Wilfried Schgoer

Monocyte migration : A novel effect and signaling pathways of catestatin

Several members of the neuropeptide family exert chemotactic actions on blood monocytes consistent with neurogenic inflammation. Furthermore, chromogranin A (CgA) containing Alzheimer plaques are characterized by extensive microglia activation and such activation induces neuronal damage. We therefore hypothesized that the catecholamine release inhibitory peptide catestatin (hCgA(352-372)) would induce directed monocyte migration. We demonstrate that catestatin dose-dependently stimulates chemotaxis of human peripheral blood monocytes, exhibiting its maximal effect at a concentration of 1 nM comparable to the established chemoattractant formylated peptide Met-Leu-Phe (fMLP). The naturally occurring catestatin variants differed in their chemotactic property insofar as that the Pro370Leu variant was even more potent than wild type, whereas the Gly364Ser variant was less effective. Specificity of this effect was shown by inhibition of catestatin-induced chemotaxis by a specific neutralizing antibody. In addition, catestatin mediated effect was blocked by dimethylsphingosine and treatment with endothelial differentiation gene (Edg)-1 and Edg-3 antisense RNA as well as by incubation with pertussis toxin and genistein indicating involvement of tyrosine kinase receptor-, G-protein- and sphingosine-1-phosphate signaling. Catestatin also stimulated Akt- and extracellular signal related kinase (ERK)-phosphorylation and catestatin-induced chemotaxis was blocked by blockers of phosphoinositide-3 (PI-3) kinase and nitric oxide as well as by inhibition of the mitogen-activated protein kinases (MAPK) system indicating involvement of these signal transduction pathways. In summary, our data indicate that catestatin induces monocyte chemotaxis by activation of a variety of signal transduction pathways suggesting a role of this peptide as an inflammatory cytokine.

The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism.

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.

Hypoxia up-regulates the angiogenic cytokine secretoneurin via an HIF-1α- and basic FGF-dependent pathway in muscle cells

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 sec‐retoneurin. 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‐depen‐dent 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‐la, 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‐1α‐ and basic fibroblast growth factor‐dependent mechanisms.—Egger, M., Schgoer, W., Beer, A. G. E., Jeschke, J., Leierer, J., Theurl, M., Frauscher, S., Tepper, O. M., Niederwanger, A., Ritsch, A., Kearney, M., Wanschitz, J., Gurtner, G. C., Fischer‐Colbrie, R., Weiss, G., Piza‐Katzer, H., Losordo, D. W., Patsch, J. R., Schratzberger, P., Kirchmair, R. Hypoxia up‐regulates the angiogenic cytokine secretoneurin via an HIF‐1α‐ and basic FGF‐dependent pathway in muscle cells. FASEB J. 21, 2906–2917 (2007)

The Angiogenic Factor Secretoneurin Induces Coronary Angiogenesis in a Model of Myocardial Infarction by Stimulation of Vascular Endothelial Growth Factor Signaling in Endothelial Cells

Background—Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated 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 extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (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 coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger 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 fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.

Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide–Dependent Mechanism

Rationale: The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells. Objective: We sought to investigate the effects of secretoneurin on therapeutic angiogenesis. Methods and Results: We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells. Conclusions: In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide–dependent mechanism.

The Liver-Selective Thyromimetic T-0681 Influences Reverse Cholesterol Transport and Atherosclerosis Development in Mice

Background Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice. Methodology/Principal Findings T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls. Conclusions/Significance The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans.

Hepatic lipase polymorphism and increased risk of peripheral arterial disease

Background.  Hepatic lipase plays a key role in the metabolism of pro‐atherogenic and anti‐atherogenic lipoproteins affecting their plasma level as well as their physico‐chemical properties. We hypothesized single nucleotide polymorphisms in the promoter region of the hepatic lipase gene to be associated with an increased risk for peripheral arterial disease (PAD).

