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Cardiovascular Research 2004 62(1):154-166; doi:10.1016/j.cardiores.2004.01.009
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Copyright © 2004, European Society of Cardiology

Non-angiogenic FGF-2 protects the ischemic heart from injury, in the presence or absence of reperfusion

Zhi-Sheng Jianga, Wattamon Srisakuldeea, Fabienne Souletb,1, Gerard Boucheb and Elissavet Kardami*,a

aInstitute of Cardiovascular Sciences, St. Boniface Research Centre, Department of Human Anatomy and Cell Science and Physiology, University of Manitoba, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6
bInstitut de Pharmacologie et de Biologie Structurale du CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex 4, France

* Corresponding author. Tel.: +1-204-235-3519; fax: +1-204-233-6723. Email address: ekardami{at}sbrc.ca

Received 20 June 2003; revised 11 December 2003; accepted 7 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Fibroblast growth factor-2 (FGF-2), given during ischemia or during reperfusion of the ischemic heart is cardioprotective, but its mitogenic activity may limit possible clinical applications. We have tested the cardioprotective potential of a non-mitogenic FGF-2 mutant (S117A) that no longer activates casein kinase 2 (CK2) in both acute and long-term studies. Methods and results: To test effects during reperfusion, the ex vivo rat heart, subjected to 30 min of global ischemia and 60 min of reperfusion was used. S117A FGF-2 administered during reperfusion protected against myocardial contractile dysfunction, activated protein kinase C and decreased the release of cytochrome C in the cytosol. To study effects on ischemic myocytes in the absence of reperfusion, myocardial infarction (MI) was induced in the rat model by irreversible left coronary ligation. S117A-, wild type (wt)-FGF-2 or saline, were administered by intramyocardial injection into the ischemic ventricular wall. One day later, infarct size (assessed by tetrazolium staining), and plasma cardiac troponin T levels (assessed by Western blotting) were significantly decreased in the S117A FGF-2-, compared to the saline-treated group. Systolic pressure, rates of contraction and relaxation and developed pressure, assessed in the Langendorff mode, were significantly improved in the S117-FGF-2 group. Improved ejection fraction and fractional shortening in the S117A-treated group were maintained up to, but not beyond, 7 days post-MI. In comparison, improvements were maintained in the wt-FGF-2-treated group at least up to 6 weeks post-MI. At 6 weeks post-MI, small vessel density (assessed by immunofluorescence-based detection) in the scar bordering viable myocardium was similar between S117A-FGF-2- and saline-treated hearts, but significantly increased in the wt-FGF-2-treated group. This was accompanied by increased coronary flow in the wt-, but not S117A-FGF-2-treated hearts, compared to controls. Conclusion: The ability of FGF-2, administered during ischemia or during reperfusion, to protect the myocardium acutely from tissue loss and dysfunction is independent of its potential for CK2 activation and angiogenesis. Non-angiogenic S117A-FGF-2 may be considered in therapies aiming for acute prevention of reperfusion-associated pathologies, especially in cases where use of mitogens is counter-indicated.

KEYWORDS Cardioprotection; Growth factors; CK2; Infarction; Reperfusion; Protein therapy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Myocardial tissue loss and contractile dysfunction, a major cause of mortality and morbidity worldwide, occurs as a consequence of irreversible ischemic injury resulting from compromised coronary blood flow to the heart. Ironically, the re-establishment of coronary flow, while essential for ultimate survival of the heart, has also been associated with the so-called ‘reperfusion injury’ that includes, in several animal models, further tissue loss and/or contractile dysfunction. In the clinic, ‘reperfusion injury’ can be encountered during reperfusion of acute myocardial infarction, during angioplasty and cardiac surgery and represents a spectrum of pathologies that include myocardial contractile dysfunction or ‘stunning’, microvascular and endothelial injury, and myocyte necrosis [1,2]. The remaining viable tissue can compensate up to a point beyond which heart failure ensues. Agents therefore that can increase the resistance of the myocardium to injury and thus salvage cardiac tissue represent a promising therapeutic option for the management of heart disease.

