Human primary chondrocytes exhibit an antiangiogenic effect despite of high secretion of VEGF

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1 Gene Therapy and Molecular Biology Vol 13, page 144 Gene Ther Biol Vol , 2009 Human primary chondrocytes exhibit an antiangiogenic effect despite of high secretion of VEGF Research Article Peter Camaj 1 *, Ivan Ischenko 1 *, Brian Ziegelaar 2, Dagmar Faktorova 3, Markus Guba 1, Karl-Walter Jauch 1, Christiane J. Bruns 1 1 Department of Surgery, Munich University Medical Center, Campus Großhadern, Marchioninistr 15, Munich, Germany 2 Department of ENT, Munich University Medical Center, Campus Großhadern, Marchioninistr 15, Munich, Germany 3 Department of Measurement and Applied Electrical Engineering, University of Zilina, Zilina, Slovak Republic *Correspondence: Peter Camaj and Ivan Ischenko, Department of Surgery, Munich University Medical Center, Campus Großhadern, Marchioninistr. 15, Munich, Germany; Tel: ; Fax: ; Peter.Camaj@med.unimuenchen.de Keywords: anti-angiogenic effect, chondrocytes, chondrosarcoma, VEGF Abbreviations: ELISA: Enzyme-linked immunosorbent assay; FCS: foetal calf serum; HIF: Hypoxia inducible factor; HRP: horseradish peroxidase; HUVEC: human umbilical vein endothelial cells; MTT: 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; OD: optical density; PHC: primary human chondrocytes; SMC: smooth muscle cells; VEGF: vascular endothelial growth factor. Received: 20 March 2009; Revised: 12 May 2009 Accepted: 21 May 2009; electronically published: May 2009 Summary Cartilage tissue is one of the few avascular tissues in humans. This fact suggests that healthy cartilage is capable to prevent the influence of angiogenesis driven by environmental stimuli such as low oxygen partial pressure and changes in ph. The aim of this work was to demonstrate and to quantify the anti-angiogenic potential of normal human chondrocytes in comparison to de-differentiated human chondrocytes and chondrosarcoma cells. We found that conditioned medium of primary human chondrocytes exhibits strong anti-angiogenic properties (inhibition of human umbilical vein endothelial cell proliferation and sprouting). The effect was specific to differentiated chondrocytes, whereas it was absent in conditioned medium from de-differentiated chondrocytes. Furthermore, we revealed that the effect was specifically anti-angiogenic but not generally anti-proliferative, when we compared the effect of the above mentioned conditioned media on proliferation of human umbilical vein endothelial cell versus smooth muscle cell. In addition, we found that the inhibition of HUVEC proliferation caused by conditioned medium of differentiated chondrocyte was even stronger than the anti-proliferative effect of Rapamycin treatment. In contrast, we could demonstrate that conditioned medium of chondrosarcoma cells SW1353 exhibits proangiogenic effects. The results from vascular endothelial growth factor ELISA revealed that the inhibition of HUVEC proliferation and sprouting caused by conditioned medium of chondrocytes was not a result of a decreased amount of secreted VEGF. In contrast to our expectations, the results demonstrated that these anti-proliferative effects of conditioned medium of differentiated chondrocytes were present despite of a very high content of VEGF together with other bioactive substances secreted by differentiated chondrocytes. This fact indicates that there might exist potential anti-angiogenic substances secreted by differentiated chondrocytes that are able to overcome the pro-angiogenic effect of VEGF. Such factors might be a very beneficial source as natural anti-angiogenic compounds bearing minimal side effects. I. Introduction Angiogenesis belongs to crucial physiological processes running in the normally developing organism as well as the pathological angiogenesis as part of carcinogenesis or inflammation processes. Some tissues in the human body such as cartilage and cornea are generally avascular. Cartilage is normally devoid of capillary networks and, with the exception of endochondral bone formation, is resistant to vascular invasion from surrounding tissues. Hypoxia is an essential environment for cartilage existence and development because it leads to an increased accumulation of cartilagespecific matrix chondroitin 4-sulfate in human dermal fibroblasts (Mizuno and Glowacki, 2005). A hypoxic environment leads to a proportional increase in glucose utilization and an increment of lactate synthesis in cultured 144

