Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila.
Rheumatology, Immunology, and Clinical Medicine Research Unit, Department of Medicine, Campus Bio-Medico University of Rome.
Rheumatology (Oxford). 2023 Mar 1;62(3):1317-1325. doi: 10.1093/rheumatology/keac377.
CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed.
By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot.
MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation.
CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.
CD248 是一种糖蛋白，在周细胞和成纤维细胞（FBs）上高度表达，与纤维化过程有关。在血管生成过程中，CD248 可以通过与多聚蛋白-2（MMRN-2）结合来促进血管退化。因此，我们研究了系统性硬化症（SSc）皮肤中的 MMRN-2 表达和分离的 SSc-FBs 中的 CD248 表达。通过与健康对照内皮细胞（HC-ECs）共培养这些细胞，评估了 CD248+SSc-FBs 的抗血管生成特性。评估了 CD248 对 HC-ECs 的凋亡作用。最后，评估了 CD248 阻止血管内皮生长因子受体 2（VEGFR2）激活的能力。
通过免疫荧光法，研究了 SSc 皮肤中的 MMRN-2 和 SSc-FBs 中的 CD248。通过 HC-ECs/SSc-FBs 共培养评估 CD248+SSc-FBs 的抗血管生成特性。采用慢病毒诱导的 CD248 短发夹 RNA 递送进行 SSc-FBs 的功能丧失研究。在存在 CD248 的情况下培养 HC-ECs，通过免疫荧光法评估凋亡，通过 Western blot 评估 VEGFR2 磷酸化。
皮肤 SSc-ECs 中 MMRN-2 表达增加，而 SSc-FBs 中 CD248 表达增加。功能上，CD248+SSc-FBs 在器官型模型中抑制血管生成，表现为 HC-ECs 总管长度减少。这种抗血管生成行为在 CD248 沉默后被逆转。此外，CD248 的存在促进了 HC-ECs 的凋亡。最后，CD248 通过减少 VEGF 刺激后 VEGFR2 的磷酸化来阻止 VEGFR2 的激活。
CD248 在体外具有抗血管生成作用，原因是管形成减少和 ECs 凋亡增加。SSc 中 CD248 表达增加可能导致 SSc 中组织水平观察到的微血管稀疏。我们的结果表明 CD248-MMRN-2 在 SSc 中具有致病作用。
