Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: The outputs of this project include the development of new knowledge, its dissemination to the research community through peer reviewed publications and to practitioners, researchers, faculty and students through presentations which include i) a talk at the Havemeyer Foundation Equine Laminitis Workshop Litchfield Beach, S. Carolina 2007, The Pathophysiology of Laminitis, ii) three talks at the 5th International Equine Conference on Laminitis and Diseases of the Foot Nov 2009, What Do Molecular Gene Approaches Tell Us; Cartilage and Lamina versus the rest; Enzymes: What role do they play, iii) two talks at the AAEP Equine Laminitis Research Workshop 2009, Gene and protein expression of polysulfated proteoglycan ECM components in the equine digital laminae and Localization; and function of sulfated proteoglycans in the equine digital laminae, iv) two talks at the Conference for Research Workers on Animal Diseases, Chicago, December 2010, Distribution of polysulfated proteoglycans in the equine digital lamellae: implications for the pathogenesis of laminitis and Depression of gene expression; and elevated ADAMTS-4 degradation of versican in equine laminitis, v) two talks at the 6th International Equine Conference on Laminitis and Diseases of the Foot. Florida Oct 2011, Expression and activity of metalloproteinases in the digital laminae of horses with starch-gruel induced laminitis; and Structural changes in the dermal and epidermal laminae of horses with starch-gruel induced laminitis, vi) annual presentations on the pathophysiology of laminitis at the USDA/NIFA PIs workshop Chicago, Nov 2007-2010, vii) annual presentations on the pathophysiology of laminitis at the Fancy Gap, immunoparasitology/Immunopathogenesis Workshop, Fancy Gap W. Virginia, Oct 2007-2011, viii) seminars presented to faculty and graduate students at the University of Pennsylvania School of Veterinary Medicine, March 2010 and 2011; Stanford University May 2010; University of Massachusetts, Nov 2011, ix) annual laminitis lectures presented to undergraduate veterinary and Animal Sciences students at the University of Massachusetts, Nov 2007-2011, and x) a lecture on the Pathophysiology of Laminitis presented to UMass alumni and veterinary practitioners at the University of Massachusetts April 2010. PARTICIPANTS: Three PhD students, John Loftus, Le wang and Erica Pawlak and two faculty members, Samuel Black and Dominique Alfandari contributed to the data reported above. A forth grad student, Almaz Taye, joined the project for 1 year to make antibodies and complementary peptides specific for exposed loops on the catalytic site and cysteine rich domains of ADAMTS-4 to support further immunohistological analyses. The project is supported by collaborations with Jim Belknap (The Ohio State University School of Veterinary Medicine), Philip Johnson (The University of Missouri School of Veterinary Medicine) and Susan Eades (The University of Georgia, School of Veterinary Medicine). Linked studies are funded by a grant from the Morris Animal Foundation to Samuel Black. In addition to providing PhD training opportunities the project provided undergraduate independent study opportunities for Jenette Lane and Danielle Alleman. TARGET AUDIENCES: Target audiences: Producers, Practitioners, Researchers, faculty and students who work on laminitis or other extracellular matrix diseases in the horse, or have an interest in mechanisms of tissue pathology. Efforts - Seminars reporting these studies were presented at the 2007 Havemeyer Equine Lameness Workshop, The 2009 and 2011 International Equine Conferences on laminitis and diseases of the foot, the 2009 and 2010 Conferences for research workers on animal diseases, the 2007, 2008, 2009 and 2010 USDA/NIFA PIs workshop, the 2007, 2008, 2009, 2010, and 2011 Fancy Gap Immunoparasitology/Immunopathogenesis Workshops, the University of Massachusetts (2011) and University of Pennsylvania School of Veterinary Medicine (2010, 2011). Lectures were also presented to undergradaute pre-vet students at the University of Massachusetts annually from 2007 - 2011. PROJECT MODIFICATIONS: The project was designed to test the hypothesis that a combination of neutrophil recruitment and MMP-9 production, and elevated endogenous matrix metalloproteinase 2 (MMP2) production are required for the development of irreversible equine laminitis and was based on earlier studies in the BWE-model of laminitis. However, during its implementation, the project evolved from primarily focusing on the relationship between inflammation and gelatinases MMP-2 and MMP-9 in the development of laminitis to encompass an extensive analysis of expression of other metalloproteinases. This resulted from the finding that neutrophils are recruited into the digital laminae of horses with CHO-induced laminitis between OG1 and OG3 lameness and hence the early developmental stages of CHO-induced laminitis is not associated with a classical inflammatory response. Furthermore, MMP-2 and MMP-9 expression in the laminae also occurred between OG1 and OG3 lameness excluding these MMPs from a role in the early developmemtal stages of the