Angiogenesis and hypertensive retinopathy -
Supernatants were collected, and total protein was quantified by bicinchoninic acid assay Pierce, Rockford, IL according to the manufacturer's protocol. All procedures were performed according to the manufacturers' instructions. Each sample μL was run in duplicate and compared with a standard curve.
Another eight mice from each group were used for immunohistochemical staining. According to a previously described method, 37 , 38 more than 50 serial 6-μm paraffin-embedded axial sections were obtained, starting at the optic nerve head.
Immunohistochemistry was performed in 6-μm retina sections. Endogenous peroxidases were quenched with 0. Eyes were enucleated and embedded in OCT compound Tissue-Tek; Sakura Fine Technical, Torrance, CA overnight for cryosectioning, and then μm serial sections were cut.
Frozen sections of mouse eyes were dried at room temperature and postfixed in cold acetone for 10 minutes. Slides were incubated with primary antibodies mouse monoclonal anti—glial fibrillary acidic protein [GFAP] antibody [GA5; Cell Signaling Technology, Beverly, MA] and rabbit anti—NGF [BioWorld, Dublin, OH] antibody overnight at 4°C.
Fluorescence pictures were taken with identical exposure settings. For negative control, slides stained without primary antibodies showed no signals. Another 40 mice underwent the OIR procedure for intraocular injections. On P12, when these OIR mice were returned to room air, they were divided randomly into two groups to receive intravitreal injections of NGF, or Ka TrkA receptor inhibitor in the right eye, and control vehicle PBS with 0.
One drop of local anesthetic was administered before injection. Intravitreal injections were performed using a 5-μL Hamilton syringe with a gauge needle, according to a previously described method. Because reflux of a certain amount of intraocular fluid is unavoidable when the pipette is removed from the injection site, the pipette was kept in place for 10 seconds to allow diffusion of the solution.
The reagent was diluted in sterile PBS. In literature reports, NGF intraocular injection has usually been conducted in an animal model to study its neuroprotective function. Technical considerations in these neonatal mice precluded testing of more concentrated solutions or more frequent administration.
Quantitation of Neovascular Cell Nuclei Anterior to the Internal Limiting Membrane. More than 50 serial 6-μm paraffin-embedded axial sections were obtained starting at the optic nerve head.
All retinal vascular cell nuclei anterior to the internal limiting membrane were counted in each section using a fully masked protocol. The mean of all 10 counted sections yielded average neovascular cell nuclei per 6-μm section per eye.
No vascular cell nuclei anterior to the internal limiting membrane were observed in normal, unmanipulated animals. Quantification of Retinal Neovascularization in the Flat-Mounted Retinas. The anterior segment was cut, and the neurosensory retina was carefully removed.
The retina was cut radially and flat-mounted in glycerin. Retinal whole mounts were examined by fluorescence microscopy BH2-RFC; Olympus, Hamburg, Germany. Total images of flat-mounted retina were produced from 9 to 12 pieces of images acquired using a fluorescence microscope BX50; Olympus, Tokyo, Japan.
Images were obtained using a high-resolution charge-coupled device camera DP30BP; Olympus with a computerized system. According to a previously described method, 45 — 48 quantitative analysis of the vasculature and vascular obliteration were performed on the flat-mounted, fluorescein dextran—perfused retinas from groups with NGF or Ka injection and from PBS-BSA or PBS controls by using a computerized system with imaging software Image-Pro Plus 5.
In brief, the area of vascular obliteration was measured by careful delineation of the avascular zones in the central retina of the fluorescein dextran—perfused retinas by two investigators using a fully masked protocol.
Similarly, the area of neovascularization tufts was carefully delineated by using confocal images focused on the preretinal plane and selecting tufts based on pixel intensities. Selected regions were then summed to generate the total area of neovascularization and calculating the total area using imaging software Image-Pro Plus 5.
Student's t -test was used to statistically compare the different experimental groups. Data are reported as mean ± SEM. Statistical analysis was performed with statistical software SAS Institute Inc. Differences among the groups were assessed by ANOVA to compare the level of NGF expression.
For the study of intravitreal injection, data were analyzed with the use of a t -test to compare the difference between two groups. All reported P values were two-sided.
Increased NGF mRNA Expression in at P17 OIR Model. Results of real-time PCR data are represented as Ct values, where Ct is the threshold cycle at which the amplified product is initially detected.
