Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Alterations of Choroidal Blood Flow Regulation in Young Healthy Subjects with Complement Factor H Polymorphism

  • Reinhard Told,

    Affiliations Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria

  • Stefan Palkovits,

    Affiliation Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

  • Helmuth Haslacher,

    Affiliation Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

  • Sophie Frantal,

    Affiliation Center for Medical Statistics, Informatics and Intelligence Systems, Medical University of Vienna, Vienna, Austria

  • Doreen Schmidl,

    Affiliation Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

  • Agnes Boltz,

    Affiliations Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria

  • Michael Lasta,

    Affiliation Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

  • Semira Kaya,

    Affiliation Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

  • René M. Werkmeister,

    Affiliation Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria

  • Gerhard Garhöfer,

    Affiliation Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

  • Leopold Schmetterer

    leopold.schmetterer@meduniwien.ac.at

    Affiliations Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria

Alterations of Choroidal Blood Flow Regulation in Young Healthy Subjects with Complement Factor H Polymorphism

  • Reinhard Told, 
  • Stefan Palkovits, 
  • Helmuth Haslacher, 
  • Sophie Frantal, 
  • Doreen Schmidl, 
  • Agnes Boltz, 
  • Michael Lasta, 
  • Semira Kaya, 
  • René M. Werkmeister, 
  • Gerhard Garhöfer
PLOS
x

Abstract

A common polymorphism in the complement factor H gene (rs1061170, Y402H) is associated with a high risk of age-related macular degeneration (AMD). In the present study we hypothesized that healthy young subjects homozygous for the high-risk haplotype (CC) show abnormal choroidal blood flow (ChBF) regulation decades before potentially developing the disease. A total of 100 healthy young subjects were included in the present study, of which 4 subjects were excluded due to problems with genotyping or blood flow measurements. ChBF was measured continuously using laser Doppler flowmetry while the subjects performed isometric exercise (squatting) for 6 minutes. The increase in ChBF was less pronounced than the response in ocular perfusion pressure (OPP), indicating for some degree of choroidal blood flow regulation. Eighteen subjects were homozygous for C, 47 subjects were homozygous for T and 31 subjects were heterozygous (CT). The increase in OPP during isometric exercise was not different between groups. By contrast the increase in ChBF was more pronounced in subjects homozygous for the high risk C allele (p = 0.041). This was also evident from the pressure/flow relationship, where the increase in ChBF in homozygous C carriers started at lower OPPs as compared to the other groups. Our data indicate that the regulation of ChBF is abnormal in rs1061170 CC carriers. So far this polymorphism has been linked to age related macular degeneration (AMD) mainly via inflammatory pathways associated with the complement system dysfunction. Our results indicate that it could also be related to vascular factors that have been implicated in AMD pathogenesis.

Introduction

Age-related macular degeneration is the leading cause of blindness in the industrialized countries [1]. Major risk factors for the disease include increasing age, smoking and a family history of AMD [2], [3], [4]. In the recent years evidence has accumulated indicating that genetic factors are associated with AMD [5], [6], [7], [8]. A polymorphism of factor H (HGNC:4883), a complement control protein, was the first gene shown to be involved in the development and progression of AMD [9], [10], [11], [12]. A single nucleotide polymorphism (SNP), rs1061170 (also known as Y402H), located within the chromosome 1q32 region and corresponding to the human complement factor H gene, was found to be associated with AMD. This supports the hypothesis that a local inflammatory process is involved in AMD pathogenesis. This was already assumed earlier based on the observation that drusen contain inflammatory materials including complement system components [13], [14].

Recently the results of a population-based study have shown that the homozygous C allele (CC) of rs1061170 entails a significant risk of mortality in Finnish nonagenarians [15]. In young healthy male subjects the relationship between SNPs in both, factor H and C-reactive protein, and early atherogenic vascular changes was studied. Interaction between C-reactive protein haplotypes and CC allele of rs1061170 were associated with increased carotid artery stiffness [16]. These results link factor H with atherosclerosis. Animal data show that CFH also plays a crucial role in the integrity of the ocular circulation. In complement factor H deficient mice C3 and C3b are progressively deposited on ocular vessels, subsequently leading to endothelial damage and restricted perfusion [17]. Alterations in the retinal and choroidal vessels were already visible in 3 month old animals and became more pronounced after 12 months.

