Fibroblast Dysfunction Is a Key Factor in the Non-Healing of Chronic Venous Leg Ulcers

ORIGINAL ARTICLE

See related commentary on pg 2361

Fibroblast Dysfunction Is a Key Factor in the Non-Healing of Chronic Venous Leg Ulcers Ivan B. Wall1,5, Ryan Moseley1, Duncan M. Baird2, David Kipling2, Peter Giles2, Iraj Laffafian3, Patricia E. Price4, David W. Thomas1 and Phil Stephens1 Chronic age-related degenerative disorders, including the formation of chronic leg wounds, may occur due to aging of the stromal tissues and ensuing dysfunctional cellular responses. This study investigated the impact of environmental-driven cellular aging on wound healing by conducting a comprehensive analysis of chronic wound fibroblast (CWF) behavior in comparison with patient-matched healthy skin normal fibroblasts (NF). The dysfunctional wound healing abilities of CWF correlated with a significantly reduced proliferative life span and early onset of senescence compared with NF. However, pair-wise comparisons of telomere dynamics between NF and CWF indicated that the induction of senescence in CWF was telomere-independent. Microarray and functional analysis suggested that CWFs have a decreased ability to withstand oxidative stress, which may explain why these cells prematurely senescence. Microarray analysis revealed lower expression levels of several CXC chemokine genes (CXCL-1, -2, -3, -5, -6, -12) in CWF compared with NF (confirmed by ELISA). Functionally, this was related to impaired neutrophil chemotaxis in response to CWF-conditioned medium. Although the persistence of non-healing wounds is, in part, due to prolonged chronic inflammation and bacterial infection, our investigations show that premature fibroblast aging and an inability to correctly express a stromal address code are also implicated in the disease chronicity. Journal of Investigative Dermatology (2008) 128, 2526–2540; doi:10.1038/jid.2008.114; published online 1 May 2008

INTRODUCTION The increase in life expectancy of the general population over the last several decades has prompted a growing body of research into age-related degenerative disorders that greatly impair quality of life in older individuals. Diseases such as cancer, Alzheimer’s disease, and diabetes are gradually replacing heart disease and respiratory disorders as the primary causes of ill health in the aged population. Agerelated decline in repair and regeneration potential in the skin might be a cause of non-healing in wounds, such as chronic venous leg ulcers, pressure ulcers, and diabetic ulcers. Indeed, a steady increase in their incidence in individuals 1

Wound Biology Group, Cardiff Institute of Tissue Engineering and Repair, Tissue Engineering and Reparative Dentistry Group, School of Dentistry, Cardiff University, Cardiff, UK; 2Department of Pathology, School of Medicine, Cardiff University, Cardiff, UK; 3Department of Surgery, School of Medicine, Cardiff University, Cardiff, UK and 4Wound Healing Research Unit, School of Medicine, Cardiff University, Cardiff, UK

5

Current address: Division of Cellular Therapy, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK Correspondence: Dr Phil Stephens, Wound Biology Group, Cardiff Institute of Tissue Engineering and Repair, Tissue Engineering and Reparative Dentistry Group, School of Dentistry, Cardiff University, Cardiff CF14 4XY, UK. E-mail: [email protected] Abbreviations: CWF, chronic wound fibroblast; MMP, matrix metalloproteinase; NF, normal fibroblast; PD, population doubling; ROS, reactive oxygen species; SA b-Gal, senescence associated b-galactosidase; STELA, single telomere length analysis Received 18 October 2007; revised 22 January 2008; accepted 20 February 2008; published online 1 May 2008

2526 Journal of Investigative Dermatology (2008), Volume 128

over the age of 65 years has been reported (Margolis et al., 2002), and owing to their frequency and ensuing disability, they are now considered to be financially the most important skin disorder in aged patients. Although there is no single, unifying theory as to why chronic wounds fail to heal, chronic inflammation (Agren et al., 2000; Smith, 2001) and bacterial infection (Robson, 1997; Dow et al., 1999) are two major contributory factors in the persistence of the wound chronicity. This chronicity may in turn lead to localized cellular/tissue aging within the wound site. It is also clear that within such aging tissue, the vasculature becomes compromised (Montagna and Carlisle, 1979; West et al., 1996), extracellular matrix composition/ turnover is dysregulated (Lavker et al., 1987; VitellaroZuccarello et al., 1992; Watson et al., 1999; Chung et al., 2001; Isnard et al., 2002), and fibroblast function and responses are altered. However, the role of the fibroblast in chronic inflammation has typically been overlooked. Nonetheless, investigators have provided recent evidence for the existence of a stromal address code (Parsonage et al., 2005) that provides the precise cues required to guide infiltrating leukocytes to the injury site. This suggests that the failure to resolve inflammation in chronic wounds is tissue-specific, as a result of fibroblast dysfunction. Such fibroblast dysfunction has been reported to be due to an increase in the proportion of cells undergoing irreversible growth arrest or replicative senescence (Dimri et al., 1995). Senescent fibroblasts, which are still metabolically active, are highly stable and express numerous extracellular & 2008 The Society for Investigative Dermatology

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

matrix-related enzymes and structural molecules including proteases, such as matrix metalloproteinases (MMPs; West et al., 1989; Millis et al., 1992; Wick et al., 1994), pro-inflammatory cytokines (Kumar et al., 1993), tissue transglutaminase (Kim et al., 2001), type III collagen (Lovell et al., 1987; Vitellaro-Zuccarello et al., 1992), and fibronectin (Dumont et al., 2000), while downregulating the main structural component of the skin, type I collagen (Chung et al., 2001; Varani et al., 2004). Decreased proliferation and increased replicative senescence have been described for fibroblasts isolated from the chronic wound site compared with normal non-wound skin fibroblasts (Stanley et al., 1997; Loots et al., 1998; Vande Berg et al., 1998, 2005; Mendez et al., 1999; Stanley and Osler, 2001). However, not all studies have shown this and other researchers have found little direct evidence of replicative senescence in chronic wound fibroblasts (CWFs) (Stephens et al., 2003). Therefore, despite our knowledge that the local environment and the inflammatory status can contribute to the non-healing phenotype of these wounds, the role of aging and altered life span of fibroblasts within the wound is still not fully understood. Gradual telomere erosion with successive cell divisions is one of the primary intrinsic triggers of replicative senescence in many somatic cell populations and thus has the potential to contribute to organismal aging (Kipling et al., 1999; Kipling, 2001). Using a high-resolution method for measuring telomere length (Baird et al., 2003), it is possible to investigate the true spectrum of telomere lengths within cells. Such findings can then be linked with differential phenotypic responses observed between chronic wound and patient-matched normal cells (Herrick et al., 1996; Mendez et al., 1998; Agren et al., 1999; Stephens et al., 2003). However, cellular aging is not solely a product of replication-dependent telomere erosion. Cells may undergo premature senescence in response to a variety of external stimuli, for example, oxidative stress (Toussaint et al., 2000; Wlaschek et al., 2003; Naka et al., 2004). Chronic wounds are associated with elevated levels of oxidative stress (Wenk et al., 2001; Allhorn et al., 2003; James et al., 2003; Yeoh-Ellerton and Stacey, 2003; Wlaschek and ScharffetterKochanek, 2005) due to the chronically inflamed environment, it has been postulated that stress-induced premature senescence, rather than ‘true’ replicative senescence, is the major factor affecting fibroblast regeneration potential in non-healing wounds (Wlaschek and ScharffetterKochanek, 2005). Furthermore, it has been reported that both keratinocytes and endothelial cells isolated from chronic wounds have an inability to upregulate chemokines and cytokines necessary for the resolution of chronic inflammation (Galkowska et al., 2006), which may help drive the continued elevation in oxidative stress. Therefore, the aim of this study has been to investigate the effects of cellular aging on fibroblast responses in a chronic wound context. Using patient-matched chronic wound and normal fibroblasts (NFs), this investigation aims to determine the aging profiles of these cells and how this impacts on genomic expression patterns and functional responses of these cells in relation to a wound healing context.

