Progress 05/15/15 to 05/14/19
Outputs
Target Audience:Veterinary and general biomedical research community, equine veterinarians, veterinary pharmaceutical industry, horse-owning public Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has supported the training of 4 veterinary research fellows (undergraduate professional students who spend time in research laboratories to obtain research experience), one of which who went on to present her work at an international veterinary specialty meeting (ACVIM Forum 2019, Phoenix AZ). It has also provided training for Dr. Teresa Burns, a junior faculty member in the Department of Veterinary Clinical Sciences at The OSU CVM; she has authorship on two published manuscripts emanating from this work, and she has subsequently assumed leadership of the PD's research laboratory upon his retirement following completion of this work. How have the results been disseminated to communities of interest?The results have been presented at a Havemeyer Foundation research workshop on equine laminitis with major researchers in the field from around the world attending. The results have also been published within the first dedicated textbook to be written on the disease, Equine Laminitis (Belknap JK, ed., Wiley Blackwell Publishing, 2017) and have recently been published in journals widely read by equine veterinarians and researchers, Equine Veterinary Journal (Lane et al EVJ 2017) and the Journal of Veterinary Internal Medicine (Watts et al JVIM 2019). Further, the results have been presented at international veterinary specialty meetings that are widely attended (ACVIM Forum 2018 - Belknap and van Eps, research presentation; ACVIM Forum 2019 - Nijveldt et al, research abstract oral presentation). We plan to submit additional abstracts for presentation at the ACVIM Forum in 2020 describing additional facets of this project, and we anticipate 3-4 additional publications in peer-reviewed journals emanating from this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The anatomy of the horse's foot is unique in that the entire weight of the animal is suspended by the hoof wall through soft tissue attachments - termed digital laminae - that attach the last bone in the equine limb (which is analogous to the bone that lies beneath a human finger nail, also called the distal phalanx) to the hoof wall. Equine laminitis is a painful, debilitating disease of horses in which the digital laminae (the soft tissue attachment structures) become injured and fail, leading to a devastating downward displacement of the distal phalanx under the force associated with the weight of the horse. Laminitis has been reported to be one of the most important causes of lameness in horses by numerous sources, including the USDA (NAHMS 2000 report on lameness). Equine metabolic syndrome is a disease in many ways quite similar to the obesity-related metabolic syndrome seen in humans; however, importantly, instead of increasing risk of heart disease and stroke in horses (as it does in humans), it is the most common cause of injury to the digital laminae leading to laminitis in industrialized nations. In the past few years, it has become apparent that high insulin levels in the bloodstream play a central role in injury to the digital lamellar tissue in horses with metabolic syndrome. Recently, an experimental model has been established called the hyperinsulinemia model (also termed the euglycemic hyperinsulinemic clamp [EHC] model), in which laminitis can consistently be induced by administering insulin to ponies or horses for approximately 48 hours. All tissues of the body rely on the normal function of the living cells that make up that tissue; these cells normally perform multiple functions in response to different signaling pathways that can be activated or inactivated depending on what is needed by the cell and tissue. Diseases occur when these signaling pathways are disrupted by different disease processes leading to dysfunction and injury to the cells and the tissue composed of the cells (e.g., heart tissue, digital lamellar tissue, etc). We used the hyperinsulinemia model to establish the signaling events occurring in the cells present in the digital lamellar tissue in equine metabolic-syndrome associated laminitis (EMSAL). We used state-of-the-art scientific techniques on lamellar tissue samples obtained from horses subjected to the hyperinsulinemia model to establish the cell signaling pathways in the lamellar tissue which are adversely affected in the hyperinsulinemia model (with a focus on signaling pathways that are known to lead to cellular /tissue injury in different diseases in animals and humans). Finally, we performed experiments on both cells cultured from equine tissue and from samples of lamellarr tissue that were kept alive in a liquid medium containing sustaining nutrients (termed "growth media"). These two in vitro/ex vivo models were used to not only further establish the primary cell signaling pathways/ mechanisms of importance that are affected by high insulin concentrations (we exposed the cells and tissue to viable concentrations of insulin), but also to begin testing of different drugs to determine if they were able to block the signaling events determined to occur in affected/ diseased lamellar tissue in this