Based on these results we hypothesized that choroidal blood flow (ChBF) regulation is abnormal in young healthy carriers, homozygous for the C risk-allele of rs1061170. This hypothesis was tested by studying the response of ChBF, as measured with laser Doppler flowmetry, during an isometric exercise-induced increase in blood pressure [18], [19], [20], [21].

Results

The baseline characteristics of the subjects are presented in Table 1. In 1 subject genotyping was not successful. In 3 other subjects no adequate laser Doppler flowmetry readings could be obtained. As such data from 96 subjects were included in the final analysis. The results of rs1061170 genotyping showed that 18 subjects were homozygous for the risk allele C, 47 subjects were homozygous for T and 31 subjects were heterozygous CT. The results did not deviate from the Hardy Weinberg equilibrium.

thumbnail
Table 1. Demographic and baseline characteristics of the subjects (n = 96, mean ± SD).

https://doi.org/10.1371/journal.pone.0060424.t001

The effect of isometric exercise on MAP and PR is shown in Figure 1. A pronounced increase in both MAP and PR was seen during isometric exercise (p<0.001 versus baseline). This response was comparable between the 3 groups (MAP: p = 0.33, PR: p = 0.088). Isometric exercise did not alter IOP (p = 0.76, data not shown). Figure 2 presents the response in OPP and ChBF during isometric exercise. As expected, OPP increased significantly during squatting (p<0.001 versus baseline). The increase in OPP was, however, comparable between the 3 groups (p = 0.23). The increase in ChBF was also significant during squatting (p<0.001 versus baseline) although less pronounced than the increase in OPP (p<0.001 versus baseline). The response in choroidal blood flow was, however, significantly more pronounced in carriers of the homozygous C allele as compared to carriers of the homozygous T allele or heterozygous CT subjects (p = 0.041).

thumbnail
Figure 1. Effect of squatting on mean arterial blood pressure (MAP) and pulse rate (PR).

Data are presented separately according to the results of rs1061170 genotyping (n = 96; means ± SD) p<0.001 versus baseline.

https://doi.org/10.1371/journal.pone.0060424.g001

thumbnail
Figure 2. Effect of squatting on ocular perfusion pressure (OPP) and choroidal blood flow (Flow).

Data are presented separately according to the results of rs1061170 genotyping (n = 96; means ± SD). Asterisks indicate significant differences between the different groups (repeated measures ANOVA), p<0.001 versus baseline.

https://doi.org/10.1371/journal.pone.0060424.g002

Figure 3 depicts the pressure/flow relationship during isometric exercise. In TT carriers ChBF values were not significantly different from baseline up to OPP values 58% above baseline. Thereafter ChBF values started to increase almost linearly. In heterozygous subjects the pressure/flow relationship was almost similar. ChBF was constant up to OPP values of 57% above baseline and increased thereafter. In CC carriers, however, the increase in ChBF took place at lower OPP values. In these subjects ChBF remained constant until an OPP change of 39% from baseline. At higher OPP values ChBF rose linearly. The 95% confidence intervals of the curves, however, still overlapped.

thumbnail
Figure 3. Pressure flow relationship during isometric exercise.

Data are sorted according to ascending ocular perfusion pressure (OPP) values and the means as well as the 95% confidence intervals are shown. The upper graph shows the comparison for homozygous C and homozygous T subjects, the lower graph for homozygous C and heterozygous CT subjects. The data are displayed separately to increase legibility.

https://doi.org/10.1371/journal.pone.0060424.g003

Discussion

When OPP is raised the choroid shows a vasoconstrictor response in order to keep blood flow constant [22]. The present study indicates significant differences in the regulatory behavior of ChBF during an increase in OPP depending on rs1061170 genotyping. This is seen decades before these rs1061170 positive subjects are at increased risk of potentially developing AMD.

