leptin对鸡胚尿囊膜血管生成的影响及其机理
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摘要
哺乳动物的胎盘是连接胎儿和母体的重要器官,胎盘上含有丰富的血管,确保胎儿生长发育所需的氧和营养物质的供应。以往的研究表明,哺乳动物leptin能够促进血管生成,但也有关于leptin抑制血管生成报道。Leptin对血管生成效应的不一致性是否与剂量有关目前还不清楚。母体血清中leptin水平影响胎儿发育,可能与胎盘血管生成有关。鸡胚尿囊膜是禽类胚胎进行物质交换的场所,其功能类似于哺乳动物的胎盘。将含有leptin的载体放在发育的鸡胚尿囊膜上,血管生成增加。我们的前期研究发现,蛋清和蛋黄内都含有leptin样免疫活性物质,蛋清内注射leptin显著降低鸡胚尿囊膜血管面积和出雏重,并有性别依赖性和品种差异。蛋黄内注射leptin是否影响鸡胚尿囊膜血管生成和胚胎发育还不清楚。目前也没有关于蛋内注射leptin影响鸡胚尿囊膜血管生成机理的相关报道。本试验以生长速度慢的温氏N414土鸡为模型,研究不同剂量leptin对尿囊膜血管生成的影响；通过蛋清和蛋黄内注射重组鼠leptin,研究leptin对尿囊膜血管生成和鸡胚发育的影响,并进一步从体内和体外研究leptin影响尿囊膜血管生成的机理。
1leptin对鸡胚尿囊膜血管生成的影响
选200枚温氏N414土鸡种蛋进行孵化,7胚龄时开窗,8胚龄时在尿囊膜上放置含有0ng、10ng、100ng、1000ng、5000ng leptin的明胶海绵,48小时后固定尿囊膜,计算血管的数量,研究不同剂量leptin对血管生成的影响。结果显示,10ng、00ng.1000ng、5000ng leptin对尿囊膜大血管和中血管的数量没有影响(P>0.05).10ng leptin显著减少鸡胚尿囊膜小血管的数量(P<0.05),100ng leptin对小血管的数量没有影响(P>0.05),1000ng和5000ng leptin显著增加小血管的数量(P<0.05)。Leptin对小血管数量影响的剂量效应仅表现在雌性,对雄性没有影响。
选取温氏N414土鸡种蛋500枚,随机分为五组,蛋清注射组于入孵前在蛋清内注射0μg或0.5μg重组鼠leptin。蛋黄注射组于入孵前在蛋黄内注射0μg、0.5μg或1μg重组鼠leptin。孵化到12胚龄时,在蛋壳上开窗,用甲醇和丙酮混合液固定尿囊膜血管,进行观察、拍照,分析血管的面积和数量,同时称胚重、测量胚长,辨别性别。出雏当天(0日龄)称重。结果显示,蛋清内注射0.5μg leptin显著降低鸡胚尿囊膜血管总面积和小血管的数量(P<0.05),这种抑制效应和胚重及出雏重的降低是一致的(P<0.05)；蛋黄内注射0.5μg leptin对鸡胚尿囊膜血管面积和血管数量没有影响(P>0.05),对胚重和出雏重也没影响(P>0.05)；蛋黄内注射1μg leptin显著降低尿囊膜血管总面积和小血管数量(P<0.05),对胚重和出雏重没有影响(P>0.05)。Leptin对尿囊膜血管生成和胚胎发育的抑制效应仅表现在雌性,对雄性没有影响。
以上结果提示,Leptin对鸡胚尿囊膜血管生成的影响与剂量有关,低剂量leptin抑制血管生成,高剂量leptin促进血管生成。蛋清内注射0.5μg leptin显著抑制尿囊膜血管生成和胚胎发育,蛋黄内注射0.5μg leptin对尿囊膜血管生成和鸡胚发育没有影响,蛋黄内注射1μg leptin显著抑制尿囊膜血管的生成,对胚胎发育没有影响。
2蛋清和蛋黄内注射leptin影响鸡胚尿囊膜血管生成的机理
为了探讨蛋清内注射0.5μg leptin和蛋黄内注射1μg leptin对鸡胚发育造成的不同影响,本试验研究了leptin影响尿囊膜血管生成的分子机理。在前一章的基础上,采集12胚龄鸡胚尿囊膜用于]mRNA表达和蛋白含量检测,采集尿囊液用于一氧化氮(nitric oxide, NO)含量测定。结果显示,蛋清内注射0.5μg leptin显著降低鸡胚尿囊膜血管内皮生长因子(vascular endothelial growth factor, VEGF) mRNA的表达和蛋白含量(P<0.05),显著下调上皮型和诱导型一氧化氮合成酶(endothelial and inducible nitric oxide synthase, eNOS和iNOS) mRNA表达和活性,抑制TNOS活性,从而减少了下游因子尿囊液中NO的含量(P<0.05)。leptin抑制尿囊膜VEGF表达和NO合成仅表现在雌性。