RESULTS CWF demonstrate an impaired ability to repopulate experimental wounds in vitro compared with NF

We have previously reported the distinct phenotypes of CWF and NF in respect to their abilities to carry out key wound healing activities (Cook et al., 2000; Stephens et al., 2003). This dysfunctional wound healing potential was further corroborated using a monolayer scratch wound assay and automated time-lapse microscopy to assess the ability of CWF and NF to repopulate a wound space. The rate of wound repopulation by all CWF samples was significantly reduced compared with that of patient-matched NF, particularly at 24 hours (Figure 1a and b; Po0.05). CWF have a decreased replicative life span compared with NF

In light of reports that the chronic wound environment can have deleterious effects on fibroblast proliferative potential (Mendez et al., 1999; Seah et al., 2005), the replicative life span of CWF and patient-matched NF from four chronic venous leg ulcer patients was investigated. Analysis of the replicative capacity of both CWF and NF demonstrated that CWF exhibited a decreased proliferative life span compared with NF before becoming senescent (Figure 2a and b; Po0.05). Whereas NF from all four patients completed a similar number of population doublings (PDs) that correlated to the classical Hayflick limit described for such cells (Hayflick, 1965), the replicative life span of CWF varied from 6 PD (patient 3) to 40 PD (patient 4). Senescence was further confirmed by morphology (larger, stellate cells with evidence of internal stress fibers; Figure 2c) and by assessing senescence-associated b-galactosidase (SA b-Gal) activity (Figure 2d). Whereas early passage (when both CWF and NF were actively proliferating) populations of CWF and NF contained a similar number of SA b-Gal-positive cells, at late passage (the point in culture when CWF had entered senescence but patient-matched NF were still replicating), CWF demonstrated significantly greater SA b-Gal activity compared with NF (Po0.05). CWF display longer telomeres at senescence than NF

As CWF demonstrated a reduced replicative life span compared with patient-matched NF, it was hypothesized that this may be due to telomeric erosion in vivo within the chronic wound environment. Single telomere length analysis (STELA) was utilized to assess telomere dynamics at the single-molecule level in populations of CWF and NF throughout their replicative life span. Notably, particularly at early passage, individual cell populations exhibited a broad range of telomere lengths (0.5 to 412 kb), indicative of the large degree of heterogeneity within each population (Figure 3). Ultrashort telomeres that were distinct from the main distribution were also evident (arrowheads). In addition to heterogeneity within cell populations, there was considerable variation in the telomere dynamics of the CWF and NF populations between patients at an early time in culture (Figure 3a). In patient 1, no significant differences in mean telomere lengths between CWF and NF were observed at early passage/PD, whereas in patients 2 and 3 CWF mean www.jidonline.org 2527

IB Wall et al.

0 hour

24 hours

40 hours

Wound area (% initial area)

CWF

100

Wound area (% initial area)

NF

100

Wound area (% initial area)

Fibroblast Dysfunction in Non-Healing Wounds

100

*

80

**

60 40 20 0

CWF1 NF1

10

20

30

40

30

40

30

40

**

80 60 40 20 0

CWF2 NF2

10

20

80 60 40 20 0

CWF3 NF3 10

*

20 Time (hours)

Figure 1. Analysis of wound repopulation by CWF and NF. Wound repopulation was assessed using a monolayer scratch wound assay and automated time-lapse cell imaging (a; broken lines represent initial wound boundary). CWFs were observed to have a decreased ability to repopulate the wound space over the duration of the experiment (a; scale bar ¼ 200 mm). Quantitation of relative wound closure for CWF vs NF (b; values ¼ mean±SD (n ¼ 2); *Po0.05; **Po0.01).

telomere lengths were longer (Po0.05 and Po0.01, respectively). However, in patient 4, mean telomere lengths were shorter in CWF (Po0.05). When the populations became senescent (late passage/PD), STELA clearly indicated that in three out of four individuals, mean telomere lengths from CWF were significantly longer than those of NF (Po0.01; Figure 3b). In the fourth individual, no significant difference in mean telomere length was apparent (P40.1). CWF display altered gene expression profiles both intrinsically and in response to an artificial wounding stimulus

As distinct differences in the proliferative status and telomere dynamics were evident for CWF, a global analysis of gene expression was undertaken to determine, at the molecular level, potential differences between CWF and NF that might help explain these findings. The serum starvation/re-stimulation model described by Iyer et al. (1999) was used on all four patient-matched populations of early passage CWF and NF with all the cells subsequently analyzed utilizing Affymetrix microarrays pre- and post-serum stimulation. Genes differentially regulated between CWF and NF independent of the effects of serum were initially characterized. Common to all four patients, 118 genes were differentially regulated between CWF and NF at a false discovery rate of 10% (Figure 4). This included 53 genes that had greater expression in CWF and 65 genes that had lower expression in CWF (see Table S1). Of the 53 genes that were upregulated in CWF, a subset of genes (Table 1) was classifiable into one 2528 Journal of Investigative Dermatology (2008), Volume 128

of three categories that are of interest with respect to wound healing, namely (i) cellular aging/cell-cycle regulation; (ii) oxidative stress-related; and (iii) cytoskeleton-related. Of the 65 genes that demonstrated lower expression in CWF compared with NF, a subset of genes (Table 2) were classified into one of six categories of genes that appeared interesting in relation to wound healing, namely (i) cellular aging/cell-cycle regulation; (ii) inflammatory response-related; (iii) protease/ protease inhibitors; (iv) growth factor signaling; (v) extracellular matrix components; and (vi) cytoskeleton-related. The second method of analysis characterized genes that were differentially regulated between CWF and NF in response to serum. Based on a patient-matched comparison of expression levels, a difference-of-differences test identified 28 genes that had 42-fold greater induction by serum in CWF than in NF and 22 genes that had 42-fold lesser induction by serum in CWF (Figure 5 and Table S1). Of those genes that were less induced in CWF, a subset of these (Table 3) included five different CXC chemokine family members and several proteases, including MMP1 and MMP12. Similarly, potentially relevant genes within the 28 genes that had 42-fold greater induction in CWF than in NF (subset shown in Table 4) included 4 genes associated with growth factor/cytokine activity and hyaluronan synthase 1. To corroborate these findings based solely on alterations at the gene expression level, three areas of interest were further investigated based on their altered gene expression profiles between CWF and NF and potential importance in the

