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1.  Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization 
Nucleic Acids Research  2014;42(20):12570-12584.
Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells.
PMCID: PMC4227755  PMID: 25352550
2.  Serum angiopoietin-2 and soluble VEGFR-2 levels predict malignancy of ovarian neoplasm and poor prognosis in epithelial ovarian cancer 
BMC Cancer  2014;14(1):696.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-696) contains supplementary material, which is available to authorized users.
PMCID: PMC4179851  PMID: 25245329
Angiopoietins; VEGFs; VEGFRs; Biomarker; Ovarian carcinoma; Prognosis
3.  Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity 
BioMed Research International  2014;2014:379340.
Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.
PMCID: PMC4016911  PMID: 24860818
4.  6-O- and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells 
Journal of Virology  2013;87(20):11148-11159.
Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.
PMCID: PMC3807288  PMID: 23926339
5.  Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells 
Molecular and Cellular Biology  2013;33(19):3749-3761.
One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.
PMCID: PMC3811871  PMID: 23878394
6.  Cell Susceptibility to Baculovirus Transduction and Echovirus Infection Is Modified by Protein Kinase C Phosphorylation and Vimentin Organization 
Journal of Virology  2013;87(17):9822-9835.
Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.
PMCID: PMC3754104  PMID: 23824807
7.  Epigenetic Upregulation of Endogenous VEGF-A Reduces Myocardial Infarct Size in Mice 
PLoS ONE  2014;9(2):e89979.
“Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.
PMCID: PMC3935957  PMID: 24587164
8.  Lipid accumulation, lipid oxidation, and low plasma levels of acquired antibodies against oxidized lipids associate with degeneration and rupture of the intracranial aneurysm wall 
Rupture of a saccular intracranial aneurysm (sIA) causes an often fatal subarachnoid hemorrhage (SAH). Why some sIAs rupture remains unknown. Since sIA walls bear some histological similarities with early atherosclerotic lesions, we hypothesized that accumulation and oxidation of lipids might occur in the sIA wall and might associate with sIA wall degeneration. Tissue samples from sIA fundi (n = 54) were studied with histochemistry and a panel of previously characterized antibodies for epitopes of oxidized LDL (OxLDL). Plasma samples from sIA carriers (n = 125) were studied with ELISA and EIA for IgG and IgM -antibodies against a panel of OxLDL epitopes.
Lipid accumulation, foam cells, and oxidized lipids were found both in unruptured and ruptured sIA walls. Lipid accumulation associated with wall degeneration (P < 0.001), as did the expression of adipophilin, a marker of lipid ingestion by cells. Lipid accumulation associated also with loss of mural cells (P < 0.001), as did the accumulation of OxLDL (P < 0.001). Plasma IgG antibody titers against OxLDL or malondialdehyde modified LDL were higher in patients with unruptured sIAs than in patients with aneurysmal SAH (P ≤ 0.001). A trend but not statistically significant differences were found in plasma IgM antibodies against oxidized lipids.
Accumulation of lipids and their oxidation in the sIA wall associates with the degeneration of the sIA wall. Acquired immunity against oxidized lipid epitopes may be protective of lipid associated sIA wall degeneration, but warrants further studies.
PMCID: PMC3893371  PMID: 24252658
Intracranial aneurysm; Intima; Oxidized LDL; IgG; Acquired immunity; Inflammation
9.  Promoting blood vessel growth in ischemic diseases: challenges in translating preclinical potential into clinical success 
Disease Models & Mechanisms  2013;6(2):312-322.
Angiogenic therapy, which involves the use of an exogenous stimulus to promote blood vessel growth, is an attractive approach for the treatment of ischemic diseases. It has been shown in animal models that the stimulation of blood vessel growth leads to the growth of the whole vascular tree, improvement of ischemic tissue perfusion and improved muscle aerobic energy metabolism. However, very few positive results have been gained from Phase 2 and 3 clinical angiogenesis trials. Many reasons have been given for the failures of clinical trials, including poor transgene expression (in gene-therapy trials) and instability of the vessels induced by therapy. In this Review, we discuss the selection of preclinical models as one of the main reasons why clinical translation has been unsuccessful thus far. This issue has received little attention, but could have had dramatic implications on the expectations of clinical trials. We highlight crucial differences between human patients and animal models with regards to blood flow and pressure, as well as issues concerning the chronic nature of ischemic diseases in humans. We use these as examples to demonstrate why the results from preclinical trials might have overestimated the efficacy of angiogenic therapies developed to date. We also suggest ways in which currently available animal models of ischemic disease could be improved to better mimic human disease conditions, and offer advice on how to work with existing models to avoid overestimating the efficacy of new angiogenic therapies.
