Modeling collagen fibril self-assembly from extracellular medium in embryonic tendon.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

Preservation of circadian rhythms by the protein folding chaperone, BiP.

Dysregulation of collagen synthesis is associated with disease progression in cancer and fibrosis. Collagen synthesis is coordinated with the circadian clock, which in cancer cells is, curiously, deregulated by endoplasmic reticulum (ER) stress. We hypothesized interplay between circadian rhythm, collagen synthesis, and ER stress in normal cells. Here we show that fibroblasts with ER stress lack circadian rhythms in gene expression upon clock-synchronizing time cues. Overexpression of binding immunoglobulin protein (BiP) or treatment with chemical chaperones strengthens the oscillation amplitude of circadian rhythms. The significance of these findings was explored in tendon, where we showed that BiP expression is ramped preemptively prior to a surge in collagen synthesis at night, thereby preventing protein misfolding and ER stress. In turn, this forestalls activation of the unfolded protein response in order for circadian rhythms to be maintained. Thus, targeting ER stress could be used to modulate circadian rhythm and restore collagen homeostasis in disease.-Pickard, A., Chang, J., Alachkar, N., Calverley, B., Garva, R., Arvan, P., Meng, Q.-J., Kadler, K. E. Preservation of circadian rhythms by the protein folding chaperone, BiP.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl-/HCO3- exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

Electron Paramagnetic Resonance Spectroscopy Investigation of Radical Production by Gold Nanoparticles in Aqueous Solutions Under X-ray Irradiation.

Nanomaterials can enhance the effect of X-rays, but the mechanisms of enhancement can be complicated. Electron paramagnetic resonance (EPR) was used here to shed light on enhancement mechanisms by detecting the originally proposed physical enhancement of the effect of X-rays by as-made large gold nanoparticles. Specifically spin trap reagent 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) was used to trap radicals produced in aqueous solutions under X-ray irradiation. Even though only BMPO hydroxyl adducts were detected at the time of EPR measurement, both hydroxyl and superoxide radicals were found to contribute to the enhancement. The measured total enhancement was 0.7-fold per weight percent (wp) of Au in water using unfiltered X-rays. The theoretically predicted physical enhancement is 0.49 fold per wp of gold in water. This information, together with scavenging experimental results and the fact that the G-values are close for both radicals, suggest that hydroxyl and superoxide radicals contributing almost equally to the total measured enhancement. Further, the enhancement was found to be linearly dependent on the amount of large gold nanoparticles in water and no additional radical was produced beyond the amount predicted by type 1 physical enhancement, indicating that hydroxyl or superoxide radicals were not produced via catalytic pathways.

Quantification of Lung Metastases from In Vivo Mouse Models.

Cancer research has made significant progress in terms of understanding and targeting primary tumors; however, the challenge remains for the successful treatment of metastatic cancers. This highlights the importance to use in vivo models to study the metastatic process, as well as for preclinical testing of compounds that could inhibit metastasis. As a result, proper quantification of metastases from in vivo models is of the utmost significance. Here, we provide a detailed protocol for collecting and handling lung tissues from mice, and guidance for subsequent analysis of metastases, as well as interpretation of data.

Identification of hypoxia-regulated proteins using MALDI-mass spectrometry imaging combined with quantitative proteomics.

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.

Hypoxia-mediated metastasis.

Metastasis is responsible for more than 90 % of deaths among cancer patient. It is a highly complex process that involves the interplay between cancer cells, the tumor microenvironment, and even noncancerous host cells. Metastasis can be seen as a step-wise process: acquisition of malignant phenotype, invasion into surrounding tissue, intravasation into blood vessels, survival in circulation, extravasation to distant sites, and colonization of new organs. Before the actual metastatic process, the secondary site is also prepared for the arrival of the cancer cells through formation of "premetastatic niches." Hypoxia (low oxygen tension) is commonly found in solid tumors more than a few millimeters cubed and often is associated with a poor prognosis. Hypoxia increases angiogenesis, cancer cell survival, and metastasis. This chapter described how hypoxia regulates each step of the metastatic process and how blocking hypoxia-driven metastasis through targeting hypoxia-inducible factor 1, or downstream effector molecules such as the lysyl oxidase family may represent highly effective preventive strategies against metastasis in cancer patients.

LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling.

INTRODUCTION: Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression. METHODS: LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients. RESULTS: Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors. CONCLUSIONS: These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression, and that these effects are driven by LOXL2-mediated activation of ErbB2. LOXL2 may also be a beneficial marker for breast cancer patients that could benefit most from anti-ErbB2 therapy.

Stress Control for Military, Law Enforcement, and First Responders: A Systematic Review.

Military personnel, police officers, firefighters, and other first responders must prepare for and respond to life-threatening crises on a daily basis. This lifestyle places stress on personnel, and particularly so on military personnel who may be isolated from support systems and other resources. The authors conducted a systematic review of studies of interventions designed to prevent, identify, and manage acute occupational stress among military, law enforcement, and first responders. The body of evidence consisted of 38 controlled trials, 35 cohort comparisons, and 42 case studies with no comparison group, reported in 136 publications. Interventions consisted of resilience training, stress inoculation with biofeedback, mindfulness, psychological first aid, front-line mental health centers, two- to seven-day restoration programs, debriefing (including critical incident stress debriefing), third-location decompression, postdeployment mental health screening, reintegration programs, and family-centered programs. Study limitations (risk of bias), directness, consistency, precision, and publication bias were considered in rating the quality of evidence for each outcome area. Overall, interventions had positive effects on return to duty, absenteeism, and distress. However, there was no significant impact on symptoms of psychological disorders such as PTSD, depression, and anxiety. Because of study limitations, inconsistency of results, indirectness, and possible publication bias, there was insufficient evidence to form conclusions about the effects of most specific intervention types, components, settings, or specific populations.

Combat and Operational Stress Control Interventions and PTSD: A Systematic Review and Meta-Analysis.

INTRODUCTION: Military personnel must prepare for and respond to life-threatening crises on a daily basis. This lifestyle places stress on personnel, and particularly so on deployed service members who are isolated from support systems and other resources. As part of a larger systematic review on the acceptability, efficacy, and comparative effectiveness of interventions designed to prevent, identify, and manage stress reactions, we assessed posttraumatic stress disorder (PTSD) outcomes. MATERIALS AND METHODS: We searched the electronic databases PsycINFO, PubMed, PTSDPubs, the Defense Technical Information Center, and Cochrane Central, as well as bibliographies of existing systematic reviews, to identify English-language studies evaluating the efficacy or comparative effectiveness of stress control interventions published since 1990. Controlled trials and cohort comparisons of interventions with military, law enforcement, and first responders were included. Two independent reviewers screened literature using predetermined eligibility criteria. Researchers individually abstracted study-level information and outcome data and assessed the risk of bias of included studies; data were reviewed for accuracy by the project leader. Changes in PTSD symptom scores from baseline to post-intervention were converted to standardized mean differences for comparison across studies. Risk ratios were calculated for PTSD case rates post-deployment. When several studies that compared an intervention group with a similar control/comparator reported the same outcome category and measure type, we conducted meta-analysis. We conducted meta-regression by adding a categorical variable, representing setting (i.e., in theater) or population (military vs. law enforcement or first responders) to the meta-analysis model to assess whether this variable was associated with the outcome across studies. The quality of the body of evidence (QoE) was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, which considers study limitations (risk of bias), directness, consistency, precision, and publication bias. RESULTS: Sixteen controlled trials and 13 cohort comparisons reporting PTSD outcomes met inclusion criteria. Eight controlled trials and two cohort studies had high risk of bias, primarily due to poor, differential, or unknown response rate at follow-up. Twenty-four of the 29 studies included military personnel. Interventions included Acceptance-based Skills training, Attention Bias Modification training, stress inoculation with biofeedback, Critical Incident Stress Debriefing, group psychological debriefing, Eye Movement Desensitization and Reprocessing for sub-clinical stress, embedding mental health providers in theater, Third Location Decompression, reintegration programs, and a 3-week post-deployment residential program for psychological resource strengthening.Meta-analyses of studies comparing a group that received a stress control intervention to a group that did not receive an intervention found no significant difference in reduction in PTSD symptom scores (moderate QoE) or PTSD case rate post-deployment (low QoE). A meta-analysis of studies comparing a specific stress control intervention to an active comparator (usually standard stress management education) found no significant effect on PTSD symptom scores (moderate QoE). CONCLUSION: Although combat and operational stress control (COSC) interventions may play a valuable role in decreasing stress, decreasing absenteeism, and enabling return to duty, a systematic review of 29 studies that included a control/comparison group found little evidence that COSC is effective in preventing PTSD or decreasing PTSD symptom scores in military personnel.

