Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16

Abstract

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

Keywords: VEGF; magnetic resonance imaging (MRI); miRNA therapy; pulmonary tumorigenesis; transgenic mice.

Figure 1. Schematic map of the hVEGF-A₁₆₅ transgene construct and detection of its integration into the transgenic mouse genome

A. hVEGF-A₁₆₅ overexpression construct controlled by mouse Clara cell-specific protein (mccsp) promoter. B. Verification of germline transmission and the transgenic patterns in the genomic DNA of the transgenic mice by Southern blot analysis. The Southern blot data showed that the foreign gene was found in similar patterns within the genomes of the transgenic founder mice and their offspring. C. The pulmonary Vegf-A₁₆₅ mRNA expression level in the offspring of transgenic mice was determined by semi-quantitative RT-PCR. The data showed that the Vegf-A₁₆₅ mRNA was expressed in the Tg 2 and Tg 3 offspring, but it was not expressed in the Tg 1 offspring. β-actin was used as an internal control. D. The Vegf-A₁₆₅ mRNA expression in specific tissues was determined using semi-quantitative RT-PCR. RT-PCR showed that Vegf-A₁₆₅ mRNA was expressed specifically in the lung tissue of transgenic mice. β-actin was used as an internal control.

Figure 2. The exterior and histopathologic staining and immunohistochemical (IHC) staining of lung tissues from wild-type and transgenic mice

A. Exterior and histopathologic section images of the lung tissues in the mice: pulmonary adenomas. (A) and (B) present the exterior of the lungs from wild-type and transgenic mice, respectively. The arrows in (B) indicate adenomas. (C) The bronchia and alveoli of lung tissue from a wild-type mouse are shown. (D) The bronchia, alveoli and adenomas of lung tissue from a transgenic mouse are shown. (E) and (F) show the 2.5X magnification images of (C) and (D), respectively. (“b” indicates the bronchia). B. Histopathologic section images of the lung tissues from the mice: fibrosis. (A) The bronchia and alveoli of the lung tissue in a wild-type mouse are shown. (B) The fibrosis (arrow) in the bronchia of the transgenic mice was due to bleeding. (C) and (D) are the 6X magnification images of (A) and (B), respectively (“b” indicates the bronchia). C. Histopathologic section images of lung tissues from the mice: inflammation. (A) The normal alveoli in a wild-type mouse are shown. (B) Inflammation of the alveoli in a transgenic mouse is shown. (C) and (D) are the 6X magnification images of (A) and (B), respectively. D. Histopathologic section images of lung tissues from the mice: abnormal bronchial epithelium. (A) The normal bronchia in a wild-type mouse are shown. (B) The proliferation of the cells (arrow) on the bronchial epithelium of a transgenic mouse is shown. (C) The flattened bronchial epithelium (arrow) of a transgenic mouse is shown. (D) The cyst (arrow) on the bronchial epithelium of a transgenic mouse is shown. (“b” indicates the bronchia, “a” indicates the alveoli). E. Detection of hVEGF-A₁₆₅ expression in the lung tissue of mice by IHC staining is shown. F. Detection of the bronchia mCCSP in the lung tissue of mice using fluorescence microscopy is shown.

Figure 3. The exterior and histopathologic sections and neovascular and angiogenic activity of the lung tissues in the wild-type mice and in the three tumorigenesis levels of transgenic mice (Tg-level-1, Tg-level-2, and Tg-level-3)

A. Exterior and histopathologic section images of the lung tissues of the mice. B. hVEGF-A₁₆₅ protein expression in the lung tissues of mice was determined by western blot analysis; mean ± SD (n = 3). C. The neovasculature of the lung tissues of the mice was analyzed using an Angiogenesis Image Analyzer. D. The angiogenic activity of the lung tissues of the mice was analyzed using chick chorioallantoic membrane (CAM).

