BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium.

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD.

Decreased Gray-White Matter Contrast of [11C]-PiB Uptake in Cognitively Unimpaired Subjects with Severe Obstructive Sleep Apnea.

BACKGROUND: Very recently, cognitively normal, middle-aged adults with severe obstructive sleep apnea (OSA) were shown to have regional cortical amyloid-ß deposits. In the normal brain, amyloid tracer (e.g., [11C]-PiB) uptake is observed in white matter (WM) but not in cortical gray matter (GM), resulting in clear GM-WM contrast. There are no reports on possible changes in this contrast in severe OSA. OBJECTIVES: Evaluate changes in the global [11C]-PiB GM-WM contrast and study if factors reflecting clinical and imaging characteristics are associated with them. DESIGN AND SETTING: Cross-sectional imaging study. PARTICIPANTS: 19 cognitively intact middle-aged (mean 44 years) patients with severe OSA (Apnea-Hypopnea Index >30/h), carefully selected to exclude any other possible factors that could alter brain health. MEASUREMENTS: Detailed neuroimaging (amyloid PET, MRI). Signs of possible alterations in amyloid tracer GM-WM contrast and kinetics were studied with static and dynamic [11C]-PiB PET and WM structures with detailed 3.0T MRI. RESULTS: Static [11C]-PiB PET uptake showed significantly decreased GM-WM contrast in 5 out of 19 patients. This was already clearly seen in visual evaluation and also detected quantitatively using retention indexes. Dynamic imaging revealed decreased contrast due to alterations in trace accumulation in the late phase of [11C]-PiB kinetics. Decreased GM-WM contrast in the late phase was global in nature. MRI revealed no corresponding alterations in WM structures. Importantly, decreased GM-WM contrast was associated with smoking (p = 0.007) and higher Apnea-Hypopnea Index (p = 0.001). CONCLUSIONS: Severe OSA was associated with decreased GM-WM contrast in amyloid tracer uptake, with significant correlation with clinical parameters of smoking and AHI. The results support and further extend the current understanding of the deleterious effect of severe OSA on proper amyloid clearance, possibly reflecting dysfunction of the brain glymphatic system.

BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF.

The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

Comparative transcriptome analysis of matched primary and distant metastatic ovarian carcinoma.

BACKGROUND: High grade serous ovarian carcinoma (HGSOC) is the most common subtype of epithelial ovarian cancers (EOC) with poor prognosis. In most cases EOC is widely disseminated at the time of diagnosis. Despite the optimal cytoreductive surgery and chemotherapy most patients develop chemoresistance, and the 5-year overall survival being only 25-35%. METHODS: Here we analyzed the gene expression profiles of 10 primary HGSOC tumors and 10 related omental metastases using RNA sequencing and identified 100 differentially expressed genes. RESULTS: The differentially expressed genes were associated with decreased embryogenesis and vasculogenesis and increased cellular proliferation and organismal death. Top upstream regulators responsible for this gene signature were NR5A1, GATA4, FOXL2, TP53 and BMP7. A subset of these genes were highly expressed in the ovarian cancer among the cancer transcriptomes of The Cancer Genome Atlas. Importantly, the metastatic gene signature was suggestive of poor survival in TCGA data based on gene enrichment analysis. CONCLUSION: By comparing the gene expression profiles of primary HGSOC tumors and their matched metastasis, we provide evidence that a signature of 100 genes is able to separate these two sample types and potentially predict patient survival. Our study identifies functional categories of genes and transcription factors that could play important roles in promoting metastases and serve as markers for cancer prognosis.

High Plasma Lipid Levels Reduce Efficacy of Adenovirus-Mediated Gene Therapy.

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE-/- mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors.

Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.

Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics.

Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Improved therapeutic effect on malignant glioma with adenoviral suicide gene therapy combined with temozolomide.

Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.

Increased invasion of malignant gliomas after 15-LO-1 and HSV-tk/ganciclovir combination gene therapy.

Recent clinical trials for malignant glioma have drawn attention to the potential therapeutic efficacy of herpes simplex virus-thymidine kinase (HSV-tk) suicide gene therapy. Nevertheless, because of the nature of these tumors, it is believed that no single treatment alone is able to combat this fatal disease. Combination therapies may provide a solution to further improve therapies against malignant gliomas. We have recently demonstrated that 15-lipoxygenase-1 (15-LO-1) is able to inhibit tumor angiogenesis as well as enhance apoptosis in tumors. As a result, we studied the potential additive/synergistic effects of 15-LO-1 gene therapy when combined with HSV-tk gene therapy for the treatment of malignant gliomas. For that, BT4C malignant glioma cells were implanted into BDIX male rats. Fourteen days after tumor cell implantation, animals were transduced using adenoviral vectors either with HSV-tk alone or in combination with 15-LO-1. The results show that the combination gene therapy neither improved inhibition of tumor growth nor did it show any benefit on survival. Instead, a profound effect on the migratory properties of the tumor cells was found, resulting in decreased survival. Similar to conventional therapies, the combination of two therapeutic genes may result in unexpected side effects, not seen when given alone.

