Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, 440 Reynolds Medical Building, College Station, TX 77843-1114, USA.
Interdisciplinary Program in Genetics, Texas A&M University, Mail Stop 2128, College Station, TX 77843, USA.
Mol Hum Reprod. 2018 Feb 1;24(2):74-93. doi: 10.1093/molehr/gax064.
Can primary human uterine microvascular endothelial cells (UtMVECs) be used as a model to study uterine angiogenic responses in vitro that are relevant in pregnancy?
UtMVECs demonstrated angiogenic responses when stimulated with proangiogenic factors, including sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), physiological levels of wall shear stress (WSS), human chorionic gonadotropin (hCG) and various combinations of estrogen and progesterone.
During sprouting angiogenesis, signaling from growth factors and cytokines induces a monolayer of quiescent endothelial cells (ECs) lining the vasculature to degrade the extracellular matrix and invade the surrounding tissue to form new capillaries. During pregnancy and the female reproductive cycle, the uterine endothelium becomes activated and undergoes sprouting angiogenesis to increase the size and number of blood vessels in the endometrium.
STUDY DESIGN, SIZE, DURATION: The study was designed to examine the angiogenic potential of primary human UtMVECs using the well-characterized human umbilical vein EC (HUVEC) line as a control to compare angiogenic potential. ECs were seeded onto three-dimensional (3D) collagen matrices, supplemented with known proangiogenic stimuli relevant to pregnancy and allowed to invade for 24 h. Sprouting responses were analyzed using manual and automated methods for quantification.
PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, Western blot analysis and immunostaining were used to characterize UtMVECs. Angiogenic responses were examined using 3D invasion assays. Western blotting was used to confirm signaling responses after proangiogenic lipid, pharmacological inhibitor, and recombinant lentiviral treatments. All experiments were repeated at least three times.
After ensuring that UtMVECs expressed the proper endothelial markers, we found that UtMVECs invade 3D collagen matrices dose-dependently in response to known proangiogenic stimuli (e.g. S1P, VEGF, bFGF, hCG, estrogen, progesterone and WSS) present during early pregnancy. Invasion responses were positively correlated with phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p42/p44 mitogen-activated protein kinase (ERK). Inhibition of these second messengers significantly impaired sprouting (P < 0.01). Gene silencing of membrane type 1-matrix metalloproteinase using multiple approaches completely abrogated sprouting (P < 0.001). Finally, UtMVECs displayed a unique ability to undergo sprouting in response to hCG, and combined estrogen and progesterone treatment.
Not applicable.
LIMITATIONS, REASONS FOR CAUTION: The study of uterine angiogenesis in vitro has limitations and any findings many not fully represent the in vivo state. However, these experiments do provide evidence for the ability of UtMVECs to be used in functional sprouting assays in a 3D environment, stimulated by physiological factors that are produced locally within the uterus during early pregnancy.
We show that UtMVECs can be used reliably to investigate how growth factors, hormones, lipids and other factors, such as flow, affect angiogenesis in the uterus.
STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH award HL095786 to K.J.B. The authors have no conflicts of interest.
原代人子宫微血管内皮细胞（UtMVECs）能否作为研究与妊娠相关的体外子宫血管生成反应的模型？
UtMVECs 在受到包括鞘氨醇 1-磷酸（S1P）、血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、生理水平壁切应力（WSS）、人绒毛膜促性腺激素（hCG）以及雌激素和孕激素的各种组合等促血管生成因子刺激时，表现出血管生成反应。
在发芽血管生成过程中，来自生长因子和细胞因子的信号诱导血管内皮细胞（ECs）单层降解细胞外基质并侵入周围组织，从而形成新的毛细血管。在妊娠和女性生殖周期期间，子宫内皮细胞被激活并经历发芽血管生成，以增加子宫内膜中血管的大小和数量。
研究设计、大小和持续时间：本研究旨在使用经过充分表征的人脐静脉内皮细胞（HUVEC）系作为对照来比较血管生成潜力，使用已确定的与妊娠相关的已知促血管生成刺激物，来检查原代人 UtMVEC 的血管生成潜力。将 ECs 接种到三维（3D）胶原基质中，补充已知的促血管生成刺激物，使其与妊娠期间局部产生的刺激物相关，并允许其侵入 24 小时。使用手动和自动方法进行定量分析发芽反应。
参与者/材料、设置、方法：使用 RT-PCR、Western blot 分析和免疫染色来表征 UtMVECs。使用 3D 侵袭测定法检查血管生成反应。使用 Western blot 确认促血管生成脂质、药理学抑制剂和重组慢病毒处理后的信号转导反应。所有实验均至少重复三次。
在确保 UtMVECs 表达适当的内皮标记物后，我们发现 UtMVECs 以剂量依赖性方式侵入 3D 胶原基质，以响应早期妊娠期间存在的已知促血管生成刺激物（例如 S1P、VEGF、bFGF、hCG、雌激素、孕激素和 WSS）。侵袭反应与磷酸肌醇 3-激酶（PI3K）/蛋白激酶 B（Akt）和 p42/p44 丝裂原激活蛋白激酶（ERK）的磷酸化呈正相关。这些第二信使的抑制显著损害了发芽（P < 0.01）。使用多种方法对膜型 1-基质金属蛋白酶进行基因沉默完全阻断了发芽（P < 0.001）。最后，UtMVECs 显示出对 hCG 以及雌激素和孕激素联合治疗产生反应的独特发芽能力。
不适用。
局限性、谨慎的原因：体外子宫血管生成的研究具有局限性，任何发现可能不完全代表体内状态。然而，这些实验确实提供了证据，证明 UtMVECs 可以在 3D 环境中用于功能性发芽测定，该环境受到妊娠早期局部在子宫内产生的生理因子的刺激。
我们表明 UtMVECs 可用于可靠地研究生长因子、激素、脂质和其他因素（例如流动）如何影响子宫中的血管生成。
研究资金/利益冲突：这项工作得到了 NIH 授予 KJB 的 HL095786 奖的支持。作者没有利益冲突。
