Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts
Abstract
enBackground
Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as “proud flesh”. Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals.
Hypothesis/Objectives
The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing.
Animals
Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses.
Methods
Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay.
Results
Cultured cells were half vimentin+ve fibroblasts and half alpha smooth muscle actin+ve and vimentin+ve myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly.
Conclusions and clinical importance
This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses.
Résumé
frContexte
Les plaies chez le cheval présentent souvent une inflammation prolongée et une vascularisation inefficace menant à une fibrose excessive et des complications appelées «proud flesh». Les protéines dérivées du virus Orf, le VEGF-E (vascular endothelial growth factor) et l'interkeukine (ovIL)-10, améliorent l'angiogenèse et contrôle l'inflammation et la fibrose des plaies cutanées des animaux de laboratoire.
Hypothèses/Objectifs
L’étude a pour but de déterminer si les cellules dermiques équines répondent à VEGF-E et ovIL-10. Il est attendu que les cellules dermiques équines expriment les récepteurs à VEGF et IL-10, le traitement aux protéines virales pourrait ainsi modifier l'expression génétique cellulaire et le comportement menant ainsi à la cicatrisation.
Sujets
Les échantillons cutanés étaient prélevés de la paroi thoracique latérale de deux chevaux sains de race pur-sang.
Méthodes
Les cellules dermiques équines ont été isolées à l'aide d'une méthode d'extraction cutanée et leur phénotype a été évalué par immunofluorescence. Les cellules ont été traitées avec des protéines recombinantes avec ou sans stimuli inflammatoires. L'expression génique a été examinée par PCR standard et quantitative par transcription inverse. Le comportement cellulaire a été évalué par test d'abrasion.
Résultats
Les cellules cultivées étaient à moitié des fibroblastes vimentin+ve et à moitié des cellules musculaires lisses alpha actin+ve et des myofibroblastes vimentin+ve. Le VEGF-E ont augmenté l'expression d’ARNm Il-10 tandis que l'expression d’ARNm de collagenase-1 et VGEF-1 était augmenté par ovIL-10. Pour les cellules exposées à un stimulus inflammatoire, les deux traitements ont ralenti l'expression d’ARNm de TNF et exacerbé l'expression de protéines chimio attractive de monocyte. Les protéines virales n'ont pas non plus influencées la migration cellulaire.
Conclusions et importance clinique
Cette étude montre que VEGF-E et ovIL-10 sont actifs sur les cellules dermiques équines et présentent des effets anti-inflammatoires et anti-fibrotiques qui pourraient améliorer la cicatrisation des plaies cutanées du cheval.
Resumen
esIntroducción
Las heridas en los caballos a menudo exhiben inflamación continuada y vascularización ineficaz, lo que lleva a una fibrosis excesiva y complicaciones clínicas tales como “carnosidad”. Proteínas derivados de virus Orf, factor de crecimiento endotelial vascular –E (VEGF) y la interleuquina (ovIL -10), mejoran la angiogénesis y el control de la inflamación y fibrosis en heridas de la piel de animales de laboratorio.
Hipótesis/Objetivos
El objetivo del estudio fue determinar si las células dérmicas equinas responden a VEGF-E y ovIL-10. Se espera que las células dérmicas equinas expresen receptores de VEGF e IL-10, así que el tratamiento con proteína viral podría alterar la expresión de genes celulares y su comportamiento de modo que favorecieran la cicatrización.
Animales
muestras de piel se recogieron de la pared torácica lateral de dos caballos de pura sangre sanos.
Métodos
células dérmicas equinas se aislaron utilizando un método de explante de piel y su fenotipo fue evaluado por inmunofluorescencia. Las células se trataron con proteínas recombinantes, con o sin estímulo inflamatorio. La expresión génica se examinó mediante PCR de transcriptasa inversa estándar y cuantitativa. El comportamiento de la célula se evaluó en pruebas de rasguño.
Resultados
Las células cultivadas fueron mitad fibroblastos vimentina + y la otra miofibroblastos actina alfa de musculo liso + y vimentina +. VEGF-E aumento de la expresión basal de IL-10 mRNA, mientras que ;a expresión de mRNA de VEGF-A y colagenasa-1 se incrementó al tratar con ovIL-10. En las células expuestas al estímulo inflamatorio, ambos tratamientos inhibieron la expresión de mRNA del factor de necrosis tumoral, y ovIL-10 incrementó la expresión de la proteína quimiotáctica de monocitos. Ninguna proteína viral afectó en gran medida la migración celular.
