Rationale:The neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone.Objective: Catestatin shares several functions with angiogenic factors. We therefore reasoned that catestatin induces growth of new blood vessels. Methods and Results:Catestatin induced migration, proliferation, and antiapoptosis in endothelial cells and exerted capillary tube formation in vitro in a Matrigel assay, and such effects were mediated via G protein, mitogen-activated protein kinase, and Akt. Catestatin-induced endothelial cell functions are further mediated by basic fibroblast growth factor, as shown by blockade of effects by a neutralizing fibroblast growth factor antibody. Furthermore, catestatin released basic fibroblast growth factor from endothelial cells and stimulated fibroblast growth factor signaling. In addition to its function on endothelial cells, catestatin also exerted effects on endothelial progenitor cells and vascular smooth muscle cells. In vivo, catestatin induced angiogenesis in the mouse cornea neovascularization assay and increased blood perfusion and number of capillaries in the hindlimb ischemia model. In addition to angiogenesis, catestatin increased density of arterioles/arteries and incorporation of endothelial progenitor cells in the hindlimb ischemia model, indicating induction of arteriogenesis and postnatal vasculogenesis. Conclusion:We conclude that catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism. (Circ Res. 2010;107:1326-1335.) Key Words: angiogenesis Ⅲ blood vessels Ⅲ endothelium Ⅲ endothelial progenitor cells C hromogranin (Cg)A, the index member of the chromogranin/secretogranin protein family, is the major soluble protein of catecholamine storage vesicles of sympathetic nerve terminals and the adrenal medulla. 1,2 CgA is a proprotein giving rise to biological active peptides like the dysglycemic hormone pancreastatin, 3 the vasodilator vasostatin, 4 and catestatin 5 (CST) ), which inhibits catecholamine release by acting as a nicotinic cholinergic antagonist, resulting in a negative-feedback mechanism. Although plasma CgA is high in human essential (hereditary) hypertension, the plasma concentration of CST is lower in both established cases and in normotensive subjects with a family history of the disease, 6 suggesting a mechanism whereby diminished CST might increase the risk for later development of hypertension. Consistent with the human findings, high blood pressure has been reported in mice after targeted ablation of the Chga gene (Chga knockout), and such high blood pressure can be "rescued" either by replacement with CST or introduction of the human ortholog in the Chga knockout background. 7 Angiogenesis, the growth of new vessels from the preexisting vasculature, is an important process in many physiological conditions including embryonic development and wound healing. However, defects in the regula- Original received February 25, 2010; revision received September 27, 2010;...

Background Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is up-regulated by hypoxia and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. Methods and Results In-vivo secretoneurin improved left ventricular function, inhibited remodeling and reduced scar formation. In the infarct border zone secretoneurin induced coronary angiogenesis as shown by increased density of capillaries and arteries. In-vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis and activated Akt and ERK in coronary endothelial cells. Effects were abrogated by a VEGF-antibody and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells and binding was blocked by heparinase indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its co-receptor neuropilin 1. In endothelial cells secretoneurin also stimulated FGF receptor-3 and IGF-1 receptor and in coronary vascular smooth muscle cells we observed stimulation of VEGF receptor-1 and FGF receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin m-RNA and protein. Conclusions Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low affinity binding sites and neuropilin 1 and stimulates further growth factor receptors like FGF receptor-3. Our in-vivo findings indicate that secretoneurin might be a promising therapeutic tool in ischemic heart disease.

No abstract

Expression of angiogenic cytokines like vascular endothelial growth factor is enhanced by hypoxia. We tested the hypothesis that decreased oxygen levels up-regulate the angiogenic factor secretoneurin. In vivo, muscle cells of mouse ischemic hind limbs showed increased secretoneurin expression, and inhibition of secretoneurin by a neutralizing antibody impaired the angiogenic response in this ischemia model. In a mouse soft tissue model of hypoxia, secretoneurin was increased in subcutaneous muscle fibers. In vitro, secretoneurin mRNA and protein were up-regulated in L6 myoblast cells after exposure to low oxygen levels. The hypoxia-dependent regulation of secretoneurin was tissue specific and was not observed in endothelial cells, vascular smooth muscle cells, or AtT20 pituitary tumor cells. The hypoxia-dependent induction of secretoneurin in L6 myoblasts is regulated by hypoxia-inducible factor-1alpha, since inhibition of this factor using si-RNA inhibited up-regulation of secretoneurin. Induction of secretoneurin by hypoxia was dependent on basic fibroblast growth factor in vivo and in vitro, and inhibition of this regulation by heparinase suggests an involvement of low-affinity basic fibroblast growth factor binding sites. In summary, our data show that the angiogenic cytokine secretoneurin is up-regulated by hypoxia in muscle cells by hypoxia-inducible factor-1alpha- and basic fibroblast growth factor-dependent mechanisms.

