Andreas Zakrzewicz

Danube Private University, Krems, Lower Austria, Austria

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Publications (44)178.15 Total impact

  • A Marki, M Boehme, S Sigrist, A Zakrzewicz, A Pries
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    ABSTRACT: The endothelial surface layer (ESL, glycocalyx) is known to be anchored to membrane bound proteins (syndecan-1,-2,-4 and glypican-1), glycoproteins (e.g. CD44) and enzymes (e.g. hyaluronan synthase-1,-2,-3). Little is known about the concentration and distribution of these anchoring elements on the endothelial surface.
    Cardiovascular Research 07/2014; 103(suppl 1):S31. · 5.81 Impact Factor
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    ABSTRACT: Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6mmol/L d-glucose), high glucose (30mmol/L d-glucose or l-glucose), 100μmol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100μmol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High d-glucose increased TRPC6 transcripts to 8.66±4.08 (p<0.05) and TRPC6 protein expression to 1.44±0.07 (p<0.05) without altering SDC-4 transcripts or protein expression. The d-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29±1.26 (p<0.05) and TRPC6 protein expression to 1.28±0.05 (p<0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high d-glucose increased ROS after 90min to 3.55±0.08 arbitrary units while 5.6mmol/L d-glucose increased ROS to 2.49±0.09 (p<0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes.
    Biochemical and Biophysical Research Communications 06/2014; · 2.28 Impact Factor
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    ABSTRACT: The aim of the study was to establish a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometre precision in a standard fluorescence microscope using a point spread function (PSF) approach, and to evaluate validity and precision for different analysis methods and optical conditions with particular application to microcirculatory flow dynamics and cell biology. Images of fluorescent particles were obtained with a standard fluorescence microscope equipped with a piezo positioner for the objective. Whole pattern (WP) comparison with a PSF recorded for the specific set-up and measurement of the outermost ring radius (ORR) were used for analysis. Images of fluorescent particles were recorded over a large range (about [Formula: see text]) of vertical positions, with and without distortion by overlapping particles as well as in the presence of cultured endothelial cells. For a vertical range of [Formula: see text], the standard deviation (SD) from the predicted value, indicating validity, was 9.3/8.7 nm (WP/ORR) in the vertical and 8.2/11.7 nm in the horizontal direction. The precision, determined by repeated measurements, was 5.1/3.8 nm in the vertical and 2.9/3.7 nm in the horizontal direction. WP was more robust with respect to underexposure or overlapping images. On the surface of cultured endothelial cells, a layer with 2.5 times increased viscosity and a thickness of about [Formula: see text] was detected. With a validity in the range of 10 nm and a precision down to about 3-5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle localizations, including the assessment of endothelial cell microenvironment.
    Biomechanics and Modeling in Mechanobiology 06/2013; · 3.33 Impact Factor
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    ABSTRACT: Alternative splicing is a key regulatory mechanism for cellular metabolism controlling cell proliferation and angiogenesis, both of which are crucial processes for tumorigenesis under hypoxia. Human cells express two tissue factor (TF) isoforms, alternatively spliced TF (asTF) and 'full length' TF (flTF). flTF is the major source of thrombogenicity whereas, the function of soluble asTF, particularly in cancer, is widely unknown. In the present study, we examined the impact of alternative splicing on the pro-angiogenic potential and the TF expression pattern of A549 cells under hypoxia. We focused our efforts toward alternative splicing factors, such as Clk1, and pro-angiogenic proliferation-regulating factors, such as Cyr61. We further examined the influence of asTF overexpression on the expression of MCP-1, Cyr61 and VEGF, as well as on cell number and pro-angiogenic properties of A549 cells. Notably, we found hypoxia to induce the expression of alternative splicing factors (Clk1 and Clk4) as well as proliferation- and angiogenesis-promoting factors (Cyr61 and flTF). asTF overexpression in A549 cells also increased both cell number and tube formation. These effects were mediated by the induction of Cyr61, MCP-1 and VEGF, as well as by integrin αvβ3. Taken together, our results suggest that the pro-angiogenic potential of A549 lung cancer cells is modulated under hypoxic conditions via modulation of TF isoform expression which in turn is controlled by alternative splicing.
