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Clinical and Experimental Ophthalmology 2008; 36: 717–720

doi: 10.1111/j.1442-9071.2008.01895.x

Original Article

Serum growth factor analysis in dry eye syndrome


Jay C Bradley MD,1,2 Rachael H Bradley PhD,1 David L McCartney MD1 and Mark J Mannis MD2
1
Texas Tech University Health Sciences Center, Department of Ophthalmology & Visual Sciences, Lubbock, Texas, and 2University of
California, Davis, Department of Ophthalmology & Visual Science, Sacramento, California, USA

ABSTRACT Key words: anterior segment, autologous serum, dry eye


syndrome, growth factor.
Background: To perform a comprehensive serum growth
factor analysis in dry eye syndrome patients and to compare
this with matched controls. INTRODUCTION
Methods: Six female dry eye syndrome patients and six age- Growth factors play a critical role in the maintenance of
and gender-matched controls were recruited. Whole blood normal corneal function and homeostasis. Imbalances in
was collected, allowed to clot and then centrifuged. Serum these growth factors in tears may be active in the pathogen-
was extracted by using sterile technique. Enzyme-linked esis of various ocular surface pathologies including dry eye
syndrome (DES).1 DES can be associated with autoimmune
immunosorbent assays were performed to quantify serum
diseases that result in chronic systemic inflammation that
growth factor levels. affects the lacrimal gland, resulting in decreased tear
Results: Levels of transforming growth factor-beta 1 and 2 production. Tear components have been studied for their
(TGF-b1 and b2), nerve growth factor (NGF), insulin-like role in the pathogenesis of DES,1 and studies have suggested
that levels of these growth factors are disrupted in disease
growth factor-1 (IGF-1), epidermal growth factor (EGF),
states.2,3
acidic and basic fibroblast growth factor (FGF), keratinocyte Prior studies have shown that autologous serum eye drops
growth factor (KGF), hepatocyte growth factor (HGF), vas- (ASE) are an effective modality for the treatment of refrac-
cular endothelial growth factor (VEGF), platelet-derived tory ocular surface disease.4–9 In patients unable to donate
growth factor-AA, AB and BB (PDGF-AA, AB and BB), adequate blood volume for ASE production, allogenic serum
brain-derived neurotrophic factor (BDNF), neurotrophin-3 eye drops have been reported as a valid alternative.8 Because
(NT-3) and glial cell line-derived neurotrophic factor of the increased use of ASE and this recent report of allo-
(GDNF) were quantified, and statistical analysis was per- genic serum eye drops, comprehensive analysis of the pro-
formed by using the Mann–Whitney U-test with the Bon- duced serum is needed to characterize further its biological
components. Because of the diminutive concentration of
ferroni correction.
many of these growth factors, the expense of immunosorbent
Conclusions: No significant difference was found between assays and the time constraint of other available testing
serum growth factor levels in dry eye syndrome patients modalities (i.e. Northern blot analysis), the spectrum and
versus controls. Our study provides comprehensive analysis quantification of serum growth factors have not previously
been comprehensively evaluated in DES. Prior studies inves-
of serum growth factor levels in autologous serum eye
tigated only a few components of ASE from patients with
drops produced from ocular surface disease patients. A ocular surface diseases, but did not provide a comprehensive
knowledge of growth factor levels in serum may be impor- analysis of the produced ASE from these patient groups or
tant because of the increasing use of autologous serum eye any comparison with an appropriately matched control
drops in refractory ocular surface diseases and for an under- group.1,7,9 In our lab, preliminary high-pressure liquid chro-
standing of how topical serum may provide benefit. matography analyses of a small number of DES patients

䊏 Correspondence: Dr Jay C Bradley, Texas Tech University Health Sciences Center, Department of Ophthalmology & Visual Sciences, 3601 4th Street, STOP
7217, Lubbock, TX 79430-7217, USA. Email: docjayb@aol.com
Received 29 March 2008; accepted 22 October 2008.
This project was presented in part at the American Society of Cataract & Refractive Surgery 2007 Symposium in San Diego, CA on 27April through 2 May.

© 2008 The Authors


Journal compilation © 2008 Royal Australian and New Zealand College of Ophthalmologists
718 Bradley et al.

