Publications by authors named "Federica Genovese"

63 Publications

From loss to recovery: how to effectively assess chemosensory impairments during COVID-19 pandemic.

medRxiv 2021 Mar 26. Epub 2021 Mar 26.

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, limited (partial) and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and its severe discomfort.
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http://dx.doi.org/10.1101/2021.03.25.21254253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010774PMC
March 2021

Proteoglycan Remodeling Is Accelerated in Females with Angina Pectoris and Diffuse Myocardial Fibrosis: the iPOWER Study.

J Cardiovasc Transl Res 2021 Mar 1. Epub 2021 Mar 1.

Department of Cardiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Angina and no obstructive coronary artery disease (CAD) have an unfavorable prognosis, possibly due to diffuse myocardial fibrosis (DMF). In DMF the proteoglycans biglycan and versican are actively remodeled by matrix metalloproteinase. We investigated biglycan and versican in females with angina and possible DMF assessed by cardiac magnetic resonance (CMR). Seventy-one females with angina and no obstructive CAD were included. Asymptomatic females served as controls. Versican and biglycan were measured and CMR was performed measuring extracellular volume. Biglycan and versican levels were higher in symptomatic females compared with controls; 31.4 ng/mL vs. 16.4 ng/mL (p < 0.001) and 2.1 ng/mL vs. 1.8 ng/mL (p < 0.001) and moderately correlated to extracellular volume (r = 0.38, p<0.001 and r = 0.26, p = 0.015). Turnover of biglycan and versican was increased in angina females compared with controls and associated with extracellular volume, supporting a link between angina with no obstructive CAD and fibrotic remodeling.
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http://dx.doi.org/10.1007/s12265-021-10106-yDOI Listing
March 2021

Imbalanced turnover of collagen type III is associated with disease progression and mortality in high-risk chronic kidney disease patients.

Clin Kidney J 2021 Feb 14;14(2):593-601. Epub 2020 Jan 14.

College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Background: Tubulointerstitial fibrosis is a major pathological feature in chronic kidney disease (CKD) and collagen type III (COL3) is a major component of the renal fibrotic scar. We hypothesized that a dysregulated turnover of COL3 is an important determinant of CKD progression. We assessed the relationship between fragments reflecting active formation (PRO-C3) and degradation (C3M) of COL3 and CKD disease progression and mortality in a prospective cohort of CKD patients.

Methods: We measured PRO-C3 and C3M in urine (uPRO-C3 and uC3M) and serum (sPRO-C3 and sC3M) of 500 patients from the Renal Impairment in Secondary Care study. Disease progression was defined as a decline in estimated glomerular filtration rate >30% or the start of renal replacement therapy within 12 and 30 months.

Results: Levels of uC3M/creatinine decreased, whereas levels of uPRO-C3/creatinine and sPRO-C3 increased with increasing CKD stage. uC3M/creatinine was inversely and independently associated with disease progression by 12 months {odds ratio [OR] 0.39 [95% confidence interval (CI) 0.18-0.83]; P = 0.01 per doubling of uC3M/creatinine} with development of end-stage renal disease [hazard ratio (HR) 0.70 (95% CI 0.50-0.97); P = 0.03 per doubling of uC3M/creatinine]. sPRO-C3 at baseline was independently associated with increased mortality [HR 1.93 (95% CI 1.21-3.1); P = 0.006 per doubling of sPRO-C3] and disease progression by 30 months [OR 2.16 (95% CI 1.21-3.84); P = 0.009 per doubling of sPRO-C3].

Conclusions: Dynamic products of COL3 formation and degradation were independently associated with CKD progression and mortality and may represent an opportunity to link pathological processes with targeted treatments against fibrosis.
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http://dx.doi.org/10.1093/ckj/sfz174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886548PMC
February 2021

The functional relevance of olfactory marker protein in the vertebrate olfactory system: a never-ending story.

Cell Tissue Res 2021 Jan 15;383(1):409-427. Epub 2021 Jan 15.

Monell Chemical Senses Center, Philadelphia, PA, 19104, USA.

Olfactory marker protein (OMP) was first described as a protein expressed in olfactory receptor neurons (ORNs) in the nasal cavity. In particular, OMP, a small cytoplasmic protein, marks mature ORNs and is also expressed in the neurons of other nasal chemosensory systems: the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. While its expression pattern was more easily established, OMP's function remained relatively vague. To date, most of the work to understand OMP's role has been done using mice lacking OMP. This mostly phenomenological work has shown that OMP is involved in sharpening the odorant response profile and in quickening odorant response kinetics of ORNs and that it contributes to targeting of ORN axons to the olfactory bulb to refine the glomerular response map. Increasing evidence shows that OMP acts at the early stages of olfactory transduction by modulating the kinetics of cAMP, the second messenger of olfactory transduction. However, how this occurs at a mechanistic level is not understood, and it might also not be the only mechanism underlying all the changes observed in mice lacking OMP. Recently, OMP has been detected outside the nose, including the brain and other organs. Although no obvious logic has become apparent regarding the underlying commonality between nasal and extranasal expression of OMP, a broader approach to diverse cellular systems might help unravel OMP's functions and mechanisms of action inside and outside the nose.
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http://dx.doi.org/10.1007/s00441-020-03349-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878404PMC
January 2021

Cross-Linked Multimeric Pro-Peptides of Type III Collagen (PC3X) in Hepatocellular Carcinoma - A Biomarker That Provides Additional Prognostic Value in AFP Positive Patients.

J Hepatocell Carcinoma 2020 9;7:301-313. Epub 2020 Nov 9.

Biomarkers & Research, Nordic Bioscience, Herlev, Denmark.

Purpose: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis is present in 80-90% of HCC patients. Cirrhosis is characterized by deposition and cross-linking of collagens that have crucial roles in HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC.

Patients And Methods: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS).

