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Cardiovascular Research 2001 49(1):69-77; doi:10.1016/S0008-6363(00)00226-1
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

PR-39, a proline/arginine-rich antimicrobial peptide, exerts cardioprotective effects in myocardial ischemia–reperfusion

Yasuhiko Ikeda1,a, Lindon H Young2,a, Rosario Scaliaa, Christopher R Rossb and Allan M Lefera,*

aDepartment of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107-6799, USA
bDepartment of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA

* Corresponding author. Tel.: +1-215-503-7760; fax: +1-215-503-2073 allan.m.lefer{at}mail.tju.edu

Received 31 May 2000; accepted 13 September 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: PR-39, a proline/arginine-rich antimicrobial peptide, has been shown to inhibit the NADPH oxidase activity of polymorphonuclear leukocytes (PMNs) by blocking assembly of this enzyme. We hypothesized that PR-39 could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production. Methods: We examined the effects of PR-39 in isolated ischemic (20 min) and reperfused (45 min) rat hearts administered PMNs at the onset of reperfusion. Results: PR-39 (4 or 10 µg/ml) given i.v. 30 min prior to ischemia–reperfusion (I–R) significantly improved left ventricular developed pressure (LVDP, P<0.01) and the maximal rate of development of LVDP (i.e. +dP/dt max, P<0.01) compared to I–R hearts obtained from rats given 0.9% NaCl. PR-39-treated PMNs (10 µg/ml) also significantly attenuated cardiac contractile dysfunction after I–R (P<0.01). Superoxide release was significantly reduced (P<0.01) in N-formylmethionyl-leucylphenylalanine stimulated PMNs pretreated with 4 or 10 µg/ml PR-39. PR-39 also significantly attenuated P-selectin expression on the rat coronary microvascular endothelium and CD18 upregulation in rat PMNs. In addition, PR-39 significantly reduced PMN vascular adherence and infiltration into the post-ischemic myocardium. Conclusion: These results provide evidence that PR-39 significantly attenuates PMN-induced cardiac contractile dysfunction in the I–R rat heart at least in part via suppression of superoxide release. This cardioprotection occurred both by inhibition of PMN and endothelial NADPH oxidase.

KEYWORDS Coronary circulation; Coronary disease; Endothelial function; Leukocytes; Reperfusion


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
It is well established that reactive oxygen species, such as superoxide, hydrogen peroxide (H2O2) and hydroxyl radical, contribute to myocardial tissue injury that follows ischemia and reperfusion [1–3]. Numerous attempts have been made to demonstrate the effectiveness of antioxidant interventions such as superoxide dismutase (SOD) and catalase. Although SOD catalyzes the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide, efficacy of exogenously administered SOD is controversial even in the presence of catalase [4–7], probably due to its short half-life, and limited permeability into cells. Therefore, we focused our attention on reducing superoxide production via inhibition of NADPH oxidase.

It is well known that NADPH oxidase is a major source of superoxide production in PMNs [8]. NADPH oxidase is a membrane-associated enzyme that catalyzes the reduction of molecular oxygen using NADPH as the electron donor. NADPH oxidase is a multicomponent enzyme that consists of several constituents, including a membrane spanning cytochrome b558 (gp91phox and p22phox) and cytosolic components (p47phox and p67phox) [9,10]. Recent studies have shown that functionally active NADPH oxidase has also been demonstrated in cultured human endothelial cells and rat coronary microvascular endothelial cells, and this enzyme is similar to that in leukocytes in that both types of oxidases require p47phox for superoxide anion generation [11,12]. Moreover, generation of reactive oxygen species via NADPH oxidase was demonstrated after anoxia–reoxygenation in cultured endothelial cells [13].

PR-39, a proline/arginine-rich antimicrobial peptide, has been shown to inhibit the NADPH oxidase activity of PMNs by blocking oxidase assembly [14]. PR-39 binds to p47phox and, thereby inhibits interaction with p22phox resulting in attenuation of superoxide production [14]. Recently, PR-39 was reported to prevent post-ischemic oxidant production, as well as leukocyte adhesion and emigration in rat mesenteric venules [15]. However, the effects of PR-39 have not yet been assessed in myocardial ischemia–reperfusion in the presence of PMNs.

