Analysis of early changes in the articular cartilage transcriptisome in the rat meniscal tear model of osteoarthritis: pathway comparisons with the rat anterior cruciate transection model and with human osteoarthritic cartilage.

OBJECTIVE: The purpose of this study was to use microarray technology to: (1) understand the early molecular events underlying the damage of articular cartilage initiated by this surgical procedure, and (2) determine whether these changes mimic those that are occurring in human osteoarthritic (OA) cartilage. DESIGN: Cartilage was harvested from both medial and lateral sides of the tibial plateaus and femoral condyles of both meniscal tear (MT) and sham surgery groups on days 3, 7 and 21 post-surgery. mRNA prepared from these rat cartilage samples was used for microarray analysis. RESULTS: Statistical analysis identified 475 genes that were differentially expressed between the sham and MT groups, at one or more of the time points that were analyzed. By integrating these genes with OA-related genes reported previously in a rat OA model and in human OA array studies, we identified 20 commonly changed genes. Six out of these 20 genes (Col5A1, Col6A2, INHBA, LTBP2, NBL1 and SERPINA1) were differentially expressed in two animal models and in human OA. Pathway analysis identified some key features of OA pathology, namely cartilage extracellular matrix remodeling, angiogenesis, and chondrocyte cell death that were recapitulated in the animal models. The rat models suggested increased inflammation and cholesterol metabolic pathways may play important role in early cartilage degeneration. CONCLUSION: We identified a large number of differentially expressed genes in the articular cartilage of the MT model. While there was lack of overall identity in cartilage gene expression between the rat models and human OA, several key biological processes were recapitulated in the rat MT OA model.

Regulation of thymidine kinase protein stability in serum-stimulated cells.

The mechanism of posttranscriptional regulation of thymidine kinase (TK) enzyme levels following serum stimulation of quiescent cells has been investigated using stably transfected Rat 3 (TK-) cells containing the human TK complementary DNA linked to a hybrid SV40/human TK promoter. These cells expressed a wild-type human TK mRNA at relatively constant levels during the first G1 and S phase after serum stimulation. In contrast, TK enzyme activity and protein levels were low during G1 and increased dramatically as the cells entered S. A comparison of the patterns of protein expression (by Western blot) and enzyme activity indicated that the specific activity of the protein did not vary between G1 and S. A combination of pulse labeling and pulse-chase experiments indicated that the increase in TK protein levels at the G1-S transition was primarily the result of a stabilization of the protein at that time. The stability of a mutant form of TK lacking 16 NH2-terminal amino acids was regulated similarly to the wild-type, indicating that this region of the protein is not required for the regulation of protein turnover. Finally, indirect immunofluorescent labeling demonstrated that TK is uniformly distributed in the cytoplasm during both G1 and S phase.

Neutrophil inhibitory factor prevents neutrophil-dependent lung injury.

Neutrophil inhibitory factor (NIF) is a recently cloned 41-kDa protein from the canine hookworm that binds CD11b/CD18 and inhibits CD11b/CD18-dependent neutrophil adhesion. We evaluated NIF's effects on neutrophil-dependent lung injury in guinea pigs. Pulmonary vascular endothelial CD54 (ICAM-1) was induced in buffer-perfused lungs by 90-min exposure to 1000 U/ml TNF-alpha. Human neutrophils (2 x 10(7)) were added to the perfusate and activated by 5 x 10(-9) PMA; in some lungs, the neutrophils were pretreated with NIF (100 nM) before their addition to the perfusate. Lung injury was assessed by wet:dry weight ratio, and neutrophil uptake by lung myeloperoxidase (MPO) activity. HUVEC exposed to TNF-alpha for 90 min were assayed for neutrophil adhesion, and we compared PMA-stimulated neutrophil adhesion to endothelial cells and fibrinogen-coated plates. PMA-induced pulmonary edema (lung wet:dry ratio increased from 8.8 +/- 0.7 to 18.8 +/- 4.4) was inhibited by NIF (10.0 +/- 1.0). Lung MPO activity concomitantly decreased from 17.1 +/- 6.1 to 8.7 +/- 1.8 U/mg dry lung tissue in the NIF-treated group, similar to controls (6.9 +/- 2.0). Endothelial monolayer experiments confirmed that NIF reduced neutrophil adherence (basal adhesion of 11 +/- 3% increased to 30 +/- 5% with TNF-alpha pretreatment of endothelial cells, an increase that was reduced to 10 +/- 4% with NIF). Moreover, NIF prevented PMA-induced neutrophil adhesion to fibrinogen, a CD11b/CD18-dependent event, but produced a smaller decrease in adherence to endothelial cells, which also involves CD11a/CD18 integrins. These studies indicate that NIF prevents neutrophil-dependent lung vascular injury by inhibiting neutrophil adhesion to the TNF-alpha-activated endothelium.