Impact of laminitis on the canonical Wnt signaling pathway in basal epithelial cells of the equine digital laminae.
ABSTRACT: The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of ?-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of ?-catenin correlates positively with that of integrin ?4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin(+)/vimentin(-), excluding mesenchymal transition as contributing to loss of the adherens junction and hemidesmosome components. We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of ? catenin and integrin ?4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.
Project description:BACKGROUND:The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. OBJECTIVES:To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. STUDY DESIGN:In vitro experiments and clinical case series. METHODS:sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). RESULTS:sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. MAIN LIMITATIONS:Absence of control group and evaluator blinding in case series. CONCLUSIONS:sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses.
Project description:Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.
Project description:Background In a laminitic horse, the maximal loading of the toe region occurs during the breakover phase. To date, no kinetic data demonstrates the effect of supportive orthopaedic therapy in horses with laminitis on breakover phase. Thus, the purpose of this study was to examine the effect of heel elevation on the breakover phase. Eight horses with acute laminitis treated medically as well as with application of a hoof cast with heel wedge (HCHW) were included in this study. Immediately following cessation of clinical signs of acute laminitis, two measurements using the Hoof™ System were taken: the first with HCHW and the second immediately following removal of the HCHW, i.e. in barefoot condition (BFC). The hoof print was divided into three regions: toe, middle hoof, and heel. Kinetic parameters included vertical force (VF), stance duration, contact area (CA) for all hoof regions during stance phase, duration of breakover, VF in the toe region at onset of breakover and location of centre of force. Results The VF and CA were higher in the heel region (63 and 61%, respectively) and decreased significantly after removal of the HCHW (43 and 28% after removal, respectively). The breakover phase in horses with HCHW lasted 2% of stance phase and was significantly shorter than that in BFC, which lasted 6% of stance phase. The VF at onset of breakover for the toe region in horses with HCHW was significantly lower than that in BFC. The centre of the force was located at the heel region in all horses with the HCHW, and at the middle the hoof region in BFC. Conclusions Heel elevation in horses with laminitis as examined on a concrete surface significantly shortens breakover phase and decreases the vertical force in the toe region during breakover. HCHW provides adequate support to the palmar hoof structures by increasing the contact area in the heel region and incorporating the palmar part of frog and sole into weight bearing, thus decreasing the stress on the lamellae. Hoof cast with heel elevation could be a beneficial orthopaedic supportive therapy for horses suffering from acute laminitis.
Project description:BACKGROUND: Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed "founder". Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated this broad hypothesis by using DIGE to assess global differences in the plasma proteome between horses with chronic laminitis and controls. RESULTS: We identified 16 differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. Clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin (PT) clotting time, although most other indices were within normal ranges. Upregulation of the intestinal apolipoprotein APOA-IV in horses with chronic laminitis was confirmed by western blot. CONCLUSIONS: Our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. Gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis.
Project description:Matrix metalloproteinases (MMP) are hypothesized to degrade structurally important components of the laminar extracellular matrix (ECM) in horses with laminitis.To compare levels of expression of stromelysin-1 (MMP-3), collagenases (MMP-1, -13), and membrane type-MMPs (MMP-14, -15, -16), and the distribution of their ECM substrates, in laminae of healthy horses and horses with carbohydrate overload laminitis.Twenty-five adult horses.Gene and protein expression were determined in extracts of laminae using real-time quantitative polymerase chain reaction and Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis. Distribution of MMP-13 and ECM components was determined using indirect immunofluorescent microscopy of nonfixed frozen sections. ECM morphology was assessed by hematoxylin and eosin staining.Of the genes studied, only those encoding MMP-1 and -13 were upregulated in CHO-induced laminitis; MMP-1 at Obel grade (OG)1 lameness and MMP-13 at OG3 lameness. Laminar MMP-1 was present as 52 kDa proenzyme only. MMP-13 was present as pro- (61 kDa) and processed (48 kDa) enzyme. MMP-13 localized to the basal epithelium of the secondary epidermal laminae and its increased expression were accompanied by the appearance in secondary dermal laminae (SDL) of multiple foci that were devoid of collagen I, fibronectin, chondroitin and keratan sulfate glycosaminoglycans, and eosin-staining material.MMP-13 is upregulated in laminae of horses with CHO-induced OG3 lameness and, by degrading components of the ECM, may contribute to the formation of ECM-free lesions (gaps or tears) that appear in the SDL with OG3 lameness.
