Wilms’ tumor is the most common type of childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with Wilms’ tumor, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with Wilms’ tumor relapse. We determined the urine proteomes at diagnosis of 49 patients with Wilms’ tumor, non–Wilms’ tumor renal tumors, and age-matched controls, leading to the quantitation of 6520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas as well as those detected in patients with cured and relapsed Wilms’ tumor. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared with cured Wilms’ tumor. In a validation cohort of 139 patients, a specific urine prohibitin ELISA demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate Wilms’ tumor relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary Wilms’ tumor specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of Wilms’ tumor cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, noninvasive monitoring of treatment response, and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms’ and other relapsed or refractory tumors.
Michael V. Ortiz, Saima Ahmed, Melissa Burns, Anton G. Henssen, Travis J. Hollmann, Ian MacArthur, Shehana Gunasekera, Lyvia Gaewsky, Gary Bradwin, Jeremy Ryan, Anthony Letai, Ying He, Arlene Naranjo, Yueh-Yun Chi, Michael LaQuaglia, Todd Heaton, Paolo Cifani, Jeffrey S. Dome, Samantha Gadd, Elizabeth Perlman, Elizabeth Mullen, Hanno Steen, Alex Kentsis
Tissue remodeling/fibrosis is a major feature of all fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). It is underpinned by accumulating extracellular matrix (ECM) proteins. Fibulin-1c (Fbln1c) is a matricellular ECM protein associated with lung fibrosis in both humans and mice, and stabilizes collagen formation. Here we discovered that Fbln1c was increased in the lung tissues of IPF patients and experimental bleomycin-induced pulmonary fibrosis. Fbln1c-deficient (–/–) mice had reduced pulmonary remodeling/fibrosis and improved lung function after bleomycin challenge. Fbln1c interacted with fibronectin, periostin and tenascin-c in collagen deposits following bleomycin challenge. In a novel mechanism of fibrosis Fbln1c bound to latent transforming growth factor (TGF)-β binding protein-1 (LTBP1) to induce TGF-β activation, and mediated downstream Smad3 phosphorylation/signaling. This process increased myofibroblast numbers and collagen deposition. Fbln1 and LTBP1 co-localized in lung tissues from IPF patients. Thus, Fbln1c may be a novel driver of TGF-β-induced fibrosis involving LTBP1 and may be an upstream therapeutic target.
Gang Liu, Marion A. Cooley, Andrew G. Jarnicki, Theo Borghuis, Prema M. Nair, Gavin Tjin, Alan C. Hsu, Tatt Jhong Haw, Michael Fricker, Celeste L. Harrison, Bernadette Jones, Nicole G. Hansbro, Peter A. Wark, Jay C. Horvat, W. Scott Argraves, Brian G. Oliver, Darryl A. Knight, Janette K. Burgess, Philip M. Hansbro
Mitochondrial dysfunction characterizes many rare and common age-associated diseases. The biochemical consequences, underlying clinical manifestations, and potential therapeutic targets, remain to be better understood. We tested the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond mitochondrial fatty acid β-oxidation, particularly in liver. This was achieved using comprehensive untargeted and targeted lipidomics in a case-control cohort of patients with Leigh syndrome French-Canadian variant (LSFC), a mitochondrial disease caused by mutations in LRPPRC, and in mice harboring liver-specific inactivation of Lrpprc (H-Lrpprc–/–). We discovered a plasma lipid signature discriminating LSFC patients from controls encompassing lower levels of plasmalogens and conjugated bile acids, which suggest perturbations in peroxisomal lipid metabolism. This premise was reinforced in H-Lrpprc–/– mice, which compared with littermates recapitulated a similar, albeit stronger peroxisomal metabolic signature in plasma and liver including elevated levels of very-long-chain acylcarnitines. These mice also presented higher transcript levels for hepatic markers of peroxisome proliferation in addition to lipid remodeling reminiscent of nonalcoholic fatty liver diseases. Our study underscores the value of lipidomics to unveil unexpected mechanisms underlying lipid dyshomeostasis ensuing from mitochondrial dysfunction herein implying peroxisomes and liver, which likely contribute to the pathophysiology of LSFC, but also other rare and common mitochondrial diseases.
