AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2729-H2736, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01330.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2729    most recent
01330.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rocic, P.
Right arrow Articles by Chilian, W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rocic, P.
Right arrow Articles by Chilian, W. M.

Optimal reactive oxygen species concentration and p38 MAP kinase are required for coronary collateral growth

Petra Rocic,1 Christopher Kolz,1 Ryan Reed,2 Barry Potter,1 and William M. Chilian1

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive oxygen species (ROS) are implicated in coronary collateral growth (CCG). We evaluated the requirement for ROS in human coronary artery endothelial cell (HCAEC) tube formation, CCG in vivo, and signaling (p38 MAP kinase) by which ROS may stimulate vascular growth. The flavin-containing oxidase inhibitor diphenyleneiodonium (DPI) or the superoxide dismutase inhibitor diethyldithiocarbamate (DETC) blocked vascular endothelial growth factor-induced HCAEC tube formation in Matrigel. We assessed the effect of DPI and DETC on CCG in a rat model of repetitive ischemia (RI) (40 s left anterior descending coronary artery occlusion every 20 min for 2 h 20 min, 3 times/day, 10 days). DPI or DETC was given intraperitoneally, or the NAD(P)H oxidase inhibitor apocynin was given in drinking water. Collateral-dependent flow (measured by using microspheres) was expressed as a ratio of normal and ischemic zone flows. In sham-operated rats, collateral flow in the ischemic zone was 18 ± 6% of normal zone; in the RI group, collateral flow in the ischemic zone was 83 ± 5% of normal zone. DPI prevented the increase in collateral flow after RI (25 ± 4% of normal zone). Similar results were obtained with apocynin following RI (32 ± 7% of that in the normal zone). DETC achieved similar results (collateral flow after RI was 21 ± 2% of normal zone). DPI and DETC blocked RI-induced p38 MAP kinase activation in response to vascular endothelial growth factor and RI. These results demonstrate a requirement for optimal ROS concentration in HCAEC tube formation, CCG, and p38 MAP kinase activation. p38 MAP kinase inhibition prevented HCAEC tube formation and partially blocked RI-induced CCG (42 ± 7% of normal zone flow), indicating that p38 MAP kinase is a critical signaling mediator of CCG.

ischemic zone; vascular endothelial growth factor

ISCHEMIA-REPERFUSION INJURY is a biphasic process in which exposure of the myocardium to prolonged hypoxia/ischemia initiates massive cell death in the affected region of the heart. This is followed by further reperfusion injury commencing on reestablishment of blood flow and is defined as further destruction, including stunning and death, of tissue still alive at the onset of reperfusion (17). However, under certain conditions, ischemia-reperfusion can lead to adaptive mechanisms that diminish and/or prevent the reoccurrence of injury. Coronary collateral development is an example of such an adaptive response to myocardial ischemia caused by chronic occlusion of major coronary arteries. It has been shown that ischemic preconditioning renders the myocardium tolerant to prolonged ischemia-reperfusion injury (10), in part through promoting collateral development (4, 16, 20).

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rat model of collateral growth/RI. Male Wistar-Kyoto rats (3–4 mo old, 300–350 g) were used for chronic (10 days) implantation of a pneumatic occluder over the left anterior descending coronary artery (LAD), as described by Toyota et al. (22). The RI protocol for rat consisted of eight 40-s occlusions, one every 20 min over a duration of 2 h and 20 min followed by a period of "rest" for 5 h and 40 min. This 8-h cycle was repeated three times per day over a period of 10 days.

