Atrial natriuretic peptide gene transfer and regulated expression in primary vascular smooth muscle cells

Abstract

In vitro tests were performed to evaluate the suitability of primary vascular smooth muscle cells (VSMCs) as targets for retroviral vector-mediated atrial natriuretic peptide (ANP) gene transfer, tetracycline (Tet)-regulated expression of ANP transgene and microencapsulation for an ex vivo approach of ANP gene therapy. Rat ANP cDNA was thus cloned from rat atrial tissue, and then sub-cloned and packaged into retroviral vectors comprising standard or Tet-regulated gene expression cassettes. After high efficiency of marker LacZ gene transfer was demonstrated, the expression of immuno-reactive ANP (irANP) was analysed. Our results showed that, unlike non-transduced or LacZ-transduced control VSMCs, LrASN/PA317-transduced VSMCs secreted a significant amount of irANP (425 ± 50 pg/ml/105cells/24 hours, at peak). Importantly enough, LNtetPrtTFrA/PA317-transduced VSMCs were shown to exhibit efficient Tet-regulated expression of irANP with nontoxic concentrations of doxycycline. The biological activity of irANP produced by such engineered VSMCs was evidenced by cyclic GMP (cGMP) activation in LrASN-transduced VSMCs or in VSMCs exposed to conditioned media harvested from VSMCs secreting irANP. Further studies showed that transduced VSMCs synthesised and secreted more irANP than either rat primary endothelial cells, or skin fibroblasts or a transformed mouse fibroblast cell line. Most importantly, micro-encapsulation of engineered VSMCs in alginate did not alter Tet-regulated expression and long-term secretion of irANP. These results suggest that encapsulated engineered VSMCs may prove instrumental in longterm in vivo studies on ANP function and in the development of an ex vivo ANP gene therapy approach for disease states such as hypertension and congestive heart failure.