Abstract

Rheumatoid Arthritis (RA) is a systemic, autoimmune, inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration in the synovial tissues, and progressive destruction of cartilage and bones. This disease often leads to chronic disability. More recently, activation of synovial fibroblasts (SFs) has been linked to innate immune responses and several cellular signalingpathways that ultimately result in the aggressive and invasive stages of RA. SFs are the major sources of pro-inflammatory cytokines in RA synovium. They participate in maintaining the inflammatory state that leads to synovial hyperplasia and angiogenesis in the inflamed synovium. The altered apoptotic response of synovial and inflammatory cells has been connected to these alterations of inflamed synovium. RA synovial fibroblasts (RASFs) have the ability to inhibit several apoptotic proteins that cause their abnormal proliferation. This proliferation leads to synovial hyperplasia. Apoptotic pathway proteins have thus been identified as possible targets for modifying the pathophysiology of RA. This review summarizes current knowledge of SF activation and its roles in the inhibition of apoptosis in the synovium, which is involved in joint damage during the effector phase of RA development.

Introduction

Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease that affects approximately 0.5% to 1% of the global population1. Synovial hyperplasia, which is the influx of inflammatory cells into synovial tissues, accelerates the destruction of bone and cartilage. The deterioration results in chronic pathology2, 3. Infiltrated immune cells cause degradation of the surface and extracellular matrix of the articular cartilage, cause bone erosion, and reduce the quality of life4, 5. The exact cause of RA is elusive. More recently, it has been demonstrated that the advancement of the disease solely relies on the activity of synovial fibroblasts (SFs) or rheumatoid arthritis specific synovial fibroblasts (RASFs) in the RA synovium2. In the RA synovium, the innate immune response and a few cellular signaling mechanisms mobilize the SFs that ultimately induce their aggressive and invasive behavior2, 6, 7. SFs provide the most important supply of pro-inflammatory cytokines in RA synovium, along with several growth factors that facilitate the prolonged inflammatory state7, 8, 9 . The latest findings elucidate the role of several inhibitors of growth factors that can inhibit the proliferation of the SFs that may result in synovial hyperplasia and promote angiogenesis within the inflamed synovium10, 11. The altered apoptotic response of synovial and inflammatory cells has been connected to these specific alterations of the inflamed synovium in RA12, 13, 14, 15. Contemporary RA studies focus on the chronic accumulation of osteoclasts and inflammatory cells in the RA synovial joints due to altered apoptosis. Furthermore, several apoptotic proteins have been identified as potential targets for modifying RA pathogenesis16, 17, 18. In this review, we have summarized the recent knowledge on SF-activation and SF-mediated mechanisms of inhibition of apoptosis inside the synovium involved in joint damage over the course of the effector phase of RA development.

Epidemiology of RA

Population-based studies have indicated that RA has a global prevalence of roughly 0.5% to 1% among adults1. According to the Global Burden of Disease study in 2010, the reported prevalence rate of RA is 0.24%, and females have twice the prevalence rate as males19, 20. However, according to a recent meta-analysis, the RA prevalence rate increased from 0.24% to 0.46% between 1980 and 201821. It was found in another study that the prevalence rate increased to 0.56% between 1980 and 201922. The prevalence rate of RA is much higher in Australia; the next highest prevalence rates are in North America and Europe. There is a relatively lower prevalence in several Asian countries19, 23. Another cross-sectional study reported that the prevalence rate of RA is 0.34% in the Indian population; however, it varies from 0.28% to 0.7% in the Indian population24, 25. These variations between regions and countries may exist as a result of differing methodological approaches as well as potential genetic and environmental risk factors. In the United States, 1 in every 12 women and 1 in every 20 men develops a rheumatic disease at some point in their lives26. Under post-COVID-19 pandemic circumstances, a UK-based population study reported that RA patients had an increased mortality rate during the pandemic compared to their pre-pandemic mortality rates, and the mortality risk was more prevalent in women27.