Figure 4. The cDNA microarray data

A. Representative images of the lung tissues of wild-type and transgenic mice (Tg-level-3/wild type) after using two-color fluorescent probe hybridization. B. Clustar display of a mouse cDNA microarray of the lung tissues of wild-type and transgenic mice (Tg-level-3/wild type). The Cy5/Cy3 ratios of 580 cDNAs that deviated by more than 2-fold are shown. C. The ingenuity pathway analysis (IPA) of the mouse cDNA microarray data obtained from wild-type and Tg-level-3 mice.

Figure 5. The expression levels of mRNA and protein in the lung tissues of wild-type mice and of three different tumorigenesis levels of transgenic mice (Tg-level-1, Tg-level-2, and Tg-level-3)

A. The quantitative mRNA expression levels of the hVegf-A₁₆₅, cyclin b1 (cell cycle), cdc2 (cell cycle), egfr (cell proliferation), mmp9 (cell migration), brca1 (oncogene), myc (oncogene), nrp1 (co-receptor), and vegfr2-kdr (receptor) genes in the lung tissues of the mice were determined using qRT-PCR. Mean ± SD (n = 3). *p < 0.05; **p < 0.01 vs. wild-type mice. B. The protein expression levels of hVEGF-A₁₆₅, CYCLIN B1, CDC2, p-CDC2, BRCA-1, MYC, and GADPH in the lung tissues of the mice were determined by western blot analysis. GADPH was used as an internal control.

Figure 6. The expression levels of CD105, mRNA, and protein in the lung tissues of wild-type mice and three different tumorigenesis levels of transgenic mice (Tg-level-1, Tg-level-2, and Tg-level-3)

A. The CD105 level in the serum of the mice was determined using ELISA kit. **p < 0.01 vs. wild-type mice. B. The quantitative mRNA expression levels of the fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1) in the lung tissues of the mice were determined using qRT-PCR. Mean ± SD (n = 3). *p < 0.05; **p < 0.01 vs. wild-type mice. C. The inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lung tissues of the mice were determined by ELISA kit. *p < 0.05; **p < 0.01 vs. wild-type mice.

Figure 7. The MRI images of tumors (arrows) in the lung tissues of wild type mice and in two different tumorigenesis levels of transgenic mice (Tg-level-2 and Tg-level-3)

Top panel: wild-type mouse. Middle panel: lower hVEGF-A₁₆₅- expressing transgenic mouse (Tg-level-2). Bottom panel: higher hVEGF-A₁₆₅- expressing transgenic mouse (Tg-level-3). Red arrows indicate the solitary nodules of lung tumor.

Figure 8. MicroRNA-16 reduces pulmonary tumorigenesis in transgenic mice

A. Histopathologic sections of lung tissues from Tg-level-3 mice are shown. B. The immunohistochemical (IHC) staining of the lung tissues of Tg-level-3 mice is shown. C. The protein expression levels of hVEGF-A₁₆₅ and GADPH in the lung tissues of Tg-level-3 mice were determined by western blot analysis. GADPH was used as an internal control. D. VEGF concentrations in the serum of Tg-level-3 mice were determined using ELISA. **p < 0.01 vs. Tg-level-3/Mock mice. E. The protein expression levels of cleavage caspase 3, cleavage caspase 8, cleavage caspase 9, cleavage PARP, and BCL2 in the lung tissues of Tg-level-3 mice were determined by western blot analyses. β-actin was used as an internal control. *p < 0.05; **p < 0.01 vs. Tg-level-3/Mock mice.

Figure 9. MicroRNA-16 reduces pulmonary tumorigenesis in human orthotopic non-small cell lung cancer xenograft model

A. Exteriors, histopathologic sections and immunohistochemical (IHC) staining of lung tissue are shown. B. VEGF concentrations in the serum of the mice were determined using ELISA. ^††p < 0.01 vs. Healthy lungs/Saline mice. **p < 0.01 vs. Tumor-bearing lungs/Saline mice. C. The CD105 level in the serum of the mice was determined using ELISA kit. ^††p < 0.01 vs. Healthy lungs/Saline mice. *p < 0.05 vs. Tumor-bearing lungs/Saline mice.