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

60 50

NF1 NF2 NF3 NF4 CWF1 CWF2 CWF3 CWF4

40 30 20 10

0

100

200

400

300

500

Time (days) Morphology

**

60

CWF

NF

Early

40 30 20

Late

Population doublings

50

10 0 CWF 100 90 80

NF % SA β-Gal activity

NF

*

CWF

CWF Early

70 % SA β-Gal

NF

60 50 40

Late

30 20 10 0 Early

Late

Figure 2. Replicative life span and senescence in populations of CWF and patient-matched NF. CWFs have a significantly reduced life span in comparison with NF (a (individual data), b (combined data); values ¼ mean±SEM (n ¼ 4); **Po0.01). Assessment of individual cultures indicates similar growth kinetics between NF strains, yet considerable variation between CWF strains. Replicative senescence was characterized by transdifferentiation from a small, bipolar morphology at early passage to one of an enlarged, polygonal appearance with prominent stress fibers at late passage (replicative senescence; c). Late passage CWF demonstrated significantly greater SA b-Gal activity compared with patient-matched NF as assessed by an increased percentage of blue cells (d; *Po0.05; scale bar ¼ 100 mm).

chronic wound environment, namely (i) oxidative stress responses; (ii) chemokine production; and (iii) directed chemotaxis of neutrophils. CWF demonstrate elevated levels of superoxide radical compared with NF

As the microarray profiling identified altered expression of oxidative stress-response genes within CWF and as elevated levels of oxidative stress are a well-reported phenomenon in chronic wounds (Wenk et al., 2001; Allhorn et al., 2003; James et al., 2003; Yeoh-Ellerton and Stacey, 2003; Wlaschek

and Scharffetter-Kochanek, 2005), the oxidative status of both CWF and NF was investigated utilizing the cytochrome c reduction assay to measure superoxide radical generation over a time course of 10 days. Despite some heterogeneity in the level of responses between patients, the general observation was that over the time course of the experiment, NF demonstrated a decline in the levels of superoxide radical generation, whereas CWF produced elevated levels (Figure 6). Notably, for all three patients investigated, CWF consistently produced significantly greater levels of superoxide than NF during the later time points of the assay (Po0.05). www.jidonline.org 2529

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

Early

kb

CWF1

NF1

CWF2

NF2

CWF3

NF3

CWF4

NF4

kb

12

12

5

5

2

2

1

1

0.5

0.5

Mean SD

4.65

3.94

5.67

4.91

5.55

3.60

5.72

2.97

3.40

3.17

3.55

1.96

1.42

2.82

P-value

0.077

0.022

0.001

6.32 2.94

0.022 12

5

5

2

2

Late

12

1

1

0.5 0.5

Mean SD P-value

5.13

4.38

4.32

3.11

5.55

1.66

3.46

3.49

2.87

1.65

2.46

1.84

1.96

0.65

1.95

2.42

0.002

0.001

0.001

0.913

Figure 3. Relative telomere length in CWF and NF. Measurement of the XpYp telomere in populations of CWF and NF using STELA at early stage of culture (a) and upon reaching replicative senescence (b). Heterogeneity in telomere length is an overwhelming characteristic of all cell populations throughout their replicative life span and many ultrashort telomeres separate from the main distribution were evident (examples indicated by arrowheads). At early passage, interpatient heterogeneity in telomere dynamics was evident, with no clear trend regarding the overall dynamics of CWF vs NF populations, but at late passage/senescence, a clear trend emerged with CWF having longer telomeres than NF in three out of four patients (P-values indicated for each blot represent CWF vs NF).

53 genes CWF>NF

65 genes CWF
CWF

level. Medium conditioned by the cells was assessed 6 and 24 hours post-serum stimulation. Production of CXCL-1 demonstrated some heterogeneity with the CWF of two of the three patients showing a significantly decreased ability to produce CXCL-1 protein (at both 6 and 24 hours) and the other patient only producing very low levels of the chemokine (Figure 7a; Po0.05 and Po0.01). For CXCL-5, CWF consistently produced less chemokine compared with NF in all patients and at all time points that reached significance in all, but in patient 2 at 24 hours (Figure 7b; Po0.01). There was no evidence of the production of CXCL-12 either by CWF or NF when assessed by ELISA (data not shown).

NF

Figure 4. Transcriptomic analysis identified 118 genes differentially expressed between CWF and NF, independent of serum exposure. These data were split into probe sets showing an upregulated (53 genes) and downregulated (65 genes) response comparing CWF with NF cells. The data were hierarchical clustered and heat map visualized.

CWF are deficient in their production of CXCLs

From the gene expression analysis, the impaired induction of five different CXC chemokine family members (CXCL-1, -2, -3, -5, and -6) and decreased serum-independent expression of a sixth (CXCL-12) was observed in CWF compared with NF. ELISA analysis of the CXCL-1, -5, and -12 was therefore undertaken to assess whether such changes at the gene level were mirrored by alterations in their production at the protein 2530 Journal of Investigative Dermatology (2008), Volume 128

CWF demonstrate a delayed ability to stimulate directed polymorphonuclear leukocytes chemotaxis

To relate the ELISA findings with respect to chemokine levels within the medium conditioned by CWF and NF to functional activity, transwell migration chambers were utilized to characterize the effects on neutrophil chemotaxis. Using medium harvested 6 hours after serum stimulation, NF-conditioned medium from two patients produced a positive neutrophil chemotactic response, which was sustained over the following 18 hours (Table 5). In contrast, CWF demonstrated a delayed ability to stimulate directed chemotaxis of the neutrophils, in that they showed a weakly positive/or absence of response at 6 hours, which only became strongly positive at 24 hours.

