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LPS-induced effects on angiotensin I-converting enzyme expression and shedding in human pulmonary microvascular endothelial cells [In Vitro Cellular & Developmental Biology](In Vitro Cellular & Developmental Biology Via Acquire Media NewsEdge) Abstract Angiotensin I-converting enzyme (kininase II, ACE, and CD143) availability is a determinant of local angiotensin and kinin concentrations and their physiological actions. Until now, it is unclear whether the decrease of pulmonary ACE activity in sepsis-described in clinical studies-is due to an enzyme compensatory downregulation (reduced ACE-mRNA expression) to shedding of ACE or endothelial damage. To address these questions, ACE distribution under septic conditions was studied in vitro by treating pulmonary microvascular endothelial cells (HPMEC) and human umbilical vein endothelial cells (HUVEC) with lipopolysaccharide from Escherichia coli (LPS). Primary isolated HUVEC and HPMEC were compared by detecting ACE activity, membrane-bound ACE, as well as shedding and mRNA production of ACE with and without LPS (1 ng/ml- 1 µg/ml). ACE mRNA expression was detected by real-time PCR, and shedded ACE was measured in cell culture supernatant by ELISA. Additionally,membrane-bound protein expression was investigated by immunohistochemistry in situ. In septic ARDS, the distribution of ACE protein was significantly reduced in all lung endothelial cells (p <0.001). After stimulation with LPS, cultivated HPMEC showed more markedly than HUVEC, a concentration-dependent reduction of ACE protein expression compared to the respective untreated controls. Real-time PCR demonstrated a reduced ACE mRNA expression after LPS stimulation, predominantly in HPMEC. Specifically, in HPMEC, a concentration-dependent increase of shedded ACE was shown 24 h after LPS treatment. HPMEC cultures are an apt model for the investigation of pulmonary ACE expression in sepsis. This study suggests that reduced pulmonary microvascular endothelial ACE expression in septic ARDS is caused by two processes: (initial) increased shedding of ACE accompanied by a compensatory downregulation of ACE- mRNA and membrane-bound protein expression. Keywords Angiotensin converting enzyme · ACE ·. Lung tissue · CD143 shedding · Human pulmonary microvascular endothelial cells Introduction Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS), refer to increased permeability pulmonary edema caused by a variety of pulmonary or systemic insults. Predisposing factors for ALI/ ARDS are diverse (Ware and Matthay 2000). The commonly associated clinical disorders can be divided into those associated with direct lung injury and those that cause indirect lung injury in the setting of a systemic process. The main direct causes are pneumonia and aspiration, while the main indirect causes include sepsis and severe trauma. Overall, the most common risk factor for ALI/ARDS is severe sepsis with a suspected pulmonary source, followed by extrapulmonary sepsis, aspiration, and severe trauma (Rubenfeld et al. 2005). The pulmonary endothelium is a major metabolic tissue involved in maintenance of pulmonary and systemic vascular homeostasis and a main target of circulating cells and humoral mediators following injury. Both clinical and experimental results show that injury to the pulmonary endothelium is accompanied by a decrease in angiotensin converting enzyme (ACE; CD143) activity in the lung homogenate, with a transitory increase in its activity in the serum (Gorin et al. 1981; Nukiwa et al. 1982; Fourrier et al. 1985; Kelley 1988). Therefore, ACE is recognized as an early and sensitive marker of pulmonary endothelial dysfunction (Siefkin et al. 1984; Fourrier et al. 1985; Johnson et al. 1985; Orfanos et al. 2000). ACE, the key renin-angiotensin-aldosterone system (RAAS) enzyme, is highly expressed on the surface of pulmonary microvascular endothelial cells (Muller et al. 2004;Aird2007). Pulmonary endothelial ACE is an ectoenzyme uniformly distributed along the luminal EC surface, with its catalytic site exposed to the blood stream; it is directly accessible to blood-borne substrates and its activity may be measured in vivo by means of indicator dilution type techniques (Orfanos et al. 1994; Orfanos et al. 2000). Due to the very high enzyme concentrations in the capillaries, monitoring pulmonary endothelial ACE activity in this type of study is equivalent in practical terms to monitoring pulmonary capillary endothelium-bound (PCEB) ACE activity (Orfanos et al. 1997; Orfanos et al. 1999). PCEB-ACE activity reduction has been among the earliest