Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-015-3030-2
NEURO-OPHTHALMOLOGY
Vascular endothelial growth factor levels in tears of patients with retinal vein occlusion M. Kasza 1 & Z. Balogh 2 & L. Biro 2 & B. Ujhelyi 2 & J. Damjanovich 2 & A. Csutak 2 & J. Várdai 2 & A. Berta 2 & V. Nagy 2
Received: 26 January 2015 / Revised: 27 March 2015 / Accepted: 20 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose We measured vascular endothelial growth factor (VEGF) levels in tear fluid and serum in patients with retinal vein occlusion (RVO). Patients and methods Eight patients with RVO due to secondary macular oedema were examined. VEGF levels were measured by enzyme-linked immunosorbent assay. All patients had a full ophthalmic examination (visual acuity, slit lamp biomicroscopy, perimetry, and fluorescein angiography). Central retinal thickness (CRT) was examined using optical coherence tomography (OCT). Tear and serum samples were collected and examinations were performed at diagnosis and 1 and 4 weeks later. Results VEGF levels in the tears of RVO eyes were significantly higher than in fellow eyes at diagnosis and after both 1 and 4 weeks (paired t test, p1=0.01, p2=0.02, p3=0.006). We found a weak but significant positive correlation between VEGF levels in tear fluid and serum of patients with RVO (r=0.21), while this correlation tended to be stronger between the fellow eyes and serum levels (r=0.33). Conclusion To the best of our knowledge, we are the first to report an increased level of VEGF in the tear fluid of patients with RVO. Alterations of VEGF levels in tears may be useful These results were presented at the 114th Meeting of the German Ophthalmology Society (DOG), September 2014, Leipzig, in conjunction with the receipt of a Travel Award. * M. Kasza [email protected] 1
Medical Centre, Hungarian Defence Forces, 1134 BudapestRóbert Károly krt. 44, Hungary
2
Department of Ophthalmology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
for determining stages of RVO. This non-invasive and objective method may also be helpful for estimating the severity of macular oedema and efficacy of treatment. Keywords Retinal vein occlusion (RVO) . Vascular endothelial growth factor (VEGF) . Tear fluid . Central retinal thickness (CRT)
Introduction In retinal vein occlusion (RVO), the central retinal vein (CRVO) or branches of the retinal vein (BRVO) suffer occlusion. Venous congestion also affects the arterial circulation; we distinguish ischaemic and non-ischaemic RVO. Occlusion in the retinal vein normally results from the combined effects of venous circulation, damage to the wall of the retinal vein at the lamina cribrosa, and increased blood coagulation (also known as the triad of Virchow). The main symptoms are slow loss of vision (usually within 1 to 2 weeks), relative afferent pupillary defects, visual field defects, and typical ophthalmoscopic findings. Diagnosis is also based on fundus examination. Because of the defects in circulation, the disease is frequently associated with papillary and macular oedema. Complications include neovascularization due to hypoxia and secondary glaucoma. Several factors contribute to neovascularization and affect the vascular permeability of newly formed vessels [1]. Vascular endothelial growth factor (VEGF) plays an essential role. In humans, VEGF has several isomers; VEGF 121 and 165 isomers of VEGF-A play a role in ocular pathology. An increase in their levels due to hypoxia and vascular congestion increases the permeability of capillary walls, leading to macular oedema [2–4], and frequently resulting in severe and irreversible visual impairment.
Graefes Arch Clin Exp Ophthalmol Table 1
Demographic characteristics, data analysis using the Mann-Whitney test
RVO subjects Controls
No. cases
Mean age ± SD
p
CRVO
BRVO
Male
Female
p
Ch↑, Tg↑
p
HT
p
DM
p
8 5
70.37±5.24 63.60±16.24
0.72
3
5
5 4
3 1
0.55
4 1
0.33
7 2
0.11
3 2
0.93
Ch↑: Hypercholesterolaemia Tg↑: Hypertriglyceridaemia HT Hypertension DM Diabetes mellitus
In our pilot study, our aim was to measure VEGF levels in the tears of RVO patients, compare them to serum VEGF levels and optical coherence tomography (OCT) findings, and analyse possible alterations over time.
