ATHEROSCLEROTIC AND THROMBOPHILIC RISK FACTORS IN PATIENTS WITH ISCHEMIC CENTRAL RETINAL VEIN OCCLUSION

ANDREA SODI, MD,* BARBARA GIAMBENE, MD, PHD,* ROSSELLA MARCUCCI, MD, PHD, FRANCESCO SOFI, MD, PHD, SANDRA FEDI, MD, ROSANNA ABBATE, MD, DOMENICO PRISCO, MD, UGO MENCHINI, MD*
Purpose: To investigate atherosclerotic and thrombophilic risk factors in patients affected by acute ischemic and nonischemic central retinal vein occlusions (CRVOs). Methods: One hundred and three patients with acute unilateral CRVO (41 ischemic and 62 nonischemic) were studied. The frequency of traditional cardiovascular risk factors was assessed, and the plasma levels of a variety of thrombophilic markers were measured. Univariate logistic regression was performed to determine risk factors for ischemic CRVO. Results: Arterial hypertension, hypercholesterolemia, postmethionine hyperhomocysteinemia (HHcy), elevated factor VIII, and reduced folic acid and B6 plasma levels were more frequent in patients with ischemic CRVO than in those with nonischemic CRVO (P = 0.030, P = 0.025, P = 0.011, P , 0.001, P , 0.001, and P = 0.044, respectively). Risk factors for ischemic CRVO were arterial hypertension (odds ratio [OR], 3.22; 95% condence interval [CI], 1.139.21; P = 0.037), hypercholesterolemia (OR, 3.03; 95% CI, 1.068.65; P = 0.042), reduced folic acid levels (OR, 6.77; 95% CI, 1.5928.79; P = 0.011), and elevated FVIII levels (OR, 6.17; 95% CI, 2.5614.82; P , 0.001). Postmethionine HHcy was associated with low folic acid levels (r = 20.413; P = 0.007; OR, 9.33; 95% CI, 2.0642.18; P = 0.005). Conclusion: The results of the present study suggest that some atherosclerotic and thrombophilic risk factors may increase the risk of having an ischemic form of CRVO. RETINA 31:724729, 2011

he natural history of central retinal vein occlusion (CRVO) is highly variable. Some cases show a favorable evolution, while others develop severe complications, as vitreous hemorrhages and neovascular glaucoma, leading to visual function impairment.1,2 The clinical course of CRVO depends on the involvement of retinal circulation, mainly related to the extent of retinal ischemia at uorescein angiography examination. Therefore, CRVOs have been classied into ischemic and nonischemic forms, even if the distinction criteria are still debated.35 The Central Vein Occlusion Study Group5 dened a CRVO
From the *Eye Clinic, University of Florence, Florence, Italy; and Thrombosis Center, University of Florence, Florence, Italy. The authors have no proprietary interests. Reprint requests: Barbara Giambene, MD, PhD, Clinica Oculistica, Dipartimento di Scienze Chirurgiche Specialistiche, Azienda Ospedaliero-Universitaria Careggi, viale Morgagni 85, 50134 Florence, Italy; e-mail: barbaragiambene@virgilio.it

as ischemic if at least 10 optic disk areas of retinal capillary nonperfusion were observed in uorescein angiography scans. This study showed that 33% of nonischemic CRVOs progress to the ischemic form in a 3-year follow-up period and that approximately 60% of ischemic CRVOs develop anterior and/or posterior segment neovascularization, being the risk of neovascularization signicantly associated with the extension of retinal ischemia.6,7 Systemic cardiovascular risk factors and thrombophilia have been reported to be highly prevalent in patients with CRVO compared with control subjects, but it is unknown if they can play a different role in the two forms of the disease.817 The present study is aimed at evaluating the frequency of traditional cardiovascular and thrombophilic risk factors in patients with acute ischemic and nonischemic CRVOs to investigate their possible inuence on the two types of disease.