Predictors of burnout among US healthcare providers: a systematic review.

OBJECTIVE: One potential barrier to optimal healthcare may be provider burnout or occupational-related stress in the workplace. The objective of this study is to conduct a systematic review to identify the predictors of burnout among US. healthcare providers. DESIGN: Systematic review using in-depth critical appraisal to assess risk of bias and present the quality of evidence in synthesised results from the prognostic studies. DATA SOURCES: We searched 11 databases, registries, existing reviews and contacted experts through 4 October 2021. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: We included all studies evaluating potential predictors and documenting the presence and absence of associations with burnout assessed as a multidimensional construct. We excluded studies that relied solely on a single continuous subscale of burnout. Data were abstracted from eligible studies and checked for accuracy by a content expert and a methodologist. DATA EXTRACTION AND SYNTHESIS: Two reviewers independently screened citations and full-text publications using predetermined eligibility criteria. RESULTS: The 141 identified studies evaluated a range of burnout predictors. Findings for demographic characteristics were conflicting or show no association. Workplace factors, such as workload, work/life balance, job autonomy and perceived support from leadership, had stronger associations with risk for burnout. Mental health factors, such as anxiety, and physical health risks may increase the risk, although the direction of these associations is unclear as few prospective studies exist to address this question. Factors such as social support appear to have a protective effect. CONCLUSION: We found the most evidence for workplace, mental health and psychosocial factors in predicting burnout but limited evidence for other potential predictors. However, more prospective studies are needed to improve our understanding about how to prevent provider burnout. PROSPERO REGISTRATION NUMBER: CRD4202014836.

The matrix in focus: new directions in extracellular matrix research from the 2021 ASMB hybrid meeting.

The extracellular matrix (ECM) is a complex assembly of macromolecules that provides both architectural support and molecular signals to cells and modulate their behaviors. Originally considered a passive mechanical structure, decades of research have since demonstrated how the ECM dynamically regulates a diverse set of cellular processes in development, homeostasis, and disease progression. In September 2021, the American Society for Matrix Biology (ASMB) organized a hybrid scientific meeting, integrating in-person and virtual formats, to discuss the latest developments in ECM research. Here, we highlight exciting scientific advances that emerged from the meeting including (1) the use of model systems for fundamental and translation ECM research, (2) ECM-targeting approaches as therapeutic modalities, (3) cell-ECM interactions, and (4) the ECM as a critical component of tissue engineering strategies. In addition, we discuss how the ASMB incorporated mentoring, career development, and diversity, equity, and inclusion initiatives in both virtual and in-person events. Finally, we reflect on the hybrid scientific conference format and how it will help the ASMB accomplish its mission moving forward.

Collagen Assembly at the Cell Surface: Dogmas Revisited.

With the increased awareness about the importance of the composition, organization, and stiffness of the extracellular matrix (ECM) for tissue homeostasis, there is a renewed need to understand the details of how cells recognize, assemble and remodel the ECM during dynamic tissue reorganization events. Fibronectin (FN) and fibrillar collagens are major proteins in the ECM of interstitial matrices. Whereas FN is abundant in cell culture studies, it is often only transiently expressed in the acute phase of wound healing and tissue regeneration, by contrast fibrillar collagens form a persistent robust scaffold in healing and regenerating tissues. Historically fibrillar collagens in interstitial matrices were seen merely as structural building blocks. Cell anchorage to the collagen matrix was thought to be indirect and occurring via proteins like FN and cell surface-mediated collagen fibrillogenesis was believed to require a FN matrix. The isolation of four collagen-binding integrins have challenged this dogma, and we now know that cells anchor directly to monomeric forms of fibrillar collagens via the α1ß1, α2ß1, α10ß1 and α11ß1 integrins. The binding of these integrins to the mature fibrous collagen matrices is more controversial and depends on availability of integrin-binding sites. With increased awareness about the importance of characterizing the total integrin repertoire on cells, including the integrin collagen receptors, the idea of an absolute dependence on FN for cell-mediated collagen fibrillogenesis needs to be re-evaluated. We will summarize data suggesting that collagen-binding integrins in vitro and in vivo are perfectly well suited for nucleating and supporting collagen fibrillogenesis, independent of FN.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells.

