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1From the Departments of Ophthalmology and Visual Sciences and 2Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.
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Abstract |
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METHODS. In pericyte-containing retinal microvessels that were freshly isolated from nondiabetic and streptozotocin-injected rats, cells with pores were identified by the uptake of YO-PRO-1. Cell viability was assayed by trypan blue dye exclusion, and cleaved caspase-3 immunoreactivity, TUNEL positivity, and nuclear morphology were used to detect apoptotic cells. Patch-clamp recordings assessed electrophysiological parameters.
RESULTS. Activation of P2X7 receptors caused large pores to form and apoptosis to occur in retinal capillaries of nondiabetic and diabetic rats. Of importance to diabetes, the agonist concentration needed to open pores and trigger apoptosis decreased markedly soon after the onset of streptozotocin-induced hyperglycemia. However, despite this increased sensitivity, diabetes minimally affected the P2X7-induced ionic currents. Thus, rather than upregulate the number of functional P2X7 receptor/channels, diabetes appears to facilitate the channel-to-pore transition that occurs during activation of these purinoceptors. In this way, normally nonlethal concentrations of P2X7 ligands may trigger apoptosis in microvessels of the diabetic retina.
CONCLUSIONS. A diabetes-induced increase in the vulnerability of retinal microvessels to the lethal effect of P2X7 receptor activation may be a previously unrecognized mechanism by which diabetic retinopathy progresses.
At present, the mechanisms by which diabetes induces apoptosis in the retinal microvasculature remain uncertain, although oxidative stress, formation of advanced glycation end products, upregulation of protein kinase C, increased polyol pathway flux, and focal leukostasis may be important.5 6 In fact, multiple lethal pathways may be activated during chronic hyperglycemia.7
In this study, we tested the hypothesis that diabetes adversely affects retinal microvessels by increasing their vulnerability to potentially lethal consequences of having their P2X7 purinoceptors activated. In capillaries of nondiabetic retinas, we have recently found that these ligand-gated cation channels mediate the vasoconstrictive action of extracellular ATP, which is likely to be a glial-to-vascular signal.8 However, in addition to this putative physiological role, our earlier study also suggested a potentially pathological effect of activating microvascular P2X7 receptors. Namely, at high agonist concentrations, ligand binding not only opens cation channels, but also results in the formation of large transmembrane pores.8 The opening of similar pores in other cell types is known to cause cell death by disrupting ionic gradients and/or by providing pathways for an efflux of vital intracellular molecules of 900 Da or less.9 10 11 12
Based on experiments using pericyte-containing microvessels freshly isolated from the adult rat retina, we now report that soon after the onset of streptozotocin (STZ)-induced diabetes, markedly lower P2X7 agonist concentrations effectively opened pores and triggered apoptosis in the retinal microvasculature. Our observations point to the previously unappreciated possibility that microvascular damage in the diabetic retina may be mediated by receptors for vasoactive molecules.
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Methods |
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Cell Viability Assay
As detailed previously,15 microvessels were exposed to 0.04% trypan blue. Cells that did not exclude trypan blue were classified as dead. The validity of this often-used viability assay did not appear to be compromised by the formation of P2X7 pores. Specifically, consistent with trypan blue (961 Da) not passing through these pores, exposure for 1 hour to the P2X7 agonist benzoylbenzoyl-adenosine triphosphate (BzATP, 100 µM), induced trypan blue staining in only approximately 2% of the microvascular cells (see the Results section, Fig. 1C ) even though P2X7 pores are induced in more than half of the cells after one hour of BzATP exposure.8 The percentage of cells stained with this dye was determined by examining isolated microvessels at x100 magnification with an inverted microscope equipped with bright-field optics. Because we could not establish with certainty whether a cell stained with trypan blue was an endothelial cell or a pericyte, subclassification of microvascular cells into these two types was not feasible. At least 200 microvascular cells per coverslip were counted. Except where noted, cell viability assays were performed on microvessels maintained under control or experimental conditions for 24 hours.
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Vessels to be assessed for cleaved capase-3 immunoreactivity were exposed to 0.3% hydrogen peroxide in PBS for 30 minutes (to block endogenous peroxidase activity), to 0.5% Triton X-100 for 60 minutes, and then to rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA) for 16 hours at 4°C. Subsequently, the coverslips were incubated with biotin-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 for 1 hour and subsequently with the avidin-biotin-peroxidase complex (ABC method; Vector Laboratories) at 1:100 for 30 minutes. After development with diaminobenzidine, at least 125 (mean = 207 ± 13) microvascular cells per coverslip (n = 21) were examined by bright-field and phase-contrast optics to detect immunopositive and nonstained nuclei, respectively.