We have reported [3–5] that FGF-2, a multifunctional and ubiquitous polypeptide that is currently being considered in the context of ‘therapeutic angiogenesis’, is such an agent of acute cardioprotection. FGF-2 belongs to the larger family of heparin-binding growth factors that has 23 members up-to-date [6]. In addition to being a mitogen for many cell types, FGF-2 can affect differentiation, migration, and survival [6,7]. Adminstration of FGF-2 to the normal heart confers a preconditioning-like cardioprotection [4,5,8,9]. Chronic overexpression of FGF-2 in transgenic models also protects from ischemic cardiomyocyte injury [10]. In addition, FGF-2 given after global ischemia and during reperfusion of the ex vivo rat heart results in significantly improved recovery of function and reduced tissue damage [3]. Finally intramyocardial delivery of FGF-2 reduced infarct size and improved cardiac function, assessed 4–24 h post-MI [3]. These acute protective effects of FGF-2 occur before any angiogenic response can be observed and very likely reflect direct protective effects on all cardiac cell types including cardiomyocytes. Other studies have reached similar conclusions [11–14].

FGF-2 binds to plasma membrane tyrosine kinase receptors causing activation of a multitude of signaling cascades; it also signals by being internalized and targeted to the nucleus [6]. The extent of overlap between signals that govern acute cardioprotection and those leading to cell cycle activation and angiogenesis is not known. One way to begin addressing this issue is to examine how mutations that affect FGF-2 mitogenicity may affect its ability to increase cell resistance to injury. Bouche et al. [15] have demonstrated that a single mutation, substituting serine 117 with alanine (S117A), diminished the ability of wt-FGF-2 to activate CK2 and stimulate cell proliferation. We have used the S117A-FGF-2 mutant to address the role of mitogenic and angiogenic potential, and ability to activate CK2, in mediating FGF-2-induced protection of the ischemic myocardium. Here we report that S117A-FGF-2 is acutely protective for the ischemic myocardium, in the presence or absence of reperfusion, and can thus be considered as a potential short-term treatment for ischemia and/or reperfusion-associated pathologies. We also show that, in the absence of reperfusion, sustained protection by FGF-2 depends on angiogenic potential.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals
Male Sprague-Dawley rats, obtained from the Central Animal Care Facility at the University of Manitoba, were used for coronary ligation and Langendorff perfusion. Studies followed the guidelines of the Canadian Council on Animal Care, which is in agreement with Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and were approved by the local Animal Care Committee of the National Research Council of Canada.

2.2. Materials
Recombinant wild type- or S117A-FGF-2 (the 18 kDa, AUG-initiated species) were produced in Escherichia coli bacteria and purified to homogeneity as published previously [5,15]. Production of the S117A-FGF-2 mutation was described previously [15].

2.3. Mitogenicity assay
Primary cultures of neonatal rat cardiomyocytes were obtained as reported previously [16]; myocytes were plated at 0.4 million cells/35 mm dish in the presence of 10% fetal bovine serum (FBS). After overnight incubation, myocytes were switched to low serum medium (0.5% FBS, in DMEM) for 48 h. Growth factors (wt- or S117A-FGF-2, 50 ng/ml), were added at this time point, and DNA synthesis (Labeling Index; fraction of myocytes incorporating BrDU in their DNA over total number of myocytes) was examined 48 h later, as described before [17].

2.4. Myocardial infarction (MI) and FGF-2 delivery
MI was produced in male SD rats (200–250 g) by permanent ligation of the left coronary artery as reported previously [3,18]. Within 10 min of coronary ligation, saline (100 µl), wt-FGF or S117A-FGF-2 (2 µg in 100 µl saline) was injected into three sites at the lower half of the ischemic (akinetic) left front ventricular wall. The chest was then evacuated and closed. At various time points after ligation (1–6 weeks), animals were anesthetized with 2% isoflurane and echocardiography was performed as described below. In another experimental series, rats were sedated and euthanized by decapitation at 24 h or 6 weeks post MI, and hearts were harvested for determination of hemodynamic function or infarct size (n=6–8). Infarct size (by tetrazolium staining) and relative plasma troponin T levels (by Western blotting) were done exactly as we described previously [3].

2.5. Echocardiography
Transthoracic echocardiography (Agilent SONOS 5500; Agilent Technologies, Andover, MA, USA) using a 12-MHz linear array transducer was performed weekly, for 1–6 weeks, as described by others [19,20]. Short axis two-dimensional views of the left ventricle at the papillary muscle level were used to obtain M-mode targeted recordings. The following echocardiographic parameters were measured in each heart following methods described by the American Society of Echocardiography [20]: Left ventricle (LV) anterior wall thickness in end-diastole (LVAWd) and in end-systole (LVAWs), LV posterior wall thickness in end-diastole (LVPWd) and in end-systole (LVPWs), LV internal chamber dimension in end-diastole (LVDd) and in end-systole (LVDs). LV fractional shortening (FS%) was calculated as (LVDd–LVDs)/LVDd x 100%.

