September 2014
Volume 55, Issue 9
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Physiology and Pharmacology  |   September 2014
Vascular Dysfunction in Ocular Blood Flow Regulation: Impact of Reactive Oxygen Species in an Experimental Setup
Author Affiliations & Notes
  • Lars Wagenfeld
    University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Hamburg, Germany
  • Sonja Weiss
    University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Hamburg, Germany
  • Maren Klemm
    University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Hamburg, Germany
  • Gisbert Richard
    University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Hamburg, Germany
  • Oliver Zeitz
    University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Hamburg, Germany
    Global Clinical Development Ophthalmology, Bayer Pharma AG, Berlin, Germany
  • Correspondence: Lars Wagenfeld, University Medical Center Hamburg-Eppendorf, Department of Ophthalmology, Martinistr. 52, D-20246 Hamburg, Germany; [email protected]
Investigative Ophthalmology & Visual Science September 2014, Vol.55, 5531-5536. doi:https://doi.org/10.1167/iovs.14-14032
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      Lars Wagenfeld, Sonja Weiss, Maren Klemm, Gisbert Richard, Oliver Zeitz; Vascular Dysfunction in Ocular Blood Flow Regulation: Impact of Reactive Oxygen Species in an Experimental Setup. Invest. Ophthalmol. Vis. Sci. 2014;55(9):5531-5536. https://doi.org/10.1167/iovs.14-14032.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Glaucoma is associated with an altered blood flow and increased levels of reactive oxygen species (ROS). Reactive oxygen species can have opposing influences on the tone of a vessel; depending on the condition and type of the vessel, ROS can induce vasodilation or vasoconstriction. In the present study, we investigated the impact of ROS on the tone of rat ophthalmic arteries under various conditions and present data on the underlying mechanisms.

Methods.: Freshly dissected rat ophthalmic arteries were pressurized in a perfusion setup to 80 mm Hg, at which a stable myogenic tone was observed. After various pretreatments (e.g., removal of endothelium, partial depolarization to −41 mV, blocking of the Na+/Ca2+-exchanger (NCX) in reverse mode by KB-R7943, or blocking of the Na+/K+-ATPase by ouabain), the vessels were exposed to ROS. Vessel diameter was continuously recorded and values before and after treatment compared.

Results.: Stable myogenic tone of vessels with and without endothelium was established at a pressure of 80 mm Hg. At the physiological resting membrane potential, ROS exposure led to a significant vasodilatation, which was significantly reduced by pretreatment with ouabain. After depolarization to −41 mV, ROS exposure led to vasoconstriction. Blocking the NCX in reverse mode using KB-R7943 completely abolished this ROS-induced vasoconstriction.

Conclusions.: At resting potential, ROS provoke dilation; however, in precontracted vessels they act synergistically and induce further vasoconstriction. In diseases involving altered blood flow through altered vascular tone (e.g., vasospasms), ROS may influence blood flow and may thereby contribute indirectly to further disease progression.