To correct differences of transcriptional activity of NGF between samples for quantification analysis, the target gene was normalized to the internal standard GAPDH gene. We used the comparative Ct method 49 and analyzed RNA expression in samples relative to that of the control in each experiment.
The comparative transcriptional activity of the NGF gene is summarized in Figure 1. There was no significant difference of NGF mRNA expression in the retina after oxygen induction at P12; however, a significant increase in NGF mRNA expression occurred at P17 in the OIR model compared with the normal control 0.
At P24, NGF mRNA expression was decreased in OIR mice; there was no significant difference between the OIR mice and the normal controls at P Figure 1.
View Original Download Slide. NGF mRNA expression in OIR model. Histograms represent quantification of real-time PCR analysis of NGF mRNA expression. The average value for each sample was normalized to the amount of GAPDH.
There was no significant difference in NGF mRNA expression in the retina after oxygen induction at P12 0. However, a significantly large increase in NGF mRNA expression occurred at P17 in the OIR model compared with normal controls 0.
At P24, NGF mRNA expression decreased in the OIR group 0. Increased NGF and VEGF Protein Expression in OIR Model. Similar to mRNA expression, the data from ELISA showed significantly increased NGF expression in the retinas of OIR mice beginning at P14 NGF levels in the retinas of OIR mice are shown in Figure 2.
Figure 3 shows representative pictures of retinal slides from the OIR model and normal controls with immunostaining. NGF-positive staining was seen in both the OIR model and normal controls. Vascular cell nuclei above the inner limiting membrane are typically indicated with arrows in retinal paraffin sections of the OIR mouse.
Figure 2. Time course of NGF content in retinas of OIR mice. Data from ELISA assay showed significantly increased NGF expression in the retina beginning at P14 and reaching the highest peak at P17 in OIR mice P Figure 3. NGF immunohistochemistry in 6-μm retinal paraffin sections. A Retinal slide from OIR mice at P B Retinal slide from normal control at P Neovascular cell nuclei above the inner limiting membrane represent areas of retinal neovascularization and are indicated with arrows A.
No vascular cell nuclei anterior to the internal limiting membrane are observed in normal, unmanipulated animals at P17 B. Figure 4. Immunofluorescence-stained pictures labeled with NGF and GFAP in μm retinal cryosections.
A — C Astrocytes and neurons localized in GCL, INL, and OPL produced NGF, in particular in the ischemic retina at P D — F Retinal cryosections from normal control. There is more intensive NGF and GFAP positive staining at P17 in OIR mice.
Green : NGF staining. Red : GFAP staining. Blue : nuclear staining. Yellow : staining denotes overlap of green and red. Data from ELISA showed that there is also significantly increased VEGF expression in retinas both at P17 Figure 5.
VEGF content in retina of OIR mice. Data from ELISA showed that there is significantly increased VEGF expression in retinas both at P17 and at P24 in OIR mice, respectively, compared with normal control P Average neovascular cell nuclei anterior to the internal limiting membrane per 6-μm histologic section per eye were determined.
Figure 3 is a representative photomicrograph illustrating neovascular cell nuclei anterior to the internal limiting membrane. Figure 6 summarizes the comparison of average neovascular cell nuclei above the internal limiting membrane among different groups. The number of neovascular cell nuclei in the NGF group is significantly increased compared with the counterpart control.
Interestingly, however, the number of neovascular cell nuclei is significantly decreased in the Ka injection or anti—NGF blocking antibody data not shown group compared with PBS control. Figure 6. Comparison of average neovascular cell nuclei above the internal limiting membrane.
The number of neovascular cell nuclei is significantly increased in the NGF group Measurement of Retinopathy Neovascularization in the Flat-Mounted Retinas.
We successfully established an oxygen-induction ischemic retina model. The retina showed the typical appearance of ischemic retinopathy at P12 and P17 Figs. Figures 7 C to 7 F illustrate the fluorescein dextran—perfused retinas from day-old mice that had been exposed to hyperoxia from P7 to P12 and had received intravitreal injections.
Supplementary Figure S1 , shows how we defined the neovascularization area in a high-resolution image. According to the profile of vasculature node, node area, and tube length, as well as blood vessel tortuosity, the neovascularization tufts area was carefully delineated with a red line by using confocal images focused at the preretinal plane and selecting tufts based on pixel intensities.
