Taina Vuorio

Lymphatic Vessel Insufficiency in Hypercholesterolemic Mice Alters Lipoprotein Levels and Promotes Atherogenesis

Objective—Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. Approach and Results—Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR−/−/ApoB100/100) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR−/−/ApoB100/100 mice and Chy×LDLR−/−/ApoB100/100 mice had higher plasma cholesterol levels compared with LDLR−/−/ApoB100/100 control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR−/−/ApoB100/100 mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR−/−/ApoB100/100 mice and Chy×LDLR−/−/ApoB100/100 mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). Conclusions—We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin–biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Avidin Fusion Protein-Expressing Lentiviral Vector for Targeted Drug Delivery

One of the main objectives of cancer therapy is to enhance the effectiveness of the drug by concentrating it at the target site and to minimize the undesired side effects to nontarget cells. We have previously constructed a fusion protein, Lodavin, consisting of avidin and the endocytotic part of the low-density lipoprotein receptor, and demonstrated its applicability to transient drug targeting in vivo. In this study we produced a lentiviral vector expressing this fusion protein and evaluated its safety and efficacy. The results showed that lentivirus-mediated gene transfer led to long-term avidin fusion protein expression on glioma cells and that the receptor was able to bind biotinylated compounds. Repeated administration was proven feasible and the optimal time frame(s) for administration of biotinylated therapeutic and/or imaging compounds was elucidated. Intravenous or intracranial injection of the virus into BDIX rats led to the production of antibodies against transgene (avidin), but repeated administration of the vector was unable to boost this effect. Neutralizing antibodies against the lentivirus were also detected. Furthermore, we showed that the anti-avidin antibodies did not significantly affect the ligand-binding capacity of the avidin fusion protein. The therapeutic efficacy of avidin fusion protein in tumor treatment was tested in vitro with biotinylated and nonbiotinylated nanoparticles loaded with paclitaxel. In vivo applicability of lentivirus was studied in the BDIX rat glioma model, in which high receptor expression was detected in the tumor area. The lentivirus-mediated delivery of the avidin fusion protein thus represents a potential approach for the repeated targeting of cytotoxic compounds to cancer cells.

Sleeping Beauty transposon vectors in liver directed gene delivery of LDLR and VLDLR for gene therapy of familial hypercholesterolemia

Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.

Cardiac Lymphatics – A New Avenue for Therapeutics?

Recent progress in lymphatic vessel biology and in novel imaging techniques has established the importance of the lymphatic vasculature as part of the cardiovascular system. The lymphatic vessel network regulates many physiological processes important for heart function such as fluid balance, transport of extravasated proteins, and trafficking of immune cells. Therefore, lymphangiogenic therapy could be beneficial in the treatment of cardiovascular diseases, for example by improving reverse cholesterol transport (RCT) from atherosclerotic lesions or by resolving edema and fibrosis after myocardial infarction. In this review we first describe recent findings on the development and function of cardiac lymphatic vessels, and subsequently focus on the prospects of pro- and anti-lymphangiogenic therapies in cardiovascular diseases.

Up-regulation of Mismatch Repair Pathway by Suicide Gene Therapy: Implications on the use of Temozolomide Treatment in Malignant Glioma

Objective: The dependency of the efficacy of temozolomide (TMZ) on cellular DNA repair activities makes it therapeutically effective in approximately half of malignant glioma (MG) patient population with a dysfunctional DNA repair system. Adenovirus-mediated Herpes simplex virus thymidine kinase and ganciclovir (AdHSV-tk/GCV) suicide gene therapy is effective in those as well as in MG patients with a functional DNA repair system. When administered together, these two therapies show evidence of synergistic cytotoxicity. However, the validity of such claims has been questioned as the exact mechanism has been unknown. Methods: The underlying mechanism was studied in rat and human MG cell lines and in an immunocompetent, orthotopic, syngeneic rat MG model. Results: The results, for the first time, revealed an up-regulation of mismatch repair (MMR) pathway in MG cells by AdHSV-tk/GCV therapy, an adjunct effect to AdHSV-tk/GCV’s pro-apoptotic therapeutic mode of action that enhanced the cytotoxicity of TMZ. When combined with AdHSV-tk/GCV therapy, initially resistant MG cells were sensitized to TMZ treatment. The enhancement of TMZ’s efficacy was also seen in vivo as a significant increase in survival and a reduction in tumor growth rate, without affecting the adverse effect profile. Conclusion: This study demonstrates a synergistic outcome of AdHSV-tk/GCV and TMZ treatment combination, underlying mechanism for the synergy and a possible improved therapeutic protocol for enhanced efficacy. The findings may have an impact on future clinical use of this treatment combination, as well as benefit other chemotherapies, which depend on MMR pathway for action.