condition. In addition, MMP-9 when detected is solely present in laminitic laminae as inactive zymogen, while MMP-2 expression is elevated in late disease only, is detected in only a portion of laminitic animals and, as a gelatinase, is more likely to be involved in tissue repair rather than damage. These findings therefore disproved the hypothesis on which the proposal was based, and caused us to perform a broader analysis of metalloproteinases in laminitis. The study showed that MMP-1, MMP-13 and ADAMTS-4 gene and protein expression is elevated in laminitic laminae, while a wide range of other metalloproteinases are not affected. Subsequent analysis of substrate processing by MMP-1 and MMP-13 showed that the enzymes cause multifocal depletion of collagens I and III and fibronectin from secondary dermal laminae and are thus responsible for gross structural damage to the laminae. Furthermore, the ADAMTS-4 substrate versican is depleted from lamellar basal epithelial cells in laminitic laminae as a result both of elevated ADAMTS-4 cleavage and versican gene suppression. Literature analysis turned up 2 papers showing that versican may regulate the mesenchymal to epithelial cell transition leading us to investigate whether depletion of versican from laminitic laminae causes dedifferentiation of laminar basal epithelial cells, which turned out to be the case. We discovered that beta-catenin and integrin-beta-4 gene and protein expression are turned off in laminar basal epithelial cells of laminitic laminae, perhaps as a result of suppressed canonical Wnt signaling. Thus, our initial objective to determine the interaction between inflammation, MMP-2 and MMP-9 in the developmental stages of CHO-induced laminitis blossomed into a broader analysis of metalloproteinase-dependent pathophysiological processes in laminitis. Much of the data developed during this research project have been published or are in press. Two final papers documenting laminitis associated changes in MMP-1 and MMP-13 enzyme and substrate, and impact of laminitis on laminar canonical Wnt signaling are in preparation.
Impacts
Funding provided by the grant allowed us to develop a tissue archive that includes laminae from healthy horses and horses with CHO-induced laminitis at developmental (DEV), Obel grade 1 (OG1), and OG3 stages of lameness and thus covers mild to severe disease. Together with additional funding provided by the Morris Animal Foundation, it supported research on changes in the expression of metalloproteinases and indicators of laminar basal epithelial cell health during laminitis. Specifically, using a combination of expressed gene analysis by RT-qPCR, protein analysis by SDS-PAGE and Western blotting, and tissue analysis by quantitative imaging of antibody-stained thin cryosections of OCT embedded frozen laminae we have discovered that failure of the equine digital laminae in CHO-induced laminitis is associated with elevated expression (gene and protein) of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-13 and A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), elevated cleavage of their substrates and a profound change in laminar basal epithelial cells that most likely results from suppressed canonical Wnt signaling and results in greatly reduced expression of attachment proteins that mediate laminar basal epithelial cell:cell and cell:basement membrane attachment thus, destabilizing the epidermal:dermal junction. Thus by the DEV stage of laminitis, expression of the gene encoding ADAMTS-4 is significantly elevated relative to that in healthy laminae; by OG1 lameness, expression of ADAMTS-4 and MMP-1 genes and proteins are substantially and significantly elevated relative to that in healthy laminae; by OG3 lameness, expression of ADAMTS-4, MMP-1, MMP-2, pro-MMP-9, and MMP-13 gene and protein expression are highly and significantly elevated relative to that in healthy laminae and this is accompanied by elevated cleavage of the proteoglycan versican, collagens I and III and fibronectin. Furthermore, versican localizes to laminar basal epithelial cells as does ADAMTS-4, and its elevated cleavage in OG3 laminae is accompanied by suppressed versican gene expression and almost complete depletion of versican from the tissue. In addition, suppressed expression of the gene encoding versican at OG3 lameness is accompanied by and significantly correlates with suppressed gene and protein expression of beta-catenin and integrin-beta-4, which respectively are required for assembly of adherens junctions which mediate epithelial cell:cell attachment, and hemidesmosomes which mediate epithelial cell:basement membrane attachment. Because beta-catenin is a key component of the canonical Wnt signaling pathway, and versican expression is regulated by this pathway, it is likely that canonical Wnt signaling is critical to laminar basal epithelial cell function and is suppressed in laminitic laminae. These findings are highly relevant to the design of therapeutics for grain overload laminitis. It remains necessary to determine whether the same pathophysiological processes operate in pasture-induced laminitis, endocrinopathic laminitis and weight bearing limb laminitis.
