凝血酶調節素對早期內皮起源細胞治療能力之益處–機制與應用

Abstract

在胚胎形成過程中，週邊血液與血管細胞有相近的位置及功能發展，這顯示存在了一個共同的來源─血管幹細胞。從人體週邊血液取得的血管母細胞，又稱為內皮起源細胞，在體外可以培養分化成為內皮細胞。內皮起源細胞能夠有效的幫助體內組織缺血時的血管新生，許多的研究都支持內皮起源細胞在組織血管新生中扮演這樣關鍵的角色，綜合許多的臨床試驗與臨床上的結果顯示，注射外源性的血管起源細胞可以促進血管再生並改善器官的功能。 凝血酶調節素是一種第一型穿膜醣蛋白，具有5個蛋白結構區域，分別是：凝集素相似區、重複表皮生長因子相似區、醣化區、穿膜區及尾部的細胞質區。凝血酶調節素是一被熟知的抗凝血分子，但後來的許多報告指出，凝血酶調節素也具有其他的生物功能，如促進細胞黏著、移行、增生及抗發炎。最近的研究檢視重組的凝血酶調節素各結構區域，指出凝血酶調節素第二合併第三結構區域(TMD23)能夠幫助血管新生。由於內皮起源細胞亦參與血管新生，這提高了人體的內皮起源細胞會表現並釋放凝血酶調節素來促進血管新生的可能性。 在這個研究中，將廣泛地探究早期內皮起源細胞處理TMD23後的變化，而生理學上血管新生的能力，會由建立後肢缺血動物模式評估。結果顯示，從週邊血單核球細胞培養的人類早期內皮起源細胞會表現凝血酶調節素，給予經培養的週邊血單核球細胞TMD23(100 ng/mL)，會增加群落形成單元數量、趨化性移行力、基質金屬蛋白酶第九型活性及細胞介白素第八因子的分泌，但會降低腫瘤壞死因子─α的分泌；分析其訊息傳遞途徑顯示，TMD23會活化Akt激酶，而抑制PI3K-Akt途徑則限制了TMD23在群落形成單元數量、趨化性移行力、基質金屬蛋白酶第九型活性及細胞介白素第八因子與腫瘤壞死因子─α分泌的影響；後肢缺血模式中，經TMD23合併早期內皮起源細胞處理的小鼠，在血管結紮手術後21天的過程中，以雷射杜卜勒血流測定儀分析，有最佳的血流恢復結果，而單獨給予TMD23或注射早期內皮起源細胞的小鼠，則有次佳的恢復結果，未給予兩種處理中任一種的小鼠，結果最差，此外，而給予TMD23的小鼠，血液及組織中腫瘤壞死因子─α表現較低。 綜上所述，人體血液循環中的內皮起源細胞會表現並釋放凝血酶調節素，而外源性的TMD23會透過PI3K-Akt途徑強化早期內皮起源細胞的促血管新生能力，若同時給予TMD23及早期內皮起源細胞，可加強在缺血組織中內皮起源細胞的血管新生治療效用。

During embryogenesis, a close regional and functional development of peripheral blood and vascular cells suggest the existence of a common origin, the putative hemangioblast. Circulating angioblasts, so-called endothelial progenitor cells (EPC), from human peripheral blood which was able to differentiate in vitro into endothelial cells (EC). EPC significantly contributed to neoangiogenesis after tissue ischemia in vivo and multiple studies have confirmed the pivotal role of EPC in tissue angiogenesis. Accumulated preclinical and clinical data suggest that introduction of exogenous vascular progenitor cells promote vascularization and improve organ function. Thrombomodulin (TM) is an type I transmembrane glycoprotein containing 5 distinct domains: an NH2-terminal lectin-like region (designated D1), a domain with 6 epidermal growth factor–like modules (D2), an glycosylation site–rich domain (D3), a transmembrane domain (D4), and a cytoplasmic tail domain (D5). TM is well known as a molecule to be an anticoagulant. Subsequent reports showed that TM has various biological roles, including contribution to cell adhesion, migration, proliferation, and anti-inflammation. Recent studies examining recombinant TM domains indicated that the TM domains 2 and 3 (TMD23) contributes to angiogenesis. Because EPCs also participate in angiogenesis, this raised the possibility that human EPCs may express and release TM to facilitate angiogenesis. In this study, a comprehensive investigation on the behavior of early EPC under TMD23 treatment will be performed. The physiological outcomes in the capacity of angiogenesis will be evaluated by the animal model of hind-limb ischemia. Results showed that TM was expressed and released by human EPCs cultured from peripheral blood mononuclear cells (PBMCs). Addition of TMD23 (100 ng/mL) to the cultured PBMCs increased the colony-forming units, chemotactic motility, matrix metalloproteinase activity, and interleukin-8 secretion but decreased tumor necrosis factor-α (TNF-α) release. Analysis of the signal pathway showed that TMD23 activated Akt. Inhibition of phosphatidylinositol-3 kinase–Akt blocked the effects of TMD23 on chemotactic motility, matrix metalloproteinase-9, interleukin-8, and TNF-α. In hindlimb ischemia mice, laser Doppler perfusion imaging of the ischemic limb during the 21 days after arterial ligation showed that the perfusion recovered best with intraperitoneal infusion of TMD23 plus local injection of early EPCs, followed by either infusion of TMD23 or injection of the cells. Animals without either treatment had the worst results. Animals treated with TMD23 also had lower circulating and tissue levels of TNF-α. In conclusion, TM is expressed and released by human circulating EPCs. Exogenous TMD23 enhances the angiogenic potential of early EPCs in vitro through activation of phosphatidylinositol-3 kinase-Akt pathway. Coadministration of TMD23 plus early EPCs augments therapeutic angiogenesis of the EPCs in ischemic tissues.