Osteoarthritis (OA) is a chronic degenerative joint disorder1 affects over 250 million people worldwide; most of them are the elderly.2 The fundamental characteristic of this disorder is the gradual progressive damage of articular cartilage, changes in the adjacent bone, and joint space narrowing. It can affect any joint, most commonly in the knees, hips, spine, and hands. Patients with OA typically experience manifestations including chronic joint pain, swelling, stiffness, joint deformities, and limited mobility, which reduce their productivity, lower their quality of life, and increase their social and economic burden.3

Several risk factors associated with knee OA have been proposed, including aging, environmental, and biomechanical factors. Furthermore, genetic factors account for 50% of the risk of OA development and prior research reveals that OA is primarily influenced by genetic risk factors due to common population polymorphisms in numerous genes.4,5 The single nucleotide polymorphism (SNP) is more than 90% of human gene polymorphisms, which is the most common and stable gene variant in the human DNA strands.6

The human angiotensin-converting enzyme (ACE) gene (Gene ID: 1636), is located on chromosome 17q23, has a length of 21kb, and consists of 26 exons and 25 introns.3 The renin–angiotensin–aldosterone system (RAAS primary)’s enzyme, ACE, is produced by the ACE gene.7 It can metabolize the vasodilator bradykinin into inactive fragments and catalytically convert angiotensin I to the potent vasoconstrictor angiotensin II (the main effector peptide of RAS).8 As a result, it plays a crucial role in maintaining blood pressure, electrolyte, and fluid homeostasis.7

However, many studies have demonstrated that the RAS system, which includes the ACE and angiotensin receptors, is expressed locally in the synovial tissue of both humans and animals and may have a role in the development of OA. Additionally, it has been demonstrated that ACE inhibitors have a protective effect against OA in a rat due to their targeting of the local RAS, suppressing the stimulation of the angiotensin II signaling pathway.9,10

Numerous polymorphisms on the ACE gene have been identified by molecular biology studies, including Insertion and/or Deletion (I/D) fragment polymorphisms of the 16th intron, and SNP rs4343 in ACE exon 17. Plasma ACE levels vary with polymorphism,3 and Exon 17's ACE rs4343 polymorphism, had the greatest impact on serum ACE concentration.11

The association between ACE gene polymorphisms and different diseases has become a hot spot in the research field; most studies12–15 focus on the pathogenesis of hypertension, diabetes mellitus, and cardiovascular disorders. Poornima et al. have found that ACE gene polymorphisms are closely associated with type 2 diabetes mellitus and gestational diabetes mellitus.16 Moreover, Narne et al. hypothesized that myocardial infarction and coronary artery disease were significantly influenced by the genetic variant of ACE.17 On the other hand, few studies in the Asian population suggested an association between ACE gene (rs4343 and rs4362 variations, and I28005D) polymorphisms and the risk of knee OA.3,16

To our knowledge, no other study has examined the relationship between SNP rs4343 in the ACE gene and the risk of knee OA. In addition, the variations in genetic susceptibility in different ethnic groups have led us to attempt to validate the association between ACE gene polymorphism (rs4343) and knee OA in another ethnicity. Thus, we conducted the present study to investigate the ACE gene rs4343 polymorphism in primary knee OA and its association with clinical findings, severity of knee OA, and angiotensin II serum level (as a product of ACE) in a cohort of Egyptian individuals.

The present study found that ACE gene rs4343 polymorphism was associated with knee OA risk. Stratified analysis further indicated significant association among those who had BMI≥25%. Furthermore, the study found GG genotype of rs4343 polymorphism was associated with greater knee specific WOMAC index, Kellgren score, and serum angiotensin II level than those with AA or GA genotypes. Moreover, serum angiotensin II levels were positively correlated to the WOMAC severity, and the radiographic severity of knee OA.

