs were obtained for some essential nutrients: 90.2% (eicosapentaenoic acid, EPA), 89.1% (docosahexaenoic acid, DHA), ≥74.6% (indispensable amino acids, IAA), and 86.8% (iodine). In both raw and boiled octopus, polyunsaturated fatty acids (252.2 and 425.1 mg/100 g), leucine (940.1 and 1613.4 mg/100 g), glutamate (1971.5 and 3257.1 mg/100 g), sodium (393.3 and 332.5 mg/100 g), and zinc (12.6 and 16.6 mg/kg) were, respectively, the most abundant fatty acids, IAA, dispensable amino acids, macro, and micro elements. Cadmium, lead, and mercury levels found in boiled octopus were 0.02, 0.10, and 0.08 mg/kg, respectively. The consumption of 150 g (usual portion) of boiled octopus is advisable because it contributes to significant daily intakes of EPA+DHA (>100%), selenium (75.6%), and iodine (12.4%), and 25% of the daily adequate intake of sodium for adults.

Chemical Composition, Nutritional Value, and Safety of Cooked Female Chaceon Maritae from Namibe (Angola).

Despite being highly appreciated and consumed, the nutritional value of Chaceon maritae from Namibe (Angola) had never been studied. In the present work, edible tissues (muscle, ovaries, and hepatopancreas) of boiled female C. maritae caught off Namibe coast in two distinct seasons were analyzed in terms of proximate chemical composition (fat, ash, protein, and moisture), fatty acid and amino acid profiles, cholesterol, essential minerals (macro and trace) and toxic elements. Results showed that, in both seasons, C. maritae muscle was a valuable source of protein, essential amino acids, polyunsaturated fatty acids, and essential elements, especially zinc, selenium, iodine, and copper. Ovaries and hepatopancreas are also good sources of protein, but were richer in fat, particularly when caught in October. Ovarian fat is rich in polyunsaturated fatty acids and that of hepatopancreas has higher values of monounsaturated and saturated fatty acids. Hepatopancreas and ovaries are also good sources of copper and, especially ovaries, of zinc. Moreover, in both seasons, all the edible tissues of C. maritae analyzed presented very low contents of heavy metals (mercury, cadmium, lead, and arsenic).

Molecular Mechanisms Mediating Antiangiogenic Action of the Urokinase Receptor-Derived Peptide UPARANT in Human Retinal Endothelial Cells.

PURPOSE: To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. METHODS: The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. RESULTS: UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. CONCLUSIONS: These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.

Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

Probiotic-loaded microcapsule system for human in situ folate production: Encapsulation and system validation.

This study focused on the use of a new system, an alginate|Ɛ-poly-l-lysine|alginate|chitosan microcapsule (APACM), able to immobilize a folate-producing probiotic, Lactococcus lactis ssp. cremoris (LLC), which provides a new approach to the utilization of capsules and probiotics for in situ production of vitamins. LLC is able to produce 95.25±26µg·L-1 of folate, during 10h, and was encapsulated in the APACM. APACM proved its capacity to protect LLC against the harsh conditions of a simulated digestion maintaining a viable concentration of 6logCFU·mL-1of LLC. A nutrients exchange capacity test, was performed using Lactobacillus plantarum UM7, a high lactic acid producer was used here to avoid false negative results. The production and release of 2g·L-1 of lactic acid was achieved through encapsulation of L. plantarum, after 20h. The adhesion of APACM to epithelial cells was also quantified, yielding 38% and 33% of capsules adhered to HT-29 cells and Caco-2 cells, respectively.

Anti-angiogenic Therapy in Cancer: Downsides and New Pivots for Precision Medicine.

