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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 8, 2000 as doi:10.1096/fj.99-0602fje. |
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The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom;
* Institute of Pharmacology and Toxicology, Second University of Naples, 80138 Naples, Italy;
Institut Cochin de Génétique Moleculaire, Unit 332, Inserm, Paris, France; and
§ Department of Biology, IBILCE-UNESP, Sao Paulo, Brazil
2Correspondence: Department of Biochemical Pharmacology, The William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail: M.Perretti{at}qmw.ac.uk
SPECIFIC AIMS
Reperfusion ofthe heart is associated with myocardial damage induced by mediators released from the myocardium or from blood-borne polymorphonuclear leukocytes (PMN). A causal role between extravasated PMNs and reperfusion injury was first demonstrated by pioneering studies performed in animals depleted of circulating PMNs as well as by more recent investigations that address the potential role of specific adhesion molecules involved in the leukocyte–vascular endothelium interaction. Inhibition of PMN accumulation to the myocardium or regulation of PMN activation is an obvious target for the development of novel therapies against myocardial injury after ischemia-reperfusion. In this study, we examined the effect of the glucocorticoid-regulated protein lipocortin 1 (LC1) in a rat model of myocardial ischemia-reperfusion.
PRINCIPAL FINDINGS
1. LC1 protects the myocardium against ischemia-reperfusion injury
Myocardial injury was produced by the occlusion of the left
anterior descending coronary artery (LADCA) for 25 min followed by
2 h reperfusion. The extent of infarct size (IS) or tissue
necrosis was assessed by staining the area of risk with
p-nitro blue tetrazolium and expressed as a function of
the total left ventricle (IS/LV). Intravenous administration of human
recombinant LC1 immediately after the 25 min ischemic period afforded a
marked protection against the myocardial injury measured 2 h later
as estimated by the IS, with almost 50% inhibition at the highest dose
tested of 50 µg per rat. The effect of LC1 was dose dependent, with a
significant protection being measured at the dose of 5 µg per rat
(equivalent to 135 pmol). This protective effect of LC1 was abolished
by protein denaturation and not mimicked by the structurally related
protein annexin V. However, the chimeric protein LC1/5 was fully active
when tested at the dose of 50 µg per rat (Fig. 1
). Treatment with the steroid dexamethasone (DEX) also significantly
reduced the infarct size. DEX was equally active when administered as a
pretreatment 90 min before beginning of the ischemic procedure, with a
calculated value of IS/LV of 44.5 ± 3.3% (n=5).
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2. LC1 reduced recruitment of PMN to the damaged myocardium
Ischemia-reperfusion was associated with a pronounced increase in
the levels of MPO, a marker used to monitor tissue infiltration by
PMNs. Administration of LC1 at the doses that produced a significant
reduction in IS produced a dose-dependent attenuation in MPO activity
as measured 2 h postreperfusion (Fig. 2
). The attenuation in MPO activity was absent in rats treated with
boiled preparation of LC1, but a significant reduction in MPO activity
was found in animals that received DEX. The causal relationship between
PMN influx and heart damage was assessed in a few rats. As measured 30
min postreperfusion, MPO activity was 410 x
103 PMN equivalent per milligram of protein
(n=4; compare to the 2 h value shown in Fig. 2
). The
IS/LV value was 9.3 ± 2.3% (n=4 in all cases,
P < 0.01 vs. 2 h values shown in Fig. 1
).
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The protective effect of LC1 was confirmed by histological examination. Histological assessment of the hearts sections (10 µm) stained with Mayer’s hematoxylin and subjected to blind analysis showed a clear reduction in intravascular and extravascular leukocytes in the hearts of LC1-treated rats vs. control infarcted animals. For example, the number of extravascular leukocytes (x103 per mm2) found in infarct hearts and those treated with LC1 were 13.90 ± 0.63 and 5.08 ± 0.36, respectively (P<0.05, n=5). Electron microscopy analysis confirmed the lower degree of leukocyte infiltration in the myocardial tissue after treatment of rats with LC1.
