Z Kardiol 89: Suppl 9, IX/96ÐIX/100 (2000)© Steinkopff Verlag 2000

C. KupattH. HabazettlB. F. BeckerP. Boekstegers

Endothelial activation – a strategic event during postischemicmyocardial inflammation

Summary Reperfusion after limitedischemia increases leukocyte adhesion,which may contribute to postischemicmyocardial and endothelial stunning.Leukocyte adhesion is enhancedimmediately after the onset of reperfu-sion, indicating rapid regulatory mech-anisms. In addition, de novo synthesisof, e.g., adhesion molecules induced byreperfusion has been described, provid-ing a prolonged postischemic inßam-matory reaction. Enhanced transcrip-tion of adhesion molecules involves NFkB activation which appears as anessential transcription factor for reper-

fusion-induced subacute endothelialactivation. Both, acute and subacuteendothelial activation seem tocontribute to the Þnal extent of leuko-cyte-dependent reperfusion injury andsuccessive treatment of both appears tobe a promising therapeutic strategy forpostischemic myocardial inßammationand its detrimental effects.

Key words Reperfusion Ð TNF a ÐPMN Ð ICAM-1 Ð selectins Ð reactiveoxygen species Ð endothelial stunningÐ myocardial stunning

Christian Kupatt, MD (Y) á P. BoekstegersInternal Medicine IUniversity Klinikum Gro§hadernMarchioninistr. 1581377 Munich, GermanyE-mail: c.kupatt@lrz.uni-muenchen.de

H. HabazettlInstitute for Surgical ResearchUniversity of MunichMunich, Germany

B. F. BeckerPhysiological InstituteUniversity of MunichMunich, Germany

Introduction

The strategic localization of the endothelium which separatesblood from tissue parenchyma largely deÞnes its physiologi-cal and pathophysiological function. Under physiological con-ditions, mechanisms potentially restricting blood perfusion,including leukocyte and platelet adhesion, are inactive orcounterbalanced. Under stress of various origins, however,endothelial cells react by changing their phenotype. Endothe-lial activation provides a proadhesive and procoagulant phe-notype, allowing for white blood cell adhesion and emigrationas well as platelet adhesion and aggregation. Importantly, theterm endothelial activation implies two distinct entities, sepa-rable by different time courses as well as underlying mecha-nisms. Acute endothelial activation takes place within secondsto minutes after stimulation. It implies transfer of P-selectin

[1], an endothelial adhesion molecule mediating the initialleukocyte contact (rolling [2]), from storage vesicles to the cellsurface. Moreover, release of leukocyte activating factors likereactive oxygen species or PAF takes place [3Ð6]. Thesefactors are capable of inducing expression of activated leuko-cyte b2-integrins (MAC-1 [7], LFA-1 [8]), which mediate Þrmadhesion by binding to endothelial adhesion molecules. Ineffect, all prerequisites of leukocyte adhesion (leukocyte acti-vation, rolling and sticking) [9, 10] are provided early in thecourse of reperfusion.

In contrast, subacute endothelial activation depends ontranscriptional activation resulting in de novo synthesis of pro-teins, which are functionally effective after a 1Ð4 h delay ofreperfusion. During this interval, adhesion molecules likeICAM-1, VCAM-1, E-selectin and again P-selectin, procoag-ulants like tissue factors [11], and cytokines like TNF a[12Ð14] or IL-1 [15], among others are formed.

C. Kupatt et al. IX/97Endothelial activation

Leukocyte adhesion is upregulated during both intervals ofendothelial activation. It is initiated by acute endothelial acti-vation leading to PMN recruitment and subsequent myocardialstunning within minutes of reperfusion [16Ð18]. Later, withthe acute trigger mechanisms fading, leukocyte recruitment isstill observed [19]. Acute and subacute endothelial activationboth contribute signiÞcantly to myocardial dysfunction: Flynnand coworkers have shown in a dog model in vivo that acuteinhibition of leukocyte rolling by a synthetic selectin-antago-nist reduced postischemic PMN recruitment, myocardial dys-function and infarct size. However, after prolonged reperfu-sion (48 h) myocardial protection was lost, if treatment had notbeen given continuously over 24 h [20].

Postischemic acute endothelial activation

Acute endothelial activation occurs rapidly after the restora-tion of blood ßow in a previously occluded coronary artery.Formation of reactive oxygen species (ROS) is a hallmark ofpostischemic reperfusion, leading to the formation of, amongothers, H2O2. Interestingly, H2O2 is capable of inducing P-selectin translocalization [21], which by interaction with itsleukocyte counterligands induces rolling as an initial,reversible step of cell-cell interaction. The fate of rollingleukocytes, either Þrm adhesion or detachment from the vesselwall, has yet to be determined by their activation status and theabundance of adhesion molecules mediating Þrm adhesionand subsequent emigration. PMNs themselves are activated byreactive oxygen species generated in a postischemic heart [22],leading to upregulation and activation of MAC-1 [4, 7]. More-over, rapid endothelial release of PAF, a potent leukocyte

activator [6], may stimulate resting leukocytes and causeinteraction with platelets [23], which only recently have beenrecognized as an additional source of leukocyte activation[24Ð26].