2.6. Hemodynamic function
The Langendorff mode was employed to assess myocardial mechanical functions (systolic pressure, SP; developed pressure, DP; end diastolic pressure, EDP; rates of contraction and relaxation, ±dP/dt), 24 hours and 6 weeks after coronary ligation as described in detail [3]. Coronary flow (ml/min) properties of hearts from the various treatment groups at 6 weeks post ligation were also determined. Perfused hearts (n=6–8) were used to test the effect of S117A-FGF-2 after 30 min of global ischemia and 60 min reperfusion, as described previously [3]. The ‘treatments’, such as wt- or S117A-FGF-2 (10 µg/10 ml buffer) were infused into the heart during the first 10 min of reperfusion, as we described previously [3–5]. This method results in a 4-fold increase in heart-associated FGF-2 [4]. The control group received perfusion buffer only.

2.7. Translocation of PKC isoforms and assessment of cytochrome C release
Cytosolic and particulate extracts were obtained from hearts subjected to 30 min ischemia, followed by reperfusion, in the presence or not of S117-FGF-2, as described previously [3]. The various cardiac fractions were analyzed by SDS/PAGE on 10% gels (20 µg/lane) and Western blotting, using antibodies specific for the various PKC subtypes: rabbit polyclonal anti-PKC{varepsilon}, -PKC{alpha}, -PKC{delta}, and -PKC{zeta} purchased from Santa Cruz Biotechnology. Calculation of the relative levels of the PKC subtypes was done as described previously [3]. Assessment of cytochrome C release. This was done exactly as described by others [21]. Briefly, ex vivo hearts were subjected to global ischemia followed by 2 h of reperfusion. Wt-or S117A-FGF-2 were administered during reperfusion as described above. At the end of reperfusion hearts were used to obtain cytosolic and particulate extracts [21]; these were analyzed by western blotting and probed for cytochrome c using specific antibodies (Pharmingen).

2.8. Coronary flow and small vessel density determinations
An assessment of overall coronary flow was obtained by collecting the effluent from isolated hearts, as described [22], harvested at 6 weeks post-MI.

Small vessel density determination was done according to published procedures [23,24]. Cryosections (7 µm thickness, eight sections per heart), obtained from hearts (n=3–4; sufficient to obtain statistically significant data) removed at 6 weeks post-MI were processed for immunolocalization of alpha-smooth muscle actin ({alpha}-SMA; to identify smooth muscle cells; mouse monoclonal antibodies from Sigma), and/or Von Willebrand Factor (vWF; to detect endothelial cells; rabbit polyclonal antibody from Sigma). Staining without the primary antibodies was used to control for non-specific fluorescence. Small vessels (arterioles, observed mostly in cross-section) were defined as round or elliptical structures of mainly 20–50 µm length or diameter, staining positive for both vWF (endothelial ‘inner’ lining) and {alpha}-SMA (‘outer’ layer, smooth muscle cells). Number of small vessels (a) in the scar area surrounding viable tissue or (b) in the viable myocardium near the infarct, were counted in 10 random fields from each section (eight sections per heart, 3–4 hearts per group) observed at x 100 magnification.

2.9. Statistical analysis
All values were reported as mean±SD. One way ANOVA with post-hoc testing was used to assess significant differences between groups. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Acute effect of S117A FGF-2 administered during reperfusion of the ischemic hearts on the recovery of contractile function, PKC activation and mitochondrial injury
Human S117A-FGF-2, and wt-FGF-2, prepared and purified to homogeneity as described previously [3,15], were compared for their effects on cardiomyocyte DNA synthesis (Fig. 1). As expected, wt-FGF-2 stimulated DNA synthesis compared to untreated controls while S117A-FGF-2 did not (Fig. 1). S117A-FGF-2 was then compared with wt-FGF-2 in ability for acute cardioprotection when administered during reperfusion.


Figure 1
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  		Fig. 1 Effect of wt-FGF-2 and S117A-FGF-2 on neonatal myocyte DNA synthesis. Cultured myocytes were stimulated with 50 ng/ml each of wt- or S117A-FGF-2, as indicated. The y-axis depicts Labeling Index, i.e. fraction of myocytes incorporating BrDU in their DNA, over total number of myocytes. **P<0.01, compared with control group, n=3. Bar=SD.