2 Camaj and Ischenko et al: Anti-angiogenic effect of human primary chondrocytes chondrocytes. The described metabolic adaptation is evidently reflected on the change of gene expression patterns induced by different hypoxic regulators such as HIF-1!. The formation of new blood vessels is a prerequisite to bone fracture healing and generally to formation of bone from cartilage and its further growth. Blood vessel invasion of cartilage is the earliest crucial step in endochondral ossification (Harper and Kalgsbrun, 1999). In the above mentioned process chondrocytes undergo well-ordered and controlled phases of proliferation, hypertrophic differentiation, mineralization of the surrounding matrix, death, blood vessel invasion, and finally replacement of cartilage by bone. Hypertrophic chondrocytes on the epiphyseal growth plate express VEGF therefore inducing angiogenesis followed by ossification (Gerber et al., 1999). Despite of the fact that some proteins responsible for the anti-angiogenic properties of cartilage were identified, the mechanisms of such effect remain unclear. The main aim of our work was to investigate the anti-angiogenic properties of chondrocyte-conditioned medium with a special emphasize on its effects on endothelial cell proliferation, sprouting and growth factor dependent survival. Here, we showed anti-angiogenic effect of the chondrocyte-conditioned medium its specificity on HUVEC cells as a target, its dependence on differential status of chondrocytes, using monitoring proliferation and sprouting of HUVEC as readout. The chondrocyte effect was semi-quantitative compared with anti-angiogenic effect of Rapamycin. Effect of malignant transformation on Antinagiogenic effect of chondrocyte was demonstrated on comparison of primary cells with proangiogenic effect of chondrosarcoma cells. Surprisingly, the strongest antinagiogenic effect was observed parallel with highest production of VEGF. II. Materials and Methods A. Cell lines and culture conditions Primary human chondrocytes were isolated from healthy human nasal septum samples obtained from surgery. The cells were released by treatment of nasal septum samples with collagenase (5,6 µg/ml) overnight and maintained in medium containing 50% D-MEM (4,5 g glucose/ml) and 50% F12 (Invitrogen, Germany) supplemented with ITS liquid media supplement (Sigma Aldrich, Germany). Human umbilical vein endothelial cells were purchased from Promocell (Germany) and cultivated in endothelial cell growth medium supplemented with an appropriate Supplement Kit (Promocell, Germany). HUVECs were maximally passaged three times after their receipt from the supplier. Smooth muscle cells were purchased from Promocell (Germany) and cultivated in endothelial cell growth medium supplemented with an appropriate Supplement Kit (Promocell, Germany). The SW1353 human bone chondrosarcoma cell line was purchased from ATCC (LGC Promochem GmbH, Germany) and cultivated in RPMI-1640 medium (Invitrogen, Germany) supplemented with 10% FCS including antibiotics. Experiments were performed in the same medium as used for the cultivation of human primary chondrocytes. All cell lines were cultivated at 37 C and 5% CO 2. B. Preparation of conditioned media of chondrocytes PHCs or SW1363 cells were cultivated in the appropriate medium over 3 days until the cell number has reached the confluence of 5"10 6 per culturing flask. One ml of medium was conditioned by cells. C. MTT cell proliferation assay 1 x 10 4 HUVECs or SMCs were plated on 96-well plates and incubated under serum-starved conditions for 24 hours to adhere. After 24 hours of attachment the original culturing medium was exchanged to conditioned or unconditioned medium (50% D-MEM + 50% F12 supplemented with ITS liquid media supplement) or endothelial cell growth medium supplemented with different growth factors. The unconditioned medium was taken in our study as a negative control of proliferation; the growth factor supplemented endothelial cell growth medium was used as a positive control of proliferation, respectively. Proliferation activities of HUVECs or SMCs were assayed 48 hour after the beginning of treatment. MTT proliferation assay was performed using the TACS MTT Cell Proliferation and Viability Assay kit (R&D Systems, Minneapolis, USA) according to the manufacturer s recommendations. Briefly, medium was exchanged to endothelial cell growth medium supplemented with the supplement pack (Promocell, Germany) containing 2% FCS. The MTT Reagent was added according to the manufacturer s recommendations to each well including controls. The plates were returned to cell culture incubator