Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis

This study addresses an important clinical issue by identifying potential candidates of vascular endothelial growth factor (VEGF) signalling through the Flk-1 receptor that trigger cardioprotective signals under ischaemic stress. Isolated working mouse hearts of both wild-type (WT) and Flk-1+/− were subjected to global ischaemia (I) for 30 min. followed by 2 hrs of reperfusion (R). Flk-1+/− myocardium displayed almost 50% reduction in Flk-1 mRNA as examined by quantitative real-time RT-PCR at the baseline level. Flk-1+/− mouse hearts displayed reduction in left ventricular functional recovery throughout reperfusion (dp/dt 605 versus 884), after 2 hrs (P < 0.05). Coronary (1.9 versus 2.4 ml) and aortic flow (AF) (0.16 versus 1.2 ml) were reduced in Flk-1+/− after 2 hrs of reperfusion. In addition, increased infarct size (38.4%versus 28.41%, P < 0.05) and apoptotic cardiomyocytes (495 versus 213) were observed in Flk-1+/− knockout (KO) mice. We also examined whether ischaemic preconditioning (PC), a novel method to induce cardioprotection against ischaemia reperfusion injury, through stimulating the VEGF signalling pathway might function in Flk-1+/− mice. We found that knocking down Flk-1 resulted in significant reduction in the cardioprotective effect by PC compared to WT. Affymetrix gene chip analysis demonstrated down-regulation of important genes after IR and preconditioning followed by ischaemia reperfusion in Flk-1+/− mice compared to WT. To get insight into the underlying molecular pathways involved in ischaemic PC, we determined the distinct and overlapping biological processes using Ingenuity pathway analysis tool. Independent evidence at the mRNA level supporting the Affymetrix results were validated using real-time RT-PCR for selected down-regulated genes, which are thought to play important roles in cardioprotection after ischaemic insult. In summary, our data indicated for the first time that ischaemic PC modifies genomic responses in heterozygous VEGFR-2/Flk-1 KO mice and abolishes its cardioprotective effect on ischaemic myocardium.

pathological conditions, such as cancer and as a pro-angiogenic cytokine for the treatment of ischaemic heart disease. VEGF binds to two receptor protein tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR), but most of the biological functions of VEGF are mediated via Flk-1 [4]. We demonstrated previously that ischaemic preconditioning (PC) induced angiogenesis in the infarcted myocardium and resulted in up-regulation of several transcription factors (STAT3, Pax-5, NFB, TFIID, SP1 etc). In addition, PC reduced VEGF-mediated vascular permeability by inhibition of c-Src in the ischaemic preconditioned group, thereby reducing ischaemic injury in a rat myocardial infarction model [5]. However, the mechanism by which activation of VEGFRs elicit these cellular events is not fully understood. Recently, attention has been directed toward studies of VEGF expression and its function in myocardial ischaemia/hypoxia [6][7][8] and relatively little is known regarding the mechanism of its receptors, Flk-1 and Flt-1. VEGF is the only known ligand for Flk-1, whereas Flt-1 is able to bind placental growth factor in addition to VEGF. Few embryological studies have demonstrated abundance of Flk-1 in human lung tissues, whereas Flt-1 was abundant in heart, lung and kidneys [9]. Several investigations demonstrated functional difference between Flk-1 and Flt-1 in endothelial cells. In the developing human heart, both receptors were expressed in the myocardial capillaries, and were known to stimulate intracellular calcium flux and VEGF stimulation. Genetically manipulated Flk-1 knockout (KO) (homozygous) studies demonstrated early embryonic death due to inhibition of vasculogenesis, whereas in another study homozygous Flt-1 disruption caused failure to assemble normal vascular channels [10]. Another important observation showed that Flt-1 was expressed in the endothelium of both large and small vessels, whereas Flk-1 expression was restricted only to small vessels [11]. Recent studies demonstrated the myocardial distribution pattern of Flk-1 and Flt-1 after rats were exposed to whole body hypoxia followed by 24 hrs of re-oxygenation. Intense staining was observed along the capillaries in addition to the coronary arteries [7]. We also documented that intensity of staining for both receptors increased significantly in the hypoxia/re-oxygenation group compared to normoxic control. We also documented significant improvement in myocardial function with increased capillary and arteriolar density after induction of survival factors VEGF, Bcl-2 and survivin in the chronic rat myocardial infarction model subjected to ischaemic PC [5].
One of our recent studies indicated reduced beneficial effects of PC in Flt-1 heterozygous KO mice compared to wild-type. This observation may be due to down-regulation of several important genes (obtained by DNA microarray analysis) such as oncogene 1 (Gro1), heat shock proteins, I Kappa B Kinase ␤ (IKK␤), colony stimulating factor (CSF-1) and annexin 7, suggesting the importance of VEGF /Flt-1 receptor signalling during ischaemic PC [12]. To gain a better understanding of the VEGF signalling through its other homologous membrane-spanning high-affinity tyrosine kinase receptor, we performed microarray analysis (Affymetrix Gene Chip Analysis) on Flk-1 +/-KO mice subjected to ischaemia reperfusion (IR) and preconditioning followed by ischaemia reperfusion (PCIR) protocols.
The results obtained from this investigation not only defined a high number of up-and down-regulated known and unknown genes in IR and PCIR when comparing wild-type (WT) and KO, but also provided functional network information. We observed many differentially expressed genes after IR (115) and PCIR (448) comparison between KO and WT mice. Several differentially regulated genes related to cardiovascular development and function, cell-cell signalling and interaction were identified. Therefore, the data obtained from our Flk-1 +/study should serve as a basis for designing future hypothesis driven signalling projects leading to a thorough mechanistic understanding of cardioprotection through VEGF signalling.