 
Subjecting the ex vivo heart to 30 min ischemia, followed by 60 min reperfusion resulted in 59% recovery of systolic pressure (SP) in the control group (Fig. 2). Administering S117A-FGF-2 during reperfusion resulted in 80% recovery, significantly (P<0.01) higher than the control group, but not significantly different to the wt-FGF-2-treated group. Very similar results were obtained for ±dP/dt. While the control group had 57% and 56% recovery in ±dP/dtmax, respectively, the corresponding values in the S117A-FGF-2 group were at 81% and 84%, significantly higher (P<0.01) than controls, but not different than the wt-FGF-2 values. Developed pressure (DP) values of the reperfused hearts, assessed under different preload settings (end diastolic pressure, EDP, of 0, 2.5, 5.0 and 7.5 mm Hg), were significantly higher in the S117A- compared to the control group, and indistinguishable from the wt-FGF-2-treated group.


Figure 2
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  		Fig. 2 Effect of S117A FGF-2 administered during reperfusion of the ex vivo heart on functional recovery after ischemia and/or reperfusion. Hearts were subjected to 30 min global ischemia and 60 min reperfusion. S117A- or wt-FGF-2 were administered during the first 10 min of reperfusion (see Materials and Methods). (A), (B) and (C) show respectively SP, ±dp/dtmax and DP. Compared with saline control, **P<0.01, n=7. Bar=SD.

 
To determine whether S117A-FGF-2 could induce protein kinase C (PKC) activation, we examined its effect on the relative cytosolic and particulate levels of various PKC isoforms; results are shown in Fig. 3. Treatment with S117A-FGF-2 resulted in increased relative levels of PKC-{alpha}, -{varepsilon}, -{zeta} and -{delta} in the particulate fraction, and correspondingly decreased levels in the cytosol, indicating translocation and, presumably, activation. Identical results were shown previously for wt-FGF-2 [3].


Figure 3
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  		Fig. 3 Effect of S117A FGF-2 administered during reperfusion of the ex vivo heart on relative subcellular distribution of PKC{alpha}, {varepsilon}, {delta}, and {zeta}. Inset, representative Western blot of subcellular fractions probed for PKC{alpha}, {varepsilon}, {delta}, and {zeta} as indicated. Densitometric values from control samples were arbitrarily converted to 1.0, and values of samples from S117A FGF-2-treated hearts were normalized accordingly. Asterisk (*) or (**) denotes significant (n=3, *P<0.05, **P<0.01) difference between S117A-FGF-2-treated and control values. Bar=SD.

 
We also compared the effect of S117A- and wt-FGF-2 on relative cytochrome c levels in the cytosol after 2 h of reperfusion. As shown in Fig. 4, both treatments significantly reduced relative levels of cytosolic cytochrome c compared to untreated ischemic-reperfused hearts. Since cytochrome c is released to the cytosol by damaged mitochondria, our data imply that FGF-2 (wt- and S117A-) protected the reperfused myocardium from myocyte cell death [25].


Figure 4
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  		Fig. 4 Effect of S117A- and wt-FGF-2, administered during reperfusion of the ex vivo heart on the relative levels of cytochrome C present in the cytosol after 2 h of reperfusion. Cytosolic Cytochrome C in the saline group is arbitrarily set as 100%, and values for the other groups are adjusted accordingly. **P<0.01, compared to the saline group, n=3. Bar=SD.

 
3.2. Acute effects of S117A FGF-2, administered in vivo
To investigate the effect of S117A-FGF2 on the ischemic myocardium in vivo, we used a model of irreversible coronary occlusion, as previously described [3]. In one experimental series, rats were killed 1 day post-MI. Myocardial damage was assessed directly, by histochemical infarct size determination. A representative image from histochemical scar detection is shown in Fig. 5, while results are summarized in Fig. 6A. The S117A-FGF-2-treated group had significantly smaller infarcts compared to the saline group. Indirect detection of myocardial damage, by measuring relative TnT levels in plasma (Fig. 6B), gave similar results: the S117A-FGF-2 group had significantly less damage compared to controls. In both cases, protection by S117A-FGF-2 was not significantly different to the wt-FGF-2-treated group (Fig. 6A,B).


Figure 5
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  		Fig. 5 Representative photographs of infarcted heart slices stained with TTC. Hearts were processed at 24 h post MI. Infarcted tissue appears pale, while viable tissue stains red. (A), (B) and (C) represent typical staining from, saline, wt-FGF-2 and S117A FGF-2 treated hearts, respectively.

