The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair
Heeseog KangAmy L. StrongYuxiao SunLei GuoConan JuanAlec C. BancroftLauren ChoiChase A. PaganiAysel A. FernandesMichael D. WoodardJuhoon LeeSowmya RameshAaron W. JamesDavid M. HudsonKevin N. DalbyLin XuRobert J. TowerBenjamin Lévi
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Abstract While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α ( Hoxa11-CreER T2 ; Hif1a fl/fl ) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreER T2 ; Hif1a fl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.Keywords:
HIF1A
Hypoxia
Cell fate determination
Hypoxia-Inducible Factors
HIF1A
Hypoxia-Inducible Factors
Hypoxia
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HIF1A
PAS domain
Hypoxia-Inducible Factors
Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factors
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PAS domain
Hypoxia-Inducible Factors
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In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages.
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Abstract Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters νe and Ktrans, representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G2–M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance. Significance: These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels.
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To investigate whether the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is required for ventilatory responses to hypoxia, we analyzed mice that were either wild type or heterozygous for a loss-of-function (knockout) allele at the Hif1a locus, which encodes the O 2 -regulated HIF-1α subunit. Although the ventilatory response to acute hypoxia was not impaired in Hif1a +/− mice, the response was primarily mediated via vagal afferents, whereas in wild-type mice, carotid body chemoreceptors played a predominant role. When carotid bodies isolated from wild-type mice were exposed to either cyanide or hypoxia, a marked increase in sinus nerve activity was recorded, whereas carotid bodies from Hif1a +/− mice responded to cyanide but not to hypoxia. Histologic analysis revealed no abnormalities of carotid body morphology in Hif1a +/− mice. Wild-type mice exposed to hypoxia for 3 days manifested an augmented ventilatory response to a subsequent acute hypoxic challenge. In contrast, prior chronic hypoxia resulted in a diminished ventilatory response to acute hypoxia in Hif1a +/− mice. Thus partial HIF-1α deficiency has a dramatic effect on carotid body neural activity and ventilatory adaptation to chronic hypoxia.
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The aim of this study was to evaluate hypoxia level at various tumor developmental stages and to compare various methods of hypoxia evaluation in pre-clinical CT26 tumor model. Using three methods of hypoxia determination, we evaluated hypoxia levels during CT26 tumor development in BALB/c mice from day 4 till day 19, in 2-3 days intervals. Molecular method was based on the analysis of selected genes expression related to hypoxia (HIF1A, ANGPTL4, TGFB1, VEGFA, ERBB3, CA9) or specific for inflammation in hypoxic sites (CCL2, CCL5) at various time points after CT26 cancer cells inoculation. Imaging methods of hypoxia evaluation included: positron-emission tomography (PET) imaging using [18F]fluoromisonidazole ([18F]FMISO) and a fluorescence microscope imaging of pimonidazole (PIMO)-positive tumor areas at various time points. Our results showed that tumor hypoxia at molecular level was relatively high at early stage of tumor development as reflected by initially high HIF1A and VEGFA expression levels and their subsequent decrease. However, imaging methods (both PET and fluorescence microscopy) showed that hypoxia increased till day 14 of tumor development. Additionally, necrotic regions dominated the tumor tissue at later stages of development, decreasing the number of hypoxic areas and completely eliminating normoxic regions (observed by PET). These results showed that molecular methods of hypoxia determination are more sensitive to show changes undergoing at cellular level, however in order to measure and visualize hypoxia in the whole organ, especially at later stages of tumor development, PET is the preferred tool. Furthermore we concluded, that during development of tumor, two peaks of hypoxia occur.
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Hypoxia, defined as in decreased oxygen tension than physiological range, triggers numerous cellular responses via hypoxia-inducible factor (HIF). As for kidney, hypoxia in tubulointerstitial area aggravates CKD progression, therefore tubular hypoxia and HIF activity as a response to hypoxia are attracting more attention. Recently, the existence of oxygen gradient in the mouse renal cortex was reported (Hirakawa Y, Kidney Int 2018). Therefore there is a need for the research on HIF activity with oxygen gradient.
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Hypoxia-Inducible Factors
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