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• W40735108 abstract "Injury to the superficial digital flexor tendon (SDFT) is common in horses and frequently associated with incomplete repair and re-injury when exercise is resumed. In normal SDFT, collagen type I predominates with collagen type III being a minor constituent. In injured SDFT, the ratio of collagen type I to III alters; both show increased synthesis but the relative increase in type III is greater. Collagen type III has smaller diameter fibrils than type I and therefore injured tendon has lower tensile strength, which increases the propensity for re-injury.Little is known about tendon metabolism and the role of tenocytes in repair. Tenocyte gap junction intercellular communication (GJIC) plays a significant role in extracellular matrix maintenance but the status of GJIC in injured SDFT is unknown. Loaded avian tendons showed upregulation of connexin43 (Cx43) gap junction (GJ) activity and increased collagen type III synthesis whilst knock-down of Cx43 GJs decreased type III and increased type I synthesis. Connexin43 antisense oligodeoxynucleotide (Cx43 asODN) was used to downregulate Cx43 in skin burns and wounds resulting in accelerated repair with less scarring.The project aims were to determine connexin (Cx) and collagen gene and protein expression in injured equine SDFT; establish an in vitro explant model of SDFT injury, determine the effects of downregulation of specific Cxs in this model oncollagen synthesis using an asODN, and evaluate uniaxial cyclic loading on Cx and collagen gene and protein expression in this model.Expression of 11 Cx isoforms in injured, contralateral and normal SDFT was investigated using reverse transcription PCR (RT-PCR); products were sequenced to confirm identity. Gene expression of identified Cx isoforms and collagen type I (Col1A1) and III (Col3A1) were quantified using real time RT-PCR (RT-qPCR). SDFT cryosections were labeled for Cx43 and Cx32 proteins and procollagen amino--propeptides I (PINP) and III (PIIINP). Confocal laser scanning microscopy (CLSM) was used to obtain z-stacks of labelled SDFT and immunofluorescence was quantified using ImageJ. Collagen content and solubility were determined by dye-binding assay. In vitro SDFT explants from uninjured horses were treated with Cx43 asODN in pluronic gel and others subjected to uniaxial cyclic strain; Cx and collagen gene and protein expression, and collagen content and solubility, were measured. Linear regression analyses were used to determine significance (P<0.05).PCR identified Cxs 26, 30, 31, 31.1, 32, 40, 43 and 50 in SDFT; none were specific to injured SDFT. Cx43 gene expression was significantly upregulated in central (P=0.006), peripheral (P=0.005) and distant (P<0.001) zones of injured SDFT. Cx32 gene expression was significantly upregulated in central (P=0.005) and peripheral (P=0.015) zones of injured SDFT. Col1A1 gene expression was significantly increased in central (P=0.001) and distant (P=0.019) zones of injured SDFT. Col3A1 gene expression was significantly increased in central (P<0.001), peripheral (P=<0.001) and distant (P=0.001) zones of injured SDFT. Cx43 protein expression, quantified as number ratio (GJ number/tenocyte) and average dot volume per nucleus (GJ volume in pixels/tenocyte) was significantly (P=0.009; 0.002) upregulated in centrally injured SDFT. Areas peripheral to SDFT injury showed significant (P=0.032; 0.011) upregulation. Central zones of contralateral SDFT trended (P=0.099; 0.096) towards significantly increased Cx43 protein expression.Cx32 protein expression was significantly (P=0.007; <0.001)  increased in injured SDFT and significantly (P=0.001; 0.026) increased in central zones of contralateral SDFT. PINP expression was significantly (P=0.012; 0.001) increased in central zones of injured SDFT. PIIINP showed markedly (P=0.002) increased volume in injured central SDFT zones.Upregulation of Cx43 and collagen gene and protein expression not restricted to ultrasonographically-detected SDFT pathology indicated diffuse metabolic alterations in injured SDFT. Trends towards significance and significant changes in Cx and collagen gene and protein expression were measured in contralateral SDFTs and probably indicative of increased loading; these metabolic alterations support clinical observation of increased incidence of injury in the contralateral SDFT following initial SDFT injury.The application of Cx43 asODN to in vitro SDFT explants variably influenced Cx43 gene and protein expression. Cx43 gene expression was significantly reduced (P=0.001) but neither number nor volume of Cx43 GJs was altered. Cx32 gene expression was not measured but Cx32 protein expression was significantly influenced by Cx43 asODN suggestive of compensatory responses or secondary to antiiv inflammatory effects of Cx43 asODN. Col1A1 expression was not significantly changed in treated explants but there was marked reduction in the number of sites synthesising PINP (P<0.001). Expression of Col3A1 and PIIINP were markedly downregulated in treated explants (P=0.001; <0.001). Loaded explants showed viscoelastic properties similar to the SDFT in vivo. Cx43 and Col3A1 gene expression was upregulated in loaded explants (P=0.039; 0.019); loading did not significantly influence Col1A1 expression. Loading increased Cx43 protein expression for GJ number and volume (P=0.029; 0.003), and the volume of sites synthesising PINP (P=0.019) and PIIINP (P=0.002).In vitro explants provided useful information concerning tenocyte and ECM metabolism. The model permitted evaluation of the effects of Cx43 asODN and mechanotransduction on Cx and collagen gene and protein expression." @default.
• W40735108 created "2016-06-24" @default.
• W40735108 creator A5034762172 @default.
• W40735108 date "2013-01-01" @default.
• W40735108 modified "2023-09-23" @default.
• W40735108 title "Modulation of gap junction expression in injured equine superficial digital flexor tendon" @default.
• W40735108 hasPublicationYear "2013" @default.
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