× Figure 1 . Schematic diagram of normal and RA synovial joint . In normal synovial joint, single layer of synovial membrane is lined by fibrous articular capsule and the bone surface within the articular capsule is covered by articular cartilage. In RA, hyperproliferation of synovial membrane fibroblasts cells, infiltration of immune cells (like T cells, B cells, macrophages, osteoclasts) lead to the activation of various proinflammatory pathways that ultimately results in pannus formation and bone erosion. Figure 1 . Schematic diagram of normal and RA synovial joint . In normal synovial joint, single layer of synovial membrane is lined by fibrous articular capsule and the bone surface within the articular capsule is covered by articular cartilage. In RA, hyperproliferation of synovial membrane fibroblasts cells, infiltration of immune cells (like T cells, B cells, macrophages, osteoclasts) lead to the activation of various proinflammatory pathways that ultimately results in pannus formation and bone erosion. × Figure 2 . NF-κB activation in RASFs . Proinflammatory cytokines (TNF-α, IL-6) trigger TLR that lead to the activation of canonical pathway of NF-κB. In this pathway, activated-IKK complex phosphorylates the inhibitory subunit of IKB that leads to degradation, which allows NF-κB (P50/P65) dimer to translocate to the nucleus and induce production of inflammatory cytokines and MMPs and also enhance the expression of YY1. YY1 act as a transcription factor that represses the miR-10a expression and downregulates IRAK/TRAF it via inhibition of miR-10a. Downregulated miR-10a accelerates IκB degradation and further continues the NF-κB activation in canonical pathway. In the alternative pathway, specific stimuli like CD40L or lyphotoxin activates NF-κB inducing kinase (NIK), which recruits IKK to phosphorylate P100 complex leading to its degradation. Processing of P100, the precursor of P52, allows P52/RelB NF-kB complex to enter into the nucleus and induces transcription of genes including chemokines, RANKL and also MMPs. The black lines indicate the canonical and alternative pathway of NF-κB activation; red line indicates the regulation of NF-κB activation in canonical pathway; red dotted line indicates the transportation of RNA molecules outside the nucleus. IRAK : Interleukin-1 receptor-associated kinase; TRAF : Tumor necrosis factor receptor associated factor; YY1 : Ying Yang 1; miR-10a : microRNA-10a; RANKL : Receptor activator of NF-κB ligand; MMPs : Matrix Metalloproteinases. Figure 2 . NF-κB activation in RASFs . Proinflammatory cytokines (TNF-α, IL-6) trigger TLR that lead to the activation of canonical pathway of NF-κB. In this pathway, activated-IKK complex phosphorylates the inhibitory subunit of IKB that leads to degradation, which allows NF-κB (P50/P65) dimer to translocate to the nucleus and induce production of inflammatory cytokines and MMPs and also enhance the expression of YY1. YY1 act as a transcription factor that represses the miR-10a expression and downregulates IRAK/TRAF it via inhibition of miR-10a. Downregulated miR-10a accelerates IκB degradation and further continues the NF-κB activation in canonical pathway. In the alternative pathway, specific stimuli like CD40L or lyphotoxin activates NF-κB inducing kinase (NIK), which recruits IKK to phosphorylate P100 complex leading to its degradation. Processing of P100, the precursor of P52, allows P52/RelB NF-kB complex to enter into the nucleus and induces transcription of genes including chemokines, RANKL and also MMPs. The black lines indicate the canonical and alternative pathway of NF-κB activation; red line indicates the regulation of NF-κB activation in canonical pathway; red dotted line indicates the transportation of RNA molecules outside the nucleus. IRAK : Interleukin-1 receptor-associated kinase; TRAF : Tumor necrosis factor receptor associated factor; YY1 : Ying Yang 1; miR-10a : microRNA-10a; RANKL : Receptor activator of NF-κB ligand; MMPs : Matrix Metalloproteinases. × Figure 3 . Regulation of apoptotic pathway in RASFs . Fas-FasL interaction recruits FADD along with pro-caspase8 which leads to the activation of caspase 8. Activated caspase 8 now activates caspase 3 or Bid. Bid disrupts the mitochondrial homeostasis with the help of pro apoptotic molecule Bax and Bak. In RA synovium, FLIP inhibits caspase 8 activation in RASFs. TNFα and growth factors also up regulates anti-apoptotic molecules like A1 and Mcl-1 respectively; along with BCL-2, these anti apoptotic molecules try to maintain the mitochondrial homeostasis. Loss of mitochondrial integrity causes release of Cyt C that leads to the activation of caspase 3. Survivin, an anti-apoptotic molecule also inhibits apoptosis in RASF by binding to caspase-9. Ursolic acid (UA), a plant derived pentacyclic triterpenoid, can induces apoptosis in RASFs s by inducing proteasomal degradation of Mcl-1. Embelin, another plant-based para-benzoquinone, can inhibits XIAP and act as a potential key molecule in RASFs apoptosis pathway. Black line indicates the normal apoptosis pathway; red line indicates the inhibition of the pathway. Figure 3 . Regulation of apoptotic pathway in RASFs . Fas-FasL interaction recruits FADD along with pro-caspase8 which leads to the activation of caspase 8. Activated caspase 8 now activates caspase 3 or Bid. Bid disrupts the mitochondrial homeostasis with the help of pro apoptotic molecule Bax and Bak. In RA synovium, FLIP inhibits caspase 8 activation in RASFs. TNFα and growth factors also up regulates anti-apoptotic molecules like A1 and Mcl-1 respectively; along with BCL-2, these anti apoptotic molecules try to maintain the mitochondrial homeostasis. Loss of mitochondrial integrity causes release of Cyt C that leads to the activation of caspase 3. Survivin, an anti-apoptotic molecule also inhibits apoptosis in RASF by binding to caspase-9. Ursolic acid (UA), a plant derived pentacyclic triterpenoid, can induces apoptosis in RASFs s by inducing proteasomal degradation of Mcl-1. Embelin, another plant-based para-benzoquinone, can inhibits XIAP and act as a potential key molecule in RASFs apoptosis pathway. Black line indicates the normal apoptosis pathway; red line indicates the inhibition of the pathway. Normal Synovial joint