 

Figure 6
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  		Fig. 6 Effect of S117A-FGF-2 on myocardial injury and contractile function one day post-MI. (A) Relative infarct size, (B) Micrograph of representative plasma western blots probed for TnT, and corresponding densitometric measurements of relative plasma TnT levels at 24 h after MI, (C) SP, (D) ±dp/dtmax, (E) DP at EDP values of 0–7.5 mm Hg. The S117A- or wt-FGF-2-treated groups are compared to the saline group, where **P<0.01. (n=6–8 for A, C, D, E; n=4 for B). Bar=SD.

 
The effect of S117A-FGF-2 treatment on myocardial function, assessed in the Langendorff mode, is shown in Fig. 6C,D,E. While SP in the saline treated infarcted hearts, at 62 mm Hg, was at about 54% of standard preischemic values, SP in the S117A-FGF-2-treated hearts showed a 97% recovery, similar to the 94% recovery of the wt-FGF-2-treated group. Similar patterns were observed for recovery of dP/dt, and DP, under different preloads (Fig. 6D,E, respectively). In all cases, the effect of S117A-FGF-2 was indistinguishable from that of wt-FGF-2.

In another experiment, rats treated with saline or S117A-FGF-2 as above, were examined by echocardiography at 1, 2 and 7 days post-MI. Another group that was not subjected to MI provided baseline values. Results are shown in Table 1: At these time points, fractional shortening (FS) in the S117A-group showed a 75%, 74% and 62% recovery, significantly higher than the 50%, 50% and 41% recovery, respectively, in the saline group. Similarly, ejection fraction (EF) in the S117A-group showed an 84%, 84% and 74% recovery, significantly higher than the 62%, 60% and 55% recovery of the saline group, respectively.


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  		Table 1 Changes of echocardiographic parameters 1, 2 and 7 days after MI (Formula±SD)

 
3.3. Long term effect of S117A-FGF-2 and wt-FGF-2, administered in vivo
To assess the durability of the protective effects of S117A-FGF-2, in comparison to wt-FGF-2, groups of rats were treated as in the previous section but were examined several weeks post-MI. In one experimental series, rats were killed at 6 weeks post-MI. Heart function was examined in the Langendorff mode and scar size assessed by morphometry; results are shown in Fig. 7. The extent of scarring was not significantly different in the S117A-FGF-2 compared to the saline group; scarring was significantly less in the wt-FGF-2 group, as we have shown before [3]. SP, ±dP/dtmax, and DP under different preload settings remained significantly higher in the wt-FGF-2 compared to the saline-group, as we showed previously [3]. In contrast, corresponding values of the S117A-FGF-2 group were not significantly different to the saline group (Fig. 7).


Figure 7
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  		Fig. 7 Effect of S117A FGF-2 on myocardial injury and contractile function 6 weeks post-MI. Hearts were assessed in the Langendorff mode. (A) Infarct size, (B) SP, (C) ±dp/dt, (D) DP of the saline-, S117A FGF-2- and wt-FGF-2-treated groups, at EDP values of 0–7.5 mm Hg. Compared with saline control, **P<0.01, n=6. Bar=SD.

 
In a separate experimental series, rats, treated as in the previous section, were monitored by ultrasound for 1–6 weeks post MI, for contractile function and heart dimensions. Results are shown in Table 2. The wt-FGF-2-treated animals displayed significantly higher values for FS and EF, maintained at all time points. In the same group, LVDs was significantly smaller, at 3, 5 and 6 weeks post-MI. At 1 week post-MI, the S117A-FGF-2-group had significantly higher values for FS and EF compared to the saline group, but not significantly different to the wt-FGF-2-treated groups; the S117A-FGF-2 group results are similar to those shown in Table 1. At the 2–6 weeks time points, however, the S117A-FGF-2-treated group was not significantly different to the saline controls. LVDs in the S117A-FGF-2 group was not significantly different to controls at any time point. Loss of improvements in the S117A- (but not in the wt-FGF-2 group) was confirmed in a separate group of rats assessed 2–8 weeks post MI by echocardiography (unpublished data).


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  		Table 2 Echocardiographic parameters in all groups in different time points (Formula±SD)

 
3.4. Effect of wt- and S117A FGF-2 on coronary flow and density of small vessels
Hearts isolated from saline, S117A- and wt-FGF-2- treated infarcted hearts at 6 weeks post-MI were assessed for coronary flow properties. As shown in Fig. 8A, wt-, but not S117A-FGF-2-treated hearts presented increased overall flow compared to saline controls.