尽管蛋清内注射0.5μg leptin对雄性鸡胚尿囊膜VEGF mRNA的表达有降低趋势(P=0.08),但对eNOS和iNOS mRNA表达和活性以及尿囊液中NO的含量没有影响(P>0.05)。蛋清内注射0.5μg leptin对鸡胚尿囊膜碱性成纤维细胞生长因子(basic fibroblast growth factor2, FGF-2) mRNA表达和蛋白含量(P>0.05)均无显著影响。蛋黄内注射1μg leptin不影响鸡胚尿囊膜VEGF mRNA表达和蛋白含量(P>0.05),也不影响eNOS和iNOS mRNA表达和活性以及尿囊液中NO含量(P>0.05)。蛋黄内注射1μg leptin显著降低雌性鸡胚尿囊膜FGF-2mRNA表达和蛋白含量(P<0.05),对雄性胚没有影响。
在哺乳动物,leptin与受体(leptin receptor, LepR)结合后,通过增强下游因子信号转导和转录激活因子-3(signal transducer and activator of transcription3, STAT3)与VEGF启动子的结合,上调VEGF mRNA的表达。尽管蛋清内注射0.5μg leptin对鸡胚尿囊膜LepR mRNA表述和蛋白含量没有影响(P>0.05),但显著降低了雌性鸡胚尿囊膜STAT3mRNA表达(P<0.05),对STAT3蛋白含量有降低趋势(P=0.06)。染色质免疫共沉淀(CHIP)分析结果显示,蛋清内注射0.5μg leptin显著降低雌性鸡胚尿囊膜STAT3与VEGF启动子的结合(P<0.05),从而抑制VEGF基因的表达。
以上结果提示：蛋清内注射0.5μg leptin是通过改变STAT3介导的VEGF-NO信号通路来影响尿囊膜血管的生成。蛋黄内注射1μg leptin是通过影响尿囊膜上FGF-2基因表达和蛋白合成来影响尿囊膜血管生成的。
3Leptin处理对尿囊膜细胞STAT3介导的VEGF-NO通路的影响
本试验培养12胚龄鸡胚尿囊膜细胞,用不同剂量leptin处理细胞,研究leptin对尿囊膜细胞STAT3介导的VEGF-NO信号通路的影响。结果显示,leptin处理24h和48h后,尿囊膜细胞的相对活力显著下降(P<0.05).1ng/mL leptin显著上调尿囊膜细胞LepR mRNA表达和蛋白含量(P<0.05),10ng/mL leptin对LepR mRNA表达和蛋白含量有上调趋势(P=0.058和P=0.063)。1ng/mL和10ng/mL leptin上调尿囊膜细胞STAT3mRNA表达和蛋白合成,促进VEGF mRNA表达和蛋白分泌,同时增加TNOS活性(P=0.07和P=0.06),促进下游因子NO合成(P<0.05)。NO的合成是通过增加eNOS mRNA表达和活性来实现的(P<0.05),leptin处理对iNOS mRNA表达和活性没有影响(P>0.05)。1ng/mL和10ng/mL leptin还显著上调尿囊膜细胞另一血管生长因子FGF2mRNA表达和蛋白分泌(P<0.05)。100ng/mL leptin对尿囊膜细胞LepR和STAT3mRNA表达和蛋白含量无显著影响,不改变VEGF mRNA表达和蛋白分泌,其下游因子eNOS和iNOS mRNA表达和活性以及NO的合成也没有受到影响(P>0.05);100ng/mL leptin对FGF-2mRNA表达和蛋白分泌没有影响。
以上结果提示：1ng/mL和10ng/mL leptin激活了尿囊膜细胞STAT3介导的VEGF-NO信号通路；并增加FGF2mRNA表达和蛋白分泌。
在体试验和体外试验都证明了leptin通过影响STAT3介导的VEGF-NO信号通路来影响血管生成；也可以通过改变FGF2来影响血管生成。Leptin对血管生成的效应在体外和体内结果正好相反,推测可能与尿囊膜细胞所处的生理状态和leptin剂量有关,也可能由体内其它激素引起。
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 angiogenesis
200fertilized 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 angiogenesis
To 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 cells
To 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.