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

Table 1. Genes that demonstrated increased expression in CWF vs NF Affymetrix probe ID

Log2 fold change

Symbol

Name

Cellular aging/cell-cycle regulation genes 203510_at

2.1

MET

Met proto-oncogene

209228_x_at

0.8

TUSC3

Tumor suppressor candidate 3

213423_x_at

0.8

201162_at

0.6

IGFBP7

Insulin-like growth factor-binding protein 7

218788_s_at

1.5

SMYD3

Set and MYND domain-containing 3

40148_at

1.0

APBB2

Amyloid b-A4 precursor protein-binding family B member 2

218976_at

1.3

DNAJC12

DnaJ homolog subfamily C member 12

209030_s_at

0.7

IGSF4

Immunoglobulin superfamily, member 4

217982_s_at

0.5

MORF4L1

Mortality factor 4-like 1

206662_at

1.8

GLRX

Glutaredoxin

209004_s_at

0.9

FBXL5

F-box and leucine-rich repeat protein 5

209005_at

0.9

209935_at

0.6

ATP2C1

Calcium-transporting ATPase type 2C, member 1

Oxidative stress-related genes

Cytoskeleton-related genes 200713_s_at

0.4

MAPRE1

Microtubule-associated protein, RP/EB family, member 1

209758_s_at

0.8

MFAP5

Microfibrillar-associated protein 5

211963_s_at

0.5

ARPC5

Actin-related protein 2/3 complex, subunit 5, 16 kDa

200996_at

0.8

ACTR3

Actin-related protein 3 homolog (ARP3) (yeast)

213101_s_at

0.7

200897_s_at

0.7

PALLD

Palladin

214043_at

2.6

PTPRD

Protein-tyrosine phosphatase receptor-type delta

200625_s_at

0.5

CAP1

Adenylate cyclase-associated protein 1

213798_s_at

0.6

212412_at

1.2

PDLIM5

PDZ and LIM domain 5

203440_at

1.8

CDH2

N-cadherin (neuronal)

CWF, chronic wound fibroblast; NF, normal fibroblast.

Discussion Dermal fibroblasts are key regulators of wound healing responses in the skin (Singer and Clark, 1999). However, in the case of venous leg ulcers, healing is impaired and evidence is starting to point to the role of the fibroblast in the pathogenesis of these non-healing chronic wounds. The dysfunctional CWF cellular activity reported in this study is in agreement with the work of others (Herrick et al., 1996; Mendez et al., 1998; Agren et al., 1999; Cook et al., 2000; Stephens et al., 2003). Some previous studies reported that CWF undergo replicative senescence very early in culture, compared with uninvolved dermal fibroblasts (Stanley et al., 1997; Mendez et al., 1998; Vande Berg et al., 1998), but others observed sustained growth for over 100 days without evidence of senescence (Stephens et al., 2003). However, we demonstrate that although the former is true, the mechanism of cellular aging that is causative of dysfunctional healing might not be due to simple telomere erosion. In this study, patient-matched CWF and uninvolved NF were extensively cultured until senescence to fully characterize replicative life

span in CWF vs NF. Although all CWF samples had reduced replicative life span, there was a large degree of heterogeneity between patients. For instance, CWF from patient 3 reflected the observations of Vande Berg et al. (1998), but CWF from patient 4 proliferated for over 100 days (akin to Stephens et al., 2003) before senescence. This was clearly a reflection of the heterogeneous wound environments that the cells were derived from despite the wounds having a similar clinical diagnosis. In contrast, NF from all patients underwent a similar number of PDs in a similar time frame. We then analyzed telomere length to determine whether CWF was biologically more aged than NF. The standard method for measuring telomeres, telomere restriction fragment analysis, only provides a crude indication of mean telomere length of the population. This study used STELA, a high-resolution method that accurately measure telomeres at the single-cell level (Baird et al., 2003). This enabled the broad spectrum of telomere lengths within the cell populations to be detected. As cell populations derived from any primary culture are heterogeneous with respect to telomere www.jidonline.org 2531

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

Table 2. Genes that demonstrated decreased expression in CWF vs NF Affymetrix probe ID

Log2 fold change

Symbol

Name

Cellular aging/cell-cycle regulation genes 207191_s_at

1.8

ISLR

Immunoglobulin superfamily containing leucine-rich repeat

209331_s_at

0.6

MAX

Myc-associated factor X

216033_s_at

0.6

FYN

FYN oncogene

209594_x_at

1.1

PSG9

Pregnancy-specific b1-glycoprotein 9

209596_at

1.1

MXRA5

matrix-remodelling associated 5

205366_s_at

2.0

HOXB6

Homeobox B6

213977_s_at

0.4

CIZ1

CDKN1A (p21cip1/waf1)-interacting zinc-finger protein 1

41220_at

0.6

MSF

Septin 9

204776_at

1.4

THBS4

Thrombospondin 4

209687_at

1.5

CXCL12

Chemokine (C-X-C motif) ligand 12 (stromal-derived growth factor 1; SDF1)

207375_s_at

0.9

IL15RA

Interleukin 15 receptor a

200675_at

0.6

CD81

CD81 antigen (target of antiproliferative antibody 1)

215346_at

1.3

CD40

CD40 antigen (TNF receptor superfamily member 5)

205828_at

2.3

MMP3

Matrix metalloproteinase 3 (stromelysin 1, progelatinase)

201487_at

1.1

CTSC

Cathepsin C

203657_s_at

1.0

CTSF

Cathepsin F

200986_at

1.4

SERPING1

Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor)

205110_s_at

2.9

FGF13

Fibroblast growth factor 13

211273_s_at

2.1

TBX1

T-box transcription factor 1

216264_s_at

0.7

LAMB2

Laminin b2 chain precursor

202007_at

0.8

NID

Nidogen (enactin)

213068_at

1.4

DPT

Dermatopontin

213428_s_at

0.4

COL6A1

Collagen type VI a1

205054_at

0.5

NEB

Nebulin

201426_s_at

0.3

VIM

Vimentin

203407_at

2.2

PPL

Periplakin

Inflammatory response-related

Protease/protease inhibitors

Growth factor signaling

ECM components

Cytoskeleton-related genes

CWF, chronic wound fibroblast; ECM, extracellular matrix; NF, normal fibroblast.

length (caused by stochastic variation in telomere erosion rates) (Martin-Ruiz et al., 2004), STELA provides more detailed information than telomere restriction fragment analysis, including ‘outlier’ cells with ultralong or ultrashort telomeres relative to the general distribution. For early passage cells, clear patient-specific heterogeneity in telomere length was evident with CWF telomeres being longer than NF in 2/4 patients, the reverse being evident in one patient and no differences in the fourth patient. However, at late passage, CWF had longer telomeres than patient-matched NF in 3/4 patients, with no difference in the fourth patient. 2532 Journal of Investigative Dermatology (2008), Volume 128

This indicates that the CWF populations underwent senescence before they had exploited the maximal capacity for telomere erosion that was evident for patient-matched NF. This suggests that simple telomeric erosion events due to end replication losses were of little consequence to aging of the CWF. These observations corroborate the argument that intrinsic telomere erosion from end replication losses only sets an outer limit for replicative life span and that more random, stochastic events caused by extrinsic stresses could be the main cause of cellular senescence (Martin-Ruiz et al., 2004). These stochastic events could also help explain the

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

large degree of heterogeneity in telomere length evident for both early passage/PD, and late passage/PD cell populations. Oxidative stress is one extrinsic factor that affects cellular aging. The cellular response to oxidative stress varies according to the level of exposure and while mild stress from low-level reactive oxygen species (ROS) accumulation accelerates the rate of telomere erosion (von Zglinicki et al., 2000; von Zglinicki, 2002; Kawanishi and Oikawa, 2004), high levels of stress from increasing concentration of ROS induce telomere-independent premature senescence (Finkel, 1999; Wright and Shay, 2001, 2002; Song et al., 2005; Zhang, 2007). As chronic wound sites exhibit increased levels of oxidative stress (Wenk et al., 2001; Allhorn et al., 2003; James et al., 2003; Wlaschek and Scharffetter-Kochanek, 2005), the ‘early onset’ senescence in 0 hour

6 hours

0 hour

6 hours

22 genes CWF
28 genes CWF>NF

NF

CWF

Figure 5. Transcriptional signature of the differential response of CWF and NF to serum. A total of 50 genes had greater than twofold differential induction in CWF vs NF. Heat map representation of the data after hierarchical clustering show that 22 genes were induced to more than twofold lesser degree in CWF than in NF and 28 genes were induced to more than twofold greater degree in CWF.