signs detected in different ALI animal models (Orfanos et al. 2000). ACE is mainly responsible for the hydrolysis of angiotensin I to angiotensin II and the breakdown of bradykinin. Increasing experimental evidence suggests that RAAS activation in the lungs and, in particular, angiotensin II enhances ALI. In this respect, van Asperen and co-workers have recently shown that injurious mechanical ventilation increased ACE in BALF of LPS-exposed rats and that ACE exerts its effect via angiotensin II enhance and not via bradykinin (Wosten-van Asperen et al. 2010). The fact that ACE and angiotensin II promote lung injury which is attenuated by ACE inhibitors and/or blocking of the angiotensin receptor 1 (AT1) (He et al. 2007; Jerng et al. 2007; Hagiwara et al. 2009) appears in apparent contrast with the constant and well-established finding that PCEB-ACE activity is reduced under ALI in both animals and humans. A possible explanation of the observed paradox might be that the observed reduction of PCEB-ACE activity seen in ALI may be related to an enzyme compensatory downregulation, aiming to limit ACE-mediated pro-inflammatory processes in the microenvironment, which would be more potent if ACE activity were higher. Alternatively, and/or in addition, reductions in PCEB-ACE activity may be related to endothelial injury with a proteolytic cleavage of PCEB-ACE (shedding) or frank endothelial losses, while viable ECs maintain or even overexpress ACE. To address these questions, we performed an in vitro study on the distribution of ACE in pulmonary microvascular endothelial cells under conditions simulating sepsis. Both human pulmonary microvascular endothelial cells (HPMEC) and the most common experimental endothelial cell type, human umbilical vein cells (HUVEC), were stimulated with bacterial endotoxin [lipopolysaccharide (LPS) from Escherichia coli ] in concentrations that induce inflammatory activation. ACE activity, membrane-bound ACE expression, and mRNA production, as well as shedding of ACE into the culture supernatant were investigated. Furthermore, the distribution of ACE was examined by immunohistochemistry in the vasculature of human lungs with and without septic ARDS. The results of these morphological studies were compared with that of the in vitro study with regard to the question whether human pulmonary microvascular endothelial cells are an apt model to investigate ACE distribution in sepsis. Material and Methods Chemicals and reagents. Hippuryl-L -histidyl-L -leucine (Hip-His-Leu), LPS, phorbol 12-myristate 13-acetate (PMA), and other reagents were obtained from Sigma (St. Louis, MO) or as specified. For the present studies, we used anti-ACE mAb CG2, which recognizes an epitope localized on the N-terminal domain of native human ACE (Danilov et al. 1994). Cell culture. The experiments were carried out on excess human tissue from the diagnostic service of the Institute of Pathology and were in accordance with the informed consent criteria of the responsible ethical commission. ACE expression on the plasma membrane of primary culture endothelial cells decreases with propagation and passage (Balyasnikova et al. 1998). Therefore, to study the effect of LPS on endothelial cell ACE expression in vitro, experiments were performed on primary isolated cells not older then passage 4. HUVEC were isolated from umbilical cord veins as previously described (Jaffe et al. 1973). The vein of an umbilical cord was washed with 20 ml of 10 mM Hepes buffer. HUVEC were dissociated from the vessel wall with 2% (w /v ) collagenase I in HEPES buffer for 10 min at 37°C. Adult human pulmonary microvascular endothelial cells were obtained from normal portions of lung specimens surgically resected from patients who underwent lobectomies for early stage lung cancer. HPMEC were isolated as described in detail elsewhere (Wagner et al. 1999). A 99% pure culture was archived by separating the endothelial cells twice with CD31 beads (Dynal, Hamburg, Germany). Both endothelial cell types were grown to confluence in the same culture medium ECGM-MV, Promocell, Heidelberg, Germany) on fibronectin-coated 6- or 96-well plates. Cells were maintained at 37°C under 5% CO2 in a humidified incubator. Cells were characterized by staining for factors VIII- and CD31- (DAKO GmbH, Hamburg, Germany) related antigens. For treatment cells were incubated with different concentrations of LPS (1 ng/ml to 1 µg/ml) and PMA (100 ng/ml), a potent PKC activator, for 24 h. The concentration of 100 ng/ml PMA functions as positive control as it was described to induce a three and fivefold increase in ACE activity in