Oncogenesis: An "Off-Target" Effect of Radiofrequency Ablation.

PURPOSE: To compare hepatocellular carcinoma (HCC) development after radiofrequency (RF) ablation, partial surgical hepatectomy, and a sham operation and to inhibit HCC recurrence after RF ablation in a mouse model of spontaneously forming HCC in the setting of chronic inflammation (ie, the MDR2 knockout model). MATERIALS AND METHODS: Animal experiments were performed according to an approved animal care committee protocol. The authors compared the survival of MDR2 knockout mice (an inflammation-induced HCC model) that underwent RF ablation, 35% partial hepatectomy (ie, left lobectomy), or a sham operation (controls) by using Kaplan-Meier survival curve analysis. Tumor load and tumor frequency in mice that underwent sham operation were further compared with those of mice treated with RF ablation at 1 month after therapy by using a two-tailed Student t test. Liver slices from mice treated with RF ablation were stained for α-smooth muscle actin and Ki-67 to establish the role of liver regeneration in the tumorigenic effect of RF ablation. Finally, tumor load and tumor incidence were evaluated in mice treated with a c-met inhibitor after RF ablation by using the Mann-Whitney U test. RESULTS: Ablation of 3.5% ± 0.02 of the MDR2 knockout mice liver induced increased tumor load (P = .007) and reduced survival (P = .03) in comparison to that of controls, with no significant difference to the 10-fold volume removal of partial hepatectomy. Seven days after RF treatment, the border zone of the coagulation zone was surrounded by α-smooth muscle actin-positive activated myofibroblasts. A significant elevation of hepatocyte proliferation was also seen 7 days after RF ablation in the distant liver (ablated lobe: P = .003; untreated lobe: P = .02). A c-met inhibitor significantly attenuated HCC development in MDR2 knockout mice treated with RF ablation (P = .001). CONCLUSION: Liver regeneration induced by RF ablation facilitates c-met/hepatocyte growth factor axis-dependent HCC tumor formation after treatment in the MDR2 knockout model. Blockage of the c-met/hepatocyte growth factor axis attenuates HCC recurrence, raising the potential for therapeutic intervention to reverse this potentially deleterious tumorigenic effect.

Radiofrequency Ablation: Inflammatory Changes in the Periablative Zone Can Induce Global Organ Effects, including Liver Regeneration.

PURPOSE: To determine the kinetics of innate immune and hepatic response to the coagulation necrosis area that remains in situ after radiofrequency (RF) ablation, the cytokine profile of this response, and its local and global effect on the whole organ in a small-animal model. MATERIALS AND METHODS: A standardized RF ablation dose (70°C for 5 minutes) was used to ablate more than 7% of the liver in 91 C57BL6 mice (wild type) according to a protocol approved by the animal care committee. The dynamic cellular response in the border zone surrounding ablation-induced coagulation and in the ablated lobe and an untreated lobe were characterized with immunohistochemistry 24 hours, 72 hours, 7 days, and 14 days after ablation (the time points at which cells migrate to necrotic tissues). After characterization of the cellular populations that reacted to the RF treatment, cytokines secreted by these cells were blocked, either by using interleukin-6 knockout mice (n = 24) or c-met inhibitor PHA 665752 (n = 15), to elucidate the key factors facilitating the wound healing response to RF ablation. Statistical significance was assessed with nonparametric analysis of variance. RESULTS: RF ablation induces a strong time-dependent immunologic response at the perimeter of the necrotic zone. This includes massive accumulation of neutrophils, activated myofibroblasts, and macrophages peaking at 24 hours, 7 days, and 14 days after ablation, respectively. In correlation with myofibroblast accumulation, RF ablation induced hepatocyte proliferation in both the ablated lobe and an untreated lobe (mean, 165.15 and 230.4 cyclin-dependent kinase 47-positive cells per ×20 field, respectively, at day 7; P < .02). Blockade of either IL-6 or c-met significantly reduced global hepatocyte proliferation (P < .05 for both), with the former reducing the accumulation of both macrophages and myofibroblasts surrounding the coagulation necrosis area (42.9 and 113.6 vs 7.3 and 46.6 macrophages and activated myofibroblasts per ×20 field, respectively; P < .036 for both). CONCLUSION: Hepatic RF ablation induces not only a local periablational inflammatory zone but also more global proliferative effects on the liver. These IL-6- and/or c-met-mediated changes could potentially account for some of the local and distant tumor recurrence observed after treatment.

Variable phenotypes of knockin mice carrying the M712T Gne mutation.

GNE myopathy is a recessive adult onset, slowly progressive distal and proximal myopathy, caused by mutations in the GNE gene. The most frequent mutation in GNE myopathy patients is the Middle Eastern founder mutation M712T. We have generated Gne (M712T/M712T) knockin mice. A high mortality rate in the first generation due to renal failure was recorded (as previously described). However, the following Gne (M712T/M712T) offspring generations could be classified into 3 phenotypic categories: severe, mild and without apparent phenotype. By further crossing between mice with no apparent phenotype, we were able to establish a colony of Gne (M712T/M712T) knockin mice with a high- and long-term survival rate, lacking any renal phenotype. These mice did not present any muscle phenotype (clinical or pathological) for up to 18 months. No correlation was found between the expression of any of the two mRNA Gne isoforms in muscle and the mouse genotype or phenotype. However, the expression of isoform 2 mRNA was significantly higher in the kidney of Gne (M712T/M712T) kidney affected mice compared with control. In contrast, the expression of UPR markers Bip, Chop and of the spliced form of XBP1, was upregulated in muscle of Gne (M712T/M712T) mice compared with controls, but was unchanged in the affected kidney. Thus, Gne defects can affect both muscle and kidney in mouse, but probably through different mechanisms.

Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery.

GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system.

Viral delivery of the therapeutic gene bone morphogenetic protein-2 (BMP-2) is a promising approach for bone regeneration. The human parvovirus adeno-associated virus (AAV) type 2 is considered one of the most encouraging viral vector systems because of its high transduction rates and biosafety ratings. Bone morphogenetic protein-2 is a highly potent osteoinductive protein, which induces bone formation in vivo and osteogenic differentiation in vitro. The exogenous regulation of BMP-2 expression in bone-regenerating sites is required to control BMP-2 protein secretion, thus promoting safe and controlled bone formation and regeneration. We have therefore constructed a dual-construct vector for the recombinant AAV (rAAV)-based recombinant human BMP-2 (rhBMP-2) gene delivery system, which is regulated by the tetracycline-sensitive promoter (TetON). Each vector was encapsidated separately, yielding two recombinant viruses. We evaluated the efficiency of rAAV-hBMP-2 to induce bone formation in ectopic and orthotopic sites. Doxycycline (Dox), an analogue of tetracycline, was orally administered to mice via their drinking water to induce rhBMP-2 expression. Bone formation was measured using quantitative imaging-microcomputerized tomography and cooled charge-coupled device imaging-to detect osteogenic activity at the cellular level, detecting osteocalcin expression. The rAAV-hBMP-2-treated mice that were given Dox demonstrated bone formation in both in vivo models compared to none in mice prevented from receiving Dox. Thus, the Tet-regulated rAAV-hBMP-2 vector is an effective means of induction and regulation of bone regeneration and repair.