Ex Vivo Quantification of Transforming Growth Factor‐Beta1 in Equine Laminitis
Authors: Gutzmann Lori D , Johnson Philip J , Mohan Rajiv R 
Published: November 2023

Background

Equine laminitis is a common, painful, crippling, and incurable disease of the foot affecting horses of all ages and breeds, and other hooved animals to a lesser extent. While laminitis can be initiated by mixed conditions (e.g. obesity, toxins, dietary carbohydrates, endocrinopathies, and overbearing injuries), the disease processes and underlying pathology are incompletely understood. Because of this, development of effective treatments has been limited or unsuccessful.

During acute injury, TGF‐β is released by platelets and macrophages, and causes migration of inflammatory cells into the wound, favoring the production of myofibroblasts. If TGF‐β becomes unregulated, overproduction of extracellular matrix occurs and can lead to fibrosis, resulting in abnormal wound repair. Other proteins that may change with shifts in inflammatory cells include α‐smooth muscle actin (α‐SMA) and desmin. While TGF‐β has been reported to be acutely increased during wound healing of the skin of the distal limb in horses, its’ role in the development of laminitis has not been reported. We hypothesized that TGF‐β and α‐SMA mRNA and protein expression would be increased in hoof tissue of acutely laminitic horses, while desmin would be unchanged or decreased.

Objective

To compare gene expression and protein levels of TGF‐β1, α‐SMA, and desmin, and map their distribution at the hoof‐lamellar interface of healthy horses and horses with carbohydrate overload‐induced (CHO) laminitis.

Animals

Archived samples from twenty‐four adult horses in three treatment groups of eight samples each. Samples from early stages of experimental laminitis (developmental (DEV) and Obel Grade 1 (OG1)) were compared with sham‐treated controls (CON).

Methods

Hoof lamellar extracts were used to determine gene expression using real‐time quantitative polymerase chain reaction (qPCR). Indirect quantitative immunofluorescent (QIF) microscopy of non‐fixed frozen sections was used to map distribution and perform semi‐quantitative analysis.

Results

Samples of cDNA do not differ between groups for TGF‐β1, α‐SMA, or desmin, when measured by qPCR. QIF shows significantly increased TGF‐β1 (p=0.025) in DEV tissues compared with CON, increased desmin in DEV and OG1 tissues compared with CON (p=0.003 and 0.0127, respectively), while α‐SMA is decreased in these same groups vs. CON (p=0.0088 and p=0.0086, respectively). TGF‐β1 protein is localized to primary and secondary epidermal lamellae (PEL, SEL), while α‐SMA and desmin are localized perivascularly in the primary and secondary dermal lamellae (PDL, SDL).

Conclusions and Clinical Relevance

Acute elevation of TGF‐β1 in equine laminitis development suggests involvement of this superfamily in disease progression. Desmin and α‐SMA are two proteins potentially affected by this cytokine through stimulation of myofibroblast production, which would then lead to fibrosis through production of proteins such as collagen and fibronectin. It appears from these data that myofibroblast production in not increased early in experimental carbohydrate‐induced laminitis, but does not rule out involvement of TGF‐β through another pathway. Since TGF‐β is involved early in the disease, in vitro manipulation of this pathway using drugs such as mitomycin‐C or pirfenidone could lead to potential therapies.

Support or Funding Information

Zoetis‐Morris Animal Foundation Fellowship (D12MS‐900) (LDG) University of Missouri, College of Veterinary Medine and Surgery (LDG, PJJ, RRM) Ruth M. Kraeuchi Missouri Endowed Chair Professor in Veterinary Ophthalmology (RRM)