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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
VEGF-induced HCAEC tube formation requires optimal ROS concentration. To assess VEGF's ability to induce HCAEC tube formation, cells were seeded on Matrigel at a density of 30,000 cells/well, allowed to attach for 24 h and then treated with VEGF (50 ng/ml). Tube formation was observed after two days and was significantly increased in response to VEGF treatment compared with control nontreated cells (19.0 ± 0.02 vs. 8 ± 1% area covered, n = 10, P < 0.05; Fig. 1). Next, we investigated the effect of agents that increase or decrease ROS on VEGF-induced tube formation. To decrease ROS generation, cells were treated with the flavin-containing oxidase inhibitor DPI (10 µM), which blocked VEGF-induced HCAEC tube formation (6 ± 1 vs. 19 ± 2%, n = 10, P < 0.05; Fig. 1). Similarly, treatment with the superoxide dismutase (SOD) inhibitor DETC (10 mM) also prevented VEGF-induced HCAEC tube formation (5 ± 2 vs. 19 ± 2%, n = 10, P < 0.05; Fig. 1). Treatment with DPI or DETC alone (without VEGF) was not significantly different from control (5 ± 1 and 5 ± 2%, respectively).


Figure 1
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 1. Human coronary artery endothelial cell (HCAEC) tube formation in Matrigel. Left: representative images of HCAEC 2 days after addition of 50 ng/ml vascular endothelial growth factor (VEGF) with or without inhibitors [10 µM diphenyleneiodonium (DPI) or 10 mM diethyldithiocarbamate (DETC)] as indicated. Right: percent of total field with tubes (n = 10 per group, means ± SE of 5 random fields per well; * and #, P < 0.05).

 
RI causes coronary collateral development in the rat model of RI in vivo. Results in Fig. 2 demonstrate impaired coronary flow in the LAD-dependent (collateral-dependent) zone in animals that underwent a sham surgical procedure and thus were not subjected to RI (collateral flow was 18 ± 6% of the normal zone flow and represents native collateral flow; n = 8). In contrast, collateral flow in animals that underwent the 10-day RI protocol was 83 ± 5% of that in the normal zone (Fig. 2), demonstrating significant collateral development in this region of the myocardium.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Collateral-dependent flow expressed as percentage of flow in the normal zone. DPI, DETC, and apocynin blocked the repetitive occlusion/ischemia (RI)-induced increase in collateral-dependent flow (n = 8 for each treatment group).

 
Blockade of superoxide production by DPI (0.2 mg·kg–1·day–1) prevented the increase in collateral flow induced by RI (25 ± 4% of that in the normal zone, n = 8, P < 0.05; Fig. 2). Similar results were obtained with apocynin (0.25 mg/ml in drinking water = 43 mg·kg–1·day–1), a more specific inhibitor of NAD(P)H oxidases (collateral flow was 32 ± 7% of that in the normal zone after RI, n = 3, P < 0.05; Fig. 2). Likewise, increasing superoxide by SOD inhibition (DETC, 1 g·kg–1·day–1) achieved similar results (collateral flow was 21 ± 2% of that in the normal zone following RI, n = 8, P < 0.05; Fig. 2).

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.


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 3. A: dihydroethidium (DHE) fluorescence in the left anterior descending coronary artery (LAD)-dependent zone in a sham-operated animal, following two periods of ischemia (40 s)-reperfusion (20 min) (RI), and RI + DPI or DETC treatment. RI increased DHE fluorescence, and this increase was abolished by DPI but was further augmented by DETC. B: superoxide levels in sham-operated, RI, and RI + DPI or RI + DETC groups measured by X-band electroparamagnetic resonance in LAD-dependent and normal zones. Similar to A, RI increased superoxide levels in the heart, and this increase was attenuated by DPI and accentuated by DETC to a greater extent in the LAD-dependent than in the normal zone. C: dihydrodichlorofluorescein (DCF) fluorescence in sham-operated, RI, and RI + DPI or RI + DETC groups. Elevated DCF fluorescence was observed following RI, but either DETC or DPI decreased the signal.

 
Because SOD inhibition might also lower myocardial H2O2 concentration, DCF (60 µg/kg) was injected into the LV (in a manner identical to DHE administration) in a separate group of animals, and DCF fluorescence was evaluated in AU to evaluate this possibility. Figure 3C shows that H2O2 levels were decreased approximately fourfold by both DPI and DETC (122 AU for RI + DPI and 125 AU for RI + DETC vs. 450 AU for RI), indicating that H2O2 concentrations below a certain level (RI alone) may result in inhibition of coronary collateral growth.