Selected regions were then summed to generate the total area of neovascularization, and the total area was calculated with imaging software Image-Pro Plus 5.
The data showed that retinal neovascularization was significantly enhanced after injection with NGF; however, a mild decrease in neovascularization was found in Ka-injected mice compared with the counterpart control Fig.
A limitation of the study was the use of only fluorescein injection, which could have underestimated nonperfused tufts; fluorescein injection plus isolectin staining may be used in neovascularization quantitative analysis to provide more accurate evaluation of the neovascularization. Figure 7.
Representative pictures of the retinal flat-mount with fluorescein-dextran perfusion. A , B Typical appearance of ischemic retinopathy at P12 and P17 of the OIR model. A Central avascular area is a typical feature of the OIR model at P B Blood vessel tufts, presumed extraretinal neovascularization, and blood vessel tortuosity are shown obviously at P C — F Fluorescein dextran—perfused retinas from P17 OIR mice that had received intravitreal injections at P C Retina of NGF-treated eye, showing markedly angiogenic features with extensive presumed extraretinal neovascularization, blood vessel tufts, and tortuosity.
D Retina of the control eye with PBS-BSA injection. E Retina of Ka-treated eye has comparatively lower angiogenesis with obvious avascular area compared with the control eye. F Retina of the contralateral control eye with PBS injection.
Figure 8. Average retinal neovascularization area. There is a significantly enhanced neovascularization after injection with NGF 1. Studies have shown that ischemia induces significant VEGF upregulation and have suggested that VEGF may be the dominant angiogenic stimulus in retinal neovascularization 50 ; however, roles for other proangiogenic and angiogenic factors, such as platelet-derived growth factor, erythropoietin, stromal cell-derived factor-1, and basic fibroblast growth factor, cannot be ruled out.
NGF abundantly produced and used by retinal ganglion cells RGCs , bipolar neurons, and glial cells 57 is well known for its role in regulating survival, growth, and functional maintenance of RGCs, photoreceptors, and other retinal neurons. The protective function of NGF intraocular administration has been clearly demonstrated in experimental models of ischemic, traumatic, hypertensive injury, 42 , 43 , 59 and NGF was recently reported to exert neuroprotective effects by inhibiting the apoptosis of RGCs.
NGF, alone or in combination with other biologically active molecules, can have an effect on endothelial cells and on angiogenic activity. S2 provides evidence to support that NGF may contribute to retinal neovascularization by acting on endothelial cells.
NGF, as a new potent angiogenic factor, has been shown to enhance VEGF production, 25 , 28 , 63 , 64 although Colafrancesco et al. Both NGF and VEGF have recently attracted special interest because they are critically involved in the survival and protection of brain and retinal cells 66 , 67 and because they have reciprocal angiogenic and neurotrophic effects on blood vessels and neurons.
Although the increased VEGF expression was not significantly correlated with NGF upregulation by a linear regression correlation analysis in our study, it is possible that the enhanced expression of retinal VEGF might be linked to the local upregulation of NGF.
Park et al. Coordinated actions of NGF and VEGF have been demonstrated in different tissues under various physiological conditions. Thus, based on the available data, we propose that there is a dual role for these angiogenic and neural factors. Early release of these growth factors in response to retinal ischemia might be a protective reaction to maintain neuron survival, but this beneficial response may have the secondary effect of inducing excessive angiogenesis in the retina.
It should be noted that vascularization also plays a pivotal role in neuronal protection. Angiogenesis is a complex process regulated by many growth factors.
The multiple regulatory components interact and modulate their individual effects and are further regulated by physiological stimuli. Our data suggest that VEGF is not the sole major mediator of OIR-associated pathologic neovascularization but that NGF also contributes to the process.
The increased release of NGF, similar to that of VEGF, is also a protective response to ischemia. The present study provides a basis for further studies on the role of NGF and other neurotrophic factors such as brain-derived neurotrophic factor and glial-derived neurotrophic factor in ischemic vasculopathies and their interactions with other angiogenic factors.
The major goal of this study was to explore the secondary effect of NGF as an enhancer of angiogenesis. Preretinal angiogenesis and intraretinal angiogenesis Supplementary Fig. S3 were enhanced in the OIR mouse after treatment with NGF, although statistical analysis regarding intraretinal angiogenesis could not be performed because of a limited number of samples.
