Gene Therapy (2010) 17, 305–314 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt
ORIGINAL ARTICLE
Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction GA Gonc¸alves1, PF Vassallo1, L dos Santos1, IT Schettert1, JS Nakamuta1, C Becker1, PJF Tucci2 and JE Krieger1 1
Department of Medicine, Heart Institute (InCor), University of Sao Paulo Med Sch, Sao Paulo, Brazil and 2Department of Physiology, Cardiac Physiology and Physiopathology Laboratory, UNIFESP, Sao Paulo, Brazil
In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in
neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in enddiastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Gene Therapy (2010) 17, 305–314; doi:10.1038/gt.2009.146; published online 10 December 2009
Keywords: gene cell therapy; VEGF; angiogenic growth factors; cardiac repair; angiogenesis gene therapy; biopolymer scaffold
Introduction Myocardial infarction (MI) is associated with high morbidity and mortality rates despite enormous progress in the development of new pharmacologic agents, percutaneous interventions to restore blood supply or direct coronary bypass grafting.1,2 The continuous demand for improvement has stimulated efforts to develop alternative approaches to stimulate neovascularization improving perfusion in damaged or ischemic myocardium or the desired task to restore muscle function using new materials or scaffolds, transplantation of cells and gene therapy-type procedures that may be used isolated or in combination.1–4 These efforts are leading to a new class of interventions, biologic cardiac repair that holds great potential to improve the outcomes of patients with cardiac ischemic disease. Vascular endothelial growth factor (VEGF) and other cytokines showed great potential in pre-clinical studies to induce neovascularization and even in small clinical studies Correspondence: Dr JE Krieger, Lab Genetics and Molecular Cardiology, Heart Institute (InCor)/University of Sa˜o Paulo Medical School, Av. Dr Ene´as C Aguiar, 44, Sa˜o Paulo, SP 05403-000, Brazil. E-mail:
[email protected] Received 1 March 2009; revised 3 August 2009; accepted 28 September 2009; published online 10 December 2009
but did not succeed in larger randomized trials.5–7 The success of the induced-angiogenic response, however, is influenced by several factors including the cytokine concentration levels reached locally and the duration of their availability. This may explain, at least in part, the inconsistent findings attributed to variable concentration of VEGF achieved at the site of the damaged tissue in response to naked DNA therapy.8,9 In contrast, sustained VEGF administration above a certain threshold is also undesired because it is associated with the production of leaky or ‘angioma-like’ immature vessels, hypotension, edema and inflammatory responses.10 Ideally, the angiogenic cytokine levels should be maintained at sufficient levels to allow only the proper increase of collateral blood flow avoiding unwanted side effects or generation of immature, non-functional vessels and this response may be tissue specific.8–15 The use of targeted viral vectors that direct gene transfer to specific cell types and reduce immunogenicity and toxicity, increase safety, and enable the systemic administration of these vectors for multiple indications including cancer, cardiovascular disease and inflammatory disease may circumvent some of the limitations.16 Another attractive approach to repair damaged cardiac tissue is transplantation of pluripotent cells or cells that behave as vectors to deploy a particular agent at the desired site. Cells from different origins and through a multitude of potential mechanisms, of which
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relative importance remains poorly understood, can improve neovascularization and mitigate cardiac dysfunction associated with cardiac ischemia.17–21 Along with others, we have shown earlier that genetically modified skeletal myoblasts (SKs) expressing VEGF used as vectors and transplanted in a relative small number can increase capillary density and decreases myocardial scaring in rats when injected 1 week before ischemia/ reperfusion injury.22 This early success occurs even though the expression of the transgene lasts only about 7–14 days when myoblasts are used as vectors.22 In this study, we hypothesized that transplantation of syngeneic fibroblasts as ‘vector’ to deliver angiogenic factors can support longer-lasting transgene expression and that association of these cells with a fibrin biopolymer would increase cell retention and the local levels of the growth factors. First, we used a proof of principle-type approach and showed that the pre-emptive transplantation of fibroblasts expressing VEGF produced similar improvements compared with the transplantation of geneticmodified myoblasts.22 Then, we provided evidence that fibroblasts expressing VEGF can act therapeutically 24 h post-MI to decrease cardiac scar area and most importantly to reduce cardiac dysfunction 4 weeks post-treatment.