for 2 to 4 hours, and when the purple precipitate was clearly visible under the microscope the MTT Detergent was added to all wells including controls. The plates were left covered in the dark for 2 to 4 hours at room temperature. The absorbance in each well OD was measured at 570 nm in a microplate spectrophotometer. D. Sprouting assay Spheroids were generated as it has been described elsewhere (Korff et al., 2001). Briefly, 1x10 3 HUVECs per well were plated on non-adherent plates suspension culture plate 96 wells U-shape (Greiner, Frickenhausen, Germany). Endothelial Cell Medium (2% FCS, Promocell Heidelberg, Germany) supplemented with 0.25% carboxymethylcellulose was used. Cells were incubated overnight at 37 C, 5% CO 2 to create spheroids. 500 spheroids suspended in PBS were mixed with acidic collagen IV 2 mg/ml (Becton-Dickinson) and 10x medium l79 (Sigma, Germany). The ph was adjusted to 7.4. A half ml of this stock solution was mixed with 0.5 ml of Endothelial Cell Medium supplemented 40% FCS and 0.5% carboxymethylcellulose. This HUVEC suspension was put into 12-well plates in the presence of collagen matrix to generate a gel. Either conditioned medium or unconditioned medium (negative control) or different growth factors diluted in PBS were applied onto the gel surface. Sprouting was microscopically evaluated as the cumulative length of sprouts after overnight incubation at 37 C, 5% CO 2. E. Determination of human vascular endothelial growth factor concentrations in cell culture supernants The level of VEGF was measured in either conditioned media or in unconditioned negative control media. Different concentrations of conditioned media were taken. The assay was performed according to the manufacturer s recommendations (Quantikine human VEGF ELISA kit, R&D Systems, Minneapolis, USA). VEGF concentrations were then calculated 145

3 Gene Therapy and Molecular Biology Vol 13, page 146 according to the parameters of the calibration curve. Calibration curves with a correlation coefficient at least were used. F. Statistical analysis The results of ELISA tests, proliferation and sprouting assays were analyzed using the paired Student s t-test (Microcal Origin, version 6.0, Microcal Software, MA, USA) with p < 0.05 considered to be significant. III. Results A. Anti-angiogenic properties of chondrocyte-conditioned medium As first we used relative proliferation activity as readout of angiogenesis. Our data showed that treatment of HUVEC with conditioned medium leads to substantial reduction of their proliferation activity (Figure 1). Already 10% content of conditioned medium leads to strong inhibition of HUVEC proliferation (Figure 1b). All these results are statistically very significant. Specificity of this effect was demonstrated on the smooth muscle cells. Treatment with both, concentrated or diluted conditioned medium does not lead to significant inhibition of the growth of these cells. Specific and statistically significant inhibition of HUVEC proliferation due to treatment with chondrocyte-conditioned medium was observed in absence of HUVEC activation as well as under activation with VEGF or EGF respectively. B. Specificity of antinagiogenic properties of chondrocytes Our data (Figure 2) show that the chondrocytes losing their differentiation phenotype due to passaging in the same time loose also their antinagiogenic abilities. Dedifferentiated chondrocytes from 3 rd passage show in some cases even proangiogenic features. All these features were observed in case of undiluted as well as 10-fold diluted conditioned medium in the absence as well as in the presence of VEGF-activation. The results are highly statistically significant. C. Sprouting of HUVEC spheroids Spheroids containing HUVECs were generated, embedded in collagen matrix and subjected to different treatments. VEGF in concentration 10, 25, 50 ng/ml was used for activation of sprouting as a positive control (Figure 3a). In majority of our previous experiments, activation with 25 ng/ml exhibit saturation of this effect and further increase of VEGF concentration did not lead to further activation of sprouting. Therefore 25 ng/ml VEGF was used for activation of sprouting to demonstrate effect of chondrocyte-conditioned medium on sprouting. Treatment with unconditioned medium having the marginal effect on sprouting was used as a negative control. The results of measurement of cumulative sprouts show that activation of sprouting caused by VEGF can be substantially diminished by treatment with 50 µl or almost completely abrogated by treatment with 100 µl of