    Oncology Reports 04/2013; · 2.30 Impact Factor
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    ABSTRACT: The impact of smoking on the local innate immune response in the oral cavity, and, commonly, on oral health is actively discussed in the scientific literature. The aim of the present study was to evaluate possible effects of smoking on gene expression of human beta-defensin-1 and -2 in human gingival tissue. Biopsies of keratinized gingival tissues were taken from donors (with written informed consent) undergoing routine surgical treatment. Prior to the sample collection, participants with clinically healthy periodontium were classified as smokers (n=9) or non-smokers (n=9). Gingival tissue was homogenized, and total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1β- and IL-6-, as well as GAPDH-mRNA. The data obtained were analysed for significant differences using the Mann-Whitney-U test. hBD-1- and hBD-2-, as well as IL-1β- and IL-6-mRNA were detected in all gingival samples. Expression of hBD-1 and -2 was significantly reduced by nearly 2.5-fold (p<0.05; Mann-Whitney) in gingival samples of smokers compared to control group specimens (non-smokers). In contrast, no significant differences of the gene expression of IL-6 and IL-1β were observed in human gingival tissue of smokers and non-smokers. The results presented here suggest that expression of human beta-defensins hBD-1 and -2, and, thus, the basal levels of innate immune defense reactions in the oral cavity are reduced by smoking.
    Journal of dentistry 08/2012; 40(11):949-54. · 3.20 Impact Factor
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    ABSTRACT: The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.
    Hypertension 05/2012; 59(6):1232-40. · 6.87 Impact Factor
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    ABSTRACT: Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.
    AJP Heart and Circulatory Physiology 01/2012; 302(1):H143-52. · 4.01 Impact Factor
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    ABSTRACT: The impact of nicotine on the local innate immune response in the oral cavity is unclear. The aim of the present study was to evaluate the possible effects of nicotine on the gene expression of human beta-defensin-1 and -2 in HaCaT keratinocytes. HaCaTs were cultured in six-well plates in Dulbecco's minimum essential medium (DMEM) supplemented with 10% FBS at a density of ×10(6). Cells were pretreated with 10 μg/ml nicotine (12 h), and then stimulated with 50 ng/ml TNF-α (during the following 12 h); or were pretreated with 50 ng/ml TNF-α, and then stimulated with 10 μg/ml nicotine; or were not pretreated but only stimulated with either nicotine or TNF-α, or a combination of both. Total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1β- and IL-6-, as well as GAPDH-mRNA. The obtained data were analysed using Tukey's B multiple comparison test for post hoc analysis. Pretreatment with nicotine caused a significant 2.5-fold inhibition of TNF-α-stimulated hBD-2 mRNA expression compared to TNF-α alone (p = 0.004). Simultaneous treatment with TNF-α and nicotine caused a significant 2-fold inhibition of hBD-2 mRNA compared to TNF-α alone (p = 0.041). The present results suggest that the pre-exposition to nicotine seems to reduce a stimulating effect of TNF-α on the gene expression of hBD-2.
    Archives of oral biology 12/2011; 57(6):814-9. · 1.65 Impact Factor
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    ABSTRACT: In skeletal muscles, the expression of neuronal NO synthase (nNOS) isoforms is uncharacterized at the protein level. We therefore conducted epitope mapping with anti-peptide-antibodies. Antibodies specific for the nNOS N-terminus recognized the 160-kDa alpha-isoform. In contrast, antibodies against the middle portion or the C-terminus of nNOS bound additionally to the truncated 140-kDa beta-isoform which lacks the PDZ-domain present in the alpha-isoform. All nNOS immunohistochemical reactivity was confined to the sarcolemma. Consistently, immunoblotting disclosed both nNOS-isoforms to be co-enriched in the membrane-associated fractions. The beta-isoform was co-immunoprecipitated with alpha-isoform antibodies in muscle extracts indicating an association of both nNOS-isoforms to direct the beta-variant to the sarcolemma.
