Rheumatoid Arthritis and the Role of VEGF Gene: An Overview

Review Article Corresponding Author: Mohsin Mumtaz Email: mohsinmumtaz085@gmail.com Abstract Purpose: The aim of this review study is to outline rheumatoid arthritis and VEGF participation in rheumatoid arthritis disease development and also to help future researchers in this field. Study Design: A Review Study. Subjects and Methods: A number of research articles were systematically reviewed. More sixty research studies related to rheumatoid arthritis and the role of VEGF Gene were examined. The results of these studies are highlighted and compared. Results: Rheumatoid Arthritis is capable of causing joint damage, moreover, lead to long term disability in severe cases. Mutation in VEGF due to the involvement of multiple factors (environmental, genetic factors etc) lead to severity in RA patients as it has a role in certain inflammatory processes, angiogenesis etc. Further research needs to be done in this field because many of the factors involve in pathogenesis of RA such as factors that cause loss of tolerance and inflammation in joints, are still unclear. Conclusion: Although old diagnostic techniques are optimized, we further need to improve the diagnostic and treatment techniques in order to diagnose the patients at early stage. This will pacify patients’ disease severity and provide certain preventive measure that will assist in preventing the aggravation of disease in family members.


Introduction
Rheumatoid arthritis is an inflammatory autoimmune disease and particularly correlating innate immunity with cognate immunity. Progression of RA involves dysregulation of homeostatic processes that lead to disturbance in pathological processes (Boissier, 2011). Females are more affected with RA as compared to males and in a population its prevalence ranges from 0.5 to 1% on the basis of regional variations (Silman & Pearson, 2002). RA mainly affects the synovial joints (lining) and can cause disability and premature death. The clinical presentation of RA involves redness, swelling, pain in joint (RA involve two or more joints) and restrictive range of motion of joint. In early diagnosis of RA (less joint destruction, no functional disability and a reduced amount of radiologic progression), improvement of affected joints can most possibly be achieved to desired level by giving treatment (involve DMARD medication) up to the first 12 weeks after appearance of early symptoms (Cho et al., 2019;Moura et al., 2015;Linden et al., 2010).
Dimeric glycoprotein (VEGF) have role in angiogenesis (formation of new blood vessels) and also play fundamental role in the migration and proliferation endothelial cells, that surrounds the joint called synovium and cause inflammation that destroy the cartilage within bone. This disease is characterized by formation of (new) blood vessels from already existing blood vessels to supply growth factors, nutrients, cytokines, oxygen to augment the inflammatory process and pannus formation (pre-natural or irregular layer of fibrous tissues) that ultimately lead to joints destruction (Azizi, Boghozian, & Mirshafiey, 2014;Carvalho et al., 2007;Hah et al., 2013;Han et al., 2004).

Literature Review Prevalence
Rate of occurrence of RA is 1 percent worldwide and it is more common in females as compared to males (Markenson, 1991). Studies conducted in Northern part of America and Europe show 0.5 -1.1% prevalence of RA while lower prevalence of RA found in Southern Europe that is 0.3 -0.7% (Carmona et al., 2002;Guillemin et al., 2005;Saraux et al., 1999;Symmons et al., 2002). Low prevalence rate found in developing countries such as in Philippines 0.17 % and in rural Africa is 0.0026 % (Brighton, Badenhorst, & Myers, 1988). The few findings reflect that RA prevalence less in developing countries because of the difference in distribution of age groups, moreover, mild RA cases may escape diagnosis due to limited health care facilities that lead to underestimation of RA prevalence in developing countries (Alamanos, Voulgari, & Drosos, 2006). The research conducted in India and Pakistan illustrated the prevalence of rheumatoid arthritis (RA) was higher in India (0.2 -1%) as compared to Pakistan (0.5%) because of diversity in Indian population (Akhter, Bilal, & Haque, 2011).

Male to female ratio of RA
RA is more frequent in females as compared to males and its ratio is 3:1. The mechanism behind why females are more susceptible to RA as compared to males is still unclear. Accordingly, hormonal changes are the major contributor in the onset of disease in women. The age at which women are more prone to RA is fifth decade at which most of the hormonal changes (estrogens level) occur (Tobón, Youinou, & Saraux, 2010).In females, both hormonal and reproductive factors have important role in the development and expression of RA. Several studies suggest that the use of oral contraceptives might offer some protective affect from developing rheumatoid arthritis or might delay its expression (Harrison & Symmons, 2000). Whereas RA incidence declines by 70% during pregnancy but its incidence significantly increases to five fold during the first 3 months of post partum(A. Silman, Kay, & Brennan, 1992). Moreover, breast feeding also show association in the progression of RA. According to recent research, risk of developing RA increases by five fold in breast feeding women as compared to non-breast feeding women. Although the mechanism is still unknown but according to recent studies it might be due to increase in the secretion of prolactin hormone (Brennan & Silman, 1994). Decrease in level of testosterone has been observed in RA affected males (Cutolo et al., 2002).