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.


Figure 4
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 4. A, top: Western blot with phospho- and total p38 MAP kinase antibodies in untreated endothelial cells or cells treated with VEGF, VEGF + DPI, or VEGF + DETC. Bottom: area x density of the Western blots (arbitrary units). Activation (phosphorylation) of p38 in VEGF-treated cells was prevented by either DPI or DETC. B: effects of p38 inhibition (SB-203580) on VEGF-induced tube formation. Inhibition of p38 completely prevented VEGF stimulation of endothelial cell tube formation.

 
p38 MAP kinase activation by ischemia-reperfusion is partially responsible for ischemia-reperfusion-induced coronary collateral growth. Figure 5A shows that p38 is specifically activated (2.7 ± 0.23-fold vs. sham, n = 3, P < 0.05) in the LAD-dependent region of the myocardium only on day 3 of the RI protocol. This is in contrast to other kinases, including Akt, which remain activated to a high degree for the duration of the protocol (data not shown). This activation is smaller in magnitude than the VEGF-induced response in HCAECs, which likely accounts for a large proportion of cardiac myocytes and fibroblasts in the tissue sample and a less robust activation of p38 in these cell types vs. in coronary endothelium. In addition, p38 activation by RI was completely blocked by DPI and DETC at the time point of maximal p38 activation by RI (day 3; Fig. 5A). In a protocol identical to that used for confirmation of DPI and DETC effects on superoxide concentration in vivo, we investigated the effect of these compounds on p38 MAP kinase activation. After 3 days of the RI protocol, animals were killed, hearts were excised, and LAD-dependent and normal zones were separated and prepared for Western blot analysis. Western blots with phospho-specific anti-p38 antibodies revealed a significant increase in p38 MAP kinase activation in response to ischemia-reperfusion (p38 = 2.7 ± 0.23-fold vs. sham = 1.0 ± 0.17, n = 3, P < 0.05) in the LAD-dependent zone, which was completely blocked by both DPI and DETC (1.0 ± 0.18- and 1.1 ± 0.3-fold, respectively, n = 3, P < 0.05; Fig. 5A). Expression (total p38 demonstrated by Western blot analysis with anti- p38 MAP kinase antibodies) was not affected by any of the interventions, and p38 was not significantly activated in the normal zone (1.2 ± 0.2-fold in RI vs. sham, n = 3; Fig. 5A).


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. A, left: Western blot analysis with antiphospho specific and antitotal p38 MAP kinase antibodies in cardiac tissue from sham-operated rats (day 0) and in rats at varying days of the RI protocol. Phospho-p38 was elevated at day 3, but not at day 6 or day 9. DETC or DPI prevented the activation at day 3. Right: comparison of phospho-p38 in the normal zone of rats undergoing RI at day 3 to that in sham-operated rats. There were no differences in activation between the normal zone shams and rats. B: collateral flow (expressed as %normal zone flow) in sham-operated, RI, and RI rats treated with the p38 inhibitor SB-203580. Inhibition of p38 attenuated the increase in collateral-dependent flow by ~50% at day 10 of the RI protocol.

 
To ascertain whether p38 MAP kinase is required for coronary collateral development in response to ischemia-reperfusion in vivo, rats were treated with SB-203580 (3.2 mg·kg–1·day–1) for the duration of the 10-day RI protocol via intraperitoneal injection once per day. p38 MAP kinase inhibition resulted in a significant (~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).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major observation of our study is that the induction of endothelial cell tube formation in vitro and coronary collateral growth in vivo require a specific intracellular concentration of ROS. ROS levels below and above (with regard to superoxide) this optimal range prohibit coronary collateral development and endothelial cell tube formation. We also demonstrate that p38 MAP kinase activation plays a pivotal role in ROS signaling for both endothelial cell tube formation and, for the first time, coronary collateral growth in vivo. Our observations and conclusions are supported by some cogent work in the literature.