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Materials and Methods One hundred and three consecutive patients with a rst episode of acute unilateral CRVO referred to the Clinica Oculistica of the Azienda OspedalieroUniversitaria Careggi in Florence were recruited. All of them gave written informed consent to the study that was approved by the local Ethics Committee and complied with the Declaration of Helsinki. All the study procedures were performed within 30 days from the onset of symptoms of CRVO. The participants underwent a detailed interview addressed to the presence of cardiovascular diseases and risk factors and a complete physical and ophthalmic examination. Color fundus photography and uorescein angiography were performed. The presence of local factors potentially leading to CRVO was excluded by orbital echography and a color Doppler imaging of retrobulbar vessels. Patients with a history of abnormal liver or renal function, thyroid diseases, and any ocular abnormalities other than CRVO were excluded. None of the patients was using drugs known to inuence the results of the thrombophilic assessment. According to the CRVO Study classication, 41 patients had ischemic CRVO and 62 patients had nonischemic CRVO.5 The subjects were classied as having hypertension and diabetes according to the guidelines of the European Society of Hypertension/European Society of Cardiology and to those of the American Diabetes Association, respectively.18,19 Dyslipidemia was dened according to the criteria of the ATP III Expert Panel of the U.S. National Cholesterol Education Program.20 Blood samples were collected into evacuated plastic tubes (Vacutainer; Becton Dickinson, Meylan, France) from the basilic vein after an overnight fasting. For determining postmethionine homocysteine (Hcy), 100 mg/(kg body weight) of l-methionine was administered in 200 mL fruit juice after the fasting blood sample, and a second sample was obtained after 4 hours. Cholesterol and triglyceride plasma values were measured by means of enzymatic and colorimetric methods, respectively. To determine Hcy plasma values and C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism, whole venous blood was collected in tubes containing EDTA 0.17 mol/L, immediately put in ice, and centrifuged within 30 minutes at 4C (2,000g for 15 minutes). Plasma samples were stored at 220C until assay. Plasma levels of total Hcy (free and protein bound) were measured by uorescence polarization

immunoassay (Homocysteine; IMX Abbott Laboratories, Oslo, Norway). Hyperhomocysteinemia (HHcy) was diagnosed when Hcy values exceeded the 95th percentile of distribution in control subjects (fasting: men 19 mmol/L and women 13 mmol/L; postmethionine: men 38 mmol/L and women 35 mmol/L). C677T MTHFR polymorphism analysis was performed through electronic microchip technology (Nanogen, San Diego, CA). For the detection of the G1691A polymorphism of the factor V (FV) gene and the G20210A polymorphism of the factor II gene, genomic DNA was extracted from peripheral blood celllymphocytes and detected by real-time polymerase chain reaction (Factor V Leiden KIT and Factor II (Prothrombin) G20210A for LightCycler Instrument; Roche Diagnostics, Mannheim, Germany). Blood was drawn directly into evacuated tubes containing sodium citrate 0.129 mol/L (1/10, vol/vol) for determination of plasminogen activator inhibitor-1 (PAI-1) activity, lupus anticoagulant, and factor VIII (FVIII) plasma levels. The samples were preserved, centrifuged at 4C (for PAI-1) or 18C (for FVIII) (2,000g for 10 minutes), rapidly frozen in liquid nitrogen, and stored at 280C. PAI-1 activity was measured by a chromogenic method (Spectrolyse [brin]; Biopool, Umea, Sweden). FVIII levels were determined by a clotting method (FVIII; DadeBehring, Marburg, Germany). PAI-1 activity and FVIII levels above the 95th percentile of distribution in control subjects (15 IU/mL and 150%, respectively) were considered over the normal range. Platelet-poor plasma for the lupus anticoagulant test was obtained by centrifuging blood samples twice (at 2,000g for 10 minutes at 18C and immediately at 6,000g for 3 minutes at 20C) and instantly used for the analysis. The tests chosen to detect the presence of lupus anticoagulant were as follows: diluted (1:50) activated partial thromboplastin time (Pathromptin SL; Dade-Behring); kaolin clotting time (kaolin; Dade-Behring); tissue thromboplastin inhibition test, using 1:1,000 dilution (Thromborel S; DadeBehring); and diluted Russells viper venom time (DRVV test; American Diagnostica, Inc, Greenwich, CT). Mixing studies with normal plasma (plateletpoor plasma from 20 normal individuals) were used to exclude clotting factor deciencies or the presence of antibodies against specic coagulation proteins. Samples shown to be abnormal were also assayed as the conrmation test according to the platelet neutralization procedure (Staclot LA, Stago, France). We considered only those patients with tests conrmed by platelet neutralization procedure as lupus anticoagulant positive.