The purpose of the present study was to examine the effects of PR-39 on cardiac contractile function in the isolated perfused rat heart following PMN-induced reperfusion injury. Our findings show that PR-39 exerts cardioprotective effects in isolated perfused rat hearts, and implicate superoxide as one of the important mediators of the reperfusion induced cardiac contractile dysfunction observed in this model of ischemia–reperfusion.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.1 Isolation of PMNs and plasma
Sprague–Dawley rats (350–400 g), used as neutrophil donors, were anesthetized with ethyl ether and were given a 14 ml intraperitoneal injection of 0.5% oyster glycogen (Sigma) dissolved in phosphate buffered saline (PBS). At 16–18 h later, the rats were anesthetized with ethyl ether, and the neutrophils were harvested by peritoneal lavage in 30 ml of 0.9% NaCl as previously described [16]. The peritoneal lavage fluid was centrifuged at 250 g for 20 min at 4°C. The PMNs were then washed in 15 ml of PBS and recentrifuged at 250 g for 10 min at 4°C. Thereafter, the PMNs were resuspended in 2.5 ml of PBS and 8–10 rat samples were pooled prior to use. The neutrophil preparations were >90% pure and >95% viable according to microscopic analysis and exclusion of 0.3% trypan blue, respectively.

Plasma was isolated from a single rat in each cardiac perfusion experiment in order to infuse along with the PMNs to more closely simulate the conditions present in vivo. Blood was collected from the aorta in citrate phosphate buffer just prior to isolating the heart (described below). The blood was centrifuged immediately in a refrigerated centrifuge at 10 000 g for 10 min. The plasma was decanted and later infused at reperfusion in ischemia–reperfusion (I–R) hearts.

2.2 Drug administration
Male Sprague–Dawley rats (275–325 g) were anesthetized with ethyl ether then given PR-39 to produce a blood concentration of 4 or 10 µg/ml into the sublingual vein 30 min prior to isolation of rat hearts. Control rats were given an equal volume of 0.9% NaCl.

2.3 Isolated rat heart preparation
Rats were reanesthetized with 60 mg/kg sodium pentobarbital intraperitoneally. Sodium heparin (1000 U) was also administered intraperitoneally. After plasma isolation, hearts were rapidly excised. The ascending aorta was cannulated, and retrograde perfusion of the heart was initiated with a modified Krebs buffer maintained at 37°C and at a constant pressure of 80 mmHg. The Krebs buffer had the following composition (in mmol/l): NaCl 120, NaHCO3 25, CaCl2 2.5, EDTA 0.5, KCl 5.9, MgCl2 1.2 and glucose 17. The perfusate was aerated with 95% O2–5% CO2 and equilibrated at a pH of 7.3–7.4. Two side arms in the perfusion line proximal to the heart inflow cannula allowed for infusion of PMNs and plasma directly into the coronary inflow line. Coronary flow was monitored by a transit time flowmeter (Model T106, Transonic Systems). Left ventricular pressure (LVDP) and the maximal rate of development of LVDP (+dP/dt max) were monitored using a pressure transducer (Model SPR-524, 2.5F, Millar Instruments) which was positioned in the left ventricular cavity. LVDP was defined as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure. Coronary flow, left ventricular pressure and +dP/dt max were recorded using a MACLAB data acquisition system (ADI Diagnostics) in conjunction with a Power Macintosh 7600 computer (Apple Computers).

2.4 Limitation of the method
Although the isolated perfused rat hearts employed in this study were perfused at a constant pressure, the left ventricle was not fluid filled nor was a fluid filled balloon introduced into the left ventricle. Thus, this was not a working heart preparation. This is a limitation of the technique and should be kept in mind when evaluating the data. Nevertheless these hearts exhibited normal initial preloads, and developed normal and consistent left ventricular systolic pressures.