PMCID: PMC3597014  PMID: 23471910
10.  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, T1ρ and T2ρ, 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 T1ρ and T2ρ processes. In the present work, the time constant characterizing water relaxation with RAFF (TRAFF) 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. TRAFF was the only parameter exhibiting significant association with cell density in three different tumor regions (border, intermediate, and core tissues). These results indicate that TRAFF may provide a marker to identify tumors responding to treatment.
PMCID: PMC3189442  PMID: 21721037
11.  LDLR–/–ApoB100/100 mice with insulin-like growth factor II overexpression reveal a novel form of retinopathy with photoreceptor atrophy and altered morphology of the retina 
Molecular Vision  2013;19:1723-1733.
The aim of this study was to characterize the ocular morphology of low-density lipoprotein receptor–deficient apolipoprotein B-100-only mice, where overexpression of insulin-like growth factor II (IGF-II) has been shown to induce glucose intolerance and increase atherosclerotic lesion progression and calcification.
Fifteen-month-old mice were examined on a normal chow diet and after 3 months of a high-fat Western diet. IGF-II-negative LDLR–/–ApoB100/100 littermates and C57Bl/6J mice served as controls. In vivo color images of the fundi were obtained, and eyes were processed either for retinal flat mounts for assessment of neovascularization or for paraffin-embedded samples for immunohistochemical analyses.
IGF-II overexpression and the resulting prediabetic phenotype did not induce microvascular damage when assessed in fundus photographs and retinal whole mounts, and the number of capillaries in IGF-II/LDLR–/–ApoB100/100 mice was not significantly different from LDLR–/–ApoB100/100 mice. However, morphology of the inner nuclear, outer plexiform, and outer nuclear layers was altered in the IGF-II/LDLR–/–ApoB100/100 mice. Moreover, photoreceptor atrophy and thinning of the outer nuclear layer were present. Caspase-3 staining was positive in the photoreceptor inner segment. In addition, retinas of the IGF-II/LDLR–/–ApoB100/100 mice displayed reduced rhodopsin positivity, consistent with the decreased number of photoreceptor cells.
This study reports a novel form of retinopathy with photoreceptor atrophy and abundant changes in retinal morphology in a mouse model of prediabetes and atherosclerosis.
PMCID: PMC3733910  PMID: 23922490
12.  rDNA-directed integration by an HIV-1 integrase—I-PpoI fusion protein 
Nucleic Acids Research  2012;41(5):e61.
Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.
PMCID: PMC3597653  PMID: 23275537
13.  Intracardiac injection of a capsid-modified Ad5/35 results in decreased heart toxicity when compared to standard Ad5 
Virology Journal  2012;9:296.
Clinical gene therapy trials for cardiovascular diseases have demonstrated the crucial role of efficient gene delivery and transfection technologies in achieving clinically relevant results. We hypothesized that the use of tropism-modified adenoviruses would improve transduction efficacy and to this end we analyzed the transduction efficiency and toxicity of standard Ad5 and tropism-modified Ad5/35 in combination with ultrasound-guided intramyocardial gene delivery.
Ultrasound-guided intracardiac injections were used to deliver 1 × 1010 pfu/ml Ad5-lacZ and Ad5/35-lacZ vectors into mouse left ventricle wall. Since Ad5/35 uses human CD46 as its primary receptor, we used transgenic hCD46Ge mice expressing human CD46 at levels comparable to man. Mice were sacrificed 6 or 14 days post-injection and immunohistochemistry and X-gal staining were used to detect transgene and viral receptor expression. Virus-induced cardiac toxicity was evaluated by a pathologist.
The intramyocardial injection was well tolerated and both Ad5-lacZ and Ad5/35-lacZ were able to give robust transgene expression after a single injection. Interestingly, while Ad5-lacZ was able to generate greater transgene expression than Ad5/35-lacZ, it also evoked more severe tissue damage with large areas of interstitial inflammatory cell infiltration and myocyte necrosis.
Ultrasound-guided intramyocardial injection is an effective and safe way to deliver vectors to the heart. The observed severe tissue damage of Ad5-lacZ greatly undermines the efficient transgene expression and suggests that Ad5/35 capsid modification can result in safer adenoviral vectors for cardiovascular gene therapy, although at the cost of some vector transduction efficacy.
PMCID: PMC3546865  PMID: 23190872
Adenovirus; Intracardiac injection; CAR; CD46; Targeting
14.  Dotted collar placed around carotid artery induces asymmetric neointimal lesion formation in rabbits without intravascular manipulations 
Neointimal formation in atherosclerosis has been subject for intense research. However, good animal models mimicking asymmetrical lesion formation in human subjects have been difficult to establish. The aim of this study was to develop a model which would lead to the formation of eccentric lesions under macroscopically intact non-denuded endothelium.
We have developed a new collar model where we placed two cushions or dots inside the collar. Arterial lesions were characterized using histology and ultrasound methods.