chondrocyte conditioned medium. Treatment with unconditioned medium negative control showed that inhibition of sprouting is caused by medium conditioning and not by medium itself. These results are statistic significant. Inhibition of spheroid sprouting is dependent by differentiation status of conditioning cells as it is demonstrated by treatment with medium conditioned by chondrocytes from passage 3. Photos on Figure 3b show the representative spheroids from all treatment groups. D. Quantification of antinagiogenic effect The next experiment (Figure 4) shows that chondrocyte conditioned medium inhibits sprouting of HUVEC-spheroids activated by 10, 25 and 50 ng/ml VEGF or 50 ng/ml EGF respectively. Qualitatively exhibited antinagiogenic effect of chondrocyte medium was partially quantified, relatively in comparison to antinagiogenic effect of Rapamycin. Relative proliferation of HUVEC was used as readout. Concentration dependency of inhibitory effect of Rapamycin on HUVEC proliferation exhibit typical U-shape curve. Parallel with this MTTmeasurement, proliferation activity of HUVEC treated with conditioned medium was determined. Treatment with unconditioned medium was used as a negative control. Results showed that antinagiogenic effect of chondrocyteconditioned medium is stronger than effect of Rapamycin under its optimal concentration. E. Effect of malignant transformation on antinagiogenic effect of chondrocytes Angiogenesis is necessary for tumor growth. Therefore chondrocyte-derived cancer cells have to change their phenotype from anti- toward pro-angiogenic. To demonstrate this, effect of the medium conditioned by chondrosarcoma cells on HUVEC proliferation was compared with effect of chondrocytes. Proliferation of HUVEC in medium supplemented with 2% FCS was used as a positive control. Our results demonstrated proangiogenic effect of the medium conditioned by chondrosarcoma cell line SW1353. Already 10% content of this conditioned medium in medium (0,5% FCS) leads to substantial activation of HUVEC proliferation. 50% content of SW1353 conditioned medium leads to so strong activation of HUVEC proliferation that saturation was already reached. Further addition of conditioned medium does not lead to increased proliferation of HUVEC. F. Production of VEGF VEGF production by chondrocytes of passage 1, 3 as well as by chondrosarcoma cells SW1353 was determined by ELISA. Unconditioned medium containing no detectable VEGF was used as a negative control. Primary human chondrocytes produced more than 3000 pg/ml VEGF per million cells. This massive VEGF production was significantly reduced during cell dedifferentiation due to cell culture passaging. Interestingly, chondrosarcoma cells SW1353 used as proangiogenic positive control produced the lowest amount of VEGF from all investigated cell cultures. Surprisingly, the cells exhibiting the highest antinagiogenic potential produce the highest amount of proangiogenic VEGF at the same time. 146

4 Camaj and Ischenko et al: Anti-angiogenic effect of human primary chondrocytes Figure 1a: Inhibition of HUVEC and SMC proliferation by conditioned medium of chondrocytes under VEGF- or EGFinduced conditions. The cells were treated with complete conditioned or unconditioned chondrocyte medium (a) or with medium contained 90% of conventional HUVEC medium and 10% of conditioned or unconditioned chondrocyte medium. Figure 2: Relative proliferation of HUVECs treated with medium conditioned by chondrocytes at different passages. The effect of medium conditioned by de-differentiated chondrocytes at the passage 3 was compared with that caused by chondrocyteconditioned medium taken from the cells at first passage. Our results demonstrate that the anti-angiogenic properties of cultivated primary human chondrocytes were cell-passagedependent. Furthermore, same loosing in the anti-angiogenic properties of de-differentiated chondrocytes was found when starved endothelial cells or cells stimulated with 25 ng/ml VEGF were used Figure 1b: Inhibition of HUVEC and SMC proliferation by conditioned medium of chondrocytes under VEGF- or EGFinduced conditions. The cells were treated with complete conditioned or unconditioned chondrocyte medium (b) SMCs were taken to demonstrate the specificity of the antiproliferative effects of conditioned medium of chondrocytes on endothelial cells. Figure 3a: Inhibition of endothelial cell sprouting by conditioned medium of chondrocytes under different cell culture conditions (a). Sprouting was microscopically evaluated as the cumulative length of sprouts after overnight incubation at 37 C, 5% CO 2. Treatment with PBS served as a negative control. Treatment of spheroids with conditioned medium of chondrocytes in the presence of 25 ng/ml VEGF led to a statistically significant inhibition of HUVEC spheroid sprouting; the effect was proportional to the volume of conditioned medium used. Furthermore, the anti-angiogenic properties of cultivated primary human chondrocytes were cell-passage-dependent. 147