1leptin对鸡胚尿囊膜血管生成的影响
选200枚温氏N414土鸡种蛋进行孵化,7胚龄时开窗,8胚龄时在尿囊膜上放置含有0ng、10ng、100ng、1000ng、5000ng leptin的明胶海绵,48小时后固定尿囊膜,计算血管的数量,研究不同剂量leptin对血管生成的影响。结果显示,10ng、00ng.1000ng、5000ng leptin对尿囊膜大血管和中血管的数量没有影响(P>0.05).10ng leptin显著减少鸡胚尿囊膜小血管的数量(P<0.05),100ng leptin对小血管的数量没有影响(P>0.05),1000ng和5000ng leptin显著增加小血管的数量(P<0.05)。Leptin对小血管数量影响的剂量效应仅表现在雌性,对雄性没有影响。
选取温氏N414土鸡种蛋500枚,随机分为五组,蛋清注射组于入孵前在蛋清内注射0μg或0.5μg重组鼠leptin。蛋黄注射组于入孵前在蛋黄内注射0μg、0.5μg或1μg重组鼠leptin。孵化到12胚龄时,在蛋壳上开窗,用甲醇和丙酮混合液固定尿囊膜血管,进行观察、拍照,分析血管的面积和数量,同时称胚重、测量胚长,辨别性别。出雏当天(0日龄)称重。结果显示,蛋清内注射0.5μg leptin显著降低鸡胚尿囊膜血管总面积和小血管的数量(P<0.05),这种抑制效应和胚重及出雏重的降低是一致的(P<0.05)；蛋黄内注射0.5μg leptin对鸡胚尿囊膜血管面积和血管数量没有影响(P>0.05),对胚重和出雏重也没影响(P>0.05)；蛋黄内注射1μg leptin显著降低尿囊膜血管总面积和小血管数量(P<0.05),对胚重和出雏重没有影响(P>0.05)。Leptin对尿囊膜血管生成和胚胎发育的抑制效应仅表现在雌性,对雄性没有影响。
以上结果提示,Leptin对鸡胚尿囊膜血管生成的影响与剂量有关,低剂量leptin抑制血管生成,高剂量leptin促进血管生成。蛋清内注射0.5μg leptin显著抑制尿囊膜血管生成和胚胎发育,蛋黄内注射0.5μg leptin对尿囊膜血管生成和鸡胚发育没有影响,蛋黄内注射1μg leptin显著抑制尿囊膜血管的生成,对胚胎发育没有影响。
2蛋清和蛋黄内注射leptin影响鸡胚尿囊膜血管生成的机理
为了探讨蛋清内注射0.5μg leptin和蛋黄内注射1μg leptin对鸡胚发育造成的不同影响,本试验研究了leptin影响尿囊膜血管生成的分子机理。在前一章的基础上,采集12胚龄鸡胚尿囊膜用于]mRNA表达和蛋白含量检测,采集尿囊液用于一氧化氮(nitric oxide, NO)含量测定。结果显示,蛋清内注射0.5μg leptin显著降低鸡胚尿囊膜血管内皮生长因子(vascular endothelial growth factor, VEGF) mRNA的表达和蛋白含量(P<0.05),显著下调上皮型和诱导型一氧化氮合成酶(endothelial and inducible nitric oxide synthase, eNOS和iNOS) mRNA表达和活性,抑制TNOS活性,从而减少了下游因子尿囊液中NO的含量(P<0.05)。leptin抑制尿囊膜VEGF表达和NO合成仅表现在雌性。尽管蛋清内注射0.5μg leptin对雄性鸡胚尿囊膜VEGF mRNA的表达有降低趋势(P=0.08),但对eNOS和iNOS mRNA表达和活性以及尿囊液中NO的含量没有影响(P>0.05)。蛋清内注射0.5μg leptin对鸡胚尿囊膜碱性成纤维细胞生长因子(basic fibroblast growth factor2, FGF-2) mRNA表达和蛋白含量(P>0.05)均无显著影响。蛋黄内注射1μg leptin不影响鸡胚尿囊膜VEGF mRNA表达和蛋白含量(P>0.05),也不影响eNOS和iNOS mRNA表达和活性以及尿囊液中NO含量(P>0.05)。蛋黄内注射1μg leptin显著降低雌性鸡胚尿囊膜FGF-2mRNA表达和蛋白含量(P<0.05),对雄性胚没有影响。
