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From the Department of Medicine I (C.K., R.H., P.R., P.B.), Klinikum Gro?hadern, Ludwig-Maximilians-University of Munich, Germany, and Unit of Pharmacology and Therapeutics (C.D., G.D., C.B., O.F.), Department of Internal Medicine, University of Louvain Medical School, Brussels, Belgium.
ABSTRACT
Objectives— The interaction of the heat shock protein 90 (Hsp90) with the endothelial NO synthase (eNOS) has been shown to account for a sustained production of NO in vitro. Here, we examined whether overexpression of Hsp90 in a pig model of cardiac infarct could preserve the myocardium from the deleterious effects of ischemia–reperfusion.
Methods and Results— Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein before left anterior descending occlusion and reperfusion. We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 33% reduction in infarct size and prevented the increase in postischemic left ventricular end diastolic pressure observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region, was increased in Hsp90-transfected animals at baseline and after pacing. All these effects were completely abrogated by administration of the NOS inhibitor NG-nitro-L-arginine methyl ester. We further documented in vivo and in cultured endothelial cells that the cardioprotective effects of Hsp90 were associated to its capacity to act as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS serine 1177 phosphorylation and threonine 495 dephosphorylation, respectively.
Conclusions— Hsp90 is a promising target to enhance NO formation in vivo, which may efficiently reduce myocardial reperfusion injury.
Percutaneous liposome-based Hsp90 gene transfer in a pig cardiac infarct model was shown to preserve the myocardium from the deleterious effects of ischemia–reperfusion. The gain in cardiac function was dependent on Hsp90-driven regulation of eNOS phosphorylation by Akt and calcineurin, thereby establishing Hsp90 as a promising target for cardiac diseases.
Key Words: Hsp90 ? nitric oxide ? eNOS ? calcineurin ? Akt
Introduction
Myocardial ischemia caused by an occluded coronary artery is treated clinically by rapid re-establishment of perfusion, which is currently viewed as uniquely effective in preventing myocyte cell death. Nevertheless, a body of evidence documents that reperfusion also leads to organ dysfunction, including endothelial and microcirculatory disturbance, arrhythmias, and myocardial stunning.1 Among the multitude of strategies aiming to prevent ischemia-reperfusion (I/R)–induced myocardial detriments, overexpression of the endothelial NO synthase (eNOS) isoform in the myocyte2 or endothelial3 compartment has proven recently to be cardioprotective. The endothelial-targeted enhancement of eNOS activity appears particularly promising because it induces vasorelaxation, inhibits platelet aggregation as well as leukocyte recruitment, and antagonizes microcirculatory disturbances.4 Yet, increase in NO formation by endothelium can exert beneficial effects on the cardiomyocyte compartment, preserving myocyte function by preventing necrosis,5 apoptosis6,7 as well as reducing myocyte vulnerability via induction of hibernation.8
However, the therapeutically desired increase in endothelial NO production does not necessarily require an increase in eNOS abundance because it may also evolve from specific post-translational modifications of endogenously expressed eNOS. Accordingly, the cardioprotective effects of insulin,6 corticosteroids,9 and vascular endothelial growth factor (VEGF)10 were recently found to stem from eNOS phosphorylation at the serine residue 1177 (Ser 1177; human sequence) on receptor-specific induction of the phosphatidylinositol 3-kinase (PI3K)/Akt–signaling pathway. We reported previously that this activation cascade implies assembly of a multiprotein complex in which heat shock protein 90 (Hsp90) acts as a scaffold, recruiting activated Akt in the eNOS vicinity.11 More recently, Hsp90 was also proposed to prevent eNOS uncoupling and detrimental production of superoxide anion by the NOS enzyme,12 particularly after chronic myocardial hypoxia.13 Furthermore, definitive credential to the existence of a dynamic Hsp90–eNOS interaction was granted recently by the mapping of respective binding sequences14 and extended to the neuronal NOS isoform.15 Finally, Hsp90 is not only acting as an adaptor molecule for assembly of the eNOS phosphorylation complex,11,14 but it also maintains the complex in its active form. Indeed, interaction with Akt prevents dephosphorylation of the kinase,16,17 thereby promoting its sustained activation and the associated antiapoptotic effects in endothelial cells (ECs).18 On the basis of this set of evidence, we hypothesized that endothelial overexpression of Hsp90 could exert cardioprotection in a pig model of myocardial I/R. Using targeted transfection of an Hsp90-encoding plasmid by a selective, pressure-regulated retroinfusion approach, we studied Hsp90 effects on the in vivo eNOS phosphorylation status and the indicators of I/R injury, such as infarct size, as well as regional and global myocardial dysfunction.
