Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells

Abstract

The therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

Figure 1

Characterization of EPCs and comparison of young (passage 4, P4)/senescent (passage 8, P8) porcine EPCs. Subconfluent EPCs were fixed with 4% paraformaldehyde (PFA) and stained with the indicated antibodies against UEA-1 (green a1). EPCs were incubated with DiI-labelled ac-LDL (red) for 4 h before PFA fixation (a2). Cell nuclei were visualized by DAPI staining (blue, a3, a7 and a11). Images were then merged (a4, a8 and a12). Cells were stained with anti-CD31 and anti-VE-cadherin (a5 and a6) antibodies. Panels a9 and a10 show cells stained with anti-vWF and anti-eNOS antibodies. Images were acquired by confocal microscopy with a 40 × (oil, na 1.30) magnification objective (SP8, Leica, Germany). Scale bar: 50 μm. P4 (young group) and P8 (senescent group) cells were evaluated for doubling time using a cell counting kit-8 (CCK-8; b), for acidic β-galactosidase activity using acidic β-galactosidase staining (c), and for telomere length using real-time PCR (d). The telomere (T) repeat copy number was normalized to the single-copy (S) gene 36B4 copy number. The T/S ratio of the control group was set as 1. n = 6 for each bar. *P < 0.05 vs. the young (P4) group. h = hours. RFU = relative fluorescence units.

Figure 2

Efficacy of ultrasonic microbubble transfection (UMT, i.e., US + B + plasmid) in young and senescent EPCs and the effect on the cell number at 48 h after treatment, as evaluated by a luciferase assay (a), by cell counting with a Coulter counter (b and d) and by ELISA for VEGF expression (c). In (c) and (d), the cells were divided into 5 groups to evaluate the treatment effects of US and microbubbles in the presence or absence of plasmid on VEGF expression and the cell number. The VEGF plasmid was used in all groups except the “US + B” group. The luminescence intensity generated by luciferase expression and the expression level of the VEGF protein (in 1000 μl of pDNA-containing growth medium per well) are shown per cell (a and c). n = 9 for each bar. Note that (b) shows the number of remaining cells per well after UMT under the conditions used in (a), and that (d) shows the number of remaining cells per well after treatment under the conditions used in (c). Additionally, note that for the UMT groups, differences between the black and grey bars are indicated. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the leftmost black bar and ^###P < 0.001 vs. the leftmost grey bar. Ctrl = control. B = microbubbles. US = ultrasound. LU = luminescence unit.

Figure 3

Comparison of the intracellular luciferase pDNA copy number and luminescence intensity in young and senescent EPCs at 2 h (h), 24 h and 48 h post UMT of luciferase pDNA. (a) The luminescence intensity, indicative of the luciferase protein level, was markedly increased in the UMT groups compared to the corresponding Ctrl groups at the same time points. In addition, in the UMT groups at 24 and 48 h, the senescent cells generated more luminescence than the young cells at the same time points. (b) Quantitative PCR analysis showed a higher luciferase pDNA copy number in the UMT groups than in the corresponding Ctrl groups at the same time points. In addition, the copy number was higher in senescent cells than in young cells at as early as 2 h post UMT. Thereafter, the copy number decreased markedly. See the text for details. n = 7 for each bar. ***P < 0.001 vs. the leftmost black bar at the same time point. ^#P < 0.05; ^###P < 0.001 vs. the leftmost grey bar at the same time point. ^••P < 0.01; ^•••P < 0.001 vs. the highest corresponding black bar and °°°P < 0.001 vs. the highest corresponding grey bar in (a) and (b). Note that for the UMT groups at the same time point, differences between the black and grey bars are indicated. All abbreviations are the same as those used in Fig. 2.

Figure 4

Comparison of VEGF protein levels in the supernatants (a) and intracellular pDNA copy numbers (b) at 2 h (h), 24 h and 48 h post UMT of VEGF pDNA into young and senescent EPCs. (a) ELISA and (b) quantitative PCR. See the text for details. n = 7 for each bar. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the leftmost black bar at the same time point. ^###P < 0.001 vs. the leftmost grey bar at the same time point. ^•P < 0.05; ^••P < 0.01; ^•••P < 0.001 vs. the highest corresponding black bar and °°°P < 0.001 vs. the highest corresponding grey bar in (a) and (b). Note that for the UMT groups at the same time points, differences between the black and grey bars are indicated. All abbreviations are the same as those used in Fig. 2.

Figure 5

Effects of UMT-mediated VEGF transfection on the angiogenic activities of transfected young and senescent porcine EPCs. At 48 h after transfection, the cells were evaluated for proliferation by a BrdU incorporation assay (a), by a migration assay (b), and by a tube formation assay (c). Representative micrographs are shown (d). Scale bar: 250 µm. Migration and tube formation were analysed with Leica QWin image analysis software (Cambridge, UK, version number: V3.5.2). See the text for details. n = 6 for each bar. *P < 0.05 vs. the leftmost black bar. ^#P < 0.05 vs. the leftmost grey bar. All abbreviations are the same as those used in Figs. 1 and 2.

Figure 6

Effects of supernatants (1000 μl/well) from young and senescent porcine EPCs 48 h post UMT-mediated VEGF transfection on the proliferation (a), migration (b) and tube formation (c) of human aortic endothelial cells. (d) Representative micrographs of tube formation in the different groups are shown. Scale bar: 250 µm. Proliferation was evaluated using a BrdU incorporation assay. Migration and tube formation were analysed with Leica QWin image analysis software (Cambridge, UK, version number: V3.5.2). n = 7 for each bar. In a, b and c: *P < 0.05; **P < 0.01; ***P < 0.001 vs. the leftmost black bar and ^#P < 0.05 vs. the leftmost grey bar. Note that for the UMT groups, differences between the grey and black bars are indicated. All abbreviations are the same as those used in Fig. 2.