Osteoarthritis (OA) is the most common chronic degenerative and disabling joint disease prevalent in middle-aged and older people worldwide.20 The exact pathogenesis of OA remains largely unclear, but it is believed that genetic polymorphisms greatly contribute to the susceptibility to complex diseases such as OA.3 The most frequent genetic variation in the human DNA chain is known as single nucleotide polymorphism (SNP), which accounts for more than 90% of all human gene polymorphism and is used to identify the genetic variables that are associated with the disease.21 A variety of polymorphisms has been discovered on the angiotensin-converting enzyme (ACE) gene, and these polymorphisms affect both ACE expression and plasma levels.22,23 Even though ACE gene has several polymorphisms, most studies16,17 focus on the insert and/or deletion (I/D) fragment polymorphisms of the 16th intron. On the other hand, only one study in the Asian population suggested an association between ACE gene polymorphisms (rs4343 and rs4362) and the risk of knee OA.3 Recently, it has been shown that the renin–angiotensin system (RAS) is directly involved in bone metabolism, and ACE inhibitors have been shown to have beneficial effects attributed to reduced level of angiotensin II and suppressed stimulation of the angiotensin II signaling pathway.9

Thus, we investigate for the first time in the Egyptian population the ACE gene rs4343 polymorphism in primary knee OA, and its association with clinical findings, severity of knee OA, and angiotensin II serum level.

Genotyping of rs4343 polymorphism in the ACE gene revealed 3 genotypes (GG, AG, and AA). AA genotype and A allele were used as the reference genotype and allele respectively. In the present study, we found that the frequency of GG genotype and G allele of the ACE rs4343 polymorphism were significantly higher in knee OA patients when compared to healthy controls. When compared with the AA genotype, the GG genotype showed 3.6-fold increased risks of developing knee OA. Similarly, the odds of getting OA in G allele carriers were 2.0 times more than those who carry the A allele. In addition, we found that GG genotype greatly increased the risk of OA among those with BMI≥25%. In accordance with our findings, a study in the Asian population done by Qing et al.3 revealed an association between ACE gene rs4343 polymorphism and the risk of knee OA. This study involved 109 knees OA patients and 114 healthy controls, and they found that the ACE gene rs4343 polymorphism was significantly higher in knee OA patients than in the controls, and the G allele might be a risk factor for knee OA.3

Using linkage and association analyses, Zhu et al.11 discovered a significant number of functional polymorphisms in the ACE gene. Recent research suggests that even silent mutations may have functional effects and raise the risk of disease in humans.24 These mutations can affect the folding, stability, and translation rate of mRNA. They may also result in abnormal mRNA splicing. The related protein's expression, structure, secretion, substrate selectivity, and/or enzymatic activity will be impacted by alterations to the mRNA molecule.24,25 Additionally, it has been hypothesized that the ACE gene rs4343 polymorphism modifies the regulatory motifs for several transcription factors, which in turn affects the levels of ACE.26 Moreover, McKenzie et al.27 showed that ACE rs4343 polymorphism could impact the level of ACE expression in serum or tissues and that the G allele might cause a considerable change in ACE expression level. Accordingly, it appears that the ACE rs4343 G allele enhances the ACE expression level and activity, which results in greater levels of angiotensin II.28

Angiotensin II (Ang II) is a physiologically active octapeptide that is produced by ACE-mediated angiotensin I cleavage and is implicated in a variety of inflammatory responses in addition to controlling blood pressure, serum electrolytes, and internal homeostasis in the human body.29,30 By interacting with two different angiotensin receptors, primarily AT1R and AT2R, angiotensin II regulates cell proliferation and apoptosis.29 Angiotensin II can also create a variety of growth factors, including platelet-derived growth factor (PDGF), insulin-like growth factor 1, and vascular endothelial growth factor (VEGF).31–33 Moreover, the production of pro-inflammatory cytokines such IL-6 (Interleukin-6), tumor necrosis factor-alpha (TNF-), and CCL2 (C-C motif chemokine ligand 2) is modulated by angiotensin II, which has been demonstrated in other studies to be crucial for the development and progression of OA. According to earlier studies, Ang II is also thought to reduce osteocalcin mRNA expression and alkaline phosphatase activity. The benefits of RAS inhibition on protecting cartilage could therefore be attributed mostly to lower Ang II levels and downstream signaling in OA.9