Aspirin regulates expression and function of scavenger receptor-BI in macrophages: studies in primary human macrophages and in mice

Scavenger receptor class B type I (SR‐BI) has been shown to be expressed in human atherosclerotic plaque macrophages, where it is believed to reduce atherosclerosis by promoting cholesterol efflux. In this study we investigated the influence of aspirin and other NSAIDs on SR‐BI expression and function in cultured human macrophages as well as in different mouse strains. Incubation of human macrophages with 0.5 mmol/l aspirin resulted in increased SR‐BI protein expression and increased uptake of HDL‐associated [3H]cholesteryl oleate without changes of SR‐BI mRNA levels. In contrast, using 5 mmol/l of aspirin, SR‐BI expression and function were significantly decreased. Sodium salicylate exerted similar effects on SR‐BI expression, whereas no effects were observed using known COX1/2 inhibitors ibuprofen and naproxen, respectively. In in vivo studies low‐dose aspirin treatment (6 mg/kg·day) induced SR‐BI expression in wild‐type and PPAR‐α knockout mice, respectively, whereas the opposite effect was observed upon high‐dose aspirin treatment (60 mg/kg·day) in these animals. We could show that COX‐independent effects of aspirin were able to enhance expression of SR‐BI in macrophages in a post‐transcriptional, PPAR‐α independent way, suggesting a novel pharmacologic effect of aspirin.—Tancevski, I., Wehinger, A., Schgoer, W., Eller, P., Cuzzocrea, S., Foeger, B., Patsch, J. R., Ritsch, A. Aspirin regulates expression and function of scavenger receptor‐BI in macrophages: studies in primary human macrophages and in mice. FASEB J. 20, 1328–1335 (2006)

Increased plasma levels of LDL cholesterol in rabbits after adenoviral overexpression of human scavenger receptor class B type I

Scavenger receptor class B type I (SR-BI), a CD36 family member, plays a key role in high-density lipoprotein (HDL) metabolism, reverse cholesterol transport, and whole body cholesterol homeostasis, and is shown to be involved in the development of atherosclerosis in mice. In this report, we describe the effects of the adenoviral overexpression of human SR-BI (hSR-BI) in New Zealand White (NZW) rabbits, a wild-type animal model that expresses cholesteryl ester transfer protein (CETP) in plasma, displays a manlike lipoprotein profile, and is susceptible to atherosclerosis. A total of 1×1012 adenoviral particles containing either hSR-BI or lacZ complementary deoxyribonucleic acid (control) were infused into the ear vein of NZW rabbits. Transgene expression was ascertained by TaqMan Real Time polymerase chain reaction measurements. Rabbits infected with Ad/hSR-BI (adenoviral plasmids containing hSR-BI) showed a faster clearance of administered [3H]HDL cholesterol and significantly decreased apolipoprotein (apo) A-I levels when compared to control rabbits, respectively. Interestingly, we found markedly increased levels of low-density lipoprotein (LDL) cholesterol exclusively in SR-BI-overexpressing rabbits. These changes were not accompanied by alterations in LDL receptor expression but by increased levels of CE transfer in these animals. By lowering HDL cholesterol and increasing plasma apoB-containing lipoprotein levels, the overexpression of SR-BI leads to a lipoprotein pattern, which is believed to enhance the development of atherosclerosis. The role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits—a CETP-expressing animal model displaying a manlike lipoprotein profile—may therefore be different from the one found in rodents.

Reduced plasma high-density lipoprotein cholesterol in hyperthyroid mice coincides with decreased hepatic adenosine 5 '-triphosphate-binding cassette transporter 1 expression

The aim of the study was to investigate the influence of severe hyperthyroidism on plasma high-density lipoprotein cholesterol (HDL-C). Recently, it was shown in mice that increasing doses of T(3) up-regulate hepatic expression of scavenger receptor class B, type I, resulting in increased clearance of plasma HDL-C. Here, we show that severe hyperthyroidism in mice did not affect hepatic expression of scavenger receptor class B, type I, but reduced hepatic expression of ATP-binding cassette transporter 1, accompanied by a 40% reduction of HDL-C. The sterol content of bile, liver, and feces was markedly increased, accompanied by up-regulation of hepatic cholesterol 7alpha-hydroxylase, and ATP-binding cassette transporter 5, which is known to promote biliary sterol secretion upon dimerization with ATP-binding cassette transporter 8. Both control and hyperthyroid mice exerted identical plasma clearance of iv injected [(3)H]HDL-C, supporting the view that severe hyperthyroidism does not affect HDL-C clearance but, rather, its formation via hepatic ATP-binding cassette transporter 1.