5 Gene Therapy and Molecular Biology Vol 13, page 148 Figure 5: Quantification of conditioned medium-induced inhibition of HUVEC proliferation. Inhibitory properties of conditioned medium of chondrocytes as well as Rapamycin were compared using the same experimental setting. The influence of conditioned medium of chondrocytes on HUVEC proliferation was as strong as that of Rapamycin when the drug was applied at clinically relevant doses (1µg/ml). Figure 3b: Inhibition of endothelial cell sprouting by conditioned medium of chondrocytes under different cell culture conditions Representative pictures of spheroids are presented (b), the arrows delineate spheroid sprouting. A-E indicates the corresponding in vitro conditions. Figure 4: Inhibition of endothelial cell sprouting by conditioned medium of chondrocytes under VEGF- or EGF-stimulation. Spheroids embedded in collagen gel were treated with conditioned medium of chondrocytes or with unconditioned control medium. Sprouting was microscopically evaluated as the cumulative length of sprouts after overnight incubation at 37 C, 5% CO2. Figure 6: Proliferation of HUVECs under SW1353-conditioned medium treatment. SW1353-conditioned medium was used in a pure form or mixed with serum-reduced HUVEC medium as shown. Serum-reduced HUVEC medium or complete medium supplemented with all growth factors served as a negative or positive control of HUVEC proliferation, respectively. The proliferation of HUVECs following treatment with conditioned medium of SW1353 cells was significantly increased and the effect was proportional to the percentage of conditioned medium. #p < 0.05 versus serumreduced HUVEC medium. 148

6 Camaj and Ischenko et al: Anti-angiogenic effect of human primary chondrocytes Figure 7: Vascular endothelial growth factor levels in differently conditioned or non-conditioned media. The VEGF concentrations were determined in various media conditioned by chondrocytes at passage 1 or 3, conditioned by chondrosarcoma cells, or in the unconditioned control medium. The chondrosarcoma cell line SW1353 secreted the lowest amount of VEGF. However, the primary human chondrocytes bearing clear anti-angiogenic properties expressed simultaneously the highest amount of VEGF. IV. Discussion The aim of this work was to demonstrate the antiangiogenic properties of normal human chondrocytes based on the capability to modulate endothelial cell proliferation and sprouting. The anti-angiogenic property of conditioned medium of chondrocytes was compared to that found in the media from human chondrosarcoma cells. To demonstrate the anti-angiogenic properties of substances produced by cartilage we performed an in vitro model consisting of cell culture of primary human chondrocytes isolated from pieces of human cartilage and HUVECs as cell sensors for the quantification of angiogenesis. First, the proliferative activity of HUVECs treated with conditioned medium from primary human chondrocytes was compared to that of HUVECs treated with unconditioned medium. The results revealed that the treatment of HUVECs with 100% of conditioned medium of chondrocytes led to a statistically significant reduction in their proliferation (Figure 1a). We hypothesized that such findings might be the result of reduced glucose, nutrients or growth factor levels in conditioned medium. To exclude this nutrient-dependent reduction in HUVEC proliferation, the cells were treated with a mixture of 90% HUVEC medium and 10% conditioned medium of chondrocytes, the unconditioned medium was included as a negative control. Indeed, the effect of such media mixture on HUVEC proliferation was found to be similar to that observed after the treatment of HUVECs with 100% conditioned medium (Figure 1b). Based on these findings, we can conclude that even 10% of conditioned medium content is sufficient to reach similar efficacy on HUVEC proliferation. Moreover, these results suggest that the antiproliferative effects on HUVECs by conditioned medium of chondrocytes were not caused by depletion of nutrients. Furthermore, we demonstrated anti-angiogenic effects of conditioned medium of chondrocytes