Project description:BACKGROUND:Laminitis is often associated with endocrinopathies that cause hyperinsulinemia and is also induced experimentally by hyperinsulinemia, suggesting that insulin initiates laminitis pathogenesis. Hyperinsulinemia is expected to activate pro-growth and anabolic signaling pathways. We hypothesize that chronic over-stimulation of these pathways in lamellar tissue results in endoplasmic reticulum stress, contributing to tissue pathology, as it does in human metabolic diseases. We tested this hypothesis by asking whether lamellar tissue from horses with naturally-occurring endocrinopathic laminitis showed expression of protein markers of endoplasmic reticulum stress. RESULTS:Three markers of endoplasmic reticulum stress, spliced XBP1, Grp78/BiP and Grp94, were upregulated 2.5-9.5 fold in lamellar tissues of moderately to severely laminitic front limbs (n = 12) compared to levels in controls (n = 6-7) measured by immunoblotting and densitometry. Comparing expression levels between laminitic front limbs and less affected hind limbs from the same horses (paired samples from 7 to 8 individual horses) demonstrated significantly higher expression for both spliced XBP1 and Grp78/BiP in the laminitic front limbs, and a similar trend for Grp94. Expression levels of the 3 markers were minimal in all samples of the control (n = 6-7) or hind limb groups (n = 7-8). Immunofluorescent localizations were used to identify cell types expressing high levels of Grp78/BiP, as an indicator of endoplasmic reticulum stress. Grp78/BiP expression was highly elevated in suprabasal epidermal keratinocytes and only observed in laminitic front limbs (10/12 laminitic samples, compared to 0/7 in sections from the hind limbs and 0/5 of controls). CONCLUSIONS:These data demonstrate that the endoplasmic reticulum stress pathway is active in naturally occurring cases of laminitis and is most active within a subset of epidermal keratinocytes. These data provide the rationale for further study of endoplasmic reticulum stress in experimental models of laminitis and the links between laminitis and human diseases sharing activation of this stress pathway. Pharmacological options to manipulate the endoplasmic reticulum stress pathway under investigation for human disease could be applicable to laminitis treatment and prevention should this pathway prove to be a driver of disease progression.
Project description:The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues.To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule.Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea).Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined.To the best of the author's knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses.
Project description:For gait classification, hoof-on and hoof-off events are fundamental locomotion characteristics of interest. These events can be measured with inertial measurement units (IMUs) which measure the acceleration and angular velocity in three directions. The aim of this study was to present two algorithms for automatic detection of hoof-events from the acceleration and angular velocity signals measured by hoof-mounted IMUs in walk and trot on a hard surface. Seven Warmblood horses were equipped with two wireless IMUs, which were attached to the lateral wall of the right front (RF) and hind (RH) hooves. Horses were walked and trotted on a lead over a force plate for internal validation. The agreement between the algorithms for the acceleration and angular velocity signals with the force plate was evaluated by Bland Altman analysis and linear mixed model analysis. These analyses were performed for both hoof-on and hoof-off detection and for both algorithms separately. For the hoof-on detection, the angular velocity algorithm was the most accurate with an accuracy between 2.39 and 12.22 ms and a precision of around 13.80 ms, depending on gait and hoof. For hoof-off detection, the acceleration algorithm was the most accurate with an accuracy of 3.20 ms and precision of 6.39 ms, independent of gait and hoof. These algorithms look highly promising for gait classification purposes although the applicability of these algorithms should be investigated under different circumstances, such as different surfaces and different hoof trimming conditions.
Project description:Horses belong to the order Perissodactyla and bear the majority of their weight on their third toe; therefore, tremendous force is applied to each hoof. An inherited disease characterized by a phenotype restricted to the dorsal hoof wall was identified in the Connemara pony. Hoof wall separation disease (HWSD) manifests clinically as separation of the dorsal hoof wall along the weight-bearing surface of the hoof during the first year of life. Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A case-control allelic genome wide association analysis was performed (ncases = 15, ncontrols = 24). Population stratification (? = 1.48) was successfully improved by removing outliers (ncontrols = 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (praw = 1.37x10-10, pgenome = 1.92x10-5). A homozygous region identified in affected ponies spanned from 79,936,024-81,676,900 bp and contained a family of 13 annotated SERPINB genes. Whole genome next-generation sequencing at 6x coverage of two cases and two controls revealed 9,758 SNVs and 1,230 indels within the ~1.7-Mb haplotype, of which 17 and 5, respectively, segregated with the disease and were located within or adjacent to genes. Additional genotyping of these 22 putative functional variants in 369 Connemara ponies (ncases = 23, ncontrols = 346) and 169 horses of other breeds revealed segregation of three putative variants adjacent or within four SERPIN genes. Two of the variants were non-coding and one was an insertion within SERPINB11 that introduced a frameshift resulting in a premature stop codon. Evaluation of mRNA levels at the proximal hoof capsule (ncases = 4, ncontrols = 4) revealed that SERPINB11 expression was significantly reduced in affected ponies (p<0.001). Carrier frequency was estimated at 14.8%. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure.
Project description:The cerebral cortex is a densely interconnected structure with neural circuits that form between cortical laminae and also between distinct cortical areas. However, the precise cell biological and developmental mechanisms that underlie the formation of these neural circuits remain unknown. Here, we visualize laminar innervation of the developing mouse cerebral cortex by layer II/III pyramidal neurons in real time, describing cytoskeletal dynamics during this process. We find that layer II/III pyramidal neurons achieve local laminar-specific innervation through the stabilization of collateral axon branches in target laminae. We also find that loss of neural activity does not abolish local laminar-specific innervation and that cells within the local environment are the likely source of cues that direct layer-specific cortical innervation.