Publications by authors named "Kirk C Hansen"

186 Publications

Mammary collagen is under reproductive control with implications for breast cancer.

Matrix Biol 2021 Nov 25. Epub 2021 Nov 25.

Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University. Portland, OR 97239, USA; Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, USA; Young Women's Breast Cancer Translational Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Mammographically-detected breast density impacts breast cancer risk and progression, and fibrillar collagen is a key component of breast density. However, physiologic factors influencing collagen production in the breast are poorly understood. In female rats, we analyzed gene expression of the most abundantly expressed mammary collagens and collagen-associated proteins across a pregnancy, lactation, and weaning cycle. We identified a triphasic pattern of collagen gene regulation and evidence for reproductive state-dependent composition. An initial phase of collagen deposition occurred during pregnancy, followed by an active phase of collagen suppression during lactation. The third phase of collagen regulation occurred during weaning-induced mammary gland involution, which was characterized by increased collagen deposition. Concomitant changes in collagen protein abundance were confirmed by Masson's trichrome staining, second harmonic generation (SHG) imaging, and mass spectrometry. We observed similar reproductive-state dependent collagen patterns in human breast tissue obtained from premenopausal women. SHG analysis also revealed structural variation in collagen across a reproductive cycle, with higher packing density and more collagen fibers arranged perpendicular to the mammary epithelium in the involuting rat mammary gland compared to nulliparous and lactating glands. Involution was also characterized by high expression of the collagen cross-linking enzyme lysyl oxidase, which was associated with increased levels of cross-linked collagen. Breast cancer relevance is suggested, as we found that breast cancer diagnosed in recently postpartum women displayed gene expression signatures of increased collagen deposition and crosslinking compared to breast cancers diagnosed in age-matched nulliparous women. Using publically available data sets, we found this involution-like, collagen gene signature correlated with poor progression-free survival in breast cancer patients overall and in younger women. In sum, these findings of physiologic collagen regulation in the normal mammary gland may provide insight into normal breast function, the etiology of breast density, and inform breast cancer risk and outcomes.
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http://dx.doi.org/10.1016/j.matbio.2021.10.006DOI Listing
November 2021

The α-globin Chain of Hemoglobin Potentiates Tissue Plasminogen Activator Induced Hyperfibrinolysis in vitro.

J Trauma Acute Care Surg 2021 Sep 16. Epub 2021 Sep 16.

Department of Surgery, School of Medicine University of Colorado Denver, Aurora Department of Surgery, Denver Health Medical Center, Denver Department of Biochemistry and Molecular Genetics Department of Pediatrics, School of Medicine University of Colorado Denver, Aurora Vitalant Research Institute, Vitalant Mountain Division, Denver, CO.

Background: Severe injury predisposes patients to trauma-induced coagulopathy (TIC), which may be subdivided by the state of fibrinolysis. Systemic hyperfibrinolysis (HF) occurs in ~25% of these patients with mortality as high as 70%. Severe injury also causes the release of numerous intracellular proteins which may affect coagulation, one of which is hemoglobin, and hemoglobin substitutes induce HF in vitro. We hypothesize that the α-globin chain of hemoglobin potentiates HF in vitro by augmenting plasmin activity.

Methods: Proteomic analysis was completed on a pilot study of 30 injured patients prior to blood component resuscitation, stratified by their state of fibrinolysis, plus 10 healthy controls. Different concentrations of intact hemoglobin (HbA), the α- and β-globin chains, or normal saline (NS, controls) were added to whole blood and tPA-challenge thrombelastography (TEG) was used to assess the degree of fibrinolysis. Interactions with plasminogen (PLG) were evaluated using surface plasmon resonance (SPR). TPA-induced plasmin activity was evaluated in the presence of the α-globin chain.

Results: Only the α- and β-globin chains increased in HF patients (p < .01). The α-globin chain but not HbA or the β-globin chain decreased the R-time and significantly increased Ly30 on CN-TEGs (p < 0.05). The PLG and α-globin chain had interaction kinetics similar to tPA:PLG, and the α-globin chain increased tPA-induced plasmin activity.