The finding of significantly higher efficiency of tube formation of retinal vascular endothelial cells in culture after treatment with NGF revealed the potential mechanism of angiogenic action of NGF. We also investigated the role of NGF to understand the process of neuron-driven angiogenesis in retinopathy.
Feit-Leichman et al. Ali 58 reported a twofold increase in NGF expression in diabetic retinas from both human and streptozotocin-induced diabetic rats. Recently, serum and tear NGF levels were found to be higher in patients with proliferative diabetic retinopathy than in nondiabetic control patients.
Our results suggest that NGF contributes to retinal neovascularization either by acting on endothelial cells or by upregulating other angiogenic factors, such as VEGF.
Our study also provides a potential explanation for the unusual neovascularization that occurs in the retinal vasculopathic complications of diabetes mellitus because the vasculopathies that affect the ischemic myocardium and limbs in this disease are, paradoxically, characterized by an insufficient angiogenic response.
In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. Although the relationship between neovascularization and neuron damage has received much attention, 75 a direct relationship remains unclear.
Other neurotrophic factors or neuronal-related factors have been reported to be specifically upregulated in vascular abnormalities or to act on vascular endothelial cells. In addition to its primary role in preventing neuronal cell death, it has a secondary effect of increasing angiogenesis.
Ischemia leads to NGF release, which protects neurons but may exacerbate abnormal vessel growth. This supports a link between retinal neovascularization and neuronal damage through cytokines, such as NGF and VEGF, those pleiotropic factors that affect both vessels and neurons.
Further studies are needed to better understand the role of cytokines in the link between retinal vascular abnormalities and neuronal tissue. Intravitreal injection with either Ka or neutralizing anti—NGF antibodies reduced neovascularization in our OIR model.
TrkA is responsible for the survival and growth properties of neurotrophins, 33 , 77 — 79 and Ka is a high-affinity TrkA receptor inhibitor. Ka can reduce NGF-induced vascular endothelial cell activity by blocking the Trk A receptor. Similarly, it has been previously shown that oral treatment with a tyrosine kinase inhibitor can reduce retinal neovascularization in the OIR mouse model.
Our aim was to elucidate whether VEGF, PEDF, P RR and AT1R could be modified during PE and during hypertension induced in rats with a history of PE.
We used female Wistar rats and subrrenal aortic coarctation to induce PE, and after delivery, we induced a second hit by Nω-nitro-L-arginine methyl ester L-NAME administration.
We measured blood pressure, proteinuria and pups development. In both models, eye fundal exploration and immunoblot for VEGF, PEDF, P RR and AT1R were performed. We found that the development of hypertension occurred faster in previously PE rats than in normal animals.
In the vitreous fluid of active DR, the concentration of VEGF was high, but the concentration of endostatin was low thick line. On the other hand, in the vitreous fluid of quiescent DR, the concentration of VEGF was low but the concentration of endostatin was high thin line.
Correlation between the concentrations of vascular endothelial growth factor VEGF and endostatin. Noma H , Funatsu H , Yamashita H , Kitano S , Mishima HK , Hori S. Regulation of Angiogenesis in Diabetic Retinopathy : Possible Balance Between Vascular Endothelial Growth Factor and Endostatin.
Arch Ophthalmol. From the Departments of Ophthalmology, Diabetes Center, Tokyo Women's Medical University, Tokyo Drs Noma, Funatsu, and Kitano , Hiroshima University Medical School, Hiroshima Drs Noma and Mishima , and Yamagata University Medical School, Yamagata Dr Yamashita , and Tokyo Women's Medical University Dr Hori , Japan.
Objective To investigate the mechanisms of regulation between vascular endothelial growth factor VEGF as a stimulator and endostatin as an inhibitor of angiogenesis in diabetic retinopathy DR. Methods One hundred fifty-nine eyes of diabetic patients were studied. Concentrations of VEGF and endostatin in vitreous fluid and aqueous humor, obtained from the eyes during ocular surgery, were measured by enzyme-linked immunosorbent assay.
The severity of DR was quantified according to the Early Treatment Diabetic Retinopathy Study retinopathy severity scale; fundus findings, including soft exudates, intraretinal microvascular abnormalities, venous abnormalities, new vessels elsewhere, new vessels on the disc, vitreous hemorrhage, and retinal detachment, were graded and evaluated.