Methods
Animal Model and Retroinfusion
All pig experiments were conducted at the Institute for Surgical Research of the University of Munich. The experimental procedures were approved by the Bavarian Animal Care and Use Committee (AZ 211 to 2531-07/01). As described previously,19 the retroinfusion catheter was advanced into the anterior interventricular vein, draining the parenchyma perfused by the left anterior descending (LAD) coronary artery. After assessment of individual systolic occlusion pressure of the venous system, retroinfusion pressure was set 20 mm Hg above the latter. Continuous pressure-regulated retroinfusion of NaCl 0.9%–diluted liposomes containing the plasmid (1 mg) encoding for either green fluorescent protein (GFP; control group) or Hsp90 (original plasmid was a gift from Dr W.C. Sessa, Yale University, New Haven, Conn)20 was conducted for 2x10 minutes at day 0 (during LAD occlusion). Next, venous and arterial sheaths were withdrawn, the vessels ligated, and suture of the muscle and the skin performed.
Ischemia and Reperfusion
Ischemia and reperfusion of these animals were conducted at day 2, allowing for expression of the transfected gene product. A balloon was placed in the LAD distal to the bifurcation of the first diagonal branch and inflated. Correct localization of the coronary occlusion and patency of the first diagonal branch was ensured by contrast agent injection under fluoroscopy. Ischemia was maintained for 60 minutes, the point after which the balloon in the LAD was deflated, initiating reperfusion of the LAD perfusion area. After 24 hours of reperfusion, animals were brought back to the operating room, anesthetized, and instrumented to evaluate regional and global myocardial function and to determine infarct size, as described previously19 (also see http://atvb.ahajournals.org).
EC Culture and Transfection
Human umbilical vein ECs (HUVECs; Clonetics) and pig coronary ECs (PCECs) obtained by the explant procedure21,22 were cultured in endothelial basal medium (Clonetics) and used at early passages for transfection (http://atvb.ahajournals.org).
NO Production and cGMP Measurements
Determination of NO levels (eg, the accumulation of nitrites in the cell-bathing medium) was performed using the Nitric Oxide Colorimetric Assay (Roche Diagnostics). cGMP determination was performed with the radioimmunoassay kit from Biotrak (Amersham).
Cardiac Tissue Processing, Immunoprecipitation, and Immunoblotting
The hearts were collected after in vivo methylene blue exclusion (negative staining of the area at risk ) and in situ tetrazolium red staining (infarct staining) so that selected cardiac tissue samples (control, infarcted, at risk) were isolated macroscopically and snap-frozen in a mixture of acetone and dry ice; this method was validated previously to be particularly efficient to preserve protein phosphorylation.23 Pig heart pieces (as well as serum-starved ECs) were then homogenized in a buffer containing phosphatase and protease inhibitors, and whole extracts were processed for immunoblotting (IB) or immunoprecipitation (IP) as described previously10,11 (also see http://atvb.ahajournals.org).
Statistical Methods
The results are given as mean±SEM; for IB experiments, data were normalized for protein amounts in the dish. Statistical analysis of results between experimental groups was performed with 1-way or 2-way ANOVA or t test (for the comparison of preischemic and postischemic left ventricular end diastolic pressure ).
