Function of p53 in angiogenesis and oxidative stress in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a complex disorder that involves the immune system, inflammation, and the growth of abnormal tissue in the joints, causing damage. The development of RA is regulated by important factors such as inflammatory cytokines, active angiogenesis, and oxidative stress, which promote the process of autoimmunity, chronic inflammation, and tissue destruction. Recent studies have shown that activation of p53, a protein that suppresses the growth of tumors, is an ongoing process that commonly occurs during inflammation. This activation contributes to regulating and controlling normal inflammatory responses. Functional mutations in the p53 gene have been detected in the synovial tissue in individuals diagnosed with RA, especially those with severe and destructive illness. These mutations may occur due to persistent inflammation caused by reactive oxygen species (ROS), leading to changes in the genetic material. The overall absence of the p53 leads to elevated hypoxia-inducible factor 1 (HIF-1) alpha subunit levels and enhances HIF-1α-mediated activation of the vascular endothelial growth factor (VEGF) gene in low-oxygen conditions, thereby promoting neovascularization. Furthermore, p53 modulates ROS production via nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), with wild-type p53 inhibiting NOX4 induction, ROS generation, and cellular migration, whereas mutant-p53 enhances NOX4 expression, ROS production and promotes cell migration. This review addresses the importance of p53 in the processes of NADPH oxidase and angiogenesis, both of which are important in developing RA.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease affecting joints, other organs, and tissues [1, 2]. RA not only causes inflammatory arthritis, but additionally causes extra-articular symptoms, which include inflammation in the skin, eyes, lungs, heart, blood vessels, and other organs [3, 4]. The extra-articular manifestations of RA can exhibit significant variability among individuals and have the potential to contribute to the overall disease burden and related effects [5, 6].

Fibroblast-like synoviocytes (FLSs) are specialized mesenchymal cells in the synovium's intimal lining layer. These cells have important functions in regulating the health of the joints [7]. FLSs in a healthy joint control the composition of the ECM and synovial fluid, which are crucial to the lubrication and supply of the cartilage. However, in RA, FLSs display distinct aggressive behaviors, actively contributing to the onset and progression of the disease [8, 9]. The extensive FLSs result in an alteration of the synovial lining into a rapidly growing and invasive cell mass called a pannus (hyperplastic pannus) [9, 10]. RA-FLSs exhibit uncontrolled growth and reduced inhibition, resembling transformed cel exhibit a number of characteristics of oncogenic transformation, including enhanced proliferation, resistance to contact inhibition, and escape from apoptosis. In the synovial milieu, these RA-FLSs exhibit elevated production of matrix-degrading enzymes (MMP-3, MMP-9) and proto-oncogenes (c-Myc), which promote cartilage invasion and pannus growth. Interestingly, somatic p53 mutations, which are commonly detected in RA synovial tissue, lead to abnormal cell cycle progression and lack of growth regulation, which in turn promotes synovial hyperplasia [9, 11–13].

RA has two inflammatory phases, the pre-vascular and vascular phase [14]. The persistence of neovascularization in RA greatly affects the development of pannus and the infiltration of leukocytes into the synovium. Neovascularization relies on several components such as growth factors, chemokines, proteases, cytokines, and cell adhesion molecules [15]. The formation of hyperplastic pannus marks the initiation of the pre-vascular inflammatory stage, during which immune cells, such as lymphocytes and macrophages, infiltrate into the joint, resulting in local invasion. The vascular phase is represented by a gradual expansion of blood vessels, leading to the growth of the pannus [16]. Because neovascularization makes it easier for the continuously proliferating synovial cells to receive oxygen and nutrients, it hastens the course of osteoarthritis. [14].

ROS functions as signaling molecules that have a crucial function in controlling cell migration, chemotaxis, apoptosis, and inflammation [17]. RA patients exhibit a greater production of ROS compared to healthy individuals. NADPH oxidase 4 (NOX4), which contributes to ROS production, is increased in RA-FLSs after being exposed to TNF-α and IL-17 stimulation [18, 19]. Besides, NOX4 amplifies the migration and infiltration of RA-FLSs by augmenting the production of ROS [20, 22]. Furthermore, activation of NOX4 in FLSs results in elevated production of vascular cell adhesion molecule 1 (VCAM1) and VEGF [19]. It has also been observed that NOX4 regulates angiogenesis under hypoxic conditions through ROS-mediated increase in HIF-1α and increase in pro-angiogenic factors such as VEGFA, glucose transporter 1 (GLUT-1), and adrenomedullin [20, 21].