Risk factors
Multiple factors (including genetics, sex and environmental factors) involve in expression of rheumatoid arthritis. Environmental factor i-e smoking factor, dietary contents, infectious microbes, silica exposure also contribute in aggravating the state of patient suffering from RA. However, some of these factors require further researches for elaboration (J. S. Smolen et al., 2018).

Environmental factors
Environmental factor can induce alteration in cellular activity by causing DNA methylation. For instance, higher methylation level found in smokers with ACPAs positive and carried risk allele HLA-DRB1 as compared to those who did not have this risk allele. However, in non-smoker this difference (methylation level) was not observed (Meng et al., 2017). Genes of potential trigger sites, as shown in figure 1, like oral, gut, lungs etc interact with environmental factors that cause initiation of self-protein citrullination and resulted in auto-antibodies production against peptides (citrullinated). Exposure of lungs to infectious agents, dietary contents, gut microbiota and harmful agents may cause the citrullination of self-protein and also ACPA maturation. PAD (peptidyl-argnine-deiminase) enzyme have role in the catalyzation of citrullination by modifying arginine (positively charged) to a polar but neutral citrulline during post translational modification. PAD enzyme secreted by macrophages and granulocytes in case of RA patients. Citrullinated proteins that include histones, fibrin, fibronectin, vimentin, Epstein-Barr Nuclear antigen 1 are disseminated throughout the body and abnormal reaction of antibodies to this wide range of protein (citrullinated) result in ACPA response. The citrullination mechanism also activate the MHC class II-dependent T cells that in turn boost B cells to enhance the production of ACPA. This stage also termed as loss of tolerance (Guo et al., 2018).

Smoking
Further research on RA demonstrates smoking was observed as a key factor in the rheumatoid arthritis disease development. Chances of RA risk increases by 2 folds in smokers that smoke 40 packs or more per year as compared to non-smokers and the risk still persist even after discontinuation of smoking. The Smoking effect is also influence by genetic background. The risk of rheumatoid arthritis increases by 21 folds in smokers having shared epitope and carrying two copies of it, as compared to non-smokers and having no shared epitope (Klareskog et al., 2006).

Dietary Factors
Dietary factor also play fundamental role in susceptibility of RA. Population consuming food rich in omega 3 fatty acid (fish, olive oil in cooking of food) have shown protection against RA. Omega 3 fatty acids have anti-inflammatory effect and reduce the inflammatory mediators through the pathway i.e. prostaglandin and leukotriene (James, Gibson, & Cleland, 2000). According to several studies people of Southern Europe showed mild RA than other population. Food with high content of vitamin K and vitamin D (in legumes and vegetables) inhibit the fibroblast proliferation and reduce inflammation in RA (Tobón et al., 2010). Vitamin D has been employed as therapeutic factor in multiple autoimmune diseases i.e. RA, SLE (Kamen et al., 2006), multiple sclerosis (Hernan, Olek, & Ascherio, 1999;Munger, Levin, Hollis, Howard, & Ascherio, 2006), type 1 diabetes (Arnson, Amital, & Shoenfeld, 2007). Vitamin D has numerous functions as it augment the regulation anti-inflammatory cytokine while suppress the proinflammatory cytokines (Canning, Grotenhuis, de Wit, Ruwhof, & Drexhage, 2001) and the vitamin D receptor have role in regulation of adaptive and innate immune response (Arnson et al., 2007).
Normal body function also depend on iron level and deficiency of iron in RA patients may lead to anaemia and may impede the normal immunological response (Baker & Ghio, 2009). Interlinking mechanism of interleukin 6, hormones that regulate iron level, cytokines (pro inflammatory), ferroprptein and hepcidin disturb the iron homeostasis and may lead to anaemia along with inflammation (Fraenkel, 2017). Properly dealing with anaemic condition can help in improving the lifestyle and physical activity of rheumatoid arthritis patients (Weiss & Schett, 2013). According to recent research, natural low fat diet might provoke the pathways that lessen the disease severity (joint pain and inflammation) and has been observed through marking decreased CRP level and improvement in the inflammatory scores (Alwarith et al., 2019).
Increased level of oxidative stress has been observed in RA patients. In literature review, increase in the production of reactive oxygen species (ROS), protein oxidation, damage of DNA, lipid peroxidation and reduction in anti-oxidant system has been marked in RA patients (Mateen, Moin, Shahzad, & Khan, 2017). In one study including 40 RA patients (only women), were supplemented with 8mg of zinc,125mg of vitamin C, 50ug of selenium, 40mg of vitamin E and 400ug of vitamin A, lessen the oxidative stress level and improved the clinical symptoms. In this study, considerable improvement in disease activity score was observed but not significant difference marked in swollen joints and painful condition of patients. The increased level of antioxidants in erythrocytes was also observed (Jalili, Kolahi, Aref-Hosseini, Mamegani, & Hekmatdoost, 2014). In another research, supplementation of antioxidant vitamins (vitamin C, vitamin A) along with trace elements (zinc, copper) had proven no significant influence in the disease activity of RA (Keysser, 2001).