Introduction
Disturbed ocular blood flow is involved in the pathogenesis of numerous ocular diseases. 13 It has been shown that choroidal and papillary perfusion is altered in primary open-angle glaucoma (POAG). There are data indicating that such changed perfusion is associated with glaucoma progression. 4 Differences between glaucoma and nonglaucoma subjects become particularly obvious if autoregulation of ocular perfusion is challenged during the measurement. Such challenges may include a change in posture, cold provocation, or exposure to flickering light. 57 This indicates that the change in ocular perfusion in glaucoma subjects is more likely to be due to an alteration of the regulation of the perfusion than to an obstruction of the vessels, which occurs, for example, in arteriosclerosis. Some authors regard these regulatory dysfunctions as vasospasms as they also occur in migraine and Raynaud syndrome. 3 While clinically many POAG patients exhibit signs of changed regulation of ocular and extraocular perfusion, whether these changes are of primary nature contributing to the disease or whether they pose an epiphenomenon remains to be proven. Certain findings suggest a primary nature (e.g., the association of particular changes in ocular hemodynamics with progression of glaucoma or even differences in perfusion at rest). 8 Beside the gaps in the present knowledge about the clinical implications of the changes in ocular perfusion observed in glaucoma, there is also a lack of understanding of molecular mechanisms accounting for these changes. 
A higher level of intraocular oxidative stress, induced by increased levels of reactive oxygen species (ROS), has been reported not only for glaucoma patients 9,10 but also for other ocular diseases with disturbed blood flow, such as age-related macular degeneration 11 and diabetic retinopathy. 12,13 It is possible that ROS influence ocular blood flow since it has been shown that oxidative stress can influence vascular tone in other vascular beds. Under certain conditions, ROS have been found to provoke vasospasms, 14 whereas, in other studies on the effects of ROS on vessels, vasodilative effects have been reported. 15  
Concerning the ocular vasculature it was shown that ROS can influence vascular tone in the retina, 16 but experimental data on the influence of ROS on ocular vasculature are rare. Our group previously described a system in which ocular vessels could be treated with defined concentrations of ROS. Oxidative stress caused a transient and reversible vasoconstriction, which had emerging parallels to vasospasms. 17 In a setup for perfusing isolated vessels, we also found vasoconstriction in vessels without endothelium, but vessels with intact endothelium showed a relaxation after ROS treatment. Therefore, we concluded that the response of the vessel to ROS depends on endothelium and the status of the vessel. 18 Beside the methods of vessel preparation used in these two previous studies, 17,18 another key difference was the way in which preconstriction was induced. Whereas in the first study, this was achieved by partially depolarizing the vessels; in the second study, it was provoked by pressurizing the vessel to induce a myogenic tone. 
The aim of the present study was to investigate ROS-induced vasoactive properties in a perfusion setup to gain more knowledge on the effects of ROS on vascular tone of ocular vessels. The fact that ROS can provoke both dilation and relaxation suggests that more than one mechanism can be activated by ROS. Since endothelium and membrane potential may play a role in determining the effect of ROS, this study focused on the effect of endothelium and membrane potential, and investigated the possible underlying mechanisms of action. 
Methods
Animals and Preparation
Adult Wistar rats were killed by asphyxiation with CO2. Ophthalmic arteries were dissected directly postmortem and placed in a cold (4°C) physiological salt solution (PSS) until use to reduce metabolism of the muscle cells. The PSS was a modified Krebs-Henseleit buffer containing: 120 mM NaCl, 5 mM KCl, 2.0 mM MgSO4, 1.2 mM NaH2PO4, 20 mM HEPES, 10 mM glucose, and 1.75 mM CaCl2. The pH was adjusted to 7.4. All experimental steps were conducted in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local authorities. Membrane potential was modified by changing the extracellular K+ concentration and calculated using the Nernst equation, which at 37°C is given by E = −61 mV x log10 ([K+]e/[K+]i). At the end of each experiment, the vessels were maximally contracted by depolarization to −4 mV by increasing the extracellular K+ to 120 mM in order to test if the smooth muscle cells of the vessel were still able to contract. 
Removal of Endothelium
To remove the endothelium, vessels were perfused with 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 42 seconds. We had shown previously that, under these conditions, the vessels showed no dilation to acetylcholine, but still developed myogenic tone. 18 In addition, they contracted further on depolarization to −4 mV, showing that muscle cells were still intact. After these experiments, some of the vessels were also examined histologically to confirm that the endothelium had been removed. This histology showed that the endothelium had been nearly completely removed, with only a few residual endothelium cells remaining (data not shown). 
Experimental Setup
Vessels were placed in a perfusion setup from DMT (Aarhus, Denmark). Each end of the vessel was tightly connected to a glass pipette. These pipettes were joined to a tube system in which the intraluminal pressures and flow rates could be measured and adjusted. The vessel and attached glass pipettes were placed in an organ bath filled with PSS. The whole system was heated to 37°C (slowly to avoid gas bubble formation). The vessel was superfused with PSS that was saturated with oxygen to ensure experimental conditions close to the in vivo situation. Using this setup, intra- and extraluminal spaces were separated. Similar setups are often used in studies on vascular physiology. 19,20 The experimental setup is schematically drawn in Figure 1
Figure 1
 
