Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA.
Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA.
Pharmacol Res. 2019 Aug;146:104268. doi: 10.1016/j.phrs.2019.104268. Epub 2019 May 9.
Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca-release (E = 195% and 143%, EC = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z' = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold E or EC compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.
需要新型的子宫收缩调节因子来治疗早产、引产和控制产后出血。因此，我们之前开发了一种高通量筛选小分子化合物的方法，用于调节原代小鼠子宫平滑肌细胞中的钙动员。本研究的目的是选择最佳的子宫平滑肌细胞用于我们的高通量药物发现测定，以及确定子宫平滑肌细胞与血管平滑肌细胞（VSMCs）之间的相似性或差异，VSMCs 是当前子宫治疗药物最常见的非靶点。分子和药理学测定用于比较来自四个来源的子宫平滑肌细胞：从足月妊娠的人和鼠子宫中分离的原代细胞、永生化的妊娠人子宫（PHM-1）细胞和永生化的非妊娠人子宫（hTERT-HM）细胞。此外，还将子宫平滑肌细胞与血管平滑肌细胞进行了比较。我们发现 hTERT-HM 和 PHM1 细胞的转录组谱与人类原代子宫平滑肌细胞最为相似（r=0.93 和 0.90）。比较原代人子宫平滑肌转录组和 VSMCs 的转录组谱，发现 498 个上调基因（p≤0.01，log2FC≥1），其中 142 个可作为子宫选择性药物靶点。在高通量 Ca 测定中，PHM1 细胞对 OT 诱导的 Ca 释放的反应与原代人子宫平滑肌细胞最相似（E=195%和 143%，EC=30 nM 和 120 nM），而所有来源的子宫平滑肌细胞均表现出良好且相似的稳健性和重现性（Z'=0.52 至 0.77）。在测试了 61 种化合物的小组后，我们发现 hTERT-HM 细胞的刺激和抑制反应与原代细胞高度相关（r=0.94 和 0.95）。此外，还鉴定出了 10 种具有子宫选择性的化合物（与 VSMCs 相比，E 或 EC 超过 5 倍）。总的来说，这项研究发现 hTERT-HM 细胞与原代人子宫平滑肌细胞最为相似，因此是大规模筛选鉴定子宫平滑肌收缩调节因子的理想替代品。此外，鉴于观察到的转录组和化合物反应的相似性，VSMCs 可以作为评估靶点和药物子宫选择性的重要对照筛选工具。