For the TUNEL (TdT-mediated dUTP nick-end) assay, microvessels were exposed to 0.5% Triton X-100 for 30 minutes and then a commercially available kit (DeadEnd; Promega, Madison, WI) was used to incorporate biotinylated dUTP at 3'-OH DNA ends using terminal deoxynucleotidyl transferase. Microvessels were then washed in PBS, exposed to 0.3% hydrogen peroxide for 30 minutes, incubated in a horseradish peroxidase-streptavidin solution (RTU; Vector Laboratories) plus 0.5% Triton X-100 for 50 hours at 4°C. After the use of the ABC method and development with diaminobenzidine, at least 125 microvascular cells (mean = 255 ± 42) per coverslip (n = 12) were examined.
Assessment of nuclear morphology was performed on microvessels that were exposed to Hoechst dye 33258 (8 µg · mL-1 in PBS) for 15 minutes at room temperature. Nuclear morphology was assessed by viewing at x400 through a UV filter of an electron microscope (model E300 or E800 Eclipse; Nikon, Tokyo, Japan) at least 125 (mean = 219 ± 19) microvascular cells per coverslip (n = 23) were examined.
Experimental Model of Diabetes
After an overnight fast, 4- to 5-week-old Long-Evans rats received an intraperitoneal injection of STZ (75 mg · kg-1) diluted in 0.8 mL citrate buffer. Animals were maintained on a 12-hour alternating light-dark cycle and received food and water ad libitum. Immediately before the harvesting of the retinal microvessels, the blood glucose level was 329 ± 18 mg · dL-1 (n = 20).
Electrophysiology
As detailed elsewhere,13 the perforated-patch configuration of the patch-clamp technique was used to monitor ionic currents from pericytes located on microvessels that had been isolated from a retina within 3 hours. A coverslip with microvessels was placed in a recording chamber, which was perfused (
2 mL · min-1) with solution A, without or with various concentrations of the P2X7 agonist BzATP. Vessels were examined at x300 magnification with an inverted microscope equipped with phase-contrast optics. Pericytes were identified by their characteristic "bump-on-a-log" location on the abluminal wall of microvessels.13 16 Pipettes, which had resistances of approximately 5 M
and contained a solution consisting of 50 mM KCl, 64 mM K2SO4, 6 mM MgCl2, 10 mM K-HEPES, 240 µg · mL-1 amphotericin, and 240 µg · mL-1 nystatin at pH 7.4 with the osmolarity adjusted to 280 mOsmol · L-1, were mounted in the holder of a patch-clamp amplifier (Dagan Corp., Minneapolis, MN) and then sealed to the cell bodies of phase-bright pericytes. As amphotericin/nystatin perforated the patch, the access resistance to the pericytes studied decreased to less than 25 M within 5 minutes. Pericytes were voltage-clamped, and currents were filtered at 1 kHz with a four-pole Bessel filter, digitally sampled every 250 ms using a data-acquisition system (DigiData 1200B; Axon Instruments, Union City, CA) and stored by a computer equipped with commercial software (pCLAMP 8; Axon Instruments; and Origin, ver. 6.1; OriginLab, Northampton, MA) for data analysis and graphics display. Voltage steps were controlled by the pCLAMP software. Membrane resistance was calculated by measuring steady state currents at -103 mV and -8 mV. The zero current potential was defined as the membrane potential of a sampled pericyte; adjustment for the calculated liquid junction potential17 was made after data collection. Circuits of an expander module (model 3910; Dagan) were used to estimate cell membrane capacitance. As detailed recently,8 the peak amplitude of the inward current induced during continuous exposure to BzATP was quantified from continuous recordings of pericytes that were voltage clamped at -103 mV, which was the equilibrium potential for potassium.