Results: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (=0.0002, 0.0001). In patients with normal AFP (<20 IU/mL), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.72 and 0.68, respectively. High PC3X and AFP predicted for poor PFS (HR=1.80, =0.032; HR=1.70, =0.031) and OS (HR=2.12, =0.024; HR=2.55; =0.003), whereas PRO-C3 did not (PFS: HR=1.19, =0.059 and OS: HR=1.12, =0.324). PC3X was independent of AFP (PFS: HR=1.74, =0.045 and OS: HR=2.21, =0.018) and combining the two improved prognostic value (PFS: HR=2.66, =0.004 and OS: HR=5.86, <0.0001).

Conclusion: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC.
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http://dx.doi.org/10.2147/JHC.S275008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665576PMC
November 2020

Biopsy-Controlled Non-Invasive Quantification of Collagen Type VI in Kidney Transplant Recipients: A Post-Hoc Analysis of the MECANO Trial.

J Clin Med 2020 Oct 7;9(10). Epub 2020 Oct 7.

Division of Nephrology, Department of Internal Medicine, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands.

The PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal associations between plasma and urine PRO-C6 and proven histological changes after kidney transplantation. The current study is a post-hoc analysis of 94 participants of the MECANO trial, a 24-month prospective, multicenter, open-label, randomized, controlled trial aimed at comparing everolimus-based vs. cyclosporine-based immunosuppression. PRO-C6 was measured in plasma and urine samples collected 6 and 24 months post-transplantation. Fibrosis was evaluated in biopsies collected at the same time points by Banff interstitial fibrosis/tubular atrophy (IF/TA) scoring and collagen staining (Picro Sirius Red; PSR); inflammation was evaluated by the tubulo-interstitial inflammation score (ti-score). Linear regression analyses were performed. Six-month plasma PRO-C6 was cross-sectionally associated with IF/TA score (Std. β = 0.34), and prospectively with 24-month IF/TA score and ti-score (Std. β = 0.24 and 0.23, respectively) ( < 0.05 for all). No significant associations were found between urine PRO-C6 and any of the biopsy findings. Fibrotic changes and urine PRO-C6 behaved differentially over time according to immunosuppressive therapy. These results are a first step towards non-invasive fibrosis detection after kidney transplantation by means of collagen VI synthesis measurement, and further research is required.
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http://dx.doi.org/10.3390/jcm9103216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600059PMC
October 2020

The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.

Atherosclerosis 2020 11 25;313:88-95. Epub 2020 Sep 25.

Cardiovascular Research - Translational Studies, Department of Clinical Sciences, Malmö, Lund University, Sweden; Department of Cardiology, Skåne University Hospital, Sweden.

Background And Aims: A vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown.

Methods: 196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1β were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-β1, β2 and β3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up.

Results: Mimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death.

Conclusions: This study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.09.011DOI Listing
November 2020

Noninvasive Assessment of Fibrosis Following Ischemia/Reperfusion Injury in Rodents Utilizing Na Magnetic Resonance Imaging.

Pharmaceutics 2020 Aug 14;12(8). Epub 2020 Aug 14.

Department of Clinical Medicine, Aarhus University, 8200 Aarhus N, Denmark.

Fibrosis is often heterogeneously distributed, and classical biopsies do not reflect this. Noninvasive methods for renal fibrosis have been developed to follow chronic kidney diseases (CKD) and to monitor anti-fibrotic therapy. In this study, we combined two approaches to assess fibrosis regression following renal ischemia-reperfusion injury (IRI): magnetic resonance imaging (MRI) and noninvasive extracellular matrix (ECM) biomarkers. MRI was used to evaluate fibrosis in bilateral IRI in rats after reperfusion at 7, 14, and 21 days. This was performed with HT and T* mapping, dynamic contrast-enhanced (DCE)-MRI, and chemical shift imaging (CSI)-Na. The degradation of laminin gamma-1 chain (LG1M) and type III collagen (C3M) was measured in urine and plasma. Fibrosis was analyzed in tissue using fibronectin (FN) and alpha-smooth muscle actin (α-SMA) using quantitative polymerase chain reaction qPCR and western blotting. We found increased fibrosis 7 days after reperfusion, which dropped to sham levels after 21 days. Single kidney glomerular filtration rate (skGFR), perfusion (DCE-MRI), and total Na kidney content correlated positively with fibrotic markers FN and α-SMA as well as noninvasive LG1M and C3M. We showed that novel MRI protocols and ECM markers could track fibrogenic development. This could give rise to a multi-parametric practice to diagnose and assess fibrosis whilst treating kidney disease without using invasive methods.
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http://dx.doi.org/10.3390/pharmaceutics12080775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463828PMC
August 2020

Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trial.

Lancet Diabetes Endocrinol 2020 09 18;8(9):762-772. Epub 2020 Aug 18.

Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.

Background: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit.

Methods: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382).

Findings: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred.

Interpretation: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression.

Funding: European Commission Seventh Framework Programme.
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http://dx.doi.org/10.1016/S2213-8587(20)30228-XDOI Listing
September 2020

More Than Smell-COVID-19 Is Associated With Severe Impairment of Smell, Taste, and Chemesthesis.