Concentrations of VEGF and endostatin in plasma were also measured by enzyme-linked immunosorbent assay. Main Outcome Measures Concentrations of VEGF and endostatin in vitreous fluid and plasma. The correlations among the clinical records and the levels of VEGF and endostatin were analyzed statistically.
The concentrations of VEGF and endostatin in the eyes were not correlated with those in the plasma. Conclusions These results show that both VEGF and endostatin are correlated with angiogenesis in DR.
Our study suggests that the regulation mechanism between VEGF and endostatin is associated with the activity of DR and may be a good candidate to develop useful therapeutic agents for proliferative DR.
NEW VESSEL formation in diabetic retinopathy DR causes visual loss with vitreous hemorrhage, retinal detachment, and neovascular glaucoma. Various cytokines and growth factors are considered to be involved in these processes and the pathogenesis of angiogenesis.
It is hypothesized that the net balance between angiogenic stimulators and inhibitors regulates the switching of the angiogenic process. Vascular endothelial growth factor VEGF acts as an endothelial cell mitogen 9 , 10 in vitro and induces increased vascular permeability 11 and angiogenesis in vivo.
Intraocular VEGF concentrations are increased during the periods of active intraocular neovascularization in patients with proliferative DR. Many endogenous inhibitors of angiogenesis, including endostatin, 13 thrombospondin, 14 , 15 interferon α and β, prolactin, platelet factor 4, 16 - 18 and angiostatin, 19 have been reported.
Endostatin is an angiogenic inhibitor produced by hemangioendothelioma. In the present study, we examined the regulation mechanism between VEGF as an angiogenic stimulator and endostatin as an angiogenic inhibitor in DR and showed that endostatin may be related to angiogenesis activity in cooperating with VEGF in DR.
Samples of aqueous humor and vitreous fluid were obtained from eyes of diabetic patients whose mean SD age was The patients included 77 men and 43 women. The mean SD duration of diabetes mellitus was Patients were excluded if they had undergone previous intraocular surgery or had a history of branch retinal vein occlusion and uveitis.
All procedures conformed to the Declaration of Helsinki for research involving human subjects. Ethics committee approval was obtained, and all participants gave informed consent.
The activity of DR is classified into active and quiescent. If there are extensive changes, including active neovascularization and proliferative membrane, fresh vitreous hemorrhage, and progressive retinal detachment, it is classified as active. If it becomes silent on photocoagulation, even with remaining neovascularization and proliferative membrane, it is classified as quiescent.
The results ensured that the levels of factors in intraocular fluid and plasma samples were within the detectable range using these assays.
The minimum detectable concentrations sensitivity using the assay kits were The preoperative and operative findings were recorded. Clinical data, including the severity of DR, were obtained by the surgeon using standardized forms at the time of surgery and were confirmed by standardized fundus color photography and fluorescein angiography performed within 3 days after the operation.
The severity of DR was graded according to the modified Early Treatment Diabetic Retinopathy Study ETDRS retinopathy severity scale. Results are presented as mean ± SD or geometric mean ± SD for data shown on the logarithmic scale. To determine the relationship between angiogenic factors and the ETDRS retinopathy severity, Spearman rank-order correlation coefficient was applied.
To test the heterogeneity of slopes of 2 linear regression lines, analysis of covariance with interaction was used. In this model, variables were analyzed on the logarithmic scale because of skewed distribution. Two-tailed P values of less than. The concentrations of VEGF in the aqueous humor geometric mean, The concentrations of endostatin in the aqueous humor mean, 2.
The concentration of VEGF in vitreous fluid was significantly correlated with the grades of soft exudate, IRMA, venous beading, venous loops, NVE, NVD, FPE, and vitreous hemorrhage Table 1.
The concentration of endostatin in vitreous fluid was significantly correlated with the grades of NVE, FPE, and retinal detachment Table 1. The VEGF levels were higher in the vitreous fluid of active DR than in the vitreous fluid of quiescent DR.
The endostatin levels were lower in the vitreous fluid of active DR than in the vitreous fluid of quiescent DR. On the other hand, in the vitreous fluid of quiescent DR, the concentration of VEGF was low but the concentration of endostatin was high. There was no significant correlation between the plasma concentration of VEGF or endostatin and the severity of DR data not shown.
