Endothelial PTP1B deficiency phenocopies aging by promoting endothelial-to-mesenchymal transition

Background and Aim: Endothelial-to-mesenchymal transition (End-MT) plays a central role in fibrosis, a hallmark of aging, including in the cardiovascular system. The transcriptional reprogramming of endothelial cells is driven by Transformgin Growth Factor beta (TGFβ) and transcriptional repressors like SNAI1. Protein tyrosine phosphatase 1B (PTP1B) has been associated with premature aging processes. However, whether PTP1B has a specific role in controlling the activity of SNAI1 and TGFβ signaling is unknown.

Materials and Methods: Human aortic endothelial cells (HAoECs) from two male donors (28 year-of-age and 73 year-of-age) were purchased from PromoCell and treated with small interfering RNA (siRNA) for 48 hours, or with PTPB inhibitors or reactive oxygen species (ROS) for 2 hours to downregulate or inactivate PTP1B, respectively. Changes in cellular markers were assessed using qPCR, western blot and immunofluorescence analysis. End-MT markers, transcription factors, and signaling proteins, were quantified using ImageJ and ImagePro software. Primary endothelial cells were isolated from mice with tamoxifen-inducible, Tie.ER^T2-Cre driven endothelial-specific deletion of PTP1B (End.PTP1B-KO). Cardiovascular collagen deposition was assessed using Carstairs and sirius red staining.

Results: Western blot analysis of HAoECs 48 hours after siRNA transfection showed an effective reduction in PTP1B protein levels by over 80%, immunofluorescence microscopy confirmed the absence of PTP1B. Loss of PTP1B resulted in a significant reduction of endothelial markers, such as CD31 and VE-cadherin. Concurrently, mesenchymal markers, such as smooth muscle alpha-actin, PDGFR-α, or PDGFR-β, were upregulated, in line with End-MT. Endothelial PTP1B deficiency was associated with elevated expression, phosphorylation and nuclear presence of SNAI1, along with higher expression of other transcription factor driving End-MT. Signaling pathway analysis revealed increased phosphorylation of protein kinase B (AKT) and inactivation of glycogen synthase kinase-3 beta in endothelial cells following PTP1B deletion. Furthermore, increased signaling activity of SMAD3, a mediator of TGF-β signaling, and NF-κB, an inflammatory transcription factor, were observed. These changes suggest that PTP1B deletion shifts cells toward a mesenchymal phenotype but also promotes pro-inflammatory pathways to increase fibrosis. In vivo studies using endothelial-specific PTP1B knockout mice confirmed these findings, showing enhanced collagen deposition in cardiac tissue. PTP1B expression levels were significantly reduced in endothelial cells obtained from an older donor compared to those from a younger donor, indicating an age-dependent PTP1B loss and supporting the clinical relevance of our data.

Conclusion: Our findings suggest that PTP1B functions as a suppressor of End-MT, fibrosis, and inflammatory signaling in endothelial cells, and that an age-associated reduction in endothelial PTP1B expression participates in vascular aging processes.