Authors:
Valentina Parma Kathrin Ohla Maria G Veldhuizen Masha Y Niv Christine E Kelly Alyssa J Bakke Keiland W Cooper Cédric Bouysset Nicola Pirastu Michele Dibattista Rishemjit Kaur Marco Tullio Liuzza Marta Y Pepino Veronika Schöpf Veronica Pereda-Loth Shannon B Olsson Richard C Gerkin Paloma Rohlfs Domínguez Javier Albayay Michael C Farruggia Surabhi Bhutani Alexander W Fjaeldstad Ritesh Kumar Anna Menini Moustafa Bensafi Mari Sandell Iordanis Konstantinidis Antonella Di Pizio Federica Genovese Lina Öztürk Thierry Thomas-Danguin Johannes Frasnelli Sanne Boesveldt Özlem Saatci Luis R Saraiva Cailu Lin Jérôme Golebiowski Liang-Dar Hwang Mehmet Hakan Ozdener Maria Dolors Guàrdia Christophe Laudamiel Marina Ritchie Jan Havlícek Denis Pierron Eugeni Roura Marta Navarro Alissa A Nolden Juyun Lim Katherine L Whitcroft Lauren R Colquitt Camille Ferdenzi Evelyn V Brindha Aytug Altundag Alberto Macchi Alexia Nunez-Parra Zara M Patel Sébastien Fiorucci Carl M Philpott Barry C Smith Johan N Lundström Carla Mucignat Jane K Parker Mirjam van den Brink Michael Schmuker Florian Ph S Fischmeister Thomas Heinbockel Vonnie D C Shields Farhoud Faraji Enrique Santamaría William E A Fredborg Gabriella Morini Jonas K Olofsson Maryam Jalessi Noam Karni Anna D'Errico Rafieh Alizadeh Robert Pellegrino Pablo Meyer Caroline Huart Ben Chen Graciela M Soler Mohammed K Alwashahi Antje Welge-Lüssen Jessica Freiherr Jasper H B de Groot Hadar Klein Masako Okamoto Preet Bano Singh Julien W Hsieh Danielle R Reed Thomas Hummel Steven D Munger John E Hayes

Chem Senses 2020 10;45(7):609-622

Department of Food Science, The Pennsylvania State University, Erickson Food Science Building, University Park, PA, USA.

Recent anecdotal and scientific reports have provided evidence of a link between COVID-19 and chemosensory impairments, such as anosmia. However, these reports have downplayed or failed to distinguish potential effects on taste, ignored chemesthesis, and generally lacked quantitative measurements. Here, we report the development, implementation, and initial results of a multilingual, international questionnaire to assess self-reported quantity and quality of perception in 3 distinct chemosensory modalities (smell, taste, and chemesthesis) before and during COVID-19. In the first 11 days after questionnaire launch, 4039 participants (2913 women, 1118 men, and 8 others, aged 19-79) reported a COVID-19 diagnosis either via laboratory tests or clinical assessment. Importantly, smell, taste, and chemesthetic function were each significantly reduced compared to their status before the disease. Difference scores (maximum possible change ±100) revealed a mean reduction of smell (-79.7 ± 28.7, mean ± standard deviation), taste (-69.0 ± 32.6), and chemesthetic (-37.3 ± 36.2) function during COVID-19. Qualitative changes in olfactory ability (parosmia and phantosmia) were relatively rare and correlated with smell loss. Importantly, perceived nasal obstruction did not account for smell loss. Furthermore, chemosensory impairments were similar between participants in the laboratory test and clinical assessment groups. These results show that COVID-19-associated chemosensory impairment is not limited to smell but also affects taste and chemesthesis. The multimodal impact of COVID-19 and the lack of perceived nasal obstruction suggest that severe acute respiratory syndrome coronavirus strain 2 (SARS-CoV-2) infection may disrupt sensory-neural mechanisms.
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http://dx.doi.org/10.1093/chemse/bjaa041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337664PMC
October 2020

Fibrogenesis and inflammation contribute to the pathogenesis of cirrhotic cardiomyopathy.

Aliment Pharmacol Ther 2020 07 11;52(2):340-350. Epub 2020 Jun 11.

Hvidovre, Denmark.

Background: Fibrogenesis and inflammation contribute to the progression of cirrhosis. However, it is unknown if these processes also contribute to the development of cirrhotic cardiomyopathy (CCM). Novel magnetic resonance imaging with quantification of the extracellular volume (ECV) provides an estimate of the fibrotic remodelling in the liver and heart.

Aim: To investigate the relationship between liver and cardiac ECV in cirrhosis and their association with collagen turnover and inflammation.

Methods: A prospective study of 52 patients with cirrhosis and 14 healthy controls. All patients underwent contrast-enhanced MRI with T1-mapping and quantification of myocardial and liver ECV, biochemical assessments of collagen turnover (PRO-C3, PRO-C5, PRO-C6, collagen type IV degradation fragment, collagen type V degradation fragment, LG1M) and inflammation (TNFα, IL-1β, IL-6, IL-8, IL-18, SDF1α, sCD163, sMR, soluble macrophage mannose receptor).

Results: Myocardial and liver ECV were increased in patients compared with healthy controls (myocardial ECV 31.2 ± 5.5% vs 27.4 ± 2.9%, P = 0.037; liver ECV 44.1 ± 9.6% vs 33.7 ± 6.7%, P < 0.001). Myocardial ECV correlated strongly with liver ECV (r = 0.48, P = 0.001) and biomarkers of collagen formation and inflammation (P < 0.005). Similarly, liver ECV correlated with biomarkers of collagen formation and inflammation (P < 0.003). In a multivariate analysis, liver ECV was predicted by biomarkers of collagen formation (PRO-C3 and PRO-C6), whereas myocardial ECV was predicted by biomarkers of collagen formation (PRO-C6) and inflammation (IL-6 and sMR).

Conclusion: Structural myocardial changes seem closely related to liver fibrosis in patients with cirrhosis. The strong associations with biomarkers of collagen formation and inflammation provide new insight into the role of inflammation and fibrogenesis in the development of structural cardiac abnormalities, potentially leading to CCM.
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http://dx.doi.org/10.1111/apt.15812DOI Listing
July 2020

Studies of human twins reveal genetic variation that affects dietary fat perception.

Chem Senses 2020 Jun 9. Epub 2020 Jun 9.

Monell Chemical Senses Center, Philadelphia, PA, USA.

