[show abstract][hide abstract] ABSTRACT: The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells.
We sought to investigate the effects of secretoneurin on therapeutic angiogenesis.
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.
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.
Circulation Research 10/2009; 105(10):994-1002. · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phospholipid transfer protein (PLTP) facilitates cholesterol efflux from cells, intravascular HDL remodelling and transfer of vitamin E and endotoxin. In humans, the relationship of PLTP to atherosclerosis is unknown. However, strong coronary risk factors like obesity, diabetes, cigarette smoking and inflammation increase circulating levels of active PLTP. The aim of the present, cross-sectional study was to analyze the relationship of PLTP to peripheral arterial disease, a marker of generalized atherosclerosis, independently of potentially confounding factors like obesity, diabetes and smoking.
We performed a case control study in 153 patients with symptomatic peripheral arterial disease (PAD) and 208 controls free of vascular disease. Smokers and patients with diabetes mellitus were excluded. A lipoprotein-independent assay was used for measurement of circulating bioactive PLTP and an ELISA utilizing a monoclonal antibody was used to analyze PLTP mass.
PLTP activity was significantly decreased in patients with PAD 5.5 (4.6-6.4)(median (25th-75th percentile)) versus 5.9 (5.1-6.9) micromol/mL/h in controls (p=0.001). In contrast, PLTP mass was similar in patients with PAD 8.5 microg/mL (7.3-9.5) and in controls 8.3 microg/mL (6.9-9.7) (p=0.665). Multivariate logistic regression analysis revealed that PLTP activity is independently associated with the presence of PAD. PLTP activity was similar in patients with and without lipid-lowering drugs (p=0.396).
Our results show that in non-diabetic, non-smoking subjects low rather than high PLTP activity is a marker for the presence of peripheral arterial disease and that distribution of PLTP between high-activity and low-activity forms may be compromised in atherosclerosis.
[show abstract][hide abstract] ABSTRACT: Ribosome biogenesis and ribosomal DNA transcription are closely correlated with the growth and proliferation of cells, with these processes being under tight epigenetic control. We have investigated the effect of ectopically expressed murine HDAC1 in reporter assays, on ribosomal DNA transcription, cell cycle progression and proliferation in transfected mammalian cells. Ectopically expressed mHDAC1 represses transcription in ribosomal reporter assays driven by ribosomal promoter elements in NIH3T3 cells as well as Cos-7 cells. Following stable transfection of NIH3T3 cells, flag-tagged HDAC1 is assembled into functional, enzymatically active HDAC-complexes that display correct nuclear localization. Induction of flag-HDAC1 expression in NIH3T3 cells caused a cell-cycle phase specific reduction in the initiation of endogenous rDNA transcription, reflected in a reduction of nascent rRNA as well as a marked depression of proliferation due to prolongation of G2-phase. This was substantiated by FACS analysis and cyclin B1 expression analysis. However, prolongation of the G2-phase in HDAC1-overexpressing cells finally led to overcompensation and thus to an increase in total ribosomal RNA. The transient downregulation of rRNA synthesis after induction of HDAC1 overexpression led to a prolongation of G2-phase. These observations were most likely a consequence of HDAC1-mediated deacetylation of upstream binding factor (UBF).
[show abstract][hide abstract] ABSTRACT: The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in "reactive stroma" adjacent to prostate cancer (PCa). It has been assumed that TGF-beta1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n=3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-beta1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-beta1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell alpha-actin, calponin and tenascin. TGF-beta1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16(Ink4A) and p21(Cip1) but increased p15(Ink4B) protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-beta1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.
Mechanisms of Ageing and Development 02/2005; 126(1):59-69. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: To further elucidate the role of scavenger receptor class B type I (SR-BI) in reverse cholesterol transport and in atherogenesis, we performed studies in the rabbit, an animal model displaying a lipoprotein profile similar to that of human, expressing cholesteryl ester transfer protein in plasma and having been demonstrated to be susceptible to atherosclerosis. In this report, we describe for the first time the isolation and characterization of rabbit cDNA fragments encoding SR-BI and scavenger receptor class B type II (SR-BII). Development of an isoform-specific Taqman Real Time PCR system and generation of isoform-specific polyclonal antibodies allowed us to measure SR-BI and SR-BII expression in various rabbit organs on mRNA and protein levels, respectively. We found the highest expression of SR-BI in adrenal gland, liver, and proximal intestine; lesser expression was found in appendix and spleen. Immunohistochemical staining of frozen sections showed SR-BI expression in the cortex but not in the medulla of adrenal gland. An increasing portal to central vein gradient of expression was found within the hepatic lobule. As shown in this report, identification and characterization of SR-BI expression in the rabbit affords a powerful tool to elucidate the role of SR-BI in cholesterol homeostasis and atherogenesis in human.
