Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, characterized by destruction of multiple joints in which the articular cartilage and bone are destroyed by proliferative synovitis. The synovial lesion is formed by proliferation of synovial lining cells, thickening of the synovial membrane with villous projections, dense infiltration of the synovia with lymphocytes, macrophages, dendritic cells, and plasma cells, and the presence of osteoclasts (1). Osteoarthritis (OA) is considered to be a degenerative disease of the hyaline articular cartilage related to mechanical damage and aging. Inflammatory infiltration of the synovial membrane with lymphoid cells is found in a proportion of OA patients (2). Loss of the perichondrium and fibrillation of the cartilaginous matrix lead to eburnation of the bone surface and deformation of the joint. Although the pathophysiologic mechanisms of RA and OA have been described extensively, the development of endothelial precursor cells in the inflamed and damaged joint along the endothelial lineage is still an enigma (3, 4).
In the peripheral blood, circulating bone marrow–derived stem cells represent <0.01% of nucleated cells, and phenotypically distinct populations of stem cells can be found (5). The majority of stem cells express the surface molecules CD34, CD133, CD117 (c-Kit), and CD90 (Thy-1) (6–8). Within the circulating CD34+ stem cell population, a subset expresses the endothelium-specific marker vascular endothelial growth factor receptor 2 (VEGFR-2) (9, 10). These cells are considered circulating endothelial precursor cells or hemangioblasts, and they give rise to endothelial colonies in culture.
CD34+,VEGFR-2+ endothelial precursor cells represent <2% of the CD34+ cell population in the peripheral blood (9, 10). In peripheral tissues, precursor cells reside in small niches involved in the permanent restoration of damaged tissue, since continuous self-renewal requires the presence of tissue-specific progenitor cells (11). They are recruited from niches to differentiate to locally required mature cell types, and they can be replaced by circulating CD34+ stem cells which are in a “steady state” of equilibrium with resident precursor cells in the niches. It has been speculated that circulating endothelial progenitor cells expressing CD34 and VEGFR-2 can migrate to the inflamed joint, be incorporated into pathologic neovascular foci, and induce vasculogenesis (3, 12). CD34+,VEGFR-2+ cells expressing CD133 have the greatest in vitro potential to differentiate along the endothelial lineage to more mature endothelial cells (11, 13). During maturation, these cells lose CD133 but still express CD34 and VEGFR-2.
New vessels can also be generated by angiogenesis, the sprouting of new capillaries from preexisting vessels, which does not require endothelial precursor cells from the circulation (14, 15). To allow subsequent sprouting, mature vessels are first destabilized (14, 15). New vessels formed by sprouting are initially immature and must develop further through remodeling and maturation. The process of blood vessel formation, growth, and stabilization is regulated by factors, such as the different VEGF isoforms, placental growth factor, angiopoietins, ephrins, transforming growth factor β, and platelet-derived growth factor, which must be very precisely orchestrated in terms of time, space, and dose to form a functional vascular network (16). Further, to ensure adequate vessel stabilization, a coordinated growth of endothelial cells, pericytes, smooth muscle cells, and fibroblasts is required (17, 18). Subendothelial pericytes and smooth muscle cells can be generated from bone marrow stromal cells that coexpress STRO-1 and α-smooth muscle actin (α-SMA) (19, 20). Under physiologic conditions, blood vessel formation is mandatory for remodeling of joint lesions, since cell regeneration, function, and survival depend on oxygen and nutrient supply, and virtually all cells must reside within 100 μm of a capillary (12, 14, 15).
In the present study, we investigated endothelial progenitor cells that arise in situ or from circulating precursors, inducing vasculogenesis in the synovial membrane of RA and OA patients. We also investigated their local differentiation along the endothelial lineage to more mature endothelial cells.
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- MATERIALS AND METHODS
During inflammatory joint disease, the damaged articular cartilage and bone can be replaced by newly differentiated cells derived from precursor cells, or through local self repair by fully differentiated cells (3, 4). To initiate tissue repair, a sufficient vascular supply is necessary, and blood vessels are mandatory for mesenchymal progenitor cells to migrate to the damaged joint. New vessels can be formed simultaneously by vasculogenesis, with circulating endothelial precursor cells migrating to the joint where they differentiate and proliferate, or by angiogenesis, which involves sprouting from preexisting vessels (12).
In the synovial tissue of 14 of 18 RA patients and in that of 11 of 15 OA patients, CD34+,CD31− precursor cells formed cell clusters with STRO-1+ cells and CD133+ precursor cells. Within these cell clusters, CD34+ precursor cells were in the center of the layer, surrounded by STRO-1+ cells and with CD133+ precursor cells on the outside. The majority of CD34+ cells in the cell clusters were STRO-1−, but they expressed CD133 with low intensity on their surfaces. CD133+ cells expressed CD34 with low intensity; STRO-1+ cells were CD34−, and some STRO-1+ cells expressed low levels of CD133.
