A novel primate model of delayed wound healing in diabetes: dysregulation of connective tissue growth factor

A novel primate model of delayed wound healing in diabetes: dysregulation of connective tissue growth factor
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  ARTICLE A novel primate model of delayed wound healing in diabetes:dysregulation of connective tissue growth factor S. E. Thomson & S. V. McLennan & A. Hennessy & P. Boughton & J. Bonner & H. Zoellner & D. K. Yue & S. M. Twigg Received: 7 July 2009 /Accepted: 22 October 2009 # Springer-Verlag 2009 Abstract  Aims/hypothesis Chronic non-healing wounds are a commoncomplication of diabetes. Prolonged inflammation anddecreased matrix accumulation may contribute. Connectivetissue growth factor (CTGF) is induced during normal woundhealing, but its regulation in diabetic wounds is unknown. Wedeveloped a primate model for the study of in vivo woundhealing in baboons with long diabetes duration.  Methods Drum implants were placed subcutaneously intothighs of diabetic and non-diabetic control baboons. After 2and 4 weeks the skin incision sites were removed for measurement of breaking strength and epithelial thickness.Drum implants were removed for analysis of granulationtissue and inflammatory cells, CTGF and tissue inhibitor of matrix metalloproteinase (TIMP-1). Degradation of addedCTGF by wound fluid was also examined.  Results Healed incision site skin was stiffer (less elastic) indiabetic baboons and epithelial remodelling was slower compared with controls. Granulation tissue from diabetic baboons was reduced at 2 and 4 weeks, with increased vessellumen areas at 4 weeks. Macrophages were reduced whileneutrophils persisted in diabetic tissue. In diabetic woundtissue at 4 weeks there was less CTGF induced, as shown byimmunohistochemistry, compared with controls. In contrast, Electronic supplementary material The online version of this article(doi:10.1007/s00125-009-1610-6) contains supplementary material,which is available to authorised users.S. E. Thomson : S. V. McLennan : D. K. Yue : S. M. Twigg ( * )Discipline of Medicine, University of Sydney,Camperdown,Sydney, NSW, Australia e-mail: stwigg@med.usyd.edu.auS. E. Thomson : A. HennessyDepartment of Renal Medicine, Royal Prince Alfred Hospital,Camperdown,Sydney, NSW, Australia A. HennessySchool of Medicine, University of Western Sydney,Camperdown,Sydney, NSW, Australia P. BoughtonSt George Clinical School, St George Hospital,Kogarah, NSW, Australia P. BoughtonDepartment of Biomedical Engineering, University of Sydney,Sydney, NSW, Australia J. Bonner Department of Endocrinology, Royal Prince Alfred Hospital,Camperdown,Sydney, NSW, Australia H. Zoellner Oral Pathology and Oral Medicine, Faculty of Dentistry,University of Sydney,Sydney, NSW, Australia H. Zoellner Westmead Centre for Oral Health, Westmead Hospital,Sydney, NSW, Australia S. V. McLennan : D. K. Yue : S. M. TwiggDepartment of Endocrinology, University of Sydney,Blackburn Building, DO6,Sydney, NSW 2006, Australia Diabetologia DOI 10.1007/s00125-009-1610-6  immunoreactive fragments of CTGF were significantlyincreased in whole tissue lysate in diabetic baboons, suggest-ing that CTGF is redistributed in diabetes from granulationtissue into wound fluid. When recombinant human CTGF wasco-incubated with wound fluid, increased CTGF degradation products were observed in both control and diabetic samples. Conclusions/interpretation This baboon model of woundhealing reflects the abnormal microenvironment seen inhuman diabetic wounds and provides insights into thedysregulation of CTGF in diabetic wounds. Keywords Animal.Complications.Cytokines. Non-human Abbreviations CTGF Connective tissue growth factor ECM Extracellular matrixGAPDH Glyceraldehyde-3-phosphate dehydrogenaseMMP Matrix metalloproteinasePDGF Platelet-derived growth factor  α  -SMA α  -Smooth muscle actinrhCTGF Recombinant human connective tissue growthfactor TIMP-1 Tissue inhibitor of matrix metalloproteinaseVEGF Vascular endothelial growth factor  Introduction Diabetes is one of the leading causes of impaired woundhealing, with many amputations performed each