The Journal of Lipid Research 03/2004; 45(2):214-22. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Secretoneurin is an abundant neuropeptide of the central, peripheral, and autonomic nervous systems, located in nerve fibers characterized by a close interaction with blood vessels and known to stimulate endothelial cell migration.
We hypothesized that secretoneurin might act as an angiogenic cytokine and tested for these effects in vivo using a mouse cornea neovascularization model and in vitro by assessing capillary tube formation in a matrigel assay. In vivo, secretoneurin-induced neovasculature is characterized by a distinct pattern of arterial and venous vessels of large diameter and length. Immunohistochemical staining for CD-31 revealed endothelial lining of the inner surface of these vessels, and recruitment of alpha-smooth muscle actin-positive perivascular cells suggests vessel maturation. In vitro, secretoneurin-induced capillary tube formation was dose dependent and specific, confirming that effects of secretoneurin occur directly on endothelial cells. Secretoneurin also stimulated proliferation and exerted antiapoptotic effects on endothelial cells and activated intracellular phosphatidylinositol 3' kinase/Akt and mitogen-activated protein kinase pathways, as demonstrated by increased phosphorylation of Akt and extracellular signal-regulated kinase.
These data show that secretoneurin represents a novel direct angiogenic cytokine and reiterate the coordinated relationship between nervous and vascular systems.
[show abstract][hide abstract] ABSTRACT: The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.
[show abstract][hide abstract] ABSTRACT: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are key enzymes in lipoprotein metabolism by mediating the transfer and exchange of phospholipids (PL) and neutral lipids between lipoproteins. Lipoprotein lipase (LPL) deficiency is associated with low HDL-cholesterol (HDL-C) levels in both, the homozygous and heterozygous state. In the present study we set out to investigate the role of lipid transfer proteins, which are known to strongly determine HDL-C levels, in LPL deficiency.
Phospholipid acceptor and donor properties of lipoproteins, PLTP activity, CETP mass, activity and cholesteryl ester (CE) transfer were determined in two homozygous and six heterozygous LPL-deficient subjects and in 10 healthy, normolipidaemic controls, respectively.
The HDL isolated from LPL-deficient subjects showed strongly increased PL-acceptance when compared with controls (homozygotes versus heterozygotes versus control: 26.46 +/- 15.26 vs. 3.41 +/- 1.61 vs. 1.89 +/- 0.33 micromol mL-1 h-1/micromol mL-1 PL; all P < 0.05). Phospholipid transfer from apolipoprotein B containing lipoproteins was increased in heterozygotes when compared with controls (46.66 +/- 23.3 vs. 28.91 +/- 18.05 micromol mL-1 h-1/micromol mL-1 PL, P = 0.05). PLTP activity, however, was similar in LPL-deficient subjects and controls. CETP mass was highest in homozygotes, whilst enzyme activity was similar in LPL-deficient subjects and controls. CE transfer was highest in homozygotes (72.5 +/- 8.8%) and lowest in controls (28.7 +/- 5.2%, P < 0.01).
In conclusion, PL and CE transfer are increased in LPL deficiency and thus, partly explain low HDL-levels in LPL-deficient subjects. Enhanced transfer seems rather to be the result of altered lipoprotein composition and concentration than altered enzyme activity. Our findings on mechanisms leading to low HDL-C levels might show another aspect in atherogenesis in LPL deficiency.
Journal of Internal Medicine 02/2003; 253(2):208-16. · 6.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: The family of transforming growth factors betas (TGF-βs) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-βs, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-β1, β2 and β3. Similar to cellular senescence, TGF-β stimulation resulted in an increase of SA-beta galactosidase (SA-β-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-β-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16INK4A, IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-βs increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-β-gal. Thus, SA-β-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-β could contribute to the increased number of SA-β-gal positive epithelial cells in BPH.
[show abstract][hide abstract] ABSTRACT: Phospholipid transfer protein (PLTP) plays an important role in plasma lipoprotein metabolism. However, PLTP is expressed in a wide range of tissues suggesting additional local functions. To analyze the tissue distribution of PLTP in an animal with high-level expression of the structurally and functionally related CETP, we have cloned the full-length cDNA of rabbit PLTP (1,796 bp). Rabbit PLTP cDNA shows high homology to human, murine, and porcine PLTP cDNA, averaging 86.1%, 80.4%, and 86.1%, respectively. Interestingly, the C-terminus contains a unique seven amino acid insertion not found in previously characterized mammalian PLTPs. In clear contradistinction to human PLTP, rabbit PLTP mRNA was prominent in brain. In situ hybridization studies revealed specific, high-level synthesis of PLTP mRNA in choroid plexus and ependyma, the organs responsible for production of cerebrospinal fluid. Consistent with these findings, PLTP activity in cerebrospinal fluid amounted to 23% +/- 3% of that in rabbit plasma. In contrast, neither CETP mRNA nor CETP activity were detectable in rabbit brain.A role of PLTP in the central nervous system could involve some of its actions previously established in vitro, like proteolysis of apolipoproteins, and be physiologically relevant for neurodegenerative disorders such as Alzheimer's disease.
