Journal of PathologyJ Pathol 2004; 203: 721–728Published online 14 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1565

Original Paper

Vascular endothelial growth factor (VEGF)-A andplatelet-derived growth factor (PDGF) play a centralrole in the pathogenesis of digital clubbingStephen Atkinson and Stephen B Fox*Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK

*Correspondence to:Dr Stephen B Fox, NuffieldDepartment of ClinicalLaboratory Sciences, University ofOxford, John Radcliffe Hospital,Headington, Oxford, OX39DU, UK.E-mail:stephen.fox@ndcls.ox.ac.uk

Received: 5 December 2003Revised: 16 February 2004Accepted: 23 February 2004

AbstractDigital clubbing is associated with many unrelated serious diseases but its pathogenesisremains a clinical enigma. It has been hypothesized that platelet clusters impacting in thedistal vasculature mediate the morphological changes of clubbing. Since the multifunctionalcytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor(PDGF) are released on platelet aggregation and are hypoxically regulated, the present studyhas examined their role in clubbing using immunohistochemistry. Basic fibroblast growthfactor (bFGF), transforming growth factor-beta 1 (TGF-β1), microvessel density, carbonicanhydrase IX (CAIX), hypoxia inducible factor (HIF)-1α, and HIF-2α were also measured.There was a significant increase in VEGF (p = 0.01), pKDR (p = 0.03), PDGF (p = 0.017),and HIF-1α and HIF-2α (p = 0.004 and p = 0.004, respectively) expression together witha significant increase in microvessel density (p = 0.03) in the stroma in clubbed digitscompared with controls. There was no difference in CAIX (p = 0.25), TGF-β1 (p = 0.66) orbFGF (p = 0.18) between affected and control groups. These findings suggest that VEGF andPDGF are released after platelet impaction and that their expression is hypoxically enhancedin the stroma after capillary occlusion. VEGF may synergize with PDGF in inducing thestromal and vascular changes present in digital clubbing.Copyright 2004 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: clubbing; VEGF; PDGF; angiogenesis

Introduction

Since its original description by Hippocrates in thefifth century BC, the strong association of digital club-bing with serious disease has been a clinical enigma.Although various hypotheses have been proposed tounify this clinical sign that is present in numerousinfectious, inflammatory, and neoplastic diseases, it isthat proposed by Dickinson and Martin in 1987 thatholds most promise [1]. These authors proposed thatin disorders that result in megakaryocyte or plateletclumps bypassing the lung capillary network (eg rightto left cardiac shunts or in bronchial carcinomas) orin diseases in which platelet aggregates arise on theleft side of the heart (eg sub-acute bacterial endo-carditis), these clusters may lodge in the peripheralvasculature of the digits. The impacted platelet parti-cles would then release platelet-derived growth factor(PDGF) and lead to the increased vascularity, per-meability, and connective tissue changes that are thehallmark of clubbing. In a follow-up study in 1991,we reported the presence of numerous platelet clustersin the vessels of clubbed but not in control samples,providing strong pathological supporting evidence [2].

However, it is only recently, with the improvedunderstanding of angiogenesis and hypoxia together

with the development of reagents able to examinethese regulatory pathways, that we have been ableto revisit the pathogenesis of clubbing. Clubbed dig-its demonstrate increased blood flow and vascular-ity in addition to connective tissue changes [3–9].Since platelets contain and release vascular endothe-lial growth factor (VEGF) on aggregation [10,11] andstimulate VEGF expression in vascular smooth mus-cle [12], we hypothesized that VEGF derived fromthese sources together with hypoxic stimulation result-ing from the regional blood flow changes in affectedvessels may play a role in the development of club-bing. PDGF, basic fibroblast growth factor (bFGF),and transforming growth factor-beta 1 (TGF-β1) havealso been reported to be hypoxically regulated [13–15]and to synergize with VEGF [16–19] and we thereforewished to assess whether these cytokines also play arole in clubbing.