A key structural feature of a synovial joint is the presence of two components: the synovial fluid and the surrounding soft connective tissues, the articular cartilage, capsule, and ligaments (Figure 1). The articulating surfaces of the bones make contact with each other in this fluid-filled space. The articulating bony surfaces in the synovial joints move efficiently over each other and allow the bones to move smoothly against one another, which enables seamless joint mobility28.

The joint provides the infrastructure that facilitates mobility. The synovial membrane encases the joints, providing structural support (via a fibrous capsule), lubrication, and nutrients to the cartilage. The walls of the synovial cavity are formed by a fibrous connective tissue just outside the area of the bones’ articulating surface, the articular capsule, and the thin synovial lining membrane28. The cells of this membrane secrete a slimy synovial fluid that provides lubrication and nutrients to articular cartilages (Figure 1).

Synovial fluid consists of two types of cells: macrophage-like synovial cells (or Type A cells) and fibroblast-like synoviocytes (FLS) (or Type B cells)29, 30. In normal synovium, type A cells are located in the intimal and sub-intimal regions and are mainly derived from the blood monocyte-macrophage lineage with characteristic Golgi apparatuses and dense vacuoles29. These cells are spherical, located at the upper part of the synovium surface, and migrate to the synovial membrane, where they remain resident cells30. Subintimal regions of normal synovium contain thin-walled lymphatic vessels. These vessels may be involved in leukocyte trafficking in the normal synovium31. The Type A synovial cells express Fc-gamma immunoglobin receptor (FcγR) and are positive for CD68 and CD 16328. These cells express major histocompatibility class II molecules (MHC-II). During the early stages of the immune response, they play an important role in antigen presentation. The type A cells can absorb and degrade extracellular constituents, cell debris, microorganisms, and many antigens within the synovia32.

Type B synoviocytes are the predominant cellular types found within the synovium. These cells are similar to fibroblast cells in that they express type IV and V collagens, vimentin, and the CD90 marker28, 30. They have rough endoplasmic reticulum throughout their cytoplasm, which indicates their prominent role in active protein trafficking within the synovia30, 32. Within the synovial lining, FLS synthesize uridine diphosphoglucose dehydrogenase (UDPGD), a key enzyme for the synthesis of hyaluronan30. FLS also release lubricin, which aids in joint-lubrication33. Cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecules-1 (ICAM-1), integrins, and the CD55 marker, are also expressed in both the intimal and sub-intimal regions of the synovium30. VCAM-1 is vital in cellular trafficking; the CD55 marker completely differentiates FLS (type B) from the type A macrophages28.

