1University Hospital Münster Vascular Signalling, Molecular Cardiology, Department of Cardiology I - Coronary and Peripheral Vascular Disease Münster, Deutschland; 2University Hospital Jena Institute for Molecular Cell Biology, Center for Molecular Biomedicine Jena, Deutschland
Background/Purpose: Protein tyrosine phosphatases (PTPs) are central regulatory proteins of intracellular signalling pathways. They control cell metabolism, proliferation, differentiation and immune response. In addition, dysregulation of PTP function has been associated with impaired glucose homeostasis, obesity and cardiovascular disease. Initial findings indicate that protein tyrosine phosphatase 1 B (PTP1B) is involved in the formation and progression of atherosclerotic plaques. However, more detailed analyses of the underlying mechanism are still lacking. We investigate PTP1B-dependent metabolic changes as a possible cause of atherosclerotic plaque progression and vascular disease.
Methods: Primary human monocyte-derived macrophages were either transfected with either siControl/siPTP1B or detached and nucleofected with a control plasmid or PTP1B overexpression plasmid. After 24 hrs, cells were exposed to either normoxic (21 % O2) or hypoxic (1 % O2) conditions. Expression levels of HIF1α target genes, like Lactate dehydrogenase (LDHA) and VEGF, were measured using RT-qPCR. Culture supernatants were collected, and levels of secreted VEGF were measured using ELISA. HUVEC-based spheroids were generated for angiogenesis assays, exposed to hypoxic macrophage culture supernatant and analysed by light microscopy.
Results: We demonstrate that PTP1B function is significantly altered in hypoxic macrophages. At the transcriptional level, PTP1B expression is suppressed by approximately 30-40 % (p=0.003). Investigating PTP1B-dependent VEGF secretion by hypoxic macrophages as a possible paracrine effect, we found that the absence of PTP1B leads to a significant increase in the transcription and release of VEGF, up to 3.5 times higher compared to normal levels (p=0.0002). Conversely, overexpression of PTP1B reduced the secretion of VEGF in response to hypoxia (p=0.008), highlighting the influence of PTP1B on the crosstalk between plaque macrophages and the endothelium. To validate these hypotheses with phenotypic evidence, we also performed angiogenesis assays and analysed neovascular density in relation to hypoxia and PTP1B expression. O2 deprivation resulted in an almost twofold increase in endothelial cell sprouting in the presence of depleted PTP1B (p=0.038). Downregulation of PTP1B levels also suppressed hypoxia-induced LDHA expression (p=0.0004), suggesting that PTP1B promotes a metabolic switch in macrophages. Consistent with our hypothesis, the overexpression of PTP1B resulted in increased LDHA levels (p=0.088).
Conclusions: PTP1B phosphatase is significantly involved in the metabolic switch of macrophages under oxygen deprivation and provides a compensatory mechanism to prevent overshooting of hypoxia-induced effects. The cells interact with neighbouring endothelial cells and, depending on the PTP1B levels, induce them to promote angiogenesis. This could potentially accelerate plaque progression and instability, which can lead to facilitated plaque rupture and total vessel occlusion as mortality-critical complications of cardiovascular disease. Our 3D spheroid experiments show the first phenotypic pattern of the influence of PTP1B on hypoxic macrophage function, which requires further validation in vivo.