Quantitative real-time RT-PCR
Reverse transcription (RT) was performed with 1 g total RNA isolated from left ventricular tissue (n ϭ 6/group) of WT and Flk-1 +/heterozygous KO mice subjected to I/R with or without PC. Real-time RT-PCR analysis was done with 10 ng of RT product using the iCycler iQ detection system (Biorad, Hercules, CA, USA) employing Syber Green I fluorescence employing ␤-actin as reference control [12,13]. Primer sequences used for real-time RT-PCR are given in Table S1 (Supplemental file).

Statistical analysis
The values for myocardial haemodynamic parameters, infarct size, apoptosis and quantitative real time PCR were all expressed as the mean Ϯ standard deviation (Ϯ SD). Differences between groups were tested for statistical significance by one-way analysis of variance (ANOVA) followed by a Bonferroni correction to test for differences between the mean values of all groups with the

Characterization of Flk-1 heterozygous KO mice
Almost 50% reduction in Flk-1 mRNA was found in hearts from heterozyogous Flk-1 KO mice ( Fig. 1A and B) assessed by both RT-PCR and real-time RT-PCR. Moreover, Flk-1 mRNA expression is significantly inhibited in the KOPCIR compared to the WTPCIR myocardium. As expected, expression of Flt-1 and VEGF mRNA are not affected in Flk-1 +/mice before or after I/R ( Fig. 1A and B); however, after PC both Flt-1 and VEGF mRNA expression in KOPCIR and WTPCIR were increased compared to I/R.

Effect of Flk-1 heterozygosity on the recovery of ventricular function after ischaemia reperfusion
There was no significant difference in baseline function among the four groups. Throughout the study, the heart rate and coronary flow were not different between the two groups (data not shown).
The functional values of each parameter, such as LVDP, dp/dtmax and AF, were significantly decreased in all groups after 30 min. of global ischaemia, as expected, compared to their respective baseline values. Post-ischaemic myocardial function was disrupted in the Flk-1 +/mice significantly as evidenced by the significant decrease in LVDP, dp/dtmax and AF compared to wild-type control. A significant decrease in LVDP ( Fig. 2A)

Effect of Flk-1 inhibition on myocardial infarct size
Infarct size expressed as percent infarction relative to total area at risk was noticeably increased in Flk-1 +/mouse hearts compared to controls (Fig. 3A)

Effect of Flk-1 inhibition on cardiomyocyte apoptosis by TUNEL Assay
Apoptotic cardiomyocytes (Fig. 3B) were detected using TUNEL staining in conjunction with staining for ␣-sarcomeric actin. Apoptotic cardiomyocytes were significantly increased in the KOIR (495) and KOPCIR (335) groups when compared to the WTIR (213) and WTPCIR (116) groups (Fig. 3C). Hence, heterozygosity for Flk-1 increased cardiomyocyte cell death due to apoptosis compared to controls. Thus, the extent of cardiac injury is much more prominent in Flk-1 +/-KO mice when subjected to ischaemia reperfusion than controls. It is also clear that PC-mediated cardioprotection is disrupted in the KO compared to controls.

