Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration.

To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (alpha(5)beta(1) integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase-dependent proteolysis of this beta(1) integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1-induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5.

Interleukin-8 gene induction in the myocardium after ischemia and reperfusion in vivo.

Neutrophil adhesion and direct cytotoxicity for cardiac myocytes require chemotactic stimulation and are dependent upon CD18-ICAM-1 binding. To characterize the potential role of IL-8 in this interaction, canine IL-8 cDNA was cloned and the mature recombinant protein expressed in Escherichia coli BL21 cells. Recombinant canine IL-8 markedly increased adhesion of neutrophils to isolated canine cardiac myocytes. This adhesion resulted in direct cytotoxicity for cardiac myocytes. Both processes were specifically blocked by antibodies directed against CD18 and IL-8. In vivo, after 1 h of coronary occlusion, IL-8 mRNA was markedly and consistently induced in reperfused segments of myocardium. IL-8 mRNA was not induced in control (normally perfused) myocardial segments. Minimal amounts of IL-8 mRNA were detected after 3 or 4 h of ischemia without reperfusion. Highest levels of induction were evident in the most ischemic myocardial segments. IL-8 mRNA peaked in the first 3 h of reperfusion and persisted at high levels beyond 24 h. IL-8 staining was present in the inflammatory infiltrate near the border between necrotic and viable myocardium, as well as in small veins in the same area. These findings provide the first direct evidence for regulation of IL-8 in ischemic and reperfused canine myocardium and support the hypothesis that IL-8 participates in neutrophil-mediated myocardial injury.

Decreased expression of tumor necrosis factor-alpha in failing human myocardium after mechanical circulatory support : A potential mechanism for cardiac recovery.

BACKGROUND: An increasing number of observations in patients with end-stage heart failure suggest that chronic ventricular unloading by mechanical circulatory support may lead to recovery of cardiac function. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine capable of producing pulmonary edema, dilated cardiomyopathy, and death. TNF-alpha is produced in the myocardium in response to volume overload; however, the effects of normalizing ventricular loading conditions on myocardial TNF-alpha expression are not known. We hypothesize that chronic ventricular unloading by the placement of a left ventricular assist device (LVAD) may eliminate the stress responsible for persistent TNF-alpha expression in human failing myocardium. METHODS AND RESULTS: Myocardial tissue was obtained from normal hearts and from paired samples of 8 patients with nonischemic end-stage cardiomyopathy at the time of LVAD implantation and removal. Tissue sections were stained for TNF-alpha, and quantitative analysis of the stained area was performed. We found that TNF-alpha content decreased significantly after LVAD support. Furthermore, the magnitude of the changes did not correlate with the length of LVAD support, although greater reductions in myocardial TNF-alpha content were found in patients who were successfully weaned off the LVAD who did not require transplantation. CONCLUSIONS: These data show for the first time that chronic mechanical circulatory assistance decreases TNF-alpha content in failing myocardium; furthermore, we suggest that the magnitude of the change may predict which patients will recover cardiac function.

Regression of fibrosis and hypertrophy in failing myocardium following mechanical circulatory support.

BACKGROUND: The cellular and structural changes that occur during long-term ventricular unloading leading to cardiac recovery are poorly understood. However, we have previously demonstrated that left ventricular assist device (LVAD) support leads to a significant decrease in intracardiac tumor necrosis factor-alpha (TNF-alpha), a protein capable of producing hypertrophy and fibrosis. METHODS: To further define the beneficial effects of long-term ventricular unloading on cardiac function, we determined the effect of mechanical circulatory support on fibrosis and hypertrophy in paired myocardial samples of 18 patients with end-stage cardiomyopathy obtained at the time of LVAD implantation and removal. RESULTS: We determined total collagen as well as collagen I and III by a semiquantitative analysis of positive immune-stained areas in pre- and post-LVAD myocardial samples. We found that total collagen content was reduced by 72% (p < 0.001), whereas collagen I content decreased by 66% (p < 0.001) and collagen III content was reduced by 62% (p < 0.001). Next, we determined myocyte size by direct analysis of cellular dimensions utilizing a computerized edge detection system in pre- and post-LVAD myocardial samples. We found that myocyte size decreased in all patients studied for an average reduction of 26% (33.1 +/- 1.32 to 24.4 +/- 1.64 microm, p < 0.001). CONCLUSION: These data demonstrate that long-term mechanical circulatory support significantly reduces collagen content and decreases myocyte size. We suggest that the reduction of fibrosis and hypertrophy observed may in part contribute to the recovery of cardiac function associated with long-term mechanical circulatory support.

