Normoxic HIF-1α Stabilization Caused by Local Inflammatory Factors and Its Consequences in Human Coronary Artery Endothelial Cells

Abstract

Knowledge about normoxic hypoxia-inducible factor (HIF)-1α stabilization is limited. We investigated normoxic HIF-1α stabilization and its consequences using live cell imaging, immunoblotting, Bio-Plex multiplex immunoassay, immunofluorescence staining, and barrier integrity assays. We demonstrate for the first time that IL-8 and M-CSF caused HIF-1α stabilization and translocation into the nucleus under normoxic conditions in both human coronary endothelial cells (HCAECs) and HIF-1α-mKate2-expressing HEK-293 cells. In line with the current literature, our data show significant normoxic HIF-1α stabilization caused by TNF-α, INF-γ, IL-1β, and IGF-I in both cell lines, as well. Treatment with a cocktail consisting of TNF-α, INF-γ, and IL-1β caused significantly stronger HIF-1α stabilization in comparison to single treatments. Interestingly, this cumulative effect was not observed during simultaneous treatment with IL-8, M-CSF, and IGF-I. Furthermore, we identified two different kinetics of HIF-1α stabilization under normoxic conditions. Our data demonstrate elevated protein levels of HIF-1α-related genes known to be involved in the development of atherosclerosis. Moreover, we demonstrate an endothelial barrier dysfunction in HCAECs upon our treatments and during normoxic HIF-1α stabilization comparable to that under hypoxia. This study expands the knowledge of normoxic HIF-1α stabilization and activation and its consequences on the endothelial secretome and barrier function. Our data imply an active role of HIF-1α in vivo in the vasculature in the absence of hypoxia.

Microglia at the blood brain barrier in health and disease

“…CX3CR1, C-X3-C motif chemokine receptor 1; P2RY12, purinergic receptor P2Y12; TGF-β1, transforming growth factor beta-1; PANX1, pannexin-1; ZO-1, zonula occludens 1; SIRT3, sirtuin 3; ATP, adenosine triphosphate; prx6, peroxiredoxin 6; Cxcl12, C-X-C motif chemokine 12; ASK1, apoptosis signal-regulating kinase 1; ROS, reactive oxygen species; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin 6; IL-1β, interleukin-1 beta; TLR4, toll-like receptor-4; HIF-1α, hypoxia inducible factor 1 subunit alpha; NLRP3, “NOD-like” receptor (NLR) family pyrin domain containing 3; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion protein 1; Ccr2, C-C motif chemokine receptor 2; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor; GLUT1, glucose transporter 1; BDNF, brain derived neurotrophic factor; TREM2, triggering receptor expressed on myeloid cells 2; HO-1, heme oxygenase-1; COX-2, cyclooxygenase-2; MPK, mitogen activated protein kinase; PI3K, phosphoinositide 3-kinase; Nrf-2, nuclear factor erythroid 2-related factor; NF-κB, Nuclear factor kappa B; JNK, c-Jun N-terminal kinase; MMP2, matrix metalloproteinase 2; INF-γ, interferon-gamma; NGF, nerve-growth factor; CD68, cluster of differentiation 68; MHCII, major histocompatibility complex 2. Sources: Resting ( Kuchler-Bopp et al, 1999 ; Salsman et al, 2011 ; Niiya et al, 2012 ; Sajja et al, 2014 ; Szalay et al, 2016 ; Davis et al, 2018 ; Xing et al, 2018 ), Stroke ( McGeer et al, 1988 ; Bandera et al, 2006 ; Lum et al, 2007 ; Krady et al, 2008 ; Guo et al, 2009 ; Zhong et al, 2010 ; Masuda et al, 2011 ; Hu et al, 2012 ; Liu et al, 2012 ; Atangana et al, 2017 ; Cheon et al, 2017 ; Xing et al, 2018 ; Cao et al, 2019 ; Rashid et al, 2019 ; Zille et al, 2019 ; Hong et al, 2020 ; Jiang et al, 2020 ; Peng et al, 2020 ; Lubart et al, 2021 ; Abdi Sarabi et al, 2022 ; Gao et al, 2022 ; Pan et al, 2023 ; Peerlings et al, 2023 ; Schwabenland et al, 2023 ; Xu et al, 2023 ), Diabetes ( Itoh et al, 1997 ; Jung et al, 2010 ; Secrest et al, 2013 ; Zhao et al, 2013 …”

“…The inflammasome inflammatory cascade may be further intensified by HIF-1α feed-back loops with cytokines like IL-6, which is upregulated in proinflammatory conditions and with HIF-1α stabilization ( Xing and Lu, 2016 ). This suggests that exacerbated inflammatory responses can persist even after the restoration of oxygen to the area of injury, as high levels of proinflammatory cytokines can re-stabilize HIF-1α, perpetuating the transcriptional signaling cascade ( Abdi Sarabi et al, 2022 ).…”