The early onset of findings in young healthy subjects might be explained by the concurrence of various aspects. As mentioned before hypoxia and local inflammatory processes involving the complement system represent one aspect of this multifactorial process. In particular, we know from animal models, that C3 and C3b complement protein deposition on vascular endothelial surfaces takes place over time in CFH deficient mice. The damage of the endothelial surface further leads to narrowing and dying out of vessels. This is also in accordance with findings in CFH deficient mice, where not only the choroid endothelium but also the overall choroid thickness itself was significantly reduced. Finally also restricted vascular perfusion, leading to local ischemia, has been shown in these mice, leading to increased oxidative stress, local release of vasoactive substances such as VEGF and the activation of the immune system [17]. The active contribution of the immune system might be questionable in the beginning, as it has been reported that remnants of phagocytosis in the retinal pigment epithelium have the potential to activate the alternative complement pathway [23]. This might indicate that vascular beds affected by this multifactorial process become impaired in their autoregulatory capacity.

A recent review has proposed a relation between complement activation and endothelial dysfunction that may provide a link between AMD and atherosclerosis [24]. Indeed markers of endothelial dysfunction such as sICAM-1, von Willebrand factor and plasminogen activator inhibitor type 1 (PAI-1) are elevated in AMD patients and are related to formation of drusen and choroidal neovascularization (CNV) [25].

Some studies have also shown an association between the rs1061170 (Y402H) polymorphism and the incidence of myocardial infarction and coronary artery disease [26], [27]. Indeed several clinical trials using complement inhibitors in both AMD and atherosclerosis are currently under way [24].

In the present study abnormalities in choroidal blood flow regulation were observed in homozygous C allele carriers of rs1061170 long before AMD potentially develops. Our results are, furthermore, compatible with an above-mentioned study showing reduced vascular elasticity in young Finnish men [16]. This raises the question whether homozygous carriers of the C allele should already be treated or closely observed before the clinical onset of the disease. So far the only proven treatment for non-exudative AMD is a combination of supplements including vitamin C, vitamin E, beta-carotene and zinc [28]. In a subgroup of participants that were at high risk for progression, an interaction between treatment and rs1061170 polymorphism was found, with carriers of the CC allele being less responsive due to the zinc component of the medication [29]. On the other hand the Rotterdam study has shown that dietary intake of very high amounts (highest tertile) of zinc, beta-carotene, lutein/zeaxanthin and omega-3 free fatty acids, reduces the risk of developing early AMD in homozygous risk allele carriers including rs1061170 [30].

Several previous studies have shown that AMD is associated with reduced choroidal blood flow [31], [32], [33], [34], [35], [36], [37]. In an experiment which used a protocol almost similar to our study a reduced regulatory capacity of the choroidal vasculature was observed in AMD patients during a squatting-induced increase in OPP [38]. Two longitudinal studies revealed that reduced choroidal blood flow is a risk factor for the development of choroidal neovascularization (CNV) in AMD. Metelitsina and co-workers [39] studied 193 eyes with AMD and followed them for a period of one to 5 years. Of those, 28 eyes developed CNV during the observation period. These eyes had reduced choroidal baseline values as compared to the eyes that did not develop CNV. Boltz et al. [40] studied 41 patients with unilateral CNV for an observation period of 3 years and followed the contralateral eye for 3 years. In this period 17 eyes developed CNV. Multivariate time-dependent Cox regression analysis revealed that these eyes had significantly lower ChBF than eyes that did not develop CNV. These clinical data are well compatible with animal experiments in mice in which the hypoxia response element (HRE) was deleted from the vascular endothelial growth factor (VEGF) promoter. As compared to wild-type mice the amount of CNV after laser-induced rupture of Bruch's membrane in HRE −/− animals was more than 10 times smaller [41], highlighting the role of hypoxia in the pathogenesis of AMD and CNV.

