Recent advances in novel therapies for lipid disorders.

The prevalence of lipid disorders is alarmingly increasing in the Western world. They are the result of either primary causes, such as unhealthy lifestyle choices or inherited risk factors, or secondary causes like other diseases or medication. Atypical changes in the synthesis, processing and catabolism of lipoprotein particles may lead to severe hypercholesterolemia, hypertriglyceridemia or elevated Lp(a). Although cholesterol-lowering drugs are the most prescribed medications, not all patients achieve guideline recommended cholesterol levels with the current treatment options, emphasising the need for new therapies. Also, some lipid disorders do not have any treatment options but rely only on stringent dietary restriction. Patients with untreated lipid disorders carry a severe risk of cardiovascular disease, diabetes, non-alcoholic fatty liver disease and pancreatitis among others. To achieve better treatment outcome, novel selective gene expression and epigenetic targeting therapies are constantly being developed. Therapeutic innovations employing targeted RNA technology utilise small interfering RNAs, antisense oligonucleotides, long non-coding RNAs and microRNAs to regulate target protein production whereas viral gene therapy provides functional therapeutic genes and CRISPR/Cas technology relies on gene editing and transcriptional regulation. In this review, we will discuss the latest advances in clinical trials for novel lipid-lowering therapies and potential new targets in pre-clinical phase.

Differential but Complementary HIF1α and HIF2α Transcriptional Regulation.

Effective vascular regeneration could provide therapeutic benefit for multiple pathologies, especially in chronic peripheral artery disease (PAD) and myocardial ischemia. The hypoxia inducible factors (HIFs) mediate the cellular transcriptional response to hypoxia and regulate multiple processes that are required for angiogenesis to ultimately restore perfusion and oxygen supply. In endothelial cells, both HIF1α and HIF2α are known to contribute to this role; however, the extent and individual roles of each of these HIFα remain unclear. To characterize the individual roles of HIFα, we sequenced the transcriptional outputs of stabilized forms of HIF1α and HIF2α, where they regulated 701 and 1,454 genes, respectively. HIF1α transcription primarily regulated metabolic reprogramming, whereas HIF2α exerted a larger role in regulating angiogenic extracellular signaling, guidance cues, and extracellular matrix remodeling factors. Furthermore, HIF2α almost exclusively regulated a large and diverse subset of transcription factors and coregulators that contribute to its diverse roles in hypoxia. Further understanding of how HIFs regulate cellular processes in hypoxia and angiogenesis could offer new avenues to modulate physiological angiogenesis to enhance revascularisation in ischemic conditions and other pathologies.

VEGF-C and VEGF-C156S in the pro-lymphangiogenic growth factor therapy of lymphedema: a large animal study.

INTRODUCTION: VEGF-C156S, a lymphangiogenesis-specific form of vascular endothelial growth factor C (VEGF-C), has been considered as a promising candidate for the experimental pro-lymphangiogenic treatment, as it lacks potential angiogenic effects. As a precursor to future clinical trials, the therapeutic efficacy and blood vascular side effects of VEGF-C and VEGF-C156S were compared in a large animal model of secondary lymphedema. Combination of lymphatic growth factor treatment and autologous lymph node transfer was used to normalize the lymphatic anatomy after surgical excision of lymphatic tissue. METHODS: Lymph vessels around the inguinal lymph node of female domestic pigs were destroyed in order to impair the normal lymphatic drainage from the hind limb. Local injections of adenoviruses (Ad) encoding VEGF-C or VEGF-C156S were used to enhance the regrowth of the lymphatic vasculature. AdLacZ (ß-galactosidase) and saline injections served as controls. RESULTS: Both VEGF-C and VEGF-C156S induced growth of new lymphatic vessels in the area of excision, although lymphangiogenesis was notably stronger after VEGF-C treatment. Also the transferred lymph nodes were best-preserved in the VEGF-C-treated pigs. Despite the enlargement of blood vessels following the VEGF-C therapy, no signs of sprouting angiogenesis or increased blood vascular permeability in the form of increased wound exudate volumes were observed. CONCLUSIONS: Our results show that VEGF-C provides the preferred alternative for growth factor therapy of lymphedema when compared to VEGF-C156S, due to the superior lymphangiogenic response and minor blood vessel effects. Furthermore, these observations suggest that activation of both VEGFR-2 and VEGFR-3 might be needed for efficient lymphangiogenesis.

Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice.

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on hypoxia-inducible factor 1 (HIF-1), which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia-responsive element reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO-mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

Preclinical safety, toxicology, and biodistribution study of adenoviral gene therapy with sVEGFR-2 and sVEGFR-3 combined with chemotherapy for ovarian cancer.

Abstract Antiangiogenic and antilymphangiogenic gene therapy with soluble vascular endothelial growth factor receptor-2 (VEGFR-2) and soluble VEGFR-3 in combination with chemotherapy is a potential new treatment for ovarian carcinoma. We evaluated the safety, toxicology, and biodistribution of intravenous AdsVEGFR-2 and AdsVEGFR-3 combined with chemotherapy in healthy rats (n=90) before entering a clinical setting. The study groups were: AdLacZ and AdLacZ with chemotherapy as control groups, low dose AdsVEGFR-2 and AdsVEGFR-3, high dose AdsVEGFR-2 and AdsVEGFR-3, combination of low dose AdsVEGFR-2 and AdsVEGFR-3 with chemotherapy, combination of high dose AdsVEGFR-2 and AdVEGFR-3 with chemotherapy, and chemotherapy only. The follow-up time was 4 weeks. Safety and toxicology were assessed by monitoring the clinical status of the animals and by histological, hematological, and clinical chemistry parameters. For the biodistribution studies, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used. Low dose (2×10(10) vp) AdsVEGFR-2 and AdsVEGFR-3 gene therapy was well tolerated, even when gene therapy was combined with chemotherapy. Notably, only transient elevation of liver enzymes and mild regenerative changes were seen in liver after the gene transfer in the groups that received high doses (2×10(11) vp) of AdsVEGFR-2 and AdsVEGFR-3 with or without chemotherapy. No life-threatening adverse effects were noticed in any of the treatment groups. The highest protein concentration of soluble VEGFR-2 (sVEGFR-2) in circulation was seen 1 week after the gene transfer. The combination of chemotherapy to gene therapy seemed to prolong the time of detectable transgene protein at least 1 week in the circulation. The expression of AdsVEGFR-2 and AdsVEGFR-3 transgenes was mainly seen in the liver and spleen as detected by qRT-PCR. According to these results, AdsVEGFR-2 and AdsVEGFR-3 gene therapy combined with chemotherapy is safe and can be brought to clinical testing in ovarian cancer patients.

History of gene therapy.

Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality.

Glioma cell density in a rat gene therapy model gauged by water relaxation rate along a fictitious magnetic field.

Longitudinal and transverse rotating-frame relaxation time constants, T(1) (ρ) and T(2) (ρ) , have previously been successfully applied to detect gene therapy responses and acute stroke in animal models. Those experiments were performed with continuous-wave irradiation or with frequency-modulated pulses operating in an adiabatic regime. The technique called Relaxation Along a Fictitious Field (RAFF) is a recent extension of frequency-modulated rotating-frame relaxation methods. In RAFF, spin locking takes place along a fictitious magnetic field, and the decay rate is a function of both T(1ρ) and T(2ρ) processes. In this work, the time constant characterizing water relaxation with RAFF (T(RAFF) ) was evaluated for its utility as a marker of response to gene therapy in a rat glioma model. To investigate the sensitivity to early treatment response, we measured several rotating-frame and free-precession relaxation time constants and the water apparent diffusion coefficients, and these were compared with histological cell counts in 8 days of treated and control groups of animals. T(RAFF) was the only parameter exhibiting significant association with cell density in three different tumor regions (border, intermediate, and core tissues). These results indicate that T(RAFF) may provide a marker to identify tumors responding to treatment.

Growth factor therapy and autologous lymph node transfer in lymphedema.