Due to the absence of a suitable model for clubbing,we have used an immunohistochemical approach tomeasure the quantity and pattern of expression ofVEGF, the phosphorylated form of the major VEGFreceptor (pKDR), together with PDGF, bFGF, TGF-β1, and microvessel density in a series of clubbed andcontrol samples. To assess the role of hypoxia, we

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722 S Atkinson et al

have also measured the expression of the transcriptionfactors hypoxia inducible factor (HIF)-1α and HIF-2α

that are induced at low oxygen tensions in addition tothe HIF-induced gene, carbonic anhydrase IX (CAIX).

Materials and methods

Patient samples and immunohistochemistry

Approval for the study was gained from the localethics committee and written informed consent fromrelatives was obtained for the use of tissues. Samplesfrom the digits of five patients (four bronchogenic car-cinoma and one fibrosing alveolitis) with unequivocaldigital clubbing (assessed by nail fold angles, alteredphalangeal depth ratios, and the Schamroth sign), allof which contained platelet clusters, together withspecimens from six control patients without clubbing,and in which no platelet clusters were present, werecollected at necropsy at the John Radcliffe Hospital,Oxford, UK. Tissue was fixed in 10% buffered forma-lin and embedded in paraffin wax. Four-micrometresections were cut on silane-coated slides and stainedwith primary antibodies (Table 1) followed by theEnVision system (Dako, UK), diaminobenzidine chro-mogen, and a haematoxylin counterstain. For the anti-hFGF-basic and anti-PDGF antibodies, a rabbit anti-goat Ig bridge (Dako, UK) was applied for 30 minat a dilution of 1 : 100 before development with theEnVision system. For each antibody, all samples werestained at the same time under the same conditionswith known positive controls. Omission of the pri-mary antibody was used as a negative control. Anti-gen retrieval comprising overnight incubation in 1 mM

EDTA (pH 8.0) in a water bath at 60 ◦C was requiredfor HIF-1α and HIF-2α. The morphological pattern ofexpression for each marker was evaluated and quanti-fied by the authors independently as outlined below.

Semi-quantitative assessment of VEGF, pKDR, andCAIX

Semi-quantitative grading was used for VEGF andpKDR using a combination of the proportion of stro-mal fibroblasts stained and the intensity of stain-ing: no staining = 0; weak/focal staining = 1; strongfocal/widespread moderate staining = 2; or strong/

Table 1. Antibodies used for immunohistochemistry

Antibody Antigen Reference

VG1 VEGF-A 6034a pKDR (VEGF-R2) 23122 HIF-1α 61190b HIF-2α 61M75 CAIX (62)Q/Bend10 CD34 63Anti-TGF-β1 TGF-β1 64Anti hFGF-basic bFGF R&D Systems, UKAnti-PDGF PDGF 65

widespread staining = 3 [20]. For CAIX, a similarassessment was performed [21] with the same scor-ing system but the immunopositive cells were locatedin the overlying epidermis.

Semi-quantitative assessment of PDGF, bFGF, andTGF-β1

Semi-quantitative grading was used for grading thestaining of pericyte and stromal fibroblasts thatis observed with these cytokines: no staining = 0;weak staining = 1; moderate staining = 2; or strongstaining = 3. Cases were considered positive for sta-tistical analysis when showing moderate or strongstaining.

Quantitative assessment of HIF-1α, HIF-2α, andmicrovessel density

Counting the proportion of stromal fibroblasts thatexpressed these hypoxic markers generated a hypoxicindex for HIF-1α and HIF-2α. Three hundred and fiftycells were counted for each sample. For microves-sel density, the three areas of maximal microves-sel number were identified at low power (×40–100)before individual microvessels were counted using ahigh-power objective (×250 — area of field of view0.17 mm2) as previously reported [22]. The averageof the three fields was converted into the number ofblood vessels per mm2 and used in the analysis.