Potential Agents
In view of previous studies, susceptibility to RA can also be induced by the active involvement of potential agents and those agents also trigger disease development in host. Several potential agents associated with RA that includes rubella virus, Epstein Barr virus, parvovirus and other microbial pathogens. Patient's sera contain high titre of these infectious agents. Although, the role of these pathogens in disease occurrence still remain controversial. Moreover, there was no direct association found between these agents and RA patients (A. Silman et al., 1997;Alan J Silman & Hochberg, 2001). Gut microbiome has a key role in the pathophysiology of rheumatoid arthritis. Intestinal membrane permeabilization allow the food particles or bacterial toxins (endotoxins) to enter into the blood stream and these endotoxins trigger the (systemic) inflammatory response (Pendyala, Walker, & Holt, 2012). The variety of gut microbe's decrease in RA infected individuals compared with control (normal) population. The diversity of rare microbes such as actinobacteria are increased in RA patients while the variety of abundant microbiome of gut is reduced (Chen et al., 2016) The absorption of these endotoxins in blood stream sometime induce the initiation of proinflammatory T cells and autoantibodies production in RA patients (Chen et al., 2016;McDougall, Bruce, Spiller, Westerdahl, & McDougall, 2002;Simpson & Campbell, 2015). The recent research suggested gum inflammation caused by Porphyromonas gingivalis also have role in the pathogenesis of RA (Lundberg, Wegner, Yucel-Lindberg, & Venables, 2010;Mikuls, 2010;Mikuls et al., 2009). The current proposition is that human peptides citrullination caused by Porphyromonas gingivalis may be involved in initiation of breakdown of self tolerance mechanism and lead to autoimmunity like RA (Lundberg et al., 2008;Wegner et al., 2010). Periodontal disease share similarities with RA pathogenesis like bone erosion along with inflammation and chronic inflammatory response while clinically they are very distinct. Both diseases (periodontal and RA) thought to be augmented by oral microbiota i.e. Aggregatibacter actinomycetemcomitans (Konig et al., 2016) and Porphyromonas gingivalis (Kharlamova et al., 2016).

Pollutants
Silica exposure have also shown association with increased risk of developing RA as it is environmental risk factor (Stolt et al., 2005). During the collapse of World Trade Centre in New York in 2001, rescue responders and fire fighters exposed to dust particulates and showed an increased risk association with autoimmune disorders including RA (Webber et al., 2015). The dust particulates contain different materials like silica, pulverized cement, asbestos, glass fibres. Occupational exposures also show association with RA. For instance, Malaysian women exposed to textile dust were shown strong association with increasing risk of developing rheumatoid arthritis (Too et al., 2016). Exposure to silica dust was a risk factor for seropositive (RF+ and ACPA+) RA patients but not a risk factor for seronegative patients. In occupations like mining, rock drilling and sand blasting exposure to silica through respiratory tract also show association in increasing the risk of RA development (Klockars, Koskela, Järvinen, Kolari, & Rossi, 1987;Sluis-Cremer, Hessel, Hnizdo, & Churchill, 1986;Steenland, Sanderson, & Calvert, 2001;Turner & Cherry, 2000). Increased exposure to dust (containing silica) can result in fibrosis and chronic inflammation of lungs, and hence provoke the humoral immunity that ultimately leads to increased risk of RA (seropositive patients). Moreover, occupational exposure to other parameters such as mineral oil (direct exposure of dust particle with skin and lungs) was also observed in epidemiological Investigation of RA (EIRA) (Sverdrup et al., 2005).

Diagnosis
Diagnosis of RA is highly specific and diagnosed by rheumatologist. However, no proper diagnostic criteria available but certain classification criteria present that involve serological assay (reactive c protein and auto antibody) and clinical manifestation. Swelling of synovial joints along with morning stiffness and tenderness in joints on examination is the important clinical feature of RA (J. S. Smolen et al., 2018). Very specific joints involve in RA that distinguish it from other joint disorders. It encompasses proximal interphalangeal and metacarpophalangeal joints of the feet, hand, ankle, wrist, elbow, shoulder, hip joints and knee (Smolen et al., 1995).