BACKGROUND: Lymphedema after surgery, infection, or radiation therapy is a common and often incurable problem. Application of lymphangiogenic growth factors has been shown to induce lymphangiogenesis and to reduce tissue edema. The therapeutic effect of autologous lymph node transfer combined with adenoviral growth factor expression was evaluated in a newly established porcine model of limb lymphedema. METHODS AND RESULTS: The lymphatic vasculature was destroyed within a 3-cm radius around an inguinal lymph node. Lymph node grafts and adenovirally (Ad) delivered vascular endothelial growth factor (VEGF)-C (n=5) or VEGF-D (n=9) were used to reconstruct the lymphatic network in the inguinal area; AdLacZ (ß-galactosidase; n=5) served as a control. Both growth factors induced robust growth of new lymphatic vessels in the defect area, and postoperative lymphatic drainage was significantly improved in the VEGF-C/D-treated pigs compared with controls. The structure of the transferred lymph nodes was best preserved in the VEGF-C-treated pigs. Interestingly, VEGF-D transiently increased accumulation of seroma fluid in the operated inguinal region postoperatively, whereas VEGF-C did not have this side effect. CONCLUSIONS: These results show that growth factor gene therapy coupled with lymph node transfer can be used to repair damaged lymphatic networks in a large animal model and provide a basis for future clinical trials of the treatment of lymphedema.

Low spin-lock field T1 relaxation in the rotating frame as a sensitive MR imaging marker for gene therapy treatment response in rat glioma.

PURPOSE: To prospectively assess the effectiveness of T1 relaxation in the rotating frame (T1 rho) dispersion and the low spin-lock radiofrequency field (B(1)) T1 rho magnetic resonance (MR) imaging relaxation time in noninvasive monitoring of gene therapy response in BT4C glioma in rats. MATERIALS AND METHODS: All animal studies were approved by the ethical committee of the National Laboratory Animal Center. Rats with BT4C gliomas (n=9) were treated with herpes simplex virus thymidine kinase gene therapy and were compared with untreated rats (n=5). Absolute T1 rho at a B(1) range of 2.0 x 10(-6) to 1.4 x 10(-4) T, T1, T2, and apparent diffusion constant were measured at 4.7 T during treatment. Statistical significance was tested by using repeated-measures analysis of variance. RESULTS: A significant (P<.05) lengthening of T1 rho was observed beginning on the 4th day of treatment, and T1 rho values increased to be approximately 80% higher than values observed before treatment. These changes preceded T1 and T2 changes and resembled those of water diffusion. The T1 rho was associated with a treatment-induced decrease in cell density; this was the only measured MR imaging property that provided significant (P<.05) Pearson correlation with cell density in the tumor border. T1 rho relaxation dispersion, however, did not offer additional benefits over those offered in one B(1) experiment in the early phase of treatment. CONCLUSION: T1 rho with low B(1) is an excellent MR imaging marker of early gene therapy response in gliomas. The low B(1) approach is not limited by specific absorption rate restrictions; this finding suggests that spin-lock methods could be applicable in clinical settings. (

Dietary (n-3) fatty acids reduce plasma F2-isoprostanes but not prostaglandin F2alpha in healthy humans.

(n-3) Fatty acids are unsaturated and are therefore easily subject to oxidization; however, they have several beneficial health effects, which include protection against cardiovascular diseases. The aim of this study was to investigate whether (n-3) fatty acids, with a controlled fat quality in the background diet, affect nonenzymatic and enzymatic lipid peroxidation and antioxidant status in humans. A total of 162 men and women in a multicenter study (The KANWU study) were randomly assigned to a diet containing a high proportion of saturated fatty acids or monounsaturated fatty acids (MUFA) for 3 mo. Within each diet group, there was a second random assignment to supplementation with fish-oil capsules [3.6 g (n-3) fatty acids/d] or placebo. Biomarkers of nonenzymatic and enzymatic lipid peroxidation in vivo were determined by measuring 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) and prostaglandin F(2alpha) (PGF(2alpha)) concentrations in plasma at baseline and after 3 mo. Antioxidant status was determined by measuring plasma antioxidant capacity with an enhanced chemiluminescence assay. The plasma 8-iso-PGF(2alpha) concentration was significantly decreased after 3 mo of supplementation with (n-3) fatty acids (P = 0.015), whereas the PGF(2alpha) concentration was not affected. The antioxidant status was not affected by supplementation of (n-3) fatty acids, but was improved by the background diet with a high proportion of MUFA. We conclude that supplementation with (n-3) fatty acids decreases nonenzymatic free radical-catalyzed isoprostane formation, but does not affect cyclooxygenase-mediated prostaglandin formation.

Water diffusion in a rat glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death in vivo: correlation with cell density.