Conclusions: the α-globin chain caused HF in vitro by binding to PLG and augmenting plasmin activity and may represent a circulating "moonlighting" mediator released by the tissue damage and hemorrhagic shock inherent to severe injury.

Level Of Evidence: III prognostic.
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http://dx.doi.org/10.1097/TA.0000000000003410DOI Listing
September 2021

Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages.

JCI Insight 2021 Nov 8;6(21). Epub 2021 Nov 8.

Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent proinflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared with CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow-derived macrophages (BMDMs) and showed similar responses. Compared with sEVs derived from CO-Fibs, sEVs derived from PH-Fibs (PH-Fib-sEVs) induced augmented expression of proinflammatory cytokines/chemokines and metabolic genes in BMDMs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of proinflammatory activation of BMDMs. "Bottom-up" proteomic analyses revealed significant enrichment of complement and coagulation cascades in PH-Fib-sEVs, including augmented expression of the complement component C3. We therefore examined whether the PH-Fib-sEV-mediated proinflammatory activation of BMDMs was complement C3 dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for proinflammatory activation of BMDMs. PH-Fib-sEVs mediated proglycolytic alterations and complement-dependent activation of macrophages toward a proinflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs served as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.
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http://dx.doi.org/10.1172/jci.insight.148382DOI Listing
November 2021

Targeted Intracellular Delivery of Trastuzumab Using Designer Phage Lambda Nanoparticles Alters Cellular Programs in Human Breast Cancer Cells.

ACS Nano 2021 Jun 30. Epub 2021 Jun 30.

Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States.

Several diseases exhibit a high degree of heterogeneity and diverse reprogramming of cellular pathways. To address this complexity, additional strategies and technologies must be developed to define their scope and variability with the goal of improving current treatments. Nanomedicines derived from viruses are modular systems that can be easily adapted for combinatorial approaches, including imaging, biomarker targeting, and intracellular delivery of therapeutics. Here, we describe a "designer nanoparticle" system that can be rapidly engineered in a tunable and defined manner. Phage-like particles (PLPs) derived from bacteriophage lambda possess physiochemical properties compatible with pharmaceutical standards, and particle tracking and cell targeting are accomplished by simultaneous display of fluorescein-5-maleimide (F5M) and trastuzumab (Trz), respectively (Trz-PLPs). Trz-PLPs bind to the oncogenically active human epidermal growth factor receptor 2 (HER2) and are internalized by breast cancer cells of the HER2 overexpression subtype, but not by those lacking the amplification. Compared to treatment with Trz, robust internalization of Trz-PLPs results in higher intracellular concentrations of Trz, prolonged inhibition of cell growth, and modulated regulation of cellular programs associated with HER2 signaling, proliferation, metabolism, and protein synthesis. Given the implications to cancer pathogenesis and that dysregulated signaling and metabolism can lead to drug resistance and cancer cell survival, the present study identifies metabolic and proteomic liabilities that could be exploited by the PLP platform to enhance therapeutic efficacy. The lambda PLP system is robust and rapidly modifiable, which offers a platform that can be easily "tuned" for broad utility and tailored functionality.
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http://dx.doi.org/10.1021/acsnano.1c02864DOI Listing
June 2021

The Inherent Dynamics and Interaction Sites of the SARS-CoV-2 Nucleocapsid N-Terminal Region.

J Mol Biol 2021 07 20;433(15):167108. Epub 2021 Jun 20.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, School of Medicine, Aurora, CO 80045, United States. Electronic address:

The nucleocapsid protein is one of four structural proteins encoded by SARS-CoV-2 and plays a central role in packaging viral RNA and manipulating the host cell machinery, yet its dynamic behavior and promiscuity in nucleotide binding has made standard structural methods to address its atomic-resolution details difficult. To begin addressing the SARS-CoV-2 nucleocapsid protein interactions with both RNA and the host cell along with its dynamic behavior, we have specifically focused on the folded N-terminal domain (NTD) and its flanking regions using nuclear magnetic resonance solution studies. Studies performed here reveal a large repertoire of interactions, which includes a temperature-dependent self-association mediated by the disordered flanking regions that also serve as binding sites for host cell cyclophilin-A while nucleotide binding is largely mediated by the central NTD core. NMR studies that include relaxation experiments have revealed the complicated dynamic nature of this viral protein. Specifically, while much of the N-terminal core domain exhibits micro-millisecond motions, a central β-hairpin shows elevated inherent flexibility on the pico-nanosecond timescale and the serine/arginine-rich region of residues 176-209 undergoes multiple exchange phenomena. Collectively, these studies have begun to reveal the complexities of the nucleocapsid protein dynamics and its preferred interaction sites with its biological targets.
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http://dx.doi.org/10.1016/j.jmb.2021.167108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214912PMC
July 2021

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

JACC Basic Transl Sci 2021 Apr 10;6(4):350-361. Epub 2021 Mar 10.

Department of Bioengineering, University of California, San Diego, La Jolla, California, USA.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.
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http://dx.doi.org/10.1016/j.jacbts.2021.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093531PMC
April 2021

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Haematologica 2021 Nov 1;106(11):2971-2985. Epub 2021 Nov 1.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.
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http://dx.doi.org/10.3324/haematol.2020.278252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8561282PMC
November 2021

Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage.

Mol Cell Proteomics 2021 Jun 3;20:100079. Epub 2021 Jun 3.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA; Cancer Center Proteomics Core, School of Medicine, University of Colorado, Aurora, Colorado, USA. Electronic address:

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.
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http://dx.doi.org/10.1016/j.mcpro.2021.100079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188056PMC
June 2021

Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors.

Int J Mol Sci 2021 Mar 25;22(7). Epub 2021 Mar 25.

Department of Biology, School of Science, National and Kapodistrian University of Athens (NKUA), 15784 Athens, Greece.

Genetic characteristics of blood donors may impact the storability of blood products. Despite higher basal stress, red blood cells (RBCs) from eligible donors that are heterozygous for beta-thalassemia traits (βThal) possess a differential nitrogen-related metabolism, and cope better with storage stress compared to the control. Nevertheless, not much is known about how storage impacts the proteome of membrane and extracellular vesicles (EVs) in βThal. For this purpose, RBC units from twelve βThal donors were studied through proteomics, immunoblotting, electron microscopy, and functional ELISA assays, versus units from sex- and aged-matched controls. βThal RBCs exhibited less irreversible shape modifications. Their membrane proteome was characterized by different levels of structural, lipid raft, transport, chaperoning, redox, and enzyme components. The most prominent findings include the upregulation of myosin proteoforms, arginase-1, heat shock proteins, and protein kinases, but the downregulation of nitrogen-related transporters. The unique membrane proteome was also mirrored, in part, to that of βThal EVs. Network analysis revealed interesting connections of membrane vesiculation with storage and stress hemolysis, along with proteome control modulators of the RBC membrane. Our findings, which are in line with the mild but consistent oxidative stress these cells experience in vivo, provide insight into the physiology and aging of stored βThal RBCs.
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http://dx.doi.org/10.3390/ijms22073369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037027PMC
March 2021

The COVIDome Explorer Researcher Portal.

medRxiv 2021 Mar 8. Epub 2021 Mar 8.

Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. In order to expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients including matched analysis of the whole blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate here the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.
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http://dx.doi.org/10.1101/2021.03.04.21252945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987038PMC
March 2021

Pediatric tri-tube valved conduits made from fibroblast-produced extracellular matrix evaluated over 52 weeks in growing lambs.