YO-PRO-1 Uptake
A coverslip with freshly isolated pericyte-containing microvessels was positioned in a chamber located on the stage of a transmission electron microscope (Eclipse TE300; Nikon) equipped for fluorescence and differential interference contrast (DIC) optics with 20x objectives. Initially, microvessels were exposed to solution A supplemented with 5 µM YO-PRO-1 (Molecular Probes, Eugene, OR), which is a 629-Da propidium di-iodide dye that on entry into a cell, binds to nucleic acids and thereby causes detectable fluorescence. After 15 minutes, microvessels were then exposed for 60 minutes at room temperature to the YO-PRO-containing solution without or with the addition of a purinergic agonist. Fluorescence was detected with excitation and emission wavelengths of 475 and 510 nm, respectively; the light source was a high-intensity mercury lamp coupled to a monochromator (Cairn Research, Faversham, UK). DIC optics facilitated detection of cells without fluorescence. At least 80 (mean = 100 ± 2) microvascular cells per coverslip (n = 37) were examined.
Chemicals
Unless noted otherwise, chemicals were obtained from Sigma/RBI (St. Louis, MO).
Statistics
Data are expressed as the mean ± SEM. Probability was evaluated by Student’s t-test or the Fisher exact test (Tables 1 2) .
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Results |
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,ß-methylene-ATP, which is a relatively specific agonist for the P2X1 receptors.18 Our finding that 1 mM ,ß-methylene-ATP did not increase cell death above the control level (Fig. 2A) supported the conclusion that the lethal effect of BzATP was mediated by P2X7, not P2X1, receptors.
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Determination of the time course for cell death during a sustained exposure of microvessels to BzATP (100 µM) revealed that approximately 80% of the cell death detected by the trypan blue assay occurred after 8 hours (Fig. 2C) . Even with a relatively brief exposure to this P2X7 agonist, most of the BzATP-induced cell death occurred after a delay of many hours. For example, between 8 and 24 hours after a 15-second exposure to 100 µM BzATP, induced cell death increased significantly (P < 0.001, n = 5) from 9% ± 6% to 28% ± 8%.
The delayed death suggested the possibility that activation of P2X7 receptors triggered programmed cell death in retinal microvessels. To test this possibility, we used cleaved caspase-3 immunoreactivity, TUNEL positivity, and Hoechst-stained spherical nuclei as criteria to detect apoptotic cells. We found that exposure to 100 µM BzATP significantly (P < 0.001) increased the percentage of microvascular cells that were positive for these apoptotic markers (Table 1) . Thus, our findings indicate that the activation of P2X7 receptors induces apoptosis in the retinal microvasculature.
P2X7-Induced Cell Death in Diabetic Microvessels
Because apoptotic cell death in the pericyte-containing microvasculature is a well-known feature of diabetic retinopathy,1 we also assessed the effect of BzATP on retinal microvessels isolated from rats made diabetic by STZ injection. As shown in Figure 3A , there was a marked shift to the left in the dose-response relationship for cell death induced in diabetic, compared with nondiabetic, microvessels. Specifically, the concentration of BzATP that was half-maximally effective in causing microvascular cell death was 45 µM for the nondiabetic microvessels, but only 0.6 µM for those of the diabetic retina. In agreement with diabetes increasing the vulnerability of retinal microvessels to apoptotic cell death triggered by BzATP, exposure to a 1-µM concentration of this P2X7 agonist induced a significant (P < 0.001) increase in apoptosis in diabetic, but not (P > 0.25) in nondiabetic, microvessels (Table 2) .
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We considered the possibility that the increased lethality of P2X7 receptor activation was a manifestation of a nonspecific vulnerability of diabetic microvessels. However, contrary to this possibility, the procedure to isolate microvessels did not result in significantly (P = 0.24) more cell death in the diabetic group; immediately after isolation, 96% ± 1% and 94% ± 1% of the microvascular cells were viable in the nondiabetic (n = 53 coverslips) and diabetic groups (n = 22, 23 ± 2 days after STZ injection), respectively. Nor did maintenance for 24 hours in solution A (without P2X7 agonists) result in more (P = 0.55) cell death in diabetic microvessels; 88% ± 2% (n = 14) and 90% ± 2% (n = 10) of the cells were viable in the nondiabetic and diabetic microvessels, respectively. Also consistent with a lack of effect of diabetes on cell death in the absence of P2X7 agonists, apoptosis was not significantly (P > 0.5) different in the nondiabetic and diabetic microvascular cells incubated in solution A (without BzATP; Table 2 ). Additional evidence suggesting that the membrane integrity of diabetic microvascular cells was not different from that in the control cells was the lack of a significant (P = 0.51) difference in the percentage of cells that were permeable to the propidium di-iodide dye YO-PRO-1. Specifically, after a 60-minute incubation in 5 µM YO-PRO-1 (without BzATP), 11% ± 1% (n = 3 coverslips) and 15% ± 5% (n = 3) of the cells were fluorescent in the nondiabetic and diabetic (26 ± 2 days after STZ injection) microvessels, respectively. Furthermore, indicative of diabetes not causing generalized damage, pericytes in nondiabetic (n = 35) and diabetic (n = 11; 27 ± 2 days after STZ injection) microvessels had similar (P 
0.14) membrane potentials (-46 ± 2 mV vs. -43 ± 2 mV), membrane resistances (557 ± 24 M vs. 595 ± 34 M), and membrane capacitances (16 ± 1 pF vs. 13 ± 2 pF). Taken together, these observations indicate that the increased P2X7-induced cell death in diabetic microvessels was not secondary to nonspecific cell injury.