Conclusiones e importancia clínica
Este estudio demuestra que el VEGF-E y la ovIL-10 son activos en las células dérmicas equinas y ejercen efectos anti-inflamatorios y anti-fibróticos que pueden mejorar la cicatrización de heridas en la piel de caballos.
Zusammenfassung
deHintergrund
Wunden bei Pferden zeigen oft eine ausgedehnte Entzündung und ungenügende Durchblutung, was zu exzessiver Fibrose und klinischen Komplikationen wie „wildes Fleisch“ führt. Aus Orf Virus gewonnene Proteine, Vascular Endothelial Growth Faktor (VEGF)-E und Interleukin (ovIL)-10, steigern die Angiogenese und kontrollieren die Entzündung und Fibrose bei Hautwunden von Labortieren.
Hypothese/Ziele
Diese Studie zielte darauf ab zu bestimmen, ob Hautzellen von Pferden auf VEGF-E und ovIL-10 reagieren. Es wird erwartet, dass equine Dermalzellen VEGF und IL-10 Rezeptoren exprimieren, daher ist es wahrscheinlich, dass eine virale Proteinbehandlung die zelluläre Genexprimierung und das Genverhalten in einer Art und Weise ändert, die der Heilung zuträglich ist.
Tiere
Es wurden Hautproben von der lateralen Thoraxwand von zwei gesunden Vollblutpferden genommen.
Methoden
Es wurden Dermalzellen von Pferden mittels Hautexplantiermethode isoliert und ihr Phänotyp mittels Immunfluoreszenz ermittelt. Die Zellen wurden mit rekombinanten Proteinen behandelt, mit oder ohne Entzündungsstimuli. Die Genexprimierung wurde mittels Standard und quantitativer Reverser Transkriptase PCR untersucht. Das Zellverhalten wurde mittels Scratch Assay evaluiert.
Ergebnisse
Die kultivierten Zellen bestanden zur Hälfte aus Vimentin+ve Fibroblasten und zur Hälfte aus alpha glatter Muskulatur Actin+ve und Vimentin+ve Myofibroblasten. VEGF-E erhöhte die basale Exprimierung von IL-10 mRNA, während VEGF-A und Kollagenase-1 mRNA Exprimierung durch ovIL-10 zunahm. In jenen Zellen, die den Entzündungsstimulatoren ausgesetzt waren, dämpften beide Behandlungen die Exprimierung der mRNA des Tumor Nekrose Faktors, und ovIL-10 erhöhte die Exprimierung von Monozyten Chemoattractant Protein. Keines der Virenproteine beeinflusste die Zellmigration drastisch.
Schlussfolgerungen und klinische Bedeutung
Diese Studie zeigt, dass VEGF-E und ovIL-10 aktiv wirken auf Hautzellen der Pferde und eine entzündungshemmende sowie eine anti-fibrosierende Wirkung ausüben, die die Wundheilung bei Pferden verbessert.
要約
ja背景
馬の創傷は持続的な炎症と血管新生不全を起こし、過剰な線維化と臨床的に「過剰肉芽(pround flesh)」と呼ばれる合併症を引き起こす。オルフウィルス由来蛋白である内皮血管増殖因子E(VEGF-E)およびインターロイキン−10(ovIL-10)は、皮膚創傷の実験動物モデルにおいて、血管新生を促進し、炎症と線維化を抑制することが示されている。
仮説/目的
本研究の目的は、馬の真皮細胞のVEGF-EおよびovIL-10に対する反応性を調査することである。馬の真皮細胞はVEGFおよびIL-10の受容体を発現しているため、ウィルス蛋白治療が、創傷治癒を助長する細胞遺伝子発現変化および細胞動態変化を起こす可能性がある。
供与動物
2頭の健康なサラブレッド馬の胸部側面壁より得た皮膚サンプルを培養した。
方法
馬の真皮細胞は皮膚移植片培養法によって分離し、免疫蛍光抗体法によって表現型を調べた。細胞は組み換え蛋白の存在下かつ炎症刺激有りもしくは無しで処理した。遺伝子発現は標準的な定量逆転写PCRによって測定した。細胞動態の評価には、スクラッチアッセイを用いた。
結果
