Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation

Abstract

Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF₁₆₅) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation.

Keywords: endothelial-like cells; foam cell; macrophages; vascular endothelial growth factor.

Figure 1. Effect of stable overexpression of VEGF on macrophages in phenotypic characteristics

(A) Quantitative analysis of mRNA expression of endothelial markers by real-time PCR. mRNA expression of FLK-1, vWF, eNOS, VE-cadherin, and Tie-2 were dramatically increased in hVEGF₁₆₅-ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. Data are normalized to β-actin and presented as fold difference relative to RAW267.4 control. (B) Western blot analysis of endothelial markers expression. Protein expression of FLK-1, vWF, and eNOS were dramatically increased in hVEGF₁₆₅-ZsGreen1-RAW264.7 cells compared with untransfected RAW264.7, ZsGreen1-RAW264.7, and exogenous VEGF-treated RAW264.7. All images are representatives of three independent experiments each performed in triplicate, and graphs depict the value of mean and S.D. * indicates P<0.01.

Figure 2. Effect of stable overexpression of VEGF on macrophages in tubular networks formation

After 24 h of culture in Matrigel, hVEGF₁₆₅-ZsGreen1-RAW264.7 formed tubular structures. But no similar structure was detected in untransfected RAW264.7, ZsGreen1-RAW264.7, or exogenous VEGF-treated RAW264.7. Scale bars =100 μm. All images are representatives of three independent experiments.

Figure 3. VEGF₁₆₅ secretion level in supernatant after 72 h serum-free culture by ELISA

(A) hVEGF₁₆₅ production increased dramatically in hVEGF₁₆₅-ZsGreen1-RAW264.7, but could not be detected in untransfected RAW264.7 and control transfected ZsGreen1-RAW264.7 (P<0.01). (B) Mouse VEGF₁₆₅ secretion level in VEGF₁₆₅-ZsGreen1-RAW264.7 is much higher than that in untransfected and control transfected groups. Data represent mean ± S.D. of three independent experiments each performed in triplicate. * indicates P<0.01.

Figure 4. Foam cell formation assay

(A) Representative images of foam cell formation assay. Untransfected RAW264.7, ZsGreen1-RAW264.7, and hVEGF₁₆₅-ZsGreen1-RAW264.7 were treated with 100 μg/ml ox-LDL for 24 h. After fixation and staining with Oil Red O, the cells were observed by light microscopy. Cytoplasmic lipid droplets accumulated in RAW264.7 or ZsGreen1-RAW264.7, but hVEGF165-ZsGreen1-RAW264.7 cells showed significantly decreased lipid accumulation. Scale bars =50 μm. (B) Quantitative analysis of lipid accumulation in cells. The cells stained with Oil Red O were treated with 1 ml 60% isopropanol for 1 h to redissolve the oil red O and absorbance was detected at 518 nm through a spectrophotometer. The absorbance of oil red O was significantly decreased in hVEGF₁₆₅-ZsGreen1-RAW264.7 compared with RAW264.7 or ZsGreen1-RAW264.7 cells. Data represent mean ± S.D. of three independent experiments each performed in triplicate. * indicates P<0.01.

Figure 5. Stable overexpression of VEGF down-regulates expression of CD36 in macrophages

(A) Quantitative analysis of mRNA expression of CD36 by qRT-PCR. After treating with 100 μg/ml ox-LDL for 24 h, mRNA expression of CD36 were significantly decreased in hVEGF₁₆₅-ZsGreen1-RAW264.7 cells compared with that in RAW264.7 and ZsGreen1-RAW264.7. (B) Western blot analysis for endothelial cell markers expression. Protein expression of CD36 was significantly decreased in hVEGF₁₆₅-ZsGreen1-RAW264.7 cells compared with RAW264.7 and ZsGreen1-RAW264.7. Data represent mean ± S.D. of three independent experiments each performed in triplicate. ^#indicates P<0.05.