探討神經起源的Thsd7a在血管新生過程中調控內皮尖端與莖稈細胞功能的分子機制

The molecular mechanism of neuronal THSD7A on endothelial tip and stalk cell selection during angiogenesis

江杰鵬(Jie-Peng Jiang)

指導教授 ： 莊永仁

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2013年)

摘要

在發育過程中內皮細胞的引導在血管網絡的擴張是非常重要的，而在生長中的新生血管芽中會發生誘導內皮細胞分化為“tip”與“stalk”細胞，這個機制參與了內皮細胞的引導過程，然而tip細胞位於新生血管芽上的頂端，能反應環境中的引導因子並引導內皮細胞的移動，同時另一特化後細胞stalk細胞位於tip細胞的後方，它能反應引導因子的訊號並且進行細胞增生，使新生血管芽得以進行生長，然而這種在新生血管芽中擁有的層級式結構組織對於正常或病變情況下血管的型態發生是非常重要的．
　　在過去我們所做的研究中發現一個在脊椎動物中具高度保留特性的蛋白“THSD7A”被稱作凝血酶敏感蛋白區域包含蛋白7，而在利用斑馬魚為模式生物的研究中，我們發現THSD7A能夠引導內皮細胞的移動和血管形成，另外我們也發現到當在斑馬魚上使用morpholino降低THSD7A的表現時，血管新生中的intersegmental vessels 和parachordal chain血管會產生缺失，然而在血管發育的過程中THSD7A是否在調控tip和stalk細胞的分化上扮演了重要的角色，這個問題始終是未知的．
　　在我們使用斑馬魚作為我們的研究工具後，我們發現經由THSD7A處理後的幼魚所產生的表現型態非常類似於notch-delta like 4 (dll4)訊息傳遞路徑缺陷的表現型，這暗示了在發育過程中THSD7A很有可能是藉由調控notch- dll4訊息傳遞來影響血管發育，在Real Time-quantitative PCR實驗中顯示notch1b和它下游所調控的VEGFR2/3都會受到降低THSD7A表現的影響而表現量下降，此外在原位雜交的實驗中也發現notch1b在小腦脊索和心臟上的表現模式會受到抑制THSD7A表現因而產生錯亂的表現模式，這些結果指出了斑馬魚的THSD7A在發育過程中會藉由notch- dll4訊息傳遞路徑來調控tip和stalk細胞的分化決定，有趣的是我們發現在THSD7A表現受到抑制的幼魚中神經的生長也會產生缺陷，顯示了THSD7A與notch訊息傳遞在神經血管交互作用中的關聯性
　　總而言之，我們的結果強烈地顯示THSD7A在血管新生過程中很有可能是調控tip和stalk細胞的分化選擇上重要的調控者，這些發現在未來血管新生相關疾病上可以提供發展THSD7A標靶治療的機會．

關鍵字

斑馬魚 ； 血管新生 ； 神經血管交互作用

並列摘要

Endothelial guidance is very important for vascular network expansion during development. It involves the induction of endothelial cell (EC) into tip and stalk cell potential at the growing angiogenic sprout. The tip cell is located at the leading edge of growing sprout, which responds to guidance cues to direct the EC migration. Meanwhile, the stalk cell trails behind the tip cell proliferates in response to guidance cues. The hierarchical organization of sprouting vessels is critical for vascular morphogenesis under normal or diseased conditions.
Previously, our group has discovered a novel protein called thrombospondin type I domain containing 7A (THSD7A) that is highly conserved among the vertebrates. During zebrafish vascular development, it is a guidance cues required for directing EC migration. We have also shown Thsd7a is related to the growth path finding of angiogenic intersegmental vessels (ISV) and parachordal chain (PAC), as morpholino (MO) knockdown of Thsd7a gene expression affects ISV sprouting and PAC patterning. However, whether Thsd7a has a role in regulating tip and stalk selection during vascular development is unknown.
Using transgenic zebrafish as the research system, we found THSD7A morphant displayed distinct phenotypes that are very similar to the phenotype after loss of notch-delta like 4 (dll4) signaling. This implies thsd7a may involve in the notch-dll4 signaling pathway during vascular development. Real Time-quantitative PCR data further showed that expression levels of notch1b and its downstream targets, VEGFR2/3, were down-regulated in the thsd7a morphants. In addition, in situ hybridization analysis on the thsd7a morphants revealed notch1b has an aberrant expression pattern in hindbrain, spinal cord and heart. These data indicated that zebrafish Thsd7a could regulate endothelial tip and stalk cell selection via notch-dll4 signaling during development. Interestingly, we found knockdown of Thsd7a also affect neuronal growth in the spinal cord, which suggest the association of Thsd7a and notch signaling in neurovascular interactions. Taken together, our data strongly suggests that Thsd7a may be a regulator on endothelial tip and stalk cell selection during angiogenesis. These findings could offer an opportunity for developing THSD7A targeting therapy in angiogenesis-related diseases in the future.

參考文獻

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