2.5 Perfused heart experimental protocol
LVDP, +dP/dt max, and coronary flow were measured every 5 min for 15 min to obtain a baseline measurement. After 15 min, the flow of Krebs buffer was reduced to zero to induce global ischemia. This global ischemia was maintained for 20 min. Coronary flow was then reestablished by returning coronary perfusion pressure to 80 mmHg. At reperfusion, hearts were infused for 5 min with 200x106 PMNs resuspended in 5 ml of Krebs buffer along with 5 ml plasma at a rate of 1 ml/min. Sham I–R hearts were not perfused with PMNs and plasma. Previous studies showed that sham I–R hearts given PMNs exhibited no changes from initial control values [17]. The hearts were allowed to reperfuse for a total of 45 min, during which time data were recorded every 5 min for the first 30 min and at the 45 min time point. In some experiments, hearts were isolated from PR-39-treated rats and perfused with normal nontreated PMNs, and in other experiments, 200x106 PMNs were incubated with PR-39 (10 µg/ml) at 30°C for 30 min in 20 ml of PBS with constant stirring. The PMNs were then centrifuged at 250 g for 10 min. The supernatant was discarded and the PMNs were resuspended in 5 ml of Krebs Buffer, and perfused into nontreated hearts subjected to ischemia–reperfusion. After each experiment, hearts were rinsed in Krebs buffer, then placed in 4% paraformaldehyde, and stored at 4°C for later histological analysis as previously described [17]. The number of infiltrated PMNs was determined by light microscopy. Moreover, we also counted intravascular PMNs that adhered to the vascular endothelium in cardiac tissue in order to determine the effect of PR-39 on PMN adherence to coronary vascular endothelium. These results are expressed as intravascular and infiltrated PMNs/mm2 area of cardiac tissue.

2.6 Measurement of superoxide release from rat PMNs
We examined whether PR-39 inhibits superoxide production in N-formylmethionyl-leucylphenylalanine (fMLP) stimulated rat PMNs. Superoxide release by PMNs was measured spectrophotometrically by the reduction of ferricytochrome C [18]. The PMNs (5x106 cells) were resuspended in PBS and incubated with PR-39 (4 or 10 µg/ml) in a total volume of 450 µl for 30 min at 37°C. Control samples did not contain PR-39. Ferricytochrome C was added (100 µmol/l, Sigma) in a total volume of 900 µl of PBS for 15 min at 37°C in spectrophotometric cells. At this point, PR-39 was also added to maintain the concentration of either 4 or 10 µg/ml. The PMNs were stimulated with 100 nM fMLP (Sigma) in a final reaction volume of 1.0 ml. Positive control samples were given human recombinant SOD (hSOD) (2 µg/ml; Grunenthal, Aachen, Germany) just prior to addition of fMLP. The absorbance at 550 nm was measured every 30 s, and superoxide anion release was determined by the {Delta} absorbance from time 0.

2.7 Quantification of CD18 expression on rat isolated neutrophils
Flow cytometric analysis of CD18 expression on the cell surface of isolated rat neutrophils was performed according to standard procedures [19]. Rat neutrophils were freshly isolated from rat whole blood according to the method of Yuan et al. [20]. Isolated neutrophils were washed twice in calcium-free Tyrode's solution containing 0.2% bovine serum albumin (BSA) and suspended in PBS. Neutrophils (5x105 cells/tube) were suspended in PBS alone, and were incubated in the presence or absence of PR-39 (10 µg/ml) for 20 min before addition of 100 nM leukotriene B4 (LTB4). Neutrophils were then incubated with an anti-CD18 antibody (WT. 3, Endogen) at 4°C for 60 min. Excess primary antibody was then removed by washing of neutrophils in PBS. A goat anti-human IgG F(ab')2 fluorescein isothiocyanate (FITC)-conjugated antibody was used as the secondary antibody at a 1:100 dilution (4°C for 30 min). The stained neutrophils were washed twice with PBS and fixed in 1% paraformaldehyde, and then analyzed by flow cytometry (FACScan, Becton-Dickinson).