When this dotted collar was placed around carotid and femoral arteries it produced asymmetrical pressure on adventitia and a mild flow disturbance, and hence a change in shear stress. Our hypothesis was that this simple procedure would reproducibly produce asymmetrical lesions without any intraluminal manipulations. Intima/media ratio increased towards the distal end of the collar with the direction of blood flow under macroscopically intact endothelium. Macrophages preferentially accumulated in areas of the thickest neointima thus resembling early steps in human atherosclerotic plaque formation. Proliferating cells in these lesions and underlying media were scarce at eight weeks time point.
The improved dotted collar model produces asymmetrical human-like atherosclerotic lesions in rabbits. This model should be useful in studies regarding the pathogenesis and formation of eccentric atherosclerotic lesions.
PMCID: PMC3485168  PMID: 23075120
Atherosclerotic lesion; Carotid collar; Rabbit; Shear stress
16.  Specific Inhibition of SRC Kinase Impairs Malignant Glioma Growth In Vitro and In Vivo 
Malignant glioma is a severe cancer with a poor prognosis. Local occurrence and rare metastases of malignant glioma make it a suitable target for gene therapy. Several studies have demonstrated the importance of Src kinase in different cancers. However, these studies have focused mainly on Src-deficient mice or pharmacological inhibitors of Src. In this study we have used Src small hairpin RNAs (shRNAs) in a lentiviral backbone to mimic a long-term stable treatment and determined the role of Src in tumor tissues. Efficacy of Src shRNAs was confirmed in vitro demonstrating up to 90% target gene inhibition. In a mouse malignant glioma model, Src shRNA tumors were almost 50-fold smaller in comparison to control tumors and had significantly reduced vascularity. In a syngenic rat intracranial glioma model, Src shRNA-transduced tumors were smaller and these rats had a survival benefit over the control rats. In vivo treatment was enhanced by chemotherapy and histone deacetylase inhibition. Our results emphasise the importance of Src in tumorigenesis and demonstrate that it can be efficiently inhibited in vitro and in vivo in two independent malignant glioma models. In conclusion, Src is a potential target for RNA interference-mediated treatment of malignant glioma.
PMCID: PMC3390242  PMID: 23344000
glioma; lentivirus; MRI; RNA interference; Src
17.  Low-grade inflammation and the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy 
Heart  2012;98(13):1007-1013.
To investigate the role of inflammation in the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy (HCM).
Clinical study.
Kuopio University Hospital and University of Eastern Finland, Kuopio, Finland.
Twenty-four patients with a single HCM-causing mutation D175N in the α-tropomyosin gene and 17 control subjects.
Main outcome measures
Endomyocardial biopsy samples taken from the patients with HCM were compared with matched myocardial autopsy specimens. Levels of high-sensitivity C-reactive protein (hsCRP) and proinflammatory cytokines were measured in patients and controls. Myocardial late gadolinium enhancement (LGE) in cardiac MRI (CMRI) was detected.
Endomyocardial samples in patients with HCM showed variable myocyte hypertrophy and size heterogeneity, myofibre disarray, fibrosis, inflammatory cell infiltration and nuclear factor kappa B (NF-κB) activation. Levels of hsCRP and interleukins (IL-1β, IL-1RA, IL-6, IL-10) were significantly higher in patients with HCM than in control subjects. In patients with HCM, there was a significant association between the degree of myocardial inflammatory cell infiltration, fibrosis in histopathological samples and myocardial LGE in CMRI. Levels of hsCRP were significantly associated with histopathological myocardial fibrosis. hsCRP, tumour necrosis factor α and IL-1RA levels had significant correlations with LGE in CMRI.
A variable myocardial and systemic inflammatory response was demonstrated in patients with HCM attributable to an identified sarcometric mutation. Inflammatory response was associated with myocardial fibrosis, suggesting that myocardial fibrosis in HCM is an active process modified by an inflammatory response.
PMCID: PMC3368494  PMID: 22447464
Hypertrophic cardiomyopathy; inflammation; fibrosis; genetics; late gadolinium enhancement; coronary angioplasty; aortic stenosis; invasive cardiology; coronary artery disease; cardiomyopathy hypertrophic; tissue characters; HCM; MRI; myocardial function; myocardial perfusion; myocardial ischaemia; myocardial infarction; arrhythmias; endocrinology
18.  Effects of Angiopoietin-2-Blocking Antibody on Endothelial Cell–Cell Junctions and Lung Metastasis 
Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized.
We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided.
Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm2, difference = 32.67 mm2, 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell–cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell–cell junctions in endothelial–tumor cell cocultures.
Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions.
PMCID: PMC3309130  PMID: 22343031
19.  VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling 
Nature Cell Biology  2011;13(10):1202-1213.
