Objective. To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses.Methods. Normal human articular cartilage from a range of donors was obtained at the time of above-theknee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1␣. Cell transfections were performed on isolated human chondrocytes.Results. Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2␣ to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1␣. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase-containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissuespecific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage.Conclusion. We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage. IntroductionOxygen is essential to life for all higher organisms. Molecular oxygen is required as an electron acceptor in the generation of cellular energy (ATP) through the process of oxidative phosphorylation, and it is also used as a substrate in various enzymatic reactions [1]. Oxygen homeostasis is, therefore, a basic requirement and complex systems have evolved to maintain this at the cell, tissue and whole organism levels. These include increased reliance on anaerobic glycolysis in the formation of ATP within the cell; increased angiogenesis and blood supply (through vasodilation) to affected organs; and systemic changes such as enhanced erythropoiesis and increased ventilation [2,3].Cartilage develops in a hypoxic environment [4], and indeed proximity to a blood supply appears to be a determining factor in the formation of bone over cartilage [5,6]. In addition, due to the absence of vasculature, articular cartilage (unlike most tissues) is maintained and functions in a low oxygen environment throughout life [7][8][9][10]. The resident cells, the chondrocytes, are the only cell type present in the tissue and appear to have developed specific mechanisms to promote tissue function in response to this chronic hypoxia, for example, by inducing increased expression of cartilage matrix components [11][12][13], and through the inhibition of angiogenesis [14]. In addition to mediating the ubiquitous hypoxia responses, hypoxia-inducible factors (HIFs) also appear to be critical to these tissue-specific responses in chondrocytes. Hypoxia-inducible factorsIn the mid-1990s a major breakthrough was made in our understanding of the molecular mechanisms mediating cellular responses to hypoxia with the discovery of HIF-1 [15]. The stability and function of HIF is regulated post-translationally by hydroxylation of specific amino acid residues. In the presence of sufficient molecular oxygen, HIF is degraded almost as soon as it is made due to hydroxylation of specific proline residues that target the HIF-α subunit for Von HippelLindau tumour suppressor protein (pVHL)-mediated proteosomal degradation. Conversely, when oxygen levels are limiting (typically <5%), hydroxylation is inhibited and HIF-α escapes degradation, and is free to heterodimerise with the constitutively expressed HIF-β subunit (also called Aryl hydrocarbon nuclear translocator (ARNT)). This complex translocates to the nucleus, binding specific consensus seq...

Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2␣ (HIF-2␣)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-␣ isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2␣ stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interferencemediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2␣ levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.Articular cartilage consists of a single cell type, the chondrocyte, which is solely responsible for the synthesis and maintenance of the extracellular matrix (1). The rigid nature of type II collagen fibrils present in the matrix confers tensile strength to the tissue, and the swelling pressure caused by water-saturated aggrecan molecules creates a compressive gellike stiffness allowing the tissue to resist deformation and giving cartilage its ability to absorb shocks (1, 2). Due to its shockabsorbing and articulating functions, articular cartilage cannot afford a blood or nerve supply. The tissue is therefore dependent on diffusion of oxygen from the synovial fluid on one side and the vascularized underlying bone on the other. As a result, articular cartilage (at least in larger animals and man) is maintained in a low oxygen environment (1-5% oxygen tension) throughout life (3-5). There is now significant evidence that hypoxia is a critical parameter in promoting the chondrocyte phenotype. It has been shown that hypoxia up-regulates key cartilage transcription factor SOX9 and increases expression of the main extracellular matrix genes in bovine and human chondrocytes (6 -10).The response of cells to hypoxia is mediated by hypoxiainducible factors (HIFs), 2 heterodimeric transcription factors consisting of an ␣ and ␤ subunit (11). Both subunits are constitutively expressed at the mRNA level; however, the ␣ subunit ...

Coronavirus disease-19 (COVID-19), resulting from infection with SARS-CoV-2, spans a wide spectrum of illness. In severely ill patients, highly elevated serum levels of certain cytokines and considerable cytolytic T cell infiltrates in the lungs have been observed. These same patients may bear low to negligible viral burdens suggesting that an overactive immune response, often termed cytokine storm, contributes to the severity of COVID-19. We report the safety and efficacy of baricitinib combined with remdesivir and dexamethasone in a retrospective review of 45 hospitalized patients with COVID-19 pneumonia at a tertiary academic medical center. Patients received 7-day course of baricitinib, 5-day course of remdesivir, and 10-day course of dexamethasone. Clinical status and biomarkers were obtained daily. Outcomes assessed include mortality, duration of hospitalization, presence of shock, need for supplemental oxygen, need for non-invasive ventilation, need for mechanical ventilation, and development of thrombosis. Obesity and multiple medical comorbidities were associated with hospitalization in the setting of COVID-19. Treated patients demonstrated rapid declines of C-reactive protein (CRP), ferritin and D-dimer with gradual improvement in hemoglobin, platelet counts, and clinical status. Only 2 of 45 (4.4%) treated patients required mechanical ventilation after initiating treatment, and there were six deaths (13.3%). Only 2 of 45 (4.4%) treated patients required mechanical ventilation after initiating treatment. There were six deaths (13.3%) and these were associated with lower BMI. These findings support the utility of immunosuppression via JAK inhibition in moderate to severe COVID-19 pneumonia.

PTHrP (parathyroid hormone-related protein) is crucial for normal cartilage development and long bone growth and acts to delay chondrocyte hypertrophy and terminal differentiation in the growth plate. After growth plate closure adult HACs (human articular chondrocytes) still produce PTHrP, suggesting a possible role for this factor in the permanent articular cartilage. However, the expression regulation and function of PTHrP in the permanent articular cartilage is unknown. Human articular cartilage is an avascular tissue and functions in a hypoxic environment. The resident chondrocytes have adapted to hypoxia and use it to drive their tissue-specific functions. In the present study, we explored directly in normal articular chondrocytes isolated from a range of human donors the effect of hypoxia on PTHrP expression and whether PTHrP can regulate the expression of the permanent articular chondrocyte phenotype. We show that in HACs PTHrP is up-regulated by hypoxia in a HIF (hypoxia-inducible factor)-1α and HIF-2α-dependent manner. Using recombinant PTHrP, siRNA-mediated depletion of endogenous PTHrP and by blocking signalling through its receptor [PTHR1 (PTHrP receptor 1)], we show that hypoxia-induced PTHrP is a positive regulator of the key cartilage transcription factor SOX9 [SRY (sex determining region on the Y chromosome)-box 9], leading to increased COL2A1 (collagen type II, α1) expression. Our findings thus identify PTHrP as a potential factor for cartilage repair therapies through its ability to promote the differentiated HAC phenotype.