2.8 Immunohistochemistry
Immunohistochemical localization of P-selectin was investigated by using the avidin–biotin immunoperoxidase technique (Vectastain ABC reagent, Vector Laboratories) according to a previously described method [21]. Tissue sections, which were prepared as mentioned above, were treated with 0.25% trypsin (Sigma) to improve reagent penetration. Blocking serum (horse) was applied to the tissue for 30 min to reduce nonspecific binding, and then the tissue sections were incubated for 24 h with specific primary antibodies. In particular, P-selectin was detected with the monoclonal antibody PB1.3 at a dilution of 1/100. PB1.3 is a monoclonal antibody that recognizes only P-selectin, which is expressed on the endothelial cell surface and does not bind to intracellular P-selectin [22]. The tissue was then incubated with the biotinylated secondary antibody, and peroxidase staining was carried out using 3,3'-diaminobenzidine. Expression of adhesion molecules was determined by microscopic observation of the brown peroxidase reaction product on the coronary microvascular endothelium of the tissue sections. Positive staining was defined as a vessel displaying brown reaction product on >50% of the circumference of its endothelium. The percent of microvessels staining positively was then calculated.

2.9 Statistical analysis
All data in the text and figures are presented as means±SEM. All data were subjected to ANOVA using post-hoc analysis with the Bonferroni/Dunn test. Probability values of <0.05 were considered to be statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
To determine whether PR-39 exerts direct effects on cardiac contractile function, we perfused nonischemic control rat hearts (i.e. sham I–R hearts) without PMNs for 80 min under normal flow conditions. Perfusion of sham I–R hearts obtained from PR-39 injected rats in Krebs buffer did not result in any sustained change in coronary flow, LVDP or +dP/dt max at the end of the observation period, indicating that PR-39 did not exert any direct effect on cardiac function (Figs. 1 and 2Go). Moreover, perfusion of untreated I–R hearts in the absence of PMNs did not result in any sustained alteration in any of the cardiac function variables measured, indicating that global ischemia per se does not provoke severe prolonged cardiac dysfunction in this model of I–R. Ischemia for 20 min followed by reperfusion produced a transient cardiac dysfunction, such that LVDP was depressed by 28±9% 15 min after reperfusion. This transient cardiac dysfunction recovered to 100±3% of control by 45 min post-reperfusion. However, I–R rat hearts perfused with PMNs experienced a more severe and sustained reduction in cardiac contractile function, remaining LVDP depressed by 49±3% and +dP/dt max by 57±4% at 45 min post-reperfusion (P<0.01). In contrast, I–R rat hearts obtained from PR-39-treated rats (10 µg/ml) exhibited a significant attenuation of cardiac contractile dysfunction in the presence of PMNs (i.e. more normalized LVDP and +dP/dt max at 45 min post-reperfusion; Figs. 1 and 2Go). LVDP decreased only 12±4% and +dP/dt max decreased only 22±6% in PR-39-treated hearts. The cardioprotection by PR-39 also showed a dose-dependent effect since the 4 µg/ml dose decreased LVDP and +dP/dt max by 26±3 and 37±6%, respectively. These hearts obtained from PR-39 injected rats exhibited a significantly higher final LVDP and +dP/dt max compared to I–R hearts obtained from untreated rats in the presence of PMNs (P<0.01, Figs. 1 and 2Go). Six additional I–R hearts perfused with PR-39-treated PMNs also exhibited a significant attenuation of cardiac contractile dysfunction (Figs. 1 and 2Go). LVDP decreased only 17±4% and +dP/dt max decreased only 30±3% at 45 min post-reperfusion (P<0.01, Figs. 1 and 2Go). These values are not significantly different from PR-39-treated hearts in which only the endothelium was treated.


Figure 1
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  		Fig. 1 Initial and final left ventricular developed pressure (LVDP) expressed as mmHg in isolated perfused rat hearts before ischemia and after reperfusion. Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant post-reperfusion contractile dysfunction, which was attenuated by PR-39. All values are expressed as means±SEM. Numbers of hearts are indicated at the bottom of the bars. **, P<0.01 from final LVDP of I–R+PMNs. NS, not significant.