The transcription factor p53 functions broadly in biological mechanisms like cell cycle arrest, apoptosis and DNA repair [22]. In addition to these mechanisms, it has been revealed that p53 can regulate the cellular oxidative stress and angiogenesis, which are involved in RA pathogenesis. In the inflammatory synovium of RA, wild-type p53 functions as a negative regulator of pathological angiogenesis and has several modes of action. VEGF, a major angiogenesis-promoting factor that is elevated in response to hypoxia and synovial inflammation, has its transcription suppressed by p53 [23]. Additionally, p53 promotes HIF-1α degradation, which lessens hypoxia-induced angiogenic signaling [24]. Furthermore, by expressing anti-angiogenic molecules such thrombospondin-1 (TSP-1), p53 prevents endothelial cell proliferation and neovascularization [25]. These anti-angiogenic properties are diminished in RA-FLSs with p53 mutations, which results in enhanced synovial angiogenesis and immune cell infiltration that sustains inflammation. p53 has two distinct, context-dependent functions in controlling oxidative stress in the synovial milieu of RA. Wild-type p53 promotes antioxidant responses, such as the overexpression of sestrins, TP53 Induced Glycolysis Regulatory Phosphatase (TIGAR), and glutathione peroxidase (GPX1), to limit ROS generation and preserve redox equilibrium under healthy or mild oxidative circumstances [26]. On the other hand, oxidative DNA damage brought on by prolonged exposure to ROS causes p53 mutations in the setting of chronic inflammation. The vicious cycle of oxidative stress, synoviocyte activation, and joint deterioration in RA is perpetuated by these mutant p53 proteins, which not only lose their antioxidant capacity but may also actively enhance ROS production by upregulating NOX4 expression [18, 27, 28].

Although the role of p53 in tumors is well-established, its function in chronic inflammatory diseases like RA (mutation types, specific regulatory mechanisms in angiogenesis/oxidative stress) remains controversial, and its association with RA clinical phenotypes has not been fully elucidated. Given the frequent detection of somatic mutations in the p53 gene RA-FLSs and synovial tissues—particularly transition mutations linked to oxidative deamination—this review systematically elucidates the dual regulatory roles of p53 in synovial angiogenesis and oxidative stress homeostasis, and how its dysfunction in RA drives pathological neovascularization, chronic inflammation, and joint destruction.

Cytokine-mediated angiogenesis in RA: the interplay of pro-inflammatory factors and VEGF/angiopoietin signaling

Angiogenesis is an important mechanism in tissue growth and repair since it promotes the formation of new blood vessels from pre-existing ones. Typically, blood vessels remain quiescent and do not undergo angiogenesis. Angiogenesis is only promoted in abnormal situations when there is an excess of cell proliferation compared to the amount of nutrients and oxygen available. This propensity frequently manifests in chronic inflammatory disorders such as RA [29]. Chemokines, cytokines, and growth factors directly modify angiogenesis in RA by predominantly affecting the VEGF/angiopoietin system. Various studies have demonstrated higher amounts of VEGF in the blood of individuals with RA compared to those who are healthy. Additionally, when these RA patients receive pharmacological treatments, their VEGF levels decrease [30]. Patients with early RA who receive treatment with anti-rheumatic drugs experience a notable reduction in the concentration of VEGF in their sera, in comparison with those who do not receive any treatment. Macrophages and FLSs in the synovial tissue of individuals with RA release various pro-inflammatory cytokines, such as IL-6, TNF-α, IL-18, IL-1β, IL-8, and macrophage migration inhibitory factor (MIF), during inflammatory circumstances [31, 32]. These cytokines have a crucial role in recruiting additional immune cells to the location of inflammation and promoting the excessive migration of leukocytes into the joint affected by RA. As a result, there is a higher need for oxygen, which causes an elevation in the levels of HIF-1α [33, 34]. The increased presence of pro-inflammatory cytokines, along with elevated levels of HIF-1α, stimulates the synthesis of pro-angiogenic factors such as VEGF, EGF, IL-1β, IL-8, CCL-28, and CCL-21 by macrophages, T cells, and FLSs within the RA joint. Pro-angiogenic substances stimulate the development of new blood vessels in joints, a process known as joint neovascularization (angiogenesis), and this process is essential for the maintenance and advancement of synovitis in RA [35].

Oxidative stress-inflammation axis in RA

A reciprocal relationship between oxidative stress and inflammation has been reported in RA. Oxidative stress causes inflammation, while heightened inflammation leads to increased oxidative stress. This continuous cycle ultimately results in synovitis, an important indicator of RA. In the synovium of RA patients, activated macrophages and T cells release pro-inflammatory cytokines such as TNF-α and IL-1β, which activate the mitogen-activated protein kinase (MAPK) pathway. Numerous genes involved in the maintenance of inflammation, including ROS, are transcriptionally triggered by MAPK-mediated activation of nuclear factor κB (NF-κB) [36]. It is important to note that one of the primary substances used in the production of ROS is hydrogen peroxide (H2O2). H2O2, by activating the NF-κB pathway, creates a feedback loop that interacts with ROS. As a result, there is an increase in oxidative stress along with altered molecular signaling in the early stages of RA [37, 38]. In addition, NF-κB enhances the production of pro-inflammatory cytokines IL-1β and TNF-α, hence increasing the activation of NF-κB and production of ROS. The resulting ROS can impair DNA, increase lipid peroxidation, lead to arteriosclerosis, destroy the extracellular matrix, stimulate bone erosion, activate osteoclasts, increase inflammation, and finally destroy joints [39].