year as a result of chronic, non-healing wounds [1]. These woundshave been associated with prolonged and dysregulatedlevels of inflammatory cells, such as neutrophils andmacrophages [2,3]. Other studies have shown that the degradative capacity of chronic wounds is increased whilst growth factor levels are reduced [4,5], collectively contributing to impaired granulation tissue formation anddelayed epithelial closure in diabetic wounds.For normal wound healing to progress, the balance between extracellular matrix (ECM) synthesis and itsdegradation is important, as is the induction of new bloodvessels [5]. These processes are tightly regulated by growthfactors such as platelet-derived growth factor (PDGF),vascular endothelial growth factor (VEGF) and connectivetissue growth factor (CTGF) and by the matrix-degradingenzymes known as matrix metalloproteinases (MMPs).Compared with normal wound healing processes, woundhealing in patients with diabetes results in decreasedconcentrations of PDGF and VEGF proteins in woundtissue [6,7]; however, the effect of diabetes on CTGF in wounds in skin and granulation tissue has not been studied.CTGF is a profibrotic growth factor in the CCN familyof proteins which is induced during normal cutaneouswound healing [8]. It has a variety of actions, includinginduction of fibroblast proliferation, migration, adhesionand ECM formation [8]. CTGF is also produced by culturedendothelial cells [9] and causes proliferation, migration andadhesion of vascular endothelial cells [10,11]. We have  previously shown, albeit in renal mesenchymal cells, that CTGF can upregulate levels of the specific MMP inhibitor TIMP-1 [12].Wound healing models using implantation of subcutane-ous devices have been developed in a variety of animals,including rats, dogs and humans [13  –  15]. These modelsallow the collection of cells, fluid and wound tissue,although typically they have only been short-term, fromdays up to a couple of weeks. The most common collectiondevice has been the sponge [13,14,16], where cells invade and lay down collagen. Other studies have involvedinsertion of porous cylinders, most commonly the Hunt   –  Schilling stainless steel mesh cylinder [17]. These cylindershave been useful for the study of wound repair as theyinduce a response that exhibits the classic phases of inflammation, connective tissue infiltration, neovascularisa-tion and matrix deposition [15,18]. Temporal changes in inflammatory cell infiltration, matrix deposition and growthfactor production can also be measured.We have previously reported changes due to diabetescomplications in our established non-human primate modelof type 1 diabetes [19  –  21]. This animal model of streptozotocin-induced diabetes has been well characterised[22,23] and develops complications comparable with those occurring in human diabetes [19,21]. Animals have diabetes duration of greater than 10 years. In the present work we examined the effects of diabetes on wound healingin these primates for the first time, by implanting sterile plastic drums subcutaneously. This novel wound modelallows the collection of granulation tissue and wound fluidfrom these drums in the absence of external contamination.Epithelial changes and tensile strength of the healedincision site can also be assessed by excising the skin at the time of removal of the subcutaneous drums. Wehypothesised that, compared with the age-matched cohort of non-diabetes controls, diabetic animals would showabnormal regulation of: cutaneous healing, inflammatorycell profiles, granulation tissue formation and tissue CTGF. Methods Baboon modelThe induction and maintenance of this colony of long-termdiabetic and control baboons has been described elsewhere Diabetologia   [22]. Briefly, male baboons were made diabetic at 2.5 years of age by intravenous injection of streptozotocin(65 mg/kg in 0.1 mol/l citrate buffer, pH 4.5). Animalswere maintained on once daily injections of a combina-tion of short- and long-acting insulin (Humulin R andHumulin NPH; Eli Lilly, West Ryde, NSW, Australia) at an average dose of 4 U/kg/day with adjustments made at intervals of 3 months according