(N = 3) displayed a pattern of upregulation and downregu- nerve growth factor (NGF), insulin-like growth factor-1
lation of various serum components, especially positive gly- (IGF-1), epidermal growth factor (EGF), acidic and basic
coproteins, as compared with normal controls (JC Bradley, fibroblast growth factor (FGF), keratinocyte growth factor
unpubl. data, 2005). (KGF), hepatocyte growth factor (HGF), vascular endothe-
In this study, we aimed to analyse this pattern by quanti- lial growth factor (VEGF), platelet-derived growth factor-
fying numerous serum growth factors using enzyme-linked AA, AB and BB (PDGF-AA, AB and BB), brain-derived
immunosorbent assays (ELISA) in DES patients and provid- neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and
ing a comparison of these levels with non-DES controls. We glial cell line-derived neurotrophic factor (GDNF). The
selected growth factors previously implicated in anterior ELISA kit for NGF was purchased from Promega (Madison,
segment homeostasis and wound healing for evaluation.2,3 WI, USA). All other ELISA kits were purchased from R&D
We also provide the first comprehensive growth factor analy- Systems (Minneapolis, MN, USA).
sis of ASE from patients with ocular surface disease. Statistical analysis was performed BY using the Mann–
Whitney U-test. Because numerous growth factor tests were
performed on a single population, the Bonferroni correction
METHODS was applied and P < 0.003 was considered to show a statis-
Preparation and storage of serum samples tically significant difference.

This study was approved by the Institutional Review Board,


and all subjects provided informed consent. Samples of RESULTS
approximately 50 mL of whole blood were obtained from six
female volunteer patients with symptomatic DES and six The average age of the patients and controls was 67.33 years
corresponding female age-matched control patients. Patients (range 46–83) and 69.33 years (range 52–84), respectively.
were recruited from the practice of one of the investigators of The levels of the above-mentioned growth factors in the
the study (DLM). A diagnosis of DES was based on Schirmer serum of DES patients and controls were quantified, and
testing with topical anaesthesia (basic secretion testing statistical analysis was performed (Table 2).
ⱕ5 mm); fluorescein, rose bengal, or lissamine green corneal
and conjunctival staining; conjunctival injection; tear break
up time <10 s; decreased tear meniscus (<0.2 mm) with tear DISCUSSION
film debris, mucus, or filamentary changes; presence of mei- Growth factors play a critical role in the maintenance of the
bomian gland disease; and associated subjective and visual ocular surface, and imbalances are associated with certain
symptoms. The dry eye severity grading scheme from the disease states including DES.1 DES can be associated with
International Dry Eye Workshop was used to further charac- autoimmune diseases such as rheumatoid arthritis, Sjogren’s
terize the DES patients (Table 1).10 All DES patients were syndrome, systemic lupus erythematosus, ocular cicatricial
symptomatic at the time of clinical examination despite pre- pemphigoid and Stevens–Johnson syndrome. These diseases
viously instituted therapies (Table 1). The above-mentioned result in chronic systemic inflammation, leading to decreased
characteristics were normal in all control subjects. tear production. Tear components have been studied for
Blood samples were allowed to clot at room temperature their role in the pathogenesis of DES,1 and studies have
(26°C) for 30 min and were then centrifuged in a swinging suggested that many of these growth factors undergo
bucket rotor at 3000 ¥ g for 15 min. Methods for preparation upregulation and downregulation in disease states.2,3
of ASE were performed according to a prior report.11 In our Dry eye syndrome is a common ocular surface disease,
lab prior to initiation of this study, the effect of clotting time which can be refractory to conventional treatments in some
and centrifugation parameters on growth factor yield in ASE cases. ASE have been used successfully for the treatment of
was investigated (JC Bradley, unpubl. Data, 2006). Our refractory DES.4–7 It is presumed that the growth factors in
results agreed with the previously reported centrifugation ASE stimulate corneal epithelial cell differentiation, prolif-
parameters, but a shorter clotting time (30 min) appeared to eration and migration in vivo based on prior in vitro studies.2–4
be optimal. The supernatant serum was carefully collected Prior studies investigated only a few components of ASE
via syringe by using sterile technique. The samples were then (EGF, vitamin A and TGF-b1) from patients with ocular
stored at -80°C until testing was performed. surface diseases, including persistent epithelial defects and
DES, and demonstrated stability of these factors during
storage for up to 3 months. These studies did not provide a
Quantification of growth factors comprehensive analysis of the produced ASE from these
Growth factors previously implicated in prior research in the patient groups or any comparison with appropriately
areas of anterior segment homeostasis and wound healing matched control groups.1,7,9 To our knowledge, quantifica-
were selected for analysis.2,3 The levels of the following tion of a comprehensive range of systemic growth factors in
growth factors in all of the serum samples were analysed by the serum of DES patients with comparison with an appro-
means of ELISA according to the manufacturer’s instructions: priately matched control group has not been previously
transforming growth factor-beta 1 and 2 (TGF-b1 and b2), performed.
© 2008 The Authors
Journal compilation © 2008 Royal Australian and New Zealand College of Ophthalmologists
© 2008 The Authors
Table 1. Clinical data of dry eye syndrome patients