CWF relative to patient-matched NF could be a consequence of increased ROS exposure. This hypothesis was supported by microarray analysis of CWF vs NF populations, which demonstrated that several genes associated with oxidative stress responses underwent increased expression or induction in CWF compared with NF populations (GLRX, IL-11, FBXL5, ATP2C1). Subsequently, measuring ROS production utilizing the cytochrome c reduction assay (Turner et al., 1998) revealed that whereas NF generated lower levels of ROS over time, CWF produced increasingly elevated levels of ROS. This is in accordance with the reports of elevated ROS generation by senescent fibroblast cell populations (Lee et al., 2002; Chiba et al., 2005; Song et al., 2005; Zdanov et al., 2006; Passos et al., 2007; Probin et al., 2007). Therefore, reportedly high levels of oxidative stress in the wound site (Wlaschek and Scharffetter-Kochanek, 2005) are probably responsible for depleting the CWF capacity to withstand ROS accumulation. Several reports have implicated the overproduction of ROS in the pathogenesis of impaired chronic wound healing and cellular senescence/aging, despite the presence of a variety of cellular and extracellular antioxidant entities (Finkel and Holbrook, 2000; von Zglinicki, 2000; Sen, 2003; Wlaschek and Scharffetter-Kochanek, 2005). Indeed, ROS overproduction can inactivate dermal enzymic antioxidants (superoxide dismutases, catalase, glutathione peroxidises, and so on) (Shukla et al., 1997; Steiling et al., 1999), deplete nonenzymic antioxidant levels (Shukla et al., 1997; James et al., 2001), and thus lead to DNA damage, telomere erosion, cellular senescence (Finkel and Holbrook, 2000; von Zglinicki, 2000), and impaired cellular responses (Moseley et al., 2004). The data presented here support and extend this hypothesis, with only four genes with established oxidative stress-/antioxidant-related functions being upregulated in CWF, including IL-11 (Waxman et al., 1998; Pascal et al., 2007), glutaredoxin (Berndt et al., 2007), calcium-transport-

Table 3. Genes whose induction was more than twofold lower in CWF than in NF Affymetrix probe ID

Log2 fold change

Symbol

Name

Inflammatory response-related 204470_at

2.1

CXCL1

Growth-related protein a (GROa)

209774_x_at

1.5

CXCL2

Macrophage inflammatory protein 2a (MIP 2a; GROb)

207850_at

2.1

CXCL3

Macrophage inflammatory protein 2b (MIP 2b; GROg)

214974_x_at

1.9

CXCL5

Epithelial-derived neutrophil-activating protein-78 (ENA78)

206336_at

1.9

CXCL6

Granulocyte chemotactic protein 2 (GCP2)

202638_s_at

1.6

ICAM1

Intercellular adhesion molecule 1 (CD54)

207526_s_at

1.2

IL1RL1

Interleukin 1 receptor-like 1

212657_s_at

1.1

IL1RN

Interleukin 1 receptor antagonist

204475_at

1.4

MMP1

Matrix metalloproteinase 1 (interstitial collagenase)

204580_at

1.7

MMP12

Matrix metalloproteinase 12 (macrophage elastase)

Proteases

CWF, chronic wound fibroblast; NF, normal fibroblast.

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IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

Table 4. Genes whose induction was more than twofold higher in CWF than in NF Affymetrix Probe ID

Log2 fold change

Symbol

Name

Growth factors/cytokines 206924_at

1.5

IL11

Interleukin 11

203373_at, 203372_s_at

1.1

SOCS2

Suppressor of cytokine signaling 2

205239_at

1.3

AREG

Amphiregulin (Schwannoma-derived growth factor)

205767_at

1.4

EREG

Epiregulin

2.0

HAS1

Hyaluronan synthase 1

ECM components 207316_at

10

CWF1 NF1

8

*

**

CXCL1 concentration (pg ml–1)

Superoxide radical (nmol per 2 hours per 105 cells)

CWF, chronic wound fibroblast; ECM, extracellular matrix; NF, normal fibroblast.

*

6 4 2 0 1

2

3

5

7

10

4,000 3,500

CWF NF

3,500

**

2,000

**

1,500 1,000

**

500 0 6 hours

24 hours

6 hours

Patient 1

CWF2 NF2

15

*

*

*

**

10 5 0 1

2

3

5

7

10

CXCL1 concentration (pg ml–1)

Superoxide radical (nmol per 2 hours per 105 cells)

**

3,000

20

Superoxide radical (nmol per 2 hours per 105 cells)

**

4,000 3,500

**

6 4 2

24 hours

Patient 3

**

**

3,000 3,500 2,000 1,500 1,000 500

*

*

*

0 24 hours

Patient 1

CWF3 NF3

8

6 hours

Patient 2

CWF NF

6 hours

10

24 hours

6 hours

24 hours

Patient 2

6 hours

24 hours

Patient 3

Figure 7. Protein levels of the chemokines CXCL-1 and CXCL-5 assessed using ELISA. Conditioned medium from CWF contained significantly reduced protein levels of CXCL-1 (a) and CXCL-5 (b) compared with NF-conditioned medium, in a patient-specific manner (values ¼ mean±SD (n ¼ 2); *Po0.05; **Po0.01).

0 1

2

3 5 Time (days)

7

10

Figure 6. Superoxide radical generation by CWF and NF measured using the cytochrome c reduction assay. In all three patients, characterized, early passage NF produced decreased levels of superoxide radical over time. However, CWF produced elevated levels, culminating in significantly greater superoxide formation than NF at late time points, correlating with an impaired ability to counteract oxidative stress (values ¼ mean±SD (n ¼ 3); *Po0.05; **Po0.01).

ing ATPase type 2C—member 1 (Ramos-Castaneda et al., 2005), and F-box and leucine-rich repeat protein (Ho et al., 2006). As the present data implies that there is a lack of major enzymic antioxidant induction in CWF, this suggests that the innate mechanisms of protecting against oxidative stress are defective. Previous studies have reported either a delayed antioxidant induction in fibroblasts until premature senes2534 Journal of Investigative Dermatology (2008), Volume 128

cence is fully reached (Chen et al., 2004) or the severely impaired induction of antioxidant defense genes in senescent fibroblast populations (Kim et al., 2003), following oxidative stress exposure. Consequently, these findings would explain the increased susceptibility of CWF to oxidative stress and telomere-independent senescence within the chronic wound environment. Importantly in this study, altered enzymic antioxidant expression/activity due to the intrinsic aging on the dermal tissues has been controlled for by directly comparing patient-matched CWF and NF. Interestingly, the upregulation of genes in CWF, such as calcium-transporting ATPase type 2C—member 1 and F-box and leucine-rich repeat protein that are associated with the recognition, degradation, and removal of malformed proteins via the ER-associated protein degradation system following ER stress (Ramos-Castaneda et al., 2005; Ho et al., 2006; Yoshida,

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

Table 5. Neutrophil chemotaxis in response to conditioned medium from CWF and NF was performed to determine whether patient-specific reductions in CXC chemokine production had any functional significance 6 hours

24 hours

CWF

NF

CWF

NF

1



++

+

++

2

+



++



3

+

++

++

++

CWF, chronic wound fibroblast; NF, normal fibroblast. Conditioned medium from all three CWF patients demonstrated a delayed ability to induce neutrophil chemotaxis.

