The major findings of the present 16-week longitudinal US study of OIA rabbits were that the knee joints had gradual increases in synovial neovascularization, lumen size, and tissue disorganization, and that they eventually developed uneven and irregular lumens. Pannus formed by neovascularization, together with synovial hypertrophy, and there was infiltration of inflammatory cells and collagen fibers. Pannus formation developed during week-8, but did not cause cartilage and bone destruction at that time. At week-12, the pannus was more obvious, and cartilage destruction was evident. At week-16, the vascular lumen was greatly enlarged, and the synovium was increasingly fibrotic.
Based on the extent of neovascularization and fibrosis in the pannus, some researchers classify the pathogenesis of RA as inflammatory, fibrous, or mixed and classify synovitis as progressing from inflammation, to pannus formation, and then fibrosis[22]. During the earlier stages of inflammation and pannus formation, the pannus is mainly inflammatory or mixed; then, due to the formation of many fibrous components, RA enters the fibrotic or sclerotic stage. During week-6, our OIA rabbits had obvious synovitis, but the proliferation of blood vessels was not yet apparent. Considering that this was the inflammatory stage, pannus formation followed at week-8 and week-12. At week-16, the fibrosis was obvious, and this corresponded to the fibrosis stage of RA. Therefore, in our OIA model, the first 8 weeks correspond to early-stage RA (inflammatory stage), weeks 8 to 12 correspond to middle-stage RA (pannus stage), and weeks 12 to 16 weeks correspond to late-stage RA (fibrosis stage). Another of our notable findings is that OIA rabbits had increasing expression of CD31 over time (P < 0.001). Because CD31 functions in angiogenesis, this indicated that the extent of synovial vascularization increased as RA progressed, with the greatest vascularization in late-stage disease. These results also suggest that vascularization index in OIA rabbits, which is based on pathological vascularization, reflects the different stages of RA.
Previous studies reported greater synovial CD31 expression in patients with RA and osteoarthritis (OA) than in healthy controls[23], and that expression was greater in RA than OA[24]. In agreement, intra-articular inhibition of local angiogenesis had a therapeutic effect on RA but no effect on OA[24]. Inhibition of angiogenesis, as an adjunct or even an independent treatment of RA, can prevent the transport of nutrients to the synovium, possibly leading to vascular degeneration and disease reversion. Thus, inhibition of angiogenesis in the synovium appears to be an effective method to inhibit the progression of RA.
Most previous clinical studies of the synovial expression of VEGF and CD31 in RA had a cross-sectional designs[24, 25], and no longitudinal studies have yet evaluated changes in the synovium during the different stages of RA. A novelty of the present study of OIA rabbits (as a model of RA) is that we used the minimally invasive technique of US-guided synovial biopsy to assess longitudinal pathological changes. Our results indicated increased VEGF expression during the early stage of pathogenesis, and that expression continued to increase over time. Moreover, VEGF expression had a strong positive correlation with MVD (CD31 positivity) and synovial pathological score. The pathogenesis in OIA rabbits is similar to the pathogenesis in RA, in that there is upregulation of synovial angiogenesis during early pathogenesis, and persistence of angiogenesis as pathogenesis proceeds. Our results therefore suggest that continued high expression of VEGF may be an important factor in the formation of pannus and the aggravation of synovitis. Therefore, early prevention of VEGF overexpression by use of a VEGF inhibitor may block the development of synovial angiogenesis and inflammation.
The results of the present study clearly showed that as disease progressed in OIA rabbits, the synovium developed more new blood vessels, and there was increased PDI grade, and increased disease score. These rabbits also had a positive correlation of MVD (CD31 positivity) with disease severity based on PDI grade. However, our observations at week-6 and week-8 (during the early stages of neovascularization), indicated that a low proportion of knees had high-grade PDI, and the correlation of PDI grade with MVD was weak. This suggests that PDI has limited sensitivity for detect neovascularization during early-stage disease. This is likely because PDI can only detect blood flow signals in vessels with diameters greater than 100 µm[7], and synovial neovascularization during early-stage disease is mainly in the capillaries. This interpretation is consistent with our pathological examinations of synovial tissues during early-stage disease. In the clinic, some patients with RA are negative in PDI evaluation soon after treatment, but experience inflammation relapse at some period after treatment. This may be related to the inability of PDI to detect microvascular changes, and result in missed diagnosis of patients with early-stage disease. Another limitation of PDI is that it is difficult to distinguish whether an increased blood flow signal in the synovium is due to an increase in the number of blood vessels or circulatory congestion.
We observed that during the early stage of US enhancement (5 to 15 s after injection of the contrast agent), most synovial vessels had "scattered dot" enhancement during week-6 and week-8, but "strip" or "multi strip" morphology was predominant during week-12 and week-16. In particular, there were significant differences in the early vascular morphology during different stages of enhancement (P < 0.05). This may be because there are mostly immature microvessels with small diameters during the early stages of modeling, but that over time the synovial blood vessels develop and become larger as the lumen increases in size. Our detailed longitudinal examinations of synovial tissues of OIA rabbits during different stages of disease confirm this interpretation. Our results thus suggest that early US enhancement of synovial vessels may provide valuable information for the early diagnosis of RA.
During early-stage RA, newly formed blood vessels in the synovium mainly consist of immature and dilated small blood vessels, and there are more blood vessels than in the normal synovium[26]. This reflects significant changes in the blood volume of the synovium. Therefore, blood volume can be used as a direct indicator of angiogenesis during RA. On the premise that the dose of the contrast agent, instrument parameters (frequency, mechanical index, etc.), and size of the ROI are fixed, the peak intensity (PI) reflects the blood volume in the synovial tissue of the designated area during a unit of time[27]. Therefore, the PI can be used as an index of synovial neovascularization. Our longitudinal analysis indicated the PI value of OIA rabbits increased from week-6 to week-8, possibly because the synovium was in the stage of pannus formation at week-8, at which time there was greater vascular density and more local blood perfusion. However, we also found that the PI value at week-16 was lower than at week-8, possibly because the synovium entered the stage of pannus fibrosis, in which there were increased fibrotic components relative to inflammatory pannus.
Interestingly, our analysis of OIA rabbits during week-6 and week-8 indicated that a small number of knee joints had PDI grades of 0. At this time, CEUS indicated blood flow in the synovium, and positive correlations of CEUS grade, PI (a CEUS-TIC quantitative index), and synovium MVD (CD31). At week-6 and week-8, the correlation coefficients of CEUS grade and PI with synovial MVD (CD31) were also significantly greater than those of PDI grade with synovial MVD (CD31). This suggests that CEUS can detect synovium neovascularization during early-stage RA. CEUS is more sensitive than PDI, and CEUS semi-quantitative and quantitative analyses are particularly important. However, CEUS also has some limitations, in that it is invasive, expensive, time-consuming, and can only evaluate a single plane or cross-sectional perfusion of one joint at a time. Therefore, from the practical and economic point of view, PDI remains the first choice for evaluation of synovial blood flow in patients with suspected RA. Based on our results, CEUS may be recommended to evaluate the synovial microcirculation in patients whose PDI results are negative but are suspected of having early RA.