    FEBS letters 09/2011; 585(20):3219-23. · 3.54 Impact Factor
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    ABSTRACT: Both, increased plasma concentrations of vascular endothelial growth factor (VEGF) and increased expression of transient receptor potential canonical type 6 (TRPC6) channels in podocytes have been associated with proteinuric kidney diseases. Now, we investigated the hypothesis that VEGF regulates TRPC6 in podocytes. TRPC6 messenger RNA (mRNA) and TRPC6 protein expression were analyzed in cultured podocytes after administration of VEGF165 using quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. YFP-tagged TRPC6 in podocytes was analyzed using confocal laser scanning microscopy. TRPC6-associated calcium influx was measured fluorometrically. Both, immunofluorescence and immunohistochemistry were performed in renal tissue from patients with diabetes mellitus and controls. Administration of VEGF165 to podocytes significantly increased TRPC6 mRNA expression and TRPC6 protein levels. The effects of VEGF165 were dose dependent and could be blocked by phosphoinositide-3-kinase inhibitors. In the presence of cycloheximide, an inhibitor of protein biosynthesis, we did not observe an effect of VEGF on TRPC6 protein levels, indicating the requirement of de novo protein synthesis. VEGF165 significantly increased TRPC6-mediated calcium influx in podocytes. Calcium influx was significantly lower in podocytes after gene knockdown using siRNA against TRPC6. Immunohistochemistry showed both increased TRPC6 channel protein and VEGF receptor type 2 (VEGFR-2) protein in podocytes from patients with diabetic nephropathy compared to control subjects. There was a significant association between VEGFR-2 mRNA and TRPC6 mRNA (n = 48; r(2) = 0.585; P < 0.0001) in human renal cortex. VEGF regulates TRPC6 in podocytes.
    Nephrology Dialysis Transplantation 08/2011; 27(3):921-9. · 3.37 Impact Factor
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    ABSTRACT: Recent studies revealed that in vivo the inner blood vessel surface is lined with an endothelial surface layer at least 0.5 μm thick, which serves as an aegis, protecting the vessel wall from arteriosclerosis. Hyaluronan seems to be a constitutive component in regard to the atheroprotective properties of this surface structure. It has been shown that arterial pulsatile laminar blood flow increases the thickness of this surface layer in vivo, while it is significantly reduced at atheroprone regions with disturbed flow. This study was undertaken to reveal whether endothelial hyaluronan synthesis via hyaluronan synthase 2 (HAS2) can be changed by different shear stress conditions in vitro, especially in regard to an undisturbed, arterial-like pulsatile flow profile. Human umbilical vein endothelial cells, exposed to constant or pulsatile shear stress in a cone-and-plate system, were analysed for HAS2 expression by real-time RT-PCR and immunoblotting, and for hyaluronan by ELISA. Hyaluronan synthase 2 mRNA and protein were found to be transiently increased in a shear stress-dependent manner via the phosphatidylinositol 3-kinase-Akt pathway. Especially pulsatile, arterial-like shear stress conditions induced enzyme and hyaluronan effectively, while lower shear stress that continuously changed its direction did not induce any differences in comparison with control cultures not exposed to shear stress. These experiments provide a link between the production of a constitutive component of the endothelial surface layer by endothelial cells and blood flow.
    Experimental physiology 05/2011; 96(9):977-86. · 3.17 Impact Factor
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    ABSTRACT: ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.
    Journal of Cellular Physiology 02/2011; 226(2):350-61. · 4.22 Impact Factor
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    ABSTRACT: The Interleukin 8 (IL-8) response of endothelial cells to lipoproteins has well known implications for the development and progression of atherosclerosis. In this study we sought for the role of zinc finger protein 580 (ZNF580) in the endothelial IL-8 response to lipoproteins. In human umbilical vein endothelial cells (HUVEC) ZNF580 and IL-8 levels were examined by real-time-RT-PCR, immunoblotting and immunostaining or ELISA, respectively. ZNF580 is located in the nucleus and regulated by LDL and HDL depending on the oxLDL/LDL-ratio but not by TNFα. IL-8 expression profiles are inversely influenced by the oxLDL/LDL-ratio, both in vitro and in vivo. Knock down of ZNF580 enhances the expression and release of IL-8 and increases monocyte arrest under flow conditions in vitro. ZNF580 is a novel factor in the lipoprotein-dependent regulation of IL-8 and monocyte arrest. Therefore it may be a new potential target for intervention in atherosclerosis.
