Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage

Enfermedad cardiovascular; Terapia celular; SecretomaMalaltia cardiovascular; Teràpia cel·lular; SecretomaCardiovascular disease; Cell therapy; SecretomeBackground Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. Methods Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). Results The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. Conclusion Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.This work has been supported under the Euronanomed 8th Joint Call-MAGGBRIS collaborative project by grants from the Spanish Ministry of Science and Innovation (PCIN-2017-090) the French national agency (ANR-ANR-17-ENM3-0005-01), the AC17/00004 grant from Instituto Carlos III (ISCIII) with FEDR funds, and the National Centre for Research and Development (NCBR 15/EuronanoMed/2018). A part of this study has been also funded in the frame of the NANOSTEM project, a Marie Skłodowska-Curie Innovative Training Network (ITN) by receiving grant from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 764958 and the Expression of Interest (EoI) for Collaborative Projects on Regenerative Medicine 2019 P-CMR[C]), and the programs 2017-SGR-1427 and 2017-SGR-765 from the Generalitat de Cataluny. Alba Grayston is supported by the fellowship FI17/00073 from ISCIII with FEDR funds. Miguel Garcia-Gabilondo is supported by the PERIS grant SLT017/20/000197 from Generalitat de Cataluny. The mass spectrometer of the Spectrométrie de Masse de l’Artois (SMART) facilities used in this study was funded by the European Regional Development Fund (ERDF), the Hauts-de-France regional council, and the Université d’Artois (France)

Progesterone Levels and Reproductive Parameters in the Periovulatory Period of Nellore Cows under FTAI protocol

Background: Fixed Time Artificial Insemination (FTAI) has achieved a significant evolution in the last 18 years, however, despite the progress achieved by modern FTAI programs, the conception rates obtained are still low. Therefore, this study aimed to evaluate the interrelation between progesterone levels in the periovulatory period and reproductive parameters of Nellore cows submitted to an FTAI protocol. Materials, Methods & Results: On a random day, called day 0 (D0), 57 cows received a P4 device associated with the intramuscular (IM) application of 2.0 mg of estradiol benzoate. On D9, the P4 devices were removed and then were administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 IUI of Equine Chorionic Gonadotrophin IM. Blood samples were collected for the determination of serum P4 concentrations on D9 and D11 of the protocol. The evaluations of follicular diameter (DFOL), follicular wall area (AFOL) and the vascularization area of the follicle wall (VFOL) were carried out on D11 using B-mode ultrasonography examination and colour Doppler, and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), of the total corpus luteum area (CLA), of the area of corpus luteum vascularization (CLV) and blood sampling for determination of post-ovulatory P4 levels (Post-P4) were performed on D24. For the analysis of the P4 concentration the chemiluminescence method was used, with a sensitivity of 0.1 ng/mL. According to the P4 concentrations on D11, cows were divided into 2 groups, LOW LEVELS OF P4 and HIGH LEVELS OF P4. The diagnosis of pregnancy was performed using transrectal ultrasonography on D45, at this point the cows were divided into 2 groups, PREGNANT and NON-PREGNANT. The correlation between DFOL and P4 dosage on D11 was moderate, negative and significant and between the AFOL and the serum P4 levels on D9, was moderate, negative and significant. As for the other correlations between follicular and luteal parameters and serum P4 levels, these were low to moderate, negative and not significant. Cows in the LOW LEVELS OF P4 group had significantly larger diameter and follicular areas than the cows in the HIGH LEVELS OF P4 group, the other follicular and luteal parameters showed no statistical difference. Of the total 57 cows that were inseminated, 30 cows became pregnant. Cows in the PREGNANT group had serum P4 levels on D9 equivalent to that obtained by the NON-PREGNANT group. However, at D11 the cows that became pregnant presented significantly lower serum P4 levels than cows that did not become pregnant. Discussion: The results of the interrelation between follicular parameters and P4 levels obtained in the present study, pointed out that the lower the levels of P4, the higher the follicular parameters, corroborating with other authors. Thus, larger preovulatory follicles provided high ovulation rates. Periovulatory serum P4 levels did not significantly affect the morphofunctional parameters of the CL. Such findings may be justified by high periovulatory P4 levels resulting from less efficient luteolysis, exert a negative effect on the results of FTAI protocols, because progesterone inhibits the release of LH pulses. It is concluded that lower periovulatory P4 levels established a favourable condition for follicular development and fertility, however, morphofunctional parameters of the corpus luteum were not affected. Keywords: follicular development, fertility, conception rate, ovarian hemodynamics, synchronization. Título: Níveis de progesterona e os parâmetros reprodutivos no período periovulatório de vacas Nelore submetidas ao protocolo de IATF. Descritores: crescimento folicular, fertilidade, índice de concepção, hemodinâmica ovariana, sincronização