Statistics

The chi-square test was used to test for independenceof categorical variables including categorized contin-uous variables. Tests of hypotheses on the locationparameter (median) were performed by using rankstatistics (Mann–Whitney) and contingency tables(Fisher’s exact) produced with the Prism 3.0 (Graph-Pad, San Diego, USA) software package.

Results

The vessels in clubbed samples were frequentlydilated and contained platelet clusters as previouslyreported (Figure 1A) [2]. CD34 staining of endothe-lium demonstrated frequent linear glomeruloid-typevascular formations (Figure 1B) within the media of,and surrounding, small to medium-sized arteriolesin clubbed samples that were not identified in con-trol samples. These arterioles were more numerousand often enlarged and tortuous in clubbed samples(Figure 1C). In two clubbed samples, numerous par-allel and linear vascular channels perpendicular tothe overlying epidermis directed towards the under-lying nail pulp were observed (Figure 1D). CD34 alsohighlighted vascular hot spots (Figure 2A) in clubbedpatients that was reflected by a significantly greatermicrovessel density in these samples than in controls(p = 0.03) (Figure 3A).

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Pathogenesis of clubbing 723

A B

C D

Figure 1. Immunohistochemistry of clubbed digits. (A) Platelet cluster in a dilated capillary showing strong immunostaining forthe platelet marker Y2/51a (CD61) (see ref 2). In clubbed samples, the vascular marker CD34 highlighted vessel glomeruloidformations (B), numerous tortuous arterioles (C), and occasional linear capillary down growths (D)

VEGF was expressed in stromal fibroblasts in allspecimens and frequently in pericytes covering smallvessels. Staining in macrophages around vascularchannels containing platelet clusters was also observedin clubbed samples but not in controls (Figure 2B).Weak VEGF immunopositivity was also observedin occasional entrapped platelet clusters (Figure 2B).There was a significant increase in the grade ofVEGF positivity (p = 0.01) (Figure 3B) in stromalfibroblasts in clubbed compared with control samples.

pKDR was prominently expressed not only inendothelial cells, but also in pericytes and stromalfibroblasts in clubbed samples, with similar tissue ele-ments staining less frequently in non-clubbed samples(Figure 2C). Expression was both nuclear and cyto-plasmic as previously reported [23]. There was a sig-nificant increase in pKDR expression in clubbed com-pared with control specimens (p = 0.03) (Figure 3C).

Expression of HIF-1α and HIF-2α showed weaknuclear immunopositivity in both clubbed samplesand controls in most tissue elements. Their expres-sion was particularly prominent in endothelial cells,pericytes, fibroblasts, and inflammatory cells surround-ing platelet clusters in clubbed compared with controltissues (Figures 2D and 2E). Analysis demonstrateda significant increase in both HIF-1α and HIF-2αin clubbed patients compared with control samples

(p = 0.004 and p = 0.004, respectively) (Figures 3Dand 3E).

Staining for CAIX was identified only in the sur-face epidermis of the nail bed and was not presentin other tissue components. Staining was membranousand observed focally in the keratinocyte cell layers incontrols (Figure 2F) but was more extensive, althoughdiscontinuous, in clubbed samples (Figure 2F). Com-parison of the immunohistochemical score showed nosignificant difference between the clubbed and controlgroups (p = 0.25) (Figure 3F).

PDGF and bFGF showed similar patterns of expres-sion in both clubbed and control samples. Expressionwas identified in pericytes of arterioles and small capil-laries (Figures 2G and 2H, respectively) together withstromal fibroblasts and tissue macrophages (Figures2G and 2H, respectively). A significant increase inPDGF (p = 0.017) but not bFGF (p = 0.18) expres-sion was observed in clubbed compared with controlsamples (Figures 3G and 3H).

