Chondrogenic progenitor cells promote vascular endothelial growth factor expression through stromal-derived factor-1

Abstract

Objective: Vascular endothelial growth factor (VEGF) is elevated in joint fluids from patients diagnosed with osteoarthritis (OA). VEGF is known to contribute to vascular tidemark invasion and osteophyte formation, which are classic features of advanced OA. Among the factors that may drive VEGF accumulation in diseased joints, stromal cell-derived factor-1α (SDF-1α) is a likely culprit, as it is enriched in synovial fluids from osteoarthritic joints and is a potent inducer of VEGF expression. Chondrogenic progenitor cells (CPCs) that overexpress SDF-1α are abundant in osteoarthritic cartilage, implicating them in elevating synovial SDF-1α levels. Here we conducted a series of experiments to determine the potential for CPCs to stimulate VEGF expression via autocrine and paracrine mechanisms.

Design: Immunohistochemistry, immunoblotting, and PCR were used to evaluate the effects of SDF-1α on VEGF expression in CPCs and chondrocytes, and the effects of CPC-conditioned medium on chondrocytes. An SDF-1α receptor antagonist and inhibitors of mitogen-activated protein kinases (MAPKs) were used to probe the pathway linking SDF-1 with VEGF expression in CPCs.

Results: SDF-1α and CPC-conditioned medium stimulated VEGF expression in chondrocytes. In both chondrocytes and CPCs, SDF-1α stimulated increased VEGF expression via C-X-C chemokine receptor type 4 (CXCR4), a cell-surface SDF-1α receptor. This response in CPCs is dependent on p38 MAPK activation, but not on ERK or c-Jun N-terminal kinase (JNK) activation.

Conclusions: By secreting SDF-1α, CPCs stimulate VEGF expression in nearby cells. The co-expression of SDF-1 and its receptor by CPCs indicates they are capable of self-sustained VEGF expression via an autocrine mechanism.

Keywords: CXCR4; Chondrogenic progenitor cells (CPCs); SDF-1α; Vascular endothelial growth factor (VEGF); p38 MAPK.

Figure 1.

Immunohistochemical staining for VEGF showed significantly increased expression of VEGF in the SDF-1α treated group (B, D-G) especially in the superficial zone of the cartilage (H), while AMD3100 impaired the SDF-1α-induced expression of VEGF (C, D-G) (10×10). P values indicate the statistical differences between groups. (IOD: integrated optical density)

Figure 2.

Effects of SDF-1α and AMD3100 on VEGF expression by CPCs. Western blot results showed that SDF-1α significantly increased VEGF protein expression relative to untreated controls, CPCs’ VEGF expression was in the same level when treated with 3 concentrations of SDF-1 (20, 50, and 100 ng/ml), around 1.6 fold higher than untreated group (A, B), while AMD3100 dramatically reduced SDF-1α-induced VEGF expression (34%, 55%,and 79% lower in 20, 50, and 100 ng/ml SDF-1 treated groups, respectively) (A, C). β-actin expression was used as an internal loading control. P values indicate the statistical differences between groups.

Figure 3.

Effects of SDF-1α and AMD3100 on VEGF mRNA levels in CPCs. The RT-PCR results indicated that SDF-1α (50 ng/ml) substantially upregulated mRNA expression of VEGF comparing to untreated CPCs (1.96 fold higher) and AMD3100 significantly decreased VEGF expression in 50 ng/ml group by 34% (A), in addition, p38 inhibitor (SB203580) tended to significantly reduce the SDF-1α response while mild decreases were also observed in other two groups treated with different kinase inhibitors (PD98059 and SP600125) (B). β-actin expression was used as an internal loading control. P values indicate the statistical differences between groups.

Figure 4.

MAPK phosphorylation in CPCs treated with SDF-1α. CPCs were incubated with SDF-1α (100 ng/ml) for the indicated times. Blots were probed with antibodies detecting total (t-) or phosphorylated (p-) p38, ERK, or JNK. Compared to untreated group, the relative absorbance ratios of p-p38 to t-p38 were significantly elevated in groups of 5mins and 15mins (2.21 and 2.06 fold higher, respectively), no significant differences were found in groups treated with longer SDF-1 (A). The relative absorbance ratio of p-ERK to t-ERK was peaked at group of 5mins (9.85 fold higher), then gradually decreased for longer time periods (7.46 and 5.48 fold higher for 15mins and 30mins, respectively), no significant differences were found in groups of 1hrs and 2hrs (B). An significantly increasing trend of relative absorbance ratios of p-JNK to t-JNK was identified in the CPC groups treated with SDF-1 for first 30 mins (2.06, 2.57, and 2.78 fold higher for 5mins, 15mins, and 30mins, respectively). no significant differences were found in 1hrs and 2hrs groups. β-actin expression was used as an internal loading control. P values indicate the statistical differences between groups.

Figure 5.

CPC-condition media stimulates chondrocyte VEGF expression. In chondrocytes that were not treated with AMD3100 (Lanes 1–5), VEGF expression increased as the proportion of CPC-conditioned media increased from 0 to 20%. Expression peaked at 40% and declined slightly at the highest CPC-CM doses (60%, 80%). In contrast, CPC-CM had no effect on VEGF expression in chondrocytes treated with AMD3100 (Lanes 6–10). β-actin expression was used as an internal loading control. (CPC-CM: CPC-condition media)