Real time-qPCR primers.

Background
The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation.
Methods
Adult male and female heterozygous Tie2 knockout mice (Tie2+/−) and wild-type controls (Tie2+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR.
Results
In Tie2+/+ mice, females had higher circulating angiopoietin-2 (138%, p<0.05) compared to males. Gene expression of angiopoietin-1 (204%, p<0.01), angiopoietin-2 (542%, p<0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2+/+ females and males. Tie2+/+ females had lower circulating NGAL (41%, p<0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected.
Interestingly, male Tie2+/- mice had 28% higher renal wet/dry weight ratio (p<0.05) compared to Tie2+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2+/- and Tie2+/+ mice in both sexes.
Conclusion
Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

**研究背景**
内皮血管生成素/Tie2系统（endothelial angiopoietin/Tie2 system）是调控内皮通透性（endothelial permeability）的关键通路，靶向Tie2可减轻雄性小鼠失血性休克（hemorrhagic shock）诱导的器官水肿（organ edema）。然而，当前研究尚未考虑内皮细胞的性别二态性（sexual dimorphism）。本研究旨在探究内皮血管生成素/Tie2系统及水肿形成是否存在性别相关差异。

**研究方法**
本研究纳入成年雄性与雌性杂合子Tie2敲除小鼠（Tie2+/−）及野生型对照小鼠（Tie2+/+），每组各9只。通过湿/干重比值（wet/dry weight ratio）及组织切片苏木精-伊红（H&E）染色评估肾与肺组织损伤。采用酶联免疫吸附试验（ELISA）检测血浆中的蛋白水平，通过实时定量聚合酶链反应（RT-qPCR）检测肺与肾组织的mRNA表达水平。

**研究结果**
在野生型Tie2+/+小鼠中，雌性小鼠的循环血管生成素-2（angiopoietin-2）水平较雄性小鼠升高138%（p<0.05）。在肾脏组织中，雌性小鼠的血管生成素-1（angiopoietin-1，升高204%，p<0.01）与血管生成素-2（升高542%，p<0.001）的mRNA表达水平均显著高于雄性小鼠，但该差异未在肺组织中出现。在两种组织中，Tie2、Tie1及VE-PTP的基因表达水平在雌雄小鼠间均无显著差异。野生型Tie2+/+小鼠的肾与肺组织湿/干重比值在雌雄间无显著差异。与雄性野生型小鼠相比，雌性野生型小鼠的循环中性粒细胞明胶酶相关脂质运载蛋白（NGAL）水平降低41%（p<0.01），但肾脏组织中NGAL与肾损伤分子1（KIM1）的基因表达未受性别影响。

值得注意的是，与同性别野生型Tie2+/+雄性小鼠相比，杂合子Tie2+/-雄性小鼠的肾组织湿/干重比值升高28%（p<0.05），但该现象未在雌性小鼠及肺组织中出现。Tie2部分敲除未影响循环血管生成素-1与血管生成素-2的水平，但与同性别野生型小鼠相比，杂合子Tie2+/-小鼠的可溶性Tie2（soluble Tie2）水平在雌雄个体中分别降低44%与53%。在两种性别中，杂合子Tie2+/-与野生型Tie2+/+小鼠的肾、肺组织中血管生成素-1、血管生成素-2、雌激素受体（estrogen receptors）及其他内皮屏障调控因子（endothelial barrier regulators）的基因表达水平均无显著差异。

**研究结论**
在杂合子Tie2敲除小鼠中，雌性性别似乎可对肾组织水肿起到保护作用，但对肺组织水肿无此效果。该现象无法通过内皮血管生成素/Tie2系统的性别二态性来解释。