Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model

Abstract

Intramyocardial injection of genes encoding angiogenic factors could provide a useful approach for the treatment of ischemic heart disease. However, uncontrolled expression of angiogenic factors in vivo may cause some unwanted side effects, such as hemangioma formation, retinopathy, and arthritis. It may also induce occult tumor growth and artherosclerotic plaque progression. Because hypoxia-inducible factor 1 is up-regulated in a variety of hypoxic conditions and it regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we propose to use HRE, found in the 3' end of the erythropoietin gene to control gene expression in ischemic myocardium. A concatemer of nine copies of the consensus sequence of HRE isolated from the erythropoietin enhancer was used to mediate hypoxia induction. We constructed two adeno-associated viral vectors in which LacZ and vascular endothelial growth factor (VEGF) expressions were controlled by this HRE concatemer and a minimal simian virus 40 promoter. Both LacZ and VEGF expression were induced by hypoxia and/or anoxia in several cell lines transduced with these vectors. The functions of these vectors in ischemic myocardium were tested by injecting them into normal and ischemic mouse myocardium created by occlusion of the left anterior descending coronary artery. The expression of LacZ gene was induced eight times and of VEGF 20 times in ischemic myocardium compared with normal myocardium after the viral vector transduction. Hence, HRE is a good candidate for the control of angiogenic factor gene expression in ischemic myocardium.

Figure 1

Structure of AAVH9LacZ and AAVH9VEGF. ITR, inverted terminal repeat.

Figure 2

HIF-1α expression in ischemic myocardium by Western blot analysis. Two animals were studied in each of the normal and ischemic groups. The protein loaded for each sample is quantified by the Black 10B method.

Figure 3

HIF-1α expression by ischemic myocardium. The arrows indicate ischemic myocardium [hematoxylin and eosin (H&E) stain, Left] that stains positively for HIF-1α (Right). No HIF-1α stain was seen on other parts of the myocardium or in normal nonischemic heart (data not show).

Figure 4

Semiquantitative RT-PCR analysis for LacZ expression with 35 PCR cycles. Mouse HPRT (mHPRT) was used as internal control and amplified with 15 PCR cycles. +, PCR with RT products; −, PCR with RNA without RT. The quantification was done by PhosphorImager analyses. The numbers indicate the individual animals in each group.

Figure 5

Fold increase of LacZ and VEGF expression in ischemic hearts. Gray and black bars represent gene expression levels in ischemic and normal hearts, respectively. The expression levels of these two genes in normal hearts were arbitrarily assigned as one. The relative fold increases of gene expression in ischemic hearts versus in normal hearts were calculated according to the PhosphorImager analyses of semiquantitative RT-PCR data.

Figure 6

Semiquantitative RT-PCR analysis for VEGF expression in AAVH9VEGF vector-inoculated hearts. VEGF was amplified with 35 cycles. Mouse HPRT (mHPRT) was amplified 15 cycles and used as internal control. +, PCR products with RT; −, PCR without RT. The quantification was done by PhosphoImager analyses. The numbers indicate each individual animal.