model. Through these methods, we were able to further our understanding of the disease processes occurring in the lamellar tissue that result in equine metabolic syndrome-associated laminitis, but to also establish candidate therapeutic targets for future evaluation in order to establish an effective, clinically feasible treatment for this common cause of severe lameness in the horse. Goal 1 - We have used immunofluorescence to localize signaling in the lamellar dermis related to growth factor signaling, although the cells showing the greatest activation of this signaling were the lamellar epithelial cells (the cells responsible for laminar failure). Two manuscripts have been published containing these findings, with additional manuscripts in the pipeline. Goal 2 - We performed RT-qPCR, immunoblotting, kinomic arrays, and immunofluorescence techniques to establish distinct growth factor-related signaling events occurring in the laminar epithelial cells (the cells which undergo structural failure in EMSAL). Two manuscripts have been published containing these findings, with additional manuscripts in the pipeline. Goal 3 - We used an equine keratinocyte culture model to establish that the phosphorylation events leading to signaling are primarily involved with one signaling cascade triggered by growth factor initiation (Akt/mTORC1), further focusing our attention on this signaling pathway (and related pathways) as a therapeutic target for laminitis and suggesting the targeted inhibitors that were used for the work of Goal 4. One manuscript is in preparation related to this work. Goal 4 - We used inhibitors of major signaling pathways downstream of growth factor receptors to determine that the most efficacious inhibitor in blocking the growth factor-related signaling that was observed in the in vivo samples (Goals 1 & 2) was the inhibition of mTORC1 signaling using a pharmaceutical that is already in widespread use in human medicine (and now available at a cost making it a practical option in equine medicine), rapamycin. We have used these data to support follow up pilot studies and obtain preliminary data on rapamycin for future investigation of this drug as a candidate to be the first pharmaceutical product that may be efficacious for lamellar protection in equine laminitis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Watts MR, Hegedus OC, Eades SC, Belknap JK, Burns TA. Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light-breed horses. J Vet Intern Med 2019, 33(3): 1483-1492.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Belknap JK and van Eps AE. Lamellar signaling events and disturbances of energy metabolism leading to different forms of laminitis. Podium presentation, ACVIM Forum 2018
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Nijveldt E, Watts MR, Eades S, Belknap JK, Burns TA. Systemic and local regulation of leptin in a model of endocrinopathic laminitis. Poster presentation, ACVIM Forum 2019.
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Progress 05/15/16 to 05/14/17
Outputs
Target Audience:Equine veteriniarians, veterinary researchers, Lay public (equine lay journals) Changes/Problems:We have obtained an immense amount of data from this project, and do not anticipate any major changes to our approaches. What opportunities for training and professional development has the project provided?This work provided training of 2 veterinaryresearch fellows, professional veterinary students who spend time in research laboratories to obtain research experience. It also provided training for a young faculty member, Dr. Teresa Burns, in the Dept. of Clinical Sciences at the veterinary school at Ohio State University; she is 2nd author on the recently accepted publication emanating from this work. How have the results been disseminated to communities of interest?The results have been presented at a Havemeyer Foundation research workshop on equine laminitis with the major researchers from throughout the world attending; the results have also been discussed in the first textbook to be publlished on the disease, Equine Laminitis (Belknap JK, ed., Wiley Blackwell Publishing, 2017), and have been recently been published as an epub (will be in print) at one of the most read journals for equine veterinarians and researchers, Equine Veterinary Journal. What do you plan to do during the next reporting period to accomplish the goals?We will finish protein biochemistry and RT-qPCR techniques on laminar explants and in vivo laminar samples, and will try again to obtain data from laser capture-microdissection techniques for Goal 1 (we've recently had difficulty with this technique). We will work on manuscripts from data emanating from the kinomic array experiments, and from the RT-qPCR data from the in vivo portion of the study. Due to the results of this study, we have also been recently funded by a research foundationto determine if local hypothermia (the only effective therapy for another type of equine laminitis, sepsis-related laminitis), will also be effective in inhibitng the growth factor related signaling (established in this current project) and structural failure in the same EHC model of equine metabolic syndrome-associated laminitis as used in the present study.
Impacts
What was accomplished under these goals?