培養細胞は半数がビメンチン陽性線維芽細胞で、半数がα平滑筋アクチン陽性かつビメンチン陽性筋線維芽細胞であった。VEGF-EはIL−10のmRNAの基礎発現量を増加させ、一方で、VEGF-Aとコラゲナーゼ−1のmRNA発現量はovIL-10によって増加した。炎症刺激を受けた細胞では、いずれのウィルス蛋白処理も腫瘍壊死因子mRNAの発現を減少させ、ovIL-10は単球走化性因子の発現を増加させた。いずれのウィルス蛋白も細胞移動には大きく関与しなかった。
摘要
zh背景
马的伤口经常出现持久性炎症,以及无法血管化,造成过度的纤维化,出现“赘肉”这种临床并发症。内皮细胞血管化生长因子(VEGF)-E和白介素(ovIL)-10,是羊痘疮病毒衍生蛋白,可增强了血管再生,并且控制实验动物皮肤伤口的炎症和纤维化。
假设/目的
研究目的是确定是否VEGF-E和ovIL-10对马真皮细胞有效。预计马真皮细胞表达VEGF-E和ovIL-10受体,这样病毒蛋白治疗可能改变细胞基因表达和运转方式,使其有利于伤口愈合。
动物
样本收集自两匹健康纯血马的胸壁两侧皮肤。
方法
使用皮肤移植方法分离马真皮细胞,用免疫荧光评估其表型。细胞用重组蛋白处理,有或没有炎性刺激。使用标准和定量反转录PCR检查基因表达。划痕试验评估细胞转运。
结果
培养的细胞是半波形蛋白+ve成纤维细胞和半α平滑肌肌动蛋白+ve,以及波形蛋白肌成纤维细胞+ve。VEGF-E 提高了IL-10 mRNA的基础表达,而ovIL-10提高了VEGF-A和胶原蛋白酶-1mRNA的表达。对于暴露于炎性刺激的细胞,两种处理抑制肿瘤坏死因子mRNA表达,以及ovIL-10加剧单核细胞趋化蛋白表达。病毒蛋白都不会很大程度影响细胞迁移。
结论和临床意义
本研究发现VEGF-E和ovIL-10在马真皮细胞上具有活性,其施加的抗炎和抗纤维化作用可以增强马的伤口愈合。
Resumo
ptContexto
Feridas em equinos frequentemente apresentam inflamação crônica e vascularização ineficiente, ocasionado fibrose excessiva e tecido de granulação. Proteínas derivadas de Orf-vírus, fator de crescimento endotelial vascular (VEGF)-E e interleucina-10 (ovIL-10), aumentam a angiogenese, controlam a inflamação e a fibrose em feridas de animais de laboratório.
Hipótese/Objetivos
O estudo teve como objetivo determinar se as células dérmicas de equinos respondem a VEGF-3 e ovil-10. É esperado que células dérmicas de equinos expressem receptores para VEGF e IL-10, desta forma, é provável que o tratamento com as proteínas virais altere a expressão gênica e o funcionamento celular, tendendo à cicatrização.
Animais
As amostas de pele foram coletadas da lateral do tórax de dois equinos da raça Puro Sangue Inglês.
Resultados
As células cultivadas foram metade fibroblastos vimentina+ve e a outra metade músculo liso alpha actina+ve e miofibroblastos vimentinave+. VEGF-E aumentou a expressão basal de mRNA de IL-10, enquanto a expressão VEGF-A e de colagenase-1 foi aumentada por ovIL-10. Em células expostas a estímulo inflamatório, ambos os tratamentos reduziram a expressão de mRNA de fator de necrose tumoral, e ovIL-10 exacerbou a expressão de proteína quimiotática de monócitos. Tampouco a proteína viral influenciou a migração celular com grandeza.
Conclusões e importância clínica
Este estudo demonstrou que VEGF-E e ovIL-10 são ativas em células dérmicas de equinos e exerce efeitos antiinflamatórios e antifibróticos que provavelmente melhorem a cicatrização em equinos.
Introduction
Skin wounds in horses are common, exerting significant financial and welfare impacts on the equine industry.1 Many wounds in horses cannot be sutured and must heal by second intention, increasing the duration and cost of management. Moreover, limb wounds often develop complications such as an excessive accumulation of granulation tissue, referred to as “proud flesh”, which compromises scar appearance and strength. This appears to result from sustained inflammation2 and inefficient tissue vascularization.3, 4 Currently there is no method proven to accelerate healing or to successfully prevent or treat proud flesh in the horse.