To learn more about the mechanisms of human dietary fat perception, 398 human twins rated fattiness and liking for six types of potato chips that differed in triglyceride content (2.5, 5, 10, and 15% corn oil); reliability estimates were obtained from a subset (n = 50) who did the task twice. Some chips also had a saturated long-chain fatty acid (hexadecanoic acid, 16:0) added (0.2%) to evaluate its effect on fattiness and liking. We computed the heritability of these measures and conducted a genome-wide association study (GWAS) to identify regions of the genome that co-segregate with fattiness and liking. Perceived fattiness and liking for the potato chips were reliable (r = 0.31-0.62, p < 0.05) and heritable (up to h2 = 0.29, p < 0.001, for liking). Adding hexadecanoic acid to the potato chips significantly increased ratings of fattiness but decreased liking. Twins with the G allele of rs263429 near GATA3-AS1 or the G allele of rs8103990 within ZNF729 reported more liking for potato chips than did twins with the other allele (multivariate GWAS, p < 1×10-5), with results reaching genome-wide suggestive but not significance criteria. Person-to-person variation in the perception and liking of dietary fat was (a) negatively affected by the addition of a saturated fatty acid and (b) related to inborn genetic variants. These data suggest liking for dietary fat is not due solely to fatty acid content and highlight new candidate genes and proteins within this sensory pathway.
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http://dx.doi.org/10.1093/chemse/bjaa036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339080PMC
June 2020

Hyperpolarized [1- C] alanine production: A novel imaging biomarker of renal fibrosis.

Magn Reson Med 2020 10 25;84(4):2063-2073. Epub 2020 May 25.

MR Research Center, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

Purpose: Renal tubulointerstitial fibrosis is strongly linked to the progressive decline of renal function seen in chronic kidney disease. State-of-the-art noninvasive diagnostic modalities are currently unable to detect the earliest changes associated with the onset of fibrosis. This study was undertaken to evaluate the potential for detecting the earliest alterations in fibrogenesis using a biofluid-based method and metabolic hyperpolarized [1- C]pyruvate imaging.

Methods: We evaluated renal fibrosis in a combined ischemia reperfusion-induced and streptozotocin-induced diabetic nephropathy rodent model by hyperpolarized [1- C]pyruvate MRI and correlated the metabolic MRI parameters with biomarkers of fibrosis measured on renal tissue and plasma/urine.

Results: The hyperglycemic rats experienced maladaptive injury repair after the ischemic insults, as shown by the elevation in the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Renal function was significantly impaired in the ischemic hyperglycemic kidney, as seen in the reduced perfusion and single-kidney glomerular filtration rate. A deranged energy metabolism was detected in the ischemic hyperglycemic kidney, as seen in the reduced fractional perfusion of lactate. Renal fibrosis biomarkers correlated significantly with the alanine production.

Conclusion: Hyperpolarized carbon-13 MRI provides a promising approach to assess renal fibrosis in an animal model of fibrotic chronic kidney disease. In particular, the metabolic supply of amino acids for fibrogenesis (alanine production) correlates well with biomarkers of fibrosis. Thus, [1- C]pyruvate-to-[1- C]alanine conversion might be a candidate for noninvasive assessment of renal fibrogenesis.
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http://dx.doi.org/10.1002/mrm.28326DOI Listing
October 2020

Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis.

Respir Res 2020 May 7;21(1):108. Epub 2020 May 7.

Nordic Bioscience A/S, Herlev, Herlev Hovedgade 205-207, DK-2730, Herlev, Denmark.

Background: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested.

Methods: Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-β1 with or without treatment with an ALK5/TGF-β1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression.

Results: TGF-β1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-β1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-β1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA.

Conclusions: TGF-β1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.
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http://dx.doi.org/10.1186/s12931-020-01369-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203825PMC
May 2020

A Fragment of Collagen Type VI alpha-3 chain is Elevated in Serum from Patients with Gastrointestinal Disorders.

Sci Rep 2020 04 3;10(1):5910. Epub 2020 Apr 3.

Nordic Bioscience, Biomarkers and Research, Herlev, Denmark.

Extracellular matrix (ECM) remodeling is a hallmark of the pathology of gastrointestinal disorders. Collagen type VI (COL6) is produced by fibroblasts, and the COL6 α3-chain has shown to be elevated in patients with ulcerative colitis (UC), Crohn's disease (CD) and colorectal cancer (CRC). Measuring COL6α3 in serum may therefore have potential as a biomarker for gastrointestinal disorders. The aims of this study were to develop and validate a competitive ELISA targeting a specific neo-epitope of COL6α3 and evaluate its associations with the gastrointestinal disorders UC, CD and CRC, in comparison to healthy controls. A monoclonal antibody was raised against a matrix metalloproteinase-2 and -9 specific cleavage site of COL6α3 (C6Mα3) and employed in a competitive enzyme-linked immunosorbent assay (ELISA). The assay was developed and technically validated. Levels of C6Mα3 were measured in serum from patients with UC (n = 58), CD (n = 44) and CRC (n = 39) and compared to healthy controls (n = 32). The levels of C6Mα3 were elevated in patients with UC, CD and CRC patients compared to healthy controls (all p < 0.0001). The area under the receiver operating characteristics (AUROC) curve for separation of patients with UC from healthy controls was 0.972 (95% CI: 0.925-1.020, p < 0.0001), with CD from healthy controls was 0.947 (95% CI: 0.885-1.009, p < 0.0001) and with CRC from healthy controls was 0.890 (95% CI: 0.809-0.972, p < 0.0001). We developed a technically robust assay targeting a fragment of COL6, which was elevated in serum from patients with UC, CD and CRC.
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http://dx.doi.org/10.1038/s41598-020-62474-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125205PMC
April 2020

Atrial fibrillation and cardiac fibrosis: A review on the potential of extracellular matrix proteins as biomarkers.

Matrix Biol 2020 09 20;91-92:188-203. Epub 2020 Mar 20.

Nordic Bioscience, Biomarkers and Research, Herlev, Denmark.