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To determine circulating vitamins (B6, B12, and folic acid), venous blood was put in tubes without anticoagulant and centrifuged at room temperature (2,000g for 15 minutes). Sera samples were stored at 220C until assay. Vitamin B6 levels were determined using a commercial high-performance liquid chromatography assay with uorescence detection (Immundiagnostik AG, Bensheim, Germany), whereas sera levels of folic acid and vitamin B12 were measured by radioimmunoassay (ICN Pharmaceuticals, Orangeburg, NY). Normal ranges for vitamin B6, folic acid, and vitamin B12 blood values were 3 ng/mL to 18 ng/mL, 3 ng/mL to 17 ng/mL, and 180 pg/mL to 970 pg/mL, respectively. Sera for testing anticardiolipin antibodies (aCL) and lipoprotein(a) (Lp(a)) were obtained by centrifuging blood collected in evacuated tubes without anticoagulant at 1,300g for 10 minutes and stored at 220C. The aCL assay was performed by an enzyme-linked immunosorbent assay (anticardiolipin IgM and IgG antibodies; IMTEC Immunodiagnostika, Berlin, Germany), and aCL levels were reported in GPL units (IgG phospholipid units) and MPL units (IgM phospholipid units). Values .20 U for both IgG and IgM were considered abnormal. Lp(a) levels were determined by an enzyme-linked immunosorbent assay (Apo(a) ELISA; Mercodia, Uppsala, Sweden). Lp(a) levels .300 mg/L were considered over the normal range. Results are given as mean 6 SD or number (n) and rate (%). Statistical comparisons between the two groups of patients were performed by Students t-test for unpaired data and chi-square test. A logistic regression analysis was done to establish the risk of ischemic CRVO according to the presence of a studied risk factor. Odds ratio and 95% condence intervals were shown. A P # 0.05 was considered as statistically signicant. All statistical analyses were done using the SPSS (Chicago, IL) software for Windows (Version 13.0). Results Demographics and the prevalence of traditional cardiovascular risk factors of the study population are

shown in Table 1. Arterial hypertension and hypercholesterolemia were more frequent in the ischemic CRVO group (P = 0.030 and P = 0.025, respectively). The plasma values of prothrombotic and hypobrinolytic markers and the prevalence of patients with abnormal values in the 2 study groups are reported in Tables 2 and 3, respectively. Both fasting and postmethionine homocysteine (Hcy), PAI-1 activity, FVIII, and Lp(a) were signicantly higher, while folic acid and vitamin B6 levels were lower in subjects with ischemic CRVO (Table 2). Postmethionine HHcy, elevated FVIII, and reduced folic acid and vitamin B6 levels were signicantly more frequent in patients with ischemic CRVO (Table 3). A logistic regression analysis was done to investigate the relationship between ischemic CRVO and the studied risk factors. At univariate analysis, arterial hypertension, hypercholesterolemia, hypertriglyceridemia, postmethionine HHcy, reduced folic acid and vitamin B6 levels, and elevated FVIII levels were associated with the occurrence of ischemic CRVO (Table 4). The correlation analysis done to evaluate the relation between postmethionine HHcy and its determinants showed that in the ischemic group, postmethionine HHcy was signicantly associated with reduced folic acid levels (r = 20.413, P = 0.007). The presence of low folic acid levels determined a 9-fold increase of having HHcy (odds ratio, 9.33; 95% condence interval, 2.0642.18; P = 0.005). MTHFR C677T polymorphism and low vitamin B6 and B12 levels resulted to not be signicantly associated with elevated levels of postmethionine Hcy. Discussion In this study, we investigated the associations between traditional and thrombophilic risk factors and ischemic CRVO. We found that arterial hypertension and dyslipidemia were signicantly more frequent in patients with ischemic CRVO. Among thrombophilic factors, we found that patients with ischemic CRVO had higher blood levels of postmethionine Hcy, FVIII, PAI-1 activity, and Lp(a) and lower levels of folic acid and vitamin B6 than those with nonischemic CRVO.