    Atherosclerosis 01/2011; 216(1):103-8. · 3.71 Impact Factor
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    ABSTRACT: Objectives: The impact of nicotine on the local innate immune response in the oral cavity is not clearly understood. The aim of the present pilot study was to evaluate the possible effects of nicotine and TNF-α on gene expression of human beta-defensine-1 and -2 in HaCaT keratinocytes. Methods: HaCaTs were cultured in DMEM medium supplemented with 10% FBS, and then split using six-well plates in equal groups. In two groups cells were pretreated either with nicotine (10 μg/ml; well 5) or TNF-α (50 ng/ml; well 6) for 12 h each. The cells in the other wells (1-4) were stimulated for 12 h: negative control (well 1), positive control (TNF-α; 50 ng/ml; well 2), nicotine (10 μg/ml; well 3), TNF-α (50 ng/ml) and nicotine (10 μg/ml; well 4). The pretreated wells 5 and 6 were stimulated with TNF-α (10 μg/ml) and nicotine (50 ng/ml) for 12 h. Total cell RNA was extracted followed by RT-reaction and cDNA synthesis. Samples were analyzed by real-time PCR. Genes of interest were: human beta-defensins-1, -2, and interleukin IL-1β. Gene expression of GAPDH served as internal standard for normalizing real-time PCR data. To compare the differences between the expression levels of studied genes, multiple comparison tests (Tukeys B) for post hoc analysis were performed. Results: Pretreatment with nicotine caused a significant inhibition (nearly 2.5 fold, p=0.004) of HBD-2 expression induced at the transcriptional level by TNF-α in HaCaT cells if compared to positive control. Simultaneous treatment with TNF-α and nicotine resulted in significantly lower (nearly 2 fold, p=0.041) gene expression of HBD-2 compared to TNF-α stimulation of controls. In contrast, nicotine caused no significant changes in the gene expression of IL-1β and HBD-1. Conclusion: The results of the present pilot investigation suggest that nicotine has an inhibitory effect on the gene expression of HBD-2.
    88th General Session & Exhibition of the IADR, Barcelona, Spain; 07/2010
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    ABSTRACT: Testicular germ cell tumour (TGCT) is the most common cause of death from solid tumours in young men and especially for platinum-refractory patients novel treatment approaches are urgently needed. Using an in silico screening approach for the detection of novel cancer drugs with inhibitory effects on the tyrosine kinase activity of growth factors (e.g., VEGFR, PDGFR), we identified two compounds (HP-2 and HP-14) with antiangiogenic and antiproliferative potency, which were evaluated in endothelial cell models and TGCT cells. HP-2 and HP-14 effectively inhibited the growth of VEGFR-2-expressing TGCT cell lines (Tera-1, Tera-2 and 2102EP) and endothelial cell models, while they failed to supress the growth of VEGFR-2-lacking tumour cells. cDNA-microarrays revealed an inhibition of the expression of several growth factor receptors and related signal transduction molecules. Vascular endothelial growth factor (VEGF)-induced cell migration was also potently inhibited. Cell cycle-regulating proteins such as p21 and p27 were upregulated, leading to an S-phase arrest. Additional in vivo evaluations confirmed the antiangiogenic potency and good tolerability of the novel substances. Our data show that the identified novel compounds inhibit the growth of TGCT cells and decrease angiogenic microvessel formation. The mode of action involves cell cycle arresting effects and changes in the expression pattern of several angiogenic genes. The novel compounds may qualify as new candidates for targeted treatment of TGCT and merit further evaluation.