Effect of the Estrus Expression on Follicular and Luteal Morphofunctional Characteristics and Fertility in Bos indicus females Synchronized for FTAI

Background: Recent studies have been conducted with the aim of improving the fertility rates in the FTAI programs in beef females. The observation of the estrus expression constitutes an important indicator of fertility in zebu females. Therefore, this work has as an objective to evaluate the impact of the estrus expression on the follicular, luteal and fertility morphofunctional characteristics of Nelore females synchronized for FTAI.Materials, Methods & Results: Sixty five lactating female Nelore (Bos taurus indicus) were used. On a random day, denominated day 0 (D0), the 65 Nelore females received a progesterone-releasing device associated to the application of 2.0 mg of estradiol benzoate intramuscularly (IM). On D9, the progesterone-releasing intravaginal devices were removed and was administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 UI of Equine Chorionic Gonadotropin IM. At this point, the animals were marked with a marking stick for the determination of the estrus expression. On D11 of the synchronization protocol, the animals were characterized in two groups: without estrus expression (WO/ ESTRUS) and with estrus expression (W/ ESTRUS). The evaluation of the follicle diameter (FOLD), of the follicle wall area (FOLA), of the follicle wall vascularization (FOLV) and the percentage of vascularization in the area of the preovulatory follicle wall (%FOLV) were conducted on D11 using B-mode ultrasonography and color Doppler and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), the total area of the corpus luteum (CLA), of the area of vascularization of the corpus luteum (CLV), of the percentage of vascularization of the in the area of the corpus luteum (% CLV) and the collection of blood for the evaluation of the serum levels of progesterone were carried out on D24. The analysis of the serum progesterone concentration were performed by the chemiluminescence method using the Access immunoassay systems Progesterone, with a sensitivity of 0.1 ng/mL. The diagnosis of gestation was carried out by transrectal ultrasonography on D45.  The animals of the WITH ESTRUS group presented superior FOLD, FOLA, FOV (P < 0.05) averages to those presented by the cows of the WITHOUT ESTRUS group. In relation to the luteal characteristics, in the WITH ESTRUS group the cows exhibited averages for CLD, CLA, CLV and P4 significantly higher than those of the WITHOUT ESTRUS group. However, the bovines of the WITH ESTRUS group presented a % CLV similar to the presented by the females of the WITHOUT ESTRUS group. As for the fertility rates, there was difference between the experimental groups WITH ESTRUS and WITHOUT ESTRUS.Discussion: The present work demonstrated the impact of the estrus expression on the follicular morphological parameters, therefore in accordance with other studies. These authors show a relation between the estrus expression and levels of estradiol, which is directly related to the sperm transport in the female reproductive tract, oocyte maturation and fertilization directly affecting the fertility of females submitted to the FTAI program. Similarly, there was an interrelation between the estrus expression and the luteal morphological parameters, corroborating with the findings of some researches, where were verified higher luteal morphological parameters associated to higher conception rates in cows that demonstrated estrus behavior in the FTAI. Therefore, the estrus detection can be used as a tool to direct matings in synchronization protocols, promoting the identification of the animals with a higher probability of conception, owing to better follicular and luteal hemodynamic conditions

Participação do sector privado na Cooperação: da teoria à prática

Comunicação apresentada no 3º Encontro Conhecimento e Cooperação, INA, Lisboa, 17 de setembro de 201

Formyl Peptide Receptors and Annexin A1: Complementary Mechanisms to Infliximab in Murine Experimental Colitis and Crohn's Disease

