| The mammalian placenta is rich in blood vessels to ensure the supply of oxygen and nutrients for fetal growth and development. The placenta angiogenesis affects fetal development. Data showed that leptin could promote angiogenesis, but there were reports on anti-angiogenic activity of leptin. The impact of leptin dose on angiogenesis is unclear. The level of leptin in mother’s serum is related to fetal development and it is possible that leptin affects placental angiogenesis. Like the mammalian placenta, the chorioallantoic membrane (CAM) of chicken embryo is rich in blood vessels and serves as an organ for gas and nutrient exchange. Therefore, the angiogenesis of CAM may affect the embryo growth and hence hatch weight in the chicken. Our studies showed that there are leptin-like immunoreactive substances deposited in the egg albumen and yolk and, injection leptin in egg albumen can affect the blood vessels area of CAM and hatching weight, in a breed and sex dependent manner. The effect of leptin injection in yolk on CAM angiogenesis and embryos development has not been reported and, the mechanisms underlying the effect of leptin on CAM angiogenesis is also unknown. The present study was, therefore, aimed at the determination of the effects and mechanisms of leptin on CAM angiogenesis and embryo development using Wen’s chicken line N414as a model, in vivo and in vitro.1Effects of leptin on chorioallantoic membrane angiogenesis200fertilized eggs were windowed at embryonic day7(E7). Gelatin sponge containing0ng,10ng,100ng,1000ng,5000ng leptin was placed on CAM at E8. After48h, CAM was fixed for assay the number of blood vessels using the Image-Pro Plus4.5software. The results showed that10ng leptin significantly decreased the number of small-sized blood vessels (P<0.05);100ng leptin had no effect on the number of small-sized blood vessels (P>0.05),1000ng and5000ng leptin significantly increased the number of the small blood vessels (P<0.05). The impact of leptin on angiogenesis was only seen in female embryos, but not in males.10ng,100ng,1000ng and5000ng leptin had no effect on the number of big-sized and median-sized blood vessels.500fertilized chicken eggs from Wen’s group (Guangdong, China) were randomly divided into five groups. Some fertilized chicken eggs were injected in albumen with0(AC group) or0.5μg (AL group) recombinant mouse leptin (498-OB-01M, R&D, USA) in100uL of phosphate-buffered saline (PBS) before incubation. Other fertilized chicken eggs were injected in yolk with0(YC group),0.5μg (YL group) or1μg (YH group) recombinant mouse leptin in100μL PBS. Injected eggs were transferred into an incubator set at38℃and70%humidity and equipped with an automatic rotator (Wansheng Company, China). On E12,40embryos per group were weighed and CAM was fixed for assay the number and area of blood vessels. The results showed that0.5μg leptin injected in albumen significantly reduced area of blood vessel and the number of small-sized vessels on CAM (P<0.05), and the suppression effects on blood vessel were consistent with lower weight at E12and at hatch (P<0.05).0.5μg leptin injected in yolk had no effects on area of blood vessel and the number of small-sized vessels on CAM (P>0.05), and no change was observed in embryonic weight and hatch weight (P>0.05).1μg leptin injected in yolk significantly reduced area of blood vessel and the number of small-sized vessels on CAM (P<0.05), but there was no change in embryonic weight and hatch weight (P>0.05). The suppression effects of leptin had been only manifested in the females, but not in males.These results suggest that effect of leptin on angiogenesis is dose-dependent. Lower dose leptin inhibits angiogenesis, while higher dose leptin stimulates angiogenesis.0.5μg leptin injected in albumen significantly inhibited CAM angiogenesis and embryos development;0.5μg leptin injected in yolk had no effect on CAM angiogenesis and embryos development;1μg leptin injected in yolk suppressed CAM angiogenesis, but not embryos development.2Mechanisms of leptin injected in albumen and yolk on chorioallantoic membrane angiogenesisTo further clarify the mechanism of the differential impact of leptin injected in albumen and yolk on CAM angiogenesis, CAM was collected for determination of mRNA and protein that are relative to angiogeneses and chorioallantoic fluid was collected to detect NO production. The results showed that0.5μg leptin injected in albumen significantly reduced the expression of vascular endothelial growth factor (VEGF) mRNA and protein (P<0.05), while endothelial and inducible nitric oxide synthase (eNOS and iNOS) mRNA expression and activity were significantly lower in AL group than that of AC group (P<0.05). Total nitric oxide synthase (TNOS) activity and nitric oxide (NO) production in allantoic fluid were lower in AL group (P<0.05). This suppression of VEGF and NO was only manifested in female embryos, but not in males.0.5μg leptin injected in albumen had no effect on FGF2mRNA expression and protein content (P>0.05).1μg leptin injected in yolk had no effect on the expression of VEGF mRNA and protein (P>0.05), while eNOS and iNOS mRNA expression and activity had no change in YH group (P>0.05). Then the activity of TNOS and NO production in YH group was the same as that in YC group (P>0.05).1μg leptin injected in yolk significantly reduced FGF2mRNA expression and protein content of female embryos in YH group (P<0.05), but not males.In mammals, leptin increased the binding of signal transducer and activator of transcription-3(STAT3) to VEGF gene promoter and subsequently the expression of VEGF mRNA. Although no difference was detected in level of LepR mRNA or protein in CAM, STAT3was significantly decreased at the level of mRNA (P<0.05) and tended to be decreased at the level of protein (P=0.063) in leptin-treated female chick embryos. The results of Chromatin immunoprecipitation (ChIP)assay also showed that the binding of STAT3to VEGF promoter was significantly decreased in female chicken embryos (P<0.05) treated with leptin. No difference between treatments was found in males.These results suggest that leptin injected in albumen inhibited CAM angiogenesis in female chicken embryos through the STAT3-mediated VEGF-NO pathway, while leptin injected in yolk affected CAM angiogenesis by changing the level of FGF-2mRNA and protein.3Effects of leptin on STAT3-mediated VEGF-NO pathway in CAM cellsTo futher study the effects of leptin on STAT3-mediated VEGF-NO pathway, chicken chorioallantoic membrane cells were cultured in FK12. Results showed that the level of LepR mRNA and protein was significantly increased (P<0.05) in cells when1ng/mL leptin was added; or tended to increase (P=0.058and P=0.063) when10ng/mL leptin was added in culture.1ng/mL and10ng/mL leptin significantly increased the level of STAT3mRNA and protein (P<0.05).1ng/mL and10ng/mL leptin significantly improved the expression of VEGF mRNA in cells and protein content in supernatant (P <0.05). The activity of TNOS in cells showed a trend of increase (P-0.07and P=0.06) and the production of NO in supernatant was significantly increased, which resulted from increased eNOS mRNA and activity, but not iNOS.1ng/mL and10ng/mL leptin significantly increased the level of FGF2mRNA in cells and protein content in supernatant (P<0.05).100ng/mL leptin had no effect on the level of mRNA and protein of LepR, STAT3, VEGF, FGF2or NOS activity.These results suggest that leptin affects STAT3-mediated VEGF-NO signaling pathway. The contradiction between in vitro and in vivo results may be caused by different conditions of the tissue/cells or different doses of leptin. |