Patient Discomfort, Visual symptoms Conjunctival Conjunctival Corneal Corneal/tear signs Lid/ TBUT Schirmer Therapy in use at
Number severity & injection staining staining meibomian (s) score time of clinical exam
frequency (severity/ glands (mm/5 min)
location)
1 Severe, frequent, Annoying, chronic, Moderate Marked Marked Low tear meniscus, Moderate 5 OU 4 OU (BST) Hourly NP artificial
Serum growth factor in dry eye syndrome

without stress limiting activity central mild debris and MGD tears, ointment at
mucus clumping bedtime, topical
cyclosporine 0.05%
2 Severe, constant, Annoying, chronic, Moderate Marked Severe Low tear meniscus, Mild 2 OU 2 OU (BST) Hourly NP artificial
without stress constant, limited punctate filamentary MGD tears, ointment at
activity erosions keratitis bedtime, topical
loteprednol 0.5%,
upper and lower
punctal occlusion
OU
3 Moderate, chronic, Annoying, chronic, Mild Marked Marked Low tear meniscus, Mild 5 OU 5 OU (BST) Hourly NP artificial
without stress activity limiting central mild debris MGD tears, ointment at
episodic bedtime

Journal compilation © 2008 Royal Australian and New Zealand College of Ophthalmologists
4 Severe, frequent, Annoying, chronic, Mild Marked Marked Low tear meniscus, Mild 5 OU 7 OD; NP artificial tears
without stress limiting activity central moderate debris MGD 5 OS (BST) every 2 h, ointment
and mucus at bedtime
clumping
5 Severe, constant, Annoying, chronic, Moderate Marked Marked Low tear meniscus, Mild 5 OU 2 OU (BST) Hourly NP artificial
without stress limiting activity central moderate debris MGD tears, ointment at
bedtime
6 Severe, frequent, Annoying, chronic, Mild Marked Marked Low tear meniscus, Mild 5 OU 5 OD; Hourly NP artificial
without stress activity limiting central moderate debris MGD 7 OS (BST) tears, ointment at
episodic bedtime

BST, basic secretion testing; MGD, meibomian gland disease; NP, non-preservative; OU, both eyes; OD, right eye; OS, left eye; TBUT, fluorescein tear break up time.
719
720 Bradley et al.

Table 2. Growth factor levels of DES patients versus age- and gender-matched controls*

Growth Factor DES patients Controls P-value


Transforming growth factor-beta 1 (TGF-b1) 37.68 ⫾ 24.18 ng/mL 44.05 ⫾ 13.99 ng/mL 0.589
Transforming growth factor-beta 2 (TGF-b2) 547.83 ⫾ 116.26 pg/mL 586.89 ⫾ 135.54 pg/mL 0.699
Nerve growth factor (NGF) 115.09 ⫾ 41.18 pg/mL 187.25 ⫾ 50.19 pg/mL 0.111
Insulin-like growth factor-1 (IGF-1) 58.14 ⫾ 16.76 ng/mL 59.69 ⫾ 25.29 ng/mL 0.937
Epidermal growth factor (EGF) 255.83 ⫾ 117.25 pg/mL 199.74 ⫾ 64.74 pg/mL 0.418
Basic fibroblast growth factor (bFGF) 6.50 ⫾ 1.05 pg/mL 8.30 ⫾ 1.75 pg/mL 0.065
Keratinocyte growth factor (KGF) 7.67 ⫾ 5.84 pg/mL 10.63 ⫾ 4.98 pg/mL 0.121
Hepatocyte growth factor (HGF) 151.20 ⫾ 93.09 pg/mL 129.32 ⫾ 46.11 pg/mL 0.394
Vascular endothelial growth factor (VEGF) 68.76 ⫾ 38.70 pg/mL 65.57 ⫾ 34.45 pg/mL 0.937
Platelet-derived growth factor-AA (PDGF-AA) 4490.80 ⫾ 1658.56 pg/mL 5396.53 ⫾ 1172.65 pg/mL 0.240
Platelet-derived growth factor-AB (PDGF-AB) 14607.07 ⫾ 7633.04 pg/mL 11927.95 ⫾ 3047.47 pg/mL 0.937
Platelet-derived growth factor-BB (PDGF-BB) 3270.97 ⫾ 1875.25 pg/mL 2437.44 ⫾ 614.34 pg/mL 0.818
Brain-derived neurotrophic factor (BDNF) 8813.12 ⫾ 2734.12 pg/mL 10153.89 ⫾ 2667.19 pg/mL 0.485

*Levels of acidic fibroblast growth factor, neurotrophin-3 and glial-derived growth factor were below detectable limits in both groups. DES,
dry eye syndrome.

Our pilot study demonstrated no statistically significant REFERENCES


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can Society of Cataract & Refractive Surgery Foundation 11. Liu L, Hartwig D, Harloff S, Herminghaus P, Wedel T,
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© 2008 The Authors


Journal compilation © 2008 Royal Australian and New Zealand College of Ophthalmologists