Non-responsiveness to anti-TNF-α therapies presents relevant rates in inflammatory bowel disease patients, presenting the need to find biomarkers involved in therapeutic efficacy. Herein, we demonstrate that higher levels of colonic formyl peptide receptor 1 and annexin A1 correlate with histological recovery in Crohn’s disease patients under remission. Using the dextran sulfate sodium colitis model in mice, we suggest that infliximab induces annexin A1 expression and secretion in activated intestinal leukocytes. Conversely, this mechanism might stimulate epithelial formyl peptide receptors, inducing wound healing and consequent histological remission. Our data indicate that assessing intestinal expressions of formyl peptide receptors and annexin A1 might provide precious information on the disease activity and responsiveness to infliximab in inflammatory bowel disease patients

Angiotensin II induced venoconstriction in normotensive and hypertensive rats: study of action mechanisms, localization and expression of AT1 and AT2 receptors.

Neste estudo, avaliamos e caracterizamos o efeito de Ang II em leito venular mesentérico (LVM) e anéis de veia porta (AVP) de wistar e SHR. A reatividade vascular para Ang II foi estudada na presença e ausência de diferentes antagonistas para elucidar os mecanismos envolvidos na venoconstrição induzida por Ang II. Nossos resultados sugerem que a Ang II induz venoconstrição através da ativação de receptores AT1 e AT2 em Wistar e receptor AT1 em SHR. Essa venoconstrição parece ser contrabalanceada pelo receptor B2 em ambas as espécies. NO contribue para este efeito em wistar e metabólitos da COX em SHR. Há redução da venoconstrição induzida por Ang II em AVP de SHR que pode estar relacionado a redução da expressão protéica de receptores AT2. Esses resultados indicam diferentes mecanismos de regulação do tônus venoso de ratos wistar e SHR em resposta a Ang II que podem ter relevância no controle do retorno venoso e débito cardíaco.In this study we have evaluated and characterized the effect of Ang II in the isolated perfused mesenteric venular bed (MVB) and portal vein rings (PVR) of SHR and wistar rats. Vascular reactivity to Ang II was studied in the absence and presence of different antagonists to elucidate the mechanisms involved in Ang II-induced venoconstriction. Our results suggest that Ang II induces venoconstriction by activation of AT1 and AT2 receptors in wistar and AT1 receptor in SHR. These venoconstriction seems to be counterbalanced by B2 receptor in both species. NO might contribute to this effect in wistar and COX metabolites in SHR. There is a decrease in Ang II induced venoconstriction in PVR of SHR that might be related to the decrease of protein expression of AT2 receptor. These results indicate different mechanisms of regulation of venous tonus of the SHR and wistar rats induced by Ang II that can be relevant in the control of the venous return and cardiac output

Influence of targeted deletion of kinins receptors in the vascular nitric oxide metabolism