Molecular structure and general overview of p53

The P53 tumor suppressor protein, encoded by the TP53 gene located on chromosome 17p13.1, is an essential regulator of cellular homeostasis and genomic integrity. P53, as a transcription factor, orchestrates cellular responses to many stress signals, including DNA damage, anchorage activation, oxidative stress, and hypoxia. Activating it may result in several outcomes, including cell cycle arrest, apoptosis, aging, autophagy, and DNA repair. The P53 operates as a pivotal element in safeguarding cells against malignant transformation and atypical proliferation [40, 41]. The P53 protein consists of 393 amino acids and mostly functions as a tetramer, which is the active form for DNA binding and transcriptional activation. This protein is separated into multiple entirely unique domains, both structurally and functionally, each contributing to its regulatory functions [42, 43]. N-Terminal Activation Domain (TAD; Residues 1–5), Commencing the transcription of target genes is crucial. This domain encompasses connection stations for many auxiliary activators and negative regulators, particularly MDM2, which facilitates the ubiquitination of P53 and directs proteasomal degradation under non-stressful settings. TAD enables engagement with the foundational transcription apparatus and permits P53 to initiate gene expression program [44]. The extensive array of proline (PRD; residues 1–4) enhances the efficacy of P53 in apoptosis and augments its capacity to regulate gene expression during stress. It additionally aids in the stabilization of proteins in reaction to DNA damage [45]. The primary region of interaction with DNA (DBD; Residues 102–292) is the specific sequence that interacts with the responsive regions of P53 on DNA and modulates the transcription of genes associated with cell cycle regulation (such as CDKN1A/P21), apoptosis (BAX, PUMA), and antioxidant responses [46]. This region is significant as it is the epicenter of somatic mutations present in over 50% of human malignancies and in the synovial tissues of RA, where oxidative stress induces transitory mutations that impair DNA and P53 transcriptional function [47, 48]. Nuclear Localization Signal (NLS; Residue 325–325), drives P53 to the nucleus, where it performs its transcriptional activity. The mutation or obstruction of this region impedes nuclear entrance and diminishes P53 functionality [49]. The Oligomerization Domain (OD; 1–8 residues) facilitates the synthesis of P53 homotetramers, which are essential for binding to high-affinity DNA and ensuring persistent transcriptional activity. Tetramerization is essential for P53-mediated gene regulation and signal amplification [50]. The C-Terminal Regulatory Domain (CTD; 1–5 residues) regulates DNA connectivity, protein–protein interactions, and post-translational modifications including phosphorylation, ubiquitination, and methylation. This domain serves as a versatile regulatory interface that assimilates cellular inputs to precisely modulate P53 activity [51].

Function of p53 in RA

RA is characterized by synoviocytes and synovial tissues that exhibit characteristics associated with cellular transformation, which may have an impact on the course of the disease. The characteristics encompass loss of contact inhibition, expression of oncogenes, independent invasion of cartilage, and compromised apoptosis [52]. Genetic changes caused by local oxidative damage are considered to have a lasting effect on RA synovial cells, altering their function and ultimately playing a role in the development of RA [47, 53].

p53 gene somatic mutations have been observed in both RA synovium and cultured FLSs. The p53 mutations exhibit dominant negative factors, leading to repression of the wild-type allele's function within the cells [54]. Most p53 mutations in RA involve transition mutations, which have also been found in human neoplastic tissues [55, 56]. Transition mutations, observed in more than 80% of the cases, are often attributed to oxidative deamination [57]. The pathological significance of these mutations in the p53 gene in RA is yet to be determined. Further research is necessary to understand the functional implications of these mutations and their contribution to the development and progression of RA [58, 59].

Dysfunction of the p53 protein can mimic numerous phenotypic alterations seen in RA, including enhanced proliferation and invasion of synovial cells [60, 61]. The p53 gene plays a significant role in various pathways implicated in RA. The immune system, angiogenesis, apoptosis, and the ROS pathway all have connections to p53 in the context of RA.

Specific mutations of p53 in RA

Somatic mutations in the TP53 gene, which produces the tumor suppressor protein P53, have been thoroughly investigated in cancer and are increasingly acknowledged in the pathophysiology of RA. In RA, p53 mutations are mostly located in FLS and synovial tissue, especially in regions of joint erosion, and are linked to aggressive, apoptosis-resistant cellular behavior and persistent inflammation. The predominant p53 mutations observed in RA are missense mutations, namely transition mutations characterized by G:C to A:T substitutions. Such mutations frequently signify oxidative DNA damage, especially via the oxidative silencing of cytosine or guanine. Such mutations are generally induced by ROS generated during chronic inflammation, a fundamental aspect of RA pathogenesis [47]. Mutations in p53 in RA closely mimic those found in neoplastic tissues, highlighting the analogous behavior observed in RA-FLS changes. These mutations impair the DNA-binding domain of p53, undermining its capacity to activate downstream genes associated with cell cycle arrest, apoptosis, and DNA repair [48, 57]. Numerous studies have pinpointed specific codons within the DNA-binding domain (residues 102–292) that are often mutated in RA. Noteworthy examples include codon 143 (Val → Ala or Val → Leu), a recognized mutational locus linked to diminished DNA-binding capacity [56]; codon 248 (ARG → TRP or ARG → GLN), a prevalent codon in RA that facilitates direct DNA interaction [54]; and codon 273 (ARG → HIS or ARG → CYS), another common mutation site that compromises the structural integrity of the DNA-binding surface [55].

