[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to explore the serum levels of eight angiogenesis biomarkers in patients with benign, borderline or malignant epithelial ovarian neoplasms and to compare them to those of healthy controls. In addition, we aimed to study how those biomarkers predict the clinical course and survival of patients with epithelial ovarian cancer.
We enrolled 132 patients with ovarian neoplasms and 32 unaffected women in this study. Serum samples were collected preoperatively at the time of diagnosis and the levels of angiogenesis biomarkers were measured with an ELISA.
Levels of Ang-1, Ang-2, VEGF, VEGF-D, VEGF/sVEGFR-2 and Ang-2/ sVEGFR-2 ratios were elevated whereas sVEGFR-2 was lower in patients with ovarian carcinoma than in women with normal ovaries, benign and/or borderline ovarian neoplasms. In ROC analysis, the area under the curve for serum Ang-2/sVEGFR-2 ratio (0.76) was greater than Ang-2 (0.75) and VEGF (0.65) but lower than for CA 125 (0.90) to differentiate ovarian cancer from benign or borderline ovarian tumors. In ovarian cancer high Ang-2/sVEGFR-2 ratio was associated with the presence of ascites, high stage and grade of ovarian cancer, with the size of primary residual tumor >1 cm and with recurrence of disease. Elevated Ang-2, VEGF, VEGF/sVEGFR-2, Ang-2/VEGF and Ang-2/sVEGFR-2 ratios and low level of sVEGFR-2 were significant predictors of poor overall survival (OS) and recurrence free survival (RFS) in univariate survival analyses.
Ovarian cancer patients had elevated levels of angiogenesis related growth factors in circulation reflecting increased angiogenesis and poor prognosis. The serum level of Ang-2 predicted most accurately poor OS and Ang-2/sVEGFR-2 ratio malignancy of ovarian neoplasms and short RFS.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Mar 2013 · Human gene therapy. Clinical development
[Show abstract][Hide abstract] ABSTRACT: We compared effects of antiangiogenic gene therapy with a combination of soluble sVEGFR-1, sVEGFR-2 and sVEGFR-3 to chemotherapy with carboplatin and paclitaxel and to antiangiogenic monoclonal anti-VEGF-antibody bevacizumab in an intraperitoneal ovarian cancer xenograft model in mice (n = 80). Gene therapy was also combined with chemotherapy. Therapy was initiated when sizable tumors were confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously (2 × 109 pfu), while chemotherapy and monoclonal anti-VEGF-antibody were dosed intraperitoneally. The study groups were as follows: AdLacZ control (n = 21); combination of AdsVEGFR-1, -2 and -3 (n = 21); combination of AdsVEGFR-1, -2, -3 and paclitaxel (n = 9); bevacizumab (n = 14); paclitaxel (n = 9) and carboplatin (n = 5). Effectiveness was assessed by survival time and surrogate measures such as sequential MRI, immunohistochemistry, microvessel density and tumor growth. Antiangiogenic gene therapy combined with paclitaxel significantly prolonged the mean survival of mice (25 days) compared to the controls (15 days) and all other treatment groups (p = 0.001). Bevacizumab treatment did not have any significant effect on the survival. Tumors of the mice treated by gene therapy were significantly smaller than in the control group (p = 0.021). The mean vascular density and total vascular area were also significantly smaller in the tumors of the gene therapy group (p = 0.01). These results show potential of the antiangiogenic gene therapy to improve efficacy of chemotherapy with paclitaxel and support testing of this approach in a phase I clinical trial for the treatment of ovarian cancer.
Full-text · Article · Nov 2012 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Despite optimal surgery and chemotherapy, the prognosis of ovarian cancer patients remains poor and new treatments are urgently needed. Solid tumors require the formation of new vessels for growth and metastasis. In the present study, we have used soluble vascular endothelial growth factor (sVEGF) receptors sVEGFR-1 and -3, soluble receptors Tie1 and Tie2 and their combinations in an ovarian cancer xenograft model. Human ovarian cancer cells were injected intraperitoneally into nude mice (n=42) and magnetic resonance imaging (MRI) was used for confirming tumors before gene delivery. Treatment with combined AdsVEGFR-1, AdsVEGFR-3 and AdsTie2 significantly decreased the size of the intraperitoneal tumors compared with the controls (AdLacZ; P=0.038) with significantly less microvessels and vascular area. Unexpectedly, treatment with combined AdsTie1 and AdsTie2 led to a dramatic shortening of the survival which was not observed in the groups receiving either of the soluble receptors alone (P=0.031). The only difference to other treatments was liver toxicity observed after the combined Tie receptor treatment. In conclusion, combined inhibition of VEGFR-1, VEGFR-3 and Tie2 pathways was safe and provided efficient therapy for ovarian cancer in mice.