Sci Transl Med 2021 03;13(585)

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

There is a need for replacement heart valves that can grow with children. We fabricated tubes of fibroblast-derived collagenous matrix that have been shown to regenerate and grow as a pulmonary artery replacement in lambs and implemented a design for a valved conduit consisting of three tubes sewn together. Seven lambs were implanted with tri-tube valved conduits in sequential cohorts and compared to bioprosthetic conduits. Valves implanted into the pulmonary artery of two lambs of the first cohort of four animals functioned with mild regurgitation and systolic pressure drops <10 mmHg up to 52 weeks after implantation, during which the valve diameter increased from 19 mm to a physiologically normal ~25 mm. In a second cohort, the valve design was modified to include an additional tube, creating a sleeve around the tri-tube valve to counteract faster root growth relative to the leaflets. Two valves exhibited trivial-to-mild regurgitation at 52 weeks with similar diameter increases to ~25 mm and systolic pressure drops of <5 mmHg, whereas the third valve showed similar findings until moderate regurgitation was observed at 52 weeks, correlating to hyperincrease in the valve diameter. In all explanted valves, the leaflets contained interstitial cells and an endothelium progressing from the base of the leaflets and remained thin and pliable with sparse, punctate microcalcifications. The tri-tube valves demonstrated reduced calcification and improved hemodynamic function compared to clinically used pediatric bioprosthetic valves tested in the same model. This tri-tube valved conduit has potential for long-term valve growth in children.
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http://dx.doi.org/10.1126/scitranslmed.abb7225DOI Listing
March 2021

Seroconversion stages COVID19 into distinct pathophysiological states.

Elife 2021 03 16;10. Epub 2021 Mar 16.

Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora, United States.

COVID19 is a heterogeneous medical condition involving diverse underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Low antibody titers associate with hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, and depletion of lymphocytes, neutrophils, and platelets. Upon seroconversion, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased D-dimer, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.
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http://dx.doi.org/10.7554/eLife.65508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963480PMC
March 2021

Alterations in extracellular matrix composition during aging and photoaging of the skin.

Matrix Biol Plus 2020 Nov 17;8:100041. Epub 2020 Jun 17.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, 12801 E 17th Ave., Aurora, CO 80045, USA.

Human skin is composed of the cell-rich epidermis, the extracellular matrix (ECM) rich dermis, and the hypodermis. Within the dermis, a dense network of ECM proteins provides structural support to the skin and regulates a wide variety of signaling pathways which govern cell proliferation and other critical processes. Both intrinsic aging, which occurs steadily over time, and extrinsic aging (photoaging), which occurs as a result of external insults such as solar radiation, cause alterations to the dermal ECM. In this study, we utilized both quantitative and global proteomics, alongside single harmonic generation (SHG) and two-photon autofluorescence (TPAF) imaging, to assess changes in dermal composition during intrinsic and extrinsic aging. We find that both intrinsic and extrinsic aging result in significant decreases in ECM-supporting proteoglycans and structural ECM integrity, evidenced by decreasing collagen abundance and increasing fibril fragmentation. Intrinsic aging also produces changes distinct from those produced by photoaging, including reductions in elastic fiber and crosslinking enzyme abundance. In contrast, photoaging is primarily defined by increases in elastic fiber-associated protein and pro-inflammatory proteases. Changes associated with photoaging are evident even in young (mid 20s) sun-exposed forearm skin, indicating that proteomic evidence of photoaging is present decades prior to clinical signs of photoaging. GO term enrichment revealed that both intrinsic aging and photoaging share common features of chronic inflammation. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015982.
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http://dx.doi.org/10.1016/j.mbplus.2020.100041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852213PMC
November 2020

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Cancer Res 2021 06 1;81(12):3255-3269. Epub 2021 Feb 1.

Department of Pharmacology, University of Colorado Comprehensive Cancer Center, University of Colorado, Anschutz Medical Campus, Aurora, Colorado.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-3892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260469PMC
June 2021

Blood donor exposome and impact of common drugs on red blood cell metabolism.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado, USA.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.
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http://dx.doi.org/10.1172/jci.insight.146175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934844PMC
February 2021

Seroconversion stages COVID19 into distinct pathophysiological states.

medRxiv 2020 Dec 7. Epub 2020 Dec 7.

Linda Crnic Institute for Down Syndrome; University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

COVID19 is a heterogeneous medical condition involving a suite of underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Seronegative COVID19 patients harbor hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, neutropenia, lymphopenia and thrombocytopenia. In seropositive patients, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased markers of platelet activation, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.
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http://dx.doi.org/10.1101/2020.12.05.20244442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743101PMC
December 2020

Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Nat Mater 2021 04 30;20(4):548-559. Epub 2020 Nov 30.