Crosstalk between p53 and pro-angiogenic signaling in RA

In the early stages of RA, inflammation within the joint structure can lead to a decrease in synovial tissue oxygen levels due to the increased metabolic demands of immune cells [53]. This can increase metabolic demand while hindering proper blood flow, leading to ongoing oxygen deficiency within the rheumatoid joint, which may stimulate VEGF production and the formation of new blood vessels (angiogenesis) [62, 63]. VEGF levels increase due to chronic inflammation, leading to higher vascular permeability and causing edema and protein leakage, likely contributing to pannus formation. VEGF binds to Flt-1 or VEGFR1 and flk-1(KDR) or VEGFR2, tyrosine kinase receptors on the endothelial cells' membrane [64, 65]. FLSs secrete VEGF four times higher than dermal fibroblasts when stimulated with hypoxia, TGF-β, or IL-1 [65, 66]. Studies suggest that numerous macrophages invade the synovium of RA individuals, exposing them to a significant hypoxic microenvironment. This leads to the upregulation of HIF-1α by macrophages, subsequently facilitating an essential angiogenic process. This process is crucial for the development of the inflammatory pannus and infiltration of leukocytes within the affected area [67]. HIF-1α and p53 are activated in response to varying degrees of hypoxia; p53 promotes the ubiquitination and degradation of HIF-1α under normal oxygen conditions. Under mild hypoxia, HIF-1α initiates pathways that promote glycolysis and angiogenesis, resulting in the relative buildup of p53. In severe hypoxia, p53 levels rise more significantly than in mild hypoxia, leading to the degradation of HIF-1α through Mdm2 and ultimately resulting in apoptosis. HIF-1α is frequently elevated in response to the hypoxic conditions, which is characteristic of inflamed RA synovial tissue [68–70].

A p53 mutation and a functional impairment of this protein in RA can lead to an elevation in HIF-1α levels, resulting in increased production of VEGF and angiogenesis [71]. Furthermore, MIF, which is upregulated in RA-FLSs, monocytes (MQs), B cells, and T cells, inhibits p53 activity and consequently enhances HIF-1α levels. This, in turn, leads to increased production of VEGF [72, 73]. Various studies have demonstrated that these pro-angiogenic mediators are interconnected with p53 signaling rather than being controlled independently. Under typical oxygen conditions, p53 suppresses angiogenesis by promoting the degradation of HIF-1α through the E3 ubiquitin ligase MDM2, thereby reducing VEGF synthesis [70, 72]. p53 inhibits angiogenesis partly by targeting HIF-1α, a crucial transcription factor in the hypoxic response. In normoxic conditions, p53 transcriptionally activates MDM2, an E3 ubiquitin ligase that inhibits p53 via feedback inhibition and promotes the ubiquitination and proteasomal degradation of HIF-1α, consequently suppressing the transcriptional activation of pro-angiogenic genes such as VEGF, GLUT1, and ANGPT2. Mutation or inhibition of p53 diminishes MDM2's action toward HIF-1α, resulting in the stabilization of HIF-1α and prolonged elevated production of VEGF, even in non-hypoxic microenvironments [70, 72]. Moreover, p53 can directly suppress VEGF transcription and enhance the production of anti-angiogenic proteins, such as thrombospondin-1 (TSP-1) [25]. Conversely, loss-of-function mutations in p53, frequently observed in RA synovium, result in HIF-1α stabilization, excessive VEGF production, and heightened neovascularization [54]. Cytokines like MIF, which are overexpressed in RA-FLSs and immune cells, have been demonstrated to functionally inhibit p53 action, resulting in the indirect buildup of HIF-1α and sustained production of VEGF. This interaction indicates that persistent inflammation in RA elevates angiogenic mediators while concurrently suppressing p53-regulated anti-angiogenic pathways Consequently, angiogenesis in RA is not solely a downstream effect of hypoxia and inflammation, but also a result of impaired p53 function, which does not inhibit the pro-angiogenic pathway. Comprehending this link establishes the molecular foundation for the subsequent section, which concentrates explicitly on the regulatory function of p53 in angiogenesis within the RA joint milieu.

The PI3K/AKT signaling pathway is crucial for regulating cell growth, survival, and function. In RA, this pathway is aberrantly activated, particularly in synovial tissue cells such as synovial fibroblasts. This activation enhances cell proliferation, confers resistance to apoptosis, and stimulates the synthesis of inflammatory cytokines like IL-1β, IL-6, and TNF-α, leading to significant joint inflammation, tissue destruction, and disease progression [74]. Additionally, PI3K/AKT activates the transcription factor HIF-1α, which responds to hypoxic conditions in the joint and promotes the production of VEGF. This factor facilitates angiogenesis in joint tissue, perpetuating inflammation and attracting more immune cells. The PI3K/AKT/mTOR pathway is a crucial intracellular circuit that significantly influences essential cellular activities, including growth, survival, metabolism, and particularly angiogenesis. In RA, this pathway is triggered due to increased inflammation in tissues impacted by hypoxia. The activation of AKT promotes the survival and proliferation of endothelial cells and enhances their motility, leading to angiogenesis. Furthermore, AKT initiates an additional pathway known as mTOR. mTOR facilitates the synthesis of proteins like HIF-1α, hence promoting increased expression of VEGF, which augments angiogenesis [75]. The p53 protein exerts an inhibitory influence on the PI3K/AKT pathway. p53 stimulates the PTEN gene, a direct inhibitor of the PI3K pathway, hence diminishing its activity and averting excessive cellular proliferation and aberrant survival. In pathological RA conditions, a malfunction of p53 results in heightened activation of the PI3K/AKT pathway [76].