to the HbA 1c level.Animal details at the time of the study are shown inTable1. All work was approved by the Sydney South West Area Health Service Animal Ethics Committee under theguidelines of National Health and Medical ResearchCouncil of Australia for use of non-human primates inresearch.To implant and remove drums, baboons were anaes-thetised using a combination of ketamine (6 mg/kg;Ketamil; Troy Laboratories, Smithfield, NSW, Australia)and medetomidine hydrochloride (60 μ  g/kg; Domitor; Novartis Animal Health, North Ryde, NSW, Australia).Effects of medetomidine were reversed by atipamezolehydrochloride (300 μ  g/kg; Antisedan; Novartis AnimalHealth, Australia).Drum implants (Fig.1a ) were 5×15 mm (depth ×diameter) polystyrene Netwells with fine polyester 74 µmmesh on the top and bottom surfaces (Corning LifeSciences, Santa Clara, CA, USA). Implants were plasma-sterilised and inserted surgically, two drums per thigh via a 3 cm skin incision. Incisions (Fig.1b) were closed usingsubcuticular absorbable sutures (Polysorb; Tyco Health-care, Norwalk, CT, USA). Antibiotic cover was provided by an injection of amoxicillin trihydrate on alternate days(10 mg/kg; Betamox LA; Norbrook Laboratories, Tulla-marine, VIC, Australia), three in total. After 2 and 4 weeks,the healed, sutured skin incision site was excised with10 mm skin margins, and stored at  − 80°C for later measurement of wound breaking strength and the thick-ness of the epithelial incision site. By the time of skinexcision, all incision sites had healed in all animals.Drums ( n =2 per time point) were then removed andwound fluid was aspirated from within the chamber for later analysis. The granulation tissue from within the drumwas divided and fixed in 10% formalin for histologicalanalysis or snap-frozen for later analysis.Incision site analysisFor the measurement of wound breaking strength, healedsutured skin incision sites were removed from storage andcut into shape using an aluminium template (Fig.1c).Width, length and thickness of skin pieces were measuredusing callipers. Tensile strength was determined at roomtemperature. Tissue ends were placed in the jaws of an Elf 3400 Tensiometer (BOSE EnduraTec, Minnetonka, MN,USA). Load and displacement until the time of skin ruptureat the healed incision site were obtained using a 45 N loadat a cross-head speed of 10 mm/min. Cross-sectional area was determined from srcinal skin thickness measurementsand values were used to calculate stress and strain andYoung ’ s modulus (tensile strength).As a marker of remodelling, the epidermal thickness of the healed incision site was measured in sections stainedwith haematoxylin and eosin (Image J; National Institutesof Health, Bethesda, MD, USA), at five regular pointsalong the suture line of the healed epidermis and at five points in unwounded skin. Values were averaged andresults expressed as the percentage increase compared withunwounded skin.Histopathology and immunohistochemistryFormalin-fixed paraffin-embedded sections (5 μ  m) werestained with haematoxylin and eosin for examination of granulation tissue appearance and Masson ’ s trichrome for calculation of the amount of granulation tissue. Analysis for numbers of neutrophils (1:500; Cat. no. 144499, Abcam,Cambridge, UK) and macrophages (1:800; Cat. no.MCA874G, Serotec Kidlington, Oxford, UK), blood vesselsize (1:200 CD31, Chemicon International, Temecula, CA,USA), collagen IV (1:400; Cat. no. 6311, Abcam), fibro-nectin (1:150; Cat. no. 341645, Calbiochem, Los Angeles,CA, USA), α  -smooth muscle actin ( α  -SMA) (1:200; Cat.no. 5694, Abcam), glyceraldehyde-3-phosphate dehydro-genase (GAPDH; Cat. no. 8245, Abcam), CTGF (1:400; in-house antiserum 196 and 197, each directed against thesame carboxy-terminal antigenic site of CTGF, as described previously [24]) and TIMP-1 (1:400; Cat. no. 770, ChemiconInternational) was by immunohistochemistry using the Table 1 Baboon characteristics at the time of the studyGroup Age (years) Diabetes duration(years)Weight (kg) Blood glucose(mmol/l)HbA 1c (%)Mean±SD Range Mean±SD Range Mean±SD Range Mean±SD Range Mean±SD RangeControl ( n =6) 13.4±0.9 11.9  –  