Results
Functional Evaluation of Hsp90 Transfection Effects on Postischemic Myocardium
To evaluate the functional impact of Hsp90 overexpression in postischemic myocardium, pigs were transfected 48 hours before ischemia with cDNA plasmids, encoding either Hsp90 or the reporter GFP. As detailed in Methods, we used a selective pressure-regulated retroinfusion device, an approach providing effective transgene expression in the area at risk of infarcted heart.10,24 GFP detection (Figure 1A) confirmed our previous observations that this retroinfusion method led mostly to the transfection of cardiac microvascular ECs and small vessels (versus myocytes). As shown in Figure 1B, the infarct area in mock-transfected animals amounted to 62±5% of the AAR, as measured after 24 hours of reperfusion. Transfection with the Hsp90 plasmid decreased the infarct size to 42±4% (P<0.05). Importantly, this effect was NO dependent because the administration of NG-nitro-L-arginine methyl ester (L-NAME; details available online at http://atvb.ahajournals.org) abrogated the protection provided by Hsp90 transfection (Figure 1B). Of note, Hsp90 transfection or L-NAME treatment did not significantly affect AAR size (Figure 1C).
Figure 1. Effects of Hsp90 transfection on infarct size. GFP- and Hsp90-transfected pigs, with or without L-NAME application, were subjected to 60 minutes of percutaneous transluminal LAD occlusion and 24 hours of reperfusion, as described in Methods. A, GFP detection revealed the selectivity of the transfection procedure for microvasular ECs (arrowheads) and coronary microvessels (arrows). Bar=25 μm. B, Hsp90 transfection effects are presented in bar graphs illustrating the extent of infarct size expressed as percentage of AAR (n=6 per group; *P<0.05 vs mock and L-NAME groups). C, The extent of the area at risk size in percentage of total left ventricular area.
Evaluation of the global myocardial function reflected these findings (Figure 2A). Indeed, postischemic LVEDP was significantly increased in control pigs, whereas such elevation was absent from Hsp90-transfected pigs. Again, this Hsp90-mediated protective effect was abolished by use of L-NAME (Figure 2A). We also assessed the regional myocardial function in the ischemic area by determining the subendocardial segment shortening (SES) at baseline and after pacing. As shown in Figure 2B, SES of the infarct area was dramatically higher in Hsp90-transfected animals than in controls at all beating rates observed. Furthermore, this local gain in function in the infarct zone was NO mediated because it was completely abrogated by L-NAME administration (Figure 2B). In the AAR, besides a slight reduction in SES in mock-transfected pigs, no significant difference was found between animal groups (Figure 2C).
Figure 2. Effects of Hsp90 transfection on post-I/R ventricular dysfunction and SES. A, LVEDP (mm Hg) determined before (PRE) and 24 hours after (POST) ischemic insult (n=6 per group; #P<0.05 vs mock group). B, Sonomicrometry crystal SES was determined at baseline and at the indicated atrial pacing rate in the infarct zone (*P<0.05 vs mock group and L-NAME–treated groups). C, In the area at risk. Mock transfected without (open bars) or with (dark gray bars) L-NAME coapplication, Hsp90 transfected without (black bars) or with (shaded bars) L-NAME coapplication (n=6 per group; #P<0.05 vs mock group).
Evaluation of Hsp90 Transfection Effects on Phosphorylation Status of eNOS in Postischemic Myocardium
To verify that Hsp90-mediated changes in eNOS activity accounted for the observed cardioprotection, we performed biochemical experiments aimed at identifying changes in phosphorylation status of eNOS (see Introduction) from whole tissue extracts.