2007), is of further significance, given that cellular senescence is also associated with the increased accumulation of oxidized cellular proteins, due to dysfunctions in the proteosome component of the ER-associated protein degradation system, under oxidative stress (Chondrogianni and Gonos, 2004; Torres et al., 2006). A second category of genes with increased expression in CWF vs NF included cytoskeletal components and regulators (MAPRE1, MFAP5, ARPC5, ACTR3, KIAA0992, PTPRD, CAP1, PDLIM5, CDH2) that ultimately controls cell adhesion and migration (Knudsen et al., 1995; Welch et al., 1997; Freeman and Field, 2000; Parast and Otey, 2000; Boukhelifa et al., 2004; Kadrmas and Beckerle, 2004). Their increased expression in CWF correlates with an aging phenotype, as cellular attachment is increased in senescent fibroblasts (Stephens et al., 2003). These findings also help explain why wound repopulation by CWF was reduced compared with that of NF. As wound repopulation in monolayer culture relies on a combination of migration and proliferation, the fact that CWF have reduced proliferative capacity (as reported here and by others) (Stanley et al., 1997; Mendez et al., 1998; Vande Berg et al., 1998) impacts on the rate of repopulation. However, the main mechanism by which the empty denuded wound space becomes repopulated is by migration of cells at the wound edge, and the data presented here correlate with the decreased migration rates of ulcer fibroblasts reported here and by others (Raffetto et al., 2001). Genes that were differentially regulated in response to serum induction (akin to a cellular wound healing response; Iyer et al., 1999) were also characterized. Genes with greater than twofold lower induction in CWF vs NF included five different CXCL chemokines (CXCL-1, -2, -3, -5, and 6), which form a co-regulated gene cluster at 4q13.3 that also includes IL-8. All five of these chemokines are closely related and are functionally similar: they all bind to CXCR2 (IL-8 receptor) at the neutrophil surface, initiating an acute inflammatory response upon injury (Baggiolini, 2001). CXCL-12 was also constitutively lower in CWF, independent of serum. Hence, this family of genes was potentially very interesting in relation to impaired wound healing. We therefore followed them up in more detail by assessing levels of the secreted products of

three of the CXCL genes using ELISA. These were selected on the basis of their previously reported function and potential importance in wound healing. CXCL-12 protein was not detected in any of the culture supernatants, but reduced levels of CXCL-1 and CXCL-5 translated to consistent reductions in protein products in CWF vs NF. This was also functionally significant in that it correlated to decreased neutrophil chemotaxis in a patient-specific manner that reflected the individual patient’s chemokine levels. This confirms that CXCL-1 and CXCL-5 production by fibroblasts is a prime inducer of neutrophil chemotaxis (Lin et al., 2007). The importance of CXC chemokines in mediating chronic inflammatory responses has been well characterized using rheumatoid arthritis as a model (Bradfield et al., 2003; Buckley, 2003; Burman et al., 2005), but unlike the impaired chemokine expression we have reported, synovial fibroblasts from rheumatoid arthritis patients overexpress these chemokines. Despite the clear difference between rheumatoid arthritis and non-healing chronic wounds, the overriding conclusion is that CXC chemokines play a crucial role in inflammatory resolution. Our observations in CWF might reflect previous, chronic overexpression of CXC chemokines in the wound site rendering the cells exhausted upon culture in vitro. Alternatively, CWF may not produce the correct signals to create an acute inflammatory response essential for healing to ensue, hence locking the wound in a perpetual state of non-resolving chronic inflammation. Both keratinocytes and endothelial cells from chronic wounds have an impaired ability to upregulate pro-inflammatory cytokines and chemokines (Galkowska et al., 2006), and researchers have postulated that the impaired function of keratinocytederived chemokines may result in delayed healing (Simka, 2007). However, our findings that chronic skin wound fibroblasts have such defective chemokine expression profiles are, to our knowledge, previously unreported. CD40 expression was also lower in CWF (Table 2) and ligation of fibroblast CD40 induces pro-inflammatory cytokines such as IL-6 and IL-8 (Sempowski et al., 1997a, b, 1998). For example, fibroblasts from the endometrium, myometrium, and cervix not only express CD40, but also upregulate synthesis of the chemoattractant MCP-1 (CCL2) in addition to IL-6 and IL-8 after CD40 ligation (King et al., 2001). This further supports the idea that CWF fail to promote and drive an acute-phase inflammatory response. Impaired induction of ICAM1 may, in combination with impaired chemokine expression, have a significant impact on inflammatory resolution. Researchers have described evidence that a stromal address code exists, consisting of three components: a chemokine, a cytokine, and a cell adhesion molecule, that collectively act as a molecular beacon for infiltrating leukocytes (Parsonage et al., 2005). A vascular address code, expressed at the luminal surface of the endothelium adjacent to the injury that recruits circulating leukocytes, has already been described (Campbell et al., 2003), and leukocyte extravasation across the vascular endothelium is dependent on them expressing a complimentary phenotype to this code. Currently, little is known about the regulatory mechanisms that occur beyond the vascular www.jidonline.org 2535

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

endothelium in the stromal compartment itself, but the emerging idea of a stromal address code that is required for guiding infiltrating cells to the site of injury is developing (Parsonage et al., 2005). In chronic wounds, healing is compromised by a protracted, non-resolving inflammatory phase, the presence of contaminating bacterial populations and high levels of oxidative stress. In this study, it was confirmed that CWF may negatively influence the healing process as they were biologically ‘older’ than patient-matched NF. Their mode of aging was not telomere-dependent, but was rather more likely a consequence of the high oxidative stress endured in the wound site. Microarray data and impaired ability to regulate superoxide radical formation indicated this. CWF also aberrantly expressed several extracellular matrix/ protease/cytoskeletal genes and were unable to repopulate experimental wounds as quickly and competently as NF, indicating that the impaired function of CWF might impact on reparative tissue formation in vivo. In addition, this study shows that CWF have an impaired ability to produce the correct stromal address code required to recruit the inflammatory cells necessary to drive an acute-phase inflammatory response that could potentially overcome the non-resolving inflammation of chronic wounds. Further research into, and manipulation of this stromal address code, along with novel antioxidant therapies could provide the key to the successful treatment of chronic wounds and other chronic degenerative disorders in the future.