the medium and in the cells, respectively, after 24 h stimulation of HUVEC (Villard et al. 1998). Cell morphology and cell number were controlled by crystal violet staining. ACE activity. The activity of membrane-bound ACE was measured in a homogenate of cultured HPMEC or HUVEC using ACE substrate (Hip-His-Leu) as described previously (Friedland and Silverstein 1976). Briefly, lysis of cells was performed with 100 µl/well of 8 mM CHAPS in PBS for 15 min at RT. Fifteen microliters of cell homogenate was added to 200 µl of ACE substrate (1 mM Hip-His-Leu) and incubated for 60 min at 37°C. The reaction was terminated with 25 µl 0.28 M NaOH, and the His-Leu product was estimated by 10 min incubation with 10 µl O-phthaldialdehyde (20 mg/ml in methanol). After addition of 20 µl 3 M HCl, the fluorescence of samples was measured using 365 nm excitation/500 nm emission filters. Membrane-bound ACE (cell enzyme-linked immunosorbent assay). The level of expression of ACE on the surface of cultured HPMEC or HUVEC treated with various concentrations of LPS for 24 h was detected by cell enzyme-linked immunosorbent assay using the anti-ACE monoclonal antibody CG2 (BMA Biomedicals, Augst, Switzerland). Cell-bound anti-human ACE antibody was detected at 492 nm by a biotin streptavidin-enhanced peroxidase reaction with 3, 3?,5, 5?-tetramethylbenzidine as substrate. Real-time PCR for ACE. Primers and the TaqMan probe for human ACE (Hs00174179_m1) and human GAPDH (Hs99999905_m1) were purchased from Perkin-Elmer Applied Biosystems.TotalRNAwasextractedfromHPMECorHUVEC cultured in 6-well plates using a QiagenRNeasy kit. The RNA was then treated by DNase I (Qiagen, Hilden, Germany) to minimize genomic DNA contamination. Reverse transcription (RT) was performed with 1 µ RNA using the Omniscript RT Kit (Qiagen, Hilden, Germany). Real-time PCR was performed with the appropriate cDNA in a 7500 real-time PCR System (Perkin-Elmer Applied Biosystems, Life Technologies, Darmstadt, Germany) using 96 samples per assay (50 µLper tube). PCR was carried out for 1 cycle at 50°C for 2 min, 60°C for 30 min, and 95°C for 5 min; the PCR protocol consisted of 40 cycles of 94°C for 15 s and 60°C for 1 min. Shedded ACE (ELISA). The complete ACE molecule contains a hydrophobic transmembrane domain that anchors ACE in the plasma membrane. The physiological mechanism for ACE shedding from the plasma membrane requires specific proteolytic cleavage of the extracellular domain of ACE by an extracellular protease, or secretase, associatedwith the plasma membrane (Hooper et al. 1997). This process leads to the appearance of the hydrophilic form of ACE in the extracellular medium. To detect the shedded ACE, both HUVEC and HPMEC grown in 96-well plates were washed three times with phosphate-buffered salt solution and incubated with LPS (1 ng/ml-1 µg/ml) diluted in culture medium. After 24 h, the aqueous medium supernatant was collected and centrifuged at 1,500 rpm for 10 min. The shedded ACE was measured in culture supernatant by commercially available enzyme-linked immunosorbent assay (ELISA) using a coupled antibody pair (Duo Set Kit, DY929 R&D, Wiesbaden, Germany). Crystal violet staining was performed to detect possible endothelial losses due to cell injury. Semiquantitative analysis of ACE immunostaining. In order to study the expression pattern of ACE expression in human septic lung tissue, paraffin-embedded samples of 41 patients (age, 34- 76 yr) with regular morphology from tumor lobectomies (control group) and 21 patients (age, 3.5 mo- 77 yr) who had died of documented multiorgan failure due to gram-negative sepsis were examined for ACE expression. As definition for septic shock, the practical modification of the definition convened in 2001 during an International Sepsis Definitions Conference by the SCCM, European Society of Intensive Care Medicine, ACCP, American Thoracic Society, and Surgical Infection Society was used (Levy et al. 2003). This modification includes exact hemodynamic definitions of septic shock (Annane et al. 2005). Microbiologically proven gram-negative sepsis as well as the presence of at least two of the internationally accepted clinical signs of sepsis in the medical history were required to assess lung specimens displaying macro- and microscopical signs of ARDS. The identification of vessel types (arteries, arterioles, capillaries, venules, and veins) was carried out by analyzing serial slides for their histomorphological characteristics. Arterioles and venules were identified by tracing them back to larger vessels from which they originated or into which they were discharging. For ACE