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Departments of 1Physiology and 2Pharmacology, Louisiana State University Health Sciences Center, New Orleans, Louisiana
Submitted 5 December 2006 ; accepted in final form 14 February 2007
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ABSTRACT |
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ischemic zone; vascular endothelial growth factor
Our laboratory has previously shown that reactive oxygen species (ROS) are critical mediators of coronary collateral development in a canine model of repetitive ischemia (15). Treatment with N-acetyl cysteine attenuated the repetitive ischemia (RI)-induced increase in coronary collateral flow. Likewise, myocardial interstitial fluid from dogs undergoing the RI protocol, but not fluid from N-acetyl cysteine-treated dogs, induced endothelial cell tube formation and vascular smooth muscle cell proliferation in cultured cells (27). Interestingly, NAD(P)H oxidase-derived ROS have been shown to play a critical role in promoting endothelial cell tube formation in vitro (26). Furthermore, in cell culture studies, low concentrations of superoxide and H2O2 induced endothelial cell migration, adhesion, and tube formation, whereas high concentrations of these ROS inhibited these processes (8).
Both in vitro and in vivo studies suggest that coronary collateral growth is mediated by a highly coordinated signaling cascade. We have previously reported (22) a requisite role for vascular endothelial growth factor (VEGF) in coronary collateral growth. VEGF has been shown to activate several signaling pathways, including ERK1/2 (21), JNK (17), and p38 (6) MAP kinases; the phosphatidylinositol 3-kinase/Akt pathway (5); and the nonreceptor tyrosine kinases c-Src (7), focal adhesion kinase (30), and Pyk2 (13). Of these, p38 MAP kinase (11, 23), Akt (11, 24), and c-Src (25) have been shown to be ROS sensitive in other cell types.
The potential role of p38 MAP kinase in coronary collateral growth is particularly interesting for a number of reasons. First, p38 MAP kinase has been shown to be specifically activated in response to hypoxia in human cancer cells (28). Second, human umbilical vein endothelial cell (HUVEC) tube formation in Matrigel was associated with rapid and transient p38 activation (12), and VEGF-induced endothelial cell migration was inhibited by specific p38 MAP kinase inhibition (29), indicating a required and beneficial effect of p38 MAP kinase on endothelial cell tube formation in vitro. The role of p38 MAP kinase has not been investigated in coronary collateral development in vivo. Third, in our preliminary studies, its activation in response to RI was ROS sensitive, but unlike the activation of other signaling molecules, p38 MAP kinase was only transiently activated.
In this study, we show that an optimal concentration of superoxide and H2O2 allows for human coronary artery endothelial cell (HCAEC) tube formation in response to VEGF and for coronary collateral growth in response to RI-reperfusion. Furthermore, we demonstrate, for the first time, that the ROS-sensitive and transient activation of p38 MAP kinase is essential for the underlying positive effect of ROS on stimulating coronary collateral growth.
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MATERIALS AND METHODS |
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Microsphere measurements of myocardial and collateral-dependent blood flow. Microspheres (5 x 105) labeled with gamma emitters 57Co (at initial surgery) or 103Ru (at end of RI protocol) were injected into the left ventricle (LV) over 20 s. Collateral (LAD)-dependent zone was identified by using fluorescence microscopy in which the area without the fluorescent microspheres (injected into the LV at the time of initial surgery while LAD is occluded) is the LAD-dependent zone. Tissue flow was calculated as a ratio between activity (cpm/g) of the tissue samples from the LAD-dependent and normal zones. For all experiments, data were analyzed by two-way ANOVA followed by t-test. Collateral flow was measured in the following groups: a sham-operated group (n = 8) that was instrumented but not subjected to RI, a control group/RI (n = 8), RI + diphenyleneiodonium (DPI, 0.2 mg·kg–1·day–1; n = 8), RI + apocynin (0.25 mg/ml in drinking water; n = 3), and RI + diethyldithiocarbamate (DETC, 1 g·kg–1·day–1; n = 8).