RA synovium

The prominent hallmark of RA is local and systemic inflammation initiated by the infiltration of immune cells2. There are two primary tissue layers in the RA synovium: the membrane lining (or intima) and the sub-intima30. RA significantly alters the structures of both areas (Figure 1). The T lymphocytes represent 30 — 50% and the B lymphocytes constitute about 5% of the sub-lining cells3. The proliferation of blood vessels and lymphoid aggregation is common in the intimal and subintimal areas of the inflamed synovium due to the hypoxic conditions34. Along with inflammatory cells such as neutrophils and lymphocytes, rheumatoid arthritis synovial fibroblasts (RASF) or rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) or type B synoviocytes modulate numerous pro-inflammatory pathways inside the rheumatoid joint8, 35. The macrophage-like cells have an extremely activated makeup and elaborate an array of pro-inflammatory cytokines, chemokines, and growth factors that stimulate RASFs3. RASFs in the synovium lining layer display many signs of cellular activation, which leads to aggressive and invasive behavior3. In primary culture, synovial cells can escape the contact inhibition and can grow in an anchorage-independent manner over multiple passages36. The ability of activated RASFs to adhere to articular cartilage and to deeply penetrate the extracellular matrix through controlling gene expression is associated with the cartilage destruction and is the most prominent trait9, 35. Even in the absence of pro-inflammatory cytokines, the activated RASFs can maintain their aggressive and invasive behavior; such activity causes long-term structural alteration within the disease joints3.

Activation of RASFs in RA Modulation of innate immunity

The activation of the innate system results in an upregulation of effector molecules involved in the aggressiveness of RASF even before clinical signs and symptoms appear. Microbial fragments can stimulate RASFs via the Toll-like receptors (TLRs), a conserved pattern recognition receptor (PRR) system37, 38. TLRs play a critical role in the early innate immune response in the face of invading microbes by sensing preserved pathogen structural patterns called PAMPs (Pathogen-Associated Molecular Patterns) or alterations in DAMPs (Danger-Associated Molecular Patterns) emitted from dead or dying cells37. Several TLR (TLR2, TLR3, TLR4, and TLR7) expressions have been discovered in RASFs out of the now 12 well-known TLRs in humans38. RASFs from the patients display high levels of specific TLR2, TLR3, and TLR4, and their stimulation triggers the production of IL-6 and matrix metalloproteinases (MMPs)39, 40. RASFs also produce a wide range of pro-inflammatory cytokines and effector molecules and exhibit a prominent pathogenic function during RA progression35. When stimulated by the appropriate PAMPs/DAMPs, RASF-TLRs initiate a number of signaling cascades, resulting in the activation of the Nuclear Factor Kappa Beta (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) pathways, including c-Jun N-terminal kinase (c-JNK) inside the disease joints. This promotes the synthesis and upregulation of several pro-inflammatory cytokines [such as Tumor necrosis factor (TNF)-α, IL-1, and IL-12], Type I interferon (Type I IFN) and numerous MMPs35, 41. Some of the genes regulated by activated NF-B include TNF-, IL-6, IL-8, inducible Nitric Oxide Synthase (iNOS), and Cyclooxygenase-2 (COX-2)36. RASFs are linked to COX-2 systems; these systems, along with other cellular signaling pathways, are involved in the regulation of synovial inflammatory pathways. TLRs are crucial to the modulation of the innate immune phases of RA development in RASFs.

Role of growth factors

Fibroblast Growth Factor (FGF) is an important stimulant for RASFs8. FGF-2 mRNA and FGF-2 proteins have been found in the pannus of RA patients, which indicates that FGF-2 is heavily involved in promoting cartilage and bone degradation, in addition to synovial hyperplasia42. Recent cell culture studies show that human RASFs are fundamental sources of FGF-2 in RA, and along with IL-17, it promotes the pathogenesis of autoimmune arthritis8. FGF-2 has an impact not only on the proliferation of RASFs but also on the induction of bone resorption by activating mature osteoclasts in a cultured cell line system43. FGF-2 also induces telomerase activity in cultured RASFs. Patients with RA have higher telomerase activity in their peripheral blood lymphocytes than healthy individuals44. The expression of various telomerase-related factors, such as Telomere Repeat-binding Factor-2 (TRF2) or the human telomerase RNA gene (hTERC), is also capable of modulating the telomerase activity in cultured RASFs, even when they are induced by pro-inflammatory cytokines such as TNFα45. Collectively, these studies indicate that FGF-2 has a direct role in upregulating the telomerase activities in cultured RASFs, which may suggest the proliferative nature of RASFs.