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−; Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
PMCID: PMC3261765  PMID: 21909098
20.  Uptake of 68gallium in atherosclerotic plaques in LDLR-/-ApoB100/100 mice 
EJNMMI Research  2011;1:14.
Atherosclerosis is a chronic inflammatory disease of artery wall characterized by infiltration of monocytes into subendothelial space and their differentiation into macrophages. Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68gallium (68Ga) in atherosclerotic plaques in mice.
Uptake of ionic 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner.
Our results revealed that the uptake of 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall (ratio 1.8 ± 0.2, p = 0.0002) and adventitia (ratio 1.3 ± 0.2, p = 0.0011). The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood.
We observed a moderate but significantly elevated 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.
PMCID: PMC3251160  PMID: 22214258
atherosclerosis; plaque; autoradiography; gallium-68; PET
21.  Left ventricular dysfunction with reduced functional cardiac reserve in diabetic and non-diabetic LDL-receptor deficient apolipoprotein B100-only mice 
Lack of suitable mouse models has hindered the studying of diabetic macrovascular complications. We examined the effects of type 2 diabetes on coronary artery disease and cardiac function in hypercholesterolemic low-density lipoprotein receptor-deficient apolipoprotein B100-only mice (LDLR-/-ApoB100/100).
Methods and results
18-month-old LDLR-/-ApoB100/100 (n = 12), diabetic LDLR-/-ApoB100/100 mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells (IGF-II/LDLR-/-ApoB100/100, n = 14) and age-matched C57Bl/6 mice (n = 15) were studied after three months of high-fat Western diet. Compared to LDLR-/-ApoB100/100 mice, diabetic IGF-II/LDLR-/-ApoB100/100 mice demonstrated more calcified atherosclerotic lesions in aorta. However, compensatory vascular enlargement was similar in both diabetic and non-diabetic mice with equal atherosclerosis (cross-sectional lesion area ~60%) and consequently the lumen area was preserved. In coronary arteries, both hypercholesterolemic models showed significant stenosis (~80%) despite positive remodeling. Echocardiography revealed severe left ventricular systolic dysfunction and anteroapical akinesia in both LDLR-/-ApoB100/100 and IGF-II/LDLR-/-ApoB100/100 mice. Myocardial scarring was not detected, cardiac reserve after dobutamine challenge was preserved and ultrasructural changes revealed ischemic yet viable myocardium, which together with coronary artery stenosis and slightly impaired myocardial perfusion suggest myocardial hibernation resulting from chronic hypoperfusion.
LDLR-/-ApoB100/100 mice develop significant coronary atherosclerosis, severe left ventricular dysfunction with preserved but diminished cardiac reserve and signs of chronic myocardial hibernation. However, the cardiac outcome is not worsened by type 2 diabetes, despite more advanced aortic atherosclerosis in diabetic animals.
PMCID: PMC3141395  PMID: 21718508
22.  TAFFEL: Independent Enrichment Analysis of gene sets 
BMC Bioinformatics  2011;12:171.
A major challenge in genomic research is identifying significant biological processes and generating new hypotheses from large gene sets. Gene sets often consist of multiple separate biological pathways, controlled by distinct regulatory mechanisms. Many of these pathways and the associated regulatory mechanisms might be obscured by a large number of other significant processes and thus not identified as significant by standard gene set enrichment analysis tools.
We present a novel method called Independent Enrichment Analysis (IEA) and software TAFFEL that eases the task by clustering genes to subgroups using Gene Ontology categories and transcription regulators. IEA indicates transcriptional regulators putatively controlling biological functions in studied condition.
We demonstrate that the developed method and TAFFEL tool give new insight to the analysis of differentially expressed genes and can generate novel hypotheses. Our comparison to other popular methods showed that the IEA method implemented in TAFFEL can find important biological phenomena, which are not reported by other methods.
PMCID: PMC3120704  PMID: 21592412
23.  Gamma-Secretase Inhibitor Treatment Promotes VEGF-A-Driven Blood Vessel Growth and Vascular Leakage but Disrupts Neovascular Perfusion 
PLoS ONE  2011;6(4):e18709.
The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimer's disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.
PMCID: PMC3077402  PMID: 21533193
24.  Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle 
Circulation research  2009;104(11):1302-1312.
The therapeutic potential of vascular endothelial growth factor C (VEGF-C) and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly employed adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the four-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.
PMCID: PMC2776655  PMID: 19443835
VEGF-C; VEGF-D; adeno-associated virus; angiogenesis; lymphangiogenesis; skeletal muscle
25.  VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts 
The EMBO Journal  2010;29(8):1377-1388.
The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.
PMCID: PMC2868571  PMID: 20224550
angiogenic sprouting; embryoid body; heterodimer; proximity ligation; VEGF receptor

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