 

Figure 2
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  		Fig. 2 Initial and final +dP/dt max expressed as mmHg/s in isolated perfused rat hearts before ischemia and after reperfusion. Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant contractile dysfunction, which was attenuated by PR-39. All values are expressed as means±SEM. Numbers of hearts are indicated at the bottom of the bars. **, P<0.01 from final +dP/dt max of I–R+PMNs. NS, not significant.

 
One of the possible mechanisms of the cardioprotective effect of PR-39 is inhibition of superoxide release from infiltrated PMNs. This may activate the coronary endothelium to promote adherence of PMNs. We therefore tested the effect of PR-39 on the release of superoxide from stimulated PMNs. PR-39 significantly reduced superoxide release from suspensions of fMLP stimulated rat PMNs at concentrations of 4 and 10 µg/ml by 22 and 46%, respectively (P<0.01, Fig. 3).


Figure 3
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  		Fig. 3 Superoxide release from 5x106 PMNs 90 s after fMLP (N-formylmethionyl leucylphenylalanine) stimulation. PMNs were stimulated with 100 nM fMLP. The change in absorbance ({Delta}) was measured 90 s after fMLP addition (peak response). hSOD, recombinant human superoxide dismutase (2 µg/ml), was employed as a positive control. All data were calculated by subtraction of hSOD values to obtain superoxide dismutase-inhibitable values. All values are means±SEM for five separate experiments.

 
We also studied effects of PR-39 on expression of the neutrophil surface adhesion molecule CD18 in vitro. Fig. 4 illustrates a representative flow cytometry histogram. Non-stimulated rat neutrophils exhibited a normal distribution. Incubation with 100 nM LTB4 induced a marked shift to the right of the PMNs characteristic of activation. However, 20 min after preincubation of rat neutrophils with 10 µg/ml PR-39, CD18 expression was attenuated in response to LTB4, suggesting that PR-39 exerts anti-neutrophil effects beside inhibition of superoxide anion release. The mean decrease in percent positive PMNs in PR-39-treated PMNs were from 88±8% to 18±6% in PMNs obtained from four rats (P<0.01), and mean channel fluorescence deceased from 89±7 to 28±6 (P<0.01).


Figure 4
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  		Fig. 4 Flow cytometry histogram of log of mean channel fluorescence vs. number of PMNs. Control unstimulated PMNs (white), LTB4-stimulated PMNs (black), and LTB4-stimulated PMNs that were preincubated with 10 µg/ml of PR-39 (gray) were incubated with an anti-CD18 monoclonal antibody and labeled with a secondary antibody. PR-39 clearly attenuated LTB4 upregulation of CD18 in rat PMNs.

 
We performed immunohistochemical analysis of P-selectin expression on the rat coronary microvascular endothelium in order to determine whether the endothelium of I–R hearts obtained from PR-39 injected rats exhibited any changes in adhesion molecule expression that could account for the low degree of PMN infiltration. Fig. 5 summarizes these results. I–R hearts perfused without PMNs exhibited a very low P-selectin surface expression on the coronary microvascular endothelium. However, I–R hearts perfused with PMNs exhibited a 3.5-fold increase in P-selectin surface expression. This was markedly attenuated (P<0.01) in I–R hearts obtained from PR-39 injected rats (10 µg/ml).


Figure 5
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  		Fig. 5 Immunohistochemical analysis of P-selectin expression in rat hearts subjected to ischemia–reperfusion. Ischemic hearts were reperfused in the presence or absence of PMNs. The presence of P-selectin-positive staining was then analyzed. All values are expressed as mean percentage values±SEM. Numbers at bottom of bars represent number of fields counted in each of three hearts per group.

