Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis

PLOS ONE RESEARCH ARTICLE Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis Cristina Vercelli ID*☯, Massimiliano Tursi☯, Silvia Miretti☯, Gessica Giusto☯, Marco Gandini☯, Giovanni Re☯, Emanuela Valle☯ Department of Veterinary Science, University of Turin, Grugliasco (TO), Italy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Vercelli C, Tursi M, Miretti S, Giusto G, Gandini M, Re G, et al. (2021) Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis. PLoS ONE 16(7): e0253840. https://doi.org/10.1371/journal. pone.0253840 Editor: Kanhaiya Singh, Indiana University Purdue University at Indianapolis, UNITED STATES Received: October 8, 2020 Accepted: June 14, 2021 Published: July 27, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0253840 Copyright: © 2021 Vercelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. ☯ These authors contributed equally to this work. * Abstract Laminitis is one of the most devastating diseases in equine medicine, and although several etiopathogenetic mechanisms have been proposed, few clear answers have been identified to date. Several lines of evidence point towards its underlying pathology as being metabolism-related. In the carbonyl stress pathway, sugars are converted to methylglyoxal (MG)— a highly reactive α-oxoaldehyde, mainly derived during glycolysis in eukaryotic cells from the triose phosphates: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. One common hypothesis is that MG could be synthesized during the digestive process in horses, and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to alterations in the hoof lamellar structure. In the present study, employing an ex vivo experimental design, different concentrations of MG were applied to hoof explants (HE), which were then incubated and maintained in a specific medium for 24 and 48 h. Macroscopic and histological analyses and a separation force test were performed at 24 and 48 h post-MG application. Gene expression levels of matrix metalloproteinase (MMP)-2 and -14 and tissue inhibitor of metalloproteinase (TIMP)-2 were also measured at each time point for all experimental conditions. High concentrations of MG induced macroscopic and histological changes mimicking laminitis. The separation force test revealed that hoof tissue samples incubated for 24 h in a high concentration of MG, or with lower doses but for a longer period (48 h), demonstrated significant weaknesses, and samples were easily separated. All results support that high levels of MG could induce irreversible damage in HEs, mimicking laminitis in an ex vivo model. Introduction Laminitis is one of the most painful and debilitating diseases in equine medicine, with significant implications in terms of horse welfare. It is characterized by deterioration of the lamellar tissue that connects the hoof wall to the underlying third phalanx in the equine hoof, with associated inflammation, heat, digital pulses, pain and lameness, and, in some cases, dorsopalmar PLOS ONE | https://doi.org/10.1371/journal.pone.0253840 July 27, 2021 1 / 15 PLOS ONE Funding: This study was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) under the programme "Dipartimento di Eccellenza ex L.232/2016" to the Department of Veterinary Science, University of Turin (to authors EV, MG, and GG); and by the ex 60% fund of the University of Turin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Effect of methylglyoxal in an ex vivo model of laminitis rotation of the third phalanx [1]. It can be classified as either acute or chronic, and it is not usually readily reversible. Laminitis can lead to permanent disability or even death [1]. The most common etiology of laminitis cases involves gastrointestinal or metabolic disease [2]. For a number of reasons, laminitis is considered a metabolism-related pathology, resembling the human diabetic foot, since hormonal influences, weight and pressure are involved [3–5]. Evidence suggests that diabetic microvascular complications, including neuropathy, are caused by the activation of mechanisms related to the polyol pathway: advanced glycation end-product (AGE) formation with subsequent activation of the receptor for AGEs (RAGE), and lead to the activation of other factors such as nuclear factor kB, protein kinase C (PKC) isoforms, and the hexosamine pathway as a result of the overproduction of superoxide by mitochondria [6]. Gastrointestinal disturbances resulting from ingestion of excess carbohydrates play a pivotal role in sepsis-related laminitis [7, 8]. Feeds rich in starch are used as highly digestible energy sources in horses. During digestion in the small bowel, starch is primarily broken down by amylase enzymes, leading to glucose liberation. Extremely large quantities of starch in the diet can result in starch overload; which means that undigested starch passes from the small to the large intestine, which can lead to a decrease in colon and caecal pH, often followed by colic laminitis [9]. In this scenario, release of toxins from the hindgut is suspected to occur, which in theory induces degradation of the lamellar basement membrane and a loss in epithelial cell adhesion, with the subsequent activation of matrix metalloproteinases (MMPs) and leukocyte infiltration into the lamellae [4]. According to some authors, AGEs accumulate in significant amounts in the hoof lamellar tissue in the acute phase of experimentally induced laminitis using the hyperinsulinemic model [10]. Moreover, Valle et al. [11] revealed increased plasma levels of pentosidine, a glycoxidative marker, in ponies with clinical equine metabolic syndrome. Methylglyoxal (MG) causes the formations of AGEs, leading to carbonyl stress [12]. AGES derived from glucose and intermediates like MG maybe the major source of intracellular and plasma AGEs [13]. Since MG is a highly reactive intermediate, it is converted into D-lactate, to prevent the formation of AGEs. Specifically, starch overload in the gut can lead to rapid, devastating changes that result in the elaboration of toxins and other substanc (...truncated)