Honorati Maria Cristina, Cattini Luca, Facchini Andrea
Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy.
Osteoarthritis Cartilage. 2004 Sep;12(9):683-91. doi: 10.1016/j.joca.2004.05.009.
To verify the involvement of proinflammatory cytokines IL-17, IL-1beta and tumor necrosis factor alpha (TNF-alpha) in cartilage vascularization by stimulating the production of vascular endothelial growth factor (VEGF) by chondrocytes isolated from patients with osteoarthritis (OA), in comparison with patients with rheumatoid arthritis (RA) and patients with femoral or humeral neck fracture (FP).
Chondrocytes isolated from patients with OA were maintained in monolayer culture for several passages. Chondrocyte dedifferentiation was monitored by the synthesis of cathepsin B by these cells. Chondrocytes freshly isolated at each subculture (subcultures 1-3) were stimulated with IL-17, IL-1beta or TNF-alpha. Supernatants were collected, immunoassayed for the production of VEGF and cathepsin B and assayed as the source of VEGF on the VEGF sensible ECV304 cell line. The cells were used to quantify intracellular cathepsin B enzymatic activity.
In differentiated conditions IL-1beta and TNF-alpha, but not IL-17, can inhibit the spontaneous secretion of VEGF by human OA, RA and FP chondrocytes, and IL-17 can restore the decrease in VEGF secretion caused by TNF-alpha. IL-17, together with IL-1beta and TNF-alpha, can enhance VEGF secretion to various extents by dedifferentiated OA chondrocytes. This change in effect with respect to primary culture was observable for all cytokines at the beginning of dedifferentiation, when the production of VEGF by chondrocytes had dramatically fallen and the cathepsin B synthesis had increased. The amount of VEGF induced by cytokines on dedifferentiated chondrocytes never reached the amount of VEGF produced by differentiated chondrocytes. VEGF produced by chondrocytes stimulated the ECV304 cell line proliferation.
These results indicate that dedifferentiated OA chondrocytes secrete VEGF after stimulation with proinflammatory cytokines. This event may be responsible for neovascularization found in OA cartilage.
通过刺激从骨关节炎（OA）患者分离出的软骨细胞产生血管内皮生长因子（VEGF），与类风湿关节炎（RA）患者和股骨或肱骨颈骨折（FP）患者相比较，验证促炎细胞因子白细胞介素-17（IL-17）、白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNF-α）在软骨血管生成中的作用。
将从OA患者分离出的软骨细胞进行单层培养传代培养数代。通过这些细胞合成组织蛋白酶B来监测软骨细胞去分化情况。对每次传代培养（传代1 - 3次）时新鲜分离的软骨细胞用IL-17、IL-1β或TNF-α进行刺激。收集上清液，免疫测定VEGF和组织蛋白酶B的产生情况，并将其作为VEGF敏感的ECV304细胞系上VEGF的来源进行测定。这些细胞用于定量细胞内组织蛋白酶B的酶活性。
在分化状态下，IL-1β和TNF-α而非IL-17可抑制人OA、RA和FP软骨细胞自发分泌VEGF，且IL-17可恢复由TNF-α导致的VEGF分泌减少。IL-17与IL-1β和TNF-α一起，可在不同程度上增强去分化的OA软骨细胞分泌VEGF。对于所有细胞因子，在去分化开始时，当软骨细胞VEGF产生显著下降且组织蛋白酶B合成增加时，相对于原代培养，这种效应变化是可观察到的。细胞因子诱导去分化软骨细胞产生的VEGF量从未达到分化软骨细胞产生的VEGF量。软骨细胞产生的VEGF刺激ECV304细胞系增殖。
这些结果表明，去分化的OA软骨细胞在促炎细胞因子刺激后分泌VEGF。这一事件可能是OA软骨中发现的新生血管形成的原因。
