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- Author or Editor: Linda A. Dahlgren x
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Abstract
Objectives—To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons.
Animals—7 horses.
Procedure—Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB).
Results—Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected by WLB in normal tendon and markedly increased in damaged tendons.
Conclusions and Clinical Relevance—Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs. (Am J Vet Res 2005;66:300–306)
Abstract
Objective—To characterize the nucleotide sequence of equine platelet-derived growth factor (PDGF)-A and -B and analyze temporal expression of these genes in equine tendon after induced tendinitis injury.
Animals—18 mature horses.
Procedures—Genes for equine PDGF-A and -B were reverse transcribed and sequenced from synovial tissue mRNA obtained from a 3-year-old horse. Collagenase-induced lesions were created in the tensile region of the superficial digital flexor tendon in 14 horses; 3 horses served as uninjured control animals. Tendons were harvested and total RNA was isolated from experimental horses 1, 2, 4, 8, and 24 weeks after collagenase injection. Temporal gene expression for PDGF-A and -B was determined by use of quantitative PCR analysis.
Results—Equine PDGF-A shared 83.8% sequence and 87.5% peptide homology with human PDGF-A, with a discrepancy of 70 bp from the human sequence. Equine PDGF-B was similar in length to the human gene, sharing 90.3% and 91.7% nucleotide and peptide identity, respectively. Expression of PDGF-A mRNA in collagenase-induced tendinitis lesions was unchanged, compared with expression for normal control tendon, and remained steady throughout the 24-week study. Expression of PDGF-B mRNA decreased over time, and the expression at 24 weeks was significantly reduced, compared with expression in normal and acutely injured tendon.
Conclusions and Clinical Relevance—Injured tendon mounts a minimal constitutive PDGF-A or -B mRNA response. Serial exogenous treatment with either PDGF isoform within the first 2 to 4 weeks after tendon injury may bolster the meager PDGF paracrine-autocrine intrinsic response to injury.
Abstract
Objective—To investigate effects of β-aminopropionitrile and a combination of insulin-like growth factor (IGF)-I and β-aminopropionitrile on metabolism of equine tendon fibroblasts.
Sample Population—Flexor tendon explants from 3 horses.
Procedure—Explants received 1 of 4 treatments (control, IGF-I, β-aminopropionitrile, and IGF-I/β-aminopropionitrile) for 10 days, and message expression for collagen types I and III was assessed by use of in situ hybridization. Histologic findings, new protein production, and quantitative determinations of glycosaminoglycan, DNA, and de novo collagen synthesis were made.
Results—Insulin-like growth factor-I stimulated an anabolic response in tendon. Collagen synthesis and glycosaminoglycan and DNA content of explants were all increased. β-Aminopropionitrile significantly suppressed collagen synthesis, which was not ameliorated by concurrent IGF-I treatment. β-Aminopropionitrile caused alterations in cell morphology characterized by large round cells with eccentric nuclei and decreased density of collagen fibers. Protein production and collagen type-III mRNA expression were reduced in these cells.
Conclusion and Clinical Relevance—Treatment with β-aminopropionitrile resulted in decreased production of protein and collagen synthesis, which could be expected to suppress tendon healing. The negative effects of β-aminopropionitrile could not be abrogated by addition of IGF-I to the medium. Treatment resulted in alterations in cell morphology and matrix consistency, which could further delay tendon healing. β-Aminopropionitrile may impair tendon healing at a cellular level by decreasing collagen production or increasing rate of degradation of existing matrix. Because of reduced crosslinking during β- aminopropionitrile treatment, in combination with transiently decreased tensile strength, alterations in collagen content and structure may weaken the healing tendon. (Am J Vet Res 2001;62:1557–1562)
Abstract
Objective—To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses.
Animals—14 healthy adult horses.
Procedure—The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis.
Results—Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease.
Conclusions and Clinical Relevance—Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated. (Am J Vet Res 2005;66:266–270)
Abstract
OBJECTIVE To compare the effects of 3 equimolar concentrations of methylprednisolone acetate (MPA), triamcinolone acetonide (TA), and isoflupredone acetate (IPA) on equine articular tissue cocultures in an inflammatory environment.
SAMPLE Synovial and osteochondral explants from the femoropatellar joints of 6 equine cadavers (age, 2 to 11 years) without evidence of musculoskeletal disease.
PROCEDURES From each cadaver, synovial and osteochondral explants were harvested from 1 femoropatellar joint to create cocultures. Cocultures were incubated for 96 hours with (positive control) or without (negative control) interleukin (IL)-1β (10 ng/mL) or with IL-1β and MPA, TA, or IPA at a concentration of 10−4, 10−7, or 10−10M. Culture medium samples were collected from each coculture after 48 and 96 hours of incubation. Concentrations of prostaglandin E2, matrix metalloproteinase-13, lactate dehydrogenase, and glycosaminoglycan were determined and compared among treatments at each time.
RESULTS In general, low concentrations (10−7 and 10−10M) of MPA, TA, and IPA mitigated the inflammatory and catabolic (as determined by prostaglandin E2 and matrix metalloproteinase-13 quantification, respectively) effects of IL-1β in cocultures to a greater extent than the high (10−4M) concentration. Mean culture medium lactate dehydrogenase concentration for the 10−4M IPA treatment was significantly greater than that for the positive control at both times, which was suggestive of cytotoxicosis. Mean culture medium glycosaminoglycan concentration did not differ significantly.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the in vitro effects of IPA and MPA were similar to those of TA at clinically relevant concentrations (10−7 and 10−10M).
Abstract
Objective—To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis.
Animals—8 horses.
Procedures—Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed.
Results—Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons.
Conclusions and Clinical Relevance—ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.