On the endothelial side, coronary postcapillary venulesconstitutively express ICAM-1, which may rapidly be upreg-ulated at the luminal site by receptor trafficking. In additionconstitutively expressed ICAM-2 may serve as a counterli-gand of b2-integrins [27].

Consistently, therapeutic strategies aimed at one of thesetargets, e.g., application of antibodies against adhesion mole-cules [16, 20, 28Ð32] as well as antagonists of leukocyte-acti-vating factors [6, 18, 33] limit the extent of PMN recruitmentacutely after the onset of reperfusion.

Postischemic subacute endothelial activation

Although subacute endothelial activation (Type II [34, 35])accounts for delayed change of the endothelial phenotype, andprovides the capability to, e.g., recruit leukocytes over a pro-longed time interval [36], this process is initiated rapidly afterpostischemic reperfusion. Transcriptional activation, assessedas DNA-binding activity of transcription factors, e.g., NF kBand AP-1, is detected within 15Ð30 min of the onset of reper-fusion in vivo [37]. The fact that a similar time course isdetected in isolated hearts provides evidence that transcrip-tional activation does not entirely rely on blood borne factorsbut also on factors present in the heart itself [14]. In parallel tocell culture experiments, where H2O2 may directly activate NFkB during posthypoxic reoxgenation [38], ROS scavengersmay inhibit subacute endothelial activation in vivo [39].

Fig. 1 ELISA of P-selectin (a)and ICAM-1 (b) expression after60 min of anoxic treatment and30 or 240 min of reoxygenation(20 % O2). P-selectin expressionincreases acutely (30 min) and, toa greater extent, subacutely (240min) after the onset of reoxygena-tion, whereas ICAM-1 expressionincreases subacutely (240 min)but not acutely after reoxygena-tion. * = p < 0.05 vs. Control, # = p < 0.05 vs. 30 min anoxictreatment. (ModiÞed after [42].)

Another likely initiator of the subacute candidate is apparentlyTNF a, which activates the NF kB and AP-1 activation cas-cades after binding to its endothelial receptors. Release ofpreformed and stored TNF a from mast cells [13] or cleavagefrom its membrane-bound precursors by a speciÞc enzyme[40] is required, since time course studies have revealed thatTNF a transcription occurs after the initial NF kB activation[14].

Upon transcriptional activation, messenger RNA andsubsequently protein content of a variety of genes increase;adhesion molecules like ICAM-1, VCAM-, E-selectin and P-selectin are synthesized de novo facilitating leukocyte recruit-ment in the subacute interval [41Ð43]. Moreover, chemotacticfactors like cytokines (TNF a, Il-1, Il-6, Il-8) [15] or ccchemokines (MCP-1) [44] are formed and released subacutelyafter the onset of reperfusion.

Given the proinßammatory environment created by suba-cute endothelial activation, functional sequelae should prevail.In fact, enhanced PMN adhesion can be observed in thesubacute interval after the onset of reoxygenation. Fittingly,adhesion molecule expression precedes the adhesion kineticswith P-selectin peaking acutely and subacutely whereasICAM-1 is upregulated only by transcription (Fig. 1) [43].Whole organ studies using ex vivo microscopy revealed sim-ilar kinetics, with a rapid increase of postischemic PMN adhe-sion at postcapillary venules compared to nonischemic con-trols. Moreover, in addition to the acute PMN recruitment, a2.5-fold increase of PMN adhesion can be found after 8 h ofreperfusion (Fig. 2).

The transcriptional factors involved in the subacute activa-tion process have been investigated by transfection with decoyelements, short (20Ð30 bp) double-stranded oligonucleotidesencoding the binding sequence for the targeted transcriptionfactor. In cell culture and in isolated hearts, NF kB or AP-1decoy transfection inhibited the subacute increase in PMNadhesion [14, 43]. In vivo, Morishita and coworkers [45, 46]

found that postischemic NF kB decoy transfection decreasedinfarct size of rat hearts after 48 h of transfection. Takentogether, decoy transfection for speciÞc transcription factorsappears as a promising strategy to target subacute endothelialactivation of postischemic hearts in vivo.

In summary, endothelial activation follows stimulation ofendothelial cells by ischemia and reperfusion/reoxygenation.Acute and subacute upregulation of adhesion molecules,cytokines and chemokines enforces postischemic inßamma-tion which induces PMN activation and adhesion, and subse-quent PMN dependent reperfusion injury. Therapeutic inter-ventions aimed at prevention of both acute and subacuteendothelial activation seems appropriate to prevent a potentialinßammatory overreaction induced by postischemic reper-fusion.

IX/98 Z Kardiol 89, Suppl 9 (2000)© Steinkopff Verlag 2000

Fig. 2 PMN adhesion to postcapillary venules (15-50 µm diameter)without ischemia (control), after ischemia (15 min) and reperfusion(2 min, acute) or after 4 h, 8 h of reperfusion and a second ischemia(15 min) and reperfusion (2 min). * = p < 0.05 vs all other groups. (Mod-iÞed after [14].)

C. Kupatt et al. IX/99Endothelial activation

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