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VEGF₁₆₅ transfection decreases postischemic NF-B-dependent myocardial reperfusion injury in vivo: role of eNOS phosphorylation¹

Internal Medicine I, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Germany;
^* Molecular Cardiology, Department of Medicine IV, University of Frankfurt, Germany; and
^# Pharmacology and Therapeutics Unit, University of Louvain Medical School, Brussels, Belgium

²Correspondence: Internal Medicine I, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany. E-mail: c.kupatt{at}lrz.uni-muenchen.de

SPECIFIC AIMS

Recent advances in understanding the signal transduction of VEGF revealed a specific phosphorylation pathway of receptor-dependent PI3-kinase, AKT, and eNOS phosphorylation. Earlier studies have shown a role of this pathway in the mediation of angiogenic functions of VEGF such as an increase in capillary density in chronic ischemic in vivo models. Considering application of long-acting vectors transducing cardiac cells with VEGF constructs, however, the role of VEGF during short-term ischemia and reperfusion is a compelling problem, since these episodes tend to occur in coronary no-option patients.

Applying liposomal transfection into coronary endothelial cells or the ischemic area of a pig heart, we investigated the role of regional myocardial VEGF transfection in the context of postischemic inflammation by investigating its effect on 1) postischemic NF-B activation and 2) subsequent leukocyte recruitment, which is closely associated with 3) subsequent infarct development and 4) loss of regional myocardial function. All results were compared with those obtained by additional NOS inhibition (L-NAME) or by transfection of a phosphomimetic eNOS construct (eNOS S1177D).

PRINCIPAL FINDINGS

1. In vivo model of liposomal gene transfection, ischemia, and reperfusion (pig)
Pigs were transfected with liposomes containing VEGF₁₆₅ or eNOS S1177D by retroinfusion of the anterior interventricular vein 48 h before LAD occlusion (60 min) and reperfusion (24 h). Thereafter, regional myocardial function was assessed as subendocardial segment shortening and infarct size was determined. Tissue from the infarct region, the noninfarcted area at risk (AAR), and a control region was analyzed for NF-B activation (EMSA), expression of iNOS, and infiltration of PMN. L-NAME was applied in one group of VEGF-transfected animals.

2. Effect of VEGF transfection on postischemic inflammation in vivo
Enhanced VEGF expression correlated with increased phosphorylation of native eNOS, attenuated NF-B activation, and decreased expression of iNOS, a proinflammatory protein. Postischemic blockade of nitric oxide synthases via L-NAME abolished the effects of VEGF, whereas transfection of eNOS S1177D mimicked the reduction of NF-B activation and subsequently decreased iNOS expression of vessels in the ischemic area (Fig. 1 ). Leukocyte recruitment in the ischemic myocardium was reduced when hearts were transfected 48 h before ischemia with either VEGF₁₆₅ or eNOS S1177D cDNA, an effect abolished by L-NAME coapplication in the VEGF₁₆₅-transfected hearts.

View larger version (43K): [in this window] [in a new window]   Figure 1. A) EMSA detected NF-B activation after 24 h of reperfusion in the area at risk (left lane), with a specific inhibition by in vitro addition of NF-B decoy ODN (s.c.) but not an unspecific ODN (u.c.). B) Compared with mock-transfected hearts, eNOS S1177D transfection blunted NF-B activation at 24 h of reperfusion. C) Similarly, VEGF-transfected hearts displayed reduced NF-B activation in EMSA, with coapplication of L-NAME abolishing the effect of VEGF transfection. D) Immunohistochemical detection of iNOS in the vessel wall of infarcted myocardium (middle panel, hemalum stain, 100x) and in greater magnification (Kernechtrot, 400x) of the same heart (right panel). Control staining with secondary antibody (left panel, 100x). Control staining with secondary antibody (left panel, 100x). Arrows indicate iNOS-positive vessels E) Analysis of vessels staining positive for iNOS in the control area, AAR, and infarct (n=3, P<0.05 vs. the control area of the respective group).

3. Effect of VEGF transfection on infarct size
Figure 2 demonstrates the effect of VEGF₁₆₅ or eNOS S1177D on infarct size in our model. Whereas mock-transfected hearts displayed an infarct size of 68 ± 6% of the area at risk, VEGF transfection was able to reduce this size to 41 ± 4%, implying a 39% reduction in infarct size. A similar effect was obtained by eNOS S1177D transfection, whereas L-NAME blocked the effect of VEGF almost entirely (63±5%). NF-B decoy ODN transfection decreased infarct size to about the same portion as either VEGF or eNOS S1177D (38±5%).

View larger version (21K): [in this window] [in a new window]   Figure 2. A) Infarct size was normalized to the area at risk AAR and given in %. Mock-transfected hearts displayed a larger relative infarct size than VEGF- or eNOS S1177D-treated hearts, similar to VEGF- + L-NAME-treated hearts. B) Area at risk normalized to the total LV revealed no difference in infarct size in all groups (n=8 per group, #P<0.05 vs. control group).