Results ‘Preventive’ study The transgene expression after intramyocardial transplantation of cardiac fibroblasts (CFs) lasted longer than SKs despite the fact that both cells were transduced by the same adenovirus vector (at least 45 days compared with 7 days) (Figure 1). Taking advantage of this finding, we then determined the efficacy of the transduced CF expressing the angiogenic factor VEGF to increase capillary density and to reduce MI scar area in a setting of cardiac ischemia without gross abnormality in cardiac function. Animals were first treated with vehicle, cells, cells transduced with an empty vector or cells transduced with a VEGF vector and, 7 days later, a small ischemia reperfusion injury was produced and the animals followed for an additional 3-week period before morphometric analysis was performed. Cardiac VEGF
protein and capillary density increased only in groups transplanted with VEGF expressing cells (VEGF protein: 2090±11.4 vs Vehicle: 123.1±5.2, Cell: 104.2±7.4 and Null 73.2±2.4 positive cells/field, Po0.01 and capillary density: 543.8±52.1 vs 349.2±0.9, 288±19.0 and 245±2.6 capillaries mm–2, Po0.01). Co-localization, revealed by immunostaining, of specific endothelial and smooth muscle cell markers was significantly greater in the VEGF group, suggesting maturation of newly formed vessels (45±3 vs 10±2, 8±1 and 16±3, vessels/field Po0.001). These responses were accompanied by a small, but significant, reduction in myocardial scar area in VEGF vs Vehicle (3.0±1.3 vs 8.0±0.8%, Po0.05).
‘Therapeutic’ study Considering the above findings as evidence for proof of principle, we then tested the therapeutic potential of this strategy to repair a more significant cardiac ischemic lesion. Moreover, we now transplanted cells using a fibrin scaffold because along with others, we have shown that the use of fibrin can significantly improve cardiac cell retention.23,24 To produce a larger MI, the left anterior descending coronary artery was permanently ligated and to minimize variation among the groups, 24 h later, using echocardiography, only animals showing 20–40% MI of the left ventricle were selected for treatment randomization. As expected, immunostaining for human vascular endothelial growth factor was detected only in groups treated with cells transduced with VEGF. Groups injected only with Vehicle, Polymer, Cell and Null, showed only a faint human vascular endothelial growth factor signal. Quantitatively, human vascular endothelial growth factor expression was: 1083±87* vs 92±11, 85±8, 85±10, 70±11 positive cells/field, *Po0.0001, in hearts receiving: VEGF, Null, Cell, Polymer and Vehicle, respectively (Figures 2a and b). Increased capillary density and development of new vessels also occurred only in groups receiving cells expressing VEGF (978±104* vs 153±21.48, 158.67±13.91, 180.83±40.83 and 152.67±28.95 capillary mm–2, and 71±9.34* vs 7.58±2.84, 7.00±2.59, 5.88±3.0 and 8.92±2.94 vessels/ field in the VEGF vs Vehicle, Polymer, Cell, and Null groups, respectively, *P ¼ 0.0001) (Figures 3a and b and 4). Hemangiomas were not observed in any of the groups.
Figure 1 Time length of transgene expression in the myocardium after transplantation of genetic-modified skeletal myoblasts (SKs) or cardiac fibroblasts (CFs). SKs (a) and CFs (b) were transduced with the reporter gene LACZ (AdCMVLACZ) and injected directly in the heart. Transgene expression lasted at least 45 days after SK transplantation compared with 7 days when the cell vector was SK. Left ventricle and right ventricle are denoted by LV and RV, respectively. Gene Therapy
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Figure 2 Immunehistochemistry reaction to detect the human vascular endothelial growth factor (hVEGF) protein. Positive cells were higher in vascular endothelial growth factor (VEGF) group compared with the others (a). *P ¼ 0.0001. The red arrows indicate positive cells (b). A full colour version of this figure is available at the Gene Therapy journal online.