The concentration of VEGF in neither the aqueous humor As for endostatin, no significant relationship was found between its concentration in aqueous humor 2. We obtained the following findings in this study.
First, in patients with DR, endostatin was detected in aqueous humor and vitreous fluid. Second, the endostatin and VEGF concentrations in aqueous humor and vitreous fluid were correlated with the severity of DR.
Third, some of the patients with severe DR showed a high endostatin concentration in vitreous fluid, but others showed a low concentration. Fourth, the slope of the regression line between the VEGF and endostatin concentrations in vitreous fluid differed significantly between active DR and quiescent DR.
There was a significant positive correlation between the severity of DR and the VEGF concentration in the aqueous humor and vitreous fluid in the present study. These results were consistent with the findings of previous reports.
These results suggest that VEGF stimulates angiogenesis in the pathology of DR. Our results and the previous reports suggest that VEGF is associated with enhanced vascular permeability, vascular occlusion, and angiogenesis. The severity of DR was also positively correlated with the endostatin concentrations in both aqueous humor and vitreous fluid.
Of the fundus findings, the grades of NVE, FPE, and retinal detachment were positively correlated with the endostatin concentration in vitreous fluid Table 1. In the aqueous humor and vitreous fluid, the VEGF concentration was positively correlated with the endostatin concentration.
These results suggest that endostatin expression is correlated with VEGF expression. The theory that the balance between stimulators and inhibitors is critical in the process of tumor angiogenesis has been proposed. Endostatin is an angiogenic inhibitor produced by hemanigioendothelioma.
Endostatin has also been reported to inhibit VEGF-stimulated endothelial cell proliferation and migration.
In this study, the endostatin concentration in intraocular fluid and blood samples was evaluated. Although the endostatin concentration in both aqueous humor and vitreous fluid showed a significant positive correlation with the severity of DR, the endostatin concentration in aqueous humor was high even in some patients with mild DR and varied widely in patients with severe DR, showing a uniform distribution independent of the severity of DR.
The endostatin concentration in vitreous fluid varied more widely as the severity of DR increased.
Xialin Liu Breakfast nutrition tips, Dingding WangYizhi AngiogebesisYan LuoWei MaWei XiaoQiang Yu; Hyperfensive Angiogenesis and hypertensive retinopathy Rstinopathy of NGF hyperrensive Retinal Neovascularization in an Oxygen-Induced Retinopathy Model. NGF mRNA annd in andd was measured Immunity strengthening nutrients Breakfast nutrition tips real-time PCR. NGF expression in Retinopathyy levels was evaluated by ELISA and immunostaining with NGF antibody. The effects of NGF on retinal neovascularization were evaluated by intravitreal injections of exogenous NGF and TrkA receptor inhibitor Ka, respectively, in an OIR model. Retinal neovascularization was measured by counting neovascular cell nuclei above the internal limiting membrane and by image quantification analysis in flat-mounted retinas perfused with fluorescein dextran. Similarly, ELISA and immunostaining assay showed significantly increased NGF expression in retina at P17 in OIR mice but no significant differences at P12 or P24 compared with normal controls. Exogenous NGF intraocular injection enhanced angiogenesis in the retina in the OIR model; however, injection with Ka, a high-affinity trkA receptor inhibitor, significantly decreased retinal neovascularization compared with that seen in the controls. Fetinopathy more information about PLOS Subject Breakfast nutrition tips, click here. Hypertension is the most Diet for injury healing cardiovascular disease and the main risk factor for stroke, Hyprtensive arterial disease, arterial Breakfast nutrition tips and kidney hypretensive. It has been reported recently that hypertensive patients and animals are characterized by decreased density of arterioles and capillaries in the tissues, called rarefaction. Rarefaction significantly increases peripheral resistance which results in elevated blood pressure, leads to vessel damage and induction of inflammation. Therefore, we hypothesized that hypertension is associated with decreased serum concentration of physiological pro-angiogenic factors and concomitant increased production of angiogenesis inhibitors.
Ich entschuldige mich, aber diesen ganz anderes. Wer noch, was vorsagen kann?
Ich denke, dass Sie den Fehler zulassen. Ich biete es an, zu besprechen. Schreiben Sie mir in PM, wir werden umgehen.