Table 6. Patient details for the cell populations utilized in this study Patient ID

Gender

Age (years)

Wound duration

F

71

47 months

2

F

75

45 months

3

M

78

448 years

4

F

68

418 months

1

F, female; M, male.

100 mg ml1 streptomycin sulfate; 0.25 mg ml1 amphotericin B), and 10% (v/v) fetal calf serum (all reagents from Invitrogen, Paisley, UK). The cultures were maintained at 37 1C in a 5% CO2 humidified atmosphere (standard culture conditions). At confluency, fibroblasts were trypsinized with 0.05 % (w/v) trypsin/0.53 mM EDTA (SigmaAldrich) and reseeded at a ratio of 1:3. The PDs of the cell populations in vitro were derived by direct counting of cell numbers at passages using the method previously described (Cristofalo et al., 1998). Fibroblast populations were cultured until senescence, as defined by the lack of growth and by the assessment of morphology and detection of SA b-Gal activity (Sigma-Aldrich). Cells were utilized for all functional assays at low (P5–7) passage (equivalent to PD10–15). As CWF from patient 3 senesced so early in culture (and hence provided only very low cell numbers), functional studies (in vitro wounding, superoxide radical generation, CXCL ELISA, and neutrophil chemotaxis assays) were conducted using only three of the original four patient samples.

MATERIALS AND METHODS Patients and tissues

In vitro wounding studies

Cultures of CWFs and patient-matched uninvolved dermal fibroblasts (NF) were obtained with approval from the Medical Ethical Committee of Cardiff University and the Local Research Ethical Committee and after written informed consent from patients (n ¼ 4) with non-healing, chronic venous leg ulcers attending the Wound Healing Clinic at the University Hospital of Wales, Cardiff. Only patients with wounds that failed to respond to conventional treatment regimes after 2 months were used in the study, and patients with diabetes, systemic immunosuppression, or clinical signs of local infection were excluded (for details of patient age and wound duration see Table 6). A 6-mm biopsy was taken from the chronic wound bed and the uninvolved outer aspect of the ipsilateral thigh. All experiments were carried out according to the Declaration of Helsinki Principles.

A standard monolayer scratch wound model was used to characterize wound repopulation. Cells were seeded into 24-well tissue culture plates (2  104 cells per well), cultured to confluence, and monolayers were wounded by scratching along the surface of the tissue culture plastic with a standard 200 ml pipette tip. Cellular monolayers were gently washed with phosphate-buffered saline, re-fed with fibroblast-serum-containing medium, and incubated under standard culture conditions on the motorized, heated, and gassed stage of a Zeiss Axiovert 200M microscope (Carl Zeiss, Welwyn Garden City, UK). Images were collected every 15 minutes for 40 hours using the Openlab software package, which was also used for post-image acquisition analysis (Improvision, Coventry, UK).

Establishment of chronic wound and patient-matched NFs Cultures were established by a single-cell suspension technique following enzymatic degradation of the specimens as previously described (Stephens et al., 1996). Briefly, tissue was incubated overnight with dispase (2 mg ml1; Sigma-Aldrich, Poole, UK) to separate epidermal tissue from the dermal tissue. Wound-bed tissue, although lacking epidermis, was treated identically. Dermal tissue specimens were then disaggregated overnight utilizing bacterial Clostridium histolyticum A collagenase (1 mg ml1; Sigma-Aldrich). Fibroblast cultures were maintained in fibroblast-serum-containing medium containing DMEM supplemented with L-glutamine (2 mM), non-essential amino acids (1  ), antibiotics (100 U ml1 penicillin G; 2536 Journal of Investigative Dermatology (2008), Volume 128

SA b-Gal activity Assessment of SA b-Gal activity was performed as described previously (Dimri et al., 1995). Briefly, fibroblasts (5  103 cells per well of an eight-well chamber slide) were cultured as monolayers for 48 hours. Cells were fixed in 4% (v/v) formaldehyde for 15 minutes and then incubated for 4 hours at 37 1C in SA b-Galstaining solution (1 mg ml1 5-bromo-4-chloro-3-indolyl b-D-galactosidase (X-Gal), 40 mM citric acid (pH 6.0), 40 mM sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, 2 mM magnesium chloride; all from SigmaAldrich). Senescent fibroblasts were identified as blue-stained cells by standard light microscopy on an Olympus OM-10 inverted microscope and photographed using a Coolpix 995 digital camera (Nikon, Kingston upon Thames, UK).

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

STELA Telomere length in early-passage and late-passage cell populations was analyzed using the DNA extraction and PCR methods previously described (Baird et al., 2003). Briefly, cells were trypsinized, washed with phosphate-buffered saline, and the genomic DNA extracted by standard proteinase K, RNase A, and phenol/chloroform protocols (Sambrook et al., 1989). DNA was solubilized by digestion with EcoRI (New England Biolabs, Hitchin, UK), quantified by Hoechst 33258 fluorometry (Bio-Rad, Hemel Hempstead, UK) and diluted to 10 ng ml1 in 10 mM Tris–HCl (pH 7.5). A ligation reaction was then carried out at 35 1C for 12 hours in a volume of 10 ml containing 10 ng of genomic DNA, 0.9 mM telorette linker ((50 -TGCTCCGTGC ATCTGGCATCTAACCCT-30 ); MWG Biotech, Ebersberg, Germany), and 0.5 U of T4 DNA ligase (GE Healthcare, Little Chalfont, UK) in 1  manufacturer’s ligation buffer. Ligated DNA was diluted to 250 pg ml1 in water and multiple PCRs were carried out for each test DNA in volumes of 10 ml containing 100–250 pg of ligated DNA, 0.5 mM telomere-adjacent (XpYpE2 (50 -TTGTCTCAGGGTCCTAGTG-30 )) and teltail (50 -TGCTCCGTGCATCTGGCATC-30 ) primers (MWG Biotech), 75 mM Tris–HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20 (all Sigma-Aldrich), 1.2 mM nucleotide triphosphate (NTPs), 1.5 mM MgCl2 (both Promega, Southampton, UK), and 1 U of a 25:1 mixture of Taq (ABgene, Epsom, UK) and Pwo polymerase (Roche Molecular Biochemicals, Welwyn Garden City, UK). The reaction mixture was subject to the following conditions on a MJ PTC-225 thermocycler (Fisher Scientific, Loughborough, UK): 25 cycles of 94 1C for 15 seconds, 65 1C for 30 seconds, and 68 1C for 10 minutes. The DNA fragments were resolved by electrophoresis in a 0.5% Tris-acetate-EDTA agarose gel and detected by Southern hybridization with a randomprimed a-32P-labeled (GE Healthcare) telomere-adjacent probe generated by PCR using primers XpYpE2 and XpYpB2 (50 -TCTGAAAGTGG ACC(A/T)ATCAG-30 ) on EcoR1-digested genomic DNA and a probe to detect the 1-kb molecular weight marker (Stratagene, Amsterdam, The Netherlands). Hybridized fragments were detected on a Molecular Dynamics Typhoon 9200 phosphorImager (GE Healthcare). The molecular weights of the DNA bands were calculated using the ImageQuant TL (GE Healthcare). Statistical comparisons were made as stated below.