4. Effect of VEGF on myocardial function
Subendocardial segment shortening (SES) of the infarct area and the area at risk was obtained after placement of sonomicrometry crystals in a standardized manner as a parameter of regional myocardial function in the infarcted and noninfarcted area at risk. Values were normalized to the values of the control (Cx perfusion) area. Subendocardial segment shortening of the infarcted area in untreated animals was < 20% of the control area under resting conditions and further declined with increased pacing rates. However, preischemic transfection with VEGF₁₆₅ resulted in significantly higher preservation of SES (>50% of the control area) under resting conditions and higher pacing rates (120/min). A similar improvement of subendocardial segmental shortening was obtained in the eNOS S1177D-transfected group as opposed to VEGF + L-NAME-treated animals.

CONCLUSION AND SIGNIFICANCE

Defining the role of VEGF in the setting of subacute myocardial ischemia and reperfusion faces certain obstacles, since continuous VEGF protein application into the ischemic myocardium is difficult to conduct and poses the problem of severe hypotension. In the present study, we used a pig model of retrograde gene transfection into the LAD perfusion area 48 h before ischemia (1 h) and reperfusion (24 h) to investigate the nonangiogenic influence of VEGF in vivo. Using a novel mode of percutaneous transluminal retrograde delivery of liposomes carrying functional genes, we found an increased expression of VEGF (concomitantly with enhanced phosphorylation of native eNOS) or eNOS S1177D in the ischemic area. VEGF₁₆₅ or eNOS S1177D transfection reduced both postischemic NF-B activation and subsequent inflammatory infiltrate in the infarct region and the noninfarcted area at risk, except for concomitant inhibition of NO formation by L-NAME in VEGF-transfected hearts. These experiments indicate for the first time that VEGF-induced eNOS phosphorylation is a relevant event in down-regulation of postischemic inflammation and its contribution to PMN-dependent myocardial reperfusion injury.

The ability of nitric oxide to rapidly reduce leukocyte adhesion at reperfused coronary endothelium cells has been observed before. Consistently, our study demonstrates that transfection of VEGF or a constitutively active eNOS mutant in vivo decreases PMN recruitment and subsequent myocardial damage. Beyond its ability to acutely prevent neutrophil adhesion ex vivo and in vivo, nitric oxide interferes with the subacute window of neutrophil recruitment regulated by transcriptional activation. The mechanism of NO-antagonizing NF-B activation has not been elucidated yet. Apparently, NO interferes with the redox sensitivity of the NF-B system. Reactive oxygen species like hypochlorous acid and hydroxyl anion released from adherent neutrophils can contribute to NF-B activation. Thus, a positive feedback loop formed by initial endothelial activation, rapid neutrophil recruitment inducing further inflammatory protein expression via NF-B, appears to form during early reperfusion. Transfection of microcirculatory venular endothelium by retroinfusion fittingly directed eNOS S1177D to the preferred site of either of both processes. Moreover, the fact that NF-B decoy ODN transfection without enhanced nitric oxide formation reduced infarct size similarly to eNOS S1177D (Fig. 2) indicates the relevance of transcriptional activation for the evolving myocardial reperfusion injury.

VEGF overexpression was able to reduce both postischemic inflammation and myocardial detriment. Does VEGF transfection use the eNOS activation pathway to exert postischemic cardioprotection? The inhibitory effect of L-NAME indicates an involvement nitric oxide synthases, which, however, could also be generated by the inducible isoform (iNOS), an enzyme found to play a protective role after murine myocardial ischemia. As depicted in Fig. 1 , the percentage of iNOS containing vessels in the postischemic regions was less elevated in VEGF- than in mock-transfected and VEGF + L-NAME-treated hearts at 24 h of reperfusion. This finding may reflect the fact that iNOS expression after myocardial ischemia is regulated in part by NF-B and down-regulated by eNOS-phosphorylating agents, e.g., simvastatin.

In summary, we have demonstrated for the first time that liposomal VEGF transfection via retroinfusion protects the postischemically reperfused heart by reducing NF-B activation and subsequent PMN-dependent loss of regional myocardial function. Nitric oxide, generated by VEGF through eNOS S1177 phosphorylation, is an essential mediator of this cardioprotective effect of VEGF. These findings extend the therapeutic relevance of VEGF beyond treatment of chronic ischemia toward cardioprotection after acute ischemic events. It remains to be examined whether other growth factors, including insulin, insulin-like growth factor, and TGF ß, known to stimulate the eNOS phosphorylation pathway, exert at least part of their cardioprotective effect in the setting of ischemia and reperfusion via silencing of NF-B as well. Concerning clinical application, it should be born in mind that overexpression of VEGF in the coronary venular endothelium is an intervention with potential side effects, if released systemically, which has to be further characterized concerning the dose and time dependency of its anti-inflammatory properties.

View larger version (16K): [in this window] [in a new window]   Figure 3. Scheme depicting VEGF effect on postischemic inflammation. After eNOS phosphorylation, the enhanced nitric oxide release decreases leukocyte adhesion by reduction of NF-B activation (left side) or by direct interference with the adhesion process (right side).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0673fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0673fje

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