Full-text · Article · Feb 2011 · Cancer gene therapy
[Show abstract][Hide abstract] ABSTRACT: Myoblast sheet transplantation is a promising novel treatment modality for heart failure after an ischemic insult. However, low supply of blood and nutrients may compromise sheet survival. The aim of this study was to investigate the effect of mitochondria-protective Bcl-2-modified myoblasts in cell sheet transplantation therapy. In the Bcl-2-expressing rat L6 myoblast sheets (L6-Bcl2), increased expression of myocyte markers and angiogenic mediators was evident compared to wild-type (L6-WT) sheets. The L6-Bcl2 sheets demonstrated significant resistance to apoptotic stimuli, and their differentiation capacity in vitro was increased. We evaluated the therapeutic effect of Bcl-2-modified myoblast sheets in a rat model of acute myocardial infarction (AMI). Sixty-four Wistar rats were divided into four groups. One group underwent AMI (n = 22), another AMI and L6-WT sheet transplantation (n = 17), and a third AMI and L6-Bcl2 sheet transplantation (n = 20). Five rats underwent a sham operation. Echocardiography was performed after 3, 10, and 28 days. Samples for histological analysis were collected at the end of the study. After AMI, the Bcl-2-expressing sheets survived longer on the infarcted myocardium, and significantly improved cardiac function. L6-Bcl2 sheet transplantation reduced myocardial fibrosis and increased vascular density in infarct and border areas. Moreover, the number of c-kit-positive and proliferating cells in the myocardium was increased in the L6-Bcl2 group. In conclusion, Bcl-2 prolongs survival of myoblast sheets, increases production of proangiogenic paracrine mediators, and enhances the therapeutic efficacy of cell sheet transplantation.
No preview · Article · May 2010 · Cell Transplantation
[Show abstract][Hide abstract] ABSTRACT: We studied antiangiogenic and antilymphangiogenic effects of sVEGFR-1 (sFlt-1), sVEGFR-2 (sFlk-1/KDR), and sVEGFR-3 (sFlt-4) gene transfers and their combinations in intraperitoneal ovarian cancer xenograft mice (Balb/c-Anu, n = 55). Gene therapy was initiated when the presence of sizable tumors was confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously via tail vein as follows: AdLacZ as a control (group I), AdsFlt-1 (group II), AdsKDR (group III), AdsFlt-4 (group IV) and two combination groups of AdsFlt-1 and AdsFlt-4 (group V) and AdsFlt-1, AdsKDR, and AdsFlt-4 (group VI). Antitumor effectiveness was assessed by sequential MRI, immunohistochemistry, microvessel density, overall tumor growth, and survival time. In combination group VI, intraperitoneal tumors were significantly smaller than in the control group at the end of the follow-up (P < 0.001). Furthermore, in group VI the microvessel density (microvessels/mm(2)) in tumor tissue and the total area of tumors covered by microvessels were significantly smaller than in the controls. One mouse in group V was cured. The combined antiangiogenic gene therapy with soluble VEGFRs reduced tumor growth, tumor vascularity, and ascites formation in ovarian cancer xenografts. The results suggest that the combined antiangiogenic gene therapy is a potential approach for the treatment of ovarian cancer patients.
Full-text · Article · Feb 2009 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is important in several pathologies, including cardiovascular diseases such as atherosclerosis and cardiac ischemia-reperfusion injury. An important mechanism for adaptation to oxidative stress is induction of genes through the antioxidant response element (ARE), which regulates the expression of antioxidant and cytoprotective genes via the transcription factor Nrf2 (nuclear factor E2-related factor 2). As Nrf2-regulated genes are induced during oxidant stress occurring, for example, in reperfusion after ischemia, we took a novel approach to exploit ARE for the development of oxidative stress-inducible gene therapy vectors. To this end, one, two or three ARE-containing regions from human NAD(P)H:quinone oxidoreductase-1, glutamate-cysteine ligase modifier subunit and mouse heme oxygenase-1 were cloned into a vector expressing luciferase under a minimal SV40 promoter. The construct, which was the most responsive to ARE-inducing agents, was chosen for further studies in which a lentiviral vector was produced for an efficient transfer to endothelial cells. Heme oxygenase-1 (HO-1), which has well-characterized anti-inflammatory properties, was used as the therapeutic transgene. In human endothelial cells, ARE-driven HO-1 overexpression inhibited nuclear factor-kappaB activation and subsequent vascular cell adhesion molecule-1 expression induced by tumor necrosis factor-alpha. We conclude that the ARE element is a promising alternative for the development of oxidative stress-inducible gene therapy vectors.
[Show abstract][Hide abstract] ABSTRACT: Excessive production of reactive oxygen species has been implicated to play an important role in a number of cardiovascular pathologies, including hypertension, atherosclerosis, myocardial infarction, ischemia/reperfusion injury, and restenosis after angioplasty or venous bypass grafting. The formation of reactive oxygen species is balanced out by antioxidant defenses, and augmenting this defense by antioxidant therapies could therefore provide a potential means to treat conditions in which the formation of reactive oxygen species exceeds the capability of natural protective mechanisms. In this review, we summarize the studies in which antioxidant gene therapy has been used successfully to treat cardiovascular diseases. We also discuss the current limitations of antioxidant gene therapy and envision future therapeutic targets and methodological approaches for an improved outcome.