    British Journal of Cancer 06/2010; 103(1):18-28. · 5.08 Impact Factor
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    ABSTRACT: The purpose of this study was to establish whether suppression of angiogenesis by nitric oxide synthase (NOS) inhibition in skeletal muscles exposed to long-term activity can be explained by changes in capillary shear stress linked to the lack of nitric oxide production. Capillary shear stress was calculated from diameters (d) and red blood cell velocities (V(rbc)) measured at rest and after acute contractions in epi-illuminated extensor digitorum longus muscles of control rats and those in which ankle flexors had been stimulated via implanted electrodes (10 Hz, 8 h x day(-1)) for 2 or 7 days without and with inhibition of nitric oxide synthase activity by N(omega)-nitro-L-arginine (L-NNA, 3-4 mg x day(-1) in drinking water). Neither chronic electrical stimulation nor L-NNA treatment altered capillary diameters. Capillary V(rbc) and shear stress (SS) were doubled in muscles after 2 days stimulation (298 +/- 22 microm x s(-1) and 11.4 +/- 1.0 dyne x cm(-2), respectively, p < .005) compared to controls (148 +/- 18 microm x s(-1) and 5.6 +/- 0.8 dyne x cm(-2)) but normalized after 7 days (153 +/- 27 microm x s(-1) and 6.2 +/- 1.0 dyne x cm(-2)), when the capillary bed is known to be enlarged. L-NNA, which increased blood pressure in all treated animals, abolished the increase in capillary SS after 2 days stimulation and decreased SS after 7 days. These data support a role for NO in the early elevation of capillary shear stress that initiates angiogenesis in stimulated muscles, likely via modulation of upstream vascular resistance, and could explain the lack of capillary growth in stimulated muscles when nitric oxide generation is suppressed.
    Microcirculation 07/2009; 13(3):249-59. · 2.76 Impact Factor
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    ABSTRACT: There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood pressure in patients. TRPC3 was detected in cultured human endothelial cells and in vascular endothelium cells from human renal tissue by immunoblotting, immunohistochemistry, and quantitative real-time reverse transcriptase-PCR. The changes of TRPC3 and vascular endothelial growth factor receptor type 2 expression in cultured human endothelial cells were measured after administration of vascular endothelial growth factor isoform 121. In cultured human endothelial cells, vascular endothelial growth factor isoform 121 significantly reduced TRPC3 expression by 57% and vascular endothelial growth factor receptor type 2 by 70%. This reduction was partly blocked by phosphatidylinositol 3-kinase inhibitors, wortmannin, or LY294002. Downregulation of TRPC3 channel expression was associated with reduced calcium influx. The changes of calcium influx could be abolished by the inhibitor of TRPC channels, 2-aminoethoxydiphenylborane, pointing to their functional importance. TRPC3 expression was significantly higher in patients with SBP more than 140 mmHg compared with patients with SBP of 140 mmHg or less (0.00181 +/- 0.00059 versus 0.00037 +/- 0.00012 arbitrary units; P < 0.01). The data support the hypothesis that TRPC3 expression in human renal tissue including vascular endothelium is associated with blood pressure regulation in humans.
    Journal of Hypertension 06/2009; 27(6):1217-23. · 4.22 Impact Factor
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    ABSTRACT: Human beta-defensins (hBDs) are antimicrobial peptides that play an important role in the innate host defense against bacterial invasion, contribute to promotion of adaptive immune responses, and show chemotactic activities. The aim of this study was to compare the gene expression of hBD-1, -2, -3, and -4 in healthy teeth and teeth with pulpitis. Samples of healthy and inflamed dental pulps were obtained from extracted third molars and during treatment of teeth with pulpitis. Gene expression was assessed by using reverse transcriptase reaction and real-time polymerase chain reaction. HBD-2 and hBD -3 were only weakly expressed in healthy and inflamed pulps. In contrast, the expression of hBD-1 and hBD -4 was significantly increased in inflamed compared with healthy pulps. These results suggest that hBD-1 and hBD-4 might play a role in the pulpal host defense.
    Journal of endodontics 05/2009; 35(4):520-3. · 2.95 Impact Factor
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    ABSTRACT: Tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-alpha. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-alpha. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-alpha-stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions.
    Circulation Research 02/2009; 104(5):589-99. · 11.86 Impact Factor
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    ABSTRACT: Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.
    The Journal of Physiology 01/2009; 587(Pt 3):655-68. · 4.38 Impact Factor

Publication Stats

668 Citations
178.15 Total Impact Points

Institutions

  • 2012
    • Danube Private University
      Krems, Lower Austria, Austria
  • 2008–2012
    • Charité Universitätsmedizin Berlin
      • Institute of Physiology
      Berlin, Land Berlin, Germany
  • 2011
    • Universität Bern
      • Institut für Anatomie
      Bern, BE, Switzerland
  • 2001–2004
    • Freie Universität Berlin
      • Institute of Immunology and Molecular Biology
      Berlín, Berlin, Germany
  • 1997–1999
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany
  • 1994
    • Harvard Medical School
      Boston, Massachusetts, United States