The Journal of Lipid Research 05/2002; 43(4):636-45. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are key enzymes in lipoprotein metabolism facilitating the transfer and exchange of cholesteryl esters, triglycerides and phospholipids between lipoproteins. In the study presented here, we investigated the influence of two hormones-the adipocyte-derived hormone leptin as well as insulin on the hepatic secretion of both, PLTP and CETP.
PLTP activity and CETP concentration-measured by exogenous substrate assay and enzyme-linked immunosorbent assay-were determined in supernatant of human hepatoma cell line HepG2 after single or combined exposure to leptin and insulin at physiological and supraphysiological concentrations, respectively. Messenger-RNA of PLTP and CETP was quantified by Northern blot analysis.
Leptin suppressed PLTP activity and CETP-concentration by up to 33% and 23%, respectively. Insulin also suppressed PLTP activity by up to 11% and CETP-concentration by up to 16%. In combination, the two hormones had additive suppressive effects for both, PLTP activity and CETP-concentration. Northern blot analysis showed no difference in m-RNA levels after exposure to leptin or insulin.
Leptin and insulin, both known to increase with body fat mass, suppress production of PLTP and CETP in HepG2 cells. When extrapolated to the in vivo situation, this suppressive effect may constitute a mechanism counteracting the potentially harmful action of lipid transfer proteins, particularly reduction of HDL-cholesterol, in conditions frequently associated with increased plasma triglyceride levels such as obesity and insulin resistance.
International Journal of Obesity 12/2001; 25(11):1633-9. · 5.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: Hyperhomocysteinemia has been accepted as an independent risk factor for atherosclerosis and atherothrombosis. In recent years, several reports have appeared in the literature linking the use of anabolic steroids with acute vascular events in bodybuilders. In this study, we investigated whether hyperhomocysteinemia could contribute to the high vascular risk in bodybuilders taking anabolic steroids. Methods and results: Twenty-three bodybuilders in different phases of their training cycle and six control athletes participated in our study. Anthropomorphic measures displayed a higher body mass index for bodybuilders in the competition phase than for bodybuilders in the work-out and build-up phases, and for control athletes. Homocysteine levels were 8.7+/-1.6 µmol/l (mean+/-S.D.) in control athletes, 8.5+/-2.8 µmol/l in work-out phase bodybuilders, and 8.3+/-1.5 µmol/l in competition phase bodybuilders, but 11.9+/-3.1 µmol/l in build-up phase bodybuilders (P<0.05 for build-up phase bodybuilders vs. control athletes, work-out phase bodybuilders, and competition phase bodybuilders, respectively). Vitamin B12 and folate levels did not differ significantly between the four groups. Conclusion: Our study shows that intake of anabolic steroids, as used typically by bodybuilders in the build-up phase, induces acute hyperhomocysteinemia and is likely to initiate an additional, potentially atherothrombotic mechanism in this group of athletes.
European Journal of Internal Medicine 02/2001; 12(1):43-47. · 2.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, we investigated the expression and localization of leptin receptors in human term placentae. On human term placenta tissue slices, digoxigenin-UTP labelled RNA-probe detected the long form of the leptin receptor ObR(L)mRNA in syncytiotrophoblasts of the villi, whereas the haematological subtype of the leptin receptor ObR/B219.1 was detected in blood cells of the intervillous space and fetal vessels. Immunohistochemistry, with two polyclonal antibodies to the N-terminus recognizing ObR(L)and ObR(S)of the leptin receptors and one to the C-terminus recognizing the long form of the leptin receptor ObR(L), localized leptin receptor protein at the apical membrane of the syncytiotrophoblasts. Our results show that the long form of the leptin receptor ObR(L)is expressed in human term placentae. We localized the long form of leptin receptor mRNA to the cytoplasm of syncytiotrophoblasts and leptin receptor proteins in human term placentae to the apical membrane of syncytiotrophoblasts. We conclude that in term placentae, leptin could mediate a growth promoting effect in the fetoplacental unit through the long form of the leptin receptor localized in the syncytiotrophoblasts. In contrast, the haematological subtype of the leptin receptor is not expressed in placental cells, but solely by blood cells in the intervillous space and fetal vessels.
[show abstract][hide abstract] ABSTRACT: The discovery of leptin, the product of the obese (ob)-gene, has broadened the horizons of research on energy balance. This hormone, produced and secreted by adipose tissue and some placental cells, finds its way to the hypothalamus, where it binds to the leptin receptors and signals satiety through the neuroendocrine axis. The fact that adipose tissue is not merely a storage depot, but also an important endocrine tissue, has revived the interest in the "lipostatic" theory of body fat regulation and has initiated many research efforts in the field of obesity, anorexia nervosa, bulimia, reproduction and haematology.
Wiener klinische Wochenschrift 04/1998; 110(6):212-9. · 0.81 Impact Factor