Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1ß), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

Multiple LEDT wavelengths modulate the Akt signaling pathways and attenuate pathological events in mdx dystrophic muscle cells.

This study is aimed at investigating the effects of LEDT, at multiple wavelengths, on intracellular calcium concentration; on transient receptor potential canonical channels; on calcium-binding protein; on myogenic factors; on myosin heavy chains; on Akt signaling pathway; on inflammatory markers; and on the angiogenic-inducing factor in dystrophic muscle cell culture experimental model. Dystrophic primary muscle cells were submitted to LEDT, at multiple wavelengths (420 nm, 470 nm, 660 nm, and 850 nm), and evaluated after 48 h for cytotoxic effects and intracellular calcium content. TRPC-1, TRPC-6, Calsequestrin, MyoD, Myogenin, MHC-slow, MHC-fast, p-AKT, p-mTOR, p-FoxO1, Myostatin, NF-κB, TNF-α, and VEGF levels were evaluated in dystrophic primary muscle cells by western blotting. The LEDT, at multiple wavelengths, treated-mdx muscle cells showed no cytotoxic effect and significant lower levels in [Ca2 +]i. The mdx muscle cells treated with LEDT showed a significant reduction of TRPC-1, NF-κB, TNF-α and MyoD levels and a significant increase of Myogenin, MHC-slow, p-AKT, p-mTOR, p-FoxO1 levels, and VEGF levels. Our findings suggest that different LEDT wavelengths modulate the Akt-signaling pathways and attenuate pathological events in dystrophic muscle cells, and a combined multiwavelength irradiation protocol may even provide a potentially therapeutic strategy for muscular dystrophies.

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity / Caracterização morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e da expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus em cativeiro

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).(AU)

Foram estudadas as caracterizações morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e a expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus de cativeiro. A avaliação morfológica dos ovários foi realizada por cortes histológicos incluídos em parafina e corados por HE. As expressões imuno-histoquímicas de CDC47, VEGF e seu receptor Flk-1, angiopoetina e seu receptor Tie-2 e recertor tireoidiano (TRα) foram realizadas pela técnica de estreptavideina-biotina-peroxidade. A apoptose foi avaliada utilizando-se kit de TUNEL. A expressão relativa dos receptores de hormônios tireoidianos (TRα e TRβ) foi avaliada pela técnica de RT-PCR tempo real. A expressão nuclear de CDC47 aumentou com a fase de maturação do oócito e foi observada nas células foliculares. Corpos apoptóticos foram observados nas células foliculares de folículos atrésicos e folículos pós-ovulatórios de ovários de peixes com 150g e 350g. A expressão de VEGF e do seu receptor Flk-1 foi também observada nas células foliculares , e a expressão de ambos aumentou com a maturidade do oócito , com uma maior intensidade no folículo maduro. A expressão de angiopoietina e do seu receptor (Tie 2) foi discreta e moderada, respectivamente. A expressão de TRα foi independente do desenvolvimento folicular. No entanto, a tilápia de 350g apresentou maior expressão de TRβ em comparação com a tilápia de 50g. Conclui-se que a atividade proliferativa e a expressão de VEGF e de seu receptor aumenta com a maturação folicular e que a expressão dos TRs aumenta com a maturidade do ovário em tilápia (Oreochromis niloticus).(AU)

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity / Caracterização morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e da expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus em cativeiro

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRß) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRß compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).(AU)

