Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures.
Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures.
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DOI：
10.1371/journal.pone.0151402
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Boyd NL
中科院分区：
文献类型：
--
作者：
Ramakrishnan VM;Tien KT;McKinley TR;Bocard BR;McCurry TM;Williams SK;Hoying JB;Boyd NL
人脂肪源性间质血管细胞（hSVF）是一种容易获得的异质细胞系统，在体内可以自发地自组装成功能性微血管。然而，血管自组装和成熟的机制尚不清楚，因此我们利用体外模型来确定潜在的体内调节机制。我们使用传代1 (P1) hSVF，因为即使在P2培养中，UEA1+内皮细胞（EC）也会迅速丢失。我们将hSVF细胞暴露于一系列血管生成抑制剂中，发现泛wnt抑制剂IWP2使hSVF- ec网络减少最显著（约25%）。为了确定哪些Wnt亚型和受体可能参与其中，我们用PCR方法筛选了与血管生成相关的hSVF亚型，在观察到的所有时间点只有WNT5A及其受体FZD4表达。免疫细胞化学证实hSVF表达Wnt5a蛋白。为了观察单独使用Wnt5a是否能恢复iwp2诱导的EC网络抑制，我们将重组人Wnt5a （0-150 ng/ml）添加到iwp2处理的培养物中。与未处理对照组相比，rhWnt5a的加入显著增加了EC网络面积，显著降低了EC网络总长度与EC网络面积的比值。为了确定Wnt5a是否介导体内微血管自组装，将含有IgG同型对照、抗Wnt5a中和抗体或rhWnt5a的3D hSVF构建物植入免疫受损小鼠皮下2w。与IgG对照相比，抗wnt5a处理显著降低了~41%的血管长度密度，而rhWnt5a处理显著增加了~62%的血管长度密度。然而，抗wnt5a或rhWnt5a对测量血管复杂性的节段和淋巴结密度没有显著影响。综上所述，这些数据表明hSVF产生的内源性Wnt5a在体内微血管自组装中起调节作用。这些发现还表明，在组织工程应用中，操纵Wnt信号可以增强对hSVF血管化的控制。
Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0–150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.