UCSF Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.
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http://dx.doi.org/10.1038/s41563-020-00849-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005404PMC
April 2021

Extracellular vesicles from young women's breast cancer patients drive increased invasion of non-malignant cells via the Focal Adhesion Kinase pathway: a proteomic approach.

Breast Cancer Res 2020 11 23;22(1):128. Epub 2020 Nov 23.

Young Women's Breast Cancer Translational Program, Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Background: Extracellular vesicles (EVs) are small membrane particles that contribute to cancer progression and metastases by transporting biologically significant proteins and nucleic acids. They may also serve as biomarkers of various disease states or important therapeutic targets. Breast cancer EVs have the potential to change the behavior of other cells in their microenvironment. However, the proteomic content of EVs isolated from young women's breast cancer patients and the mechanisms underlying the influence of EVs on tumor cell behavior have not yet been reported.

Methods: In our current translational studies, we compared the proteomic content of EVs isolated from invasive breast cancer cell lines and plasma samples from young women's breast cancer (YWBC) patients and age-matched healthy donors using mass spectrometry. We analyzed the functionality of EVs in two dimensional tumor cell invasion assays and the gene expression changes in tumor cells after incubation with EVs.

Results: We found that treatment with EVs from both invasive breast cancer cell lines and plasma of YWBC patients altered the invasive properties of non-invasive breast cancer cells. Proteomics identified differences between EVs from YWBC patients and healthy donors that correlated with their altered function. Further, we identified gene expression changes in non-invasive breast cancer cells after treatment with EVs that implicate the Focal Adhesion Kinase (FAK) signaling pathway as a potential targetable pathway affected by breast cancer-derived EVs.

Conclusions: Our results suggest that the proteome of EVs from breast cancer patients reflects their functionality in tumor motility assays and may help elucidate the role of EVs in breast cancer progression.
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http://dx.doi.org/10.1186/s13058-020-01363-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681773PMC
November 2020

Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-Mediated ARDS.

Ann Surg 2020 Nov 18. Epub 2020 Nov 18.

Department of Surgery, School of Medicine University of Colorado, Aurora, CO.

Objectives: Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased metabolites prime PMNs and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the pulmonary sequestration of neutrophils (PMNs), which serves as the first event in the acute respiratory distress syndrome (ARDS).

Summary Background Data: Intracellular metabolites accumulate in the plasma of severely injured patients.

Methods: Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that signficantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a two-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS.

Results: After controlling for confounders, four metabolites significantly increased: creatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS (p<0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous (IV) succinate-induced PMN sequestration in the lung, a first event, and followed by IV lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. Succinate receptor (SUCNR1) inhibition abrogated PMN priming, PMN sequestration, and ARDS.

Conclusion: Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally.
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http://dx.doi.org/10.1097/SLA.0000000000004644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128932PMC
November 2020

Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.

J Proteome Res 2020 11 26;19(11):4455-4469. Epub 2020 Oct 26.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado 80045, United States.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
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http://dx.doi.org/10.1021/acs.jproteome.0c00606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640979PMC
November 2020

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Haematologica 2021 10 1;106(10):2726-2739. Epub 2021 Oct 1.

University of Virginia, Charlotesville, VA.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.
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http://dx.doi.org/10.3324/haematol.2020.266676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485689PMC
October 2021

Matrix reverses immortalization-mediated stem cell fate determination.

Biomaterials 2021 01 16;265:120387. Epub 2020 Sep 16.

Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA. Electronic address:

Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins as top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of the matrix protein influence on surrounding stem cell differentiation.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944411PMC
January 2021

Serum Proteomics in COVID-19 Patients: Altered Coagulation and Complement Status as a Function of IL-6 Level.

J Proteome Res 2020 11 14;19(11):4417-4427. Epub 2020 Aug 14.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, United States.