Activation of B1 receptor in the vascular endothelium triggers diverse signaling pathways that results in elevation of intracellular Ca2+ and Nitric Oxide Synthase (NOS) activation, followed by NO production and vasodilation. Although much has been investigated about the B1-induction and functionality during inflammation, the importance of B1 subtype in normal vessels remains unclear. To clarify this question, the present study analyzed endothelial function and endothelial NO generation in B1 receptor knockout (B1 -/-) and Wild Type (WT) mice. Mesenteric arteriolar bed was perfused with Krebs solution and vascular responses to Acetilcholine (ACh), sodium nitroprusside (SNP) and norepinephrine (NE) were evaluated by a data acquisition system. Plasmatic NO levels (μmol/L) were analyzed by NO derivatives nitrate and nitrite using NO Analyzer (NOATM280, Sievers Instruments) and vascular NO generation was assessed in mesenteric arterioles slices using DAF -2 DA, a fluorescent cell permeable dye for NO (arbitrary units, a.u.). NOS activity (pmol/mg.min) was measured by the biochemical conversion of L-[3H] arginine to L-[3H] citrulline in homogenates of mesenteric vessels in the presence of optimal levels of substrate and co-factors. Primary endothelial cells were incubated with DAF-2 DA and images obtained in a confocal microscope were analyzed by optic densitometry (a.u.). Cells were stimulated with ACh [1 mmol/L] in presence or absence of the NOS substrate Larginine, or the co-factor tetrahydrobiopterin (BH4), or the antioxidant compound ascorbic acid. Production of superoxide anion (a.u.) was assessed in endothelial cells incubated with dihydroethidine, a fluorescent cell permeable dye for superoxide anion, in the presence or absence of BH4 or ascorbic acid. Mesenteric arterioles from B1 -/- exhibited a severe impairment of ACh-vasodilation for all tested doses, with no changes in the response to SNP and NE. Circulating NO was markedly decreased in B1 -/- (49.6 ± 10.5*; n=6) vs WT (141.9 ± 17.3; n=6 ), accompanied by reduced basal NO release in mesenteric arterioles from B1 -/- (0.16 ± 0.03*; n=6) when compared to WT (0.58 ± 0.08; n=4). NOS activity was elevated in mesenteric homogenates from B1 -/- (3.4 ± 0.58*; n=4) in comparison to WT (1.9 ± 0.05; n=5). ACh-induced NO release was markedly reduced in primary cultured endothelial cells from B1 -/- (35.8 ± 3.1*; n=4) in comparison to WT cells (66.9 ± 3.2; n=4). NO release in endothelial cells from B1 -/- was reversed by incubation with BH4 (54.3 ± 1.7; n=4) and ascorbic acid (101.8 ± 6.0; n=4), but not by L-arginine, while incubation of endothelial cells from WT with BH4, ascorbic acid or L-arginine had no effect. Elevated production of superoxide anion in endothelial cells from B1 -/- (77,1 ± 2,5*; n=4) in comparison to WT (29,3 ± 6,9; n=4) was reversed by incubation with ascorbic acid (35,3 ± 6,4; n=3). The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation. Reduced NO availability may be preceded by exacerbation of NO inactivation by superoxide anion, which can leads to inactivation of BH4 in vascular endothelium, resulting in NOS uncoupling and NOS derived production of superoxide anion.A ativacao de receptores B1 de cininas no endotelio vascular desencadeia vias de sinalizacao que resultam na elevacao do Ca+2 intracelular e ativacao da enzima oxido nitrico sintase (NOS), seguido por producao de NO e vasodilatacao. Embora a inducao do receptor B1 e sua funcao durante a inflamacao tenha sido abordada por diversos estudos, a importancia de receptores B1 na homeostase vascular em condicoes fisiologicas nao esta totalmente elucidada. Para esclarecer essa questao, o presente estudo analisou a funcao endotelial e a producao de NO em camundongos nocaute do receptor B1 (B1 -/-) e selvagens (WT). O leito arteriolar mesenterico foi perfundido por solucao Krebs e respostas vasculares para Acetil-colina (ACh), nitroprussiato de sodio (SNP) e norepinefrina (NE) foram analisadas por um sistema de aquisicao de dados. Niveis plasmaticos de NO (ƒÊmol/L) foram analisados por deteccao dos derivados nitritos/nitratos atraves de metodo de quimioluminescencia e a producao vascular de NO foi avaliada em cortes histologicos de arteriolas mesentericas incubadas com DAF-2 DA, um marcador fluorescente de NO (unidades arbitrarias, u.a.). A atividade da NOS (pmol/mg.min) foi mensurada atraves da conversao bioquimica de L-[3H]arginina para L-[3H]citrulina em homogenatos de vasos mesentericos na presenca de substrato e co-fatores. Celulas endoteliais primarias foram incubadas com DAF-2 DA e as imagens obtidas em microscopio confocal foram analisadas por densitometria optica (u.a.). Celulas foram estimuladas com ACh [1 mmol/L] na presenca ou ausencia de Larginina, o substrato da NOS, ou tetrahidrobiopterina (BH4), co-fator da NOS, ou acido ascorbico, composto antioxidante. Producao de anion superoxido (u.a.) foi avaliada em celulas endoteliais incubadas com di-hidroetidina, um marcador fluorescente de anion superoxido, na presenca ou ausencia de BH4 ou acido ascorbico. Arteriolas mesentericas de B1 -/- exibiram severo comprometimento da vasodilatacao mediada por ACh, sem alteracoes na resposta ao NPS e NE. Os niveis circulantes de NO foram consideravelmente reduzidos em B1 -/- (49,6 } 10,5*; n=6) vs WT (141,9 } 17,3; n=6 ), acompanhado por reducao da producao basal de NO em arteriolas mesentericas de B1 -/- (0,16 } 0,03*; n=6) quando comparado a WT (0,58 } 0,08; n=4). A atividade da NOS foi elevada em amostras de B1 -/- (3,4 } 0,58*; n=4) em comparacao a WT (1,9 } 0,05; n=5). A producao de NO mediada por ACh foi significantemente reduzida em celulas endoteliais de B1 -/- (35,8 } 3,1*; n=4) quando comparado a celulas de WT (66,9 } 3,2; n=4). A producao de NO em celulas endoteliais de B1 -/- foi revertida por incubacao com BH4 (54,3 } 1,7; n=4) e acido ascorbico (101,8 } 6,0; n=4), mas nao por L-arginina, enquanto incubacao de celulas endoteliais de WT com BH4, acido ascorbico ou L-arginina nao teve efeito. A producao elevada de anion superoxido em celulas endoteliais de B1 -/- (77,1 } 2,5*; n=4) quando comparado a WT (29,3 } 6,9; n=4) foi revertida pela incubacao com acido ascorbico (35,3 } 6,4; n=3). O severo comprometimento da vasodilatacao mediada pelo endotelio acompanhado por reducao da biodisponibilidade de NO, apesar do aumento da atividade da NOS, sugere a exacerbacao da inativacao de NO em endotelio de B1 -/-. A producao elevada de anion superoxido em endotelio de B1 -/- provavelmente e responsavel pela exacerbacao da inativacao de NO nestes animais. Adicionalmente, a inativacao de BH4 por peroxinitrito pode acarretar em desacoplamento da NOS e producao de anion superoxido pela enzima.TED