Collagen area and perimeter scar Microscopically, permanent coronary ligation resulted in a significant increase in collagen area and scar perimeter, which were influenced by the treatments. Collagen area reduced significantly in the groups treated with cells expressing VEGF (collagen content: 15.43±2.02* vs 35.12±7.05, 31.28±5.03, 30.07±6.21 and 35.13±6.21 for VEGF vs Vehicle, Polymer, Cell, and Null groups, respectively, *Po0.05) (Figure 5a). The scar perimeter area followed the same trend as seem for the collagen content (23.34*±1.97 vs 43.38±2.03, 37.79±1.28, 38.84±4.71 and 36.57±3.6 for VEGF vs Vehicle, Polymer, Cell, and Null, respectively, *Po0.001) (Figure 5b). Basal hemodynamics by echocardiography There were no major significant differences between infarcted groups concerning chamber dilatation or ventricular wall thickening as evaluated by echocardiogram (Table 1). Functional evaluation showed a moderate degree of systolic dysfunction analyzed by fractional shortening in infarcted rats compared with Shamoperated (Sham). There was no difference between infarcted gene-treated (VEGF), and infarcted non-treated groups (Vehicle, Polymer, Cell and Null).
Figure 3 Capillary density and vasculogenesis after 4 weeks of cell transplantation. Capillary density assessed PAS staining was higher in vascular endothelial growth factor (VEGF) group compared with other groups (a). Vasculogenesis assessed by colocalization using double fluorescent immunostaining for von Willebrand factor (vWF) and a-smooth muscle actin (SMA) was also increased in the VEGF group (b). *P ¼ 0.001.
Basal and pharmacologic stress hemodynamics by direct measurements As shown in Table 2, direct hemodynamic assessment under basal conditions also failed to identify important and consistent changes in MI groups as indicated by left ventricular end-diastolic pressures, +dP/dtmax, heart rate, and the ejective parameters of cardiac output (CO), stroke volume (SV) and stroke work (SW). Only LVESP Gene Therapy
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Figure 4 Vasculogenesis assessed by co-localization using double fluorescent immunostaining for von Willebrand factor (vWF) and a-smooth muscle actin (SMA) was also increased in the vascular endothelial growth factor (VEGF) group. The right arrows indicate the co-localization.
was statistically lower in rats receiving Cell (106±4 mm Hg, Po0.05) and Null (111±2 mm Hg, Po0.01) than in rats receiving Sham (122±6 mm Hg). In contrast, there were important changes on the main hemodynamic parameters related to cardiac performance in response to sudden pharmacologic afterload stress as presented in Figure 6. Percent changes of SV and CO decreased in all infarcted non-VEGF-treated hearts (SV: Vehicle: 60.0*±3.2; Polymer: 54.1*±2.7; Cell: 62.1*±2.2; and Null: 56.0*±3.1% of change, Po0.01, and CO: Vehicle: 59.7*±4.3; Polymer: 53.8*±3.1; Cell: 63.3*±2.5; and Null: 56.2*±3.1% of change, Po0.01) compared with either Sham (SV: 7.5±3.4%; and CO: 13.5±4.1% of change) or hearts transplanted with cell expressing VEGF (SV: 12.0±4.8; and CO: 12.7±5.2% of change) (SV changes in Figure 6a). Pressure overload was accompanied by a significant rise in the +dP/dtmax in the Sham-operated group (67.6±7.1%). Although not significant, infarcted non-VEGF-treated animals tended to present smaller inotropic response (Vehicle: 37.3±16.9; Polymer: 41.3±7.5; Cell: 33.6±9.9; and Null: 31.2±13.1 of increase) compared with gene-treated group that enhanced this parameter to levels similar to control (VEGF: 57.6±14.0% of increase) (Figure 6b). In all infarcted non-VEGF-treated hearts groups, a significant increase in left ventricular end-diastolic pressures was observed in response to pressure overload (13.0±1.5, 11.8±1.6, 14.8±1.9 and 12.6±2.3 mm Hg for Vehicle, Gene Therapy
Polymer, Cell and Null, respectively) compared either to Sham (2.7±0.7 mm Hg of increase) or VEGF-treated hearts (VEGF: 3.8±1.3 mm Hg of increase) (Figure 6c). Bilateral vagothomy was effective in preventing changes on heart rate as a result of the barorreflex, and the degree of bradycardia remained unchanged for all groups (Sham: 4.3±1.3%; Vehicle: 0.3±2.6%; Polymer: 0.0±1.4%; Cell: 3.3±2.3%; Null: 0.8±0.5%; VEGF: 0.6±0.9% of change, P ¼ 0.39). SW generation during afterload stress increased in Sham-operated (57.7±4.9%) rats and in the VEGF group (42.5±6.9%). In contrast, all remaining infarcted non-treated groups showed a negative change in SW (33.0±5.5, 30.5±4.2, 40.8±3.8 and 32.2±4.4% change for Polymer, Cell and Null, respectively, Po0.05 vs Sham or VEGF) (Figure 6d). To further examine these significant results obtained in the animals receiving cells expressing VEGF, we plotted the individual curves for the changes in SW generation against the level of pressure overload from each experimental group (Figure 7). Significant and positive correlations were noted only in Sham (Po0.0001 with Pearson’s r ¼ 0.82, mean slope ¼ 0.83±0.18) and VEGF (Po0.0001 with Pearson’s r ¼ 0.60, mean slope ¼ 0.66±0.15) transplanted animals while negative correlations were observed in the remaining infarcted control rats (mean slopes ¼ Vehicle: 0.30±0.04; Polymer: 0.43±0.06; Cell: 0.54±0.06; and Null: 0.46±0.08, Po0.001 all vs Sham and VEGF).