Phagocytes in ischemia injury.

We are now developing the means to evaluate components of this inflammatory response that may facilitate healing. A key event in the change in the inflammatory response is the development of a cytokine cascade that promotes phenotypic changes in the infiltrating leukocytes, which endow them with the ability to promote fibroblast proliferation and collagen deposition, the hallmarks of healing.

The implications for cardiac recovery of left ventricular assist device support on myocardial collagen content.

BACKGROUND: To define the beneficial cellular changes that occur with chronic ventricular unloading, we determined the effect of left ventricular assist device (LVAD) placement on myocardial fibrosis. METHODS: We obtained paired myocardial samples (before and after LVAD implantation) from 10 patients (aged 43 to 64 years) with end-stage cardiomyopathy. We first determined regional collagen expression of an explanted heart by a computerized semiquantitative analysis of positive picro-sirius red stained areas. RESULTS: We found that there was no statistically significant difference in collagen content between regions of the failed heart studied. Next we determined collagen content in these paired myocardial biopsies pre- and post-LVAD implantation. All 10 patients had significant reductions in collagen content after LVAD placement with a mean reduction of 82% (percent of tissue area stained decreased from 32% +/- 4% to 4% +/- 0.8%, P < 0.001). CONCLUSION: In summary, these data demonstrate that chronic mechanical circulatory support significantly reduces fibrosis in the failing myocardium.

Tumor necrosis factor-α in the failing human heart: Does it play a role in disease progression?

An increasing body of clinical and experimental evidence suggesting that TNF(α) may play a pathogenetic role in failing hearts continues to accumulate. Perhaps the most direct evidence for the role of TNF(α) in heart failure will come from the analysis of the phase I study in which a soluble recombinant human TNF receptor: Fc fusion protein was utilized in patients with moderate to severe heart failure Enrollment in that trial was recently completed; the results will soon be available for analysis. But perhaps more importantly, the knowledge gained from studying the role of TNF(α) in cardiac function draws attention to a series of molecules previously unrecognized as potential mediators in the pathogenesis of heart failure. Various cytokines and TNF(α), in particular, represent new targets for therapeutic intervention in patients with heart failure.

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein.

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.

Induction of interleukin-6 synthesis in the myocardium. Potential role in postreperfusion inflammatory injury.

BACKGROUND: Neutrophil-induced injury of myocardial cells requires the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte surface and is mediated by ICAM-1-CD11b/CD18 adhesion. We have previously shown that interleukin-6 (IL-6) cytokine activity, present in cardiac lymph, induces ICAM-1 on isolated cardiac myocytes. Furthermore, in previous in vivo studies, we have also shown ICAM-1 mRNA induction in the myocardium within the first hour of reperfusion in the previously ischemic viable zone. We hypothesized that induction of IL-6 synthesis in the myocardium was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells. METHODS AND RESULTS: In this study, cloned canine IL-6 cDNA was used as a molecular probe to study the regulation of IL-6 in an awake canine model of myocardial ischemia and reperfusion. IL-6 mRNA was induced in ischemic and reperfused segments of myocardium preferentially in segments previously exposed to severe ischemia. Peak levels of IL-6 mRNA were reached within 3 hours of reperfusion. At the same time, IL-6 mRNA and ICAM-1 mRNA were found in the same myocardial segments. In contrast to hearts that were ischemic for 1 hour and reperfused for 3 hours, nonreperfused hearts after 4 hours of persistent ischemia demonstrated minimal induction of ICAM-1 or IL-6 despite similar degrees of injury and blood flow reductions during ischemia. After 24 hours of persistent ischemia, levels of IL-6 mRNA were comparable to those observed in hearts that were ischemic for 1 hour and subsequently reperfused for 24 hours. CONCLUSIONS: Our results demonstrate induction of IL-6 mRNA in the myocardium and that this synthesis is accelerated by reperfusion. Evidence is also provided to show that peak IL-6 mRNA precedes that of ICAM-1 mRNA. These findings are compatible with our hypothesis that IL-6 is important in the induction of ICAM-1 in the area of ischemia. In addition, these studies suggest that the necessary factors to promote adhesive interactions between transmigrated neutrophils and cardiac myocytes are present in reperfused myocardium.

Nucleotide specificity of canine cardiac sarcoplasmic reticulum. Differential alteration of enzyme properties by detergent treatment.