Over 5 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 as of May 29, 2020, a third of which are in the United States alone. These infections are associated with the development of a disease known as COVID-19, which is characterized by several symptoms, including persistent dry cough, shortness of breath, chills, muscle pain, headache, loss of taste or smell, and gastrointestinal distress. COVID-19 has been characterized by elevated mortality (over 100 thousand people have already died in the US alone), mostly due to thromboinflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. While the levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) have been associated with the severity of the disease, little is known about the impact of IL-6 levels on the proteome of COVID-19 patients. The present study provides the first proteomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to markers of inflammation and renal function. As a function of IL-6 levels, we identified significant dysregulation in serum levels of various coagulation factors, accompanied by increased levels of antifibrinolytic components, including several serine protease inhibitors (SERPINs). These were accompanied by up-regulation of the complement cascade and antimicrobial enzymes, especially in subjects with the highest levels of IL-6, which is consistent with an exacerbation of the acute phase response in these subjects. Although our results are observational, they highlight a clear increase in the levels of inhibitory components of the fibrinolytic cascade in severe COVID-19 disease, providing potential clues related to the etiology of coagulopathic complications in COVID-19 and paving the way for potential therapeutic interventions, such as the use of pro-fibrinolytic agents. Raw data for this study are available through ProteomeXchange with identifier PXD020601.
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http://dx.doi.org/10.1021/acs.jproteome.0c00365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640953PMC
November 2020

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

J Clin Invest 2020 11;130(11):5721-5737

Department of Surgery.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.
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http://dx.doi.org/10.1172/JCI129249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598051PMC
November 2020

Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Sci Adv 2020 Jul 1;6(27):eaba4526. Epub 2020 Jul 1.

McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole ( = 6) or omeprazole alone ( = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α cell infiltrate compared to control animals. The metaplastic tissue in control animals ( = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.
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http://dx.doi.org/10.1126/sciadv.aba4526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329334PMC
July 2020

Evidence for structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients.

medRxiv 2020 Jun 30. Epub 2020 Jun 30.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen.
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http://dx.doi.org/10.1101/2020.06.29.20142703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340206PMC
June 2020

Metabolomics of Endurance Capacity in World Tour Professional Cyclists.

Front Physiol 2020 5;11:578. Epub 2020 Jun 5.

Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

The study of elite athletes provides a unique opportunity to define the upper limits of human physiology and performance. Across a variety of sports, these individuals have trained to optimize the physiological parameters of their bodies in order to compete on the world stage. To characterize endurance capacity, techniques such as heart rate monitoring, indirect calorimetry, and whole blood lactate measurement have provided insight into oxygen utilization, and substrate utilization and preference, as well as total metabolic capacity. However, while these techniques enable the measurement of individual, representative variables critical for sports performance, they lack the molecular resolution that is needed to understand which metabolic adaptations are necessary to influence these metrics. Recent advancements in mass spectrometry-based analytical approaches have enabled the measurement of hundreds to thousands of metabolites in a single analysis. Here we employed targeted and untargeted metabolomics approaches to investigate whole blood responses to exercise in elite World Tour (including Tour de France) professional cyclists before and after a graded maximal physiological test. As cyclists within this group demonstrated varying blood lactate accumulation as a function of power output, which is an indicator of performance, we compared metabolic profiles with respect to lactate production to identify adaptations associated with physiological performance. We report that numerous metabolic adaptations occur within this physically elite population ( = 21 males, 28.2 ± 4.7 years old) in association with the rate of lactate accumulation during cycling. Correlation of metabolite values with lactate accumulation has revealed metabolic adaptations that occur in conjunction with improved endurance capacity. In this population, cycling induced increases in tricarboxylic acid (TCA) cycle metabolites and Coenzyme A precursors. These responses occurred proportionally to lactate accumulation, suggesting a link between enhanced mitochondrial networks and the ability to sustain higher workloads. In association with lactate accumulation, altered levels of amino acids before and after exercise point to adaptations that confer unique substrate preference for energy production or to promote more rapid recovery. Cyclists with slower lactate accumulation also have higher levels of basal oxidative stress markers, suggesting long term physiological adaptations in these individuals that support their premier competitive status in worldwide competitions.
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http://dx.doi.org/10.3389/fphys.2020.00578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291837PMC
June 2020

COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.