Patients and methods During the 2 years of recruitment in 2013 and 2014, we diagnosed RVO in 15 patients at the outpatient care unit of the Department of Ophthalmology, University of Debrecen. We excluded patients who had concomitant diseases that might have altered our results. Two patients had malignant tumours, one had rheumatoid arthritis, two patients were diagnosed at least 3 months after the onset of symptoms (they already had secondary glaucoma and rubeosis of the iris), and one patient had chronic obstructive pulmonary disease. These patients were not entered into the study. One patient was present only at the baseline examination and did not participate in the follow-up visits. Thus, ultimately, eight patients (age 70.37±5.24 years; three female and five male) fulfilled our inclusion criteria and participated in the study. Three patients had CRVO (one female, two male), and five patients had BRVO (two female and three male). Our control group consisted of five healthy volunteers (age 63.60±16.24 years; one female and four male) without any concomitant diseases. Demographic and comorbidity data were compared using the Mann-Whitney test. All data are displayed in Table 1. We defined the baseline visit (Visit 1) at the time of the diagnosis, within 3 weeks after the onset of symptoms. Of the eight patients, two arrived within 24 h, two within 1 week, one Table 2 Mean VEGF level in tears of RVO and fellow eyes
after 2 weeks, and three within 3 weeks after the onset of symptoms. None of the patients had bronchial asthma, COPD, an immunological disorder, a malignant tumour, or any condition associated with hypoxia or inflammation. With regard to ocular history, no patients had ocular surface or corneal pathology, uveitis, iris rubeosis, active inflammation, or any vitreoretinal pathology. Our patients underwent a careful ophthalmological examination, including best-corrected visual acuity, perimetry, tonometry, ophthalmoscopy, optical coherence tomography (Spectralis OCT), and fluorescein angiography. At the baseline visit at diagnosis (Visit 1), we evaluated the patients and obtained serum and tear samples. At 1 week (Visit 2) and 4 weeks (Visit 3), we evaluated tear samples in all eight patients. Tear samples were obtained by capillary flow with no nasal stimulation, previous installation of drugs, or vital dyes, and by the same examiner. No anaesthetic drops were used; samples were collected non-traumatically from the inferior meniscus without touching the cornea, conjunctiva, or eyelids. The amount of the tear sample (μl) and collection time (120 sec) were recorded, and samples were centrifuged. All samples were stored at −70 °C in the presence of the cOmplete ULTRA Mini protease inhibitor (Roche Diagnostics, Basel, Switzerland) until measurement. VEGF levels were quantified using the Human VEGF Quantikine® ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according the manufacturer’s instructions. Samples were thawed at room temperature immediately before use. Plasma samples were measured directly; tear samples were diluted up to 200 μl with Calibrator Diluent RD5K (a component of the kit), as amounts collected were small. For statistical analysis, the SPSS version 22.0 software program
Study eye
Visit 1 Visit 2 Visit 3
Fellow eye
Mean VEGF level ± SD (μg/ml)
p
Mean VEGF level ± SD (μg/ml)
p
0.47±0.48 0.99±0.76 0.95±0.49
0.023 0.050 0.874
0.37±0.48 0.73±0.69 0.64±0.44
0.038 0.113 0.596
A significant increase in VEGF levels was observed over time (Visit 1 at baseline, Visit 2 after 1 week, Visit 3 after 3 weeks)
Graefes Arch Clin Exp Ophthalmol Table 3 Changes in central retinal thickness (CRT) in RVO eyes measured by optical coherence tomography (OCT)
Study eye Mean CRT±SD (μm)
p
Visit 1
429.75±91.23
0.01
Visit 2 Visit 3
497.62±98.99 447.37±105.34
0.57 0.11
(IBM Corp., Armonk, NY, USA) was used. We used paired t tests and Pearson correlation analysis, and the level of significance was defined at p
NEURO-OPHTHALMOLOGY