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Discussion
Figure 5 Microscopically, permanent ligation resulted in significant increase in collagen area (a) (*Po0.05) and perimeter scar area (b) (*Po0.001), which was influenced by transplantation of cells expressing vascular endothelial growth factor (VEGF).
The results obtained in this study show that transplantation of genetically modified cells expressing an angiogenic factor increases angiovasculogenesis and, most importantly, attenuate the progression of the scar size and the cardiac dysfunction in myocardial ischemia in the rat. This evidence is particularly important considering that results from direct viral vector-based gene delivery therapies remain inconsistent and there are serious immunological concerns that can lead to life threatening systemic inflammatory-like reactions.2,25 It is believed that variable success of direct injections of naked DNA to damaged tissue results, at least in part, from the fact that concentration and availability of growth factors expressed may not be constant. These observations justify our rationale for using gene delivery by ex vivo modification of cells as source of local factors for longer periods of time. Transplantation of ex vivo genetically modified CF in rats before MI lead to a high percentage of mature blood vessels as compared with control groups. This response may depend on the local concentration achieved, the time span as well as the presence of other poor controlled factors elicited by the tissue ischemia, which may explain why the response is variable and sometimes immature capillaries are observed.26,27 Ozawa et al.28 showed that there is a discrete threshold of VEGF dosage (70 ng per 106 cells day–1), below which stable capillaries are induced and above which hemangioma growth occurs. Thus, continuous delivery of VEGF, maintained below a certain threshold, can lead to normal angiogenesis29 and thus, we may
Table 1 Basal hemodynamic parameters (mean±s.e.m.) obtained by echodopplercardiography from all groups
LV diastolic diameter (cm) LV systolic diameter (cm) Shortening fraction (%) E/A ratio
Sham
Vehicle
Polymer
Cell
Null
VEGF
P-value
0.793±0.05 0.560±0.05 58±2.4 1.9±0.3
0.869±0.05 0.658±0.09 54±6.2 1.75±0.02
0.811±0.04 0.627±0.05 51.9±2 1.87±0.24
0.845±0.07 0.632±0.09 47.9±8.2 2.14±0.22
0.923±0.05 0.751±0.07 44.9±6.8 1.72±0.25
0.778±0.07 0.569±0.06 50.2±6.4 1.61±0.1
40.05 40.05 40.05 40.05
Abbreviations: LV, left ventricle; RV, right ventricle; VEGF, vascular endothelial growth factor. Analysis by one-way ANOVA and *denotes Po0.05.
Table 2 Basal hemodynamic parameters obtained by direct assessment
LVEDP (mm Hg) LVSP (mm Hg) +dP/dtmax (mm Hg s–1) dP/dtmax (mm Hg s–1) HR (b.p.m.) CO (ml min–1) SV (ml beat–1) SW (gm-m beat–1)
Sham
Vehicle
Polymer
Cell
Null
VEGF
P- value
2.6±0.7 122±6 9523±414 6257±568 347±26 45.7±7.5 0.13±0.02 0.21±0.02
5±1 123±3 9122±568 6093±215 365±46 51±7 0.14±0.01 0.23±0.03
5±1 125±3 8465±517 6009±314 360±22 47±5 0.13±0.02 0.21±0.03
6±2 111±2* 7971±470 5666±401 327±51 49±6 0.14±0.02 0.2±0.02
9±2 106±4* 7733±1005 5723±465 369±16 53±4 0.14±0.01 0.2±0.02
4±1 120±3 9654±990 6381±575 369±5 51±3 0.14±0.01 0.22±0.02
0.0672 0.0022 0.5251 0.7521 0.9410 0.3543 0.4948 0.4175
Abbreviations: b.p.m., beats per minute; CO, cardiac output; +dp/dtmax, maximal rate of pressure rise; dP/dtmax, decline; HR, heart rate; LVEDP, left ventricular end-diastolic pressure; LVSP, left ventricular systolic pressure; SV, stroke volume; SW, stroke work; VEGF, vascular endothelial growth factor. *Po0.05. Analysis by one-way ANOVA. Gene Therapy
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Figure 6 Changes in hemodynamics induced by pressure pharmacologic stress presented as percentage change of baseline values (except for left ventricular end-diastolic pressures (LVEDP), expressed as mm Hg of change). Positive or negative values represent increases or decreases from baseline, respectively. (a) Stroke volume (SV), (b) +dP/dtmax, (c) LVEDP, and (d) Stroke work (SW). One-way analysis of variance (ANOVA) and *denotes Po0.05 vs Sham. #Po0.05.