Measurement of superoxide and H2O2. Superoxide production was evaluated by using dihydroethidium (DHE) in vitro and in vivo and electroparamagnetic resonance (EPR) in vivo. H2O2 production was evaluated by dihydrodichlorofluorescein (DCF) in vivo. For HCAEC cell culture, DHE was administered during the last 20 min of treatment. Cells were then observed immediately under a fluorescent microscope. For in vivo studies, DHE or DCF were injected into the LV (60 µg/kg) for 20 min before two consecutive periods of ischemia-reperfusion (40 s occlusion followed by 20 min reperfusion and another 40 s occlusion and 20 min reperfusion). Animals were then killed. The heart was removed, frozen in optimum cutting temperature compound on dry ice, and stored at –70°C until sectioning. Sections (5 µm) were made in a cryomicrotome and were mounted on glass slides. DHE or DCF fluorescence was detected with excitation/emission at 518/605 nm (for DHE) or 480/515 nm (for DCF). All images were analyzed at the same microscope settings, and relative fluorescence readings were obtained by Metamorph Software on three hearts (5 consecutive sections per heart). A Bruker EMX spectrometer was used for X-band EPR measurements of superoxide by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H) as a spin-trap. Animals underwent two consecutive periods of ischemia-reperfusion (as above). Animals were then killed, hearts were removed, LAD-dependent and normal zones were separated, and CP-H (238 µg/100 mg tissue) was added to the tissue samples immediately. Tissue was then homogenized by sonication on ice and was frozen in liquid nitrogen until EPR measurements. Superoxide concentration was calculated from arbitrary units (AU; 3.4 x 106 AU/nM).
HCAEC cell culture. Cells were purchased from Clonetics and were cultured at low passages (passages 3–8) in Clonetics EGM-2 BulletKit medium (Lonza) that contains 25% FBS, 0.2% hydrocortisone, 2% human FGF-B, 0.5% IGF-I, 0.5% ascorbic acid, 0.5% human EGF, and 0.5% GA-1000.
Endothelial tube formation. HCAECs (passages 3–10) were seeded on Matrigel (BD Biosciences)-coated 24-well plates according to manufacturer instructions in EGM-2 BulletKit medium at a density of 30,000 cells/well. Cells were allowed to attach for 24 h before the addition of 50 ng/ml VEGF, DPI (10 µM), DETC (10 mM), or the p38 MAP kinase inhibitor (SB-203580; 20 µM). All treatment groups consisted of 10 wells. The extent of tube formation was quantified after two days by using Scion Image software. An electronic grid was superimposed on microscopic images, and the number of squares containing tubes were counted and averaged from five randomly selected fields for each well to obtain the percentage of total field that contained tubes.
Western blot analysis. Proteins from cells and myocardial tissue were extracted in a lysis buffer containing 0.1% SDS and 1% Triton as previously described (18). For extraction from tissue, hearts were excised, LVs were dissected, and the LAD-dependent zone was separated from the normal zone and snap-frozen in liquid nitrogen before homogenization in lysis buffer. Equal amounts of protein (60 µg) were separated by SDS-PAGE and were transferred to Hybound-ECL nitrocellulose membranes. Phospho-specific anti-p38 and anti-total p38 antibodies (Cell Signaling) were used for Western blot analysis. Bands were visualized by enhanced chemiluminescence (Amersham) and were quantified by using National Institutes of Health Image software. Because none of the treatments altered p38 MAP kinase expression, its total levels were used to check for equal lane loading and to normalize phosphorylation levels. All experiments were performed in triplicate (n = 3 animals/group).
Data analysis. ANOVA followed by t-tests with Bonferroni inequalities was used for statistical analysis. A probability value of P < 0.05 was used to determine statistical significance.
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RESULTS |
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To confirm the effects of ischemia-reperfusion and efficacy of DPI and DETC treatments on superoxide generation in vivo, in some animals DHE was given by LV injection (60 µg/kg) for 20 min before two consecutive periods of ischemia-reperfusion. Ischemia-reperfusion caused an increase in superoxide generation in the LAD-dependent zone compared with sham (RI = 450 ± 13 vs. sham = 124 ± 6 AU, n = 3, P < 0.05; Fig. 3A). When DPI was administered for two days before DHE injection, superoxide production was markedly lower (RI + DPI = 118 ± 9 vs. RI = 450 ± 13 AU, n = 3, P < 0.05). In contrast, DETC treatment for two days resulted in an increase in superoxide concentration above that produced by ischemia-reperfusion (RI + DETC = 861 ± 16 vs. RI = 450 ± 13 AU, n = 3, P < 0.05; Fig. 3A). Identical results were observed following both one and three consecutive periods of ischemia-reperfusion (data not shown). Furthermore, in some animals CP-H was added and tissue samples were subjected to X-band EPR to obtain a quantitative measurement of superoxide. In all treatment groups the observed superoxide concentrations in the LAD-dependent zone are in precise agreement with the DHE fluorescence intensity (0.12 nM for sham, 0.53 nM for RI, 0.10 nM for RI + DPI, and 1.18 nM for RI + DETC; Fig. 3B). Superoxide concentrations in the normal zone were 0.10 nM for sham, 0.36 nM for RI, 0.29 nM for RI + DPI, and 0.64 nM for RI + DETC (Fig. 3B). Thus RI increased superoxide concentrations in both the LAD-dependent and the normal zones, but this increase was greater in the LAD-dependent zone. Likewise, the effect of DETC was more pronounced in the LAD-dependent than in the normal zone, whereas the effect of DPI was pronounced in the LAD-dependent but negligible in the normal zone.
