Background/Purpose: The endothelial-to-mesenchymal transition (EndoMT) transdifferentiation process can be promoted by several proinflammatory mediators in many pathological conditions. Recently, it was suggested a crucial role of EndoMT in the pathogenesis of vasculopathy in systemic lupus erythematosus (SLE). However, it remains unclear which proinflammatory mediators and signaling pathways promote EndoMT. Phosphatase Tyrosine Protein 1B (PTP1B) regulates signaling pathways of proinflammatory cytokines that are known to be involved in EndoMT. Previous works have shown that type I Interferons (IFNs) are key players in the progression of vascular damage and atherogenesis in SLE, but the role of proinflammatory cytokines such as TNF-α in EndoMT remains to be investigated.

Objective:

To elucidate the capacity of TNF-α as EndoMT inducer and the putative role of PTP1B in this process. 

Methods: Human aortic endothelial cells (HAEC) and human glomerular microvascular endothelial cells (HGMVEC) were used as model for in vitro induction of EndoMT. These endothelial cells (ECs) lines were incubated with human recombinant TNF-α (25 ng/ml) for 2 and 4 days. Expression of specific endothelial markers (eNOS, PECAM-1, VE-cadherin) and mesenchymal (SNAIL1, N-cadherin, α-SMA) markers were analyzed by Western blot (WB). Canonical activation of TNF-α-induced NF-κB pathway in EndoMT was assessed by phosphorylation of p65-Ser536, degradation of IkBa and nuclear translocation of p65 by WB and immunofluorescence (IF), respectively. PTP1B expression changes during EndoMT were evaluated by WB. CRISPR-Cas9 gene editing assay will be performed to generate PTPN1 knockdown ECs in vitro and corroborate its role in EndoMT.

Results: TNF-α induced EndoMT in both HAEC and HGMVEC by downregulation of endothelial markers (eNOS, PECAM-1, VE-cadherin) and upregulation of mesenchymal markers (SNAIL1, N-cadherin, α-SMA). TNF-α induced phosphorylation of NF-κB (p65-Ser 536) and degradation of IkB-α. Nuclear translocation of p65 was observed as early as 5 min after treatment and remained in the nucleus up to 1 h. TNF-α-induced EndoMT provoked an increase in PTP1B levels during the 4 days of EndoMT process. Additionally, PTP1B inhibition by CRISPR-Cas9 system is going to be investigated.

Conclusion: We established a model of TNF-α-induced EndoMT in the HAEC and HGMVEC endothelial cell lines. We observed decreased of endothelial markers and upregulation of mesenchymal markers. Concomitant with these changes of EndoMT markers, we found that PTP1B increases its expression. Furthermore, we found that NF-κB canonical signaling pathway is activated during this process. These findings unveil a putative important pathway in SLE vasculopathy and suggest novel avenues for intervention.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-protein-tyrosine-phosphatase-1b-ptp1b-in-endothelial-to-mesenchymal-transition-endomt-promoted-by-inflammation-implications-for-sle/