speculate that the tissue levels obtained in this study most likely did not reach this threshold. Considering potential clinical implications, it is important to evaluate whether the hemodynamic parameters are indicative of functional recovery of myocardial remodeling after infarction. It may be noted that no perceptible changes were detected in the hemodynamics of infarcted groups compared with Sham using both direct and indirect methods under basal conditions. Although impaired systolic and diastolic function may be evident in rats with large MI after 30 days of coronary occlusion,30–32 rats and other mammals with MI varying in size can often present normal or similar to normal hemodynamic parameters making it difficult to appropriately characterize impairment of ventricular performance.33–35 Ventricular performance in ischemic hearts is mainly dependent on extension of myocardial injury, time of evolution and experimental conditions,33 and may be more obvious when tested under stress such as volume or pressure overload.34–36 Consistent with this, our results showed evident impairment in cardiac perforGene Therapy
mance on pharmacological stress in all animals submitted to experimental MI. Most importantly, it showed a clear improvement in cardiac performance only in the groups receiving cells expressing VEGF compared with the other MI groups. Non-transfected fibroblasts or injection of fibrin scaffold in combination or isolated could not mimic the positive response. These observations are consistent with a small, if any, non-specific contribution from the injected cells or the fibrin scaffold for the positive response observed with transplantation of cells expressing VEGF, especially considering the small number of transplanted cells, which was on the order of 106 cells per animal injected directly into the myocardium. As expected, during the pharmacologic stress, Sham-operated group showed an increased work generation associated with a discrete decline in CO and SV, indicating an efficient ventricular performance. Surprisingly, the MI rats transplanted with cells expressing VEGF showed positive SW in response to pharmacologic stress comparable to Sham animals. Accordingly, positive correlations between pressure overload and SW
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Figure 7 Linear regressions from each animal from values for changes in stroke work (SW) generation (percentage change of baseline values) plotted against the levels of pressure overload from each experimental group.
generation were seen only in Sham and in infarcted rats treated with VEGF. There was striking discrimination (negative vs positive relationship) between groups with impaired or preserved performance, suggesting that probably this was essentially a consequence of VEGF therapy delivery system. Two important aspects emerge from these observations: will these beneficial effects be translated in decreased morbidity and mortality in these animals and most importantly, will these effects apply to cardiac ischemia in man? The first can be tested more easily but the latter most likely will need further investigation using an intermediate model, such as the swine model, that more closely resembles the pattern of cardiac tissue damage observed in man that unlikely the rat contains patchy areas of dead cells, fibrous tissue and live underperfused tissue that requires neovascularization. In addition, important aspects regarding the ‘dose-response curve’ of the VEGF for these beneficial effects, the site of action of this cytokine and the time course of this response remain to be determined. Taken together, our data indicate that transplantation of CF expressing human vascular endothelial growth factor using fibrin biopolymer induces neovascularization, attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Thus, the therapeutic potential of this intervention should be further examined as an alternative therapy for ischemic cardiac repair.
