Long term effects of pcDNA3-VEGF165 intramyocardial transfer after experimental myocardial infarction in the rat scar tissue extracellular matrix.

Fabio D Mataveli, MD1*, Sang W Han, PhD2, Aline Mendes, MSc3, Rose Kanashiro, PhD4, Paulo Tucci, MD,PhD4, Helena B Nader, PhD3, Antônio Carlos Lopes, MD,PhD1 and Maria Aparecida S Pinhal, PhD3. 1Internal Medicine, Federal University of São Paulo-UNIFESP, São Paulo, SP, Brazil, 04039-032; 2The Center for Gene Therapy - CINTERGEN, Federal University of São Paulo-UNIFESP, São Paulo, SP, Brazil, 04044-010; 3Molecular Biology, Federal

University of São Paulo, São Paulo, SP, Brazil, 04044-020 and 4Cardiovacular Physiology, Federal University of São Paulo, São Paulo, SP, Brazil, 04055-010.

After the acute occlusion of a coronary artery, a considerable number of growth factors (GF) are released at the infarct site, triggered by the inflammatory process and the ischemic condition. In this process to compensate regional ischemia, glycosaminoglycans (GAGS) and proteoglycans (PGs) play an important role by stabilizing and protecting GFs from inactivation, promoting GFs binding to its receptors, and facilitating receptor dimerization and signaling (1,2,3). The sulfated GAG can be differentiated regarding the type of hexosamine and uronic acid moieties, degree and position of sulfation, as well as the type of the inter- and intra-disaccharide glycosydic linkages, such as heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (CS). The diversity in structure and specific cellular and tissue distribution indicate that these glycoconjugates could perform different biological roles. Indeed, pivotal functions have been postulated for proteoglycans and their glycosaminoglycan moieties, such as organization and modulation of the extracellular matrix, tissue morphogenesis and differentiation, cellular adhesion and recognition, among others (4). The expression of the Condroitin, Dermatan and Heparan Sulfate (CS, DS and HS), Hyaluronan (HA), the HSPG Syndecan-4 and the enzymes Heparanase and Cathepsin-B in the infarction scar tissue were studied.

INTRODUCTION

MATERIALS and METHODSYoung female Wistar-EPM rats were submitted to to the ligation of the left anterior coronary artery by lateral thoracotomy and heart exposure (5). In the treatment group, a total of 250 g of pcDNA3-VEGF165 in 150 L of PBS was immediately injected intramyocardially at three distinct points drawing an equilateral triangle around the area irrigated by the left anterior descending artery. Six weeks latter, all rats underwent echocardiogram evaluation, and then sacrificed. Rats were divided into 5 groups: small size MI (SSMI, n=6), large size MI (LSMI, n=8), phVEGF165 transfer SSMI (VEGF SSMI, n=5), phVEGF165 transfer LSMI (VEGF LSMI, n=10) and control (n=3) group. HS, DS and CS were identified by agarose gel electrophoresis and quantified by densitometry. HA was quantified by a noncompetitive fluorescence-based assay. HPLC assays were performed to detect low and high molecular weight HA. For immunostaining, sections were incubated with the primary antibodies directed against either heparanase (1:100) and syndecam-4 (1:50) overnight at 4C and cathepsin B (1: 80) for 1 hour at 4C. For HA, sections were incubated with HABP(1: 80) for 1 hour at 4C. Statistical significance was evaluated by ANOVA for comparison between the 5 groups. Interaction was investigated in the experimental group to determine correlation between infarct size and VEGF165 gene transfer in the expression of GAG. A value of p<0.05 was interpreted to denote statistical significance.

RESULTS

GAG content is increased in the VEGF-LSMI group and CS is over-expressed in comparison to DS and HS. The glycosaminoglycans extracted from the tissues were initially analyzed by agarose gel electrophoresis. Figure 1 indicates that the CS band is present in small amounts in the VEGF-SSMI (S) group while a large band is visualized for the VEGF-LSMI group (L). This perception was confirmed by posterior quantification and correction of results based on dry weight tissue samples (Table 1). Measurement of HS, DS and CS content in the VEGF LSMI group showed a significant increase (p<0.01) of all three sulfated GAG in comparison to the other three groups (Fig 2 A). When the HS, DS and CS contents were compared in same sample, CS was over-expressed in terms of mg CS/ g dried tissue, only in the VEGF LSMI group.