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.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-32788 (to W. M. Chilian), HL-65203 (to W. M. Chilian), HL-73755 (to W. M. Chilian), and COBRE RR-18766 (to P. Rocic and W. M. Chilian) and by an American Heart Association Scientist Development Grant (to P. Rocic).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Rocic, NEOVCOM, Dept. of Physiology and Pharmacology, 4209 State Route 44, Rootstown, OH 44272-0095, (e-mail: procic{at}neoucom.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amin M, Volpert O, Woods J, Kumar P, Harlow L, Koch A. Migration inhibitory factor mediates angiogenesis via mitogen-activated protein kinase and phosphatidylinositol kinase. Circ Res 93: 321–329, 2003.[Abstract/Free Full Text]
  2. Angermayr B, Fernandez M, Mejias M, Gracia-Sancho J, Garcia-Pagan J, Bosch J. NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperaemia in portal hypertensive rats. Gut 56: 560–564, 2007.[Abstract/Free Full Text]
  3. Boyd P, Doyle J, Gee E, Pallan S, Haas T. MAPK signaling regulates endothelial cell assembly into networks and expression of MT1-MMP and MMP-2. Am J Physiol Cell Physiol 288: C659–C668, 2005.[Abstract/Free Full Text]
  4. Chen W, Gabel S, Steenbergen C, Murphy E. A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res 77: 424–429, 1995.[Abstract/Free Full Text]
  5. Gerber H, McMurtrey A, Kowalski J, Yan M, Keyt B, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273: 30336–30343, 1998.[Abstract/Free Full Text]
  6. Gratton J, Morales-Ruiz M, Kureishi Y, Fulton D, Walsh K, Sessa W. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J Biol Chem 276: 30359–30365, 2001.[Abstract/Free Full Text]
  7. He H, Venema V, Gu X, Venema R, Marrero M, Caldwell R. Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src. J Biol Chem 274: 25130–25135, 1999.[Abstract/Free Full Text]
  8. Huang S, Zheng R. Biphasic regulation of angiogenesis by reactive oxygen species. Pharmazie 61: 223–229, 2006.[ISI][Medline]
  9. Kersten J, McGough M, Pagel P, Tessmer J, Warltier D. Temporal dependence of coronary collateral development. Cardiovasc Res 34: 306–312, 1997.[Abstract/Free Full Text]
  10. Koerselman J, van der Graaf Y, de Jaegere P, Grobbee D. Coronary collaterals: an important and underexposed aspect of coronary artery disease. Circulation 107: 2507–2511, 2003.[Free Full Text]
  11. Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant S, Lambeth J, Griendling K. Novel gp91(phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 88: 888–894, 2001.[Abstract/Free Full Text]
  12. Lee O, Bae S, Bae M, Lee Y, Moon E, Cha H, Kwon Y, Kim K. Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models. Br J Cancer 82: 385–391, 2000.[CrossRef][ISI][Medline]
  13. Liu Z, Ganju R, Wang J, Schweitzer K, Weksler B, Avraham S, Groopman J. Characterization of signal transduction pathways in human bone marrow endothelial cells. Blood 90: 2253–2259, 1997.[Abstract/Free Full Text]
  14. Marikovsky M, Ziv V, Nevo N, Harris-Cerruti C, Mahler O. Cu/Zn superoxide dismutase plays important role in immune response. J Immunol 170: 2993–3001, 2003.[Abstract/Free Full Text]
  15. Matsunaga T, Warltier D, Weihrauch D, Moniz M, Tessmer J, Chilian W. Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide. Circulation 102: 3098–3103, 2000.[Abstract/Free Full Text]
  16. Maulik N, Das D. Potentiation of angiogenic response by ischemic and hypoxic preconditioning of the heart. J Cell Mol Med 6: 13–24, 2002.[CrossRef][ISI][Medline]
  17. Park J, Lucchesi B. Mechanisms of myocardial reperfusion injury. Ann Thorac Surg 68: 1905–1912, 1999.[Abstract/Free Full Text]