Strong TGF-β1 expression was observed in mono-cytes entrapped within the platelet clusters present inthe clubbed samples, with weaker positivity in the per-icytes surrounding affected vessels (Figure 2I). Occa-sional TGF-β1-positive stromal macrophages werealso observed in both clubbed and control samples

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Figure 2. (A) CD34-positive vessel hot spots were observed in clubbed tissues. (B) Expression of VEGF was up-regulated infibroblasts and was also identified in macrophages (lower left inset) and in platelet clusters (upper right inset) in clubbed samplescompared with control (lower right inset). (C) pKDR expression was prominently expressed in fibroblasts (black arrow) and inendothelial cells (white arrow) in clubbed tissues (inset shows control with negligible expression). (D) HIF-1α and (E) HIF-2αexpression was observed in fibroblasts (black arrows) together with strong expression of both HIFs in endothelium (white arrows)and pericytes (vertical arrows). (F) There was occasional strong membrane expression of CAIX in the sub-ungual epidermis butnot in other tissue elements in clubbed samples (inset control). (G) PDGF and (H) bFGF was expressed in pericytes of vessels(broad arrows) and in stromal fibroblasts (narrow arrows). TGF-β1 was expressed strongly in entrapped monocytes in plateletclusters (broad arrow) and weakly in pericytes (narrow arrow) in clubbed cases

but TGF-β1 was not identified in stromal fibrob-lasts. No significant difference in TGF-β expressionbetween clubbed and controls was observed (p =0.66) (Figure 3I).

Discussion

Classical pathological studies have shown that club-bing manifests not only connective tissue, but also vas-cular changes [3]. Although several cytokines includ-ing growth hormone [24,25], tumour necrosis factor-α,hepatocyte growth factor [26], and TGF-β [27] havebeen proposed to be involved in clubbing, Dickinsonand Martin proposed that PDGF was the most likely

candidate [1]. However, the increasing recognition thatVEGF is strongly expressed in platelets [10,11,28]and has many non-endothelial effects [29,30], includ-ing influencing collagen synthesis [31], suggested thatthis angiogenic factor may at least partly mediate thedevelopment of clubbing. This hypothesis was sup-ported by a recent study in lung cancer patients show-ing up-regulation of VEGF in serum and plasma inpatients with lung cancer and clubbing compared withcancer patients without clubbing [32].

VEGF and PDGF are released from platelets onaggregation [33] and VEGF is also increased inthrombus turnover [34]. Our thesis that their releaseon platelet impaction together with their hypoxicinduction in the surrounding tissue elements is due

Figure 3. Graphs of microvessel density, VEGF, KDR, and other cytokines in clubbed and control samples. (A) Box and whiskergraph demonstrating microvessel density (mm2) in clubbed and unclubbed samples. (B) Column graph demonstrating VEGF gradein clubbed and unclubbed samples. (C) Column graph demonstrating pKDR grade in clubbed and unclubbed samples. (D) Boxand whisker graph demonstrating HIF-1α in clubbed and unclubbed samples. (E) Box and whisker graph demonstrating HIF-2αin clubbed and unclubbed samples. (F) Column graph demonstrating CAIX grade in clubbed and unclubbed samples. (G) Columngraph demonstrating PDGF grade in clubbed and unclubbed samples. (H) Column graph demonstrating bFGF grade in clubbed andunclubbed samples. (I) Column graph demonstrating TGF-β1 grade in clubbed and unclubbed samples

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to simultaneous occlusion of capillaries is in keepingwith the significant increase in VEGF, PDGF, andHIFs observed in the stroma of clubbed patientsreported herein. In support of this sequence of eventsis the enhanced glycolysis (the glycolytic pathwaybeing another HIF target) in the fingertips of a patientwith clubbing using positron emission tomography[35]. The absence of a difference in the CAIX levelsbetween the groups is likely to be due to its restrictedexpression in the epidermis and from regulation byseveral transcription factors in addition to HIF [36,37].

