Endothelial cell-targeted pVEGF165 polyplex plays a pivotal role in inhibiting intimal thickening after vascular injury

Abstract

Upregulation of vascular endothelial growth factor (VEGF) expression can inhibit intimal thickening after vascular injury. However, the lack of efficient gene delivery systems leads to insufficient VEGF expression, which prevents its application in gene therapy. In the present study, to improve the delivery of the plasmid vector with the VEGF gene (pVEGF165) to the injured vessel wall, we explored the potentially important difference between endothelial cell-targeted and nontargeted polymeric carriers. The α_vβ₃ integrin is overexpressed on activated endothelial cells but not on normal quiescent vessels. In this study, CDG2-cRGD, synthesized by conjugating an α_vβ₃ integrin-binding cyclic arginylglycylaspartic acid (cRGD) peptide with the Generation 2 polycation polyamidoamine (PAMAMG2)-g-cyclodextrin (termed as CDG2), was developed as a targetable carrier. It was observed that the specific integrin–ligand interactions greatly enhanced cellular internalization of CDG2-cRGD in human umbilical vein endothelial cells (HUVECs), which are notoriously difficult to transfect. Consequently, HUVECs were found to show remarkably high levels of VEGF165 expression induced by the CDG2-cRGD polyplex. Interestingly, VEGF165 overexpression in vivo was more complex than that in vitro, and in vivo assays demonstrated that the stimulus response to balloon injury in arteries could obviously upregulate VEGF165 expression in the saline-treated group, although it was not enough to prevent intimal thickening. In gene-transfected groups, intravascular delivery of pVEGF165 with the CDG2-cRGD polyplex into rabbits after vascular injury resulted in a significant inhibition of intimal thickening at 4 weeks, whereas the low therapeutic efficacy in the nontargeted CDG2-treated group was only comparable to that in the saline-treated group. It is becoming clear that the conflicting results of VEGF165 gene therapy in two gene-transfected groups are reflective of the pivotal role of the cRGD-conjugated carriers in achieving the beneficial therapeutic effects of vascular gene therapy.

Keywords:

• pVEGF165
• α_vβ₃ integrin binding
• intimal thickening
• vascular gene therapy

Supplementary materials

Characterization of cRGD-conjugated PAMAMG2-g-cyclodextrin (CDG2-cRGD)

CDG2-cRGD was synthesized by a two-step cross-linking procedure. Supplementary Figure S1 was the ¹H-NMR spectra of α-CD, PAMAM G2, cRGD, CDG2, and CDG2-cRGD. Figure S1A showed the proton signals of α-CD (5.14 ppm (s, C(a)H of α-CD), 4.07-3.89 ppm (m, C(c) H, C(f) H and C(e) of α-CD), 3.69-3.58 ppm (m, C(b), and C(d)H of α-CD)). These proton signals of α-CD became almost negligible in CDG2 because of the restriction of the molecular motion by grafting PAMAM G2 to α-CD core, while proton signals of PAMAM G2 were distinctly observed (3.43-3.25 ppm (bm, C(i) H), 2.86-2.68 (m, C(j)H and C(g)H), 2.50-2.2.25 ppm (m, C(h) H)) (Figure S1B and D). For CDG2-cRGD, new proton signals at 6.48–7.32 ppm (7H(k), br s, ArH Phe, 2×NH) were detected due to the conjugation of cRGD (Figure S1C and E).

EGFP expression in transfected cells

HEK293T, U87 and HUVEC cells were seeded into 24-well plates and preincubated for 24 hours. The transfection experiments were conducted at approximately 70% confluence. 100 μL of pEGFPC1 polyplex and 0.4 mL of serum-free medium were added. The final pEGFPC1 concentration was 2.0 μg/well. After transfection for 4 hours, the medium was replaced with fresh serum-containing medium and the cells were incubated for another 48 hours to enhance green fluorescent protein expression. The green fluorescence of transfected cells was observed by fluorescence spectroscopy (Olympus IX71 fluorescence spectroscope).

Figure S1 ¹H-NMR spectra of (A) α-CD, (B) PAMAMG2, (C) cRGD, (D) CDG2, and (E) CDG2-cRGD.

Abbreviations: CD, cyclodextrin; CDG2, PAMAMG2-g-cyclodextrin; cRGD, cyclic arginylglycylaspartic acid peptide; D₂O, deuterium oxide; ¹H-NMR, proton nuclear magnetic resonance; PAMAMG2, Generation 2 polyamidoamine.

Figure S2 The relative transfection efficiencies of CDG2-cRGD conjugates.

Notes: EGFP expression after transfection with the conjugates was evaluated with FACS analysis in several different cell lines with varying levels of α_vβ₃ integrin receptor expression, which was observed by fluorescence spectroscopy. 200× magnification.

Abbreviations: CD, cyclodextrin; CDG2, PAMAMG2-g-cyclodextrin; cRGD, cyclic arginylglycylaspartic acid peptide; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; HEK 293T, human embryonic kidney cells; HUVEC, human umbilical vein endothelial cell; PAMAMG2, Generation 2 polyamidoamine; U87, human glioma cells.

Acknowledgments

The authors gratefully acknowledge the National Natural Science Foundation of China for extending financial support (project number 31170918) for this research.

Disclosure

The authors report no conflicts of interest in this work.