The p53 protein and the mTOR pathway interact through various feedback loops and regulatory mechanisms. In healthy individuals, p53 promotes autophagy (a process that removes damaged cells) that can suppress the mTOR pathway and stop stressed cell growth. p53 can suppress the mTOR signaling pathway through many mechanisms. p53 promotes the LKB1 gene, which plays a role in cellular energy management. LKB1 subsequently activates AMPK, which directly inhibits mTORC1 activity by suppressing cell growth factors. Furthermore, p53 restricts the Rheb-mTOR pathway's activity by inducing REDD1 (Regulated in Development and DNA Damage Response 1), resulting in the inhibition of mTORC1 [18, 77]. The AMPK pathway is normally regarded as a positive regulator of p53 during metabolic stress; yet, in specific circumstances, such as chronic inflammation or disorders like RA, this pathway may indirectly suppress p53 activity through various methods. A crucial factor in this process is the activation of the enzyme SIRT1, which is augmented by AMPK. SIRT1 diminishes the transcriptional activity of p53 through deacetylation, hence restricting p53's capacity to initiate cell cycle arrest or apoptosis. Furthermore, under some conditions, AMPK activation may enhance the activity of the protein MDM2, which serves as a natural inhibitor of p53 and induces p53 destruction in the proteasome by ubiquitination. In individuals with RA, heightened activity of the AMPK pathway adversely affects the regulation of p53, leading to diminished apoptosis and heightened uncontrolled cell proliferation in synovial tissue, ultimately culminating in elevated levels of VEGF and angiogenesis [78–80]. In addition, the AMPK pathway, which increases in activity in RA, causes further suppression of p53, and on the other hand, the PTEN pathway, which is lowered, leads to greater activity of the mTOR pathway. All these variables collectively induce VEGF, the principal factor for angiogenesis [81].

MicroRNAs originating from extracellular vesicles (EVs) derived from RA-FLS have a significant impact on the process of angiogenesis in vascular endothelial cells (ECs). Overexpression of miR-1972 decreases p53 levels and promotes mTOR phosphorylation, ultimately stimulating angiogenesis via the p53/mTOR pathway [82].

miR-191, which is overexpressed in hypoxic RA-FLS, has the potential to interact with the 3′-untranslated region (3′-UTR) of the ten-eleven translocation 1 (TET1) to downregulate its expression. This interaction is expected to result in the suppression of cell proliferation by influencing the p53 signaling pathway. Elevated miR-191 expression was associated with decreased TET1 levels, resulting in sustained methylation of CpG-rich regions at the transcription start site of the p53 gene, ultimately leading to reduced p53 expression. Additionally, miR-191 is involved in HIF-2α-triggered blood vessel formation, with hypoxia-induced miR-191-C/EBPβ signaling. So, miR-191 can induce angiogenesis by downregulating p53 and inducing HIF-2α [83, 84].

p53 can trigger the TSP-1 promoter region, which acts as an anti-angiogenic factor. Therefore, another way that wild-type p53 inhibits the development of new blood vessels is by regulating the production of TSP-1 [85]. Moreover, recent studies show that p53 induces the expression of the alpha (II) collagen prolyl-4-hydroxylase [alpha (II)PH], which results in the production of anti-angiogenic collagen type 4 and 18 fragments [25].

It has been established that αVβ3 antagonists exhibit anti-angiogenic effects in many in vivo models [86–89]. αvß3 integrin plays a crucial role in the development of new blood vessels in different situations, as shown by multiple studies conducted in in vivo models [90]. Integrin αvß3 and p53 have been demonstrated to play a role in the regulation of angiogenesis. In healthy individuals, the αvß3 integrin facilitates endothelial cell migration and neovascularization, while p53 typically functions as an anti-angiogenic agent, inhibiting excessive angiogenesis. Due to mutations and functional deficiencies in p53 among RA patients, it is incapable of inhibiting αvß3 integrin-mediated angiogenesis, resulting in persistent inflammation inside the synovium. Integrin αvß3 modulates p53 through FAK and PI3K/AKT signaling pathways, resulting in alterations in p53-mediated cell survival or death, hence impacting the persistence of synovial fibroblasts in inflamed rheumatoid arthritis joints. It safeguards RA synovial fibroblasts from p53-mediated apoptosis. Activation of the αvß3 receptor also causes the translocation of NF-κB transcription factor into the nucleus, which increases the survival of endothelial cells [91, 92].

Concerning p53 mutations in RA-FLSs, which result in impaired function of p53 and its regulatory function in angiogenesis by inhibition of angiogenic factors and induction of anti-angiogenic factors, it seems that impaired function of p53 in RA-FLSs can contribute to neovascularization, pannus formation, and progression of RA (Fig. 1).

Fig. 1.