DISCUSSION
This study examined the therapeutic potential of LC1 against ischemia-reperfusion injury in the heart. LC1, a member of the annexin or lipocortin superfamily of phospholipid and calcium binding proteins, is endowed with a potent leukocyte anti-migratory activity. Recently, we have demonstrated that administration of LC1 to experimental animals reduced the extravasation of blood-borne neutrophils in simple models of acute inflammation, apparently by interfering with the process of neutrophil interaction with the activated endothelium. This study is the first to show that intravenous treatment of rats with LC1 can protect against myocardial damage associated with ischemia-reperfusion. We propose that the inhibitory effects produced by LC1 were functionally related to a reduced recruitment of blood-borne PMNs into the damaged tissue.
Administration of human recombinant LC1 immediately after reopening of
the LADCA produced a dose-dependent reduction in the tissue damage
measured 2 h later in the rat heart. The effect of the protein was
pronounced, with a measured 
50% inhibition of the injury area at
the highest dose tested (50 µg per rat or equivalent to 130 pmol).
The protective effect of LC1 was dose dependent, but the denatured
protein was inactive as was the structurally related protein annexin V.
The data obtained with annexin V are of particular importance. This
protein has in fact been described to possess anti-thrombotic and
anti-coagulant actions. In addition, annexin V has been found in the
rat heart and its levels have been reported to increase in human plasma
after acute myocardial infarction. Our data, however, suggest that
annexin V may be a marker associated solely with the damage produced in
the myocardium by ischemia-reperfusion and that the protein is not
effective in altering the outcome of this experimental pathology. Most
significantly, the protection obtained with the chimeric protein LC1/5
(containing LC1 amino terminus on the core of the inactive annexin V)
indicated that the amino-terminal region of LC1 is the pharmacophore
responsible for the anti-migratory activity displayed by the entire
protein. Other groups have confirmed this observation. Indeed, peptides
derived from the LC1 amino terminus region are effective in reducing
the extent of tissue damage produced in the splanchnic region by
occlusion and reopening of the superior mesenteric artery.
Ischemia-reperfusion injury of the rat heart was associated with an increase in MPO activity, a marker used to monitor tissue infiltration by PMNs. Thus, a causal link between recruitment of white blood cells to the myocardium and subsequent damage was demonstrated. The increase in myocardial MPO activity was dose-dependently reduced by treatment of rats with LC1. These observations were confirmed by histological examination. Examination of hearts subjected to ischemia-reperfusion, but not those collected from sham-operated animals, showed adherent of white blood cells to the vessel wall below the endothelium but above the basement membrane, with some emigrated into the tissue. However, in hearts obtained from rats treated with LC1, a reduction in extravasated leukocytes was observed. These data, coupled with changes in MPO activity, strongly suggest that the protection afforded by LC1 was associated with a reduction in leukocytes recruitment to the infarct area.
It is now known that LC1 synthesis is modulated by glucocorticoids, so we tested the effect of DEX in this study. Given at the dose of 0.1 mg/kg, which is highly effective in blocking neutrophil adhesion to the inflamed endothelium of postcapillary venules in the rat, DEX produced a similar attenuation of heart injury when administered before or immediately after ischemia. Treatment with DEX is likely to increase LC1 levels in circulating leukocytes, thereby augmenting the inhibitory action of endogenous LC1 on neutrophils adherent to the endothelium. Further investigation is required to study the potential role of endogenous LC1 in a systematic manner.
In conclusion, LC1 inhibits the leukocyte-dependent damage of an infarcted myocardium. The molecular mechanism(s) responsible for these actions of LC1 remains elusive. However, we have recently described the novel property of the protein to cause detachment of adherent leukocytes from inflamed vessels. In addition, PMN adhesion to the endothelium up-regulates the expression of the putative LC1 ‘receptor’. Therefore, we propose that reduction in PMN adhesion to the myocardial vessels, likely through a phenomenon of cell detachment, is the major mechanism underlying the protection afforded by LC1 in these experimental conditions (see Scheme 1).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-0602fje 
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