Endothelial nitric oxide synthase induces heat shock protein HSPA6 (HSP70B') in human arterial smooth muscle cells.

Endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide (NO) production in blood vessels. One of the pleitropic functions of eNOS derived NO is to inhibit vascular smooth muscle cell proliferation in the blood vessel wall, and whose dysfunction is a primary cause of atherosclerosis and restenosis. In this study there was an interest in examining the gene profile of eNOS adenoviral (Ad-eNOS) transduced human coronary artery smooth muscle cells (HCASMC) to further understand the eNOS inhibitory effect on smooth muscle cell proliferation. To this aim a whole genome wide analysis of eNOS transduced HCASMCs was performed. A total of 19 genes were up regulated, and 31 genes down regulated in Ad-eNOS transduced HCASMCs compared to cells treated with an empty adenovirus. Noticeably, a cluster of HSP70 gene family members was amongst the genes up regulated. Quantitative PCR confirmed that transcripts for HSPA1A (HSP70A), HSPA1B (HSP70B) and HSPA6 (HSP70B') were elevated 2, 1.7 and 14-fold respectively in Ad-eNOS treated cells. The novel gene HSPA6 was further explored as a potential mediator of eNOS signaling in HCASMC. Immunoblotting showed that HSPA6 protein was induced by Ade-NOS. To functionally examine the effect of HSPA6 on SMCs, an adenovirus harboring the HSPA6 gene under the control of a constitutive promoter was generated. Transduction of HCASMCs with Ad-HSPA6 inhibited SMC proliferation at 3 and 6 days post serum growth stimulation, and paralleled the Ad-eNOS inhibition of SMC growth. The identification in this study that HSPA6 overexpression inhibits SMC proliferation coupled with the recent finding that inhibition of HSP90 has a similar effect, progresses the field of targeting HSPs for vascular repair.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model.

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

Utrophin upregulation in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a devastating, progressive muscle wasting disease for which there is currently no effective treatment. DMD is caused by mutations in the dystrophin gene many of which result in the absence of the large cytoskeletal protein dystrophin at the sarcolemma. Over-expression of utrophin, the autosomal paralogue of dystrophin, as a transgene in the mdx mouse (the mouse model of DMD) has demonstrated that utrophin can prevent the muscle pathology. Thus, up-regulation of utrophin in DMD muscle is a potential therapy for DMD. In this review we discuss recent advances in our understanding of the regulatory pathways controlling utrophin expression and the various approaches that have been applied to increasing the level of utrophin in the mdx mouse. These results are very encouraging and suggest that pharmacological up-regulation of utrophin may well be a feasible approach to therapy for DMD.