We first examined the relative abundance of Hsp90 in control and ischemic regions of the heart after transfection. As shown in Figure 3A (top), Hsp90 protein amounts were increased dramatically in the transfected myocardium (also see Figure 3B). Next, we blotted the membranes with antibodies against phospho-Ser 1177 (P-Ser 1177) and phospho-threonine 495 (P-Thr 495) eNOS. As suggested by previous studies,11,14,17 the extent of Ser 1177 phosphorylation was largely related to the level of Hsp90 expression (Figure 3A, second panel). Indeed, although the expression of eNOS was not different in Hsp90-transfected versus mock-transfected hearts (Figure 3A, bottom), the extent of phosphorylation on Ser 1177 was significantly increased in transfected animals. Levels of Ser 1177 phosphorylation were 2.1- and 2.7-fold higher in the AAR and in the infarct zone, respectively, than in the control zone of Hsp90-transfected hearts (Figure 3B). Interestingly, a mirror pattern was obtained for Thr 495 because the phosphorylation of eNOS on this residue was undetectable in the AAR and infarct regions of the Hsp90-transfected myocardium, whereas in the nonischemic region of the same hearts, Thr 495 was consistently found phosphorylated (Figure 3A, third panel and Figure 3B). In mock-transfected animals, the extent of Ser 1177 and Thr 495 phosphorylations were not altered regardless of heart regions (Figure 3A and 3B).
Figure 3. Effects of Hsp90 transfection on posttranslational regulation of eNOS in postischemic myocardium. Fragments of cardiac tissues from control (CTL) area (nonischemic), AAR, and infarct zones were collected from mock- and Hsp90-transfected pigs. A, Corresponding lysates were immunoblotted with antibodies directed against Hsp90, eNOS, and the indicated phosphorylated residues. B, Bar graph shows the densitometric analyses of Hsp90, P-Ser 1177 eNOS, and P-Thr 495 eNOS immunoblots obtained from 4 different pig heart extracts (*P<0.05; **P<0.01 vs corresponding CTL group); note that the lower affinity of the Hsp90 antibody for the native (pig) protein (vs the human recombinant one) could lead to a partial overestimation of the amounts of transfected Hsp90. C, Lysates from infarct zones were immunoprecipitated with eNOS or Hsp90 antibodies and then immunoblotted, as indicated, with antibodies directed against eNOS, Hsp90, Akt, and calcineurin. These experiments were reproduced from extracts obtained from 3 different pig hearts. D, cGMP levels in cardiac tissue from mock- (open bar) and Hsp90-transfected animals without (black bar) or with (shaded bar) L-NAME coapplication (n=6; 3 samples per animal; *P<0.05 vs mock transfection and Hsp90 L-NAME–treated animals).
We then examined whether changes in Ser 1177 and Thr 495 phosphorylation could be related to the increased interaction of eNOS with Hsp90 and the consecutive recruitment of the kinase Akt and the phosphatase calcineurin (PP2B). Indeed, Akt has been reported previously to promote eNOS Ser 1177 phosphorylation in an Hsp90-dependent manner (see Introduction), whereas calcineurin was shown to dephosphorylate eNOS on Thr 49525 and to interact with Hsp9026 (although this latter finding was obtained in studies unrelated to the NO field).
We first showed that antibodies directed against eNOS could coimmunoprecipitate Hsp90 and, inversely, that Hsp90 antibodies could precipitate eNOS from infarct region extracts of Hsp90-transfected but not mock-transfected myocardium (Figure 3C), indicating that a robust interaction occurred between eNOS and Hsp90 in transfected animals. We further documented that significantly higher amounts of Akt and calcineurin were detected in eNOS IP from Hsp90-transfected hearts (454±54% and 196±17%, respectively, versus mock-transfected hearts; P<0.01; n=3; Figure 3C). Finally, we verified whether observed changes in eNOS phosphorylation after Hsp90 transfection were associated with increased NOS activity. Indeed, we found that the cGMP content was almost 2-fold higher (P<0.05; n=6) in Hsp90-transfected hearts (versus mock-transfected hearts) and that this was strictly NO mediated because the cGMP increase was completely prevented in L-NAME–treated Hsp90-transfected pigs (Figure 3D).