Role of p53 in normal and Rheumatoid arthritis (RA) angiogenesis. Wild-type p53 inhibits angiogenesis by increasing the expression of thrombospondin-1 (TSP-1) and the transcription factor E2F, which is crucial for the progression of the cell cycle. Nevertheless, Wild-type p53 restricts angiogenesis by degrading hypoxia-inducible factor 1 alpha (HIF-1α) and basic fibroblast growth factor (bFGF), recognized as angiogenic proteins. Dysregulated p53 activity in immune cells and Fibroblast-like synoviocytes (FLSs) leads to enhanced production of cytokines and macrophage migration inhibitory factor (MIF), accordingly contributing to the inflammatory environment in RA. MIF enhances HIF-1α levels by suppressing its action, leading to the stimulation of vascular endothelial growth factor (VEGF) synthesis. However, in low-oxygen settings, mut-p53 collaborates with HIF-1 to enhance the expression of specific genes that play a role in angiogenesis. Moreover, p53, which typically controls the cell cycle and death, is inhibited by the PI3K/Akt/mTOR signaling pathway and αvß3 integrin

Complex associations between ROS and p53 in the Onset of RA

ROS originates from multiple sources, such as mitochondria and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, Nox). The Nox family encompasses seven isoforms, namely Nox1–5 and Duox1–2. Among these, Nox1, Nox2, Nox4, and Nox5 have been detected in human vascular tissues [93]. Nox4 is uniquely expressed in all three major cell types found in the vasculature: endothelial cells, smooth muscle cells, and fibroblasts. Unlike other Nox isoforms, Nox4 which is constitutively active, demonstrates a preference for generating H2O2 rather than superoxide anions. It has been proposed that Nox4 might function as an oxygen sensor, playing a role in sensing and responding to changes in oxygen levels [94].

In recent investigations, ROS have been studied as significant modulators of signaling pathways within the immune system [67]. Indeed, it is increasingly evident that elevated production of ROS is a common occurrence during the initiation and progression of the inflammatory response. The detrimental effects of ROS highlight its role in propagating inflammation and potentially contributing to the development of various inflammatory diseases [95–97].

Exposure to environmental mutagens can lead to the degradation of genomic DNA. Over an extended period, genotoxic molecules can surpass the DNA repair mechanisms, resulting in lasting changes to susceptible genes. Studies have also linked DNA damage to chronic RA. This mutagenesis does not cause malignancy; instead, it enhances the disease's aggressiveness [47]. Evidence indicates that ROS and RNS, produced endogenously in areas of chronic inflammation, exhibit genotoxic characteristics. Most p53 mutations in both erosion and non-erosion FLSs are predominantly missense mutations, especially transition mutations. Such mutations are generally linked to oxidative damage [48]. Chronic inflammation leads to increased ROS production, which can result in somatic mutations. Somatic mutations in the p53 gene have been detected in the RA synovium and cultured FLSs [98]. Extended exposure to ROS, specifically hydrogen peroxide and hydroxyl radicals—causes oxidative alterations to DNA, including 8-oxoguanine (8-oxoG) lesions. If unaddressed, these lesions result in G:C to T:A translocations, which are characteristic of oxidative mutagenesis. In RA-FLS, persistent inflammation results in elevated levels of ROS that hinder DNA repair processes, including base excision repair (BER). The oxidative environment promotes translational mutations at CpG locations within the p53 gene, specifically at codons 143, 248, and 273. These mutations impair DNA binding, resulting in dominant negative effects and possible gain-of-function characteristics, which further augment ROS generation, angiogenesis, and synovial invasion [48].

Several studies have shown that the wild-type and mutant forms of the p53 regulate the production of ROS through NOX4. The wild-type p53 inhibits NOX4 induction, ROS generation, and cell migration; in contrast, mutant p53 increases NOX4 expression and facilitates ROS production and cell migration [94, 99, 100]. It has been revealed that NOX4 is upregulated in RA-FLSs following IL-17 and TNF-α stimulation. This upregulation of NOX4 leads to the enhanced migration and invasion of RA-FLSs through a pathway involving ROS, VCAM1, and VEGF. These findings suggest that NOX4 plays a critical role in the pathogenesis of RA and may serve as a potential therapeutic target for the treatment of RA and other inflammatory diseases [19]. Given the regulatory role of p53 on NOX4 expression and p53 status in RA-FLSs, it seems that p53 may be a better target for RA treatment.

In situations of oxidative stress in RA, elevated levels of DNA-dependent protein kinases and deficiencies in ATM and p53 within T cells, cause disruptions in DNA repair mechanisms. This, in turn, leads to heightened levels of DNA damage and cell death in naïve CD4⁺ T cells, resulting in premature aging of the immune system and a possible bias toward autoimmune responses. In RA patients, dysfunctional T cells exhibit indicators of inflammation activation and maintain chronic inflammation within the synovium [1, 101, 102]. Both animal and human studies have shown that oxidative stress has the potential to influence the development of early-stage RA and the breakdown of T-cell tolerance through various mechanisms [103, 104]. This includes heightened production of ROS, the creation of danger-associated molecular patterns (DAMPs), induction of mutations in both genomic and mitochondrial DNA, and potential immune system remodeling through the reduction of activity in key molecules such as SIRT1, p53, PTEN, FoxOs, and antioxidants. Additionally, the oxidative modification of self-antigens, such as type II collagen and C1q, which might initiate a process of “oxidative post-translational modification intolerance”, could contribute to the development of RA [102, 104].

