Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis

Abstract

STATEMENT OF FINDINGS: We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.

Figure 1

RNase protection assay with the set of coagulation/fibrinolytic genes. (a) Typical eight-band protection pattern resulting from the analysis of 5 μg of mouse-skin total RNA. (b) Kidney RNA prepared from various knockout (KO) mice (PAI1-, UPA-, or PAR1-deficient mice) and from wild-type (WT) mice, analyzed using RPA. (c) Kidney RNA prepared from lipopolysaccharide (LPS)-treated or untreated mice, analyzed using RPA. (d) RNA from murine hypoxic or normal endothelial cells, analyzed using RPA. In all these RPA experiments, 5 μg of total RNA/lane was analyzed.

Figure 2

Synovial levels of mRNA from procoagulant and fibrinolytic genes in AIA, as analyzed by RPA. (a) Autoradiographic signals of synovial RNA samples from left (L; control) and right (R) knees, 4 h after arthritis induction. The single sample shown for each condition shows the typical, reproducible pattern obtained from the RPA analysis of at least three different RNAs, each from different animals. (b) Same as (a), but 7 d after arthritis induction.

Figure 3

Time course of accumulation of mRNA from procoagulant and fibrinolytic genes during AIA. RNA levels are expressed as arthritic/normal knee value ratios, after correcting for GAPDH signal. The 0 time point is given the value 1. Values are the mean ± SEM of at least three different mRNA values from three different mice.

Figure 4

RNA from procoagulant and fibrinolytic genes in paws of mice with CIA, as analyzed by RPA. (a) Autoradiographic signals of RNA samples from eitherarthritic paws (CIA) or normal paws from nonimmunized (NI) animals. The single sample shown for each condition represents the typical, reproducible pattern obtained from the RPA analysis of four RNAs, each from a different animal. (b) Expression of genes studied during CIA, shown as the ratio of mRNA in arthritic to that in normal paws (CIA/NI), after correction for GAPDH signal. Values are means ± SEM of at least four different mRNA values from four different mice.  ^*P < 0.05, CIA versus NI values.

Figure 5

Activity of procoagulant and TF and concentrations of plasma TAT in mice with induced arthritis (CIA or AIA) and controls. (a) PCA (expressed in seconds taken for thrombus formation) in tissue extracts. Arthritic tissue extracts (A) were prepared from arthritic, swollen paws at day 42 after the first immunization (CIA model) or from arthritic right knee joints at day 3 after arthritis induction (AIA model). Nonarthritic (NA) tissue extracts were from paws of normal, nonimmunized mice (CIA model) or normal left knees (contralateral to induced arthritis; AIA model). (b) TF activity in arthritic (A) and nonarthritic (NA) tissue extracts (the same extracts as were used for PCA), measured using a chromogenic assay. (c) Plasma concentrations of TAT complexes as determined in arthritic (A) mice in comparison with nonimmunized, nonarthritic (NA) controls. Citrated plasmas from arthritic mice were prepared at day 42 after the first immunization (CIA model) or at day 3 after arthritis induction (AIA model). Values are means ± SEM of at least four samples from four mice. –^*P < 0.05, arthritic versus nonarthritic values.