Vascular endothelial growth factor levels in tears of patients with retinal vein occlusion M. Kasza 1 & Z. Balogh 2 & L. Biro 2 & B. Ujhelyi 2 & J. Damjanovich 2 & A. Csutak 2 & J. Várdai 2 & A. Berta 2 & V. Nagy 2
Received: 26 January 2015 / Revised: 27 March 2015 / Accepted: 20 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose We measured vascular endothelial growth factor (VEGF) levels in tear fluid and serum in patients with retinal vein occlusion (RVO). Patients and methods Eight patients with RVO due to secondary macular oedema were examined. VEGF levels were measured by enzyme-linked immunosorbent assay. All patients had a full ophthalmic examination (visual acuity, slit lamp biomicroscopy, perimetry, and fluorescein angiography). Central retinal thickness (CRT) was examined using optical coherence tomography (OCT). Tear and serum samples were collected and examinations were performed at diagnosis and 1 and 4 weeks later. Results VEGF levels in the tears of RVO eyes were significantly higher than in fellow eyes at diagnosis and after both 1 and 4 weeks (paired t test, p1=0.01, p2=0.02, p3=0.006). We found a weak but significant positive correlation between VEGF levels in tear fluid and serum of patients with RVO (r=0.21), while this correlation tended to be stronger between the fellow eyes and serum levels (r=0.33). Conclusion To the best of our knowledge, we are the first to report an increased level of VEGF in the tear fluid of patients with RVO. Alterations of VEGF levels in tears may be useful These results were presented at the 114th Meeting of the German Ophthalmology Society (DOG), September 2014, Leipzig, in conjunction with the receipt of a Travel Award. * M. Kasza [email protected] 1
Medical Centre, Hungarian Defence Forces, 1134 BudapestRóbert Károly krt. 44, Hungary
2
Department of Ophthalmology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
for determining stages of RVO. This non-invasive and objective method may also be helpful for estimating the severity of macular oedema and efficacy of treatment. Keywords Retinal vein occlusion (RVO) . Vascular endothelial growth factor (VEGF) . Tear fluid . Central retinal thickness (CRT)
Introduction In retinal vein occlusion (RVO), the central retinal vein (CRVO) or branches of the retinal vein (BRVO) suffer occlusion. Venous congestion also affects the arterial circulation; we distinguish ischaemic and non-ischaemic RVO. Occlusion in the retinal vein normally results from the combined effects of venous circulation, damage to the wall of the retinal vein at the lamina cribrosa, and increased blood coagulation (also known as the triad of Virchow). The main symptoms are slow loss of vision (usually within 1 to 2 weeks), relative afferent pupillary defects, visual field defects, and typical ophthalmoscopic findings. Diagnosis is also based on fundus examination. Because of the defects in circulation, the disease is frequently associated with papillary and macular oedema. Complications include neovascularization due to hypoxia and secondary glaucoma. Several factors contribute to neovascularization and affect the vascular permeability of newly formed vessels [1]. Vascular endothelial growth factor (VEGF) plays an essential role. In humans, VEGF has several isomers; VEGF 121 and 165 isomers of VEGF-A play a role in ocular pathology. An increase in their levels due to hypoxia and vascular congestion increases the permeability of capillary walls, leading to macular oedema [2–4], and frequently resulting in severe and irreversible visual impairment.
Graefes Arch Clin Exp Ophthalmol Table 1
Demographic characteristics, data analysis using the Mann-Whitney test
RVO subjects Controls
No. cases
Mean age ± SD
p
CRVO
BRVO
Male
Female
p
Ch↑, Tg↑
p
HT
p
DM
p
8 5
70.37±5.24 63.60±16.24
0.72
3
5
5 4
3 1
0.55
4 1
0.33
7 2
0.11
3 2
0.93
Ch↑: Hypercholesterolaemia Tg↑: Hypertriglyceridaemia HT Hypertension DM Diabetes mellitus
In our pilot study, our aim was to measure VEGF levels in the tears of RVO patients, compare them to serum VEGF levels and optical coherence tomography (OCT) findings, and analyse possible alterations over time.
