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Abstract：
BACKGROUND: Early-onset bone dysplasia is a common manifestation of hypophosphatasia (HPP), an autosomal inherited disease caused by ALPL mutation. ALPL ablation induces prototypical premature bone ageing characteristics, resulting in impaired osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMMSCs). As angiogenesis is tightly coupled with osteogenesis, it also plays a necessary role in sustaining bone homeostasis. We have previously observed a decrease in expression of angiogenesis marker gene CD31 in the metaphysis of long bone in Alpl+/- mice. However, the role of ALPL in regulation of angiogenesis in bone has remained largely unknown.
METHODS: Exosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively.
RESULTS: We verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs.
CONCLUSIONS: The ALPL-ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL-deficient bone defects.
METHODS: Exosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively.
RESULTS: We verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs.
CONCLUSIONS: The ALPL-ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL-deficient bone defects.
摘要：
背景：早发性骨发育不良是低磷酸盐症（HPP）的常见表现，由ALPL突变引起的常染色体遗传性疾病。ALPL消融诱导原型骨过早老化特征，导致人骨髓间充质干细胞（hBMMSCs）成骨分化能力受损。由于血管生成与骨生成紧密相关，它在维持骨稳态方面也起着必要的作用。我们先前已经观察到Alpl+/-小鼠中长骨干末端的血管生成标记基因CD31的表达降低。然而,ALPL在调节骨内血管生成中的作用在很大程度上还不清楚.
方法：通过超速离心分离和鉴定来自正常和HPPhBMMSCs的外泌体，透射电子显微镜,和纳米颗粒尺寸测量。通过免疫荧光评估ALPL对HPP患者hBMMSCs血管生成能力的影响，管形成,伤口愈合和迁移试验。exo-ELISA和WesternBlot用于评估HPP中hBMMSCs的外泌体分泌，和VEGF的蛋白表达，PDGFBB,血管抑素和内皮抑素分别在外泌体中。
结果：我们验证了ALPL消融导致hBMMSCs的促血管生成能力受损，减少人脐静脉内皮细胞的迁移和管形成，外泌体的数量和蛋白质组成随ALPL表达而变化。机械上,ALPL功能的丧失增强了ATP的释放。额外的ATP,反过来,导致ATP受体P2X7水平显著升高，从而促进外泌体分泌，导致促进血管生成的能力下降。相反，P2X7的抑制通过防止ALPL缺陷型hBMMSC中抗血管生成外泌体的过度释放而增加血管生成诱导能力。
结论：ALPL-ATP轴通过P2X7受体控制外泌体分泌来调节hBMMSCs的促血管生成能力。因此，P2X7可能被证明是加速ALPL缺陷骨缺损中新生血管形成的有效治疗靶标。
方法：通过超速离心分离和鉴定来自正常和HPPhBMMSCs的外泌体，透射电子显微镜,和纳米颗粒尺寸测量。通过免疫荧光评估ALPL对HPP患者hBMMSCs血管生成能力的影响，管形成,伤口愈合和迁移试验。exo-ELISA和WesternBlot用于评估HPP中hBMMSCs的外泌体分泌，和VEGF的蛋白表达，PDGFBB,血管抑素和内皮抑素分别在外泌体中。
结果：我们验证了ALPL消融导致hBMMSCs的促血管生成能力受损，减少人脐静脉内皮细胞的迁移和管形成，外泌体的数量和蛋白质组成随ALPL表达而变化。机械上,ALPL功能的丧失增强了ATP的释放。额外的ATP,反过来,导致ATP受体P2X7水平显著升高，从而促进外泌体分泌，导致促进血管生成的能力下降。相反，P2X7的抑制通过防止ALPL缺陷型hBMMSC中抗血管生成外泌体的过度释放而增加血管生成诱导能力。
结论：ALPL-ATP轴通过P2X7受体控制外泌体分泌来调节hBMMSCs的促血管生成能力。因此，P2X7可能被证明是加速ALPL缺陷骨缺损中新生血管形成的有效治疗靶标。