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VEGF induces p38 MAP kinase activation in vitro and p38 MAP kinase inhibition prevents VEGF-induced HCAEC tube formation. To elucidate some of the signaling mechanisms that underlay these processes, HCAEC cultures were treated with 50 ng/ml VEGF for 20 min, harvested, and prepared for Western blot analysis with phospho-specific antibodies. One of the molecules that showed an increase in activation in response to VEGF was p38 MAP kinase (p38 = 4.5 ± 0.23 vs. control = 1 ± 0.19, n = 3, P < 0.05; Fig. 4A). Additionally, a 45-min pretreatment with both DPI and DETC completely abolished VEGF-induced p38 MAP kinase activation (1.4 ± 0.43 and 1.3 ± 0.18, respectively, n = 3, P < 0.05) demonstrating the redox sensitivity of its activation in response to VEGF. Importantly, VEGF, DPI, or DETC treatment did not alter the expression of the kinase. To establish a causal role for p38 in HCAEC tube formation, we determined the effect of p38 MAP kinase inhibition by its specific inhibitor, SB-203580 (20 µM), on VEGF-induced tube formation. Figure 4B shows that p38 MAP kinase inhibition prevents VEGF-induced HCAEC tube formation (0 ± 0% vs. 19 ± 2%, n = 10, P < 0.05), without any apparent effect on HCAEC survival or proliferation.
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50%) decrease in coronary collateral development (42 ± 7% of that in the normal zone, n = 8, P < 0.05; Fig. 5B).
Together, these results show that the redox-sensitive p38 MAP kinase is partially responsible for ischemia-reperfusion-induced coronary collateral development, although it remains unclear in which cell type its activation is most important. We confirmed the blockade of p38 MAP kinase by intraperitoneal administration of SB-203580 (3.2 mg·kg–1·day–1) in a modified RI protocol (3 days) and found an almost complete (90%) inhibition of the kinase in both the LAD-dependent and the normal zones, whereas activation of several other kinases, including Akt and ERK1/2, was not affected (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In support of the present study, it has been reported that short and repetitive exposure to hypoxia-reoxygenation produces ROS that are associated with angiogenesis (9, 16, 27). A role for ROS in VEGF-induced tube formation in cultured endothelial cells has been demonstrated (1, 26). Here we show, for the first time, that coronary collateral development in the rat model of RI is critically dependent on an optimal concentration of ROS generated in the myocardium by our RI protocol. Specifically, we show that a specific concentration range of superoxide is required for coronary collateral growth and that elevated superoxide levels are detrimental to the process (Fig. 4). Our data are in agreement with in vivo studies demonstrating that SOD inhibition resulted in marked reduction of tumor growth and metastasis, which was dependent on vascular angiogenesis (14). Too-low concentrations of both superoxide and possibly H2O2 were not conducive to coronary collateral growth, because SOD inhibition results in both elevation in superoxide levels and a decrease in H2O2 levels in the myocardium, whereas treatment with DPI decreased concentrations of both of these ROS. Thus the results in this study do not answer the question of whether this process is inhibited by too-high superoxide concentrations alone, by too-low H2O2 concentrations, or by a combination of both.
Our results implicate superoxide derived from flavin-containing oxidases to be critical for coronary collateral growth, because DPI blocks this process in response to ischemia-reperfusion in vivo. Furthermore, inhibition of NAD(P)H oxidases by apocynin yielded nearly identical results (Fig. 2). Ushio-Fukai et al. (26) established a role for NAD(P)H oxidase in endothelial cell tube formation, and Angermayr et al. (2) demonstrated that NAD(P)H oxidase modulates angiogenesis and portal collateral development in rat. These results, together with our own, suggest that NAD(P)H oxidase-derived ROS play a critical role in regulation of coronary collateral growth.