ROS can activate p53 either directly through stress-activated kinases or indirectly through DNA damage caused by increased ROS levels. p53 regulates intracellular redox and is also influenced by ROS, exhibiting varying effects based on the intensity of the stress. ROS can induce DNA damage, hence activating p53 via the ATR/CHK1 and ATM/CHK2 signaling pathways. Furthermore, p53 may assume an antioxidant function by regulating ROS levels, especially with diminished quantities of ROS. At elevated levels of ROS, p53 may facilitate ROS accumulation, potentially inducing cell death (apoptosis) or senescence. p53 regulates necroptosis, a form of cell death, by controlling mitochondrial H2O2 levels via peroxiredoxin 3 and sulfiredoxin, affecting ROS generation [105–108].

SIRT1, a well-conserved group of deacetylases, is activated by nicotinamide dinucleotide (NAD+) and plays an important role in angiogenesis, inflammation, and oxidative stress [109]. SIRT1 activates cell cycle arrest and anti-stress response genes in response to oxidative stress by forming a complex with the FOXO family of forkhead transcription factors. This process promotes cell survival. SIRT1 regulates the transcriptional activity of p53 by deacetylating lysine 382 (K382) in the C-terminal domain. Deacetylated p53 has diminished DNA-binding affinity and reduced efficacy in activating genes associated with apoptosis (BAX, PUMA) and antioxidant defense (SOD2, TIGAR). Deacetylated p53 has diminished DNA-binding affinity and reduced efficacy in activating genes associated with apoptosis (BAX, PUMA) and antioxidant defense (SOD2, TIGAR). In RA synovium, diminished SIRT1 expression correlates with hyperacetylated yet malfunctioning p53, resulting in decreased apoptotic clearance of RA-FLS, heightened ROS buildup, and elevated inflammatory cytokine production. Moreover, the inhibition of SIRT1 can indirectly enhance NF-κB activation and intensify inflammation [110, 111]. Decreased expression of SIRT1 could lead to elevated proliferation and activation of endothelial cells, as well as increased angiogenesis, through the acetylation of p53 and P65. SIRT1, being a pivotal molecule, significantly regulates oxidative stress. Decreased SIRT1 expression results in elevated ROS levels and angiogenesis through various mechanisms. The increased level of ROS production is directly correlated with angiogenesis, and this significant issue deserves attention. Decreased SIRT1 function might contribute to RA development and progression by encouraging abnormal angiogenesis through the upregulation of the matricellular protein, cysteine-rich angiogenic protein-61 (CRP61). Moreover, SIRT1 suppresses the aggressiveness and inflammatory response of FLS via inhibiting the NF-κB pathway [110, 112, 113]. SIRT1 safeguards cells against oxidative stress by augmenting the transcription of genes responsible for antioxidant enzyme synthesis, such as SOD2, and by inhibiting the activation of signaling pathways that result in ROS generation. SIRT1's involvement in angiogenesis is intricate and contingent upon context. It can facilitate angiogenesis in specific contexts, such as wound healing, while concurrently inhibiting it in other scenarios, such as tumor progression or in smooth muscle cells [114, 115].

Research has also revealed that the migration and invasion of FLSs are increased in individuals with RA due to a promotion in MDM2 levels [116]. MDM2 plays a crucial role in regulating p53 degradation and acts as a negative regulator of p53. It achieves this by binding to p53 and promoting its degradation through ubiquitination [117]. It has also been found that the stability and increase in MDM2 elevate the effects of DNA damage in cells and suppress the response of p53 to oxidative stress, which results in increasing oxidative stress and cell growth [118].

The interplay between P53 protein and ROS constitutes a complicated and reciprocal loop, wherein either can activate the others under various physiological situations, including oxidative stress, cell cycle regulation, and apoptosis. Given the role of mutant-p53 in ROS production through induction of NOX4 and NOX4 overexpression in RA-FLSs, it seems that impaired function of p53 contributes to the overproduction of ROS in RA synovium which can leads to new mutations in other genes and RA development (Fig. 2).

Fig. 2.

Role of p53 in normal and RA oxidative stress. Reactive oxygen species (ROS) are produced by the process of oxidative phosphorylation (OXPHOS) as well as NADPH oxidase complexes (NOXs). Furthermore, during inflammatory conditions, there is a significant increase in the synthesis of nitric oxide (NO), which plays a crucial role in maintaining the normal function of endothelial cells and blood vessels. These conditions can potentially induce mutations in the p53 gene and enhance the half-life of its aberrant protein. The aggressive behavior of FLSs in RA is caused by the upregulation of mutant p53 and the suppression of apoptosis. Furthermore, the presence of abnormal p53 proteins enhances the production of NOX4, resulting in heightened migration and invasion of RA-FLSs. On the other hand, normal p53 decreases the synthesis of NOS and the resulting NO by interacting with the NOS2 promoter region, therefore preventing the development of inflammation

Progress in clinical trials related to p53 and relevance to RA

While there are presently no clinical studies specifically addressing p53 pathways in RA, notable advancements have been achieved in the formulation of p53-targeted treatments in oncology that could potentially be repurposed or modified for chronic inflammatory disorders like RA. Numerous small molecule medicines designed to restore wild-type p53 function in tumors harboring mutant p53 have commenced clinical study.