Dissection of Hsp90-Mediated Reciprocal Regulation of eNOS Ser 1177 Phosphorylation and Thr 495 Dephosphorylation in ECs
The causal link between Hsp90–eNOS interaction and eNOS phosphorylation/dephosphorylation changes were evaluated in isolated ECs. We first used HUVECs that were transfected either with the same Hsp90-encoding construct as used in vivo or the empty vector and exposed, for half of the dishes, to geldanamycin, a drug known to alter eNOS–Hsp90 interaction.11,20
As expected, Hsp90 overexpression strongly promoted its association with eNOS, Akt, and calcineurin (Figure 4A, compare lanes 1 and 3). In parallel, we observed that Ser 1177 phosphorylation as well as Thr 495 dephosphorylation were stimulated in Hsp90-transfected HUVECs (Figure 4B, compare lanes 1 and 3). More importantly, we found that these eNOS phosphorylation changes as well as the Hsp90–eNOS interaction were exquisitely sensitive to the action of geldanamycin (Figure 4A and 4B, compare lanes 3 and 4); similar results were obtained when using radicicol, another Hsp90 inhibitor that does not redox cycle (data not shown). Importantly, geldanamycin had no effect on either Akt or calcineurin recruitment to the Hsp90 complex (Figure 4A, bottom), in agreement with the existence of numerous distinct binding domains within Hsp90.16 Figure 4C shows that, in agreement with IB data, geldanamycin prevented eNOS activation increase in Hsp90-overexpressing ECs.
Figure 4. Reciprocal regulation of eNOS Ser 1177 phosphorylation and Thr 495 dephosphorylation in ECs. HUVECs transfected with a plasmid encoding for Hsp90 or with the empty vector were incubated in the presence or absence of 1 μg/mL geldanamycin and then rapidly collected. A, Corresponding lysates were immunoblotted with antibodies directed against Hsp90, Akt, and calcineurin, either directly or after IP with eNOS or Hsp90 antibodies (indicated as IgG). B, Lysates were also immunoblotted with antibodies directed against eNOS and the indicated phosphorylated residues. These experiments were repeated 2x with similar results. C, Bar graph represents the amounts of nitrites accumulated for 6 hours in the medium bathing the indicated cell populations (n=4; **P<0.01 vs sham CTL group).
In a second series of experiments, we used ECs derived directly from pig coronary arteries and examined Hsp90-induced eNOS phosphorylation status changes after short (10 minutes) and longer (60 minutes) VEGF exposure and also in basal conditions. We found that in unstimulated conditions, Hsp90 transfection led to Ser 1177 eNOS phosphorylation but failed to induce Thr 495 dephosphorylation (Figure 5A, compare lanes 1 and 4). When these experiments were repeated on ECs stimulated for 10 minutes with VEGF, the extent of eNOS associated with Hsp90 increased and, although the Ser 1177 eNOS phosphorylation also increased, Thr 495 eNOS phosphorylation was not further decreased; this was observed in sham- and Hsp90-transfected cells (Figure 5A and 5B). Interestingly, we found that longer VEGF exposure (60 minutes) was required to lead to Thr 495 eNOS dephosphorylation (Figure 5A, lanes 3 and 6). This was associated with an increased interaction between eNOS and Hsp90 and a small decrease in Ser 1177 eNOS phosphorylation when compared with the 10-minute VEGF exposure (Figure 5A and 5B).