PURPOSE: To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)-thymidine kinase gene therapy-induced programmed cell death (PCD) in vivo. MATERIALS AND METHODS: The trace of the diffusion tensor (Dav = 1/3TraceD), T2, and spin density were determined by MRI and the apparent diffusion coefficient (ADC) of water by diffusion nuclear MR (NMR) spectroscopy using largely varying b values and diffusion times (tD) at 4.7 T. Cell count and apoptotic cells were quantified by histological means. RESULTS: Decline in cell count was strongly associated with increase in both Dav and T2. Spin density ratio between tumor and contralateral parietal cortex increased with a very similar time course as Dav and T2, indicating net water gain into the eradicating tumor. Diffusion spectroscopy showed a nonmonoexponential signal decay at all tD values ranging from 14-192 msec. During PCD, the ADC of the component yielding fast diffusion coefficient (D1), as acquired with tD > or = 47 msec, increased with kinetics similar to those of Dav (tD = 4.8 msec). The fractional size of D1 increased by 10% to 15% throughout the entire tD range. Apparent water residence time of the slow diffusion component, D2, shortened from a value of 38.3 +/- 1.7 msec on day 0 to 33.4 +/- 0.5 msec by day 8. CONCLUSION: The present results show that reduced cell density and increased water content, leading to altered water microenvironment, are associated with increased water diffusion coefficient in eradicating gliomas as a result of PCD.

Comparison of the effects of alpha-tocopherol, ubiquinone-10 and probucol at therapeutic doses on atherosclerosis in WHHL rabbits.

Oxidative modification of lipoproteins may trigger and maintain atherogenesis. We compared the effects of different antioxidants (alpha-tocopherol, probucol, ubiquinone-10) at doses similar to those used in humans in Watanabe Heritable Hyperlipidemic (WHHL) rabbits for 12 months. Aortic lesions were analyzed for their extent and cellular composition of lesions, mean thickness of fibrous caps and density of smooth muscle cells therein, content of antioxidants, non-oxidized and oxidized lipids. Compared to controls, probucol significantly lowered the extent and macrophage content of lesions and increased the existence and smooth muscle cell density of fibrous caps. alpha-Tocopherol supplementation increased the aortic content of vitamin E, but had no decreasing effect on either the accumulation of macrophage-specific antigen in the aorta or lesion size. Nevertheless, both probucol and alpha-tocopherol significantly decreased in vitro LDL oxidizability, measured under typically strong oxidative conditions. Ubiquinone-10 supplement increased lesion size and the fraction of lesions containing fibrous caps; however, LDL oxidizability remained unaffected by ubiquinone-10 treatment. None of the antioxidants tested lowered oxidized lipids within aortic tissue; however, long-term treatment with probucol provided the most effective anti-atherosclerotic effect, while alpha-tocopherol may be pro-atherogenic and ubiquinone-10 exerts ambivalent effects. Our data suggest that (i) widely used oxidation measures, such as ex-vivo LDL oxidizability, do not reflect the degree of atherosclerosis; and (ii) long-term beneficial effects of relatively low doses of antioxidants may be outweighed by high levels of plasma cholesterol in WHHL rabbits.

Early gene therapy-induced apoptotic response in BT4C gliomas by magnetic resonance relaxation contrast T1 in the rotating frame.

The design and evaluation of therapeutic gene transfection protocols and vectors are under extensive development. Magnetic resonance imaging (MRI) techniques can aid considerably in the development of experimental treatment approaches, as well as in determining treatment response by observing gross tissue morphology. However, through a unique set of contrast parameters, namely T1, T2, and diffusion, more information about tissue status can be obtained while delineating and classifying tumor characteristics in more detail. We show here that T1 relaxation in the rotating frame, T1rho, provides unique in vivo MRI contrast. Ganciclovir treatment of HSV-tk+BT4C gliomas, which effectively eradicates these tumors, resulted in significantly prolonged T1rho relaxation times in MRI already after 3 days of treatment, whereas conventional contrast parameters were elevated after 6-8 days of therapy. Interestingly, the prolonged T1rho values were observed while an increase in tumor volume was still taking place. The regions of elevated T1rho relaxation coincided with high apoptotic activity as determined by histology, suggesting that T1rho MRI contrast could be used as a novel early indicator of cytotoxic cell damage in gliomas.