Table 1. Demographics and Prevalence of Traditional Cardiovascular Risk Factors in the Study Population Ischemic CRVO Age, years, mean 6 SD Male, n (%) Arterial hypertension, n (%) Diabetes, n (%) Current smoking habit, n (%) Hypercholesterolemia, n (%) Hypertriglyceridemia, n (%) 67.4 6 7.7 21 (51.2) 25 (61.0) 4 (9.8) 11 (26.8) 21 (51.2) 20 (48.8) Nonischemic CRVO 67.4 6 8.2 33 (53.2) 23 (37.1) 5 (8.1) 15 (24.2) 17 (27.4) 18 (29.0) P 0.990 0.998 0.030 0.953 0.944 0.025 0.068

RISK FACTORS IN RETINAL VEIN OCCLUSION  SODI ET AL Table 2. Plasma Values of the Thrombophilic Risk Factors in the Study Population Ischemic CRVO, Mean 6 SD Fasting Hcy, mmol/L Postmethionine Hcy, mmol/L PAI-1, IU/mL FVIII (%) Lp(a), mg/dL Folic acid, ng/mL Vitamin B6, ng/mL Vitamin B12, pg/mL 14.9 37.2 15.3 195.2 291.4 6.0 4.4 425.9 6 6 6 6 6 6 6 6 4.8 7.9 10.1 67.6 118.2 4.2 2.0 144.1 Nonischemic CRVO, Mean 6 SD 12.5 31.5 12.1 148.1 249.2 9.9 6.5 414.4 6 6 6 6 6 6 6 6 3.5 9.2 8.3 30.0 90.4 3.6 2.3 179.8

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P 0.003 0.003 ,0.001 ,0.001 0.043 ,0.001 ,0.001 0.731

Hcy, homocysteine; PAI-1, plasminogen activator inhibitor-1; FVIII, factor VIII; Lp(a), lipoprotein(a).

Considering the frequency of patients with these parameters out of the range of normal values, the differences between the two groups were still signicant for postmethionine Hcy, factor VIII, folic acid, and vitamin B6. These results may have relevant clinical implications because three of these risk factors are easily modiable even if this hypothesis should prompt specic researches. Many studies817 have demonstrated that patients with retinal vein occlusions (RVOs) are more likely to have cardiovascular diseases and risk factors than control subjects. The association of arterial hypertension with ischemic RVO has been reported by the Eye Disease Case-Control Study Group.8 Hayreh et al11
Table 3. Thrombophilic Risk Factors: Prevalence of Patients with Abnormal Values Ischemic Nonischemic CRVO, CRVO, n (%) n (%) Fasting HHcy Postmethionine HHcy Elevated PAI-1 levels Elevated FVIII levels LA positive Elevated aCL levels Elevated Lp(a) levels G1691A polymorphism, G20210A polymorphism Reduced folic acid levels Reduced vitamin B6 levels Reduced vitamin B12 levels MTHFR C677T polymorphism CC TC TT 12 23 17 30 5 10 13 4 2 17 (29.3) (56.1) (41.5) (73.2) (12.2) (24.4) (31.7) (9.8) (4.9) (41.5) 10 18 18 19 8 9 20 4 6 6 (16.1) (29.0) (29.0) (30.6) (12.9) (14.5) (32.3) (6.5) (9.7) (9.7)

found a higher prevalence of arterial hypertension and diabetes in ischemic CRVO in comparison with nonischemic CRVO. The association of dyslipidemia and RVO has been reported by several authors,2125 even if, at the best of our knowledge, this is the rst study in which the association with the ischemic and nonischemic forms was evaluated. Interestingly, in a recent small retrospective study, we observed an association of dyslipidemia with the recurrence of RVO.26 Thus, the results of the present research support the possible usefulness of treating patients with CRVO with antidyslipidemic drugs early after the clinical presentation of the disease to try to modify the functional prognosis, even if this approach needs a specic validation. The lack of association between ischemic CRVO and diabetes in our patients might be because of a demographic difference between studied populations.
Table 4. Univariate Logistic Regression Analysis Odds Ratio (95% Condence Interval) Arterial hypertension Hypercholesterolemia Hypertriglyceridemia Fasting HHcy Elevated PAI-1 levels LA positive Elevated aCL levels Elevated Lp(a) levels G1691A polymorphism G20210A polymorphism MTHFR TT + TC vs CC Postmethionine HHcy Reduced folic acid levels Reduced vitamin B6 levels Elevated FVIII levels 2.65 2.78 2.32 2.57 1.73 0.94 1.86 0.97 1.56 0.48 1.14 3.12 6.61 4.08 6.17 (1.175.97) (1.216.36) (1.025.29) (0.996.62) (0.763.97) (0.283.09) (0.685.09) (0.422.27) (0.376.66) (0.092.49) (0.492.59) (1.377.13) (2.3218.82) (1.1614.30) (2.5614.82)

