Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation
Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation
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DOI：
10.1186/s12885-015-1420-9
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发表时间：
2015-05-23
期刊：
影响因子：
3.8
通讯作者：
Metheny-Barlow, Linda J.
中科院分区：
文献类型：
--
作者：
Choudhary, Mayur;Naczki, Christine;Metheny-Barlow, Linda J.
背景：壁细胞和内皮细胞（EC）之间适当的功能联系会导致血管EC变得静止。肿瘤血管上的壁细胞表现出对EC的附着减少，这使得血管不稳定和增殖。肿瘤阻止壁细胞和EC之间正确结合的机制尚不清楚。由于间隙连接（GJ）在细胞间的接触和沟通中发挥着重要作用，我们研究了GJ的丧失是否在肿瘤诱导的壁细胞dissociation.Methods中发挥作用：通过流式细胞术或酶标仪定量荧光标记细胞的直接共培养试验，评估了壁细胞对内皮细胞增殖的调节。通过降落伞试验评估间隙连接功能。通过Western和共聚焦显微镜评估暴露于癌细胞条件培养基的壁细胞中的连接蛋白43（Cx43）蛋白;通过定量实时PCR评估mRNA水平。表达载体或siRNA用于过表达或敲低Cx43。结果：用降落伞染料转移实验证明，MDA-MB-231乳腺癌细胞条件培养液可抑制血管平滑肌细胞（vSMC）与EC之间的GJ通讯。GJ组分连接蛋白43（Cx43）的蛋白质和mRNA在肿瘤条件培养基的壁细胞中下调;来自非致瘤性MCF 10A细胞的培养基没有影响。通过Cx43 siRNA敲低、用阻断肽处理或暴露于肿瘤条件培养基而导致的GJ通信的丧失降低了壁细胞在共培养测定中抑制EC增殖的能力，而vSMC中Cx43的过表达恢复了GJ和内皮抑制。植入Cx43杂合子小鼠的乳腺肿瘤细胞显示肿瘤生长没有变化，但通过CD 31染色确定的肿瘤血管形成显著增加，沿着NG 2染色检测到的壁细胞支持减少。我们的数据表明：i）功能性Cx43是壁细胞诱导的内皮静止所必需的，和ii）肿瘤对Cx43 GJ的下调释放内皮以响应血管生成信号。这些数据定义了一个新的和重要的作用，维持Cx43功能的调节血管静止，并建议其损失可能有助于病理性肿瘤血管生成。
Background: Proper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation.Methods: Mural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43.Results: Using parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support detected by NG2 staining.Conclusions: Our data indicate that i) functional Cx43 is required for mural cell-induced endothelial quiescence, and ii) downregulation of Cx43 GJ by tumors frees endothelium to respond to angiogenic cues. These data define a novel and important role for maintained Cx43 function in regulation of vessel quiescence, and suggest its loss may contribute to pathological tumor angiogenesis.