Figure 5. Effects of Hsp90 transfection on VEGF-induced regulation of eNOS phosphorylation in pig coronary ECs. PCECs transfected with a plasmid encoding for Hsp90 or with the empty vector were exposed to VEGF for the indicated time. A, Corresponding lysates were immunoblotted with antibodies directed against Hsp90, total or phosphorylated eNOS, either directly or after IP with eNOS antibodies. B, Bar graph shows the densitometric analyses of Hsp90 IB from eNOS IP and of P-Ser 1177 eNOS and P-Thr 495 eNOS immunoblots (*P<0.05; **P<0.01 vs corresponding basal condition; n=3).
Discussion
In the present study, we found that targeted overexpression of Hsp90 reduced 2 important facets of myocardial reperfusion injury: infarct size and myocardial dysfunction (Figures 1 and 2 ). The NOS inhibitor L-NAME entirely abolished these effects, indicating that NO accounted for most of the Hsp90-mediated cardioprotection.
Importantly, we also provided mechanistic insights into how Hsp90 can stimulate eNOS activity from in vivo and in vitro experiments. Although the stimulatory effect of eNOS phosphorylation on Ser 1177 by Akt has already been largely documented,11,27–29 this report is the first to demonstrate the occurrence of the dephosphorylation of eNOS on Thr 495 in animal studies. More interesting is identification of the role of Hsp90 as a key determinant of the intimate relationship between the phosphorylation on one site (ie, Ser 1177) and the dephosphorylation on the other (ie, Thr 495). Stimulatory effects of each eNOS activity post-translational modification have been reported by different groups.30 Accordingly, Ser 1177 phosphorylation is proposed to improve the electron flux through the enzyme and to increase its affinity for calmodulin,31,32 whereas dephosphorylation of Thr 495 is thought to suppress the steric inhibition for calmodulin association to its binding site.33,34
Recent data suggest that in intact cells, eNOS dephosphorylation on Thr 495 is associated with an increased superoxide anion production. This phenomenon, known as eNOS uncoupling, was documented to be overcome by the eNOS concomitant phosphorylation on Ser 1177. Therefore, the major role of Thr 495 eNOS phosphorylation could consist of regulating the fidelity of electron flux coupled to NO synthesis in basal conditions.35 On agonist challenge, phosphorylation of other residues and recruitment of other partners in the eNOS complex would then take the lead. In our study, by directly examining changes in native eNOS post-translational regulation, we found that the scaffolding function of Hsp90 that we previously identified for Akt also applies to calcineurin (Figures 3 and 4). These Hsp90-driven protein–protein associations provide an explanation for reciprocal regulation of eNOS on distant phosphorylation sites. Indeed, although a reciprocal regulation between both phosphorylation sites was suspected,25,33,36 only speculation on the putative proximity of both sites in the eNOS tertiary structure was available so far.37 In fact, time course studies suggest that Hsp90-induced eNOS phosphorylation on Ser 1177 occurred before Thr 495 dephosphorylation (Figure 5), thereby confirming another study in which adenoviral Hsp90 expression led to an NO production increase independently of a Thr 495 phosphorylation decrease. 14 Still, we showed that eNOS dephosphorylation on Thr 495 occurred at later stages associated with a further increased recruitment of Hsp90 in the eNOS complex.
Identification of Hsp90 as a key regulator of the eNOS phosphorylation status, taken together with previous studies documenting the interaction between Hsp90 and various phosphatase(s), further elucidates the mechanism of enzymatic eNOS activation. Besides its scaffolding effects, Hsp90 is also known to protect Akt from dephosphorylation (ie, deactivation) by PP2A16 but also to promote activation of PP2B.26 Altogether, these findings indicate that in vivo activation of eNOS by Hsp90 overexpression is mainly regulated by Akt-dependent Ser 1177 phosphorylation and also involves calcineurin-mediated Thr 495 eNOS dephosphorylation. However, further studies are required to evaluate whether the latter influences the net increase in bioactive NO and how this is influenced by the time or absolute abundance of Hsp90. Nevertheless, in our experimental conditions, the scaffold capacity of Hsp90 appears particularly suited to sustain eNOS activation, such as that required for cardioprotection against I/R injury; this might extend to mediation of acute effects of preconditioning, which have been attributed to postischemically enhanced eNOS activity.5 In fact, eNOS phosphorylation on Ser 1177 has been documented to be associated with the cardioprotection provided by VEGF,10 statins,38,39 corticosteroids,9 insulin,6 and even cyclosporine.40 For each of these strategies, Hsp90 has been either identified directly as the effector allowing activation of the prosurvival PI3K/Akt pathway10,11,17 or at least as an obligatory chaperone for involved receptors (eg, see hormone-bound glucocorticoid receptors).41 Therefore, Hsp90 appears as a common, primary target for various therapeutic stimuli involving Akt activation and NO production.