Publications
- Pawlak E, Wang L, Johnson PJ, Belknap, J.k., Alfandari, D., Black, S.J. 2011. Distribution and processing of ADAMTS-4, aggrecan, versican and hyaluronan in the equine digital laminae. Am. J. Vet. Res;in press.
- Wang L, Pawlak E, Johnson PJ, Nuova, G., Taye, A., Belknap, J.K., Alfandari, D., Black S.J. 2011. Cleavage by ADAMTS-4 and gene repression deplete versican from the digital laminae of horses with starch gruel-induced laminitis. Am J Vet Res;in press.
- Wang L, Pawlak E, Johnson PJ, Belknap, J.K., Alfandari, D., Black S.J. 2011. Expression and activity of metalloproteinases in the digital laminae of horses with starch gruel-induced laminitis. J Equine Vet Sci. 31(10):566.
- Pawlak E, Wang L, Johnson PJ, Belknap, J.K., Alfandari, D., Black, S.J. 2011. Structural changes in the dermal and epidermal laminae of horses with starch gruel-induced laminitis. J Equine Vet Sci 31(10):567.
- Leise BS, Faleiros RR, Watts M, Johnson PJ, Black SJ, Belknap JK. 2011. Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis. Equine Vet J. 2011 Jan;43(1):54-61
- Faleiros RR, Johnson PJ, Nuovo GJ, Messer NT, Black SJ, Belknap JK. 2011. Laminar leukocyte accumulation in horses with carbohydrate overload-induced laminitis. J Vet Intern Med. 25(1):107-15
- Loftus, J.P., Williams, J.M., Belknap, T.K., Black, S.J. 2010. In vivo priming and ex vivo activation of equine neutrophils in Black Walnut Extract-induced equine laminitis is not attenuated by systemic lidocaine administration. Vet. Immunol. Immunopathol. 138:60-9
- Pawlak E, Wang L, Alfandari D, Johnson P.J., Belknap J.K., Black S.J. 2010. Localization and function of sulfated proteoglycans in the equine digital laminae. J Equine Vet Sci 30(2):99.
- Coyne, M.J., Cousin, H., Loftus, J.P., Johnson, P.J., Belknap, J.K., Gradil, C.M., Black, S.J., Alfandari, D. 2009. Cloning and expression of ADAM related metalloproteinases in equine laminitis. Vet Immunol Immunopathol 129(3-4):231-41.
- Loftus J.P., Johnson, P., Belknap, J.K., Pettigrew, A., Black, S.J. 2009. Leukocyte derived and endogenous matrix metalloproteinases in the laminae of horses with naturally-acquired and experimentally-induced laminitis. Vet Immunol Immunopathol. Vet Immunol Immunopathol 129 (3-4):221-30.
- Black, S.J. 2009. Extracellular matrix, leukocyte migration and laminitis. Vet Immunol Immunopathol Jun 15;129(3-4):161-3. Epub 2008 Nov 7.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Our previous analyses using samples of equine digital laminae collected from animals with starch-gruel induced laminitis demonstrated elevated expression of the gene encoding ADAMTS-4 during the developmental stages of laminitis and before increased expression of MMP9 or MMP2, or recruitment of inflammatory leukocytes into the laminae. During this funding period, we focused on the role of ADAMTS-4 in laminitis. ADAMTS-4 cleaves large polysulfated proteoglycans, namely aggrecan and versican. These have chondroitin sulfate and keratan sulfate side chains, form macromolecular complexes with hyaluronan, become hydrated gels and protect against compression. Using RT-qPCR and primers that span introns, genes encoding aggrecan, versican, chondroitin synthase and hyaluronan synthase were found to be constitutively expressed in normal equine digital lamellae but not, or to a much lesser extent in lung and liver tissue. ADAMTS-4 was detected by Western blotting of 0.5% NP40 protein extracts of lamellae subjected to SDS-PAGE, whereas the large sulfated ptoteoglycans were detected by Western blotting of guanidine hydrochloride extracts of lamellae after digestion with chondroitinase ABC and keratanase followed by SDS-PAGE. Aggrecan, versican, chondroitin-4 