Figure 8
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  		Fig. 8 Effect of saline, S117A FGF-2 and wt-FGF-2 on heart vessels at 6 weeks post-MI: (A) Percent (%) change in heart coronary flow (CF). (B) and (C) Small vessel (arteriolar) density within infarct bordering viable myocardium, or, within viable myocardium bordering the infarct, respectively. Compared with saline group **P<0.01, n=3. Bar=SD.

 
We also determined the density (number per visual field) of small vessels (arterioles, displaying a diameter/apparent length mostly at 30–80 µm; immuno-positive for both {alpha}-SMA (smooth muscle cells, outer layer) and vWF (endothelial cells, inner layer) in the scar area bordering viable myocardium, and in the myocardium surrounding the scar at 6 weeks post MI. Results are summarized in Fig. 8B. Please note that under the conditions used, staining for vWF elicited a weak green background fluorescence staining that helped delineate viable myocardium in contrast to the bright green staining of the endothelium (appearing yellow at sites of apparent overlap with the anti-{alpha}-SMA stain). Relative small vessel density was increased in the wt-, but not the S117A-FGF-2-treated group, compared to the saline controls, at both sides of the infarct border. Representative small vessel images from the wt- and S117A-FGF-2-treated hearts are shown in Fig. 9. Single cells staining for {alpha}-SMA alone (arrows in Fig. 9, presumed myofibroblasts) also appeared to be increased in the infarct of the wt-FGF-treated group; this has not been explored further in this work.


Figure 9
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  		Fig. 9 Representative micrographs of blood vessels (white circles) detected by immunofluorescence staining for {alpha}-Smooth Muscle Actin (red) and von Willembrand Factor (bright green), including nuclear (blue) counterstaining. Viable myocardium (M) stains light green. (A) and (B): Vessels within infarct bordering viable myocardium in S117A- or wt-FGF2- treated hearts, respectively. Small arrows indicate presumed myofibroblasts. Sizing bar=100 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We have shown that: (a) Potential for CK2 activation and for mitogenesis/angiogenesis are not required for acute cardioprotection of ischemic myocytes by FGF-2, either in vivo in the absence of reperfusion, or ex vivo, in the presence of reperfusion. (b) The non-angiogenic S117A-FGF-2, administered during reperfusion, retains the ability to activate PKC and to prevent mitochondrial damage. (c) The protective effect of S117A-FGF-2 towards the ischemic, non-reperfused myocardium is maintained up to one week post MI. (d) In the absence of reperfusion, sustained protection by FGF-2 is linked to its potential for CK2 activation and mitotic stimulation.

4.1. Acute cardioprotection by wt- and S117A-FGF-2; the role of CK2
FGF-2 activates the tyrosine kinase receptor FGFR1, as well as erk1/2 and PKC in the heart. All of these signals are implicated in both cardioprotection as well as mitotic stimulation [6,7]. In addition, FGF-2 activates the ubiquitous kinase CK2, an event necessary for its mitogenicity [15]. It is not known whether CK2 activation, that has also been linked to cell survival [26,27] is also mediating acute and/or long-term cardioprotection by FGF-2. To address these questions we investigated the cardioprotective properties of an FGF-2 mutant (S117S-FGF-2) that retains several of the wt-FGF-2 properties such as ability to bind heparin and activate FGFR1 as well as erk1/2, but is incapable of CK2 activation and mitogenic stimulation [15]. To assess cardioprotection of already ischemic myocardium, in the presence or absence of reperfusion we used two model systems: the isolated perfused heart, in which S117A-FGF-2 was administered during early reperfusion; and an in vivo model of irreversible coronary ligation that results in large infarcts [18]. We have shown that thus administered wt-FGF-2 is retained by the myocardium in a basement-membrane-like distribution around cardiomyocytes, and that it activates the cardiomyocyte tyrosine kinase receptor in situ [3–5,28]. The S117A-FGF-2 has similar properties [15]. A wt-FGF-2-treated group, acting as a positive control, was included in most studies. We were thus able to show that S117A-FGF-2 protected from myocardial dysfunction as well as tissue loss acutely, in both models, in a manner indistinguishable to that of wt-FGF-2. We can thus conclude that potential for CK2 activation and mitogenic stimulation are not required for the acute cardioprotective effect of FGF-2, and that S117A-FGF-2 can be added to the small list of agents that are effective when administered during reperfusion of the ischemic heart.