Orf virus (OV) causes a contagious skin disease in sheep and humans with lesions reminiscent of equine proud flesh, with marked inflammatory infiltrate, vascular oedema, epidermal hyperplasia and fibrosis, but that resolve spontaneously without scarring.5, 6 The key to lesion resolution may lie in viral proteins that manipulate the wound healing response in cell culture and in laboratory animals.7, 8 Specifically, OV expresses an interleukin-10 (ovIL-10) that controls inflammation and fibrosis,8 and a vascular endothelial growth factor (VEGF)-E that promotes angiogenesis, thereby accelerating healing.7 The ovIL-10 shares 64–80% amino acid identity with its mammalian equivalent and is functionally similar to ovine IL-10.6 VEGF-E shares 25–43% identity with mammalian VEGFs and, unlike its counterparts, is selective for the reparative receptor, VEGFR2, and does not interact with the pro-inflammatory receptor, VEGFR1.6
The aim of this study was to determine if equine dermal cells respond to ovIL-10 and VEGF-E, prior to embarking on an in vivo trial of their effects on equine wound healing. It was hypothesized that equine dermal cells express receptors to IL-10 and VEGF, and that the viral proteins may regulate cellular gene expression and behaviour in a manner conducive to healing.
Materials and methods
Recombinant proteins
Recombinant FLAG-tagged VEGF-E and ovIL-10 were expressed in 293-EBNA cells and then purified by affinity chromatography as described previously.7, 8
Equine skin samples
Skin samples were obtained from two healthy thoroughbred horses involved in a parallel study (institutional approval MUAEC 14/84). Horses were sedated with detomidine hydrochloride (Dormosedan, Zoetis; Auckland, New Zealand) and butorphanol tartrate (Ilium Butorgesic, Troy Laboratories; Auckland, New Zealand) (0.01/0.04 mg/kg; IV) and local anaesthesia performed using 2% lidocaine hydrochloride (Lopaine, Ethical Agents Ltd.; Wiri, New Zealand). Two skin biopsy samples (4 cm2) per horse were placed in Dulbecco's modified Eagle's medium (DMEM, Life Technologies; Carlsbad, CA, USA) with 5% fetal bovine serum (FBS), 50 ng/mL gentamicin and 2.5 μg/mL amphotericin B, then stored for 24 h at 4°C.
Cell culture
Cells were isolated from skin samples using an explant method.9 Skin samples were cleaned with 10% povidone-iodine and 70% ethanol, then fat and blood vessels removed. Samples were cut into 1 mm2 fragments then placed upright 1 cm apart in culture wells coated previously with 0.1% gelatin for 30 min. For 7 days, the explants were partially submerged in DMEM with 20% FBS and antimicrobial agents and incubated at 37°C/5% CO2. Cell colonies were trypsinized, combined and then maintained at a density of 1 × 104–1 × 105 cells/mL by sub-culturing when 90% confluent. Cells used in assays were two to four passages old.
Immunofluorescent analysis
Cells were grown to confluence on coverslips and then fixed overnight in 0.5% Zinc-Salts fixative. Cells were incubated with antibodies against vimentin (D21H3, XP® AlexaFluor 488 conjugate, Cell Signaling Technology; Danvers, MA, USA; 1:50 dilution) and smooth muscle actin (αSMA, 1A4, Cy3 conjugate, Sigma-Aldrich; St Louis, MO, USA; 1:400 dilution) in tris-buffered saline containing 0.1% bovine serum albumin (BSA, Invitrogen; Carlsbad, CA, USA) for 2 h at room temperature. Isotype controls were used in parallel to show the specificity of the staining (rabbit IgG AlexaFluor 488 conjugate, Sigma-Aldrich). For the final 30 min, 4′,6-diamidino-2-phenylindole (DAPI, 75 nM, Invitrogen) was added. Coverslips were mounted onto slides using SlowFade Gold (Invitrogen) and stored at 4°C. Stained cells were photographed at 40× magnification on an upright fluorescence microscope (BX-51, Olympus, Tokyo, Japan) with cellSens software (Olympus). Stained cells were counted in 10 fields of view using ImageJ's particle analysis function. The average percentage of DAPI+ve cells stained for vimentin or αSMA was then calculated.
PCR analysis
Cells (3 × 106) were harvested using 0.25% trypsin, pelleted by centrifugation, then RNA isolated using the TriRNA Pure Kit (Geneaid; New Taipei City, Taiwan) with on-column DNase treatment (Quanta Bio; Beverly, MA, USA). Synthesis of cDNA was carried out with SuperScript III (Invitrogen) and standard PCR conducted using the Expand High Fidelity PCR System (Roche; Basel, Switzerland). Each reaction mix contained 5% of the cDNA sample and 100 mM of each primer (Supporting Information Table S1) and underwent the cycling protocol: denaturation, 94°C for 15 s; annealing, 60°C for 30 s; elongation, 72°C for 45 s. The PCR products were subjected to gel electrophoresis and bands visualized under UV light using Chemi-Doc XRS and Quantity One software (Bio-Rad; Hercules, CA, USA).