[Show abstract][Hide abstract] ABSTRACT: Progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. However, it remains unclear whether umbilical cord blood (UCB)-derived progenitor cells can provide therapeutic effects in ischemic muscles and whether ex vivo gene transfer can be used for improving the effect. In this study, the use of a lentiviral vector led to efficient transduction of both UCB-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Our method resulted in a long-term transgene expression and did not alter the differentiation potential of either HSCs or MSCs. In addition, we studied the therapeutic potential of CD133(+) and MSC progenitor cells transduced ex vivo with lentiviruses encoding the mature form of vascular endothelial growth factor D (VEGF-D(DeltaNDeltaC)) or the enhanced green fluorescent protein (eGFP) marker gene in a nude mouse model of skeletal muscle ischemia. Progenitor cells enhanced the regeneration of ischemic muscles without a detectable long-term engraftment of either CD133(+) or MSC progenitor cells. Our results show that, rather than directly participating in angiogenesis or skeletal myogenesis, UCB-derived progenitor cells indirectly enhance the regenerative capacity of skeletal muscle after acute ischemic injury. However, VEGF-D gene transfer of progenitor cells did not improve the therapeutic effect in ischemic muscles.
Full-text · Article · Jan 2008 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long-term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1).
We investigated whether there is any significant difference between the efficiencies of U6 and H1 in LV-mediated RNAi using green fluorescent protein (GFP) as a target gene by flow cytometry and real-time reverse-transcription polymerase chain reaction (RT-PCR) in endothelial cells. Also, we compared the efficiencies of U6 and H1 in the GFP transgenic mouse brain after stereotactic LV injection.
We show that the U6 promoter is more efficient than H1 in GFP silencing in vitro, leading to 80% GFP knockdown at an average of one integrated vector genome per target cell genome. The silencing is persistent for several months. In addition, the U6 promoter is superior to H1 in vivo and leads to stable GFP knockdown in mouse brain for at least 9 months.
These results show that LV-mediated RNAi is a powerful gene-silencing method for the long-term inhibition of gene expression in vitro and in vivo.
Full-text · Article · Apr 2006 · The Journal of Gene Medicine
[Show abstract][Hide abstract] ABSTRACT: The role of vascular endothelial growth factors (VEGFs) in large arteries has been proposed to be either vasculoprotective or proatherogenic. Because VEGF family members are used for human therapy, it is important to know whether they could enhance atherogenesis. We tested the effects of the members of the VEGF gene family on atherogenesis in LDL-receptor/apolipoprotein (apo) B48 double-knockout (LDLR/apoB48) mice using systemic adenoviral gene transfer.
Six groups of LDLR/apoB48-deficient mice (n=110) were kept 3 months on a Western-type diet. After 6 weeks of diet, mice were injected via tail vein with recombinant adenoviruses expressing VEGF-A, -B, -C, or -D or LacZ (1 x 10(9) PFU) or rhVEGF-A protein (2 microg/kg) and euthanized 6 weeks later. Also, older mice (n=36) were injected after 4 months on the diet and euthanized 6 weeks later (total time on the diet, 22 weeks) to evaluate the effects of gene transfers on the development of more mature lesions. Aortas were analyzed for the presence of macroscopic lesions, cross-sectional lesion areas, neovascularization, and cellular composition of the lesions. All groups had equivalent plasma cholesterol and triglyceride levels. Gene transfers with recombinant adenoviruses or administration of rhVEGF-A protein had no statistically significant effects on en face atherosclerotic lesions in the aorta, cross-sectional lesion area, neovascularization, or cellular composition of the lesions.
This study shows no proatherogenic effects of adenovirus-mediated gene transfers of VEGF-A, -B, -C, or -D in the LDLR/apoB48-deficient hypercholesterolemic mice, in which lipoprotein profile and atherosclerosis closely resemble those in human disease.
[Show abstract][Hide abstract] ABSTRACT: Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S40|[ndash]|S40; doi: 10.1016/j.ymthe.2005.06.104
98. Stable Lentivirus-Mediated RNA Interferen Comparison of U6 and H1 Promoters in Endothelial Cells and GFP Knockdown in Mouse Brain
Petri I. Makinen1, Jonna K. Koponen1, Anna-Mari Turunen1, Tarja M. Malm2, Jari Koistinaho2, Mikko P. Turunen1 and Seppo Yla-Herttuala11Department of Biotechnology and Molecular Medicine, A.I.Virtanen Institute, Kuopio, Finland2Department of Neurobiology, A.I.Virtanen Institute, Kuopio, Finland
[Show abstract][Hide abstract] ABSTRACT: We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.
No preview · Article · Apr 2004 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2(S)-M2 and the other has a rtTA responsive promoter driving the expression of beta-galactosidase gene (lacZ). The rtTA2(S)-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.
[Show abstract][Hide abstract] ABSTRACT: In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.
[Show abstract][Hide abstract] ABSTRACT: Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.