Schematic drawing of the experimental setup, based on Wagenfeld et al. 18 With permission from Springer Science+Business Media: Wagenfeld L, von Domarus F, Weiss S, Klemm M, Richard G, Zeitz O. The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium. Graefes Arch Clin Exp Ophthalmol. 2013;251:2339–2344. Copyright Springer-Verlag Berlin Heidelberg 2013.
Figure 1
 
Schematic drawing of the experimental setup, based on Wagenfeld et al. 18 With permission from Springer Science+Business Media: Wagenfeld L, von Domarus F, Weiss S, Klemm M, Richard G, Zeitz O. The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium. Graefes Arch Clin Exp Ophthalmol. 2013;251:2339–2344. Copyright Springer-Verlag Berlin Heidelberg 2013.
Measurements
The vessel diameter and the corresponding intraluminal pressure were recorded continuously (acquisition frequency: 1 Hz) using a digital edge-detection system together with data acquisition software (MyoView; DMT; Fig. 2). Further experiments were performed in vessels with stable myogenic tone. For presentation and better comparability, values before and after manipulation were normalized, and changes were expressed in percent as follows: ([vessel diameterafter – vessel diameterbefore] / vessel diameterafter) × 100. 
Figure 2
 
Measurement of vessel diameter. (A) Vessel mounted in the pressure myograph. (B) Digital edge detection.
Figure 2
 
Measurement of vessel diameter. (A) Vessel mounted in the pressure myograph. (B) Digital edge detection.
Experimental Design: Myogenic Tone
Previously, to establish the optimum settings for subsequent experiments, we evaluated the myogenic tone in our vessels under various conditions. 18 Vessels were pressurized in steps of 10 mm Hg from 0 to 180 mm Hg for 5 minutes each. The first cycle was run with Ca2+ in the solution to record the vessel diameter with intact myogenic tone (T1). After removing all Ca2+, the pressure steps were repeated to evaluate the diameter during passive dilation (T2). The myogenic tone was calculated as: (diameterT2 – diameterT1) / diameterT2 × 100 and expressed in percent. 18 The underlying data of that study indicated that at 80 mm Hg all vessels developed stable myogenic tone; therefore, this pressure was used for the experiments described here. 
Experimental Design: ROS Effects
After the vessels were precontracted by myogenic tone, they were exposed to hydroxyl radicals as described previously. 17,18,21 Hydroxyl radicals were generated using the Fenton reaction from H2O2 and the iron redox chelate Fe3+/nitrilotriacetate (FeNTA): 
The sum of reactions (1) and (2) is the Fenton reaction: 
Since ROS have a high reactivity toward most organic materials, H2O2 was infused close to the preparation over a separate inflow with a volume-controlled pump. Vessels were exposed to radicals for 20 seconds. Final concentrations in the extracellular buffer were 4 mM H2O2 and 30 μM FeNTA. 
Pharmacological Blocking
Blocking of the Na+/K+-ATPase.
For some experiments, vessels were exposed to 10−5M ouabain (4995; Merck Millipore, Billerica, MA, USA)—a cardioactive glycoside that blocks the Na+/K+-ATPase—before ROS were added. 
Blocking of the Na+/Ca2+-Exchangers in Reverse Mode.