The involvement of fibrosis as an underlying pathology in heart diseases is becoming increasingly clear. In recent years, fibrosis has been granted a causative role in heart diseases and is now emerging as a major contributor to Atrial Fibrillation (AF) pathogenesis. AF is the most common arrhythmia encountered in the clinic, but the substrate for AF is still being debated. Consensus in the field is a combination of cardiac tissue remodeling, inflammation and genetic predisposition. The extracellular matrix (ECM) is subject of growing investigation, since measuring circulatory biomarkers of ECM formation and degradation provides both diagnostic and prognostic information. However, fibrosis is not just fibrosis. Each specific collagen biomarker holds information on regulatory mechanisms, as well as information about which section of the ECM is being remodeled, providing a detailed description of cardiac tissue homeostasis. This review entails an overview of the implication of fibrosis in AF, the different collagens and their significance, and the potential of using biomarkers of ECM remodeling as tools for understanding AF pathogenesis and identifying patients at risk for further disease progression.
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http://dx.doi.org/10.1016/j.matbio.2020.03.005DOI Listing
September 2020

Reduced -GlcNAcylation and tubular hypoxia contribute to the antifibrotic effect of SGLT2 inhibitor dapagliflozin in the diabetic kidney.

Am J Physiol Renal Physiol 2020 04 2;318(4):F1017-F1029. Epub 2020 Mar 2.

MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences, Budapest, Hungary.

Diabetic kidney disease is a worldwide epidemic, and therapies are incomplete. Clinical data suggest that improved renal outcomes by Na-glucose cotransporter 2 inhibitor (SGLT2i) are partly beyond their antihyperglycemic effects; however, the mechanisms are still elusive. Here, we investigated the effect of the SGLT2i dapagliflozin (DAPA) in the prevention of elevated -GlcNAcylation and tubular hypoxia as contributors of renal fibrosis. Type 1 diabetes was induced by streptozotocin in adult male Wistar rats. After the onset of diabetes, rats were treated for 6 wk with DAPA or DAPA combined with losartan (LOS). The effect of hyperglycemia was tested in HK-2 cells kept under normal or high glucose conditions. To test the effect of hypoxia, cells were kept in 1% O for 2 h. Cells were treated with DAPA or DAPA combined with LOS. DAPA slowed the loss of renal function, mitigated renal tubular injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), and reduced tubulointerstitial fibrosis. DAPA diminished high glucose-induced protein -GlcNAcylation and moderated the tubular response to hypoxia through the hypoxia-inducible factor pathway. DAPA alone was as effective as combined treatment with LOS in all outcome parameters. These data highlight the role of ameliorated -GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia, and fibrosis, a vicious trio that could be targeted by SGLT2i in kidney diseases of other origins as well.
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http://dx.doi.org/10.1152/ajprenal.00021.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242633PMC
April 2020

Collagen turnover profiles in chronic kidney disease.

Sci Rep 2019 11 5;9(1):16062. Epub 2019 Nov 5.

Nordic Bioscience, Herlev, Denmark.

Renal fibrosis is a hallmark of chronic kidney disease (CKD) caused by an imbalance between formation and degradation of extracellular matrix proteins. We investigated the collagen turnover profile of 81 non-dialysis CKD stage 2-5 patients by measuring peptides reflecting formation and degradation of collagen type (COL) I, III, IV, and VI. Based on the collagen turnover profile, we identified four clusters of patients. Cluster 1 contained one patient with prostate cancer, who had a distinct collagen turnover. The other clusters generally had severe (Cluster 2), moderate (Cluster 4), or mild CKD (Cluster 3). Cluster 4 patients were characterized by higher levels of COL III, COL IV, and COL VI (all p < 0.001) degradation fragments in plasma, while patients in Clusters 2 and 4 had higher levels of COL VI formation (p < 0.05). COL IV fragments in plasma were lower in Cluster 2 (p < 0.01). Urinary COL III fragments decreased from Cluster 3 to 4, and from Cluster 4 to 2 (both p < 0.001). We show that patients with similar kidney function have a different collagen remodeling profile, suggesting that different phenotypes exist with different disease activity and potentially disease progression. Biomarkers of collagen remodeling could provide additional information to traditional markers of renal function.
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http://dx.doi.org/10.1038/s41598-019-51905-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831687PMC
November 2019

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

J Inherit Metab Dis 2020 07 5;43(4):737-747. Epub 2020 Feb 5.

Department of Musculoskeletal Biology, University of Liverpool, Liverpool, UK.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CL ) (P < 0.001) and FE (P < 0.001). CL and FE were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.
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http://dx.doi.org/10.1002/jimd.12181DOI Listing
July 2020

Markers of Collagen Formation and Degradation Reflect Renal Function and Predict Adverse Outcomes in Patients With Type 1 Diabetes.

Diabetes Care 2019 09 1;42(9):1760-1768. Epub 2019 Jul 1.

Steno Diabetes Center Copenhagen, Gentofte, Denmark.

Objective: Patients with type 1 diabetes (T1D) have a higher risk of developing chronic kidney disease, cardiovascular events (CVEs), and mortality than the general population. We hypothesized that two previously published biomarkers, namely PRO-C6, a biomarker of collagen type VI formation, and C3M, a biomarker of collagen type III degradation, may be associated with impaired renal function and have prognostic value for adverse renal, CVE, and mortality in patients with T1D.

Research Design And Methods: PRO-C6 and C3M in serum (sPRO-C6, sC3M) and urine (uPRO-C6, uC3M) were measured by ELISA in 663 patients with T1D ranging from normoalbuminuric to macroalbuminuric. Association of the biomarkers with mortality, CVEs, heart failure, decline in estimated glomerular filtration rate (eGFR) ≥30%, and end-stage renal disease (ESRD) were tested in Cox proportional hazards models after log transformation and adjusted for relevant clinical characteristics. Hazard ratios (HRs) were reported per doubling of biomarker levels.

Results: High levels of sPRO-C6 were independently associated with a higher risk of all-cause mortality (HR 2.26 [95% CI 1.31-3.87], < 0.0031). There was an association with higher risk of CVEs ( = 94) and heart failure ( = 28) but not after adjustment ( ≥ 0.58). In relation to renal outcomes, adjusted sPRO-C6 was associated with a higher risk of eGFR decline ≥30% in T1D, with eGFR >45 and >30 mL/min/1.73 m, and with a higher risk of ESRD (all ≤ 0.03). Higher uPRO-C6 was associated with a lower risk of decline in eGFR.