Several limitations need to be considered when interpreting the present data. Most importantly we have focused on one specific polymorphism associated with AMD. Several other complement pathway-related genes including complement factor B, complement component 2 and C3 were linked to the disease, as well as variants in the ARMS2, PLEKHA1, and HTRA 1 genes [7]. Deeper analysis has also shown that CFH intronic SNPs are more significantly associated with AMD than rs1061170. Structural variations and SNPs in the RCA gene cluster including two common deletions: DCNP147, which removes all of CFHR3 and CFHR1, and DCNP148, which removes CFHR1 and CFHR4 in addition to a large segment of flanking non-coding sequence, were linked to the disease. The present study was, however, not designed to study the potential impact of other AMD-associated SNPs on choroidal blood flow regulation, which would require larger sample sizes. In addition, we did not measure plasma complement components or activation fragments that have been shown to be associated with AMD indicating ongoing complement activation [42]. The mechanism by which rs1061170 polymorphism increases the risk for AMD is not fully understood. Most likely it alters the binding of CFH to sulfated glycosaminoglycans thereby inactivating C3b that becomes deposited [43]. In addition, the rs1061170 polymorphism reduces the ability of CFH to bind to malondialdehyde thereby inducing oxidative stress and enhanced lipid peroxidation [44]. Finally, although the time course of ChBF was different between groups, the 95% confidence intervals still overlapped in the pressure/flow curve, although a clear tendency towards a difference was seen. A larger sample size would be required to show a difference in the choroidal pressure-flow relationship between groups.

In conclusion the data of the present study show that a common rs1061170 polymorphism is associated with choroidal blood flow dysregulation in healthy young subjects. This polymorphism has been shown to be linked to AMD in a variety of previous studies where the main focus was directed towards inflammatory processes triggered by complement system dysfunction. Our study shows that rs1061170 may also be associated with vascular dysregulation and ischemia/hypoxia, which has been implicated in the pathogenesis of AMD.

Materials and Methods

Ethics Statement

The present study was performed in adherence to the Declaration of Helsinki and the Good Clinical Practice (GCP) guidelines. The study protocol was approved by the Ethics Committee of the Medical University of Vienna. (Clinicaltrials.gov: NCT00708929, http://www.clinicaltrials.gov/ct2/show/NCT00708929).

Changes to the original study protocol

The approved study protocol originally included a second group. These would have been women and men aged between 46 and 65. But due to difficulties in performing the measurements during squatting and the very poor data quality, this group was discontinued. Flicker light stimulation data are published separately.

Experimental Design

A total of 100 healthy male and female subjects aged between 18 and 45 years were enrolled in this study. The sample size of this pilot study was based on the genotype frequency for homozygous risk allele carriers of the rs1061170, which is approximately 14% in previous studies [45], [46].

Subjects were recruited from May 2010 till July 2011. The nature of the study was explained to all subjects and they gave written consent prior to participation. Each subject passed a screening examination including medical history and physical examination. Subjects were excluded if any abnormality was found during screening, unless the investigators considered the abnormality to be clinically irrelevant. Moreover, visual acuity was assessed using ETDRS charts and an ophthalmic examination, including slit lamp biomicroscopy and indirect funduscopy, was performed. Inclusion criteria were normal ophthalmic findings, ametropia of less than 3 diopters and anisometropia of less than 1 diopter.

All measurements were performed at the Department of Clinical Pharmacology/Medical University of Vienna, Austria and after a resting period of at least 20 minutes in a sitting position. Stability of blood pressure and pulse rate was verified by repeated measurements before the actual experiments were started.

The isometric exercise experiments comprised a three minutes continuous baseline recording of ChBF in a sitting position, followed by a six minutes recording with isometric exercise, which consisted of squatting in a position where the upper and the lower legs formed approximately a right angle in order to increase mean arterial blood pressure. At the beginning and at the end of the ChBF recording IOP was assessed. Blood pressure and pulse rate were measured every minute throughout the experiment.