Materials and methods Adenovirus construction Recombinant replication deficient first-generation adenovirus harboring the human vascular endothelial growth factor, an empty vector or the LACZ gene were produced following the manufacturer’s instructions (CLONTECH Laboratories, Mountain View, CA, USA) and described in detail elsewhere.37,38 CF and SK preparation and infection isolation, cultivation and cell transduction of CFs and SKs were performed as described earlier.39,40 Gene expression analysis Cells transduced with the LACZ gene were assessed at different time points: 7 (n ¼ 3), 15 (n ¼ 3), 30 (n ¼ 3) and 45 (n ¼ 3) days after intramyocardial injection. SKs or CFs (106 cells per rat) were injected into the left ventricular wall. Tissue samples were homogenized and prepared for a chemiluminescence assay with a commercially available kit (Promega, Madison, WI, USA) using a luminometer (Monolight 2010, Analytical Luminescence Lab, San Diego, CA, USA). Fibrin polymer Fibrin polymer was prepared by combining fibrinogen and thrombin at the time of injection. Fibrinogen was obtained by separating the plasma from 50 ml of rat whole blood and then adding 5 ml of 3.8% sodium citrate solution. Fibrinogen was isolated using the cryoprecipitation technique and diluted to a final concentration of Gene Therapy
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90 UI ml–1. Human thrombin (Baxter Healthcare, Inc., Deerfield, IL, USA) was used to catalyze fibrin polymerization. Cells were resuspended in 80 Pl of the fibrinogen solution and, a few seconds before the injection in the tissue; 20 Pl of thrombin (250 U ml–1) was added to the syringe containing the cell suspension. This combination allowed a suitable time window of 20 s to perform the myocardial injection before polymerization.
Cell transplantation and myocardial infarction All procedures involving animals were approved by the Institutional Review Board of the University of Sa˜o Paulo Medical School, Brazil (SDC INCOR: 2256/03/050 – prot: 397/03). ‘Preventive’ study. Male Lewis rats were submitted to MI as described earlier.22 This method produced a discrete infarct area without compromising cardiac function. After 3 weeks, animals were anesthetized with sodium pentobarbital and hearts perfused with 10% formalin for morphometric and immunohistochemical analysis. Animals were divided into four experimental groups: Vehicle (DMEM/0.1 ml, n ¼ 10); Cell (106cells/ 0.1 ml, n ¼ 8); Null, (106cells transduced with an empty vector/0.1 ml, n ¼ 14) and VEGF, (106cells transduced with adenovirus harboring the human vascular endothelial growth factor/0.1 ml, n ¼ 18). ‘Therapeutic’ study. Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Twenty-four hours after the procedure animals were subject to an echocardiogram to estimate infarction size (animals with infarction size 20–40% of left ventricle were included in this study), 106 cells or placebo are injected per animal into the free ventricular wall. Animals were divided into five groups: Sham, animals subjected to surgery without coronary occlusion or therapy, n ¼ 6, (used for functional evaluation); Vehicle, (DMEM/0.1 ml n ¼ 6); Polymer, injection of fibrin polymer (0.1 ml n ¼ 6); Cell (106cells plus polymer/0.1 ml, n ¼ 6); Null (0.5 106 cells transduced with an empty vector plus polymer /0.1 ml, n ¼ 6) and VEGF (0.5 106cells transduced with AdVEGFEGFP plus polymer/0.1 ml, n ¼ 5). After 4 weeks, animals were subjected to direct and indirect hemodynamic analysis. At the end, the animals were anesthetized with sodium pentobarbital and hearts perfused with 10% formalin for morphometric and immunohistochemical analysis. Histology Hearts were fixed in 10% formalin for 24 h, embedded in paraffin and cut into 3 mm sections that were mounted onto slides and stained with Masson’s trichrome for scar area measurement (‘preventive’ study); Picrossirius Red for collagen area and perimeter scar measurement (‘therapeutic’ study); and periodic Schiff acid for capillary density quantification. A computerized image acquisition system (Leica Imaging Systems, Bannockburn, IL, USA) was used to measure scar areas. Infarct size was quantified by the percentage of left ventricular area or perimeter containing scar tissue. Capillary density evaluation We estimated capillary density in slides treated with periodic acid Schiff using a 10 10 grid optically superGene Therapy
imposed on each of 25 non-overlapping fields at 400 magnification distributed in a random manner at the antero–septal side of the left ventricle. The capillary density was evaluated adjacent to the cardiac scars as a total of capillaries in the area of the fields, calculating the number of capillaries per field (units mm–2) in all experimental groups in a blinded manner.22