Matthieu Ruiz, Alexanne Cuillerier, Caroline Daneault, Sonia Deschênes, Isabelle Robillard Frayne, Bertrand Bouchard, Anik Forest, Julie Thompson Legault, The LSFC Consortium, Frederic M. Vaz, John D. Rioux, Yan Burelle, Christine Des Rosiers
Podoplanin, a small mucine-type transmembrane glycoprotein, has been recently shown to be expressed by lymphangiogenic, fibrogenic and mesenchymal progenitor cells in the acutely and chronically infarcted myocardium. Podoplanin binds to CLEC-2, a C-type lectin-like receptor 2 highly expressed by CD11bhigh cells following inflammatory stimuli. Why podoplanin expression appears only after organ injury is currently unknown. Here, we characterize the role of podoplanin in different stages of myocardial repair after infarction and propose a podoplanin-mediated mechanism in the resolution of post-MI inflammatory response and cardiac repair. Neutralization of podoplanin led to significant improvements in the left ventricular functions and scar composition in animals treated with podoplanin neutralizing antibody. The inhibition of the interaction between podoplanin and CLEC-2 expressing immune cells in the heart enhances the cardiac performance, regeneration and angiogenesis post MI. Our data indicates that modulating the interaction between podoplanin positive cells with the immune cells after myocardial infarction positively affects immune cell recruitment and may represent a novel therapeutic target to augment post-MI cardiac repair, regeneration and function.
Maria Cimini, Venkata Naga Srikanth Garikipati, Claudio de Lucia, Zhongjian Cheng, Chunlin Wang, May M. Truongcao, Anna Maria Lucchese, Rajika Roy, Cindy Benedict, David A. Goukassian, Walter J. Koch, Raj Kishore
Atrial dysfunction is highly prevalent and associated with increased severity of heart failure. While rapid excitation-contraction coupling depends on axial junctions in atrial myocytes, the molecular basis of atrial loss of function remains unclear. We identified approximately 5-fold lower junctophilin-2 levels in atrial compared with ventricular tissue in mouse and human hearts. In atrial myocytes, this resulted in subcellular expression of large junctophilin-2 clusters at axial junctions, together with highly phosphorylated ryanodine receptor (RyR2) channels. To investigate the contribution of junctophilin-2 to atrial pathology in adult hearts, we developed a cardiomyocyte-selective junctophilin-2–knockdown model with 0 mortality. Junctophilin-2 knockdown in mice disrupted atrial RyR2 clustering and contractility without hypertrophy or interstitial fibrosis. In contrast, aortic pressure overload resulted in left atrial hypertrophy with decreased junctophilin-2 and RyR2 expression, disrupted axial junctions, and atrial fibrosis. Whereas pressure overload accrued atrial dysfunction and heart failure with 40% mortality, additional junctophilin-2 knockdown greatly exacerbated atrial dysfunction with 100% mortality. Strikingly, transgenic junctophilin-2 overexpression restored atrial contractility and survival through de novo biogenesis of polyadic junctional membrane complexes maintained after pressure overload. Our data show a central role of junctophilin-2 cluster disruption in atrial hypertrophy and identify transgenic augmentation of junctophilin-2 as a disease-mitigating rationale to improve atrial dysfunction and prevent heart failure deterioration.
Sören Brandenburg, Jan Pawlowitz, Benjamin Eikenbusch, Jonas Peper, Tobias Kohl, Gyuzel Y. Mitronova, Samuel Sossalla, Gerd Hasenfuss, Xander H.T. Wehrens, Peter Kohl, Eva A. Rog-Zielinska, Stephan E. Lehnart
Heterozygous missense mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysms and dissections. To assess how LOX mutations modify protein function and lead to aortic disease, we studied the factors that influence the onset and progression of vascular aneurysms in mice bearing a Lox mutation (p.M292R) linked to aortic dilation in humans. We show that mice heterozygous for the M292R mutation did not develop aneurysmal disease unless challenged with increased hemodynamic stress. Vessel dilation was confined to the ascending aorta although both the ascending and descending aortae showed changes in vessel wall structure, smooth muscle cell number and inflammatory cell recruitment that differed between wild-type and mutant animals. Studies with isolated cells found that M292R-mutant Lox is retained in the endoplasmic reticulum and ultimately cleared through an autophagy/proteasome pathway. Because the mutant protein does not transit to the Golgi where copper incorporation occurs, the protein is never catalytically active. These studies show that the M292R mutation results in LOX loss-of-function due to a secretion defect that predisposes the ascending aorta in mice (and by extension humans with similar mutations) to arterial dilation when exposed to risk factors that impart stress to the arterial wall.
Vivian S. Lee, Carmen M. Halabi, Thomas J. Broekelmann, Philip C. Trackman, Nathan O. Stitziel, Robert P. Mecham
Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.
Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein
The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.
Lalita Oonthonpan, Adam J. Rauckhorst, Lawrence R. Gray, Audrey C. Boutron, Eric B. Taylor
Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.
Lance W. Middleton, Zhewei Shen, Sushama Varma, Anna S. Pollack, Xue Gong, Shirley Zhu, Chunfang Zhu, Joseph W. Foley, Sujay Vennam, Robert T. Sweeney, Karen Tu, Jewison Biscocho, Okyaz Eminaga, Rosalie Nolley, Robert Tibshirani, James D. Brooks, Robert B. West, Jonathan R. Pollack
Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFβ and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFβ- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.
Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y. S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin
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