Absence of Protein Tyrosine Phosphatase-1B in vascular fibroblasts promotes cell proliferation, migration and pathological vascular remodeling

Background and Aim: Protein Tyrosine Phosphatase-1B (PTP1B) is a member of the protein tyrosine phosphatase family and involved in the control of tyrosine kinase receptor signaling. PTP1B is most prominently known for its role in insulin signaling and metabolism. Previous work has implicated PTP1B in the control of platelet-derived growth factor receptor (PDGFR), however, its significance for vascular remodeling processes is unknown. Our group has shown that deletion of PTP1B in vascular SMCs results in perivascular fibrosis, the mechanisms involving phosphorylation-dependent changes in transcription factors controlling SMC differentiation. The aim of this study is to examine the role of PTP1B in vascular fibroblasts and its relevance for vascular remodeling processes.

Methods: Mice with fibroblast-specific deletion of PTP1B (Fibro.PTP1B-KO) were generated by crossing PDGFRB.Cre-ERT2 recombinase transgenic mice with PTP1B-flox/floxtransgenic mice. For lineage tracing experiments, mice (Rosa26-mTmG) expressing enhanced membrane-tethered green fluorescent protein (mEGFP) under control of the PDGFRB.Cre-ERT2 promoter were used. Vascular injury, platelet activation and growth factor release were induced by application of 10 % ferric chloride to the carotid artery of 12-week-old male mice. To investigate the regulatory role of PTP1B on specific cellular functions, primary cells were isolated from the aorta and carotid arteries of uninjured mice and 3 weeks later, cultivated in DMEM and analyzed between passage 0 and 1.

Results: Primary aortic cells with genetic PTP1B deficiency exhibited higher expression of PDGFRB and increased proliferation, as shown by findings of significantly elevated cyclin D1 mRNA expression, PCNA protein levels, numbers of Ki-67-immunopositive cells, and higher EdU incorporation. Moreover, flow cytometry analysis of propidium iodide incorporation suggested increase G1-to-M cell cycle progression in the absence of PTP1B. qPCR findings of elevated mRNA levels of the apoptosis inhibitors Bcl-XL and the apoptosis regulator Bcl2 further supported a role for PTP1B in cell cycle regulation. Migration in response to a scratch wound injury also was significantly more pronounced in SMCs isolated from Fibro.PTP1B-KO mice compared to wildtype littermate controls. Transmigration assays using the Boyden chamber suggested that paracrine factors did not play a role in these observations. MTS assay did not reveal differences in cell viability, TUNEL assay and annexin V immunostaining, as well as qPCR analysis of apoptosis markers no differences in cell death between Fibro.PTP1B-KO mice and Fibro.PTP1B-WT controls. Flow cytometry analysis of PDGFRB reporter cells isolated from aorta revealed that more than 70% of the cells express mEGFP. In vivo, morphometric analyses of serial carotid artery cross-sections showed that absence of PTP1B in fibroblasts significantly enhanced the neointima area, intima-to-media ratio and vascular lumen stenosis. Lineage tracing experiment using PDGFRB reporter mice showed that the majority of cells within the neointima originated from PDGFRB Cre recombinase transgenic cells.

Conclusion: The findings of this ongoing study suggest that absence of PTP1B in vascular fibroblasts promotes pathological vascular remodeling by enhancing fibroblast proliferation and migration. Our current work is focusing on determining the specific mechanisms underlying these observations.