on starved HUVECs following activation with VEGF or EGF treatment, respectively (Figure 1). These results allow us to hypothesize that the principle of action of some compounds within the conditioned media of chondrocytes does not have to be based on blockade of receptors, since even in the presence of at least two different activated receptors the anti-angiogenic properties of the conditioned medium of chondrocytes remain similar. Smooth muscle cells are usually present in an intimate contact with endothelial cells in their natural environment. However, treatment of SMCs with conditioned medium from primary human chondrocytes led only to minor changes in their proliferation rate (Figure 1a, b). Therefore we can conclude that the antiproliferative effects of conditioned medium of chondrocytes are not based on unspecific inhibition of the proliferative activity of cells in general, the inhibitory effects are particularly attributable to endothelial cells. It is well established in the previous studies that primary human chondrocytes lack their natural phenotype under in vitro conditions after multiple passages of cultivation (Schnabel et al., 2002). Our results showed that the anti-angiogenic properties of cultivated primary human chondrocytes were cell-passage-dependent when examined by HUVEC proliferation (Figure 2). Changing differentiation status of passaged chondrocytes was confirmed by monitoring the substantial changes of collagen-pattern and the cell morphology towards fibroblastic phenotype (data not shown). This observation led us to conclude that the anti-angiogenic activity of chondrocytes is coupled to gene expression that is specific to the differentiation status of the cells. We assume that dedifferentiated cells loose the anti-angiogenic potential and even the opposite pro-angiogenic activity can occur. Furthermore, lost of anti-angiogenic properties of dedifferentiated chondrocytes was found as well when starved endothelial cells or cells stimulated with 25 ng/ml VEGF were used. To further evaluate the anti-angiogenic properties of conditioned medium of chondrocytes in a physiological three-dimensional model we chose the endothelial cell spheroid-sprouting assay in vitro. Interestingly, the effect of conditioned medium of chondrocytes on spheroid sprouting was less pronounced than that found on HUVEC proliferation. Treatment of HUVEC spheroids with 50 ng/ml VEGF increased the sprouting of endothelial cells compared to that observed with 25 ng/ml VEGF, however, the difference did not reach significance (Figure 3a). Furthermore, treatment of spheroids with conditioned medium of chondrocytes in the presence of 25 ng/ml VEGF led to a statistically significant inhibition of sprouting, and the effect was proportional to the volume of conditioned medium used (Figure 3, a and b). The inhibitory effect on spheroid sprouting was nearly absent when the highest volume (100 µl) of conditioned medium 149

7 Gene Therapy and Molecular Biology Vol 13, page 150 of chondrocytes from cells at passage 3 was taken. Interestingly, the difference between the sprouting of spheroids treated with unconditioned medium or with medium conditioned by chondrocytes at passage 3 was negligible (Figure 3, a and b). Finally, the results obtained from the more biologically credible spheroid model fully confirmed our previous findings that chondrocytes loose their anti-angiogenic properties after cell culture passaging. The same assay showed that antinagiogenic properties of chondrocytes are obvious after both activations with VEGF or EGF respectively (Figure 4). Using MTT proliferation assay we found that the anti-angiogenic effect of conditioned medium of chondrocytes as shown by HUVEC proliferation was as strong as the anti-angiogenic capacity of the mtor inhibitor Rapamycin when the drug was applied at the clinically relevant doses (1µg/ml; Figure 5). To date, the U-shaped therapeutic curve of Rapamycin is widely known as a typical dose-dependent bend of efficacy of antiangiogenic compounds (Tjin et al., 2006). We assume that conditioned medium of chondrocytes includes a lot of unknown anti-angiogenic substances which should be further identified, isolated and characterized. The usage of other anti-angiogenic substances such as VEGF-blocking antibodies or antibodies against the VEGF receptor might be a useful strategy to analyse the anti-angiogenic properties of conditioned medium of chondrocytes. It is widely accepted that tumor progression is always associated with pathological angiogenesis and that this process is highly dependent