RA is a chronic disease characterized by persistent inflammation, and the main pathological manifestations are synovial inflammatory hyperplasia, pannus formation, and joint destruction. Angiogenesis, as the basic event of pannus formation, has attracted extensive attention whether contributing to synoviocytes tumor-like proliferation, inflammatory cell infiltration, or articular cartilage destruction [17,18]. The nutrients, oxygen and a variety of signal factors produced by the neovasculature fuel the hypoxia and closed synovial microenvironment undoubtedly, which activates the interaction and signaling crosstalk between cells, prompting abnormal proliferation and aggravation of inflammatory phenotypes in various types of cells [19, 20].
Appropriate animal models are the basis of experimental research and new drug development. The AA rat model shares many similar features with RA in clinical manifestations, pathology, and immunological changes, making it a more ideal animal model to study the pathological mechanisms and evaluate drug efficacy of RA [21.22]. In addition, the AA rat model was successfully established, the characteristics of AA model were described in detail from the whole to the joint, and the evaluation indexes of the model were determined, including systemic evaluation, arthritis index, paw swelling level, movement score. In this study, the cell co-culture model and the TNF-α stimulated cell model were compared to select the suitable model for in vitro study. The cell co-culture model is widely used in vitro because of its similarity with the real internal environment [23]. FLS, as the main effector cell of RA joint synovium, contributes to and participates in synovium inflammation. VEC is not only an important part of vascular structure formation and angiogenesis, but also the material basis for the survival of FLS, which is closely related to the occurrence and development of RA [24,25]. FLS extracted from synovial tissue were used to establish a co-culture model with HUVECs to simulate synovial environment. TNF-α acts on VEC as a classical pro-inflammatory factor, which not only increase the secretion of inflammatory cytokines and angiogenic mediators (such as IL-1 and VEGF), but also damage the function of endothelial cells or lead to vascular dysfunction, resulting in vascular injury and local ischemia and hypoxia [26,27]. Therefore, TNF-α is a good stimulator acting on HUVECs to simulate inflammation and vascular injury. Our experiments have demonstrated that FLS and VEC in synovial tissue of AA rats are abnormally activated, including abnormal proliferation and angiogenesis. The comparison of in vitro models showed that there was no significant difference between them in the detection of experimental related indexes (including cell proliferation, secretion of angiogenic factors and PTEN expression). Finally, TNF-α was selected for the in vitro experiment, considering that FLS can not be guaranteed to be in the inflammatory state and the operability of the experiment in the in vitro co-culture experiment.
Angiogenesis, the formation of new capillaries from pre-existing vessels, is associated with inflammation and inflammatory diseases and is also a potential target for the treatment of RA. The pathogenicity of angiogenesis in RA, resulting in enhanced endothelial surface and persistent inflammatory cell infiltration, has been described to play an important role in the pathogenesis of this disease [7,28]. The angiogenesis process of RA is a programmed event. Angiogenic mediators produced by various types of cells (including FLS, macrophages, etc) in the synovial environment activate VEC. The endothelial basement membrane and perivascular extracellular matrix are degraded by proteolytic enzymes. After that, VEC proliferate abundantly and migrate into the interstitial tissue due to the differentiation into invasive tip cells, called sprouting. The formation of capillary loops by sprouting VEC (tip cells) to synthesize new basement membranes helps to maintain the structural and functional integrity of nascent blood vessels and ultimately the formation of new capillaries [29–31]. Therefore, cell proliferation, migration and tubulogenesis in vitro were comprehensively studied to evaluate the vascularization ability of the cells in this study.
As more and more angiogenic mediators that activate VEC are identified, such as growth factors, proteolytic enzymes, integrins, and adhesion molecules, researchers mostly focus on this [32,33]. Previous studies by our group also focused on VEGF and found that VEGF binds to its surface receptors to activate downstream pathways of the cell and plays a role in mediating angiogenesis [15,16]. However, the discovery of novel approaches to target multiple cascades or to select upstream cascades of many pro-angiogenic factors may provide promising strategies for RA treatment, because the functions of angiogenic mediators are mostly cross regulated. As early as the 1990s, researchers found the important role of genetic factors regulating angiogenic mediators in tumor angiogenesis. For example, Bouck first reported that the inactivation of the p53 gene by mutation or deletion downregulateed the expression of thrombospondin, which induces angiogenesis [34–36]. Thus, one of the main consequences of tumor suppressor gene inactivation is to promote tumor angiogenesis, which makes people re-recognize the importance of tumor suppressor genes (and oncogenes) in the study of angiogenesis. In this study, PTEN was proved to have a negative regulatory effect on HUVEC angiogenesis in an inflammatory environment.