Measurements

Systemic hemodynamics.

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP) were monitored on the upper arm by an automated oscillometric device. Pulse rate (PR) was automatically recorded from a finger pulse-oxymetric device (HP-CMS patient monitor, Hewlett Packard, Palo Alto, CA, USA). The performance of this system has been reported previously [47].

Laser Doppler flowmetry.

Continuous measurement of ChBF was performed by laser Doppler flowmetry as described by Riva et al. [18], [48]. With this technique, the vascularized tissue is illuminated by coherent laser light. From the laser Doppler power spectrum hemodynamic parameters can be determined based on a theory of light scattering in tissue. The following blood flow parameters were obtained: blood flow, velocity and volume. Velocity is the mean velocity of the red blood cells moving in the sampled tissue proportional to the mean Doppler frequency shift. Volume is the number of moving red blood cells in the sampled tissue proportional to the amount of Doppler shifted light. Blood flow was calculated as the product of velocity and volume. In the present study, a laser Doppler flowmeter, which has been described in detail previously, was used for the ChBF measurements [49], [50].

Intraocular pressure.

A slit-lamp mounted Goldmann applanation tonometer was used to measure intraocular pressure (IOP).

Genotyping of rs1061170.

DNA was isolated continuously from fresh EDTA-anticoagulated whole blood using a Gentra® Puregene® Blood Kit (Qiagen GmbH, Hilden, Germany). Samples were subsequently stored within the MedUni Vienna Biobank facility at −80°C until measurement. Genotyping was performed by means of real-time polymerase chain reaction (RT-PCR) on an ABI 7900HT Fast-Realtime thermocycler (Applied Biosystems, Rotkreuz, Switzerland) using sequence-specific, fluorescence-labeled TaqMan® probes with a minor groove binder and a non-fluorescent quencher. Oligonucleotide sequences were obtained from Goverdhan et al. [51]. RT-PCR was conducted in 384-well plates with a total volume of 5 µL per reaction consisting of 2.5 µL TaqMan® Genotyping Mastermix (Applied Biosystems), 500 nM of each primer (VBC Genomics, Vienna, Austria), 200 nM of each probe (Applied Biosystems) and 10 ng DNA. PCR conditions were 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Data was analyzed using SDS 2.3 sequence detection software (Applied Biosystems).

Data analysis

Ocular perfusion pressure was calculated as OPP  = 2/3*MAP-IOP [52]. We obtained IOP levels at baseline and at the end of the squatting period. From these data the IOP values during every single minute of squatting were calculated using linear regression analysis.

A repeated measures ANOVA model was used to analyze data. Grouping of subjects was done according to the results of rs1061170 genotyping. Differences between subjects were calculated based on the interaction between time and group. Post hoc analyses were done using planned comparisons. For this purpose the time effect was used to characterize the effect of squatting on the outcome parameters.

In addition, pressure-flow relationships were calculated. For this purpose the data were expressed as %change in OPP and choroidal blood flow values over baseline. The OPP values were then sorted according to ascending values and grouped into 9 intervals. A statistically significant deviation from baseline flow was defined when the 95% confidence interval did not overlap with the baseline value any more.

For data description percent changes from baseline were calculated. A p-value <0.05 was considered the level of significance. Statistical analysis was carried out using CSS Statistica for Windows® (Statsoft Inc., Version 6.0, Tulsa, California).

Author Contributions

Conceived and designed the experiments: RT HH SF RMW GG LS. Performed the experiments: RT SP DS AB ML SK RMW. Analyzed the data: RT HH SF RMW GG LS. Contributed reagents/materials/analysis tools: RT SP DS AB ML SK. Wrote the paper: RT SP HH SF DS AB ML SK RMW GG LS.

