Effects of NO on chondrogenic differentiation

As we were able to demonstrate chondrogenic differentiation and expression of chondrocyte specific genes in the hMSCs pellet model (chapter 4.1) we were interested if NO has an influence on chondrogenic differentiation. Therefore hMSCs were differentiated in the presence of NO donors.

In the first experimental setting hMSCs from two bone marrow donors were pelleted and cultured in the presence of 2,5µM DETA NONOate. The NO-donor was present in the culture medium during the whole differentiation time (up to 3 weeks), medium with freshly prepared NO donor was changed every 48h. DETA NONOate spontaneously dissociates in a pH-dependent, first-order process with a half-life of 20 hours at 37°C, pH 7.4, to liberate 2 moles of NO per mole of parent compound. Decomposition of NONOates is nearly instantaneous at pH 5 (Hrabie et al., 1993). The concentration of DETA NONOate was chosen in connection with reported levels of NO in synovial fluid of patients with OA: 3.4 ± 2.0 µM (Brenner et al., 2004).

During the differentiation course chondrogenic pellets were harvested after 6, 13, 20 and 27 days of differentiation. After RNA isolation PCR analysis concerning SOX-9, HIF-1α, VEGF, iNOS, COX-2, PCS, collagen II, collagen I, collagen X and aggrecan expression levels was performed.

In this preliminary experiment there were almost no differences in the expressions levels of studied genes between cells differentiated with and without NO-donor. Slight increases in SOX-9, collagen II, VEGF and PCS transcript levels could be detected if DETA NONOate was present in the differentiation medium (data not shown). Collagen 10 levels were lower, when NO was present during the differentiation time (data not shown).

We believed, that lack of a distinct effect of NO on chondrogenic differentiation could be due to the low concentration of DETA NONOate. Therefore in the next experimental setting concentration of DETA NONOate was increased to 50µM during the whole differentiation course of hMSCs originating from two bone marrow donors. Additionally

after 6, 13, 20, 27 days of differentiation in the presence of NO donor cells were stimulated for further 24h with IL-1β (NO-donor was still present in the culture medium).

Again analysis of expression levels of several genes important for chondrocyte physiology was performed.

Collagen 1 expression: The expression of this collagen was quite stable during the whole differentiation course. DETA NONOate had no effect on collagen 1 expression.

The decrease in collagen 1 transcripts level was observed after incubation with IL-1β on the day 14 and 21 (Figure 36).

Col 1 expression

0,00 0,50 1,00 1,50 2,00

7 14 21 28

days

relative expression

control IL-1 1nM NONO 50µM NONO+IL-1

Figure 36. The effect of DETA NONOate and IL-1β on the relative gene expression levels of collagen 1 during chondrogenic differentiation. The expression level of control cells after 7 days of chondrogenesis was set as one and fold-exchange was calculated. PCR was performed in triplicates. For standardization of the gene expression levels determined by TaqMan analysis mRNA derived cDNA signal in each sample was calculated relative to 18s ratio as an internal control. Data are presented as mean ±SD.

Collagen 2 expression: Collagen 2 was differently expressed during the differentiation cycle (Figure 37). Expression values detected under the basal differentiation conditions were very low. Suprisingly the expression rate in cells differentiated in the presence of 50µM DETA NONOate was much higher. The incubation of cells with IL-1β led to suppression of collagen 2 expression.

Col 2 expression

Figure 37. The effect of DETA NONOate and IL-1β on the relative gene expression level of collagen 2 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

Collagen 10 expression: DETA NONOate had no effect on collagen 10 expression (Figure 38). Number of collagen 10 transcripts in the first three weeks of chondrogenic differentiation was significantly down-regulated if the cells had been exposed to IL-1β.

Col 10 expression

Figure 38. The effect of DETA NONOate and IL-1β on the relative gene expression levels of collagen 10 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

iNOS expression: iNOS was not expressed in hMSC monolayer nor basal neither after stimulation with IL-1β and NONOate incubation. After 7 days of chondrogenic differentiation low levels of iNOS transcripts were found also in the control cells, however IL-1β was able to increase this level 30- fold (Figure 39). Interestingly the highest iNOS expression has been observed in cells which were differentiated in the presence of DETA NONOate and then stimulated for 24h with IL-1β. This tendency was observed in the first three weeks of differentiation. In the 4^th week of differentiation expression of iNOS after 24h of stimulation with IL-1β in the cells differentiated with NO-donor was decreased in comparison to control cells.

iNOS expression

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7 14 21 28

days

relative expression

control IL-1 1nM NONO 50µM NONO+IL-1

Figure 39. The effect of DETA NONOate and IL-1β on the relative gene expression levels of iNOS during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

COX-2 expression: The expression of COX-2 was highest on the7^th day of differentiation, afterwards basal and IL-1 stimulated expression of COX-2 gene declined. DETA NONOate decreased basal and IL-1β stimulated amounts of COX-2 mRNA transcripts (Figure 40).

COX-2 expression

Figure 40. The effect of DETA NONOate and IL-1β on the relative gene expression levels of COX-2 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

IL-1 expression: IL-1 expression was strongly upregulated in hMSC monolayer after stimulation with IL-1β (data not shown). Interestingly, after beginning of chondrogenic differentiation there were several samples where any IL-1β transcripts were detected although the cells had been stimulated (day 14^th).

IL-1 expression

Figure 41. The effect of DETA NONOate and IL-1β on the relative gene expression levels of IL-1 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

However, in other stimulated cells high expression of IL-1β was observed. Therefore it is difficult to evaluate the results obtained with DETA NONOate. Clearly, there was no expression of IL-1β in unstimulated chondrogenic pellets (Figure 41).

IL-6 expression: This cytokine was not expressed in unstimulated cells independently on the differentiation status and presence of NO-donor.

After treatment with IL-1β levels of IL-6 mRNA transcripts significantly increased. DETA NONOate had no effect on the rate of IL-6 transcription (Figure 42).

IL-6 expression

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7 14 21 28

days

relative expression

control IL-1 1nM NONO 50µM NONO+IL-1

Figure 42. The effect of DETA NONOate and IL-1β on the relative gene expression levels of IL-6 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

RUNX-2 expression: High levels of RUNX-2 expression were observed in stem cells before differentiation (data not shown). In the first week of differentiation the expression of this transcription factor declined to about 30% of control, but with the course of differentiation increasing levels of RUNX-2 were detected. Regulation of RUNX-2 expression could only be seen on the 21^st day of chondrogenic differentiation; a slight increase in mRNA levels in pellets differentiated in the presence of DETA NONOate was observed and suppression of RUNX-2 expression by IL-1β; NO donors had no influence on this suppression (Figure 43).

RUNX-2 expression

Figure 43. The effect of DETA NONOate and IL-1β on the relative gene expression levels of RUNX-2 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.

ADAMTS 5 expression: Expression of this aggrecanase was observed in undifferentiated hMSCs and was slightly up-regulated with IL-1β (data not shown).

In the first week of chondrogenic differentiation the expression level of ADAMTS 5 was very low, but continously increased with the differentiation time. The expression of ADAMTS 5 was elevated by the stimulation with IL-1β, but there was no effect noticed with DETA NONOate (Figure 44).

ADAMTS 5 expression

Figure 44. The effect of DETA NONOate and IL-1β on the relative gene expression levels of ADAMTS 5 during chondrogenic differentiation. Experiments were performed in the same way as for Col 1.