and -6 sulfate, keratan sulfate and hyaluronan were also detected by immunofluorescent staining of thin sections of lamellae frozen in OCT; the specificity of stains was confirmed by peptide blocking and glycosaminoglycan degradation by specific enzymes. Aggrecan and hyaluronan were largely concentrated in the secondary epidermal lamellae, while chondroitin and keratan sulfates displayed varying and unique distributions throughout the tissue. Versican was distributed throughout the primary and secondary dermal lamellae in association with fibrous collagen but not hyaluronan. Few or no changes were remarked in the distribution of collagen or the large polysulfated proteoglycans, laminin and versican in the front hoof lamellae up to Obel 1 lameness. However, by Obel 3, focal changes were noted in the distribution of collagen, versican, chondroitin sulfate and keratan sulfate proteoglycans within the secondary dermal lamellae. PARTICIPANTS: Two PhD students, Erica Pawlak and Le Wang, and two faculty members, Samuel Black and Dominique Alfandari contributed to the data reported above. A third grad student who worked on the project, John Loftus, has completed his PhD studies on laminitis in my lab and is now studying for a DVM at Cornell University School of Veterinary Medicine. In addition the project is supported by collaborations with Jim Belknap (The Ohio State University School of Veterinary Medicine), Philip Johnson (The University of Missouri School of Veterinary Medicine) and Susan Eades (The University of Georgia, School of Veterinary Medicine). Linked studies are funded by a grant from the Morris Animal Foundation to Samuel Black. TARGET AUDIENCES: Target Audience - Researchers who work on laminitis or other extracellular matrix diseases in the horse, Veterinarians and Producers. Efforts - Seminars reporting these studies were presented at the 5th International Equine Conference on Laminitis and Diseases of the foot, at AAEP Equine Laminitis Research Workshop and at CRWAD. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Gene expression and product analyses indicate that the equine digital lamellae contain large polysulfated proteoglycans and hence are functionally specialized to resist compressive forces, e.g., arising as a result of positional changes in the coffin bone during normal movement. In addition, elevated expression of the gene encoding ADAMTS-4 is a consistent feature of laminitis, occurring early in the disease process and sustained thereafter. Despite this, we did not find evidence of increased destruction of the lamellar ECM large polysulfated proteoglycans by ADAMTS-4 but rather found evidence of focal destruction of fibrous collagen consistent with migration and matrix metalloproteinase activities of inflammatory leukocytes and resulting in redistribution of proteoglycans in the secondary dermal laminae. We propose this to be the major process leading to loss of lamellar ECM structure in laminitis.
Publications
- Loftus J.P., Johnson, P., Belknap, J.K., Pettigrew, A., Black, S.J. 2009. Leukocyte derived and endogenous matrix metalloproteinases in the laminae of horses with naturally-acquired and experimentally-induced laminitis. Vet Immunol Immunopathol. Vet Immunol Immunopathol Jun 15;129(3-4):221-30. Epub 2008 Nov 7.
- Black, S.J. 2009. Extracellular matrix, leukocyte migration andlaminitis. Vet Immunol Immunopathol Jun 15;129(3-4):161-3. Epub 2008 Nov 7.
- Coyne, M.J., Cousin, H., Loftus, J.P., Johnson, P.J., Belknap, J.K., Gradil, C.M., Black, S.J., Alfandari, D. 2008. Cloning and expression of ADAM related metalloproteinases in equine laminitis. Vet Immunol Immunopathol 15;129(3-4):231-41. Epub 2008 Nov 25.
