Angiogenesis is an important component in the pathophysiology of rheumatic diseases, but although it has been described to be more enhanced in PsA than in RA patients [3], the molecular mechanisms that regulate this phenomenon are still unclear. Many pro-angiogenic factors have been associated with PsA, but those were mostly studied in the synovial tissue or the synovial fluid. Here, we evaluated the serum concentrations of pro- and anti-angiogenic factors and demonstrated that the balance between them in active PsA patients, more than in the RA or HC, has shifted toward the pro-angiogenic factors. Specifically, we show that PsA patients with active disease exhibit higher serum levels of CD147, relative to RA patients or HC. Using an in vitro co-culture system, we show that CD147 mediates the interactions between fibroblasts and monocytes and enhances angiogenesis through the increased expression of VEGF and MMP-9, placing it as an important, but not sole mediator of PsA pathophysiology.
Our results showing enhanced cytokine levels in serum from active PsA patients (Fig. S1) are consistent with previous studies that have shown a central role for IL-17, IL-6, TGF-β, and TNF-α in PsA patients [8]. Since these inflammatory cytokines also have an important pro-angiogenic role [8], their enhanced serum levels in active PsA patients further supports the presence of enhanced angiogenesis reported in PsA patients.
CD147 is an important pro-angiogenic factor, as it can participate in the regulation of both VEGF and MMPs. Its enhanced secretion by monocytes derived from RA patients was previously described [15]. Here, we demonstrate elevated CD147 levels in serum obtained from active RA patients relative to HC, and even higher levels in serum samples obtained from PsA patients regardless of disease activity. This may reflect the enhanced angiogenesis observed in PsA patients reflected by the formation of long, tortuous, and dilated vessels in the synovium, in comparison to RA patients who have thickened, straight, and evenly branched vessels in the synovial tissue [8, 26]. This suggests that CD147 plays a role in rheumatic diseases in general, and in PsA in particular, in accordance with the important role of angiogenesis in the pathogenesis of PsA. This is also supported by similar studies demonstrating elevated serum levels of CD147 in patients with psoriasis [27] and elevated angiogenesis and expression of CD147 in fibroblast-like synoviocytes (FLS) [28]. The important role played by CD147 in angiogenesis is also highlighted by our in vitro studies, which emphasize the need for interaction between at least two cell types to enhance CD147 concentrations and its ability to induce the potent pro-angiogenic factors VEGF and MMP-9. Moreover, in view of the role CD147 plays in chemotaxis and Th17 cell differentiation in lymphocytes or neutrophils derived from RA patients [19, 29], its targeting by an antibody might prove beneficial in human PsA patients.
The elevated CD147 levels found in both PsA groups were not mirrored by these patients’ VEGF serum levels, which were similar to those seen in the active RA or HC groups. This may reflect the high variability of the VEGF assay or the suppressive effects of medications taken by the PsA patients in remission. The role played by additional regulatory factors [such as Hypoxia-Inducible Factor-1 (HIF-1), Signal Transducer and Activators of Transcription 3 (STAT3), Mitogen-Activated Protein Kinase (MAPK), and p53] in VEGF expression suggests a complex regulatory process involving this mediator in disease pathogenesis [30].
MMPs primarily degrade extra-cellular matrix (ECM) proteins, and as such, they play an important role in damage to the bone and cartilage characteristic of rheumatic diseases. Additionally, MMPs also regulate angiogenesis, as they facilitate endothelial cell migration and proliferation [10]. PsA and RA are both associated with elevated levels of MMP-9 and other MMPs [10]. Despite this, we demonstrated reduced levels of MMP-9 in all the patients relative to HC. Different drugs, such as methotrexate (MTX), sulfasalazine (SSZ), several statins, infliximab, and other TNF-α blockers, were shown to reduce serum levels of MMP-9, [15, 31,32,33,34,35,36,37]. Thus, the reduction in MMP-9 levels seen in the PsA and RA patient groups in our study may be partially explained by their baseline treatment. Of note, we conducted several experiments to rule out the possibility that the reduction in MMP-9 was due to the ELISA kit we used, a kit that recognizes pro- and active-MMP-9, but not MMP-9 bound to TIMP-1. We repeated the test with a kit that recognizes only the bound form of MMP-9 and used a fluorescently labeled substrate to evaluate MMP-9 activity, all with similar results to those we obtained with the initial kit (data not shown).