In another set of experiments, the vessels were precontracted by partial depolarization to −41 mV Nernst potential by increasing extracellular K+ to 30 mM. Some experiments with partially depolarized vessels were also performed after pretreatment with 10−6M KB-R7943 (420336; Merck Millipore), which inhibits the influx/reverse mode of Na+/Ca2+-exchangers (NCXs). 22  
Statistics
The recorded diameters before and after ROS treatment were exported to spreadsheet (Microsoft Excel 2011; Microsoft Corp., Redmond, WA, USA) and statistical software (SPSS 21; IBM Corp., Armonk, NY, USA). Values before and after certain manipulations were compared using a Wilcoxon-test for nonparametric values. The change of diameters (normalized to the premanipulation value) provoked by ROS was compared between the groups using a Mann-Whitney U-test. 
Results
Having previously established a pressure level for stable myogenic tone at 80 mm Hg, the main experiments were conducted with myogenic tone developed at this pressure. The mean passive vessel diameter at 80 mm Hg was 308.4 ± 14.8 μm; in the presence of calcium in the perfusing solution the mean diameter was 259.0 ± 15.5 μm, yielding in a mean myogenic tone of 16.0% ± 1.8% (n = 7) in vessels with endothelium. After removal of the endothelium, the mean vessel diameter was 326.2 ± 23.1 μm under active dilation and 383.1 ± 15.9 μm for passive dilation, resulting in a myogenic tone of 14.9% ± 6.4% at 80 mm Hg (n = 6). In all of our experiments the vessel diameter, indirectly representing the myogenic tone, stayed stable for at least 15 minutes prior to ROS exposure (all values, measured continuously every second, were within ± 3% compared to baseline; not significant). Under control conditions, the potential for maximum constriction was preserved (ranging from 14.7% ± 3.9% to 26.3% ± 5.1%). 
ROS Effect at Physiological Resting Membrane Potential
At the physiological resting membrane potential in vessels with myogenic tone at 80 mm Hg and no further intervention, ROS exposure led to a significant vasodilatation, which was independent of the presence of endothelium. 
ROS Effect at Membrane Potential of −40 mV
On increasing the extracellular K+ to 30 mM, which resulted in a depolarization to −40 mV, a slight constriction was seen in vessels with or without endothelium. This was significant in vessels with endothelium (with endothelium: −6% ± 1.7%; P = 0.009; n = 10, without endothelium: −12% ± 4.4%; P = 0.07; n = 6). However, under these precontracted conditions, exposing the vessels to ROS no longer led to dilation, but to constriction. 
Further experiments were performed to gain an insight into the potential mechanisms for constriction and dilation. Since it was previously shown that the reverse mode of the NCX accounts for calcium overload in radical-exposed cardiomyocytes and neurons, 22 we blocked the NCX reverse mode in some cases using KB-R7943. In addition, since ROS may stimulate the Na+/K+-ATPase, 23 and thereby may lead to repolarization and loss of vasotonus, we inhibited the Na+/K+-ATPase with ouabain. 
Blocking of NCX in Reverse Mode
In precontracted vessels exposed to ROS, KB-R7943 completely abolished ROS-induced vasoconstriction. 
Blocking of the Na+/K+-ATPase
In the presence of ouabain, there was still a trend toward slight dilation of the nonprecontracted vessel, but this was significantly reduced compared with experiments without ouabain. 
Results are shown in Figure 3
Figure 3
 