Treatment
The treatment of RA involves methotrexate and steroids (glucocorticoids). Optimal dose (25mg once in a week) of methotrexate should be given to patient that substitutes the folate in order to alleviate or pacify the adverse effects of disease. However, higher dose (more than used in RA) of methotrexate cause anti-proliferative effects but it does not follow the folate pathway (Ede et al., 2001). In early diagnosis, glucocorticoid along with methotrexate is given to the patient within 6 months to recover from severe disease activity. The efficiency of these two steroids never surpassed the effectiveness of synthetic disease modifying anti-rheumatic drugs (DMARDs) (J. Nam et al., 2014).

Genes Involved in RA Pathogenesis
Multiple genes involve in the pathogenesis of RA. These genes include HLA gene with shared epitope and some other non-HLA genes with single nucleotide polymorphism such as VEGF, CTLA4,STAT4,PAD14,PTPN22,TRAF1,IRF5,IL23R and CCR6. More than 30 loci involve in the pathogenesis of RA, according to the recent genome wide association studies (Kurkó et al., 2013).

VEGF Gene
The VEGF gene is located on human chromosome 6p21.3 and comprised of eight exons with alternate splicing (Zhang et al., 2013). Heparin binding protein encoded by VEGF gene that exist as disulphide linked homo-dimer, this growth factor (VEGF) induce vascular endothelial cells migration and proliferation, therefore, play crucial role in angiogenesis (new blood vessels formation) (Merete Lund . VEGF have also important role in certain pathological conditions that involve embryo development, healing of wound, endometrium formation, placenta, corpus luteum, neovascularisation, cardiovascular diseases, tumor formation and certain autoimmune diseases (Carvalho et al., 2007). It has been dissertated that endothelial cells activated by VEGF produce chemokines for example IL-8 and MCP-1, as shown in figure 2 (T.-H. Lee, Avraham, Lee, & Avraham, 2002;Marumo, Schini-Kerth, & Busse, 1999). In synovial membrane, these chemokines function as recruiting the monocytes around the endothelial cells and existed macrophages, in synovial membrane produce IL-6 and TNF-α by interacting with either endothelial cells (activated) or VEGF. In return, these IL-6 and TNF-α augment the capacity of synoviocytes and macrophages to secrete VEGF and endothelial cells stimulation thus induce the cell contact macrophage mediated activation, generating a positive feedback mechanism. Hence, VEGF may serve as fundamental link between synoviocytes and endothelial cells (Yoo, Kwok, & Kim, 2008). VEGF induces pro-inflammatory changes that encompass accumulation of leukocytes, remodelling of blood vessels, collagen deposition and also increase in vascular permeability in chronic inflammation (Merete Lund Hetland et al., 2008;E. J. Nam et al., 2005;Yoo et al., 2008). Recently, it is documented that VEGF is critically required for rheumatoid synoviocytes survival (Kim et al., 2006). In previous studies, it has been demonstrated recombinant VEGF ligation to its receptors, induced by sodium nitroprusside (SNP) or serum starvation, prevent the synoviocytes apoptosis. The pAkt pathway and pERK activity rapidly stimulated by VEGF and then induce the Bcl-2 expression in RA synoviocytes. Moreover, Sodium nitroprusside induced Bcl-2 down regulation and translocation of Bax, completely blocked by VEGF, from cytosol to mitochondria (Yoo et al., 2008).
VEGF level in patient's serum determine the severity of disease or disorder. The expression level of VEGF was observed more in synovial fluid and in serum of rheumatoid arthritis patients as compared to osteoarthritis patients (S. Lee et al., 2001;Ozgonenel et al., 2010). The over expression of VEGF is related with disease activity, inflammatory changes, damage in joints and in angiogenesis (E. J. Nam et al., 2005). VEGF up regulation induced by inflammatory cytokines and hypoxic state persists in RA patients (Taylor, 2005).

Conclusion and Suggestion
Rheumatoid Arthritis is capable of causing joint damage. Moreover, it leads to longterm disability in severe cases. Mutation in VEGF due to the involvement of multiple factors (environmental, genetic factors etc) lead to severity in Rheumatoid Arthritis patients as it has a role in certain inflammatory processes, angiogenesis etc. Further research needs to be done in this field because many of the factors involved in pathogenesis of RA such as factors that cause loss of tolerance and inflammation in joints, are still unclear. Although old diagnostic techniques are optimized, we further need to improve the diagnostic and treatment techniques in order to diagnose the patients at early stage. This will pacify patients' disease severity and provide certain preventive measure that will assist in preventing the aggravation of disease in family members.