Key Words: angiogenesis Ⅲ endothelium Ⅲ gene therapy Ⅲ hypoxia Ⅲ peripheral vascular disease C ardiovascular diseases represent the leading cause of death worldwide. 1 In the case of peripheral arterial disease, a substantial number of patients who develop critical limb ischemia are not eligible for surgical or interventional revascularization and, despite optimal medical therapy, have reduced quality of life and life expectancy. 2,3 This group of patients would require new therapeutic methods to increase collateral blood flow to areas of decreased tissue perfusion distal to arterial occlusion.Angiogenesis, the growth of new blood vessels from the preexisting vasculature, plays an important role in wound healing, tumor growth, diabetic retinopathy, tissue ischemia, and inflammatory diseases. 4 Cytokines mediating this process include vascular endothelial growth factor (VEGF) 5 and basic fibroblast growth factor (b-FGF). 6 For stability of newly formed vessels, recruitment of pericytes and smooth muscle cells (SMCs) is important; such recruitment is known as arteriogenesis, which is mediated by cytokines such as monocyte chemoattractant protein-1 and platelet-derived growth factor (PDGF)-BB. 7,8 Postnatal vasculogenesis, which is the mobilization and incorporation of bone marrow-derived endothelial progenitor cells (EPCs), also plays an important role in the growth of new vessels. 9 Recently, application of endothelial cytokines as proteins or genes has been of scientific and clinical interest to treat limb or myocardial ischemia, a procedure called therapeutic angiogenesis. 10 -14 We recently demonstrated that the neuropeptide secretoneurin (SN) induces angiogenesis and postnatal vasculogen- MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Construction of the Human SN Expression PlasmidSynthetic oligonucleotides encoding SN and a signal peptide were cloned into the expression vector pAAV-MCS (Stratagene). The plasmid vector is described in the Online Data Supplement. Animals, Mouse Hindlimb Ischemia Model, SN Gene Therapy, and Bone Marrow Transplantation ModelMice at ages between 12 and 18 months were subjected to unilateral hindlimb surgery and injected with plasmid DNA expressing SN or green fluorescent protein (GFP) into thigh and calf muscles immediately after surgery.Bone marrow transplantation was performed as described. 19 After hindlimb ischemia, injection of SN plasmid or saline was performed. Blood Flow Measurement and In Vivo Assessment of Limb Function and Ischemic DamageBlood flow measurements were performed using a laser-Doppler perfusion imaging (LDPI) analyzer. Blood perfusion is expressed as LDPI index representing the ratio of left (ie, operated, ischemic leg) versus right (ie, unoperated, not-ischemic leg) limb blood flow. Movement score and analysis of necrosis are described in the Online Data Supplement. Immunofluorescence and Fluorescence-Activated Cell Sorting AnalysisECs were stained for CD31, and arteries/arteriole...

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B), a member of the LDL receptor family, is frequently inactivated in multiple malignancies including lung cancer. LRP1B is therefore considered as a putative tumor suppressor. Due to its large size (4599 amino acids), until now only minireceptors or receptor fragments have been successfully cloned. To assess the effect of LRP1B on the proliferation of non-small cell lung cancer cells, we constructed and expressed a transfection vector containing the 13.800 bp full-length murine Lrp1b cDNA using a PCR-based cloning strategy. Expression of LRP1B was analyzed by quantitative RT-PCR (qRT-PCR) using primers specific for human LRP1B or mouse Lrp1b. Effective expression of the full length receptor was demonstrated by the appearance of a single 600 kDa band on Western Blots of HEK 293 cells. Overexpression of Lrp1b in non-small cell lung cancer cells with low or absent endogenous LRP1B expression significantly reduced cellular proliferation compared to empty vector-transfected control cells. Conversely, in Calu-1 cells, which express higher endogenous levels of the receptor, siRNA-mediated LRP1B knockdown significantly enhanced cellular proliferation. Taken together, these findings demonstrate that, consistent with the postulated tumor suppressor function, overexpression of full-length Lrp1b leads to impaired cellular proliferation, while LRP1B knockdown has the opposite effect. The recombinant Lrp1b construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis.

ObjectiveTo investigate low-density lipoprotein receptor-related protein 1b (LRP1b) expression in human tissues and to identify circulating ligands of LRP1b.Methods and resultsUsing two independent RT-PCR assays, LRP1b mRNA was detected in human brain, thyroid gland, skeletal muscle, and to a lesser amount in testis but absent in other tissues, including heart, kidney, liver, lung, and placenta. Circulating ligands were purified from human plasma by affinity chromatography using FLAG-tagged recombinant LRP1b ectodomains and identified by mass spectrometry. Using this technique, several potential ligands (fibrinogen, clusterin, vitronectin, histidine rich glycoprotein, serum amyloid P-component, and immunoglobulins) were identified. Direct binding of LRP1b ectodomains to fibrinogen was verified by co-immunoprecipitation. ApoE – carrying lipoproteins were shown to bind to LRP1b ectodomains in a lipoprotein binding assay. Furthermore, binding as well as internalization of very low density lipoproteins by cells expressing an LRP1b minireceptor was demonstrated.DiscussionLRP1b expression in humans appears to be confined to few tissues, which could point out to specialized functions of LRP1b in certain organs. Most of the newly identified LRP1b ligands are well-known factors in blood coagulation and lipoprotein metabolism, suggesting a possible role of LRP1b in atherosclerosis.

Adult primary focal segmental glomerulosclerosis (FSGS) remains a therapeutic challenge for the treating physician. With the advent of novel immunosuppressive measures, our arsenal of therapeutic options increased considerably. The aim of this review was to summarize reports published over the last two decades which reported on treatment outcome. Most reports included patients with a steroid-resistant (SR) disease course, yet the cohort with the highest unmet need, since persistent nephrotic range proteinuria is associated with a poor renal prognosis and portends a high risk of developing end-stage renal disease. While in first-line treatment, steroid treatment remains the recommended standard with an overall remission rate of 50% and higher, optimal treatment strategies for steroid-dependent/multirelapsing (SD/MR) and SR patients have to be defined. In both entities, calcineurin inhibitors showed good efficacy, while mycophenolate mofetil was less effective in SR cases compared to those with SD/MR. The same was true for rituximab, a monoclonal antibody targeting B-cells. In resistant cases, addition of extracorporeal treatment options or treatment with alkylating agents may be considered. To shape the future for treatment of FSGS, international collaborations to conduct larger clinical trials are needed to identify potential novel efficacious immunosuppressive or immunomodulatory therapies.