staining, an alkaline phosphatase anti-alkaline phosphatase method was applied using a monoclonal mouse-antibody against ACE, clone CG2 (BMA Biomedicals). As in situ control for cell damage, the endothelial marker CD 31 was stained (Dako, Hamburg, Germany). Statistical analysis. For cell culture experiments, means and standard deviations were calculated from several independent measurements. Testing for significant differences between means was carried out using one-way ANOVA at a probability of error of 5%, 1%, and 0.1%. For immunohistochemistry staining intensity (evaluated by two examiners) and the percentage of positively stained endothelial cells per vessel were expressed by a score based on the internationally accepted immunoreactivity score (Remmele and Schicketanz 1993). For comparison and statistical analysis of the mathematical means, SPSS software (version 12.0) was used. A p value of p < 0.05 was defined as statistically significant. Results ACE activity. The activity of ACE was measured in a homogenate of cultured HPMEC or HUVEC using the ACE substrate (Hip-His-Leu) after 24 h treatment with increasing concentrations of LPS. For both cell types, HPMEC and HUVEC, ACE activity of the LPS treated cells, expressed as percentage of non-treated control cells, decreased in a concentration-dependent manner (Fig. 1). No significant differences in ACE activity reduction were measurable between HPMEC and HUVEC after 24 h treatment with 0.001-1 µg/ml LPS. Membrane-bound ACE. The level of expression of ACE on the surface of cultured cells was detected by cell enzyme-linked immunosorbent assay. Figure 2 shows the surface expression of ACE for LPS- and PMA-treated cells calculated as percentage of non-treated control cells. Two of three HPMEC isolates and both HUVEC donors investigated showed a concentration-dependent decrease in anti-ACE binding after stimulation with LPS compared to the untreated control. Comparing HUVEC and HPMEC, the loss of membranous ACE was significantly (p <0.01) higher in HPMEC cells. Treatment of HPMEC with 1 µg/ml LPS for 24 h caused a membranous ACE staining of 80.8 ± 24.6% of untreated control. In HUVEC, this concentration yielded a reduction down to 89.7±6.7% of untreated control. The positive control PMA (100 ng/ml) induced an increase of ACE labeling for both cells types. Two of three HPMEC isolates increased membranous ACE after PMA treatment highly significantly (p <0.001) to 192±9.4% of untreated control. Real-time PCR. Relative quantitation of ACE mRNA expression in cells using rtPCR demonstrates a concentration-dependent reduction in ACE mRNA expression after 24 h stimulation with 0.001-1 µg/ml LPS for all three HPMEC isolates investigated (Fig. 3). After treatment with 1 µg/ml LPS for 24 h, the ACE mRNA expression of HPMEC was reduced to 0.5-fold of the non-treated control. In contrast, ACE mRNA expression in HUVEC cells was not significantly reduced until addition of 1 µg/ml LPS. A lower concentration of 10 ng/ml LPS on the other hand caused an increase of ACE mRNA expression in HUVEC. The positive control PMA (100 ng/ml) significantly (p <0.001) increased ACE mRNA in HUVEC to 1.6-1.8-fold of untreated control. HPMEC reacted differently in that two of the three donors showed a highly significantly (p <0.001) reduced ACE mRNA expression upon treatment with 100 ng/ml PMA. Shedded ACE. In addition to the differences found between HPMEC and HUVEC in mRNA expression, both cell types differed in shedding of ACE into the culture supernatant after 24 h treatment with 0.001-1 µg/ml LPS. HPMEC showed an increased shedding of ACE with increasing concentrations of LPS whereas minimal amounts of ACE were detected in the culture supernatant of HUVEC (Fig. 4). Exposure to 1 µg/ml LPS caused an average release of 1,990 ± 275 pg/ml ACE to the culture supernatant of HPMEC. On the contrary, for HUVEC on average 221 ± 38 pg/ml, shedded ACE was not measurable until treatment with 1 µg/ml LPS. Cell morphology after exposure to LPS and PMA was not altered compared to controls (crystal violet staining data not shown). Immunohistochemistry. A vessel type-specific ACE immunostaining was found in lung specimens with regular morphology which was identical in the sepsis group. Endothelial cells of alveolar capillaries displayed the strongest ACE immunostaining, followed by arterioles and arteries, while endothelial cells of venules and veins showed next to no staining. In the septic specimens, ACE immunostaining was significantly reduced (p =0.001) (Fig. 5). A decrease of ACE immunostaining due to damaged endothelium and/or endothelial cell losses was excluded