PI3K is an intracellular phosphatidylinositol kinase that phosphorylates PIP2 to produce PIP3 which in turn activates Akt [37]. PTEN reverses this process by removing phosphate groups. PI3K-Akt signaling plays a role in a variety of cellular functions including proliferation, survival, migration, invasion and cell metabolism [38,39]. Upstream components of the PI3K-Akt signaling pathway such as PTEN and Ras are commonly mutated in many human cancers, especially playing an important role in regulating normal vascularization and pathological angiogenesis. Direct evidence for the involvement of PI3K and Akt in regulating angiogenesis in vivo was observed by forced expression of PI3K and Akt by the RCAs retroviral vector system [11]. Over-expression of PI3K or Akt induces angiogenesis, whereas over-expression of PTEN inhibits angiogenesis in chicken embryos, indicating that PI3K-Akt signal is required for normal embryonic angiogenesis. In addition, PI3K is activated by growth factors and angiogenesis inducers, such as vascular endothelial growth factor (VEGF) and angiopoietins, activates Akt or other targets, and induces HIF-1 and VEGF expression to regulate angiogenesis[40,41]. PTEN deficient endothelial cells showing increased excessive angiogenesis have been demonstrated in the vasculogenesis of tumors [42,43]. Akt regulates multiple downstream targets, including nitric oxide synthase (NOS), nuclear factor kappaB (NF-кB), glycogen synthase kinase 3(GSK-3), and Jun N-terminal kinase (JNK). However, the specific target of Akt induced angiogenesis remains to be determined. Based on the comprehensive research of RA disease and PTEN signal, we proposed for the first time that PTEN-PI3K-Akt signal is involved in the regulation mechanisms of RA angiogenesis. In the HUVECs model in vitro, we found that the expression levels of PTEN protein and mRNA were significantly decreased, and the expression levels of PI3K-Akt signal axis related protein and mRNA were significantly increased after TNF-α stimulation. In order to further verify the involvement of signal axis, we established a PTEN over-expression lentiviral vector. The experimental results are consistent with our expectation that the over-expression of PTEN inhibited the proliferation, migration and tubulogenesis of HUVECs in vitro. These findings support targeting the PTEN-PI3K-Akt signal as an effective strategy for the treatment of RA angiogenesis.
Inhibitors targeting the PI3K-Akt pathway have been developed, and these drugs can reduce VEGF secretion and angiogenesis as predicted. The traditional PI3K-Akt inhibitors LY294002 and wortmannin showed antiangiogenic activity, but these inhibitors are not suitable for human because of their toxicity and crossover inhibition of other lipid and protein kinases [44]. The treatment of traditional Chinese medicine has attracted more and more attention as an effective treatment with the emergence of new technological approaches. GE, as a iridoid glycoside obtained from Gardenia jasminoides Eills by modern extraction and separation technology of traditional Chinese medicine, is often used in the treatment of RA [14]. In recent years, our team has performed a lot of work around the study of the therapeutic effect and mechanism of GE on RA. Previous studies have shown that GE can restore the balance of pro/anti-angiogenic factors and inhibit VEGF-SphK1-S1P signal axis to exert anti-angiogenic effects [15]. This study supports the role of GE in up-regulating PTEN expression to inhibit synovial microvascular angiogenesis. Especially in the in vitro model, the cellular biological function of HUVECs was significantly inhibited. Furthermore, we found that the anti-angiogenic effect of GE may be regulated by PTEN-PI3K-Akt signal. The results obtained by lentivirus vector and specific inhibitors are consistent with our expectation that regulating PTEN-PI3K-Akt signal can significantly reduce the angiogenesis of HUVECs, which is consistent with the therapeutic effect of GE (Fig. 8).