14.3 –  24.0±1.1 22.9  –  25.0 4.3±0.5 3.9  –  5.2 3.9±0.4 3.3  –  4.3Diabetic ( n =7) 13.8±0.81 12.8  –  14.9 11.3±0.81 10.3  –  12.4 19.3±1.9 16.3  –  22.0 29.9±6.3 22.5  –  39.7 8.9±1.0 7.9 − 10.7Diabetologia   ABC method (Vectastain ABC Kit; Vector Laboratories,Burlingame, CA, USA) as reported previously [21]. Tofurther assess the results of granulation tissue CTGFimmunohistochemistry, a completely different primaryanti-CTGF antibody (XY-1) was used in place of anti-CTGF 196 antibody. XY-1 was generated in New ZealandWhite rabbits (Chiron Mimotopes, Melbourne, VIC,Australia) against full-length recombinant human CTGF(rhCTGF) protein [25] and the resulting antiserum wasconfirmed using a previously described method [21] tospecifically detect CTGF in baboon renal tissue byimmunohistochemistry (not shown).Quantification of stainingSections stained with Masson ’ s trichrome were examinedmicroscopically and amounts of granulation tissue present were determined using Image Pro Plus (Media Cybernetics,Silver Spring, MD, USA). The amount of granulation tissuewas expressed as a percentage of total tissue area obtainedfrom three sections 50 µm apart. Sections stained for CTGF, TIMP-1, fibronectin and collagen IV were scoredfrom 0 to 3 by two independent observers blinded to tissuesource, 0 indicating no staining and 3 intense staining.Macrophages and neutrophils were counted at ×100magnification in 20 fields using two sections 50 μ  m apart and results were averaged. Lumen areas of blood vesselswere measured at ×20 magnification using Axiovision 4(Carl Zeiss Vision). All vessel lumens in three separatesections per animal were measured.Tissue RNA isolation and real-time quantitative PCR Pure total RNA was isolated freshly from wound tissue(~100 mg) and prepared as cDNA, as described previously[12,24]. Primers were designed against highly conserved regions of multiple species including >99% homology withhuman amplicons. Primer sequences are shown in theElectronic supplementary material (ESM) Table1. Thermalcycles for amplification were as follows: 2 min at 50°C,5 min at 95°C and 45 cycles of 95°C for 10 s, 55°C for 20 sand 72°C for 20 s. The delta/delta method was used tocompare gene expression between animals with diabetesand non-diabetic controls. Results were corrected for  36B4 (also known as RPLP0 ) as housekeeper.Analysis of whole wound tissueCTGF protein was extracted from wound tissue using RIPA buffer. Whole tissue lysate was loaded (50 μ  g per lane)onto SDS-PAGE gels under reducing conditions thentransferred onto polyvinylidene fluoride membranes andassessed by western immunoblotting using anti-CTGF primary antibody (197, 1:1,000) as described previously[26]. Immunoreactive band intensities were assessed byImage Analysis, corrected for the housekeeper loadingcontrol, GAPDH.Degradation of rhCTGF by wound fluidWound fluid samples (5 µl from each baboon) were pooledinto control and diabetic groups. The rhCTGF was produced as described previously [12,26]. Pooled wound fluid (15 µl) was mixed with either PBS (15 µl) or rhCTGF(15 µl), each containing 0.1% BSA (Sigma). In other samples, rhCTGF (15 µl) alone was incubated with PBScontaining 0.1% BSA (15 µl). All samples (total 30 µl),except one rhCTGF sample, as indicated in Fig.8b, wereincubated at 37°C for 24 h and then loaded onto a SDS-PAGE gradient gel. The effect of wound fluid on rhCTGFdegradation was analysed by western immunoblottingagainst CTGF using 197 antibody [25]. Total immunoreac-tivity was determined by Image Analysis and expressed asindividual molecular mass band intensities and the sum of all immunoreactive bands.Statistical analysisDifferencesbetweengroupswereanalysedbyANOVAusing NCSS (Number Cruncher Statistical Analysis, UT, USA),except for blood vessel lumen size, for which differences between the groups were analysed using a two-sample t  test  6 mm    1   0  m  m 4 mmSuturedincisionsite a b c Fig. 1 The baboon model and drum implant. a Drum implant used for tissue collection. b Drum implants in situ after surgical insertion. c H-shaped template used to excise skin of standard dimensions and containing the healed