 
The marked deficit in post-reperfusion cardiac performance can be largely attributed to the presence of PMNs at the time of reperfusion, since the same I–R protocol in the absence of PMNs produced only very small alterations in cardiodynamics at 45 min post-reperfusion. PMN adhesion and infiltration in I–R hearts occurred to a significant degree in hearts perfused with PMNs compared to all other groups (Fig. 6a and b). However, the total number of adhered and infiltrated PMNs in post-reperfused hearts obtained from PR-39 injected rats (10 µg/ml) was 54% less than that observed in I–R hearts obtained from 0.9% NaCl injected rats (P<0.01, Fig. 6a). Moreover, PMNs adhered to the vascular endothelium in hearts obtained from PR-39 injected rats (10 µg/ml) was 50% less than that observed in I–R hearts obtained from 0.9% NaCl injected rats (P<0.01, Fig. 6b). Similarly, ischemic–reperfused hearts perfused with PR-39-treated PMNs exhibited a significant attenuation of PMN adhesion and infiltration (P<0.01, Fig. 6a and b). These anti-adherence observations in PR-39-treated PMNs and hearts are a major factor contributing to the attenuated infiltration of PMNs into the reperfused myocardium.


Figure 6
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  		Fig. 6 Histological assessment of (a) total intravascular and infiltrated PMNs and (b) intravascular adhered PMNs to the coronary vascular endothelium in isolated perfused rat hearts taken from three rats per group. Ten areas were analyzed per heart. All values are mean numbers of PMNs/mm2±SEM. PR-39 significantly attenuated the number of PMNs (a) infiltrated into post-reperfusion cardiac tissue (b) as well as PMNs adhered intravascularly.

 
Thus, PR-39 exerted marked dose-dependent cardioprotective effects in the isolated perfused rat heart characterized by maintenance of left ventricular cardiac function and attenuated infiltration of PMNs into the ischemic–reperfused rat myocardium. These effects of PR-39 were associated with (a) reduced expression of adhesion molecules on the surface of both neutrophils and coronary endothelium, (b) attenuated release of superoxide from rat PMNs, and (c) reduced adherence of PMNs to the coronary vascular endothelium.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study demonstrated that PR-39 exerts significant effects on PMN-mediated post-reperfusion cardiac contractile dysfunction. The cardioprotective effects of PR-39 were characterized by a significant preservation of post-reperfusion LVDP and +dP/dt max compared to those of hearts subjected to PMN perfused I–R hearts obtained from rats receiving only 0.9% NaCl. There are several possibilities to account for the cardioprotective effects of PR-39.

First, reactive oxygen species are produced in isolated perfused rat hearts subjected to ischemia–reperfusion [23,24]. Reactive oxygen species have been shown to depress cardiac contractile function in isolated rat hearts [25,26]. Although the molecular mechanism whereby oxygen metabolites induce cardiac dysfunction remains unclear, the effects of reactive oxygen species (e.g. superoxide) may occur via nonspecific injury to cellular components via protein denaturation, enzyme inactivation, and peroxidation of the polyunsaturated fatty acids [27]. Moreover, it has been reported that isolated rat cardiac myocytes are sensitized by hypoxia–reoxygenation and are subsequently injured by reactive oxygen species [3]. Since reactive oxygen species themselves exert deleterious effects against a variety of cellular components, it is possible that inhibition of superoxide production via inhibition of NADPH oxidase could be cardioprotective in the setting of myocardial ischemia–reperfusion.