In the tibial end of the OA rats’ group, Tang et al.10 discovered that the expression of RAS components such as ACE and the angiotensin 1receptor (AT1R) was significantly enhanced whereas AT2R was decreased. Interestingly, captopril treatment prevented RAS activation and markedly decreased the expression of ACE, AT1R, and Ang II, while increasing AT2R in the captopril-treated OA rat group compared to the untreated OA rat group. In a rat OA model, captopril also has protective effects on cartilage, since it slowed down cartilage degeneration. Silveira et al.,34 who discovered that OA rats had considerably higher levels of ACE expression and that losartan treatment reduced OA symptoms and corrected histological joint changes in osteoarthritic rats, made similar findings. On the other hand, Wang et al.35 showed that the severity of arthritis was considerably reduced after intra-articular injection of rats with the AT2R agonist (CGP42112), indicating that the upregulation of AT2R may be another mechanism that has a therapeutic role in osteoarthritic rats. Therefore, because it targets the local RAS and reduces OA-induced joint and bone damage, therapeutic inhibition of AT1R or ACE (ACE inhibitors) and AT2R agonists may potentially have a protective impact on OA. Based on our results, there was a significant difference in serum angiotensin II levels between knee OA patients and healthy controls. Our data also showed significant differences among the ACE rs4343 AA, AG, and GG genotypes regarding the circulating angiotensin II serum level, where the GG genotype has the highest concentration followed by the AG genotype followed by AA genotype. These data mean that the presence of the G allele, whether in homozygous or heterozygous form, can affect ACE gene expression and serum angiotensin II level, leading to an increase in the risk of knee OA. In accordance with our study, Ghafil et al.36 who reported that the subjects with ACE polymorphism (rs 4343) GG and GA genotypes have higher plasma angiotensin II levels. On the other hand, some other studies have concluded that despite the effect of this polymorphism on serum ACE levels, there is no influence on angiotensin II levels or aldosterone production.37,38

In addition, our results showed a significant difference between ACE rs4343 genotypes regarding severity of disease (GG the high, GA the intermediate, and AA the low), indicating that GG genotype carriers are more prone to have a severe form of the disease. Moreover, a positive correlation of serum angiotensin II levels with WOMAC severity, and KL grades was detected in the present study, This increase of serum angiotensin II level with the increased severity of knee OA could be explained by the fact that, it is secreted in response to inflammation and increased secretion occur with the degree of inflammation. Unfortunately, we were not able to find similar studies regarding the association of ACE rs4343 genotypes and serum angiotensin II with knee OA radiographic severity in any other populations to date to compare them with the results of the current study. Moreover, the reliance on self-reported data may introduce limitations.

There were few studies focused on the role of ACE gene polymorphism in knee OA, also there were paucity of studies in different ethnic groups with the ACE gene rs4343 polymorphism and knee OA. Therefore, it is necessary to confirm these results by conducting further studies of a larger size in Egyptians and in other diverse ethnic populations.

Also, as a result of absence of wide genomic mapping in Arabian Countries, large-scale GWAS (Genome Wide Association Study) combined with WGS (whole genome sequencing) studies, will be very interesting to validate these results. Another possible limitation of the present study is relatively small sample size of the enrolled subjects with unequal genders proportion in the studied group with female predominance; however, this may be explained by the sex differences in the incidence of OA with large sex bias of OA incidence in females.39 Thus, these results should be validated in more well-designed studies considering the relatively limited sample sizes of subgroup analyses.

Our results revealed a possible genetic association between ACE gene rs4343 polymorphism and knee OA, and the GG genotype of rs4343 increased the risk of knee OA in Egyptian population. The interaction between ACE gene rs4343 polymorphism and obesity further increased the risk of knee OA. Moreover, the higher serum angiotensin II level may be involved in the pathogenesis of knee OA. However, due to the limitations of our study, further validation studies with larger size participants are needed to confirm our results.

All procedures performed in this study involved human Participants were in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments and approved by the Research Ethics Committee of the Faculty of Medicine for Girls, Al-Azhar University, and informed consent was obtained from all individuals for being included in the study.

The Research Ethics Committee of the faculty of medicine for girls, Cairo, Al-Azhar University reviewed and approved this work (approval number 2021121165, December 2021).

All authors have contributed significantly and equally to the design of this work, data acquisition, analysis, and interpretation. In addition to the writing and revising of this manuscript, all authors approved the final version before submission.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare that there is no conflict of interest regarding the publication of this article.