Dissociation of different biological activities of wt-FGF-2 by structural modifications has been reported [29]. Removal of a small sequence (res. 28–32) from the native molecule maintained its mitogenicity and its ability to translocate to the nucleus, but reduced its ability to stimulate production of tissue plasminogen activator [29]. On the other hand, an equivalent modification on another well studied member of the FGF family, FGF-1, prevented translocation to the nucleus and induced loss of mitogenicity [30]. At the same time the modified FGF-1 retained other activities such as vasodilation and protection of tissues (including the heart) from ischemic injury [13]. Our results on non-mitogenic FGF-2 are in broad agreement with the data from non-mitogenic FGF-1, in that it is possible to dissociate mitogenicity and cardioprotection based on molecular modifications of the growth factor. The molecular interactions and signals affected by the different modifications are likely to be different. Both FGF-2 and FGF-1 have two sites of interactions with the tyrosine kinase receptors (FGFR) that mediate their broadly similar biological activities. Modifications in non-mitogenic FGF-1 affect site 1 of interaction with FGFR; the S117 mutation in FGF-2 is not expected to have this effect since S117 is not considered to contribute to either site of interaction with FGFR [15]. Indeed S117A-FGF-2 was as potent as wt-FGF-2 in stimulating FGFR [15]. Finally S117A-FGF-2 (unlike the modified FGF-1) retains ability to localize to the nucleus and thus to potentially interact with nuclear targets [15].

In further addressing the mechanism of wt- and S117A-FGF-2-induced acute cardioprotection we hypothesized that the mutant would be as potent as the wild type factor in activating PKC. We have established previously that PKC mediated FGF-2 cardioprotection irrespectively of whether this factor was given before ischemia [4,5] or during reperfusion [3]. We found that S117A-FGF-2, administered in reperfusion, caused translocation (and presumably activation) of various PKC isoforms, including PKC-{varepsilon}, to the particulate fraction, in a manner indistinguishable to wt-FGF-2, and can thus conclude that the mutant molecule activates PKC and that this event is very likely mediating its protective effects. Activation of PKC is a central event in cardioprotection, linked directly to mitochondrial preservation, and regulation of mitochondrial K+ (ATP) channel [31]. Since mitochondria are a target of PKC-mediated cardioprotection, we anticipated that S117A-, as well as wt- FGF-2-treatment would result in mitochondrial preservation, and decreased cytochrome c release to the cytosol. This was indeed the case (Fig. 4). We can thus conclude that the mechanism of acute cardioprotection by S117A- as well as wt-FGF-2 is linked to PKC activation and preservation of mitochondrial integrity. Reperfusion is reported to induce apoptotic cell death, a feature of which is cytochrome c release [32]. All things considered and since FGF-2 is known to prevent cardiomyocyte apoptotic death [33], we suggest that S117A- and wt-FGF-2-induced cardioprotection during reperfusion likely reflects prevention of apoptosis. It is noteworthy however that protection from apoptosis during reperfusion has been linked to the PI3/Akt pathway [34] that is not activated by FGF-2 [35].

S117A-, like wt-FGF-2 protected from the extensive cardiomyocyte loss that occurs during irreversible coronary occlusion. We believe that this reflects a direct effect of the factor on cardiac cells, including myocytes, preventing cell death. We were unable to detect TUNEL positive cells in the infarcted and peri-infarcted zone for up to 48 h post MI (unpublished observations). We conclude that S117A-, like wt-FGF-2, protected cardiomyocytes, from irreversible ischemic injury that did not present overt apoptotic features. More detailed investigation, beyond the scope of this work, is however required to resolve this issue.

4.2. The role of CK2 in long-term cardioprotection by FGF-2
Treating the permanently occluded, ischemic myocardium with S117A-FGF-2 did not support cardioprotection past the first week, showing that sustained protection required intact CK2 activity. Because the wt-FGF-2-induced long term cardioprotection was associated with increased overall cardiac flow properties as well as increased small vessel density (indicative of increased vascular response) at both sides of the infarct border, while the absence of long-term protection by S117A-FGF-2 was associated with absence of a vascular effect we conclude that long term cardioprotection by FGF-2 is dependent on its angiogenic properties, and includes improved tissue perfusion. Previous studies have demonstrated that mitogenic FGF-2 can increase arteriogenesis as well as capillarization [10,36]. It is expected that CK2-dependent mitogenicity is an important feature of the angiogenic potential of wt-FGF-2. Nevertheless, the exact cellular events involved in the FGF-2-induced vascular changes in our system merit further study. We hypothesize that FGF-2, a chemoattractant and a powerful mitogen for endothelial and smooth muscle cells would promote neovascular sprouting from pre-existing vessels, as well as likely contribute to increased size of pre-existing arterioles. In addition, wt-FGF-2 (but not S117A-FGF-2) would promote proliferation of stem-cells reaching the site of injury, either from the circulation [37] or from the heart itself [38] and thus contribute to an overall enhanced regenerative response. Our findings of increased vascularity both within the infarct near the border zone and the myocardium bordering the infarct are in agreement with a recent report by Iwakura et al. [39] who also used intramyocardial FGF-2 delivery in a rat infarct model.