Another significant growth factor involved in the proliferation of RASFs is Transforming Growth Factor-β (TGF-β). TGF-β1 is a multi-functional cytokine that promotes angiogenesis and regulates cellular development. Studies demonstrate that RASFs exhibit a high level of TGF-β expression39. According to a recent study, the stimulatory effect of TGF-β enhances the MMP2 expression in RASFs. TGF-β causes epithelial–mesenchymal transition (EMT) in RASFs by activating the TGF-β/Smad signaling pathway, which ultimately contributes to the migratory phenotypes of RASFs39. Multiple recent studies reveal that a ubiquitous phosphatase, non-receptor Protein Tyrosine Phosphatase 14 (PTPN 14), and a tumor-promoting transcription co-activator, Yes-Associated Protein (YAP), promote TGF-β-dependent SMAD3 nuclear localization in RASFs. This may also promote the pathogenic behavior of the RASFs in relation to synovial hyperplasia46. Platelet-Derived Growth Factor (PDGF) is another strong stimulator of RASFs. Numerous isoforms of it can be expressed within the RA synovium47. In RASFs, PDGF causes the loss of contact inhibition and anchorage-independent growth36. A recent study indicates that the phosphorylation of PDGF receptors (PDGFR) along with TGF-β co-operation triggers the formation of matrix-degrading invadosomes in RA synoviocytes48.

Activation by NF-κB pathway

One of the key phases concerning RASFs’ proliferation is the NF-κB activation pathway49, 50, 51. It plays an important role in the inflammatory pathway by controlling the expression of many pro-inflammatory gene clusters. This transcription factor is dimeric in nature, formed by p50 and p65 subunits; however, several combinations with alternative subunits happen conjointly. Normally, a class of inhibitory proteins called inhibitor of NF-κB kinases (IKKs) sequesters transcription factor NF-κB proteins within the cytoplasm, which masks the nuclear localization sequence of NF-κB dimers and holds them in an inactive state in the cytoplasm51. Activation of NF-κB needs diverse stimuli, including ligands of various cytokine receptors, PPRs, or TNF receptors (TNFRs), as well as B-cell receptors (BCRs) and T-cell receptors (TCRs)51. Phosphorylation of IKK occurs in response to stimulation, which is an important step in NF-κB activation (Figure 2). Two kinase subunits, IKKα (IKK1) and IKKβ (IKK2), and a regulatory subunit, IKKγ (NEMO), comprise the IKK complex51. Phosphorylation of the inhibitory subunits releases the NF-κB dimers and allows the dimers for nuclear translocation, where they attach to the target gene’s promoter50, 51. The NF-κB signaling pathway is activated by two mechanisms. The canonical route is activated by a variety of stimuli, including lipopolysaccharides (LPSs) and pro-inflammatory cytokines such as TNF- and IL-1, PPRs, and growth factors. Alternatively, certain stimuli, such as B-cell-activating lymphotoxin or CD40 ligand (CD40L), can activate the non-canonical pathway52. However, the activation of NF-κB dimers depends on the phosphorylation and processing of precursor protein p100 in the non-canonical pathway. Both pathways cause the translocation of NF-κB dimers to the nucleus in order to induce NF-κB-targeted gene transcription52.

Both IKK1 and IKK2 are constitutively expressed by RASFs. Activation of the IKK complex results in sequential degradation of IκB and accelerated NF-κB nuclear binding; however, IKK1 is the key molecule for NF-κB activation in RASFs49. There is recent evidence that microRNA-10a (miR-10a) also accelerates the IκB degradation pathway in RASFs6. As a result, NF-κB is released and reaches the nucleus of RASFs, where it binds to the Ying Yang 1(YY1) promoter and upregulates its expressionFigure 2. The YY1 protein also functions as a transcription factor, repressing miR-10a expression by binding to the miR-10a promoter region. Downregulating miR-10a expression promotes NF-B activation, which results in the production of a large amount of cytokines (TNF-, IL-1, IL-6, and IL-8) and MMPs6. NF-κB additionally activates a vast spectrum of downregulating genes and effector molecules (such as COX-2) that further contribute to synovial proliferation and inflammation16. Bioactive plant components such as paeonol inhibited both the NF-κB p65 subunit phosphorylation and IκB degradation in a cell culture-based study53. According to an additional study, icariin (a potent and bio-active phytocomponent from Epimedium) can inhibit TNF-α stimulated RASF proliferations in a concentration-dependent manner by inhibiting the TLR2/ NF-κB pathway54. According to an additional recent study, several phenolic constituents can inhibit the IκB degradation and phosphorylation NF-κB p65 subunit at the protein level in a TNF-α stimulated human RASF cell line55. These findings illustrate the potential of NF-κB signaling pathway modulation as a future therapeutic agent for RA progression.