We previously demonstrated that, in contrast to the hydrolysis of ATP, the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium. Based on a variety of qualitative and quantitative considerations (cf. Tate, C. A., Bick, R. J., Chu, A., Van Winkle, W. B., and Entman, M. L. (1985) J. Biol. Chem. 260, 9618-9623), we suggested that the hydrolysis of ATP and GTP appears to be effected by the same enzyme. In the present paper, we examined the sensitivity of both enzymatic activities to low concentrations of detergent. With nonsolubilizing concentrations of the nonionic detergent, octaethylene glycol monododecyl ether, the hydrolysis of GTP was rendered partially calcium-sensitive resulting from a slightly increased total (Ca2+ + Mg2+)-GTPase activity and a markedly inhibited calcium-independent (Mg2+-dependent) GTPase activity. Calcium-dependent ATPase activity was increased with octaethylene glycol monododecyl ether, mimicking the effect of the ionophore, A23187. Calcium-dependent ATPase activity and detergent-induced calcium-dependent GTPase activity were similar in (a) calcium sensitivity, (b) sensitivity to mersalyl, and (c) pressure inactivation through dilution and centrifugation, all of which differed from the untreated calcium-independent GTPase activity. Calcium-dependent ATPase activity differed from calcium-dependent GTPase activity with (a) a higher nucleotide affinity, (b) a lower vanadate sensitivity, and (c) a calcium sensitivity for phosphoenzyme formation. Thus, the detergent-induced perturbation of the GTPase resulted in an enzyme with many characteristics qualitatively and quantitatively similar to the calcium ATPase.

Time-dependent loss of Mac-1 from infiltrating neutrophils in the reperfused myocardium.

Numerous studies have shown that polymorphonuclear neutrophils (PMNs) infiltrate the myocardium immediately after reperfusion of infarcted tissue. Studies with mAbs in vivo and cellular studies in vitro suggest that PMN-induced injury of the cardiac myocyte involve Mac-1 adhesion to myocyte ICAM-1. In this study we demonstrate that PMNs that have infiltrated the ischemic area begin to lose Mac-1 within the first 3 h. By the fifth hour of reperfusion, minimal CD11b staining is seen on PMNs using immunostaining, whereas CD11a remained unchanged. Immunoreactivity of postreperfusion cardiac lymph with R15.7 (anti-CD18) or MY904 (anti-CD11b) was positive in all animals but not for CD11a (R7.1), indicating a specific loss of Mac-1. Immunoprecipitation with either R15.7 or MY904 resulted in identical peptides (a doublet at 190 kDa and a band at 80 kDa), suggesting that both alpha and beta subunits of Mac-1 heterodimer were released. Immunoprecipitation of control PMN lysates revealed bands of 198 kDa and 91 kDa slightly greater than those from the released Mac-1. An in vitro model of homotypic aggregation showed a similar loss of Mac-1 from PMNs; immunoprecipitates of the supernatant demonstrated peptide bands identical with those found in postischemic cardiac lymph. The appearance of soluble Mac-1 in vitro was prevented by anti-CD18 mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and in vitro, activated PMNs lose Mac-1 in a process that may be dependent upon adhesion and subsequent proteolysis.

Myocardial ischemia and reperfusion: a murine model.

Myocardial ischemia followed by reperfusion promotes a complex series of inflammatory reactions as noted in a variety of large animal studies. With development of genetically altered mice, there is intense interest in developing murine models to study mechanisms operative in cardiovascular disease. We developed a mouse model to study coronary artery occlusion and reperfusion effects and the method required to perform these studies both acutely and chronically. In mice, we applied a left anterior descending coronary artery occlusion either permanently or for 30 or 60 min followed by reperfusion allowing flow through the previously occluded coronary artery bed. Reperfusion was documented visually as well as by using Doppler ultrasound and histopathological techniques. The area at risk (AAR) and infarct size (IS) were assessed by EVans blue dye and triphenyltetrazolium chloride staining with computerized planimetry using an image analysis software program. The infarct as percentage of AAR and IS as percentage of the left ventricle in 13 mice with permanent occlusion was 68.6 +/- 4.4 and 28.0 +/- 2.8%, respectively. Reperfusion after occlusions of 60 and 30 min resulted in a significant decrease in IS as a percentage of the AAR compared with permanent occlusion. Histological examination of the ischemic and reperfused myocardium shows infiltration of leukocytes into the ischemic region as well as contraction bands classically associated with reperfusion. This new model allows assessment of AAR, IS, cardiac function, and pathophysiology in the mouse. With the current technology to develop genetically altered mice for overexpression or targeted mutations of various genes, this model is used to understand the complex pathophysiology of ischemia and reperfusion injury.