Conclusions: In patients with T1D, higher sPRO-C6 was an independent predictor of both decline in eGFR and development of ESRD and of all-cause mortality. Higher uPRO-C6 was also associated with a lower risk of decline in eGFR.
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http://dx.doi.org/10.2337/dc18-2599DOI Listing
September 2019

Pressure overload induced right ventricular remodeling is not attenuated by the anti-fibrotic agent pirfenidone.

Pulm Circ 2019 Apr-Jun;9(2):2045894019848659

1 Department of Cardiology, Aarhus University Hospital, Denmark.

Cardiac fibrosis contributes to the development of heart failure in pulmonary hypertension. We aimed to assess the development of fibrosis and the effects of treatment with the anti-fibrotic agent pirfenidone in pressure overload induced right ventricular (RV) failure. Wistar rat weanlings were randomized to pulmonary trunk banding (PTB) or sham surgery. One week after the procedure, PTB rats were randomized into two groups with either six weeks on standard chow or treatment with pirfenidone mixed in chow (700 mg/kg/day). RV hemodynamic effects were evaluated by echocardiography, cardiac magnetic resonance imaging (MRI), and pressure-volume measurements. Sections from the isolated RV, left ventricle, and septum were sampled systematically; stereological point grids and the nucleator were used to estimate volume of fibrosis and cardiac hypertrophy, respectively. PTB caused RV failure in all rats subjected to the procedure. The volume fraction of fibrosis in the RV increased threefold in PTB rats corresponding to a sixfold increase in total volume of RV fibrosis. Volume fraction of fibrosis and total volume of fibrosis also increased in the septum and in the left ventricle. Pirfenidone reduced body weight but did not improve RV hemodynamics or reduce cardiac fibrosis. RV cardiomyocyte profile area was increased twofold in PTB rats without any effect of pirfenidone. RV pressure overload after PTB induced not only RV but also septal and left ventricular fibrosis assessed by stereology. Treatment with pirfenidone reduced body weight but did not reduce the development of cardiac fibrosis or delay the progression of RV failure.
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http://dx.doi.org/10.1177/2045894019848659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540527PMC
April 2019

Markers of Basement Membrane Remodeling Are Associated With Higher Mortality in Patients With Known Atherosclerosis.

J Am Heart Assoc 2018 11;7(21):e009193

3 Experimental Cardiovascular Research Unit Department of Clinical Sciences, Malmö Lund University Malmö Sweden.

Background Patients with atherosclerosis have a high risk of cardiovascular events and death. Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix proteins in the intima. We hypothesized that dysregulated remodeling of the basement membrane proteins may be associated with clinical outcomes in patients with atherosclerosis. Methods and Results Neoepitope fragments of collagen type IV (C4M) and laminin ( LG 1M) were assessed by ELISA s in serum from 787 endarterectomy patients. Matrix metalloproteinase s were measured using proximity extension assay and correlated to C4M and LG 1M levels using Spearman correlations. A total of 473 patients were followed up for 6 years using national registers, medical charts, and telephone interviews. The incidence of cardiovascular events, cardiovascular mortality, and all-cause mortality were associated to levels of C4M and LG 1M using Kaplan-Meier curves and Cox regression analyses. A total of 101 patients had cardiovascular events, 39 died of cardiovascular mortality, and 64 patients died from all-cause mortality. C4M levels were increased in patients with symptomatic carotid atherosclerotic disease before surgery ( P=0.048). High C4M and LG 1M levels were associated with increased risk of all-cause mortality ( P=0.020 and 0.031, respectively) and predicted all-cause death together with glomerular filtration rate and diabetes mellitus. Conclusions High LG 1M and C4M levels were associated with all-cause mortality, together with glomerular filtration rate and diabetes mellitus. These novel biomarkers need further evaluation but might be tools to identify high-risk patients.
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http://dx.doi.org/10.1161/JAHA.118.009193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404182PMC
November 2018

A non-invasive biomarker of type III collagen degradation reflects ischaemia reperfusion injury in rats.

Nephrol Dial Transplant 2019 08;34(8):1301-1309

Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

Background: Maintenance of kidney function in kidney allografts remains a challenge, as the allograft often progressively develops fibrosis after kidney transplantation. Fibrosis is caused by the accumulation of extracellular matrix proteins like type I and III collagen (COL I and III) that replace the functional tissue. We assessed the concentrations of a neo-epitope fragment of COL III generated by matrix metalloproteinase-9 cleavage (C3M) in two rat models resembling the ischaemic injury taking place following kidney transplantation.

Methods: We measured C3M in urine (U-C3M) and plasma (P-C3M) samples of rats subjected to unilateral nephrectomy followed by sham operation (NTx) or ischaemia reperfusion injury (NTxIRI) as well as in rats subjected to bilateral ischaemia reperfusion injury (BiIRI). Levels of U-C3M were normalized to urinary creatinine and were correlated to plasma creatinine, blood urea nitrogen, messenger ribonucleic acid (mRNA) of markers of kidney injury, and mRNA and protein levels of markers of tissue repair and fibrosis.

Results: Levels of U-C3M were significantly elevated 7 days after ischaemia reperfusion in the NTxIRI. BiIRI animals showed higher levels of U-C3M after 7 and 14 days of reperfusion but not at 21 days. P-C3M did not change in any of the models. There was a significant correlation between U-C3M and mRNA levels of fibronectin, COL I alpha 1 chain (COL Ia1) and neutrophil gelatinase-associated lipocalin (NGAL), and protein levels of alpha smooth muscle actin (αSMA), fibronectin and COL III in NTxIRI but not in NTx animals. Levels of U-C3M increased significantly in the BiIRI animals subsequent to reperfusion, and mirrored the histological alterations. Furthermore, U-C3M was associated with the extent of fibrosis, and remained elevated even after plasma creatinine levels decreased.