在哺乳动物,leptin与受体(leptin receptor, LepR)结合后,通过增强下游因子信号转导和转录激活因子-3(signal transducer and activator of transcription3, STAT3)与VEGF启动子的结合,上调VEGF mRNA的表达。尽管蛋清内注射0.5μg leptin对鸡胚尿囊膜LepR mRNA表述和蛋白含量没有影响(P>0.05),但显著降低了雌性鸡胚尿囊膜STAT3mRNA表达(P<0.05),对STAT3蛋白含量有降低趋势(P=0.06)。染色质免疫共沉淀(CHIP)分析结果显示,蛋清内注射0.5μg leptin显著降低雌性鸡胚尿囊膜STAT3与VEGF启动子的结合(P<0.05),从而抑制VEGF基因的表达。
以上结果提示：蛋清内注射0.5μg leptin是通过改变STAT3介导的VEGF-NO信号通路来影响尿囊膜血管的生成。蛋黄内注射1μg leptin是通过影响尿囊膜上FGF-2基因表达和蛋白合成来影响尿囊膜血管生成的。
3Leptin处理对尿囊膜细胞STAT3介导的VEGF-NO通路的影响
本试验培养12胚龄鸡胚尿囊膜细胞,用不同剂量leptin处理细胞,研究leptin对尿囊膜细胞STAT3介导的VEGF-NO信号通路的影响。结果显示,leptin处理24h和48h后,尿囊膜细胞的相对活力显著下降(P<0.05).1ng/mL leptin显著上调尿囊膜细胞LepR mRNA表达和蛋白含量(P<0.05),10ng/mL leptin对LepR mRNA表达和蛋白含量有上调趋势(P=0.058和P=0.063)。1ng/mL和10ng/mL leptin上调尿囊膜细胞STAT3mRNA表达和蛋白合成,促进VEGF mRNA表达和蛋白分泌,同时增加TNOS活性(P=0.07和P=0.06),促进下游因子NO合成(P<0.05)。NO的合成是通过增加eNOS mRNA表达和活性来实现的(P<0.05),leptin处理对iNOS mRNA表达和活性没有影响(P>0.05)。1ng/mL和10ng/mL leptin还显著上调尿囊膜细胞另一血管生长因子FGF2mRNA表达和蛋白分泌(P<0.05)。100ng/mL leptin对尿囊膜细胞LepR和STAT3mRNA表达和蛋白含量无显著影响,不改变VEGF mRNA表达和蛋白分泌,其下游因子eNOS和iNOS mRNA表达和活性以及NO的合成也没有受到影响(P>0.05);100ng/mL leptin对FGF-2mRNA表达和蛋白分泌没有影响。
以上结果提示：1ng/mL和10ng/mL leptin激活了尿囊膜细胞STAT3介导的VEGF-NO信号通路；并增加FGF2mRNA表达和蛋白分泌。
在体试验和体外试验都证明了leptin通过影响STAT3介导的VEGF-NO信号通路来影响血管生成；也可以通过改变FGF2来影响血管生成。Leptin对血管生成的效应在体外和体内结果正好相反,推测可能与尿囊膜细胞所处的生理状态和leptin剂量有关,也可能由体内其它激素引起。
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 angiogenesis
200fertilized 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 angiogenesis
To 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 cells
To 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.
引文
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