In summary, we demonstrated that Hsp90 overexpression in the ischemic region of infarcted hearts can preserve the myocardium from the I/R deleterious effects through an exquisite stimulation of the endothelial NO pathway. Our data further indicate that the mechanism involved in this feature of Hsp90 comprised eNOS Ser 1177 phosphorylation by Akt as well as Thr 495 dephosphorylation by calcineurin. This reciprocal regulation emphasizes the relevance of strategies aiming to interfere with this upstream key activator of NO-mediated pathways, particularly in pathophysiological situations in which myocardial protection is desirable.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (C.K., P.B.) and by grants (to O.F.) from Fonds de la Recherche Scientifique Médicale and the Fonds National de la Recherche Scientifique (FNRS). O.F. and C.D. are FNRS research associates.
References
Verma S, Fedak PW, Weisel RD, Butany J, Rao V, Maitland A, Li RK, Dhillon B, Yau TM. Fundamentals of reperfusion injury for the clinical cardiologist. Circulation. 2002; 105: 2332–2336.
Brunner F, Maier R, Andrew P, Wolkart G, Zechner R, Mayer B. Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Cardiovasc Res. 2003; 57: 55–62.
Jones SP, von Haperen R, de Crom R, Kawashima S, Lefer DJ. Vascular endothelial cell overexpression of the eNOS gene attenuates the extent of myocardial reperfusion injury. Circulation. 2002; 106 (suppl): II–56.
Jones SP, Lefer DJ. Myocardial reperfusion injury: insights gained from gene-targeted mice. News Physiol Sci. 2000; 15: 303–308.
Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol. 2001; 33: 1897–1918.
Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002; 105: 1497–1502.
Iwata A, Sai S, Nitta Y, Chen M, Fries-Hallstrand R, Dalesandro J, Thomas R, Allen MD. Liposome-mediated gene transfection of endothelial nitric oxide synthase reduces endothelial activation and leukocyte infiltration in transplanted hearts. Circulation. 2001; 103: 2753–2759.
Heusch G, Post H, Michel MC, Kelm M, Schulz R. Endogenous nitric oxide and myocardial adaptation to ischemia. Circ Res. 2000; 87: 146–152.
Hafezi-Moghadam A, Simoncini T, Yang E, Limbourg FP, Plumier JC, Rebsamen MC, Hsieh CM, Chui DS, Thomas KL, Prorock AJ, Laubach VE, Moskowitz MA, French BA, Ley K, Liao JK. Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med. 2002; 8: 473–479.
Kupatt C, Hinkel R, Vachenauer R, Horstkotte J, Raake P, Sandner T, Kreuzpointner R, Muller F, Dimmeler S, Feron O, Boekstegers P. VEGF165 transfection decreases postischemic NF-kappa B-dependent myocardial reperfusion injury in vivo: role of eNOS phosphorylation. FASEB J. 2003; 17: 705–707.
Brouet A, Sonveaux P, Dessy C, Balligand JL, Feron O. Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells. J Biol Chem. 2001; 276: 32663–32669.
Pritchard KA Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, Sessa WC. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem. 2001; 276: 17621–17624.