Effect of Diabetes on P2X7-Induced Pore Formation
Recently, we demonstrated that activation of P2X7 receptors in retinal microvessels is associated with the formation on large transmembrane pores.8 Because the opening of pores may have lethal consequences due to the leakage of vital intracellular molecules and the disruption of ionic gradients, we assessed the link between P2X7-induced pore formation and microvascular cell death.
If P2X7-induced pores play an essential role in microvascular cell death, then the increased lethality of BzATP should be associated with increased pore formation. To test this prediction, we assessed the effect of this P2X7 agonist on the permeability of microvascular cells to YO-PRO-1 (629 Da), which we and other investigators have used to detect P2X7-induced pores.8 12 19 20 Consistent with a casual relationship between pores and death, exposure to 10 µM BzATP induced YO-PRO permeability in substantially more (P < 0.001) diabetic than nondiabetic microvascular cells (Fig. 4A) . This increase in BzATP-induced YO-PRO-1 uptake in diabetic microvessels correlated with the increase in BzATP-induced cell death in these vessels (Fig. 3A) .
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Effect of Diabetes on P2X7-Induced Currents
In another series of experiments, we compared the amplitudes of the BzATP-induced current in nondiabetic and diabetic microvessels. If the number of functional P2X7 receptor/channels was significantly greater in the diabetic microvessels, then the current induced by this P2X7 agonist should be larger. However, diabetes did not significantly (P 0.44) affect the amplitude of the currents induced by 1 to 100 µM BzATP (Fig. 4C) . This lack of an effect of diabetes on the ionic current generated by the activation of P2X7 receptors, contrasts with the diabetes-induced increase in P2X7-mediated apoptotic cell death (Fig. 3 , Table 2 ) and pore formation (Figs. 4A 4B) . Thus, it appears that the number of functional P2X7 receptor/channels was not substantially increased in diabetic microvascular cells, even though P2X7-induced YO-PRO-1 uptake and cell death were markedly greater.
A parsimonious explanation for these observations is that, rather than upregulating the number of P2X7 receptor/channels, diabetes facilitates the process by which the activation of these receptor/channels results in the formation of lethal pores. In addition, because pores would be expected to increase the membrane conductance, our inability to detect an effect of diabetes on the BzATP-induced current also suggests that the opening of a relatively small population of pores in the microvascular cells of the retina results in a detectable uptake of YO-PRO-1 and the triggering of apoptosis.
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Discussion |
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Our working hypothesis is that chronic hyperglycemia due to diabetes enhances the opening of lethal pores during exposure of retinal microvessels to P2X7 ligands. Consistent with one aspect of this model, Solini et al.21 observed that maintenance of cultured fibroblasts in high glucose increased P2X7-induced apoptosis. Unfortunately, these investigators did not assay for the presence of pores in the fibroblasts, although they did observe that elevated glucose levels caused the P2X7 receptors to aggregate into ringlike structures within the plasma membrane. However, a causal relationship between receptor aggregation, pore formation, and cell death remains to be established.22
At present, the mechanism by which hyperglycemia enhances P2X7-induced pore formation in retinal microvessels remains uncertain. One possibility is that an increased number of functional P2X7 receptor/channels results in the formation of more pores. Although this is likely to be the mechanism by which P2X7-induced pore formation and cell death are enhanced when monocytes become macrophages,19 it does not appear to account for our observations. Inconsistent with an increase in functional receptor/channels in diabetic microvascular cells, we observed that the ionic current induced by the P2X7 agonist BzATP was not significantly affected by diabetes. Similarly, Bringmann et al.23 also noted a lack of correlation between the density of functional P2X7 receptor/channels and the opening of transmembrane pores in Müller (glial) cells.