Both endostatin and Tsp-1 are known as potent anti-angiogenic factors, and we expected them both to show reduced levels in the inflammatory groups relative to HC, reflecting the enhanced angiogenic process found especially in PsA patients. To our surprise, endostatin exhibited elevated levels in PsA patients regardless of disease activity. These results are in agreement with a study demonstrating elevated serum levels of endostatin in patients with systemic sclerosis where elevated endostatin reflected the degree of vascular injury [38]. Since, to the best of our knowledge, our study is the first to measure serum levels of endostatin in PsA and RA patients, we can only assume that these elevated levels point to additional functions of endostatin, such as its ability to inhibit fibroblast proliferation or attenuate osteoclast formation [39, 40]. Likewise, Tsp-1 serum levels were also not previously studied in PsA patients, and conflicting data regarding Tsp-1 serum levels come from studies in other rheumatic diseases (e.g., RA, SLE) [41, 42]. Our results show decreased Tsp-1 levels in PsA and RA patients relative to HC and are in agreement with the decreased levels found in SLE patients [42], requiring further exploration.
Notably, the factors tested in this study are mediators not only of angiogenesis, but also of inflammation, cell proliferation, and leukocyte recruitment and activation [8]. Thus, it is difficult to separate their pro-angiogenic role from their other functions in vivo. Therefore, we turned to the in vitro co-culture system, where their angiogenic effect, and particularly the effect of CD147, could be studied. We show that in an in vitro co-culture of fibroblasts and monocytes, CD147 plays an important role in mediating interactions between these two cell types, as the addition of recombinant protein mimicking homophilic CD147 interactions induced secretion of VEGF and MMP-9, and CD147 inhibition with the anti-CD147 antibody lowered this secretion. Likewise, the inhibiting effect of the anti-CD147 antibody on the formation of tube-like structures and on the migration and proliferation of endothelial cells demonstrates that these interactions and their resulting increased VEGF and MMP-9 levels directly affect endothelial cells, the cell most intimately involved in angiogenesis.
The fact that CD147 inhibition did not result in complete inhibition of these factors suggests that other mediators may also affect VEGF and MMP-9 levels. The discrepancies between the in vivo influence and the in vitro systems (e.g., changes in the MMP-9 levels) may arise from the lack of influence from other cell types and remote organs (such as the inflicted skin and synovium) in the in vitro system.
Lastly, the pro-angiogenic state of a PsA patient may be expressed by calculating the ratio of CD147 and Tsp-1. We show here that CD147 is an important mediator of the interactions between fibroblasts and monocytes and that this interaction, in turn, leads to the induction of the potent pro-angiogenic factors VEGF and MMP-9, rendering it a pro-angiogenic protein by itself. In contrast to CD147, Tsp-1 is known to be a strong anti-angiogenic factor [43]. This ratio, which is elevated in active inflammation and reduced in disease remission, may reflect the state of angiogenesis in these conditions. This is supported by the significant correlations found between this parameter and other pro-angiogenic factors in this study. However, further studies are needed to demonstrate the relevance of this ratio in clinical practice.
Our study has some limitations. The small size of the patient cohorts, especially of the control groups, may have masked the significance of our findings in some instances. Our active PsA patients used tobacco more than the patients in remission, and this could enhance angiogenesis in this group [44]. Most of our PsA and RA patients were treated. We did not have access to fibroblast-like synoviocytes (FLS) or to synovial fluids, and therefore, we used the fibroblast cell line HT1080 in our in vitro experiments. Since the concentrations of many of the tested factors are regulated locally, a future study should verify our in vivo findings in co-cultures of FLSs and monocytes.