P 0.178 0.011 0.234 ,0.001 0.844 0.315 0.875 0.812 0.607 ,0.001 0.044 0.302 0.885

P 0.019 0.016 0.044 0.050 0.190 0.900 0.200 0.900 0.500 0.900 0.800 0.006 ,0.001 0.028 ,0.001

9 (21.9) 1 (2.4)

4 (6.4) 6 (9.7)

14 (34.2) 21 (51.2) 6 (14.6)

23 (37.1) 28 (45.2) 11 (17.7)

HHcy, hyperhomocysteinemia; PAI-1, plasminogen activator inhibitor-1; FVIII, factor VIII; LA, lupus anticoagulant; aCL, anticardiolipin antibodies; Lp(a), lipoprotein(a); MTHFR, methylenetetrahydrofolate reductase.

HHcy, hyperhomocysteinemia; PAI-1, plasminogen activator inhibitor-1; LA, lupus anticoagulant; aCL, anticardiolipin antibodies; Lp(a), lipoprotein(a); MTHFR, methylenetetrahydrofolate reductase; FVIII, factor VIII.

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Most available studies12,13,2736 have demonstrated an association between both fasting and postmethionine HHcy and RVO. Some authors27,29,36 have also investigated the prevalence of HHcy in a small series of patients with ischemic and nonischemic CRVOs, but data are not conclusive yet. This is the rst study in which an association between postmethionine HHcy and ischemic CRVO was found. This result advocates the possible role of methionine loading test in the evaluation of the risk of developing ischemic CRVO and suggests intervention studies to evaluate the effects of normalizing Hcy values on the clinical outcome of the disease. A role for B-group vitamins, namely folic acid and vitamins B6 and B12, as independent risk factors for arterial and venous thrombotic events has been repeatedly suggested, and an association between low folic acid levels and the occurrence of RVO has been demonstrated in a meta-analysis.13,3740 In a recent study,41 folic acid in the lowest tertile, independent of Hcy levels, was found to be signicantly related to an increased risk of having RVO, conrming the pathogenetic role of the deciency of this vitamin on the occurrence of this disease. The present nding of an association of low folic acid levels with ischemic CRVO might have therapeutic implications. Both genetic and acquired, namely nutritional, factors are determinants of fasting and postmethionine Hcy levels.4244 No previous data are available about the role of different causes of HHcy in different types of RVO. Our data suggest a prevalent role for nutritional factors in the determination of HHcy in ischemic RVO. A limitation of the present study was that nutritional habits of the examined subjects were not registered, missing the possibility to directly evaluate the role of diet on Hcy circulating values. High levels of FVIII in patients with RVO have been reported in several investigations.4548 The association of higher levels of FVIII with ischemic CRVO is a likely expression of higher endothelial damage and hypercoagulability. This nding indirectly supports the usefulness of the treatment with lowmolecular weight heparins in acute RVO, at present validated by only two relatively small studies.49,50 In conclusion, our present study showed that several traditional and thrombophilic risk factors are frequently reported in CRVO, but only some of them revealed to be associated with the ischemic form of the disease. Because some of them are modiable, intervention studies are necessary to understand if they are only markers of risk or true risk factors and if their correction in patients with acute ischemic CRVO would be able to modify the clinical outcome of the illness. The modication of the vascular risk factors

may not only reduce the incidence of further cardiovascular disorders but also have benecial consequences on the course of CRVO. In fact, the rate of sight-threatening complications can be reduced by limiting the progression from nonischemic to ischemic forms of CRVO.51,52 Finally, our data might suggest an anticoagulant approach in the acute phase of the disease. Further investigations are awaited to conrm our data and validate therapeutic options in patients with ischemic RVO. Key words: atherosclerotic risk factors, folic acid, homocysteine, ischemic central retinal vein occlusion, thrombophilia. References
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