Objectives CD248 is a glycoprotein highly expressed on pericytes and fibroblast (FBs), and it is implicated in fibrotic process. During angiogenesis, CD248 could promote vessel regression, binding the multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 on isolated SSc-FBs. Anti-angiogenic property of CD248+ SSc-FBs was evaluated coculturing these cells with healthy control endothelial cells (HC-ECs). CD248 apoptotic role on HC-ECs was evaluated. Finally, CD248 ability to prevent the VEGFR2 activation was assessed. Methods By immunofluorescence (IF), MMRN-2 was investigated on SSc-skin and CD248 on SSc FBs. CD248+ SSc-FBs anti-angiogenic property was evaluated by HC-ECs/SSc-FBs cocultures. Lentiviral induced CD248 short-hairpin RNAs delivery was employed for loss of function studies on SSc-FBs. HC-ECs were cultured in presence of CD248 to assess both apoptosis by IF and VEGFR2 phosphorylation by WB. Results MMRN-2 expression was increased in skin SSc-ECs whereas CD248 was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by reduction in total tubes length of HC-ECs. This anti-angiogenetic behaviour was reverted by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented the VEGFR2 activation, by reducing its phosphorylation after VEGF stimulation. Conclusion CD248 was anti-angiogenic in vitro due to reduction the tube formation and to induction of ECs apoptosis. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at tissue level during SSc. Our results suggest the pathogenic role of CD248-MMRN-2 in SSc.

中文翻译：

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CD248 的成纤维细胞表达可能会加剧系统性硬化症期间的微血管损伤

目的 CD248 是一种在周细胞和成纤维细胞 (FB) 上高表达的糖蛋白，与纤维化过程有关。在血管生成过程中，CD248 可以促进血管退化，结合 multimerin-2 (MMRN-2)。因此，我们研究了 MMRN-2 在系统性硬化症 (SSc) 皮肤中的表达以及 CD248 在分离的 SSc-FB 中的表达。通过将这些细胞与健康对照内皮细胞 (HC-EC) 共培养，评估了 CD248+ SSc-FB 的抗血管生成特性。评估了 CD248 对 HC-EC 的凋亡作用。最后，评估了 CD248 阻止 VEGFR2 激活的能力。方法 通过免疫荧光 (IF)，研究了 SSc 皮肤上的 MMRN-2 和 SSc FB 上的 CD248。通过 HC-ECs/SSc-FBs 共培养评估 CD248+ SSc-FBs 抗血管生成特性。慢病毒诱导的 CD248 短发夹 RNA 递送用于 SSc-FB 的功能丧失研究。在存在 CD248 的情况下培养 HC-EC，以评估 IF 的细胞凋亡和 WB 的 VEGFR2 磷酸化。结果 MMRN-2 表达在皮肤 SSc-EC 中增加，而 CD248 在 SSc-FB 中增加。在功能上，CD248+-SSc-FB 抑制了器官型模型中的血管生成，这是通过减少 HC-EC 的总管长度来评估的。这种抗血管生成行为被 CD248 沉默恢复。此外，CD248 的存在促进了 HC-EC 的凋亡。最后，CD248 通过在 VEGF 刺激后降低其磷酸化来阻止 VEGFR2 激活。结论 CD248 由于减少管形成和诱导 ECs 凋亡而在体外具有抗血管生成作用。SSc 中 CD248 表达增加可能导致 SSc 期间在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 中的致病作用。