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

Circadian control of the secretory pathway maintains collagen homeostasis.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

Nutrition and involuntary weight loss: a pilot study of an educational intervention for nursing home surveyors.

OBJECTIVE: To describe the knowledge and attitudes of nursing home (NH) surveyors before and after a brief educational intervention related to nutrition and involuntary weight loss in nursing home residents. DESIGN: A questionnaire covering knowledge and attitudes about nutrition was given 1 month before and 6 months after a targeted educational intervention. PARTICIPANTS AND SETTING: State of Maryland nursing home surveyors. MEASUREMENTS: A 24-item questionnaire of NH surveyor knowledge (11 items) and attitudes (13 items) regarding issues related to nutrition and involuntary weight loss in NH residents. RESULTS: Overall surveyors' knowledge scores increased from 68% (SD, 17%) pre-intervention to 76% (SD, 18%) post-intervention (P = .11). Knowledge related to the lack of the effect of tube feeding on survival in NH residents with end-stage dementia was the only knowledge item that improved significantly with the intervention (39% correct pre-intervention and 68% correct post-intervention, P = .04). There were no changes in attitudes toward the diagnosis or treatment of nutrition after the intervention. CONCLUSION: Overall, NH surveyor knowledge related to nutrition and involuntary weight loss varied widely across topic areas. Neither knowledge nor attitudes were substantially affected by a brief educational intervention. Development of effective educational interventions for NH surveyors should be a priority for stakeholders in NH care.

Pre-clinical evaluation of small molecule LOXL2 inhibitors in breast cancer.

Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.

Hypoxia modulates the phenotype of osteoblasts isolated from knee osteoarthritis patients, leading to undermineralized bone nodule formation.

OBJECTIVE: To investigate the role of hypoxia in the pathology of osteoarthritic (OA) bone by exploring its effect on the phenotype of isolated primary osteoblasts from patients with knee OA. METHODS: OA bone samples were collected at the time of elective joint replacement surgery for knee or hip OA. Normal bone samples were collected postmortem from cadaver donors. Primary osteoblasts were isolated from knee OA bone chips and cultured under normoxic or hypoxic (2% O2 ) conditions. Alkaline phosphatase activity was quantified using an enzymatic assay, and osteopontin and prostaglandin E2 (PGE2 ) production was assayed by enzyme-linked immunosorbent assay. Total RNA was extracted from bone and osteoblasts, and gene expression was profiled by quantitative reverse transcription-polymerase chain reaction. RESULTS: Human OA bone tissue sections stained positively for carbonic anhydrase IX, a biomarker of hypoxia, and exhibited differential expression of genes that mediate the vasculature and blood coagulation as compared to those found in normal bone. Culture of primary osteoblasts isolated from knee OA bone under hypoxic conditions profoundly affected the osteoblast phenotype, including the expression of genes that mediate bone matrix, bone remodeling, and bone vasculature. Hypoxia also increased the expression of cyclooxygenase 2 and the production of PGE2 by OA osteoblasts. Osteoblast expression of type II collagen α1 chain, angiopoietin-like 4, and insulin-like growth factor binding protein 1 was shown to be mediated by hypoxia-inducible factor 1α. Chronic hypoxia reduced osteoblast- mineralized bone nodule formation. CONCLUSION: These findings demonstrate that hypoxia can induce pathologic changes in osteoblast functionality consistent with an OA phenotype, providing evidence that hypoxia is a key driver of OA pathology.