Role of MMPs and matrix degradation

MMPs constitute 5 subfamilies of enzymes that conjointly degrade all extracellular matrix components56. Collagenases (MMP-1, MMP-13), stromelysins (MMP-3), and membrane type (MT) MMP-14 are abundantly expressed within the RA synovium56, 57. Multiple components, including pro-inflammatory cytokines such as IL-1β, TNF-α, growth factors, NF-κB, and TLR ligands, activate their synthesis and expressions in the RASFs53, 57. Within the RASFs, MT1-MMP (MMP-14) is the most important MMP related to cartilage destruction58. MT1-MMP performs a crucial role in the aggressive makeup of RASFs, is enormously expressed on the RA synovium, and is accountable for the invasion of RASFs into cartilage58.

Tissue inhibitors of MMPs (TIMPs), a physiologic inhibitor produced by synovium chondrocytes and fibroblast cells, tightly regulate MMP gene expression in RASFs56. All TIMPs can inhibit all the MMPs through non-covalent interactions involving a conserved cysteine residue at position 1, which raises the possibility of targeting TIMPs as therapeutic agents56. However, the normal amounts of TIMPs found in RA joints, are insufficient to counteract the matrix-degrading effects of MMPs and cathepsins59. A study involving a canine RA model has demonstrated decreased synovial TIMP-1 expression in comparison to MMP-3 level59. However, the over-expression of the TIMPs may have the beneficial effect of decreasing the RASF-mediate cartilage destruction60. It has been reported in a cell culture study that the treatment with anti-malarial drugs such as artesunate can increase the mRNA and protein expression of TIMP-2, in contrast to MMP-2 and MMP-9 expression, in a dose-related manner60. These studies could lead to possible therapies targeted at the RASF-mediated joint-destructive events in RA. In addition to MMPs, cathepsin K, produced through RASFs, contributes extensively to the degrading processes and the resorption of bones inside the rheumatoid joints61, 62. The cathepsin family contains eleven members of proteinases, among which cathepsins B, H, K, L, and S are the notable proteolytic enzymes capable of degrading native collagens and other components of the ECM63. Pro-inflammatory cytokines such as IL-6 can stimulate cathepsin K expression in murine cell lines64. Cathepsin B also promotes TIMP degradation in RA patients and activates PDGF-mediated migration and invasion of RASFs via MAPKs and c-JNK65, 66. In anti-citrullinated protein antibody (ACPA)-positive RA patients, it has been found that the cathepsin K concentration increases in bone marrow samples more than in the peripheral blood. This suggests the local production of the protease enzyme62. In summary, it is well documented that RASFs are the prominent effector cells in producing MMPS and cathepsins that stimulate the progressive degradation of cartilages and bone, which might be the primary hallmarks of RA.