by staining of CD31 (data not shown), which was uniformly expressed along the vascular tree and in the capillary networks. Discussion Several groups have attempted to use ACE as an indicator of pulmonary vascular injury (Atochina et al. 1997; Orfanos et al. 2000; Cziraki et al. 2002). Increase of ACE activity in the plasma and a reduction of activity in lung tissue have been reported as consequences of endotoxin-induced lung damage in the mouse and rat (Hollinger 1983; Votta-Velis et al. 2007). Orfanos and co-workers described a reduced ACE expression in ALI in humans mainly caused by trauma, pneumonia, or multiple trauma and in a few cases by sepsis. Whether decreased serum ACE in patients with septic ARDS (Fourrier et al. 1985;Marshalletal.2002) is due to endothelial damage, shedding, reduced enzyme activity, and/or reduced ACE expression has not yet been studied in detail. According to their localization in the vasculature endothelial cell, ACE expression varies (Metzger et al. 2011). Once again, we could demonstrate here the existence of a vessel-type specific ACE expression pattern for pulmonary vessels (Muller et al. 2004). Our data are in accordance with a recent report of Metzger et al. (2011)who found all EC of lung capillaries and small and large pulmonary arteries strongly expressed ACE in contrast to EC of the large pulmonary veins, which completely lacked the enzyme. The functional significance of this distribution in respiratory disease is not yet clear, but may contribute to the pathology of ALI. While in septic lung tissue the vessel-type specific ACE expression pattern persisted, the labeling intensity was significantly reduced. To explain this reduced ACE expression, we investigated primary isolated HPMEC, the cell type that simulates the local systems component of RAAS, under septic conditions in vitro. In this study, we focused on the effects of sublethal doses of LPS (1 ng/ml- 1 µg/ml) on ACE distribution in HPMEC. According to our in vitro results, we hypothesize that reduced pulmonary endothelial ACE expression in sepsis is caused by two LPS-induced processes: (1) a transient increased shedding of ACE and (2) a reduced ACE-mRNA expression. These two processes have to be discussed as reasons for our findings of reduced ACE immunostaining in septic lung tissue. Initially, augmented shedding of ACE is shown in several animal studies after endotoxic lung injury (Cookson et al. 1985; Votta-Velis et al. 2007). According to Cookson et al. (1985), intraperitoneal injection of E. coli endotoxin in mice caused a significant but transient increase in serum ACE after 6 h which was then followed by a fall. The yet missing description of a transient ACE increase in human serum can be explained by the fact that septic patients are normally not hospitalized at the very onset of sepsis so that this process is not observed. In in vivo studies, a damaged endothelial surface has been discussed as a cause of decreased ACE serum levels in septic patients (Fourrier et al. 1985; Marshall et al. 2002). In this context, Fourrier and co-workers (1985) explained the return of decreased to normal ACE serum levels within the first 3 days of sepsis as evidence of endothelial repair. In any case, destructive endothelial damage, for a long time discussed as the reason for decreased serum ACE, is according to our findings not the main mode of the endotoxin effect. We could exclude endothelial damage by positive CD31 staining of pulmonary endothelium in septic lungs. Additionally, no endothelial losses were detected in endothelial cultures treated with concentrations up to 1 µg/ml LPS, although decreased ACE expression was induced. In vitro studies on the effect of endotoxin on human ACE expression were carried out in macrovascular endothelium (Watanabe et al. 1992; Watanabe and Jaffe 1993; Balyasnikova et al. 1998), although it is well established that ACE distribution during inflammation largely takes place in the microcirculation (Orfanos et al. 2000). For this reason, we compared the effects of LPS on HPMEC and the commonly used macrovascular HUVEC. In regard to ACE activity, HPMEC and HUVEC reacted similarly on LPS exposure with a dose-dependent reduction. ACE activity in intact HPMEC and HUVEC monolayers incubated with LPS (1 µg/ml) decreased markedly with time and was inhibited by 15% after 24 h when compared with untreated control cells. Watanabe and co-authors additionally demonstrated a time and E. coli serotype specificity of ACE activity reduction in HUVEC cells (Watanabe et al. 1992). After treatment with LPS, HPMEC and HUVEC monolayers reduced ACE immunostaining