Goal 1.Using the euglycemic hyperinsulinemic clamp [EHC] model of endocrinopathic laminitis, determine thein vivoeffect of hyperinsulinemia on growth factor-related signaling in the cellular constituents laminar dermis which may play a role in dysregulation of the adjacent laminar basal epithelial cell (LBEC) layer leading to laminar failure. 60% accomplished. We have used innunofluorescence to localize signaling in the dermis related to growth factor signaling, although the cells most prominent for undergoing this signaling were the laminar epithelial cells (the cells responsible for laminar failure). Goal 2. Using the EHC model, determine thein vivoeffect of hyperinsulinemia on signaling related to growth factor receptor activation and cellular adhesion events in the LBEC layer 80% accomplished. We have performed RT-qPCR, immunoblotting, kinomic arrays, and immunofluosrescence techniques to establish distinct growth factor-related signaling events occurring in the laminar epithelial cells-the cells which undergo structural failure in in EMSAL. Goal 3 Using an equine skin keratinocyte cell culture model , determine the signaling induced in the equine epithelial cell by exposure to insulin and other growth factors documented to be upregulated in the laminar dermis and LBEC layer in the in vivo model described above. 90% complete. We have used equine keratinocyte culture model to establish that the phosphorylation events leading growth factor signaling are primarily due to one signaling cascade downstream of growth factor initiation (Akt/mTORC1), further focusing our attention to this signaling as a therapeutic target for laminitis therapy and giving us targets for inhibitors used in Goal 4. Goal 4:Using the same equine skin keratinocyte cell culture and ex vivo laminar explant models, determine the efficacy of different inhibitors of growth factor signaling pathways to block laminar signaling events documented to occur in thein vivo(EHC model) andin vitro/ex vivo(keratinocyte cell cuture/laminar expant) models. 90 % complete. We have used inhibitors of major signaling pathways downstream of growth factor receptors to determine that the most efficacious inhibitor in blocking the growth factor related signaling established to occur in the in vivo samples (Goals 1&2) is the inhibition of mTORC1 signaling using a pharmaceutical being used in human medicine and now at a cost making it a practical option in equine medicine, rapamycin. We are currently obtaining preliminary data on rapamycin for future investigation of this drug as possibly the first pharmaceutical product that may be efficacious in laminar protection in equine laminitis.
Publications
- Type:
Books
Status:
Published
Year Published:
2017
Citation:
Belknap JK, editor. Equine Laminitis. Wiley Blackwell, 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Lane HE, Burns TA, Hegedus OC, Watts MR, Weber PS, Woltman KA, Geor RJ, McCutcheon
LJ, Eades SC, Mathes LE, Belknap JK*. Lamellar events related to insulin-like growth factor-1 receptor signaling in two models relevant to endocrinopathic laminitis. Equine Vet J, 2017; doi:10.1111/evj.12663.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Talk given: Belknap, JK. Insulin and growth factor-related signaling within the equine epithelial cell and equine lamellae in models of endocrinopathic and sepsis-related laminitis
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Progress 05/15/15 to 05/14/16
Outputs
Target Audience:veterinary and general biomedical researchers, equine veterinarians, veterinary pharmaceutical industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training for another summer research fellow (a professional veterinary student), and continued to provide research experience to a young faculty member at OSU College of Veterinary Medicine, Teresa Burns. How have the results been disseminated to communities of interest?The results have primarily been distributed through scientific meetings and manuscripts. What do you plan to do during the next reporting period to accomplish the goals?Further work on equine epithelial cell culture will be performed in the next period, as will further examination of growth factor-related signaling using lamellar samples from the EHC animal protocol.
Impacts
What was accomplished under these goals?
Further immunoblotting and immunofluorescence was performed resulting in the submission of the included journal manuscript. Results were obtained for the kinomic array protocol for both lamellar explants and lamellar samples from the EHC animal protocol; a manuscript is being written currently on these data. Real time qPCR data was obtained demonstrating increased mRNA concentrations of cytokines in lamellae from animals maintained in a hyperinsulinemic state.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Lamellar events related to insulin-like growth factor-1 receptor signaling in two models relevant to equine metabolic syndrome, Equine Veterinary Journal, submitted.
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