JCI Insight 2020 07 23;5(14). Epub 2020 Jul 23.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado, USA.

BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.
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http://dx.doi.org/10.1172/jci.insight.140327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453907PMC
July 2020

COVID-19 infection results in alterations of the kynurenine pathway and fatty acid metabolism that correlate with IL-6 levels and renal status.

medRxiv 2020 May 16. Epub 2020 May 16.

Previous studies suggest a role for systemic reprogramming of host metabolism during viral pathogenesis to fuel rapidly expanding viral proliferation, for example by providing free amino acids and fatty acids as building blocks. In addition, general alterations in metabolism can provide key understanding of pathogenesis. However, little is known about the specific metabolic effects of SARS-COV-2 infection. The present study evaluated the serum metabolism of COVID-19 patients (n=33), identified by a positive nucleic acid test of a nasopharyngeal swab, as compared to COVID-19-negative control patients (n=16). Targeted and untargeted metabolomics analyses specifically identified alterations in the metabolism of tryptophan into the kynurenine pathway, which is well-known to be involved in regulating inflammation and immunity. Indeed, the observed changes in tryptophan metabolism correlated with serum interleukin-6 (IL-6) levels. Metabolomics analysis also confirmed widespread dysregulation of nitrogen metabolism in infected patients, with decreased circulating levels of most amino acids, except for tryptophan metabolites in the kynurenine pathway, and increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and kidney dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis in COVID-19 patients. Metabolite levels in these pathways correlated with clinical laboratory markers of inflammation and disease severity (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen). In conclusion, this initial observational study of the metabolic consequences of COVID-19 infection in a clinical cohort identified amino acid metabolism (especially kynurenine and cysteine/taurine) and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.
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http://dx.doi.org/10.1101/2020.05.14.20102491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274252PMC
May 2020

Inflammation, immunity, and vascular remodeling in pulmonary hypertension; Evidence for complement involvement?

Glob Cardiol Sci Pract 2020 Apr 30;2020(1):e202001. Epub 2020 Apr 30.

University of Colorado, Denver, Anschutz Medical Campus, USA.

Pulmonary (arterial) hypertension (PH/PAH) is a life-threatening cardiopulmonary disorder. Experimental evidence suggests involvement of inflammatory and autoimmune processes in pathogenesis of PH/PAH, however the triggering and disease-promoting mechanisms remain unknown. The complement system is a key arm of innate immunity implicated in various pro-inflammatory and autoimmune diseases, yet, surprisingly little is known about the role of complement in PH/PAH pathogenesis. The preponderance of the existing data associates complement with PH/PAH via analysis of plasma and does not study the lung directly. Therefore, we aimed to resolve this by analyzing both the mechanisms of local lung-specific complement activation and the correlation of dysregulated plasma complement to clinical outcome in PAH patients. In our recent studies, reviewed herein, we show, for the first time, that  immunoglobulin-driven activation of the complement cascade, specifically its alternative pathway, in the pulmonary perivascular areas, is a key mechanism initiating pro-inflammatory processes in the early stage of experimental hypoxic PH (a form of "sterile inflammation"). In human patients with end-stage PAH, we have demonstrated that perivascular deposition of immunoglobulin G (IgG) and activation of the complement cascade are "longitudinally" persistent in the disease. We also showed, using unbiased network analysis, that plasma complement signaling, including again the Alternative pathway, is a prognostic factor of survival in patients with idiopathic PAH (IPAH). Based on these initial findings, we suggest that vascular-specific, immunoglobulin-driven dysregulated complement signaling triggers and maintains pulmonary vascular remodeling and PH. Future experiments in this area would facilitate discoveries on whether complement signaling can serve both as a biomarker and therapeutic target in PH/PAH.
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http://dx.doi.org/10.21542/gcsp.2020.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232865PMC
April 2020
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