Immunohistochemistry Cross-sections embedded in paraffin were first treated with antigenic exposure, and then blocked with 2% milk solution in phosphate-buffered saline. Tissue sections were then incubated with anti-human VEGF antibody (1:150; R&D) for 1 h at 37 1C. Sections were incubated with a biotinylated goat secondary antibody (1:400, Vector Laboratories; Burlingame, CA, USA) and then with streptavidin peroxides followed by the peroxidase substrate diaminobenzidine tetrahydrochoride according to manufacturer’s instructions. Negative control slides were incubated with normal goat serum; and the secondary antibody alone. Dual-fluorescence immunostaining was performed for blood vessel quantification. For that, cross-sections were pre-incubated with 5% bovine serum albumin followed by human anti-rabbit von Willebrand factor (1:20, Dako Laboratories, Glostrup, Denmark) and goat anti-mouse smooth muscle actin (1:400, Sigma Laboratories, Saint Louis, MO, USA) overnight at 4 1C. Sections were subsequently incubated with the secondary antibody anti-mouse CY3 (1:700, Sigma Laboratories) and anti- rabbit Fitc (1:50 Dako Laboratories) for 45 min as well as with DAPI to identify the cell nuclei. For each tissue section the number of blood vessels (in which red fluorescence for smooth muscle actin co-localized with green fluorescent for von Willebrand factor and blue fluorescent for DAPI) were counted from four randomly selected microscopic fields under a 200 objective. A total of four different tissue sections from three animals in each group were used to measure the final blood vessel number. Echodopplercardiography examination Doppler echocardiogram was performed by one observer; using a HP SONOS 5500 instrument (Philips Medical System, Amsterdam, Netherlands) under anesthesia with a 12-MHz transducer at a depth of 2 cm as described earlier.41 In brief, end-diastolic and systolic left ventricle diameters were measured from the transverse parasternal view using M-mode images. Systolic function was estimated using the fractional area shortening. End-diastolic and systolic transverse ventricular areas were obtained by tracing the endocardial border of basal, middle and apical images. Diastolic function was analyzed using parameters derived from mitral diastolic inflow and ventricular outflow tract velocity curves by pulsed-wave Doppler. Peak of E and A-wave velocities were measured, and E/A ratios calculated. Hemodynamic study under pharmacologic stress The direct hemodynamic analysis was performed as described before.42 In brief, invasive hemodynamic evaluation was performed on a heated rodent operating table (37 1C), under adjusted anesthesia (urethane 1.2 g kg–1), and oxygen-enriched mechanic ventilation. Left femoral vein was accessed to supplement anesthesia and drugs or saline administration. Bilateral vagothomy
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was produced to prevent changes on heart rate as a result of the barorreflex in response to pharmacologic stress. A Millar micro manometer (MikroTip 2F, Millar Instruments Inc., Houston, TX, USA) was inserted from the right carotid artery to the left ventricle cavity to access intraventricular pressure. A blood flow ultrasound probe (Transonic Systems Inc., Ithaca, NY, USA) was positioned on the ascending aorta to access CO (excluding coronary flow), through right thoracotomy. Data were acquired by the software acknowledge for windows (Biopac Systems, Goleta, CA, USA) to get systolic (LV systolic pressure) and left ventricular enddiastolic pressures, rate of LV pressure rise (dP/dt), heart rate, CO and SV. SW was estimated offline as a product of SV developed pressure (LVSPLVEDP) constant 0.0136. After baseline evaluation, a sudden afterload stress was induced by sudden pressure overload with a vasoconstrictive phenylephrine bolus injection (25–75 mg kg–1 body weight) through the left femoral vein. Phenylephrine doses were adjusted for each animal to produce comparable blood pressure increases (60–80% of baseline).
Statistical analysis Results were expressed as mean±standard error of the mean (s.e.m.). One-way analysis of variance with post hoc Tukey’s test was used to compare groups as appropriate. Linear regression analyses with comparison of slopes were used to evaluate the effect of afterload stress on cardiac function and Po0.05 was considered significant.
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Conflict of interest The authors declare no conflict of interest. 14
Acknowledgements This study was funded by grants from public Brazilian Agencies Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP #01/00090), Ministerio da Ciencia e Tecnologia/Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/Ministe´rio da Saude/Departamento Ciencia e Tecnologia (MCT/CNPq/MS/DECIT #552324/20005-1 and 10120104096700). JSN, CB, and GAG were recipients of fellowships from FAPESP (04/06784-4, 03/02671-8 and 03/02672-4, respectively).
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