- Yin C, Pettigrew A, Loftus JP, Black SJ, Belknap JK. 2009 Tissue concentrations of 4-HNE in the black walnut extract model of laminitis: indication of oxidant stress in affected laminae. Vet Immunol Immunopathol. Jun 15;129(3-4):211-5. Epub 2008 Nov 11.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Our previous analyses of metalloproteinase activity in the laminae of horses with mild (black walnut extract-induced) and severe (starch-induced and naturally acquired) laminitis supported the hypothesis that a combination of inflammation and elevated endogenous matrix metalloproteinase 2 (MMP2) production are required for the development of irreversible laminitis. To test the hypothesis, lamellar pathology and metalloproteinase activity were analyzed at a developmental time point (DEV) and at Obel grade 1 (OG1)-lameness in horses following administration of a gastric bolus of starch gruel, a regime that leads to irreversible laminitis in 70% of cases. Control horses (CON) received water. ACTIVITIES - i) Horses (n = 16) were provided a bolus of starch gruel (85% cornstarch and 15% wood flour, 17.6 g/kg body wt.) by nasogastric tube. Control horses (n = 8) received 6 liters of deionized water. via a nasogastric tube. ii) Twenty four hours after administration of the starch gruel bolus (DEV point) or water (CON) 8 horses were anaesthetized and tissues were collected and flash frozen in liquid nitrogen for biochemical and molecular genetic analyses, or fixed in formalin for immunohistochemistry, or in OCT for immunofluorescent analyses. Fore and hind limb laminae, skin, gut, liver, lung, kidney, cartilage and blood were processed. iii) Eight starch gruel-treated horses that developed OG1-lameness between 36 and 48 hours after treatment were similarly processed as were the 8 control horses. Leukocyte derived matrix metalloproteinase 9 (MMP9) and endogenous MMP2 zymogen and processed forms in extracts of lamellae were measured by zymography after SDS-PAGE. In addition, expression of genes encoding membrane type I MMP (MT1MMP), ADAMTS4 (a secreted disintegrin and metalloproteinase [ADAM] with thrombospondin motifs; (gene family member 4), ADAMTS5, ADAM10, ADAM17 were measured in the tissues using qPCR. Six veterinary medicine students were trained to help with sample collection and processing, while 2 graduate students were trained to carry out zymographic analyses and qPCR. Results obtained to date were reported at the Fancy Gap Immunology Workshop, October 10th 2008. PARTICIPANTS: PIs - Samuel J. Black, Dominique Alfandari are faculty of the Department of Veterinary and Animal Sciences, University of Massachuestts. Collaborators - Jim Belknap, Ohio State University School of Veterinary medicine and Phillip Johnson, Missouri University School of Veterinary medicine provided materials for the study. Trainees - John Loftus competed a PhD working on this project and is now attending Cornell University School of Veterinary Medicine. Le Wang has enrolled in our PhD program and has worked on the project during the 2007 academic year. TARGET AUDIENCES: The target audience is researchers, veterinarians and owners who want to develop therapeutic agents the will prevent the onset of laminitis in high risk animals. Our effort has been directed at describing pathological processes that mediate damage to the digital laminae, i.e., the tisue that suspends the horse's axial skeleton within the hoof and which fail in laminitis. These efforts include the training of undergraduate and graduate students, veterinarians and producers in the structure and function of the extracellular matrix and inflammatory processes that affect this tissue component. Training involves laboratory skills acquisition, theoretic knowledge acquisition and instruction in data presentation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
RESULTS - Results obtained in the study so far are both straight forward and informative. Firstly, neither MMP2 gene nor protein expression are elevated at the developmental (DEV) or OG1 stages of starch gruel-induced laminitis. Indeed, little or no MMP-2 activity was detected. Consequently, MMP-2 does not contribute to the developmental stages of irreversible laminitis. Secondly, neither Pro-MMP9 nor MMP9 was detected in laminae at the DEV time point but pro-MMP9 was detected at OG1. Furthermore, immigrant neutrophils were also detected in the laminae at the OG1 but not the Dev time point, determined by a neutrophil-specific antibody that reacts with a 100kDa neutrophil secondary granule protein which remains to be identified. These data show that inflammation of the laminae co-incides with lameness but is not a prelude to lameness. Thirdly, a screen of genes encoding metalloproteinases showed that expression of ADAM-TS4 was elevated by 160 fold at the Dev time point and declined thereafter although expression remained elevated relative to that in control laminae. Expression of the gene encoding ADAM-TS5 was also elevated although only 2.5 fold relative to controls. Expression of genes encoding ADAM-10, ADAM-17, MMP-9, MMP-2 and MT1-MMP did not change after administration of the starch gruel. These data suggest that ADAM-TS4, also known as aggrecanase, may be a key enzyme in pathologic remodeling of extracellular matrix leading to laminitis and that MMP-9 and MMP-2 are unlikely to contribute to developmental stages of the disease in the starch-gruel model. IMPACTS: The data obtained so far strongly suggest against a role for MMP2 and MMP9 in the developmental stages of starch-induced irreversible laminitis, i.e., at a time when targeted inhibition of involved enzymes would be most likely to have an effect. Rather, they raise the possibility that ADAMTS4 may play a role in the early pathologic remodeling of the laminae and consequently that one or more substrates of ADAMTS4 may be required for integrity of the laminae. On-going studies aim to investigate this new hypothesis.
Publications
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