  18. Sabri A, Govindarajan G, Griffin T, Byron K, Samarel A, Lucchesi P. Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle. Circ Res 83: 841–851, 1998.[ISI][Medline]
  19. Schmidt M, Smerup M, Konstantinov I, Shimizu M, Li J, Cheung M, White P, Kristiansen S, Sorensen K, Dzavik V, Redington A, Kharbanda R. Intermittent peripheral tissue ischemia during coronary ischemia reduces myocardial infarction: first demonstration of remote ischemic perconditioning. Am J Physiol Heart Circ Physiol (December 15, 2006). doi:10.1152/ajpheart.00617.2006.
  20. Skyschally A, Schulz R, Gres P, Korth H, Heusch G. Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid. Am J Physiol Heart Circ Physiol 284: H698–H703, 2003.[Abstract/Free Full Text]
  21. Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene 18: 2221–2230, 1999.[CrossRef][ISI][Medline]
  22. Toyota E, Warltier D, Brock T, Ritman E, Kolz C, O'Malley P, Rocic P, Focardi M, Chilian W. Vascular endothelial growth factor is required for coronary collateral growth in the rat. Circulation 112: 2108–2113, 2005.[Abstract/Free Full Text]
  23. Ushio-Fukai M, Alexander R, Akers M, Griendling K. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 273: 15022–15029, 1998.[Abstract/Free Full Text]
  24. Ushio-Fukai M, Alexander R, Akers M, Yin Q, Fujio Y, Walsh K, Griendling K. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 22699–22704, 1999.[Abstract/Free Full Text]
  25. Ushio-Fukai M, Griendling K, Becker P, Hilenski L, Halleran S, Alexande R. Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21: 489–495, 2001.[Abstract/Free Full Text]
  26. Ushio-Fukai M, Tang Y, Fukai T, Dikalov S, Ma Y, Fujimoto M, Quinn M, Pagano P, Johnson C, Alexander R. Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res 91: 1160–1167, 2002.[Abstract/Free Full Text]
  27. Weidong G. Reactive oxygen species are critical mediators of coronary collateral development in canine model. Am J Physiol Heart Circ Physiol 285: H1582–H1589, 2003.[Abstract/Free Full Text]
  28. Xu L, Pathak P, Fukumura D. Hypoxia-induced activation of p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase signaling pathways contributes to expression of interleukin 8 in human ovarian carcinoma cells. Clin Cancer Res 10: 701–707, 2004.[Abstract/Free Full Text]
  29. Yang S, Xin X, Zlot C, Ingle G, Fuh G, Li B, Moffat B, de Vos A, Gerritsen M. Vascular endothelial cell growth factor-driven endothelial tube formation is mediated by vascular endothelial cell growth factor receptor-2, a kinase insert domain-containing receptor. Arterioscler Thromb Vasc Biol 21: 1934–1940, 2001.[Abstract/Free Full Text]
  30. Zachary I, Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell 71: 891–894, 1992.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Circ. Res.Home page
S. H. Schirmer, J. O. Fledderus, P. T.G. Bot, P. D. Moerland, I. E. Hoefer, J. Baan Jr, J. P.S. Henriques, R. J. van der Schaaf, M. M. Vis, A. J.G. Horrevoets, et al.
Interferon-{beta} Signaling Is Enhanced in Patients With Insufficient Coronary Collateral Artery Development and Inhibits Arteriogenesis in Mice
Circ. Res., May 23, 2008; 102(10): 1286 - 1294.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. W. Kim, A. Lin, R. E. Guldberg, M. Ushio-Fukai, and T. Fukai
Essential Role of Extracellular SOD in Reparative Neovascularization Induced by Hindlimb Ischemia
Circ. Res., August 17, 2007; 101(4): 409 - 419.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2729    most recent
01330.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rocic, P.
Right arrow Articles by Chilian, W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rocic, P.
Right arrow Articles by Chilian, W. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.