In addition to these cell clusters, single CD133+ cells were found in the lumen of blood vessels and in perivascular areas within the synovial membranes of RA patients. It is fascinating to find and show a single circulating CD133+ cell in a vessel, since this cell population is extremely rare in the blood circulation. Morphometric analysis of RA synovium showed that higher numbers of CD133+ precursors and STRO-1+ cells were found, particularly in synovial membranes with lymphocyte ingress. Interestingly, in the synovial tissue of 11 of the 15 OA patients, precursor cells expressing CD34, CD133, or STRO-1 were found, and synovial tissue extracts showed message for CD34 and CD133. The intensities of the PCR bands corresponding to CD34 and CD133 mRNA expression were similar in RA and OA patients, since ∼3–5 times more OA synovial tissue was needed to yield the same amount of total RNA as that isolated from RA synovium. Further, the total number of cells/mm2 was 3–4 times greater in RA synovial membranes than in OA tissues. These results indicate that vasculogenesis is initiated in the synovial membranes of both RA and OA patients. In the synovial tissue of the remaining 4 OA patients, however, CD133+ cells and STRO-1+ mesenchymal progenitor cells were not detectable, and no CD133 message was observed.
The majority of CD34+ precursor cells within the cell layer expressed high levels of the chemokine receptor CXCR4, the receptor of SDF-1. The chemokine SDF-1 plays a central role in selective homing and recruitment of circulating stem cells and activated CD4+ T cells to the inflamed joint (25). SDF-1 can stimulate integrin-mediated arrest of CD34+,CXCR4+ stem cells on vascular endothelium under shear flow by increasing the adhesiveness of the integrins very late activation antigen 4 (VLA-4), VLA-5, and lymphocyte function–associated antigen 1 (26).
Furthermore, CD34+ progenitor cells and CD133+ endothelial precursor cells in the synovial tissue of RA and OA patients expressed high levels of VEGFR-2. VEGFR-2 mediates the major growth and permeability actions of VEGF, the most critical driver of vascular formation under various physiologic and pathologic conditions (15, 16, 27, 28). VEGF consists of 4 major isoforms (VEGF121, VEGF165, VEGF189, and VEGF206) assembled by alternative splicing. In the synovial tissue of RA and OA patients, VEGF121 is constitutively expressed, while the expression of VEGF165 is detected in only 41% of RA synovia and is not found in OA (28). Selective up-regulation of VEGF165 and its signaling via VEGFR-2 can play an important role in synovial angiogenesis and vasculogenesis in RA. Herein we have shown that high levels of VEGFR-2 were expressed on the surface of two populations of precursor cells in synovial tissues of RA and OA patients, one population being CD34+ and the other being CD133+.
In contrast, the majority of STRO-1+ cells in the synovial tissue were VEGFR-2− but expressed α-SMA, a marker for subendothelial pericytes and smooth muscle cells, on their surfaces. This confirms the findings of previous studies in which STRO-1+ cells were found within the walls of the microvasculature of the human thymus (where a subset of them also expressed α-SMA) and in normal and hyperplastic pannus of RA patients (20, 29–31). STRO-1+,α-SMA+ pericytes can be recruited from neighboring resident mesenchymal cells through replication, migration, and differentiation of other pericytes downstream of the growing vascular bud, or they can arise directly from endothelial cells or their progenitors. They are needed to coat the microvessels and stabilize them by preventing vessel pruning (18). Pericytes are one of the most elusive cell types in the body, and their significance as potential progenitor cells has been postulated repeatedly.
In general, STRO-1+ mesenchymal progenitors have an enormous plasticity; they can differentiate to chondroblasts, osteoblasts, adipocytes, and smooth muscle cells, and a fully differentiated chondrocyte can dedifferentiate in culture and then shift to an osteogenic phenotype (32–35). This highlights the nonirreversible nature of differentiation of cells from the mesenchymal cell lineage, otherwise seen as end points of various pathways. There is a physiologic need for plasticity of connective tissue cells in inflammation, namely, the need to adapt different tissues that reside next to one another during tissue repair.
In conclusion, we can say that endothelial progenitor cells are present in the synovial membranes of RA and OA patients and can form cell clusters to generate new vessels. De novo formation of blood vessels can help to prevent disease by allowing bone marrow–derived mesenchymal cells to migrate to the inflamed joint to induce tissue repair. New vessel growth, however, is not always desirable. Extensive vessel formation facilitates the proliferation of the pannus in RA by providing the growing cell mass with sufficient blood supply. Leukocyte infiltration into synovial tissue depends on generation of new blood vessels, vessel formation is induced and modulated by cytokines and chemokines. Halting of blood vessel development could be an avenue of research potential new therapies.