Hypoxia and angiogenetic factors

Hypoxia is another prominent feature that triggers RASFs’ involvement in increased synovitis, angiogenesis, bone erosion, and cartilage destruction in RA pathogenesis. In RA patients, Angiotensin Converting Enzyme (ACE), which catalyzes the synthesis of angiotensin II from its inactive precursor, angiotensin I, is elevated in RASFs67. Increased tissue ACE can raise the angiotensin II concentration, which leads to synovial hypoxia67. Hypoxia-inducible factor (HIF) is one of the key regulators of synovial tissue hypoxia68. In normal conditions, HIF-1α protein is hydroxylated by oxygen-dependent prolyl hydroxylase domain (PHD) protein and by factors inhibiting HIF-1α (FIH1) protein. These factors inactivate NF-κB transcriptional activation and cause degradation of HIF-1α68, 69. In hypoxic conditions, PHD activity reduces, which permits HIF-1α to phosphorylate the IKK subunits; this leads to the activation of NF-κB-induced gene expression68. In RASFs, HIF-1α promotes IL-8, MMPs, and vascular endothelial growth factor (VEGF) expression, which lead to cartilage destruction and angiogenesis70. When synovial tissues from RA patients are cultured in a hypoxic environment, they had elevated amounts of MMP-1 and MMP-3 expression and relatively low levels of TIMP-1 expression71. Consequently, over-expression of HIF-1α causes significant up-regulation of MMPS in cultured RASFs, which may lead to the cartilage invasion72. Furthermore, suppression of HIF-1α by RNA interference (RNAi) also suppresses the MMP-13 expression in cultured RASFs, which confirms the direct regulation of HIF-1α-induced MMP-13 expression72. Inducing HIF-1α through TLR signaling increases the production of inflammatory cytokines (IL-6, IL-8, TNF-α), MMPs (MMP-1, MMP-3, MMP-9), and VEGF contributes to the progression of RA73. In a recent study of a collagen-induced arthritis Wistar rat model, researchers found that inhibiting HIF-1α using RNAi reduces the expression of inflammatory cytokines (such as IL-6, IL-1, and TNF-α) in both synovial cell culture and peripheral blood serum69.

To restore oxygen delivery to the RASFs’ tissue, angiogenesis is initiated as a part of the cellular adjustments. The increased blood supply is critical for transporting immune cells to the site of inflammation and providing nutrition to the pannus74. VEGF is a key regulator of angiogenesis in RASFs70. A positive feedback regulation between HIF-1α and VEGF triggers angiogenesis in hypoxic conditions68, 73. Both HIF-1α and HIF-2α can stimulate VEGF expression in the RASFs65. In addition to HIF1-α and VEGF, pro-inflammatory mediators can also promote angiogenesis in RASF. This promotion leads to the expression of cytokines (IL-6, IL-8), chemokines (CXCL10, CXCL17), MMPs (MMP-1), and adhesion molecules (ICAM-1, VCAM-1), which may trigger the synovial cellular infiltration and invasive behavior of the RASFs73, 75. According to a recent study, Antcin K (a phytosterol isolated from Antrodia cinnamomea, medicinal mushroom) significantly inhibited VEGF expression in cultured RASFs by downregulating the phospholipase C(PLC)-γ and protein kinase C (PKC)-α pathway76. These findings imply that hypoxia has an important role in promoting angiogenesis, cell infiltration, cartilage damage, and disease progression. They also imply that targeting HIF1 and VEGF could be a novel strategy for future medication for RA.

RASFs and matrix degradation

The control of osteoclastogenesis by RASFs influences bone erosion77, 78. Receptor activator of NF-κB (RANK) and its ligand (RANKL), a TNF receptor-family protein, initiate the bone-degrading pathway, osteoclast development, and bone resorption78. RANK is highly expressed in osteoclast precursor cells, mature osteoclasts, dendritic cells, and synovial fibroblasts77, 78. When the RANK pathway is activated, monocyte-macrophage progenitor cells become osteoclasts, and mature osteoclasts become able to activate79. RANK activates different intracellular signaling cascades in response to RANKL-stimulation by interacting with Tumor Necrosis Factor Receptor-Associated Factors (TRAFs). In particular, TRAF6 leads to the activation of its downstream pathway, which includes rapid activation of MAPKs, NF-κB, and activator protein-1 (AP-1)79.

Inflammatory cytokines such as IL-6, TNF-α, IL-22 can also modulate the RANKL expression in RASFs. This expression promotes further proliferation of RASFs11, 80, 81. C-reactive proteins (CRP), biomarkers for the assessment of RA progression, also have stimulatory effects on RANKL production, which was observed in the synovial fluid and serum samples obtained from RA patients82. RANKL mRNA has been found in the synovial lining layer of RA patients and in the synovium of rats in both collagen-induced and adjuvant-induced arthritis83, 84. The expression of RANKL mRNA increased when RASFs were stimulated by VEGF85. Another study indicated that the selective secretion of IL-17 cytokine from Type 17 helper T cells (Th17) can stimulate RANKL production in cultured RASFs at both the gene and protein levels77. The expression pattern of RANKL mRNA increased in a dose-dependent manner when cultured RASFs were stimulated with varying dosages of IL-1777. However, a recent study demonstrated that pre-treatment of RASFs with IL-25 can decrease osteoclastogenesis by reducing IL-22-induced RANKL expression at the mRNA level