Our data offer direct support to the notion that FGF-2 cardioprotection (i.e., preservation of cardiac tissue and functionality) is composed of two independent components: a direct and acute cytoprotective component, shared by S117A-FGF-2, and an ‘indirect’ component that includes angiogenesis, ensuring proper tissue perfusion and long-term survival.

Loss of cardioprotection by S117A-FGF-2, detected after one week post-MI by ultrasound (2–6 weeks), and by direct assessment of scar size at 6 weeks could be explained by the occurrence of infarct-border myocyte cell death during this period. We suggest that S117A- (as well as wt-FGF-2) interactions with affected cardiac cells at or near the border of the infarct area helps maintain them in a state of ‘reversible injury’, up to a certain time point. It may be that thus affected cells can survive on very little, perhaps in a ‘hibernation’-like mode. If reperfusion (by angiogenesis or by surgical re-establishment of flow) does not occur within this critical time window, infarct border cells no longer survive. According to this scenario, angiogenesis by FGF-2 and resulting perfusion occurs in a timely fashion to ensure recovery and survival of border myocytes. An additional or parallel scenario can however be envisaged: increasing evidence indicates that recruitment and/or activation of stem cells occurs during the repair period after MI [40]. As already mentioned, it is possible that increased local FGF-2 mitogenic activity contributes to a stem-cell-derived repair/regeneration response that would include angiogenesis as well as new muscle formation. FGF-2 is a potent mitogen for stem cells (including skeletal muscle satellite cells [41] and heart-derived stem cells [38]) while S117A-FGF-2 would not play that role. Infarct size (2–6 weeks) would reflect not only the magnitude of the original insult but also the quality of the repair/regeneration response. It is likely that the significant and sustained improvement of the permanently occluded MI-heart by wt-FGF-2 is not only a matter of protecting from cell death and inducing increased vascularization but also a matter of overall improved repair/regeneration, requiring its mitogenic activity.

4.3. Therapeutic potential of S117A FGF-2
Early clinical trials for treatment of coronary artery disease by therapeutic angiogenesis using wt-FGF-2 produced encouraging results [42–46], although the latest double-blind phase II clinical trial failed to show significant improvements [47]. These studies have established the relative safety of using wt-FGF-2. Nevertheless these trials have excluded several groups of patients, such as those at risk for cancer development, fibrotic disease, or atherosclerosis. This is quite prudent in view of potential ‘side effects’ of wt-FGF-2. Prolonged exposure of skeletal muscle or myocardium to high local levels of the FGF family of peptides can cause hemangiomalike tumors and vascular malformations [48], although even in those animal experimental studies demonstrating tumor development there has been no evidence of malignant transformation [20,49]. FGFs are present in atherosclerotic plaques [50]. Under various experimental circumstances their administration increases neointimal smooth muscle cell proliferation and neointimal mass [51,52], and this might play a role in plaque instability [52] and promote restenosis [53], which is an important cause of complications in coronary heart disease patients.

Irrespectively of the final verdict on therapeutic angiogenesis, the acute protective properties of wt-FGF-2, shared by S117A-FGF-2, represent another therapeutic modality in the setting of reperfusion-associated pathologies. In this context, S117A-FGF-2, retaining similar protective potential as the native molecule, would not pose risks associated with stimulation of proliferative growth and would thus allow treatment of a wider group of patients. Absence of mitogenicity might allow repeated treatments with S117A-FGF-2, since it would represent a safer alternative for several patient groups. We suggest that admimistration of S117A-FGF-2 as an adjunct treatment together with thrombolytics and angioplasty would significantly reduce ongoing and reperfusion-induced injury to the heart and result in improved outcome. Since acute coronary occlusion is a major cause of mortality, a significant number of patients may benefit from such treatment.


   Acknowledgements
 
This work was funded by the Canadian Institutes for Health Research (EK). Dr. Z-S Jiang was supported by a postdoctoral fellowship from the Manitoba Health Research Council, and by the IMPACT-CIHR programme.


   Notes
 
1 Current address: Scripps Research Institute, La Jolla, CA, USA. Back

Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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