Secondly, it has been established that ischemia followed by reperfusion in the presence of PMNs results in a marked cardiac dysfunction in this preparation [16,17,28]. This process involves leukocyte–endothelium adhesion, which is mediated by the expression of cell adhesion molecules on the surfaces of both PMNs and endothelial cells [29]. Moreover, It has been reported that superoxide-induced leukocyte–endothelial interactions were mediated by cell adhesion molecules such as P-selectin or CD18 in vivo [30]. Therefore, our data of immunohistochemical analysis of P-selectin expression on the rat coronary microvascular endothelium and in vitro effects of PR-39 on expression of the neutrophil surface adhesion molecule CD18 may explain the mechanism of inhibition of PMN-endothelium interaction in the present study. Our histology data also indicated less adhered PMNs onto the microvascular endothelium in PR-39-treated hearts. Since endothelium-derived superoxide release is a very early effect of reoxygenation of the hypoxic isolated perfused rat heart occurring within the first minutes of reperfusion [31], and because reactive oxygen species can upregulate cell adhesion molecules (e.g. P-selectin) on the surface of the endothelium [32], inhibition of superoxide production via NADPH oxidase may be cardioprotective. On the other hand, Korthuis et al. [15] reported that pretreatment with PR-39 markedly diminished leukocyte adhesion to post-ischemic rat mesenteric venules and prevented phorbol myristate acetate-induced intercellular adhesion molecule-1 expression in cultured endothelial cells. They stated that NF-{kappa}B translocation, which is important for expression of cell adhesion molecules on the endothelium, was attenuated by PR-39 [15]. Therefore, the cardioprotective effects of PR-39 may be due in part to inhibition of cell adhesion molecule expression independently of inhibition of NADPH oxidase. In this regard, PR-39 has been shown to rapidly enter microvascular endothelial cells and bind to a variety of intracellular proteins, an action independent of its effect on inhibition of superoxide release [33].

Thirdly, although it is clear that reducing PMN adherence to the vascular endothelium can lead to a significant reduction in PMN infiltration into post-reperfused cardiac tissue, there may be other cardioprotective effects of PR-39. Korthuis et al. [15] reported that PR-39 attenuated neutrophil chemotactic responses to platelet activating factor. This inhibitory effect on neutrophil chemotaxis may also contribute to lower PMN infiltration into PR-39-treated post-reperfused hearts. Since transmigrated PMNs provoke tissue injury by the release of cytotoxic substances, including reactive oxygen species, inflammatory cytokines, and proteolytic enzymes [34], this effect of PR-39 may exert cardioprotective effects.

There is some uncertainty about the locus of action of PR-39. Therefore, we performed two different methods of heart perfusion. First, PR-39 was injected into rats, which were cardiac donors for the perfused heart experiments, suggesting that the effects of PR-39 are likely due to reduced superoxide production via inhibition of endothelial NADPH oxidase. In this connection, Al-Mehdi et al. [35] reported PR-39-induced inhibition of NADPH oxidase in endothelial cells. Secondly, ischemic–reperfused hearts were perfused with PMNs, which were preincubated with PR-39, suggesting that the effects of PR-39 can also be due to reduced superoxide production via inhibition of NADPH oxidase in PMNs. This is consistent with our data showing PR-39 inhibits the release of superoxide from stimulated PMNs, and is also consistent with previous results [15].

Although our data implicate superoxide as one of the important mediators of post-ischemic cardiac contractile dysfunction, there is still some question regarding the role of neutrophils as the major cell type generating post-ischemic cardiac contractile dysfunction. Some investigators have reported that post-ischemic cardiac contractile dysfunction was not attenuated by depletion of neutrophils [36,37]. This may be due to the degree of neutrophil depletion in these studies. However, several other groups clearly point to neutrophils as playing a major role in post-reperfusion contractile dysfunction [38,39]. We showed that ischemic–reperfused hearts without PMNs showed only a brief and mild cardiac contractile dysfunction, which was markedly enhanced and prolonged by infusion of PMNs. These data clearly indicate that PMNs are important cell types responsible for enhancing post-ischemic cardiac contractile dysfunction.

In summary, our results are the first to show a cardioprotective effect of PR-39 in myocardial ischemia–reperfusion. PR-39 attenuates PMN-induced cardiac contractile dysfunction in isolated ischemic–reperfused rat hearts. These effects of PR-39 are likely due to reducing PMN adherence to coronary vascular endothelium, thereby leading to a significant reduction in PMN infiltration into post-reperfused cardiac tissue. These cardioprotective effects appear to be related to inhibition of superoxide production via NADPH oxidase inhibition on both endothelial cells and neutrophils.

Time for primary review 29 days.


   Notes
 
1 Y.I. is a Research Fellow of the Japanese Society of Clinical Pharmacology and Therapeutics. Back

2 L.H.Y. is a Postdoctoral Trainee of the National Heart, Lung and Blood Institute of the NIH (HL-07599). Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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