Nitric oxide has been implicated in collateral development so that reduced NO bioavailability or inhibition of NO synthase resulted in decreased collateral growth (15). DETC, which increases superoxide concentrations, would potentially result in decreased NO bioavailability and leaves the question of NO involvement open for discussion. However, treatment with DPI markedly reduces superoxide concentrations in the heart (Fig. 3, A and B), thus potentially increasing or at least not altering NO concentrations. Therefore, we believe that an optimal concentration of superoxide and H2O2 but not NO is critical for coronary collateral growth.
Another proposed explanation for the increase in collateral flow is an increase in coronary dilation in response to ischemia-reperfusion. We doubt that this occurs because coronary circulation is maximally dilated by ischemia. We have confirmed this in a previous study (22) by maximally dilating coronary arteries with dipyridamole during the initial occlusion. Dipyridamole did not increase coronary flow beyond that during occlusion. In addition, in previous work published by our laboratory (22), microcomputed tomography images clearly show an increase in coronary collateral development following the RI protocol. Another point we are compelled to make is that an increase in the collateral-dependent region must reflect collateral growth, i.e, an increase in the caliber of the collateral vessels. The native collateral circulation is high in resistance (collateral flow is 20% of normal zone flow because of the high resistance of these vessels) because of the small caliber of the vessels. Therefore, the only way for flow to quadruple following the RI protocol is through growth of these vessels.
p38 MAP kinase has been shown to be redox dependent in cultured aortic smooth muscle (23), and VEGF has been shown to activate p38 MAP kinase in endothelial cultures (6), but these studies fall short of investigating the redox sensitivity of p38 MAP kinase in response to VEGF. Here, we show that p38 MAP kinase activation in response to VEGF is ROS sensitive (Fig. 4). In contrast, Amin et al. (1) found that the antioxidant resveratrol did not affect p38 phosphorylation in HUVECs. The reason for this discrepancy is unclear but may include differences in ROS dependency of p38 activation between HCAECs and HUVECs or whether the dose of resveratrol was sufficient to affect the redox state of cells. Functional consequences of p38 MAP kinase inhibition in endothelial cell tube formation likewise remain unknown. In fact, both proangiogenic (29) and antiangiogenic (3) effects of p38 MAP kinase activation have been reported in vitro. In this study, we demonstrate that specific inhibition of p38 MAP kinase activation prevents VEGF-induced HCAEC tube formation in Matrigel (Fig. 4B).
Importantly, we show a role for p38 MAP kinase in coronary collateral growth. Near-complete inhibition (>90%) of p38 MAP kinase by SB-203580 in vivo led to a partial (50%) attenuation in coronary collateral development in response to LAD occlusion (Fig. 5B). These results demonstrate that collateral growth is partially but critically dependent on p38 MAP kinase and indicate that p38 MAP kinase activation in response to RI depends on an optimal level of ROS. Since 80% of cells, by volume, in the whole heart are cardiac myocytes, the observed p38 activation in these preparations is likely to be primarily related to its activation in myocytes. However, p38 activation may be critical in both endothelial cells, where its activation could directly regulate endothelial cell migration, and in cardiac myocytes, where it may affect the expression of growth factors and matrix-degrading enzymes to facilitate migration of vascular cells.
Another relevant question is whether regional ischemia is the driving force for coronary collateral growth. In our study, RI induced an increase in myocardial superoxide both in the LAD-dependent and the normal zone. The increase in superoxide in the normal zone may be related to the myriad observations of remote preconditioning (19). However, we are compelled to emphasize that superoxide levels generated by RI in the normal zone were lower then those in the LAD-dependent zone. In addition, the effect of DETC on further increasing superoxide concentrations was far greater in the LAD-dependent than in the normal zone, and the effect of DPI on decreasing superoxide concentrations in the normal zone was negligible. Importantly, p38 MAP kinase activation in the normal zone was not altered by RI. Thus we believe that although alterations in ROS levels in the remote region of the myocardium may contribute to regulation of coronary collateral growth, regulation of ROS concentrations and especially redox-dependent signaling in the ischemic zone is more significant.
The novel findings in this study, specifically 1) the demonstration of a requirement for a specific concentration range of ROS for coronary collateral growth and 2) the partial but definitive dependence of coronary collateral growth on p38 MAP kinase, provide an important step toward understanding the molecular mechanisms that regulate coronary collateral growth.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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