10-year safety follow-up in patients with local VEGF gene transfer to ischemic lower limb.

Vascular endothelial growth factor (VEGF)-mediated gene therapy (GT) has shown promising results as a novel method in the treatment of severe cardiovascular diseases. VEGF GT has proved to be safe and well tolerated in short-term studies, but there is only very limited data available on long-term effects. In this study we examined the effects of VEGF GT on patients having received VEGF-A gene transfer for the treatment of symptomatic (that is, claudication or critical lower limb ischemia) peripheral arterial occlusive disease. Out of 54 patients, 25 (46%) were interviewed for this study and 26 (48%) had died during the follow-up. Interviewed patients were treated with adenoviral (n=8, mean age 76 (62.7-90.6) years) or plasmid/liposome (n=8, mean age 84.2 (71.9-94.7) years) vectors compared with a randomized control group (n=10, mean age 80.5 (70.7-88.1) years) using a local balloon catheter device. The follow-up time was 10 years. Causes of death were clarified from hospital records. There were no statistically significant differences between the study groups in the causes of death or in the incidence of cancer (VEGF-Adv 0/10 vs VEGF-p/l 1/8 vs Control 1/7, P=0.5), diabetes (3/10 vs 3/8 vs 2/7, P=1.00) or diabetic retinopathy (0/10 vs 1/8 vs 0/7, P=0.45). In addition, we found no differences in the number of amputations of the treated leg (0/10 vs 3/8 vs 1/7, P=0.17). We conclude that transient VEGF-A-mediated GT did not increase the incidence of cancer, diabetes, retinopathy or any other diseases during the 10-year follow-up time. No significant differences were detected in the number of amputations or causes of death. This study supports our previous findings that local adenovirus and plasmid/liposome-mediated VEGF-A GT is safe and well-tolerated treatment in elderly patients with cardiovascular diseases.

Long-term VEGF-A expression promotes aberrant angiogenesis and fibrosis in skeletal muscle.

Vascular endothelial growth factor A (VEGF-A) induces strong angiogenesis and it has been widely used in proangiogenic gene therapy studies. However, little is known about long-term effects of VEGF-A expression in skeletal muscle. Here the long- term effects of adeno-associated virus (AAV) encoding human VEGF-A(165) (AAV-VEGF-A) gene transfer in normal and ischemic rabbit hindlimb skeletal muscles were studied. AAV-LacZ was used as a control. In one-year follow-up, a remarkable increase in skeletal muscle perfusion compared with AAV-LacZ was observed measured with Doppler and contrast pulse sequence ultrasound. Angiogenesis was also seen in histology as enlarged and sprouting capillaries. In addition to favorable angiogenic effects, aberrant vascular structures with CD31 positive cell layers were seen inside muscle fibers after AAV-VEGF-A gene transfer. Importantly, we found increased amounts of extracellular matrix with a high number of macrophages and fibrosis in AAV-VEGF-A transduced muscles. No changes in skeletal muscle morphology were detected in AAV-LacZ transduced muscles. Our results indicate that local AAV-VEGF-A gene transfer efficiently promotes long-term angiogenesis in large animal model. However, non-regulated expression of VEGF-A causes unfavorable changes in muscle morphology, which suggests the need for regulation of the transgene expression in long-term AAV-mediated VEGF-A gene transfer applications.

Cotargeting of VEGFR-1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice.

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.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Antegrade flow and peripheral resistance determine the level of endogenous arteriogenesis in patients with superficial femoral artery occlusion.

OBJECTIVES: Occlusion in a limb artery leads to impaired blood supply and ischaemia. Collateral artery growth (arteriogenesis) is one of the most effective natural response mechanisms to compensate this pathologic situation. However, it is unknown if clinically important features, like poor run-off, have an impact on compensatory vessel growth. METHODS: Study population of this retrospective study consisted of 70 patients who suffered from lower limb ischaemia and underwent bypass surgery because of an occlusion of the superficial femoral artery. Clinical data were collected and pre- and postoperative angiograms were reviewed. Number of collateral vessels bypassing the occluded segment was counted. Features of inflow and outflow vessels were recorded. RESULTS: The mean number of collaterals was 13 + or - 0.5 per patient. In univariate analysis, short daily walking distance, chronic critical leg ischaemia, low ankle brachial index, low number of patent calf arteries and stenosed inflow arteries predicted low number of collateral arteries. In the multivariate analysis, only the quality of inflow and the number of patent calf vessels demonstrated an independent association (P < 0.05) with the number of collaterals. CONCLUSIONS: Ankle-brachial index, grade of symptoms and daily walking capacity could be used to predict collateral density. Importantly, a good antegrade flow and peripheral runoff seem to have a significant effect on collateral density, implying an impact on the activation of arteriogenesis.

Expression of urokinase-type plasminogen activator receptor is increased during epileptogenesis in the rat hippocampus.