Although occlusion of capillaries, with its expectedconcomitant reduction of blood flow, is contraryto the published data (for a review see Shneerson[38]), the small calibre of the vessels affected, theincreased vascularity, the glomeruloid structures, andthe tortuous arterioles that we observed in clubbeddigits would account for the reported increase in bloodflow.

Clubbing is not usually associated with an inflam-matory infiltrate [3]. However, we observed small col-lections of VEGF-expressing macrophages in clubbedsamples, akin to that reported in tumours, that mayhave been recruited through hypoxically inducedVEGF receptor, flt-1 [39–41].

Thus, VEGF from the various cellular sourceswould be able to bind to KDR, leading to endothe-lial phosphorylation, and account for the formation ofglomeruloid vascular structures (which have also beendescribed in animal models of VEGF-induced angio-genesis [42]), the tortuous arterioles, and the increasedmicrovessel density in clubbed patients. VEGF wouldalso increase vascular permeability, leading to oedemaand, through fibrin leakage [43], stromal changes.The connective tissue would then be altered furtherthrough fibroblast pKDR-mediated changes in colla-gen metabolism [31].

A central role for VEGF signalling may not initiallyappear to be in agreement with current thinking. How-ever, there are numerous reports of KDR expressionon cells other than endothelial [23], including smoothmuscle cells [44,45] and mesangial cells [31], in addi-tion to a variety of tumours and cell lines [46–52].Indeed, many of the stromal changes that have beendescribed in clubbing show similarities to those in neo-plasia.

Dickinson and Martin’s hypothesis suggested thatPDGF was a likely candidate in the pathogenesisof clubbing and our observations of its increase inclubbing support the suggestion that it may play arole. PDGF from platelet clusters, and its hypoxicenhancement in other tissue elements, would aidthe generation of newly formed sub-ungual vessels[53] and lead to further up-regulation of VEGF invascular smooth muscle cells [54]. Indeed, the PDGFexpression profile in fibroblasts in clubbing mirrorsthat reported in idiopathic pulmonary fibrosis [55],which shows similar stromal changes.

By virtue of its role in angiogenesis, stromaleffects [56], and regulation by hypoxia in some cell

types [53,57] (but not smooth muscle cells [16]),bFGF is another candidate factor that might medi-ate the changes reported in clubbing. However, weobserved no difference in expression between clubbedand control samples, which is in keeping with HIF-independent bFGF transcription [58]. Nevertheless,bFGF is able to synergize with VEGF and endothelialcells are more responsive in a hypoxic environmentthrough enzymatic alteration of bFGF receptor bind-ing sites [59]. Thus, we cannot exclude a role forbFGF as only small increases may have an ampli-fied effect in the context of the hypoxic environment.Similarly, although the paucity of TGF-β1 expressionin this study makes it unlikely that it is involved inclubbing, TGF-β1 from monocytes entrapped in theplatelet clusters may induce VEGF in pericytes [16].

The search for a common pathogenesis of digitalclubbing despite its many associated disparate dis-eases has been elusive. The findings reported in thisstudy extend our previous observations [2] and pro-vide insight into the potential regulatory mechanismsof clubbing. Although based on limited numbers, dueto the issues related to acquiring tissue from affectedsites, our findings suggest that VEGF from the con-tinued impaction of megakaryocytes and platelets inthe digital vasculature, potentiated by hypoxia, pro-vide a persistent positive autocrine and paracrine loopvia KDR to drive the cellular and stromal changes inclubbing. PDGF is also likely to contribute to the stro-mal changes, including facilitating the maturation ofnewly formed microvessels. Successful treatment ofthe primary pathology would remove the megakary-ocytes/platelet particles from the axial stream of thecirculation and lead to the reported regression ofthese changes. It will be of interest to note specifi-cally whether clubbing is reversed in trials of VEGFblockers, VEGF kinase inhibitors, and anti-platelettherapies.

Acknowledgements

This work was supported by the Medical Research Fund, theUniversity of Oxford, and the Pathological Society of GreatBritain and Ireland.

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