Primary solid tumors originate close to pre-existing tissue vasculature, initially growing along such tissue blood vessels, and this phenomenon is important for the metastatic potential which frequently occurs in highly vascularized tissues. Unfortunately, preclinic and clinic anti-angiogenic approaches have not been very successful, and multiple factors have been found to contribute to toxicity and tumor resistance. Moreover, tumors can highlight intrinsic or acquired resistances, or show adaptation to the VEGF-targeted therapies. Furthermore, different mechanisms of vascularization, activation of alternative signaling pathways, and increased tumor aggressiveness make this context even more complex. On the other hand, it has to be considered that the transitional restoration of normal, not fenestrated, microvessels allows the drug to reach the tumor and act with the maximum efficiency. However, these effects are time-limited and different, depending on the various types of cancer, and clearly define a specific "normalization window." So, new horizons in the therapeutic approaches consist on the treatment of the tumor with pro- (instead of anti-) angiogenic therapies, which could strengthen a network of well-structured blood vessels that facilitate the transport of the drug.

PJ-34 inhibits PARP-1 expression and ERK phosphorylation in glioma-conditioned brain microvascular endothelial cells.

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

An in vitro retinoblastoma human triple culture model of angiogenesis: a modulatory effect of TGF-ß.

Retinoblastoma is the most common intraocular tumour in children. In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumour and cells constituting retinal microvessel, Transwell co-cultures constituted by human retinal endothelial cells (HREC), pericytes (HRPC), and human retinoblastoma cell line Y-79 were performed. Y-79 enhanced HREC proliferation, reduced by the introduction of HRPC in triple culture. In HREC/HRPC cultures, TGF-ß in media increased, decreasing in triple cultures. High VEGF levels in triple cultures witnessed the establishment of a strongly in vitro angiogenic environment. Y-79 induced in HREC an increase in c- and iPLA2, phospho-cPLA2, inducible COX-2 protein expressions, PLA2 activities and prostaglandin E2 (PGE2) release. These effects were attenuated when HRPC were introduced in triple culture. Moreover, antibody silencing of TGF-ß demonstrated a strong correlation between the signalling pathway triggered by TGF-ß of pericytal origin and the phospholipase activation and the modulation of PGE2 release. Inhibiting VEGFA effect, the HRPC loss in triple culture decreased, showing its modulatory effect on their survival. Relying on the data here presented, sustaining the pericytal survival in a tumour retinal environment could ensure the integrity of microvessels and the TGF-ß supply, essential for controlling aberrant endothelial pruning and angiogenesis.

Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?

In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumor and cells constituting microvessel, a triple culture model in which endothelial cells (EC), pericytes (PC) and glioma C6 cells were cultured together. In the present work, we observed that C6 enhanced EC proliferation. This effect was reduced by cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2), cyclooxygenase-2 (COX-2), PI3-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA2s. In EC, C6 induced an increase in iPLA2, cPLA2 and COX-2 total protein expression. Moreover, the increase in endothelial cPLA2 phosphorylation was attenuated by kinase inhibitors. Both EC proliferation and signal protein phosphorylation were attenuated when PC were in triple culture. In EC/C6 supernatants, and, in a lesser extent, in EC/PC co-cultures, an enhancement in prostaglandins E2 (PGE2) was found. The presence of PC in triple-cultures caused a decrease in production of PGE2 respect to EC/C6 double-cultures. In all systems, AACOCF3 and BEL significantly reduced PGE2 secretion. In Matrigel-based assays, emerging branch points from EC cell bodies and tubule-like structures were observed. C6 conditioned EC/PC co-cultures in constituting poorly organized tubules. Transfection of EC with c- and iPLA2 siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKCα, ERK kinase phosphorylation, PLA2s and COX-2 activation, and PGE2 production in EC stimulated by tumor cells are coincident phenomena and could represent therapeutic targets in chemoprevention of glioma. Moreover, PC exhibited an important "modulating" role in the initial stages of angiogenesis driven by a brain tumor.

Role of phospholipases A2 in diabetic retinopathy: in vitro and in vivo studies.

Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A2 (PLA2) in diabetic retinopathy using an in vitro blood-retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA2 activity, VEGF and PGE2 levels and cell proliferation were measured, with or without PLA2 inhibition. Diabetes was induced in rats by STZ injection and PLA2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA2 activity, PGE2 and VEGF in EC/PC co-cultures; inhibition of PLA2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA2. PLA2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA2 inhibitor. In conclusion, the present findings indicate that PLA2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA2 may be an interesting target in managing diabetic retinopathy.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier.

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film.

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.