Urokinase-type plasminogen activator receptor (uPAR) is functionally a pleiotropic mediator involved in cell adhesion, proliferation, differentiation and migration as well as in matrix degradation, apoptosis, and angiogenesis in cancer tissue. Comparable cellular alterations occur in the brain during post-injury tissue repair. As the first step to assess the role of uPAR in brain tissue remodeling, we tested a hypothesis that uPAR expression is altered in the hippocampus during epilepsy-related circuitry reorganization. Epileptogenesis was triggered by inducing status epilepticus (SE) with electrical stimulation of the amygdala in rats. To monitor the development of SE and the occurrence of spontaneous seizures animals were continuously video-EEG monitored until sacrificed (1, 2, 4 or 14 days after SE). The hippocampal expression of uPAR was studied with real time qPCR and immunohistochemistry. Double-immunohistochemistry and confocal microscopy were used to investigate the expression of uPAR in astrocytes, microglia and neurons. We show that in the normal hippocampus the expression of uPAR was low and confined to small population of astrocytes and interneurons. In animals undergoing SE, uPAR expression increased dramatically, peaking at 1 and 4 days after SE. According to double-immunohistochemistry, uPAR was highly expressed in parvalbumin positive interneurons in the hippocampus and dentate gyrus, and in a subgroup of somatostatin and neuropeptide Y positive hilar interneurons. Increased uPAR expression during post-injury phase supports its contribution to tissue remodeling in the brain. Surviving hilar interneurons that are known to be denervated due to loss of afferent inputs in post-SE brain provide a target for future studies to investigate the contribution of uPAR in reinnervation of these cells, and to identify the signaling cascades that mediate the effects of uPAR.

(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.

Differences in retinal neovascular tissue and vitreous humour in patients with type 1 and type 2 diabetes.

AIMS: The aim of the study was to evaluate the histopathology of neovascular tufts and vitreous samples collected from patients with diabetes. METHODS: Vitreous samples and neovascular tufts were collected from patients with type 1 (n = 13) and (n = 17) type 2 diabetes with proliferative retinopathy, and from controls with a macular hole (n = 5). Neovessels were analysed using immunohistochemistry and vitreous samples with an enzyme-linked immunosorbent assay (ELISA). The main outcome measure was to examine differences in the levels of growth factors in patients with type 1 and type 2 diabetes with proliferative retinopathy. RESULTS: Vascular endothelial growth factor (VEGF)-A was most strongly present in the samples from patients with type 1 diabetes. In type 2 diabetes, VEGF-D was more abundantly present than in type 1 diabetes. Angiopoietin (ANG)-2 was also abundantly present. Macrophages and nuclear factor kappa B (NFkappaB) were found, indicating the presence of an inflammatory process in the neovascular tissues. CONCLUSIONS: VEGF-A and ANG-2 are equally important in the neovascular process in both type 1 and type 2 diabetes. VEGF-D is abundantly present in type 2 diabetes. In order to achieve better control of diabetic retinopathy, it might be beneficial to develop treatments that prevent the actions of ANG-2 and VEGF-D.

Eight-year safety follow-up of coronary artery disease patients after local intracoronary VEGF gene transfer.

Vascular endothelial growth factor (VEGF) has been shown to stimulate angiogenesis and myocardial perfusion. The short-term safety of VEGF gene therapy is excellent. However, there are only limited results regarding the long-term effects. The Kuopio Angiogenesis Trial (KAT) studied the efficiency and short-term safety of the local VEGF-A(165) gene transfer in 103 patients with coronary artery disease. Three patient groups received either VEGF as an adenoviral (n=37), or as a plasmid/liposome vector (n=28), or as a placebo (n=38), during coronary angioplasty and stenting (percutaneous coronary intervention, PCI)AQ1. The aim of this study was to examine the long-term effects and safety of VEGF gene therapy. Patients were interviewed by telephone or with a questionnaire on their current status of health, coronary and other cardiovascular events and symptoms, working ability, exercise tolerance, other diseases, such as cancer and diabetes, as well as their personal experience of the treatment. Causes of death were clarified from hospital records. The total follow-up time was 8.1 years (range 6.9-9.7 years). Overall 82% of the patients were reached across the study. Eight (7.5%) of the patients died during the follow-up, but there was no significant difference in mortality between the groups (3/32 vs 2/26 vs 3/31 VEGF-adenovirus vs VEGF-plasmid/liposome vs placebo, respectively; P=0.88). The incidence of major adverse cardiovascular events (MACEs) (10 vs 11 vs 15; P=0.85), cancer (1 vs 4 vs 2; P=0.38) or diabetes (2 vs 2 vs 2; P=0.97) did not differ between the groups. Local intracoronary VEGF gene transfer is safe and does not increase the risk of MACE, arrhythmias, cancer, diabetes or other diseases.

Extended release of adenovirus from silica implants in vitro and in vivo.

Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 degrees C and months at +4 degrees C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.