on the levels of nutrients and oxygen. This statement is valid for progressing tumors originating from chondrocytes as well, although it is known that these cells do have strong anti-angiogenic properties. To examine the angiogenic/anti-angiogenic properties of chondrocyte-derived tumor cells we used conditioned medium of SW1353 chondrosarcoma cells and again analysed the proliferative activity of HUVECs by MTT proliferation assay. Interestingly, the proliferation of HUVECs following treatment with conditioned medium of SW1353 cells was significantly higher when compared to the proliferation of HUVECs under non-conditioned terms (Figure 6). Furthermore, the anti-proliferative effect on HUVECs was directly proportional to the concentration of conditioned medium of SW1353 cells, however, more than 50% conditioned medium of SW1353 cells did not lead to a further increase in HUVEC proliferation (Figure 6). On the other hand, a 10% content of conditioned medium of SW1353 cells in HUVEC medium did already lead to an evident activation of HUVEC proliferation. Furthermore, serum-reduced HUVEC medium (0.5% FCS) mixed 1:1 with conditioned medium of chondrosarcoma cells induced HUVEC proliferation even stronger than that being FCSand growth factor-supplemented and was used as a positive control (Figure 6). Our results support the hypothesis that transformation of primary human chondrocytes into chondrosarcoma cells lead to a change of inhibition of angiogenesis towards a rather pro-angiogenic phenotype. Since VEGF is known as one of the most important regulators of angiogenesis we evaluated the VEGF concentrations in various media conditioned by chondrocytes at passage 1 or 3, conditioned by chondrosarcoma cells, or in unconditioned control medium. Interestingly, the results from the VEGF ELISA showed an absolutely inverse picture in comparison to the results of HUVEC proliferation or the sprouting assays. The chondrosarcoma cell line secreted the lowest amount of VEGF. However, the primary human chondrocytes bearing clear anti-angiogenic properties expressed simultaneously the highest amount of VEGF (Figure 7). If these cells were passaged, they loose the ability to secrete VEGF. Thus, our next aim will be to identify these genes, which are responsible for the anti-angiogenic properties of chondrocytes reflected in our experiments. Furthermore, it seems important to describe changes in gene expression in cells present at transitional state, in which the antiangiogenic phenotype of normal cartilage has been changed to pro-angiogenic one of hypertrophic cartilage or chondrosarcoma. During this process the anti-angiogenic genes are turned off and the expression of pro-angiogenic genes is activated. Such analyses in gene expression leading to a better understanding of global regulation of cartilage physiology might be helpful in the development of anti-angiogenic drugs natural origin exhibiting minimal side effects. V. Conclusion Conditioned medium of primary human chondrocytes exhibits strong anti-angiogenic properties. The effect was specific to differentiated chondrocytes, whereas it was absent in conditioned medium from de-differentiated chondrocytes. Antiproliferative effect of conditioned medium is specifically directed toward HUVEC and no effect on smooth muscle cells was observed. In addition, we found that this effect was even stronger than the antiproliferative effect of Rapamycin treatment. By contrast, we could demonstrate that conditioned medium of chondrosarcoma cells SW1353 exhibits pro-angiogenic effects. The results from ELISA revealed that the inhibition of HUVEC proliferation and sprouting caused by conditioned medium of chondrocytes was not a result of a decreased amount of secreted VEGF. Acknowledgements The authors thank Prof. Alexander Berghaus and Dr. Andreas Naumann for the providing with cartilage tissue samples. We thank Dr. Gerald Schmid for the help with spheroid assay. We also thank Michael Brückel for the excellent technical assistance. This research was supported by the grant of the clinical research group KFO 128/1-1 of the Deutsche Forschungsgemeinschaft (DFG), the Wilhelm-Sander-Stiftung (Nr ) and the research grant BR 1614/3-1of the Deutsche Forschungsgemeinschaft (DFG). References Mizuno S, Glowacki J (2005) Low oxygen tension enhances chondroinduction by demineralised bone matrix in human dermal fibroblasts in vitro. Cells Tissues Organs 180(3): Harper J, Kalgsbrun M (1999) Cartilage to bone-angiogenesis leads the way. Nat Med 5(6):

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