Overview of vessel diameter changes provoked by ROS after certain pretreatments.
Figure 3
 
Overview of vessel diameter changes provoked by ROS after certain pretreatments.
Discussion
As hypothesized, ROS can influence the myogenic tone and vascular diameter. Depending on the actual membrane potential of the vessel, radicals can trigger either dilation or constriction. The endothelium does not seem to play an important role for relaxation or dilation in response to ROS. 
Oxidative stress occurs if oxygen and free electrons (energy) are available in excess. Because of the high blood flow and direct influence of light, oxidative stress is well known to occur in the choroid and retina. 24 Moreover, cycles of ischemia and reperfusion are also typical in such situations: During ischemia free electrons accumulate in the respiratory chain. After transition to reperfusion, oxygen availability increases over a short period of time, and free electrons can convert oxygen to a radical. Hydrogen peroxide is known to play a role in the regulation of coronary blood flow, dilating the vessels at low concentration levels, while acting as an antagonist to nitric oxide at higher levels. 25 The physiological role of ROS could, therefore, be a factor for vasodilation, which has been described in various previous articles. It has been hypothesized that ROS react as an endothelium-derived hyperpolarizing factor (EDHF), 26,27 although it is unclear whether the action is promoted by the ROS or H2O2. 26,28 The EDHF leads to a hyperpolarization that results in a reduced influx of Ca2+ into the muscle cells. This may be promoted by an activation of the Na+/H+-exchanger (NHE), 29 which produces a rise in intracellular Na+. The increased levels of Na+ lead to at least two effects. The first is that Na+/K+-ATPase is activated to eliminate intracellular Na+, leading to a hyperpolarization and subsequent relaxation of the smooth muscle cells. That ouabain blocks the relaxation in our experiments supports this theory, as it blocks the Na+/K+-ATPase and, therefore, eliminates a part of the downstream action. The second effect is that NCX is activated in reverse mode to lower intracellular Na+ levels. This also results in increased intracellular Ca2+ levels, which is normally overruled by the hyperpolarization but leads to a constriction under predepolarized conditions. The involvement of the NCX in reverse mode with resulting increased intracellular Ca2+ is supported by the finding that blocking the NCX in reverse mode using KB-R7943 leads to reduced constriction on ROS treatment. 
While our data could support the theory that ROS react as an EDHF (released from the endothelium), it seems unlikely that the ROS interact with the endothelium for this action, because we see little difference between vessels with or without endothelium. The ROS could act as endothelium-independent vasoactive substances. We cannot answer the question of whether ROS or H2O2 promotes an activation of the NHE as we used H2O2 to generate ROS in the organ bath. Also, ROS can be catalyzed to H2O2 in living cells and ROS are very instable chemical formations that interact directly with surrounding molecules. 30 Our experiments were designed to generate ROS directly in the organ bath close to the muscle cells of the vessels, but we cannot rule out that H2O2 could promote a part of the action. 
In precontracted vessels, ROS led to a vasoconstriction that was blocked by KB-R7943 in our experiments. Both facts—that precontacted vessels react with further contraction and that KB-R7943 blocks this action—support the theory that the mechanism of action is as an EDHF. The fact that vessels that are precontracted by depolarization constrict to ROS indicates that the activation of the Na+/K+-ATPase cannot change the membrane potential sufficiently. 
Besides this switch in ROS effect on vessel tone, it is also possible that a longer influence of ROS on the vessel can modify the effect of ROS on vascular tone by changing how the Na+/K+-ATPase reacts to ROS. 31  
Reactive oxygen species have been shown to play an important role in endothelium-dependent contraction (EDC) and can potentiate EDC under certain disease conditions such as hypertension. 32 Our findings show that ROS can also lead to vasoconstriction in precontracted vessels. Both findings show that the presumed physiological role of ROS in promoting vasodilation can be reversed under certain disease conditions. While all experiments on the influence of ROS have been tested at a level where a stable myogenic tone was produced, the limitation to one pressure level is one drawback of the presented study. 
Conclusions
This work shows that ROS influence vascular tone. The effect of ROS depends on membrane potential. At resting potential, ROS provoke dilation; in precontracted vessels they act synergistically and induce further vasoconstriction. In diseases such as glaucoma, which is associated with both altered blood flow due to vasospasms and increased oxidative stress, ROS may influence blood flow and may, thereby, contribute indirectly to further disease progression. 
Acknowledgments
Supported by grants from the Ernst und Berta Grimmke Stiftung, Ernst und Claere Jung Stiftung, and the Werner Otto Stiftung (LW, OZ). 
The authors alone are responsible for the content and writing of the paper. 
Disclosure: L. Wagenfeld, None; S. Weiss, None; M. Klemm, None; G. Richard, None; O. Zeitz, Bayer AG (E) 
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Footnotes
 LW and SW contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Schematic drawing of the experimental setup, based on Wagenfeld et al. 18 With permission from Springer Science+Business Media: Wagenfeld L, von Domarus F, Weiss S, Klemm M, Richard G, Zeitz O. The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium. Graefes Arch Clin Exp Ophthalmol. 2013;251:2339–2344. Copyright Springer-Verlag Berlin Heidelberg 2013.
Figure 1
 
Schematic drawing of the experimental setup, based on Wagenfeld et al. 18 With permission from Springer Science+Business Media: Wagenfeld L, von Domarus F, Weiss S, Klemm M, Richard G, Zeitz O. The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium. Graefes Arch Clin Exp Ophthalmol. 2013;251:2339–2344. Copyright Springer-Verlag Berlin Heidelberg 2013.
Figure 2
 
Measurement of vessel diameter. (A) Vessel mounted in the pressure myograph. (B) Digital edge detection.
Figure 2
 
Measurement of vessel diameter. (A) Vessel mounted in the pressure myograph. (B) Digital edge detection.
Figure 3
 
Overview of vessel diameter changes provoked by ROS after certain pretreatments.
Figure 3
 
Overview of vessel diameter changes provoked by ROS after certain pretreatments.
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