Foram estudadas as caracterizações morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e a expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus de cativeiro. A avaliação morfológica dos ovários foi realizada por cortes histológicos incluídos em parafina e corados por HE. As expressões imuno-histoquímicas de CDC47, VEGF e seu receptor Flk-1, angiopoetina e seu receptor Tie-2 e recertor tireoidiano (TRα) foram realizadas pela técnica de estreptavideina-biotina-peroxidade. A apoptose foi avaliada utilizando-se kit de TUNEL. A expressão relativa dos receptores de hormônios tireoidianos (TRα e TRß) foi avaliada pela técnica de RT-PCR tempo real. A expressão nuclear de CDC47 aumentou com a fase de maturação do oócito e foi observada nas células foliculares. Corpos apoptóticos foram observados nas células foliculares de folículos atrésicos e folículos pós-ovulatórios de ovários de peixes com 150g e 350g. A expressão de VEGF e do seu receptor Flk-1 foi também observada nas células foliculares , e a expressão de ambos aumentou com a maturidade do oócito , com uma maior intensidade no folículo maduro. A expressão de angiopoietina e do seu receptor (Tie 2) foi discreta e moderada, respectivamente. A expressão de TRα foi independente do desenvolvimento folicular. No entanto, a tilápia de 350g apresentou maior expressão de TRß em comparação com a tilápia de 50g. Conclui-se que a atividade proliferativa e a expressão de VEGF e de seu receptor aumenta com a maturação folicular e que a expressão dos TRs aumenta com a maturidade do ovário em tilápia (Oreochromis niloticus).(AU)

Expressão gênica de fatores angiogênicos na placenta de ratas submetidas ao estresse / Expression of the angiogenic factors in the placenta of stressed rats

Objetivos: Avaliar a influencia do estresse sobre os pesos maternos, placentários e fetais, as alterações histológicas placentárias e a expressão gênica dos fatores angiogênicos em ratas prenhes. Métodos: De setembro de 2007 a julho de 2008, foi realizado um estudo experimental do tipo casocontrole, em que 6 ratas prenhes foram submetidas a estímulos estressantes crônicos e agudos enquanto que 6 animais constituíram o grupo controle. No 20º dia de prenhez, todas as ratas foram sacrificadas. Os seguintes dados foram analisados e comparados entre os grupos: a. pesos maternos, placentários e fetais; b. alterações histológicas placentárias; e, c. expressão gênica dos fatores angiogênicos (VEGF-A e PlGF), dos seus receptores (VEGFR-1 e VEGFR-2) e do fator induzido por hipoxia (HIF-1). Resultados: Os pesos maternos, placentários e fetais foram significativamente menores no grupo de ratas prenhes-estressadas em relação ao controle. Histologicamente foram encontradas a presença de núcleos picnóticos no grupo prenhe-estressado. Observou-se expressão gênica significativamente maior de VEGF-A no grupo rata prenhe-estressada assim como redução significante da expressão gênica de PlGF, VEGFR-1, VEGFR-2, quando comparadas ao controle. Não houve alterações entre os grupos para o gene HIF-1. Conclusões: No modelo animal de estresse estudado, observou-se alterações significativas no peso placentário e da expressão gênica dos fatores angiogênicos placentários em ratas submetidas ao estresse.

Objectives: to evaluate the influence of the stress on the maternal, placental and fetal weights, on the histological findings and on the genetic expression of the angiogenic factors in pregnant rats. Methods: From September 2007 to Julie 2008, an experimental case-control study was conduced in which 6 pregnant rats were submitted to chronic and acute stress while six other were considered as controls. In the 20th day of gestation, all animals were sacrified. The following data were evaluated and compared between both groups: a. maternal, placental and fetal weights; b. histological findings; and c. genetic expression of angiogenic factors (VEGF-A and PIGF), receptors (VEGFR-1 and VEGFR-2) and hypoxic-induced factor (HIF- 1). Results: Maternal, placental and fetal weights were statistically smaller in the stressed animals comparing to controls. Histological analysis revealed picnotic nuclei in the placentas of stressed rats. A statistically significant increase in the genetic expression of VEGF-A as well as a reduction of the expression of the PlGF, VEGFR-1, VEGFR-2 were observed in the placentas of the stressed group in comparison to controls. There was no difference of expression of the gene HIF-1 between both groups. Conclusions: In the present animal model of stress, significant alterations in the placental weights, histology and genetic expression of the angiogenic factors among pregnant rats under stress.