Group EF HS DS CS HA SSMI 61,3833 0,2003 0,8696 0,2746 0,7623 (n=6) (22,7835) (0,4) (0,1218) (0,1497) (0,3991) LSMI 33,7875 0,3970 0,6711 0,3605 3,8450 (n=8) (10,7580) (0,1318) (0,1928) (0,2192) (1,3653) VEGF SSMI 68,44 0,1784 0,6370 0,0365 5,2017 (n=5) (16,8365) (0,2314) (0,3683) (0,0544) (0,8604) VEGF LSMI 33,05 2,5948 4,1216 4,9061 13,6155 (n=10) (10,2166) (1,6902) (1,7395 ) (3,5793) (14,0230) NL 68,863 0,0448 0,0458 0,0005 0,11491 (n=3) (7,1316) (0,0309) (0,0374) (0,0009) (0,0504)

HA content is increased in the VEGF LSMI group Quantification of HA presented a significant (p < 0.05) increase in the HA content of the VEGF LSMI group in comparison to other groups (Fig 2A). Samples analyzed by HPLC showed two different chromatographic peaks with a retention time coinciding with those of high and low molecular weight HA control samples, and a third peak corresponding to HA with a molecular weight lower than 70 KDa (fig 3). Approximately 99% of all HA was of low molecular weight, regardless the size of the infarct (Figure.3). Strongest immunostaining for hyaluronan is observed in the VEGF LSMI group, meeting the results obtained previously with the fluorimetric enzyme-linking immunosobent-like assay (Fig 4).

Strong immunostaining for Heparanase was observed in the non-phVEGF165 transfer group in comparison to treated groupThe non-treated group showed a more consistent immunostaining for Heparanase than the gene transfer group (Fig. 4). Higher expression of this enzyme has been related to EC proliferation and tumor growth, while heparanase inhibition was found to be related to partial extracellular matrix preservation, myocytes survival, enhanced revascularization and enhanced recovery of cardiac function after acute MI (7,8). Intensity of immunostaining is related to the size of the MI, since stronger heparanase immunostaing was observed in both groups of LSMI, treated and non treated (Fig 4).

Immunostaing for Cathepsin B is more prominent in the VEGF LSMIThe gene transfer group showed a stronger immunostaing for Cathepsin B when compared to the normal control group (no staining) and non gene transfer group (Fig 4). The VEGF LSMI group presented the most remarkable immunostainning for Cathepsin B among the five groups.

Immunostaing for Syndecan-4Syndecan-4 is one of the major HSPGs generated within the cardiovascular system, and it is predominantly expressed in cardiac myocytes in the normal heart (1,2). Increase sydecan-4 gene expression was already observed in the fist 24 hours and one week after myocardial infarction (1, 2) The gene transfer group showed an increased syndecan-4 expression by immunostaining analysis compared with normal control, SSMI and LSMI groups. (fig.4)

CONCLUSIONSTaken together, the results obtained in this experiment show a different point of view in cardiac remodeling. It is widely accepted that up to three weeks after MI production scar formation and healing is essentially complete in rats (6). Our experiment shows that six weeks after MI the scar tissue ECM is still active and under remodeling process. Interestingly, in the VEGF LSMI group, the situation in the ECM improved dramatically, suggesting that this group is under an even more active healing process. More studies are needed to fully explore the role of ECM in regulating the action of the GF expressed in gene transfer therapy in the pos MI cardiac remodeling process.

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Fig.1. Agarose gel electrophoresis of the Gags extracted from the myocardial infracted area.(A) SSMI; (B) LSMI; (C) VEGF SSMI and LSMI groups. (L) large size myocardial infarction; (S) small size myocardial infarction.

Fig 2. (A) Statistical difference among experimental groups in HS, DS, CS and HA content. ( confidence interval = 95%). VEGF LSMI shows a significant difference of p<0.01 in comparison to the other groups in the expression of HS, DS and CS and p<0.05 in HA expression. (B) Interaction between size of the infarcted area and phVEGF 165 transfer in the expression of GAG. ( Blue line = VEGF transfer; Green line= Control )

Table 1. GAG Quantification Values in mg/g dry tissue.

Fig. 4. Immunohistochemistry of Hyaluronan, Syndecam-4, Heparanase and Cathepsinin the normal and infarcted myocardium. Normal control, SSMI, VEGF SSMI, LSMI and VEGF LSMI. Magnification: 40X .

Fig. 3.High performance liquid chromatography (HPLC) profile of the infarcted area HA showing two different chromatographic peaks with a retention time coinciding with those of high (HMW) and low molecular weight (LMW) HA controls. SSMI group and LSMI group.

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