Molecular evidence for induction of intracellular adhesion molecule-1 in the viable border zone associated with ischemia-reperfusion injury of the dog heart.

BACKGROUND: Acute inflammation may play a role in injury during reperfusion following myocardial ischemia. Studies in vitro suggest that intracellular adhesion molecule-1 (ICAM-1) mediates neutrophil adherence to cardiac myocytes and neutrophil-mediated injury. We have shown cytokine activity in postischemic cardiac lymph sufficient to maximally express ICAM-1 on myocytes and that ICAM-1 mRNA is found in the previously ischemic myocardium early in reperfusion. METHODS AND RESULTS: In the present study, we used in situ hybridization techniques to detect ICAM-1 mRNA and examine the cells of origin, relation to cell injury, and relation to inflammatory infiltration after 1 hour of ischemia and varying times of reperfusion. By 1 hour of reperfusion, ICAM-1 mRNA was detected in much of the ischemic myocardium, except in areas of contraction band necrosis. At 2 and 3 hours, a clear demarcation of necrotic areas surrounding ischemic areas of viable myocardium with ICAM-1 mRNA staining was present, and ICAM-1 mRNA staining increased with time. Nonischemic areas had no visible ICAM-1 mRNA staining in the first 3 hours. By 24 hours of reperfusion, ICAM-1 mRNA was present in both control and ischemic segments (excluding the necrotic areas) compatible with a generalized circulation of cytokines persistent at 24 hours. In the absence of reperfusion, ICAM-1 mRNA staining was not seen in the first 3 hours and was markedly reduced at 24 hours. The interface of viable and necrotic cells also contained the most extensive inflammatory infiltration. CONCLUSIONS: Evidence is presented that induction of ICAM-1 mRNA has highly specific localization to ischemic but viable myocardium. Induction of ICAM-1 mRNA transcription in early reperfusion may render the viable "border zone" susceptible to neutrophil-induced injury.

Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions.

BACKGROUND: Previous work from our laboratory demonstrated that interleukin (IL)-6 plays a potentially critical role in postreperfusion myocardial injury and is the major cytokine responsible for induction of intracellular adhesion molecule (ICAM)-1 on cardiac myocytes during reperfusion. Myocyte ICAM-1 induction is necessary for neutrophil-associated myocyte injury. We have previously demonstrated the induction of IL-6 in the ischemic myocardium, and the current study addresses the cells of origin of IL-6. METHODS AND RESULTS: In the present study, we combined Northern blot analysis and in situ hybridization to demonstrate IL-6 gene expression in cardiac myocytes. Isolated ventricular myocytes were stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, preischemic lymph, and postischemic lymph. Unstimulated myocytes showed no significant IL-6 mRNA expression. Myocytes stimulated with preischemic lymph showed minimal or no IL-6 mRNA expression, whereas myocytes stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, or postischemic lymph showed a strong IL-6 mRNA induction. Northern blot with ICAM-1 probe revealed ICAM-1 expression under every condition that demonstrated IL-6 induction. We then investigated the expression of IL-6 mRNA in our canine model of ischemia and reperfusion. Cardiac myocytes in the viable border zone of a myocardial infarction exhibited reperfusion-dependent expression of IL-6 mRNA within 1 hour after reperfusion. Mononuclear cells infiltrate the border zone and express IL-6 mRNA. CONCLUSIONS: Isolated cardiac myocytes produce IL-6 mRNA in response to several cytokines as well as postischemic cardiac lymph. In addition to its production by inflammatory cells, we demonstrate that IL-6 mRNA is induced in myocytes in the viable border zone of a myocardial infarct. The potential roles of IL-6 in cardiac myocytes in an infarct border are discussed.

Role of early reperfusion in the induction of adhesion molecules and cytokines in previously ischemic myocardium.

Our studies in vitro demonstrate that neutrophil mediated injury of isolated cardiac myocytes requires the presence of ICAM-1 on the surface of the myocyte and CD11b/CD18 activation on the neutrophil. In post-ischemic cardiac lymph, there is rapid appearance of C5a activity during the first hours of reperfusion. Interleukin-6 activity is present throughout the first 72 h of reperfusion and is sufficient to induce ICAM-1 on the surface of the cardiac myocyte. In situ hybridization studies suggest that ICAM-1 mRNA is found in viable myocardial cells on the edge of the myocardial infarction within 1 h of reperfusion. ICAM-1 protein expression on cardiac myocytes is seen after 6 h of reperfusion, and increases thereafter. Non-ischemic tissue demonstrates no early induction of ICAM-1 mRNA or ICAM-1 protein on myocardial cells. In our most recent experiments, we have determined that reperfusion is an absolute requirement for the early induction of myocardial ICAM-1 mRNA in previously ischemic myocardial cells. To further assess this, we have cloned and sequenced a canine interleukin-6 (IL-6) cDNA. The data suggest that early induction of IL-6 mRNA is also reperfusion dependent as it could be demonstrated in the same ischemic and reperfused segments in which ICAM-1 mRNA was found. Peak expression of IL-6 mRNA occurred much earlier than that for ICAM-1 mRNA. Similar experiments were then performed with a molecular probe for interleukin-8 (IL-8). This chemokine is a potent neutrophil stimulant and has a higher degree of specificity for neutrophils than classic chemoattractants such as C5a.(ABSTRACT TRUNCATED AT 250 WORDS)

Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival.

Cardiac muscle regeneration after injury is limited by "irreversible" cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis, in vitro and in vivo. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.

Acute inflammatory reaction after myocardial ischemic injury and reperfusion. Development and use of a neutrophil-specific antibody.

Reperfusion of the infarcted canine myocardium after 1 hour of ischemia is associated with an acute inflammatory infiltrate at the border of the infarct. In this paper, we demonstrate that early margination and emigration of neutrophils originate in thin-walled (approximately 5 micrometers) venous cisterns that average 200 micrometers in length and vary from 10 to 70 micrometers in width and show strong constitutive expression of both ICAM-1 and P-selectin; this class of vessels (venous cisterns) appears to be a unique feature in heart. A monoclonal antibody (SG8H6) with specificity for canine neutrophils was developed that allowed much more sensitive immunohistochemical detection of neutrophils in tissue and allowed us to follow tissue infiltration with time. Samples from 1 hour of reperfusion revealed dense margination and substantial emigration of neutrophils associated with the venous cisterns and collecting venules. By 2 hours, there was intense local emigration to the extravascular space between cardiac myocytes. By 3 hours, the infiltrate extended deeper into the infarct, and there was a continuous border zone of neutrophil infiltration that overlapped a region where intact cardiac myocytes strongly expressed ICAM-1 mRNA and extended into the necrotic tissue. At later times, neutrophil migration into infarcted tissue continued to progress. Neutrophil transmigration into reperfused myocardium is more extensive than previously described, and its extravascular distribution during early reperfusion is primarily in the viable border zone of the myocardium where myocyte ICAM-1 mRNA is found. These data are compatible with the hypothesis that extravascular neutrophils may participate in reperfusion injury.

Molecular evidence for a border zone vulnerable to inflammatory reperfusion injury.

Acute inflammation has been suggested as a potential mechanism for some of the injury associated with reperfusion of the ischemic myocardium. This hypothesis implies that viable myocardial cells adjacent to the lethally injured cells are vulnerable to injury induced by the neutrophil influx observed to attend reperfusion. In our previous work, we demonstrated that the presence of ICAM-1 on the surface of cardiac myocytes is required for neutrophils to directly damage them; blocking monoclonal antibodies to either ICAM-1 on cardiac myocytes or Mac-1 on activated neutrophils completely precluded neutrophil-induced myocyte injury. We also demonstrated that postischemic cardiac lymph (cardiac extracellular fluid) contained leukotactic factors (primarily C5a) and cytokines present in concentrations sufficient to maximally induce Mac-1 on the surface of neutrophils and ICAM-1 on the surface of isolated dog cardiac myocytes. The present study sought to further these observations by examining the site of potential ICAM-1 induction as a function of time of reperfusion, degree of ischemia, and viability of myocardial cells. Our evidence suggests that ICAM-1 mRNA is induced very early after reperfusion only in the previously ischemic myocardium and is not seen in the nonischemic myocardium during the early hours of reperfusion. Moreover, ICAM-1 mRNA induction is seen most intensely in the ischemic area directly bordering the necrotic area (which, after 1-hr reperfusion, does not contain any ICAM-1 mRNA) and immediately abutting the site of maximal influx of neutrophils. Thus, the induction of ICAM-1 and the influx of neutrophils (presumably activated by the chemotactic factors that guided their migration) exists on the border between viable and necrotic cells. This provides the first direct molecular evidence for a jeopardized border zone on the edge of myocardial infarction during reperfusion. As previously demonstrated, this reaction is wholly dependent upon tissue injury of the ischemic myocardium and therefore represents an example of a mechanism of injury extension induced as a reaction to a primary injury. The degree of specificity of this reaction demonstrated by the subendocardial sparing directly adjacent to ischemic cells suggests finely modulated mechanisms by which this process is controlled.