IL-1β/MMP9 activation in primary human vascular smooth muscle-like cells: Exploring the role of TNFα and P2X7

Background: Vascular smooth muscle cells exhibit phenotypic plasticity in response to microenvironmental stimuli and contribute to vascular remodelling through mechanisms only partially understood. In atherosclerosis, P2X-purinoceptor7 (P2X7) has been related to interleukin-1 beta (IL-1 beta) and metalloproteinase 9 (MMP9). The hypoxia-inducible factor-1alpha (HIF1 alpha) was associated to remodelling. Here the activation of IL-1 beta and MMP9 was studied in relationship to P2X7 and HIF1 alpha in cells exploited from human carotid plaque and internal mammary artery. Methods and results: Migrating cells expressed HIF1 alpha-regulated canopy FGF-signalling regulator 2 and CD117, and led to primary cells with SMC-like phenotype (VSMC), P2X7(+). We investigated in VSMC the effects of hypoxia, of treatment with tumour necrosis factor-alpha (TNF alpha) and/or with P2X7 antagonist, A740003. Quantitative RT-PCR showed that hypoxia unaffected IL-1 beta and down-regulated MMP9 mRNAs, without activating HIF1 alpha. TNF alpha increased IL-1 beta mRNA via NLR Family Pyrin Domain-Containing 3, with production of proIL-1 beta but no rise of mature IL-1 beta. Zymography demonstrated that A740003 triggered MMP9 secretion fromVSMC. Combination of A740003 with TNF alpha abrogated this effect. Combination was ineffective on IL-1 beta activation elicited by TNF alpha, but down-regulated HIF1 alpha mRNA. A740003 induced the intracellular P2X7 aggregation and differently perturbed lysosome and mitochondria network compared to TNF alpha. Conclusions: Cells migration from human arteries leads to partially differentiated VSMC analogous to neointimal cells within atherosclerotic lesions. Down-regulated HIF1 alpha in stimulated VSMC translates in resilience in atherosclerotic lesions. P2X7-independent partial activation of IL-1 beta elicited by TNF alpha underlines complexity of the cytokine secretion. Data also supported P2X7 as modulator of MMP9 secretion, important for atherosclerosis progression. (c) 2018 Elsevier B.V. All rights reserved.