Shi Y, Baker JE, Zhang C, Tweddell JS, Su J, Pritchard KA Jr. Chronic hypoxia increases endothelial nitric oxide synthase generation of nitric oxide by increasing heat shock protein 90 association and serine phosphorylation. Circ Res. 2002; 91: 300–306.
Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, Tsuruo T, Sessa WC. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res. 2002; 90: 866–873.
Song Y, Cardounel AJ, Zweier JL, Xia Y. Inhibition of superoxide generation from neuronal nitric oxide synthase by heat shock protein 90: implications in NOS regulation. Biochemistry. 2002; 41: 10616–10622.
Sato S, Fujita N, Tsuruo T. Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci U S A. 2000; 97: 10832–10837.
Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res. 2001; 89: 866–873.
Shiojima I, Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res. 2002; 90: 1243–1250.
Boekstegers P, Peter W, Degenfeld Gv, Nienaber CA, Abend M, Rehders TC, Habazettl H, Kapsner T, Lüdinghausen Mv, Werdan K. Preservation of regional myocardial function and myocardial oxygen tension during acute ischemia in pigs: comparison of selective synchronized suction and retroinfusion of coronary veins to synchronized coronary venous retroperfusion. J Am Coll Cardiol. 1994; 23: 459–469.
Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature. 1998; 392: 821–824.
Nicosia RF, Villaschi S, Smith M. Isolation and characterization of vasoformative endothelial cells from the rat aorta. In Vitro Cell Dev Biol Anim. 1994; 30A: 394–399.
Nerheim PL, Meier JL, Vasef MA, Li WG, Hu L, Rice JB, Gavrila D, Richenbacher WE, Weintraub NL. Enhanced cytomegalovirus infection in atherosclerotic human blood vessels. Am J Pathol. 2004; 164: 589–600.
Petroff MG, Kim SH, Pepe S, Dessy C, Marban E, Balligand JL, Sollott SJ. Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes. Nat Cell Biol. 2001; 3: 867–873.
Kupatt C, Wichels R, Deiss M, Molnar A, Lebherz C, Raake P, von Degenfeld G, Hahnel D, Boekstegers P. Retroinfusion of NFkappaB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs. Gene Ther. 2002; 9: 518–526.
Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem. 2001; 276: 16587–16591.
Someren JS, Faber LE, Klein JD, Tumlin JA. Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms. Biochem Biophys Res Commun. 1999; 260: 619–625.
Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 399: 597–601.
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.
Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PR, Kemp BE, Pearson RB. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol. 1999; 9: 845–848.
Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1–R12.
Takahashi S, Mendelsohn ME. Calmodulin-dependent and -independent activation of endothelial nitric-oxide synthase by heat shock protein 90. J Biol Chem. 2003; 278: 9339–9344.
McCabe TJ, Fulton D, Roman LJ, Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem. 2000; 275: 6123–6128.
Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: E68–E75.
Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett. 1999; 443: 285–289.
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr., Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
Thomas SR, Chen K, Keaney JF Jr. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway. J Biol Chem. 2002; 277: 6017–6024.
Greif DM, Kou R, Michel T. Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. Biochemistry. 2002; 41: 15845–15853.
Lefer DJ, Scalia R, Jones SP, Sharp BR, Hoffmeyer MR, Farvid AR, Gibson MF, Lefer AM. HMG-CoA reductase inhibition protects the diabetic myocardium from ischemia-reperfusion injury. FASEB J. 2001; 15: 1454–1456.
Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med. 2000; 6: 1004–1010.
Rezzani R, Rodella L, Dessy C, Daneau G, Bianchi R, Feron O. Changes in Hsp90 expression determine the effects of cyclosporine A on the NO pathway in rat myocardium. FEBS Lett. 2003; 552: 125–129.
Bamberger CM, Wald M, Bamberger AM, Schulte HM. Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin. Mol Cell Endocrinol. 1997; 131: 233–240.