References

  1. 1. Coleman AL, Yu F (2008) Eye-related medicare costs for patients with age-related macular degeneration from 1995 to 1999. Ophthalmology 115: 18–25.
  2. 2. Chakravarthy U, Wong TY, Fletcher A, Piault E, Evans C, et al. (2010) Clinical risk factors for age-related macular degeneration: a systematic review and meta-analysis. BMC Ophthalmol 10: 31.
  3. 3. Chen J, Smith LE (2012) Protective inflammasome activation in AMD. Nat Med 18: 658–660.
  4. 4. Boltz A, Lasta M, Schmidl D, Kaya S, Garhofer G, et al. (2010) Risk factors for age-related macular degeneration. Spektrum Der Augenheilkunde 24: 296–304.
  5. 5. Bergeron-Sawitzke J, Gold B, Olsh A, Schlotterbeck S, Lemon K, et al. (2009) Multilocus analysis of age-related macular degeneration. Eur J Hum Genet 17: 1190–1199.
  6. 6. Tong Y, Liao J, Zhang Y, Zhou J, Zhang H, et al. (2010) LOC387715/HTRA1 gene polymorphisms and susceptibility to age-related macular degeneration: A HuGE review and meta-analysis. Mol Vis 16: 1958–1981.
  7. 7. Anderson DH, Radeke MJ, Gallo NB, Chapin EA, Johnson PT, et al. (2010) The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited. Prog Retin Eye Res 29: 95–112.
  8. 8. Donoso LA, Vrabec T, Kuivaniemi H (2010) The role of complement Factor H in age-related macular degeneration: a review. Surv Ophthalmol 55: 227–246.
  9. 9. Edwards AO, Ritter R 3rd, Abel KJ, Manning A, Panhuysen C, et al (2005) Complement factor H polymorphism and age-related macular degeneration. Science 308: 421–424.
  10. 10. Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, et al. (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308: 419–421.
  11. 11. Wegscheider BJ, Weger M, Renner W, Steinbrugger I, Marz W, et al. (2007) Association of complement factor H Y402H gene polymorphism with different subtypes of exudative age-related macular degeneration. Ophthalmology 114: 738–742.
  12. 12. Klein R, Knudtson MD, Lee KE, Klein BE (2009) Serum cystatin C level, kidney disease markers, and incidence of age-related macular degeneration: the Beaver Dam Eye Study. Arch Ophthalmol 127: 193–199.
  13. 13. Mullins RF, Russell SR, Anderson DH, Hageman GS (2000) Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J 14: 835–846.
  14. 14. Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, et al. (2001) An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res 20: 705–732.
  15. 15. Jylhava J, Eklund C, Jylha M, Hervonen A, Lehtimaki T, et al. (2009) Complement factor H 402His variant confers an increased mortality risk in Finnish nonagenarians: the Vitality 90+ study. Exp Gerontol 44: 297–299.
  16. 16. Jylhava J, Eklund C, Pessi T, Raitakari OT, Juonala M, et al. (2009) Genetics of C-reactive protein and complement factor H have an epistatic effect on carotid artery compliance: the Cardiovascular Risk in Young Finns Study. Clin Exp Immunol 155: 53–58.
  17. 17. Lundh von Leithner P, Kam JH, Bainbridge J, Catchpole I, Gough G, et al. (2009) Complement factor h is critical in the maintenance of retinal perfusion. Am J Pathol 175: 412–421.
  18. 18. Riva CE, Titze P, Hero M, Movaffaghy A, Petrig BL (1997) Choroidal blood flow during isometric exercises. Invest Ophthalmol Vis Sci 38: 2338–2343.
  19. 19. Luksch A, Polska E, Imhof A, Schering J, Fuchsjager-Mayrl G, et al. (2003) Role of NO in choroidal blood flow regulation during isometric exercise in healthy humans. Invest Ophthalmol Vis Sci 44: 734–739.