Affymetrix microarray analysis RNA was extracted from serum-induced cells from all four patients following the protocol described by Iyer et al. (1999). Briefly, cells were seeded at a density of 640 cells cm2 in 20 cm diameter dishes and cultured under standard conditions for 24 hours. Cells were then serumstarved for 48 hours to induce quiescence. Cells were then re-stimulated with serum and harvested at 0 and 6 hours for RNA extraction. Cells were washed with phosphate-buffered saline, incubated with TRIzol (Invitrogen) at room temperature for 10 minutes, and RNA was extracted according to standard phenol/chloroform extraction protocols (Sambrook et al., 1989). RNA concentration was quantified on an automated RNA quantitation machine (Bio-Rad) and RNA quality determined using the RNA Nano LabChip kit (Agilent Technologies, Wokingham, UK). Samples were then processed by the Central Biotechnology Service, Cardiff University, Cardiff, USA, following the detailed protocol for sample preparation and microarray processing available from Affymetrix (http://www.affymetrix.com). Briefly, firststrand cDNA was synthesized from 5 mg total RNA using a T7-(dT)24 primer (Genset Corp., Nottingham, UK) and reverse-transcribed with

the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen). After second-strand synthesis, the resulting cDNA was subjected to an in vitro transcription reaction using a Bioarray kit (Enzo Diagnostics, New York, NY) to generate biotinylated cRNA. This was subsequently fragmented and hybridized to the Affymetrix U133A GeneChip, which contains B22,000 probe sets to detect the transcripts of B14,000 distinct human genes plus additional expressed sequences tags. Following hybridization and scanning, the resultant image files (.CEL) were analyzed using the MAS 5.0 statistical algorithm (Affymetrix) to calculate an expression summary value for each probe set on each GeneChip. The resulting expression data were scaled to a target intensity of 100. Differentially expressed genes were identified by applying analysis of variance to the expression values for each probe set, using the patient identifier, cell type (CWF vs NF) and serum treatment (±) as the three sets of factors. The resulting P-values were corrected for multiple hypothesis testing using the false discovery rate method (Benjamini and Hochberg, 1995). The expression values were further visualized and explored by hierarchical clustering (cosine similarity applied to log-transformed, median-centered data) and heat map visualization. Difference of the differences testing was undertaken using expression summaries generated using the RMA algorithm (Irizarry et al., 2003). For each patient NF or CWF sample, the (log2) fold change between each pair of samples with and without serum was first calculated and then mean values for the NF and CWF groups were calculated. Probe sets were selected where the mean induction (or repression) by serum was twofold greater in the CWF group when compared with the NF group. All analyses were performed in R (R DCT, 2006) and the scripts for all analyses are available from the authors on request. The expression values and .CEL files will be available at Gene Expression Omnibus (Edgar et al., 2002).

Superoxide radical generation Superoxide radical generation was measured as described by Turner et al. (1998). Cells were seeded into 24-well plates at a density of 5  104 cells per well (three cell-containing wells and one cell-free well per day) in 1 ml of fibroblast-serum-containing medium. At various time points over 10 days, cells were washed with phosphatebuffered saline (3  ) and re-incubated with serum-free, phenol redfree DMEM containing 80 mM cytochrome c (horse heart type III; Sigma-Aldrich) under standard culture conditions for 2 hours. Cytochrome c-containing DMEM was then removed from each well and cytochrome c reduction (a direct function of superoxide radical generation), measured at 550 nm on a Beckman DU 800 UV/Visible Spectrophotometer (Beckman Coulter, High Wycombe, UK), against a blank consisting of cytochrome c-DMEM, derived from the cellfree well. Remaining viable cells were trypsinized, counted and, using a molar extinction coefficient of 21,000 mol cm1 l1, data were expressed as nanomoles of superoxide radical generation per 2 hours per 105 cells.

ELISA quantitation of chemokine protein ELISA kits (R&D Systems, Abingdon, UK) were used to detect protein levels of the chemokines CXCL-1, -5, and -12. Serial dilutions of standards were made and 200 ml of the standard, sample, or control was added per well to a 96-well plate provided with the kit. Wells were incubated for 90 minutes at room temperature. Each well was www.jidonline.org 2537

IB Wall et al. Fibroblast Dysfunction in Non-Healing Wounds

washed with 400 ml wash buffer for 1 minute (3  ). Then, 200 ml of the appropriate CXCL conjugate was added to each well. Wells were then incubated for 60 minutes at 4 1C. After washing with wash buffer (3  ), 200 ml of the manufacturer’s substrate solution was added to each well and incubated at room temperature for 15 minutes in a dark environment. A 50 ml volume of manufacturer’s stop solution was added to each well, and the optical density of each well was determined spectrophotometrically at 450 nm. The concentration of CXCL proteins in each sample was determined from the standard curves.

Neutrophil chemotaxis Chemotaxis was monitored by the migration of freshly isolated human neutrophils separated as previously described (Laffafian and Hallett, 1995), through Corning Costar transwell filter inserts (12 mm diameter, 3.0 mm pore size; Sigma-Aldrich). The transwell filter separated an upper chamber, containing 106 neutrophils in 0.1 ml, from the lower chamber, formed by the wells in 12-well plates. The lower chamber contained the negative control, cell suspension buffer (DMEM); a positive control, f-met-leu-phe (1 mM; SigmaAldrich) in DMEM; or fibroblast-conditioned medium (600 ml). After incubation (30 minutes, 37 1C, 5% CO2), cells loosely attached to the underside of the filter were dislodged by tapping, and the cells in the lower chamber concentrated by centrifugation (1,000  g for 1 minutes) before resuspension and counting. The relative number of neutrophils in the lower chamber was scored semiquantitatively.

Statistical analyses Statistical analyses of PDL and SA b-Gal data were conducted using Mann–Whitney U-test for non-parametric data and STELA data was analyzed using an independent sample t-test. All other data was analyzed using Student’s unpaired t-test. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We acknowledge the help and assistance of the following people: Professor Keith Harding and clinical staff of the Wound Healing Research Unit, School of Medicine, Cardiff University, for obtaining tissue samples; Daniel Kirwilliam and Megan Musson, Department of Pathology, School of Medicine, Cardiff University, provided the microarray analysis service/ analysis; Jan Rowson, Department of Pathology, School of Medicine, Cardiff University, provided advice about STELA; Professor Terje Espevik, Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway, for quantifying chemokine protein levels; and Dr David Moles, UCL Eastman Dental Institute, advised on statistical analyses.

SUPPLEMENTARY MATERIAL Table S1. Names and Affymetrix probe IDs of genes differential regulated between disease and patient-matched normal fibroblasts.

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