Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF ₁₆₅ Plasmid Gene Delivery to Chronically Ischemic Skeletal Muscle

Abstract

Abstract-Current methods of gene delivery for therapeutic angiogenesis are invasive, requiring either intraarterial or intramuscular administration. A noninvasive method of gene delivery has been developed using ultrasound-mediated destruction of intravenously administered DNA-bearing carrier microbubbles during their microcirculatory transit. Here we show that chronic ischemia could be markedly improved by ultrasound-mediated destruction of microbubbles bearing vascular endothelial growth factor-165 (VEGF 165 ) plasmid DNA. Using a model of severe chronic hindlimb ischemia in rats, we demonstrated that ultrasound mediated VEGF 165 /green fluorescent protein (GFP) plasmid delivery resulted in a significant improvement in microvascular blood flow by contrast-enhanced ultrasound, and an increased vessel density by fluorescent microangiography, with minimal changes in control groups. The improvement in tissue perfusion was attributed predominantly to increases in noncapillary blood volume or arteriogenesis, with perfusion peaking at 14 days after delivery, followed by a partial regression of neovascularization at 6 weeks. Transfection was localized predominantly to the vascular endothelium of arterioles in treated ischemic muscle. RT-PCR confirmed the presence of VEGF 165 /GFP mRNA within treated ischemic muscle, being highest at day 3 postdelivery, and subsequently decreasing, becoming almost undetectable by 6 weeks. We found a modulation of endogenous growth factor expression in VEGF-treated ischemic muscle, consistent with a biologic effect of ultrasound mediated gene delivery. The results of our study demonstrate the utility of ultrasonic destruction of plasmid-bearing microbubbles to induce therapeutic arteriogenesis in the setting of severe chronic ischemia.

Nocturnal Hemodialysis Is Associated with Restoration of Early-Outgrowth Endothelial Progenitor-Like Cell Function

“…In line with the complexity of the angiogenic process, multiple signaling molecules play stagespecific roles in coordinating new blood vessel growth (43). Ischemic hindlimb muscle was collected 28 days postligation, at a time when the levels of factors such as VEGF-A, Ang2, and SDF-1␣ that are activated early in angiogenesis have returned back to baseline (23). In contrast, Ang1 expression is often maintained for weeks after ischemia onset (23).…”

“…Male athymic nude rats (6 to 8 weeks, Charles River, Montreal, Canada) were obtained and maintained at the St. Michael's Hospital Animal Research Vivarium in a temperaturecontrolled (22°C) room with ad libitum access to commercial irradiated standard rat chow and autoclaved water. Rats underwent unilateral left-sided chronic hindlimb ischemia induction as described previously (23). Briefly, rats were anesthetized with inhaled isoflurane (2% to 3%).…”

“…Perfusion in the adductor muscles was assessed during continuous intravenous infusion of lipid microbubbles (1 ϫ 10 7 min Ϫ1 ) as described previously (23).…”

Biophysical insight into mechanisms of sonoporation

“…Studies using microbubbles driven by ultrasound in the megahertz frequency range have shown initial success in delivering therapeutic payloads in in vitro (6-10) and animal models (11)(12)(13). The delivery efficiency, however, remains inferior to that demonstrated via viral methods (4), in large part due to an incomplete physical, mechanistic understanding of the manner in which acoustic cavitation alters endothelial membrane permeability.…”

Crucial factors and emerging concepts in ultrasound-triggered drug delivery

“…Most studies involve pDNA, associated with a microbubble [47] or co-administred [48,49] with them. Due to its negative electrostatic loading pDNA will not penetrate into cells, but if it is located near a microbubble imploding in the vicinity of a cell it may profit from the temporal permeablization of the cell membrane (sonoporation) [50].…”