Patients and methods During the 2 years of recruitment in 2013 and 2014, we diagnosed RVO in 15 patients at the outpatient care unit of the Department of Ophthalmology, University of Debrecen. We excluded patients who had concomitant diseases that might have altered our results. Two patients had malignant tumours, one had rheumatoid arthritis, two patients were diagnosed at least 3 months after the onset of symptoms (they already had secondary glaucoma and rubeosis of the iris), and one patient had chronic obstructive pulmonary disease. These patients were not entered into the study. One patient was present only at the baseline examination and did not participate in the follow-up visits. Thus, ultimately, eight patients (age 70.37±5.24 years; three female and five male) fulfilled our inclusion criteria and participated in the study. Three patients had CRVO (one female, two male), and five patients had BRVO (two female and three male). Our control group consisted of five healthy volunteers (age 63.60±16.24 years; one female and four male) without any concomitant diseases. Demographic and comorbidity data were compared using the Mann-Whitney test. All data are displayed in Table 1. We defined the baseline visit (Visit 1) at the time of the diagnosis, within 3 weeks after the onset of symptoms. Of the eight patients, two arrived within 24 h, two within 1 week, one Table 2 Mean VEGF level in tears of RVO and fellow eyes
after 2 weeks, and three within 3 weeks after the onset of symptoms. None of the patients had bronchial asthma, COPD, an immunological disorder, a malignant tumour, or any condition associated with hypoxia or inflammation. With regard to ocular history, no patients had ocular surface or corneal pathology, uveitis, iris rubeosis, active inflammation, or any vitreoretinal pathology. Our patients underwent a careful ophthalmological examination, including best-corrected visual acuity, perimetry, tonometry, ophthalmoscopy, optical coherence tomography (Spectralis OCT), and fluorescein angiography. At the baseline visit at diagnosis (Visit 1), we evaluated the patients and obtained serum and tear samples. At 1 week (Visit 2) and 4 weeks (Visit 3), we evaluated tear samples in all eight patients. Tear samples were obtained by capillary flow with no nasal stimulation, previous installation of drugs, or vital dyes, and by the same examiner. No anaesthetic drops were used; samples were collected non-traumatically from the inferior meniscus without touching the cornea, conjunctiva, or eyelids. The amount of the tear sample (μl) and collection time (120 sec) were recorded, and samples were centrifuged. All samples were stored at −70 °C in the presence of the cOmplete ULTRA Mini protease inhibitor (Roche Diagnostics, Basel, Switzerland) until measurement. VEGF levels were quantified using the Human VEGF Quantikine® ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according the manufacturer’s instructions. Samples were thawed at room temperature immediately before use. Plasma samples were measured directly; tear samples were diluted up to 200 μl with Calibrator Diluent RD5K (a component of the kit), as amounts collected were small. For statistical analysis, the SPSS version 22.0 software program
Study eye
Visit 1 Visit 2 Visit 3
Fellow eye
Mean VEGF level ± SD (μg/ml)
p
Mean VEGF level ± SD (μg/ml)
p
0.47±0.48 0.99±0.76 0.95±0.49
0.023 0.050 0.874
0.37±0.48 0.73±0.69 0.64±0.44
0.038 0.113 0.596
A significant increase in VEGF levels was observed over time (Visit 1 at baseline, Visit 2 after 1 week, Visit 3 after 3 weeks)
Graefes Arch Clin Exp Ophthalmol Table 3 Changes in central retinal thickness (CRT) in RVO eyes measured by optical coherence tomography (OCT)
Study eye Mean CRT±SD (μm)
p
Visit 1
429.75±91.23
0.01
Visit 2 Visit 3
497.62±98.99 447.37±105.34
0.57 0.11
(IBM Corp., Armonk, NY, USA) was used. We used paired t tests and Pearson correlation analysis, and the level of significance was defined at p