  20. 20. Fuchsjager-Mayrl G, Luksch A, Malec M, Polska E, Wolzt M, et al. (2003) Role of endothelin-1 in choroidal blood flow regulation during isometric exercise in healthy humans. Invest Ophthalmol Vis Sci 44: 728–733.
  21. 21. Polska E, Luksch A, Schering J, Frank B, Imhof A, et al. (2003) Propranolol and atropine do not alter choroidal blood flow regulation during isometric exercise in healthy humans. Microvasc Res 65: 39–44.
  22. 22. Schmidl D, Garhofer G, Schmetterer L (2011) The complex interaction between ocular perfusion pressure and ocular blood flow – relevance for glaucoma. Exp Eye Res 93: 141–155.
  23. 23. Zhou J, Jang YP, Kim SR, Sparrow JR (2006) Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium. Proc Natl Acad Sci U S A 103: 16182–16187.
  24. 24. Machalinska A, Kawa MP, Marlicz W, Machalinski B (2012) Complement system activation and endothelial dysfunction in patients with age-related macular degeneration (AMD): possible relationship between AMD and atherosclerosis. Acta Ophthalmol 90: 695–703.
  25. 25. Schaumberg DA, Christen WG, Buring JE, Glynn RJ, Rifai N, et al. (2007) High-sensitivity C-reactive protein, other markers of inflammation, and the incidence of macular degeneration in women. Arch Ophthalmol 125: 300–305.
  26. 26. Kardys I, Klaver CC, Despriet DD, Bergen AA, Uitterlinden AG, et al. (2006) A common polymorphism in the complement factor H gene is associated with increased risk of myocardial infarction: the Rotterdam Study. J Am Coll Cardiol 47: 1568–1575.
  27. 27. Volcik KA, Ballantyne CM, Braun MC, Coresh J, Mosley TH, et al. (2008) Association of the complement factor H Y402H polymorphism with cardiovascular disease is dependent upon hypertension status: The ARIC study. Am J Hypertens 21: 533–538.
  28. 28. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol 119: 1417–1436.
  29. 29. Klein ML, Francis PJ, Rosner B, Reynolds R, Hamon SC, et al. (2008) CFH and LOC387715/ARMS2 genotypes and treatment with antioxidants and zinc for age-related macular degeneration. Ophthalmology 115: 1019–1025.
  30. 30. Ho L, van Leeuwen R, Witteman JC, van Duijn CM, Uitterlinden AG, et al. (2011) Reducing the genetic risk of age-related macular degeneration with dietary antioxidants, zinc, and omega-3 fatty acids: the Rotterdam study. Arch Ophthalmol 129: 758–766.
  31. 31. Friedman E, Krupsky S, Lane AM, Oak SS, Friedman ES, et al. (1995) Ocular blood flow velocity in age-related macular degeneration. Ophthalmology 102: 640–646.
  32. 32. Grunwald JE, Hariprasad SM, DuPont J, Maguire MG, Fine SL, et al. (1998) Foveolar choroidal blood flow in age-related macular degeneration. Invest Ophthalmol Vis Sci 39: 385–390.
  33. 33. Pauleikhoff D, Spital G, Radermacher M, Brumm GA, Lommatzsch A, et al. (1999) A fluorescein and indocyanine green angiographic study of choriocapillaris in age-related macular disease. Arch Ophthalmol 117: 1353–1358.
  34. 34. Ciulla TA, Harris A, Kagemann L, Danis RP, Pratt LM, et al. (2002) Choroidal perfusion perturbations in non-neovascular age related macular degeneration. Br J Ophthalmol 86: 209–213.
  35. 35. Uretmen O, Akkin C, Erakgun T, Killi R (2003) Color Doppler imaging of choroidal circulation in patients with asymmetric age-related macular degeneration. Ophthalmologica 217: 137–142.
  36. 36. Grunwald JE, Metelitsina TI, Dupont JC, Ying GS, Maguire MG (2005) Reduced foveolar choroidal blood flow in eyes with increasing AMD severity. Invest Ophthalmol Vis Sci 46: 1033–1038.