APR-246 (Eprenetapopt) is a significant chemical, a methylated derivative of PRIMA-1 that reactivates mutant p53 by restoring its functional conformation. APR-246 has exhibited potential in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with TP53 mutations, demonstrating safety and partial clinical efficacy in phase II/III trials [119, 120]. While initially investigated in oncology, the strategy for reinstating wild-type p53 function may also prove beneficial in RA synovial fibroblasts, which possess analogous p53 missense mutations and exhibit modified phenotypes. Moreover, MDM2 inhibitors like Nutlin-3 and its derivatives (Idasanutlin/RG7388) have commenced clinical studies for solid tumors and hematological malignancies. These medicines inhibit the connection between MDM2 and p53, hence averting p53 degradation and augmenting its tumor suppressor actions [121]. Considering that MDM2 is increased in RA-FLSs and facilitates p53 downregulation and apoptosis resistance, MDM2 inhibition may represent a viable treatment strategy for RA. Furthermore, preclinical investigations have demonstrated that NOX4, HIF-1α, and SIRT1—the downstream or regulatory targets of p53—are subject to pharmacological modulation. Inhibitors of NOX1/4, such as GKT137831 (Setanaxib), have progressed to phase II studies for fibrotic and inflammatory conditions, including primary biliary cholangitis and diabetic nephropathy. These experiments demonstrate the feasibility of clinically translating p53-related oxidative stress pathways outside oncology [122]. Although no clinical trials have currently utilized these medicines for RA, the common molecular irregularities-such as p53 mutations, oxidative DNA damage, and neovascularization-indicate that p53-modulating approaches merit exploration in RA models and, eventually, human investigations. Future clinical trials may explore these medicines as monotherapy or in conjunction with current biologics or small molecule inhibitors to attain synergistic immunomodulation and tissue protection.

Conclusion and future perspectives

Concerning the p53 role in angiogenesis through different mechanisms and ROS production through NOX4 regulation and given the impaired function of p53 in RA-FLSs, it seems that p53 may play a role in neovascularization involved in FLS invasion and RA development. Furthermore, mutant-p53 induces NOX4 expression and ROS production which can trigger new mutations in p53 and the other genes. So, the reactivation of p53 may overcome these mechanisms. The functional inactivation of p53—resulting from somatic mutations, post-translational changes, or upstream inhibitory regulators—results in dysregulated angiogenesis via HIF-1α/VEGF signaling and enhances ROS overproduction through the overexpression of NOX4. These pathways promote chronic inflammation, pannus development, synovial hyperplasia, and gradual joint degradation in RA. The tumor suppressor function of p53 in oncology is well-established, although its atypical involvement in chronic inflammatory disorders such RA requires further investigation. Data accumulation indicates that reinstating or replicating wild-type p53 functionality in rheumatoid arthritis synovium may mitigate the vascular and oxidative aspects of disease advancement.

Considering the emerging mechanistic connections between p53 dysfunction and rheumatoid arthritis pathobiology, several promising therapeutic strategies warrant future exploration, including p53 reactivation, targeting downstream effectors such as NOX4 and HIF-1α, modulating regulatory pathways, and employing combinatorial approaches. Small medicines like PRIMA-1 (p53 reactivation and induction of widespread apoptosis) and its methylated derivative APR-246 (Eprenetapopt) have demonstrated effectiveness in reactivating mutant p53 in cancer mice by reinstating its wild-type conformation. Preclinical evaluation of these drugs in RA-FLS models may elucidate whether p53 reactivation might reverse invasive fibroblast characteristics or inhibit synovial angiogenesis. As p53 inhibits NOX4-induced ROS generation, medications aimed at NOX4 (such as GKT137831, a NOX1/NOX4 inhibitor) may partially mimic the antioxidant role of p53. Likewise, HIF-1α inhibitors (e.g., PX-478) might diminish pathological angiogenesis resulting from p53 deficiency. These compounds may function as supplementary treatments for rheumatoid arthritis patients exhibiting heightened oxidative and hypoxia indicators. Pathways both upstream and downstream of p53, including SIRT1, PI3K/AKT/mTOR, and MDM2, provide more therapeutic targets. SIRT1 activators (e.g., resveratrol, SRT1720) may augment the antioxidant ability of p53, while MDM2 inhibitors (e.g., Nutlin-3) may stabilize p53 by inhibiting its endogenous degradation. Integrating p53-targeted drugs with existing rheumatoid arthritis treatments (e.g., TNF-α or IL-6 inhibitors) may enhance therapeutic efficacy, especially in patients experiencing suboptimal outcomes despite biologic therapy. Such combinations may potentially decrease the necessary dosage of immunosuppressants while improving disease remission. Future studies should concentrate on creating medications that can improve or restore wild-type p53 function in the synovial tissues of RA. To guarantee localized activity and reduce systemic toxicity, this involves designing selective delivery mechanisms. Furthermore, p53-targeted medicines may have synergistic effects and enhance patient outcomes when used in conjunction with current RA medications. For personalized medical strategies, research into biomarkers that can forecast therapy response will be essential. Lastly, to confirm the effectiveness and safety of p53-targeted treatments in RA patients, a transition from preclinical research to carefully planned clinical trials would be necessary.

Author contribution

BR-J, EF, MM, AJ wrote the main manuscript text AND MA, ASV prepared figures.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.