Conclusions: These results demonstrate that degradation of COL III increases after ischaemia reperfusion injury, and that U-C3M may be a non-invasive marker of tissue repair and fibrosis in the ischaemic kidney.
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http://dx.doi.org/10.1093/ndt/gfy345DOI Listing
August 2019

Serological Assessment of Activated Fibroblasts by alpha-Smooth Muscle Actin (α-SMA): A Noninvasive Biomarker of Activated Fibroblasts in Lung Disorders.

Transl Oncol 2019 Feb 30;12(2):368-374. Epub 2018 Nov 30.

Nordic Bioscience A/S, Herlev, Denmark.

Objectives: Remodeling of the extracellular matrix (ECM) is a key event in different lung disorders, such as fibrosis and cancer. The most common cell type in the connective tissue is fibroblasts, which transdifferentiate into myofibroblasts upon activation. All myofibroblasts express α-SMA, which has been found to be upregulated in lung fibrosis and cancer. We evaluated the potential of α-SMA as a noninvasive biomarker of activated fibroblasts in lung fibrosis and cancer.

Methods: A monoclonal antibody was raised against the N-terminal of α-SMA, and a novel competitive enzyme-linked immunosorbent assay (ELISA) measuring α-SMA was developed and technically characterized. Levels of α-SMA were measured in the fibroblast model, "scar-in-a-jar", and in serum from patients with idiopathic pulmonary fibrosis (IPF), chronic obstructive lung disorder (COPD) and non-small cell lung cancer (NSCLC) belonging to two different cohorts.

Results: The novel α-SMA assay was developed and validated as technically robust. Based on the scar-in-a-jar results, α-SMA was only present in the fibroblasts activated by TGF-β. In cohort 1, levels of α-SMA were significantly higher in IPF, COPD and NSCLC patients compared to healthy controls (P = 0.04, P = 0.001 and P <0.0001, respectively). The area under the receiver operating characteristics (AUROC) for separation of healthy controls from IPF patients was 0.865, healthy controls from COPD patients was 0.892 and healthy controls from NSCLC patients was 0.983. In cohort 2, levels of α-SMA were also significantly higher in NSCLC patients compared to healthy controls (P = 0) and the AUROC for separating NSCLC and healthy controls was 0.715.

Conclusions: In this study we developed and validated a robust competitive ELISA assay targeting the N-terminal of α-SMA. The level of α-SMA was upregulated when adding TGF-β, indicating that α-SMA is increased in activated fibroblasts. The level of α-SMA in circulation was significantly higher in patients with IPF, COPD and NSCLC compared to healthy controls. This assay could potentially be used as a novel noninvasive serological biomarker for lung disorders by providing a surrogate measure of activated fibroblasts.
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http://dx.doi.org/10.1016/j.tranon.2018.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277250PMC
February 2019

RAAS inhibitors directly reduce diabetes-induced renal fibrosis via growth factor inhibition.

J Physiol 2019 01 2;597(1):193-209. Epub 2018 Nov 2.

MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.

Key Points: Increased activation of the renin-angiotensin-aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes-induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia-induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis.

Abstract: In diabetic kidney disease (DKD) increased activation of renin-angiotensin-aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin-induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet-derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)-induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha-smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK-2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia-induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non-antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.
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http://dx.doi.org/10.1113/JP277002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312411PMC
January 2019

A novel biomarker of laminin turnover is associated with disease progression and mortality in chronic kidney disease.

PLoS One 2018 1;13(10):e0204239. Epub 2018 Oct 1.

Department of Renal Medicine, Queen Elizabeth Hospital, Birmingham, United Kingdom.

Background: Patients with chronic kidney disease (CKD) have increased risk of development of end-stage renal disease (ESRD) and early mortality. Fibrosis is the central pathogenic process in CKD and is caused by dysregulated extracellular matrix (ECM) remodeling. The laminin γ1 chain (LAMC1) is a core structural protein present in the basement membrane of several organs, including the kidneys. We hypothesized that dysregulation of LAMC1 remodeling could be associated with a higher risk of adverse clinical outcomes in patients with CKD.

Methods: A novel immunoassay targeting LG1M, a specific MMP-9-generated neo-epitope fragment of LAMC1, was developed and used to measure the levels of the fragment in urine and serum from 492 patients from the Renal Impairment in Secondary Care (RIISC) study, a prospective cohort of patients with high-risk CKD. Patients were monitored for a median follow-up time of 3.5 years. Associations between serum and urine LG1M levels and progression of CKD at 12 months were assessed by a multivariable logistic regression model. The association with ESRD or mortality was assessed by Kaplan-Meier survival curves and Cox proportional hazards regression.

Results: Forty-six (11%) of the 416 patients who reached 12-month follow-up had progression of CKD; during the study follow-up, 125 patients (25.4%) developed ESRD and 71 patients (14.4%) died. Serum and urine levels of LG1M correlated with baseline eGFR (r = -0.43, p<0.0001 and r = -0.17, p = 0.0002, respectively). Serum levels of LG1M were higher in patients with one-year progression of CKD compared to those who did not progress (p<0.01). Baseline serum levels of LG1M were associated with development of ESRD (HR 3.2, 95% CI 1.99-5.2 for patients in the highest LG1M tertile compared to patient in the lowest tertile). Baseline urinary levels of LG1M (uLG1M) were significantly associated with mortality (HR 5.0, 95% CI 2.8-8.9, p<0.0001 for patients in the highest LG1M tertile compared to patients in the lowest tertile). Urine LG1M was retained in the model for prediction of mortality (HR per standard deviation of uLG1M: 1.01, 95% CI 1.00-1.02, p = 0.001).

Conclusions: LG1M, a marker of basement membrane remodeling, is increased in serum and urine of patients with CKD and levels are associated with one-year disease progression, development of ESRD, and mortality.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204239PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166934PMC
March 2019

Microvillous cells in the olfactory epithelium express elements of the solitary chemosensory cell transduction signaling cascade.

PLoS One 2018 13;13(9):e0202754. Epub 2018 Sep 13.

Monell Chemical Senses Center, Philadelphia, Pennsylvania, United States of America.

