title: Leukocyte-derived and endogenous matrix metalloproteinases in the lamellae of horses with naturally acquired and experimentally induced laminitis.
abstract: BACKGROUND
  Inflammation and dysregulation of endogenous matrix metalloproteinase (MMP) production are implicated in the development of equine laminitis. In this study, we examine quantitative relationships among levels of leukocyte-derived proMMP-9 and MMP-9, lamellar proMMP-2 and MMP-2, and expression of proMMP-2 processing enzymes, MT1-MMP/PACE4, as steps towards determining whether inflammation and dysregulation of endogenous MMP production are independent or co-dependent processes.
  
  
  METHODS
  Archived samples of lamellae from horses with naturally acquired laminitis (n = 12), and from horses administered a pro-laminitic gastric bolus of starch gruel were used, the latter horses falling into two groups: (i) responders (CHO-R, n = 7), which developed Obel grade 3-lameness and (ii) non-responders (CHO-NR, n = 4), which did not become lame.
  
  
  METHODS
  Lamellar tissue extracts were analyzed by gelatin zymography to determine gelatinase content and by a myeloperoxidase ELISA to quantify relative monocyte/neutrophil content in the tissue. Real-time PCR was employed to measure gene expression of MT1-MMP and PACE4.
  
  
  CONCLUSIONS
  Extracts of lamellae from control horses, CHO-NR and horses with chronic (non-aggravated) laminitis had similarly low levels of pro and processed MMP-9 and MMP-2. In contrast, proMMP-9 was significantly elevated in extracts of lamellae from CHO-R and horses with naturally acquired acute and aggravated chronic laminitis. Lamellar MMP-2 was also increased significantly in the CHO-R and aggravated chronic laminitis groups, although not in the horses with naturally acquired acute laminitis. Concentrations of proMMP-9 correlated directly with myeloperoxidase content in lamellar extracts, suggesting production/induction by inflammatory leukocytes. In contrast, concentrations of proMMP-2 and MMP-2 were unrelated to concentrations of myeloperoxidase or proMMP-9 suggesting that leukocyte infiltration and dysregulation of endogenous MMP-2 are independent processes most likely with distinct inducers. Neither MT1-MMP nor PACE4 gene expression was elevated relative to controls in any group; this is discussed with respect to proMMP-2 processing in disease. In addition, variability in relative concentrations of lamellar MMPs observed among horses with Obel grade 3-lameness is discussed in the context of laminitis risk assessment and disease outcome.
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Loftus
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Johnson
authors.forename: James K
authors.initials: JK
authors.lastname: Belknap
authors.forename: Amanda
authors.initials: A
authors.lastname: Pettigrew
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Black
mesh_terms: D000818:Animals; D002241:Carbohydrates / Q000633:toxicity; D005260:Female; D005534:Foot Diseases / Q000139:chemically induced / Q000201:enzymology / Q000662:veterinary*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006724:Hoof and Claw / Q000201:enzymology* / Q000473:pathology; D006734:Horse Diseases / Q000139:chemically induced / Q000378:metabolism*; D006736:Horses; D007249:Inflammation / Q000139:chemically induced / Q000201:enzymology / Q000662:veterinary; D007962:Leukocytes / Q000201:enzymology*; D008297:Male; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D020782:Matrix Metalloproteinases