Background/Purpose: Macrophages in atherosclerotic plaques get exposed to a hypoxic micro milieu, triggering a switch in their metabolism. This ensures energy production of the cells despite the oxygen deficit and induces the sprouting of new vessels, which fosters plaque progression. Protein tyrosine phosphatases are critical regulatory proteins that interfere with various intracellular signalling pathways and significantly influence the activity of target proteins through tyrosine dephosphorylation. Even though PTP1B tyrosine phosphatase is known to influence cell metabolism, detailed investigations on its potential role in regulating macrophage metabolism are absent. In this study, we investigated the specific role of PTP1B and its impact on macrophage cell metabolism in response to a hypoxic microenvironment.

 

Methods: Primary monocytes were isolated from thrombocyte reduction filters. After differentiation into macrophages, a transient knockdown (KD) and overexpression of PTP1B were carried out. Cells were then exposed to either normoxic (21% O2) or hypoxic (1% O2 conditions). The expression of the HIF target genes was analysed using RT-qPCR and confirmed by Western Blot. Relevant targets were further validated using ELISA. In addition, the activity of PTP1B was analysed using malachite-green-based assays, and the extent of PTP1B oxidation was evaluated using direct oxidised-cysteinyl labelling. Seahorse analyses were carried out to understand the metabolic alterations.

 

Results: We demonstrate that PTP1B expression is downregulated in hypoxic macrophages. After hypoxia treatment, there was a moderate suppression of PTP1B in approximately 60% of cases. Interestingly, we found evidence that the activity of PTP1B was significantly impaired (40%, p=0.003, n=6) in hypoxic macrophages. Biotin labelling assays showed that the cysteine residue in the catalytic centre of PTP1B is oxidised under oxygen deprivation. Measurements of VEGF levels in the supernatant of the cells by ELISA also suggested a correlation between VEGF release and PTP1B expression: VEGF levels were increased with PTP1B KD compared to the control group indicating that PTP1B potentiates HIF-1 alpha effects. In contrast, PTP1B overexpression decreased VEGF levels in the supernatant. A closer examination of the glycolytic enzymes hexokinase-2 (HK2) and lactate dehydrogenase A (LDHA) revealed a differential regulation. LDHA was suppressed by a mean of approximately 40% upon depletion of PTP1B in hypoxia (p=0.0004, n=8) and upregulated by about 30% upon overexpression of PTPT1B (p=0.088), whereas opposite conclusions could be drawn for HK2. HK2 expression was already augmented about 1.5-fold under normoxic conditions with PTP1B KD (p=0.0248, n=8) and reduced by about 1.2 with overexpression (p=0.016). Consistent with this, the metabolic analysis revealed that PTP1B depletion reduced the hypoxia-driven glycolytic switch.

 

Conclusions: The results reveal that PTP1B is an essential regulatory protein controlling macrophage cell metabolism. In terms of neovascularisation and angiogenesis, it seems to amplify the effects induced by oxygen starvation through HIF-1 alpha. However, concerning the metabolism and energy production of hypoxic cells, PTP1B balances and prevents the overshoot of signalling pathways induced by HIF-1 by attenuating the HIF-1-induced metabolic switch. It remains to be explored what influence the aforementioned factors exert on atheroprogression.