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Updated: Sep 2, 2025
Murine Dermal Fibroblast Isolation by FACS
Published on: January 7, 2016
Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis
Paola Cipriani1, Piero Ruscitti1, Ilenia Di Cola1
1Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila.
Objectives:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
CD248, a protein implicated in fibrosis, was found to be increased in systemic sclerosis (SSc). This protein suppressed blood vessel formation and induced cell death in endothelial cells, suggesting a role in SSc pathogenesis.
Area of Science:
- Cell Biology
- Vascular Biology
- Fibrosis Research
Background:
- CD248 is a glycoprotein expressed on fibroblasts and pericytes, involved in fibrotic processes.
- CD248 binds to multimerin-2 (MMRN-2) and can promote vessel regression during angiogenesis.
- Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities.
Purpose of the Study:
- To investigate the expression of MMRN-2 and CD248 in SSc.
- To evaluate the anti-angiogenic properties of CD248 in SSc fibroblasts (SSc-FBs).
- To assess the effect of CD248 on endothelial cell (EC) apoptosis and VEGFR2 activation.
Main Methods:
- Immunofluorescence (IF) was used to detect MMRN-2 in SSc skin and CD248 in SSc-FBs.
- Co-culture experiments with SSc-FBs and healthy control ECs (HC-ECs) assessed anti-angiogenic properties.
- Lentiviral short-hairpin RNA (shRNA) was used for CD248 knockdown in SSc-FBs.
- Apoptosis and VEGFR2 phosphorylation in HC-ECs were analyzed.
Main Results:
- MMRN-2 and CD248 expression were elevated in SSc skin and SSc-FBs, respectively.
- CD248-expressing SSc-FBs suppressed angiogenesis, evidenced by reduced HC-EC tube length.
- CD248 induced HC-EC apoptosis and inhibited VEGF-induced VEGFR2 phosphorylation.
Conclusions:
- CD248 exhibits anti-angiogenic effects in vitro by inhibiting tube formation and inducing EC apoptosis.
- Increased CD248 in SSc may contribute to microvascular rarefaction.
- CD248 and MMRN-2 likely play a pathogenic role in SSc.