Gene expression changes in the Flk-1 heterozygous and WT mice by Affymetrix Microarray analysis
Microarray gene profiling was conducted with RNA isolated from the left ventricles of wild-type and KO mice to identify genes involved in ischaemic PC-mediated VEGF signalling. Microarray analysis was performed on RNA isolated from each group to   Fig. S1). The differentially expressed genes were further filtered using a Volcano plot (Fig. 4) (Fig. 4A) and 1394 differentially expressed probe sets in KOPCIR versus WTPCIR (Fig. 4B) Fig. 6A compares differentially expressed genes in-between the two different comparisons, which showed 45 common genes were differentially regulated. Further classification of the 45 genes according to biological function (Fig. 6B) was determined from the Netaffx gene ontology tool, Affymetrix analysis shows that 11 genes are involved in catalytic activity, 25 genes in binding activity and five in signal transduction activity. A pie chart shows commonly regulated functions (Netaffx gene ontology tool, Affymetrix) for the different comparisons (Fig. 6).

Biological network and functional analysis
In order to further refine the functional properties of the genes down-regulated, the total down-regulated genes in both comparisons (1156 genes) were loaded in the IPA tool for the network, identified genes were also mapped to genetic networks in the IPA database and ranked by score. The score reflects the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. A score of more than 10 was used as a cut-off for identifying gene networks. The list of networks generated (Table 5 and 6) and important networks related to cardiovascular function in each comparison is given (Fig. 7A and B). Network analysis revealed two important genetic networks related to cardiovascular development, which is shown in Fig. 7A     In this study, we have confirmed that PC-induced VEGF signalling is disrupted by knocking down Flk-1, and is characterized by a decrease in haemodynamics, an increase in infarct size and apoptosis. Several high-throughput technologies have been used to investigate the effect of ischaemic PC-mediated myocardial changes [14,15]; but, to our knowledge, this is the first microarray study of global gene expression in Flk-1 +/mice using highdensity oligonucleotide microarrays. Left ventricular RNA samples from six mice in each group were analysed for differential expression by hybridizing with Affymetrix 430 2.0 Mouse arrays. Our data uncovered several pathways induced by VEGF signalling through Flk-1 and provide a framework for comparing whole-heart gene expression changes associated with ischaemic PC.

Validation of differentially expressed genes by realtime RT-PCR
Gene chip data analysis with the help of Genespring software analysis revealed 448 genes and 115 genes differentially expressed in WTIPCIR versus KOPCIR and WTIR and KOPCIR comparisons which showed the involvement of several genes related with ischaemic PC-mediated VEGF-Flk-1 signalling. Identification of the differentially expressed genes and cluster analysis [16] of these genes are the important initial steps, but further analysis of these genes by IPA software for networks/functional analysis allowed us to look into more informative and convincing evidence of changed biological processes due to ischaemic PC. Pathway analysis showed several genes such as presenelin -1, AKT, AP1, FGF1, HIF1A [17], which is important in cell signalling. Na + /K + ATPase activity in the myocardium plays an important role in generating the rapid upstroke of the action potential and drives several ion exchange and transport processes crucial for normal cellular functions [18]. In addition, phosphatidylinositol 3-kinase is found in the downregulated list, and is composed of 85 kD and 110 kD subunits. The 85 kD subunit lacks PI3-kinase activity and acts as an adaptor, coupling the 110 kD subunit (p110) to activated protein tyrosine kinases. Phosphatidylinositol 3-kinase, catalytic, ␣ polypeptide has been reported to play an important role in VEGF-mediated angiogenesis [19]. Homozygous KO for this gene leads to embryonic lethality. Hetrozygous mice were viable and fertile [20], but showed severely blunted signalling via insulin receptor substrate (IRS) proteins, which is a key mediator of insulin, IGF1 and leptin action. Evidence also suggested [21] a critical role for p110␣ in growth factor and metabolic signalling. Our study shows that p110␣ plays an important role in several signalling pathways, including myocardial angiogenesis.    [26] in the myocardium promoting angiogenesis by activating target genes such as VEGF, LDH and several other genes. VCAM-1 is a member of the immunoglobulin superfamily of adhesion molecules [27], and is expressed in fibroblast-like cells. The intensity of VCAM-1 expression correlates with the degree of inflammation. The interaction of VCAM-1 and its ligands, the integrins, may play significant role in angiogenesis. Pathway analysis also demonstrated down-regulation of GR which is a homodimeric flavoprotein which maintains the cellular thiol redox state by catalyzing the reduction of glutathione disulpide (GSSG) to glutathione. Its activity is present in both the cytosol and mitochondria [28].
In conclusion, use of gene chip technology allowed us for the first time to identify several target genes downstream of VEGF/Flk-1 signalling in PC myocardium. Moreover, biological network and pathway analysis revealed several other related genes indirectly affected due to down-regulation of directly affected genes in the KO. Finally, to our knowledge, this is the first report in VEGF receptor-2 KO mice (Flk-1 +/-) in which several important genes related to cardioprotection and angiogenesis have been documented and should help to facilitate the design of effective future therapies.