Evaluation of a human iPSC-derived BBB model for repeated dose toxicity testing with cyclosporine A as model compound

International audienceThe blood-brain barrier (BBB) is a highly restrictive barrier that preserves central nervous system homeostasis and ensures optimal brain functioning. Using BBB cell assays makes it possible to investigate whether a compound is likely to compromise BBBs functionality, thereby probably resulting in neurotoxicity. Recently, several protocols to obtain human brain-like endothelial cells (BLECs) from induced pluripotent stem cells (iPSCs) have been reported. Within the framework of the European MSCA-ITN in3 project, we explored the possibility to use an iPSC-derived BBB model to assess the effects of repeated dose treatment with chemicals, using Cyclosporine A (CsA) as a model compound. The BLECs were found to exhibit important BBB characteristics up to 15 days after the end of the differentiation and could be used to assess the effects of repeated dose treatment. Although BLECs were still undergoing transcriptional changes over time, a targeted transcriptome analysis (TempO-Seq) indicated a time and concentration dependent activation of ATF4, XBP1, Nrf2 and p53 stress response pathways under CsA treatment. Taken together, these results demonstrate that this iPSC-derived BBB model and iPSC-derived models in general hold great potential to study the effects of repeated dose exposure with chemicals, allowing personalized and patient-specific studies in the future

Role of vascular Kinin B-1 and B-2 receptors in endothelial nitric oxide metabolism

Kinin B-1 and B-2 receptors play an essential role in inflammatory process and cardiovascular homeostasis. the present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B-1 (B-1(-/-)) and B-2 receptor (B-2(-/-)) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B-1(-/-) and B-2(-/-) in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B-1(-/-) and B-2(-/-). in contrast, in the presence of L-arginine, Ca2+ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B-1(-/-) and B-2(-/-). the present study demonstrated that targeted deletion of B-1 or B-2 receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. the severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B-1(-/-) and B-2(-/-) vessels. the present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Biol Sci, BR-09972270 Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Clin Anal & Toxicol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-09972270 Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Clin Anal & Toxicol, São Paulo, BrazilFAPESP: 2007/59039-2FAPESP: 2008/06676-8Web of Scienc