Quantitative RT-qPCR analysis
Confluent monolayers of cells were washed with phosphate-buffered saline (PBS) then incubated overnight in DMEM basal medium with 0.1% BSA and antibiotics. Cells were washed again then basal medium containing 200 ng/mL VEGF-E, 20 ng/mL ovIL-10 or media alone was added. Additional cells were treated as above and with 50 μg/mL polyinosinic:polycytidylic acid (PolyI:C, Sigma-Aldrich) to induce an inflammatory response. After 2 h cells were harvested using 0.25% trypsin, pelleted by centrifugation, before RNA was isolated and cDNA synthesized as described above. Quantitative PCR was conducted using cDNA equivalent of 5 ng RNA, 200 mM of each primer (Supporting Information Table S1), SYBR GreenER qPCR SuperMix (Invitrogen) on the 7500 Fast PCR machine (Applied Biosystems; Foster City, CA, USA). Primer efficiencies were calculated and gene expression levels determined relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and to the levels in cells treated with media only.
Scratch assay
Confluent monolayers of cells were scratched down the centre of the well with a 200 μL pipette tip. Media and damaged cells were removed with PBS washes. Cells were then incubated with basal medium containing 1% FBS, 200 ng/mL VEGF-E, 20 ng/mL ovIL-10 or with medium alone. FBS was used as a positive migration control as it contains growth factors and chemokines that stimulate fibroblast migration. Medium containing BSA lacks such stimuli and was used as a negative control. Replicate images (×3) were taken of each scratch at 0, 6, 12, 24 and 48 hours at 20× magnification using the Olympus TH4-200 inverted microscope and cellSens software. Scratch areas were measured using ImageJ and expressed as a ratio of the original scratch area.
Results
The cells stained 96±4% positive for vimentin, whereas 48±9% of all cells were also positive for αSMA (Figure 1), indicating that the cell line was a mix of fibroblasts and myofibroblasts. The cells expressed mRNA for VEGF and IL-10 receptors necessary for viral protein activity (Figure 2). Within 2 h, equine dermal fibroblasts showed altered gene expression in response to VEGF-E or ovIL-10 treatment. VEGF-E increased basal expression of equine IL-10 mRNA (Figure 3a, P = 0.0001, ANOVA with Sidak's means comparison test), whereas ovIL-10 increased the expression of equine VEGF-A (Figure 3b, P = 0.03) and collagenase-1/matrix metalloproteinase (MMP)-1 mRNA (Figure 3c, P = 0.002).
Neither viral protein influenced expression of key mediators of fibrosis, αSMA, collagen (Col)1α2 or transforming growth factor beta (TGFβ)1 (Figure 3d–f). In response to the inflammatory stimulus PolyI:C, mRNA of tumour necrosis factor (TNF) and monocyte chemoattractant protein (MCP)-1 was increased (Figure 3g–h). Both viral proteins prevented increases in TNF mRNA (Figure 3g, P = 0.0001), whereas ovIL-10 exacerbated expression of MCP-1 mRNA (Figure 3h, P = 0.008).
Finally, in the scratch assay, ovIL-10 significantly increased cell proliferation and migration after 48 h (Figure 4, P = 0.012, RM-ANOVA with Sidak's means comparison test), whereas no differences were observed between VEGF-E- and control-treated cells.
Discussion
Orf virus IL-10 and VEGF-E positively influence skin repair in other species.7, 8 The findings of this study suggest that equine dermal cells can respond to the viral proteins and that ovIL-10, particularly, exerts effects on fibroblasts. The ovIL-10 may modulate the inflammatory and fibrotic environment of equine wounds by directly dampening expression of TNF and enhancing MCP-1 expression.10 Upregulation of VEGF-A and collagenase-1 expression by ovIL-10 implies that it may also promote granulation tissue formation and remodelling. Future investigations will ascertain if extended exposure to ovIL-10, like mammalian IL-10s,11 dampens fibroblast differentiation in vitro and, ultimately, if it can prevent fibrosis in equine wounds.
The study findings indicate that VEGF-E also has some beneficial effects on equine fibroblasts, by inducing expression of anti-inflammatory IL-10 and directly dampening TNF expression. In contrast to other mammalian VEGFs,10, 12 VEGF-E does not appear to promote fibroblast activation, proliferation or migration, effects likely to promote fibrosis in equine wounds. Further studies will determine if VEGF-E has beneficial effects on other cells present in healing wounds, such as keratinocytes or endothelial cells, and if it can promote epithelialization, vascularization and, ideally, closure of equine wounds.
Acknowledgements
The authors thank Nicola Real and Gabriella Stuart for technical assistance.