An alternative mechanism by which diabetes may increase the lethal effect of P2X7 agonists without increasing the number of receptor/channels is to facilitate the transition from activated P2X7 receptor/channels to opened transmembrane pores. Although we think that this scenario is likely, it is difficult at present to test this hypothesis directly. A major challenge is that the steps linking the activation of P2X7 receptors with the opening of pores are not well understood.24 Elucidation of this process in nondiabetic cells should set the stage for the subsequent characterization of the mechanism by which diabetes facilitates the formation of pores during the activation of P2X7 receptor/channels.
Our experiments led us to conclude that only a relatively small number of transmembrane pores are opened in diabetic microvessels during activation of their P2X7 receptor/channels. Evidence supporting this conclusion is that diabetes did not cause a detectable increase in the BzATP-induced current, even though pores provide high-conductance pathways for ionic fluxes.25 Thus, the diabetes-mediated increase in P2X7-induced YO-PRO uptake and apoptotic cell death must not require the opening of a large number of transmembrane pores. This is in agreement with the suggestion of others26 that the number of P2X7-induced pores is likely to determine the mechanism of cell death. If many pores are opened, then death is induced rapidly (within minutes), due to osmotic imbalance.25 27 Conversely, more subtle changes in the intracellular milieu caused by the opening of a few pores causes delayed apoptotic death, as we observed in the retinal microvasculature.
The enhanced formation of lethal transmembrane pores during the activation of P2X7 receptor/channels may be a mechanism by which apoptosis is triggered in the microvasculature of the diabetic retina. Potentially exacerbating cell death by this mechanism, the concentration of extracellular ATP may be increased due to the release of this nucleotide by platelets whose aggregation is enhanced in the diabetic retina.28 However, because extracellular ATP levels have not been measured at sites adjacent to microvascular cells in either the normal or the diabetic retina, the possibility remains that the concentration of this nucleotide may be reduced in the diabetic retina. If ATP levels are markedly decreased, then ATP-induced death in the diabetic retinal microvasculature may be minimal in vivo. Clearly, although probably not feasible at present, measurement of the luminal and abluminal concentration of ATP in retinal capillaries will help to test our hypothesis that an enhancement of the P2X7 channel-to-pore transition plays a role in diabetic retinopathy.
Our ideas concerning P2X7-mediated apoptosis in the retinal microvasculature are based on experiments using freshly isolated microvessels. One benefit of studying microvessels in isolation is that the concentration of purinergic agonists and antagonists could be precisely controlled. In addition, the duration of exposure to these chemicals could be closely regulated. Furthermore, our use of isolated microvessels eliminated confounding effects mediated through the P2X7 receptors that are located in nonvascular retinal cells.29 30 31 However, it remains to be demonstrated that the apoptotic response triggered in isolated microvessels during P2X7 receptor activation also occurs in vivo. In addition, caution must be used when extrapolating results of our experiments using rat microvessels to the human condition. For example, the P2X7 receptors expressed by rats and humans appear to have different ligand affinities,32 and the STZ model in rodents does not fully mimic diabetes in humans. Consequently, the results of future studies assessing P2X7-mediated transmembrane pore formation and microvascular apoptosis in vivo will be of keen interest.
Despite the noted caveats, the use of freshly isolated microvessels provides a useful experimental preparation to help generate and test new hypotheses concerning mechanisms by which diabetes affects the retinal microvasculature. From our experiments, we propose that diabetes increases the sensitivity of the retinal microvasculature to the lethal effect of P2X7 receptor activation. This may be a previously unrecognized mechanism that plays a role in the progression of diabetic retinopathy. If so, then pharmacologic interference with P2X7-mediated apoptosis may be of some benefit in the prevention of sight-threatening complications of diabetes.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 25, 2003; revised November 9, 2003; accepted November 14, 2003.
Disclosure: T. Sugiyama, None; M. Kobayshi, None; H. Kawamura, None; Q. Li, None; D.G. Puro, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Donald G. Puro, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105; dgpuro{at}umich.edu.
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