The nasal cavity hosts an array of chemoresponsive cells, including the extended olfactory system and several other cells involved in detection of and responses to irritants. Solitary chemosensory cells (SCCs), which respond to irritants and bacteria, express the transient receptor potential channel TRPM5 an essential element of the taste transduction-signaling cascade. Microvillous cells (MVCs), non-neuronal cells situated in the apical layer of the main olfactory epithelium, also express TRPM5, but their function has not yet been clarified. TRPM5-positive MVCs, like SCCs, show a cholinergic phenotype expressing choline acetyl transferase (ChAT), but none of the other elements of the bitter taste transduction cascade could be detected. We reexamined TRPM5-positive MVCs with more sensitive gene expression and staining techniques to clarify whether they rely only on TRPM5 and ChAT or express other elements of the taste/SCC transduction cascade. Analyzing existing RNA sequencing data from whole olfactory mucosa and isolated olfactory sensory neurons, we determined that several elements of the taste/SCC transduction cascade, including taste receptors, are expressed in the olfactory mucosa in cells other than olfactory sensory neurons. Immunostaining confirmed the presence TRPM5 and ChAT in a subset of cells of the olfactory mucosa, which also showed the expression of PLCB2, gustducin, and T1R3. Specifically, these cells were identified as TRPM5-positive MVCs. Furthermore, we examined whether MVCs are innervated by trigeminal fibers, similarly to SCCs. Using antibodies against trigeminal nerve markers calcitonin gene-related peptide and substance P, we determined that, despite the cholinergic phenotype, most MVCs in the olfactory mucosa lacked consistent trigeminal innervation. Our findings indicate that MVCs, like SCCs, express all the elements of the bitter taste transduction cascade but that, unlike SCCs, they possess only sparse trigeminal innervation. The cholinergic phenotype of MVCs suggests a modulatory function of the surrounding olfactory epithelium, through the release of acetylcholine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202754PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136699PMC
February 2019

Cardiac magnetic resonance using late gadolinium enhancement and atrial T1 mapping predicts poor outcome in patients with atrial fibrillation after catheter ablation therapy.

Sci Rep 2018 09 11;8(1):13618. Epub 2018 Sep 11.

Department of Internal Medicine II, University Hospital Bonn, Rheinische Friedrich-Wilhelms University, Bonn, Germany.

To determine the pre-procedural value of different fibrotic biomarkers and comprehensive cardiac magnetic resonance (CMR) for the prediction of poor response to ablation therapy in patients with atrial fibrillation (AF). Left atrial (LA) late gadolinium enhancement (LGE) and native LA T1 relaxation times were assessed using CMR. Plasma levels of relaxin, myeloperoxidase and serum levels of matrix metalloproteinase (MMP)-mediated cardiac specific titin fragmentation and MMP-mediated type IV collagen degradation were obtained. Poor outcome was defined by the recurrence of AF during 1-year follow-up. 61 patients were included in final analysis. Twenty (32.8%) patients had recurrence of AF. Patients with a recurrence of AF had a higher percentage of LA LGE (26.7 ± 12.5% vs. 17.0 ± 7.7%; P < 0.001), higher LA T1 relaxation times (856.7 ± 112.2 ms vs. 746.8 ± 91.0 ms; P < 0.001) and higher plasma levels of relaxin (0.69 ± 1.34 pg/ml vs. 0.37 ± 0.88 pg/ml; P = 0.035). In the multivariate Cox regression analysis, poor ablation outcome was best predicted by advanced LGE stage (hazard ratio (HR):5.487; P = 0.001) and T1 relaxation times (HR:1.007; P = 0.001). Pre-procedural CMR is a valuable tool for prediction of poor response to catheter ablation therapy in patients with AF. It offers various imaging techniques for outcome prediction and might be valuable for a better patient selection prior to ablation therapy.
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http://dx.doi.org/10.1038/s41598-018-31916-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134059PMC
September 2018

Higher Collagen VI Formation Is Associated With All-Cause Mortality in Patients With Type 2 Diabetes and Microalbuminuria.

Diabetes Care 2018 07 11;41(7):1493-1500. Epub 2018 Apr 11.

Steno Diabetes Center Copenhagen, Gentofte, Denmark.

Objective: Type 2 diabetes is a common risk factor for the development of chronic kidney disease (CKD). Enhanced de novo collagen type VI (COL VI) formation has been associated with renal fibrosis and CKD. We investigated the hypothesis that PRO-C6, a product specifically generated during COL VI formation, is prognostic for adverse outcomes in patients with type 2 diabetes and microalbuminuria.

Research Design And Methods: In a prospective, observational study, we measured PRO-C6 in the serum (S-PRO-C6) and urine (U-PRO-C6) of 198 patients with type 2 diabetes and microalbuminuria without symptoms of coronary artery disease. Patients were followed for a median of 6.5 years, and end points were a composite of cardiovascular events ( = 38), all-cause mortality ( = 26), and reduction of estimated glomerular filtration rate (eGFR) of >30% (disease progression [ = 42]). Cox models were unadjusted and adjusted for the conventional risk factors of sex, age, BMI, systolic blood pressure, LDL cholesterol, smoking, HbA, plasma creatinine, and urinary albumin excretion rate.

Results: Doubling of S-PRO-C6 increased hazards for cardiovascular events (hazard ratio 3.06 [95% CI 1.31-7.14]), all-cause mortality (6.91 [2.96-16.11]), and disease progression (4.81 [1.92-12.01]). Addition of S-PRO-C6 to a model containing conventional risk factors improved relative integrated discrimination by 22.5% for cardiovascular events ( = 0.02), 76.8% for all-cause mortality ( = 0.002), and 53.3% for disease progression ( = 0.004). U-PRO-C6 was not significantly associated with any of the outcomes.

Conclusions: S-PRO-C6 generated during COL VI formation predicts cardiovascular events, all-cause mortality, and disease progression in patients with type 2 diabetes and microalbuminuria.
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http://dx.doi.org/10.2337/dc17-2392DOI Listing
July 2018