  37. 37. Metelitsina TI, Grunwald JE, DuPont JC, Ying GS (2006) Effect of systemic hypertension on foveolar choroidal blood flow in age related macular degeneration. Br J Ophthalmol 90: 342–346.
  38. 38. Pournaras CJ, Logean E, Riva CE, Petrig BL, Chamot SR, et al. (2006) Regulation of subfoveal choroidal blood flow in age-related macular degeneration. Invest Ophthalmol Vis Sci 47: 1581–1586.
  39. 39. Metelitsina TI, Grunwald JE, DuPont JC, Ying GS, Brucker AJ, et al. (2008) Foveolar choroidal circulation and choroidal neovascularization in age-related macular degeneration. Invest Ophthalmol Vis Sci 49: 358–363.
  40. 40. Boltz A, Luksch A, Wimpissinger B, Maar N, Weigert G, et al. (2010) Choroidal blood flow and progression of age-related macular degeneration in the fellow eye in patients with unilateral choroidal neovascularization. Invest Ophthalmol Vis Sci 51: 4220–4225.
  41. 41. Vinores SA, Xiao WH, Aslam S, Shen J, Oshima Y, et al. (2006) Implication of the hypoxia response element of the Vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development. J Cell Physiol 206: 749–758.
  42. 42. Reynolds R, Hartnett ME, Atkinson JP, Giclas PC, Rosner B, et al. (2009) Plasma complement components and activation fragments: associations with age-related macular degeneration genotypes and phenotypes. Invest Ophthalmol Vis Sci 50: 5818–5827.
  43. 43. Clark SJ, Bishop PN, Day AJ (2010) Complement factor H and age-related macular degeneration: the role of glycosaminoglycan recognition in disease pathology. Biochem Soc Trans 38: 1342–1348.
  44. 44. Weismann D, Hartvigsen K, Lauer N, Bennett KL, Scholl HP, et al. (2011) Complement factor H binds malondialdehyde epitopes and protects from oxidative stress. Nature 478: 76–81.
  45. 45. Thakkinstian A, Han P, McEvoy M, Smith W, Hoh J, et al. (2006) Systematic review and meta-analysis of the association between complement factor H Y402H polymorphisms and age-related macular degeneration. Hum Mol Genet 15: 2784–2790.
  46. 46. Zee RY, Diehl KA, Ridker PM (2006) Complement factor H Y402H gene polymorphism, C-reactive protein, and risk of incident myocardial infarction, ischaemic stroke, and venous thromboembolism: a nested case-control study. Atherosclerosis 187: 332–335.
  47. 47. Wolzt M, Schmetterer L, Rheinberger A, Salomon A, Unfried C, et al. (1995) Comparison of non-invasive methods for the assessment of haemodynamic drug effects in healthy male and female volunteers: sex differences in cardiovascular responsiveness. Br J Clin Pharmacol 39: 347–359.
  48. 48. Riva CE, Cranstoun SD, Grunwald JE, Petrig BL (1994) Choroidal blood flow in the foveal region of the human ocular fundus. Invest Ophthalmol Vis Sci 35: 4273–4281.
  49. 49. Geiser MH, Riva CE, Diermann U (1999) [Measuring choroid blood flow with a new confocal laser Doppler device]. Klin Monbl Augenheilkd 214: 285–287.
  50. 50. Geiser MH, Riva CE, Dorner GT, Diermann U, Luksch A, et al. (2000) Response of choroidal blood flow in the foveal region to hyperoxia and hyperoxia-hypercapnia. Curr Eye Res 21: 669–676.
  51. 51. Goverdhan SV, Hannan S, Newsom RB, Luff AJ, Griffiths H, et al. (2008) An analysis of the CFH Y402H genotype in AMD patients and controls from the UK, and response to PDT treatment. Eye (Lond) 22: 849–854.
  52. 52. Robinson F, Riva CE, Grunwald JE, Petrig BL, Sinclair SH (1986) Retinal blood flow autoregulation in response to an acute increase in blood pressure. Invest Ophthalmol Vis Sci 27: 722–726.