in a concentration-dependent fashion compared to the untreated control. The loss of membranous ACE was significantly (p < 0.01) higher in HPMEC cells compared to HUVEC cells. Additionally, LPS-activated HPMEC strongly decreased ACE mRNA level transcription, an effect associated with the decreased ACE immunostaining seen by HPMEC after 24 h LPS treatment. Although the reduction in ACE activity was not distinctly different between micro- and macrovascular cells, in the case of ACE shedding, both cell types behaved in an entirely different manner upon LPS treatment. A concentration of 10 ng/ml LPS already caused a release of ACE from the cell surface of HPMEC while only a 100-fold higher concentration of LPS induced a shedding of ACE by HUVEC to a comparable extent. The concentration of 1 µg/ml LPS caused a ninefold elevated amount of ACE in the culture supernatant of HPMEC. The maximal rate of ACE shedding after 1 µg/ml LPS treatment recorded in our in vitro experiments was 545±61 pg soluble ACE/24 h/cm2 of confluent HPMECs. Taking into account that the surface area of capillary endothelium in the lungs of a healthy individual is evaluated at 120 m2 (Simionescu 1980), such a shedding together with a decreased mRNA production of ACE would represent a great loss of local ACE activity. Muzykantov et al. (1991) have found in vivo in rats that LPS-induced mild or middle pulmonary edema is accompanied by a decrease in specific pulmonary uptake of radiolabeled ACE antibody. Additionally, the ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 h after endotoxin infusion. Similar results were found by the group of Atochina et al. (1992) working with platelet activating factor-induced pulmonary alterations. They assume that endothelial injury and enhanced ACE shedding may be one of the reasons for reduced ACE antibody uptake in injured lung endothelium (Atochina et al. 1992). The results obtained for human septic lung tissue in this study are in accordance with both animal studies. In addition, the reason for the reduced ACE activity and staining in HPMEC cells after LPS treatment in the present study was not endothelial damage but a reduced mRNA expression of ACE in combination with increased shedding of ACE. Thus, measurement of ACE expression and shedding by pulmonary microvascular endothelial cells is a potent candidate as specific marker of lung endothelial activation and/or injury upon exposure to LPS and other insults. Recently, investigators have focused on the mechanism of ACE shedding (Ehlers et al. 2012). Although there has been biochemical characterization of the basic properties of ACE sheddase activity, ACE sheddase has not as yet been identified. It is known that ACE shedding is primarily metalloproteinase dependent and can be blocked by inhibitors of the MMP or ADAM families. English and co-workers recently found that LPS activates ADAM9-dependent shedding of ACE from HUVEC (English et al. 2012). The dynamics between the membrane-bound and soluble forms of ACE in the local and systemic renin-angiotensin systems are complex, and a better understanding of the role played by the ACE sheddase(s) is still required. For these reasons, choosing the right organ-specific vessel type is imperative in studying endothelial ACE shedding in vitro. We suggest that HPMEC cells are a suitable in vitro model to investigate the regulatory control of pulmonary endothelial ACE expression in more detail. Conclusion In summary, we propose that two mechanisms, namely increased shedding and reduced endothelial mRNA expression of ACE, are responsible for reduced pulmonary ACE expression in sepsis. These facts not only explain reduced vasoconstriction in sepsis but also persistent vasodilatation due to reduced degradation of bradykinin. Investigating the regulatory control of pulmonary microvascular endothelial cells could provide new insight into the physiology of pulmonary ACE distribution and might lead to new therapeutical approaches. Acknowledgments The authors wish to thank Mrs. M. Moisch and Mrs. A. Sartoris for their excellent assistance with the cell culture and the real-time PCR. References Aird W. C. 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Hermanns (*) IKFE GmbH, Cell Biology, Parcusstr. 6, 55116Mainz, Germany e-mail: [email protected] A. M. Müller Department of Pediatric Pathology, University Clinic, Bonn, Germany M. Tsokos Institute of Legal Medicine, University Clinic Charite Berlin, Berlin, Germany C. J. Kirkpatrick Institute of Pathology, University Medical Center of the J. Gutenberg University Mainz, Mainz, Germany (c) 2014 Society for In Vitro Biology |