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Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL 2 – 4 . Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr −/− background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr −/− mice, Ldlr −/− E06-scFv mice had 57–28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis. A single-chain variable fragment of the antibody E06, which binds to the phosphocholine headgroup of oxidized phospholipids, blocks the uptake of oxidized low-density lipoprotein by macrophages, and reduces inflammation and atherosclerosis in hypercholesterolaemic mice.

Lipids on the move VEGF-B, a vascular endothelial growth factor that is highly expressed in heart, skeletal muscle and brown adipose tissue, has been found to have an unexpected role in targeting lipids to peripheral tissues. VEGFs are familiar as major angiogenic regulators, but the detailed role of VEGF-B in blood vessel function had been unclear. Mice lacking VEGF-B accumulate lower amounts of lipids in muscle, heart and brown adipose tissue, and instead shunt them to white adipose tissue. The involvement of VEGF-B in redistributing lipids suggest possible novel strategies for modulating lipid accumulation in diabetes, obesity and cardiovascular diseases. VEGF–B is shown to have an unexpected role in targeting lipids to peripheral tissues. VEGF–B controls endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. Bioinformatic analyses suggest that the uptake of these lipids is tightly coupled with lipid use by mitochondria. Mice that do not have VEGF–B accumulate less lipids in muscle, heart and brown adipose tissue, and instead shunt them to white adipose tissue. The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology 1 . However, the detailed role of VEGF-B in blood vessel function has remained unclear 2 , 3 . Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood 4 . Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb -/- mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Anti-angiogenesis: VEGFR-3 shows its paces The vascular endothelial growth factor (VEGF) receptor subtype VEGFR-3 is expressed only on lymphatic endothelium in adults. A new study, however, finds that VEGFR-3 is upregulated in the vasculature of tumours by Notch signalling. VEGFR-3 inhibition interferes with tumour angiogenesis and tumour growth, in particular in combination with inhibitors of VEGFR-2, suggesting that it represents a novel anti-angiogenic target for cancer therapy. The vascular endothelial growth factor (VEGF) receptor subtype VEGFR-3 is only expressed on lymphatic endothelium in adults. However, VEGFR-3 is upregulated in the vasculature of tumours by Notch signalling. VEGFR-3 inhibition interferes with tumour angiogenesis and tumour growth, in particular in combination with inhibitors of the VEGFR-2, suggesting that it represents a novel anti-angiogenic target for cancer therapy. Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration 1 . Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells 2 . VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium 3 . However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds 4 , 5 . Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.

Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed. As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic p.H1047R variant and fibroblasts. Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

Genetic variation in the 9p21.3 chromosomal region has one of the strongest associations known for coronary artery disease (CAD) that often leads to myocardial infarction (MI). This risk locus encodes a long noncoding RNA, ANRIL, which has been suggested to regulate the neighboring cyclin‐dependent kinase inhibitors 2A and B (Cdkn2A/B), the key regulators of cell proliferation. In this study, we aimed to clarify the role of the 9p21.3 risk locus in acute and chronic myocardial ischemia in mice. Mice carrying a deletion equivalent to the human CAD risk interval (Chr4Δ70kb/Δ70kb) and wild type mice were exposed to MI and followed until 5 days or 4 weeks. In the wild type mice, expression of a lncRNA, Ak148321, was increased after MI, and Cdkn2a was upregulated in chronic ischemia. Chr4Δ70kb/Δ70kb downregulated both Cdkn2a/b, but this did not affect the survival or cardiac pathology after MI. These results suggest that the 9p21.3 locus is activated in response to cardiac ischemia. However, deficiency in the risk locus does not play a role in the cardiac pathophysiology in mice, supporting the studies suggesting the risk locus being more involved in the development of CAD, rather than the subsequent MI.

Coronary heart disease (CHD) is the leading cause of death around the world. Based on the roles of vascular endothelial growth factor (VEGF) family members to regulate blood and lymphatic vessels and metabolic functions, several therapeutic approaches have been attempted during the last decade. However proangiogenic therapies based on classical VEGF-A have been disappointing. Therefore, it has become important to focus on other VEGFs such as VEGF-B, which is a novel member of the VEGF family. Recent studies have shown the very promising potential of the VEGF-B to treat CHD and heart failure. The aim of this review article is to present the role of VEGF-B in endothelial biology and as a potential therapeutic agent for CHD and heart failure. In addition, key differences between the VEGF-A and VEGF-B effects on endothelial functions are demonstrated.

Examine features of blood and lymphatic vessels in ovarian tumors and their significance to prognosis of ovarian cancer. A total of 139 women with epithelial ovarian tumors were included: 86 malignant, 17 borderline and 36 benign. Density, percentage, mean size and number of blood microvessels in tumors were measured by immunohistochemistry with antibodies against CD34 and CD105. Lymphatic vessel density was assayed using the D2-40 antibody against podoplanin. Angiogenesis was most profuse in malignant tumors. Small size of lymph vessels predicted 26% shorter 5-year survival of ovarian cancer patients. Further, high percentage of lymphatic vessels in tumors was associated with lymph node metastasis, and high density with cancer recurrence. Lower number of microvessels, as assessed by CD34 staining, predicted shorter progression-free survival. Additionally, the large size of microvessels assessed by CD34 and the high number of vessels assessed by CD105 were related to residual tumor >1 cm at primary surgery and also, large vessel size was associated with stage III, as assessed by CD105 staining. CD34 and CD105 define different characteristics of microvessels. Parameters of lymph vessels may predict the prognosis of ovarian cancer.

Diabetes mellitus is a lifelong, incapacitating metabolic disease associated with chronic macrovascular complications (coronary heart disease, stroke, and peripheral vascular disease) and microvascular disorders leading to damage of the kidneys (nephropathy) and eyes (retinopathy). Based on the current trends, the rising prevalence of diabetes worldwide will lead to increased cardiovascular morbidity and mortality. Therefore, novel means to prevent and treat these complications are needed. Under the auspices of the IMI (Innovative Medicines Initiative), the SUMMIT (SUrrogate markers for Micro- and Macrovascular hard end points for Innovative diabetes Tools) consortium is working on the development of novel animal models that better replicate vascular complications of diabetes and on the characterization of the available models. In the past years, with the high level of genomic information available and more advanced molecular tools, a very large number of models has been created. Selecting the right model for a specific study is not a trivial task and will have an impact on the study results and their interpretation. This review gathers information on the available experimental animal models of diabetic macrovascular complications and evaluates their pros and cons for research purposes as well as for drug development.