cutaneous incision site at 2 or 4 weeksDiabetologia   assuming equal variance. All data are mean±SD and valuesof  p <0.05 were considered significant. Results Animal characteristicsPhysical characteristics of the baboons at the time of tissuesampling are shown in Table1. The diabetic baboons hadlower body weight, higher HbA 1c and higher blood glucoselevels than control animals and their diabetes duration wasmore than 10 years.Effect of diabetes on the wound breaking strengthand incision site epidermal thicknessYoung ’ s modulus was greater in the diabetic baboonscompared with the control baboons (Table2, p <0.05),indicating the healed incision site was stiffer and lessextendable in the diabetic baboons. In control animals theepidermal thickness at the incision site was significantlygreater at 2 weeks than 4 weeks. In contrast, this decreasein thickness with time was not observed in the diabeticanimals, in which the thickness was significantly greater than in controls at 4 weeks (Fig.2, Table2). Effect of diabetes on granulation tissue formation,inflammatory cell response, CTGF and TIMP-1immunostaining  Formation of granulation tissue After 4 weeks there wasless granulation tissue in the drums of the diabetic baboonscompared with controls (Fig.3a   –  d, quantified in Fig.4a ). Under H&E staining at higher magnification the tissue alsoappeared coarser, less well formed and more disorganised indiabetic animals than in controls (Fig.3e  –  h). Fibronectin andcollagen IV staining appeared darker in control than diabetictissue, as seen at 4 weeks (Fig.3i, jandk, lrespectively). There was a trend for fewer blood vessels in diabetic tissue(Fig.4b). Blood vessel lumens at 4 weeks were significantlylarger (control 610±218 pixels, diabetic, 1263±389 pixels;  p =0.01), and the vessel walls appeared to be thinner (Fig.4c,d) in tissue from the diabetic baboons. The α  -SMA stainingin control and diabetic animals was mainly localised toendothelial cells rather than fibroblasts (Fig.4e, f ).  Pattern of inflammatory cell response The pattern of change in inflammatory cell infiltrate appeared to bedifferent between the groups. In control animals theneutrophil and macrophage numbers decreased betweenthe 2- and 4-week time points (Fig.5a andbrespectively). This decrease was not observed in the diabetic animals andthe macrophage number at 2 weeks was lower than incontrols at the same time point (  p <0.05) (Fig.5b). CTGF and TIMP-1 detection By immunohistochemistry,the amount of CTGF protein was greater in control tissuecompared with diabetic tissue at both 2 and 4 weeks, thedifference reaching significance at 4 weeks (Figs6a   –  dand7a ) (  p <0.05). The same overall trend was observed withXY-1 anti-CTGF antibody (staining score, mean±SD:control, 2 weeks, 2.17±1.04, diabetic, 2 weeks, 1.69±0.31; control, 4 weeks, 2.40±0.38, diabetic, 4 weeks, 1.92±0.75; not significant). (See Electronic supplementarymaterial [ESM] Fig.1). A similar pattern was observedfor TIMP-1 protein levels, differences failing to reachstatistical significance (Figs6e  –  hand7b). There was no difference in CTGF  mRNA levels in the tissue (Fig.7a ),suggesting post-transcriptional regulation and the possiblecompartmentalisation of CTGF across wound tissue andfluid. The TIMP-1 (also known as TIMP1 ) mRNA levelswere also not different between groups (Fig.7d). Interest-ingly, reduced macrophage number at 2 weeks in diabetictissue correlated positively with a low level of wound tissueCTGF protein at 4 weeks ( r  =0.65, p <0.05). Table 2 Analysis of the healed incision siteTime point Young ’ smodulus (N/m 2 )Epidermal thicknessat incision site (mm)Epidermal thickness of unwounded skin (mm)Epidermal thickness (increasecompared with unwounded skin) (%)Control group2 weeks 0.51±0.16 0.139±0.024 0.052±0.016 2694 weeks 1.35±0.42* 0.085±0.014* 0.054±0.008 158Diabetic group2 weeks 0.64±0.24 0.133±0.024 0.049±0.012 2734 weeks 2.04±0.21* † 0.130±0.040 † 0.056±0.049 232Results are mean±SD*  p <0.05 vs data for 2 weeks in the same group; †  p <0.05 vs control at same time point (ANOVA)Diabetologia 

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