Evaluation of Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1) Serum Levels as Predictors for Ankylosing Spondylitis Severity, and the Study of Nano-Calcium as a Novel Therapeutic Approach Targeting These Molecules in Iraqi Patients

INTRODUCTION
Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the sacroiliac joints and spine [1]. It is a chronic, progressive disease that is associated with intervertebral ligament ossification and limited mobility [2]. As occurs with all types of arthritis, men are three times more susceptible than women to developing AS, with the disease’s onset usually occurring before the age of forty. The simple presence of the HLA-B27 antigen, present in ~7% of the general population but in 90% of AS patients, provides evidence of the genetic correlation between AS and Crohn’s. AS has a prevalence of 1–2% in HLA-B27-positive all adults [3,4]. Although the disease’s genesis is uncertain, several antigens of the invading organisms has been demonstrated in these people [5]. This has led to the hypothesis that, in susceptible HLA-B27-positive individuals, AS may be triggered by an infection, possibly within the gastrointestinal system. AS is characterized by chronic inflammation that affects various joints and organs, contributing to disease progression and complication [7]. Every person typically has a different pattern of sickness activity and presentation. The body’s immune system may have been activated by a previous bacterial infection or a combination of infectious microbes, which could have caused the first inflammation [8]. Even though the primary bacterial infection may have long since passed, once triggered, the body’s immune system cannot shut itself down [9]. An inflammatory autoimmune illness is characterized by persistent tissue inflammation brought on by the body’s immune system continuing to activate even when there is no active infection [10–11]. A mild lumbar discomfort that lasts for three months and is accompanied by morning stiffness that is eased by exercise is typically the subtle beginning of AS [12]. One-third of the patients have peripheral joint arthritis [13]. Iritis is the most problematic extrarticular manifestation; it usually occurs unilaterally and is accompanied by photophobia pain. Although common, colon and ileal inflammation is typically asymptomatic [14]. Currently, the most common tests for AS patients include genetic testing for HLA-B27, imaging of the sacroiliac joints using plain radiographs and/or magnetic resonance imaging (MRI)[15], and measurements of acute phase reactants like erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). However, there are restrictions on each of these tests [16]. The aim of this study was to ascertain the levels of Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) in AS patients; the results of which could be useful in follow-up treatment of AS.
In recent years, nanotechnology has emerged as a promising modern trend in the medical and therapeutic fields, particularly in the treatment of chronic inflammatory diseases. Calcium nanoparticles are characterized by their high absorption and cellular interaction capacity, giving them anti-inflammatory effects and the potential to directly influence endothelial cells and reduce their activation. Studies have shown that nanoparticles extracted from biological materials such as eggshells can possess effective properties in modulating the immune response and reducing inflammatory markers [17]. Therefore, investigating the effect of these nanoparticles on adhesion molecules associated with the progression of ankylosing spondylitis may contribute to opening new therapeutic avenues and improving the quality of life for patients.

MATERIALS AND METHODS
Methodology
The study was conducted in the rheumatology unit of Baghdad Teaching Hospital from June 2023 to September 2024. A total of 100 male and female participants, aged 18 years or older, were enrolled in the study. Group 1 (G1) included 50 healthy individuals, while Group 2 (G2) comprised 50 patients diagnosed with ankylosing spondylitis (AS). Venous blood samples (10 milliliters) were collected from each participant in both groups. Ankylosing spondylitis (AS) was diagnosed by a rheumatologist based on HLA-B27 positivity and assessments using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI). Participants with recently diagnosed conditions, long-term illnesses, rheumatic diseases, or metabolic and endocrine abnormalities were excluded from the study. After that, all the blood samples were processed to check for erythrocyte sedimentation rate (ESR, mm/h), C-reactive protein (CRP, mg/L), Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). The CRP levels were measured with an Abbott Architect C16000 machine from the USA. The VCAM-1 and ICAM-1 levels were found using ELISA kits from Biotech in the USA.Serum samples were separated by centrifugation at 3000 rpm for 10 minutes and stored at –20°C until use, Nano-calcium stock solution (1 mg/mL) was prepared and diluted to obtain working concentrations of 25, 50, and 100 µg/mL using PBS, Serum samples were mixed with nano-calcium (900 µL serum + 100 µL Nano-Ca) and incubated at 37°C for 24 hours. 
Following incubation, the supernatant was collected for ELISA measurement of VCAM-1, ICAM-1, and CRP, and results were compared with untreated serum (control).
Following in vitro CaNPs treatment (50 µg/ml, non-cytotoxic concentration)
Serum levels of VCAM, ICAM, ESR and CRP were determined using ELISA kits (SunLong, China) following the manufacturer’s procedure.

Preparation of Calcium Nanoparticles (CaNPs) from Eggshells [18]
1. Clean the shells and remove the membrane.
2. Boil and sterilize the shells.
3. Dry them.
4. Burn them in an oven (600-900°C).
5. Grind them to nanoparticle size (ball milling).
6. Re-treat them with solutions (hydrochloric acid followed by sodium chloride).
7. Final drying. (CaNPs)
The nanoparticles were characterized by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR).

Statistical analysis
A lot of the time, numbers are what statistical analysis is used for. It also lets us describe data and quickly draw conclusions about both continuous and categorical data. Part of the process is getting data that will be used to see if there is a link between two sets of statistics. All of the study’s data are shown as percentages and frequencies. We used the dependent t-test (two-tailed) and the independent t-test (two-tailed) for factors that were spread out in a nice way. That is why we used the Mann-Whitney U test, the Wilcoxon test, and the Chi-square test to find factors that were not spread out regularly. People used to think that P < 0.05 meant something statistically important.

RESULTS AND DISCUSSION
Evaluation of Erythrocyte Sedimentation Rate and C-Reactive Protein Levels in Ankylosing Spondylitis Patients
The study results showed significant differences between the two groups in terms of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. The mean erythrocyte sedimentation rate in the control group (healthy subjects) was 4.06 ± 2.36 mm/h, compared to a significant increase in AS patients to 27.29 ± 18.19 mm/h, and this difference was highly statistically significant (p<0.001). Likewise, C-reactive protein levels were significantly higher in the patients group, with a mean of 19.33 ± 4.25 mg/L, compared with 1.12 ± 1.98 mg/L in the control group, with a statistically significant difference (p<0.001). Such results echo the significant elevation of the inflammation factors in AS patients i.e. cementing their importance in the evaluation of disease severity and disease monitoring.
The study’s findings revealed that serum levels of vascular cell adhesion molecules (e.g., VCAM-1) and intercellular adhesion molecules (e.g., ICAM-1) in AS patients (G2 group) were markedly higher than those of healthy individuals (G1 group). For example, the content of VCAM-1 in the control group was (1014.3±136.5) ng/ml, increased significantly in AS patients (2888.2±722.15) ng/ml, statistically significant difference (p<0.001). Patients had significantly elevated levels of ICAM-1, with concentration of (2.6±0.39) ng/ml compared to control group (1.7±0.12) ng/ml, p<0.001. As expected, these results are linked to the part these molecules play in inflammation and the development of the disease. This means that these molecules could be important diagnostic tools or useful markers of how bad the disease is in people with AS.

Correlation Between VCAM-1, ICAM-1, ESR, and CRP in Ankylosing Spondylitis Patients
The results of the Pearson Correlation analysis, as shown in Table 2, indicated that there were strong positive correlations between all the variables studied (CM, FM, and GBP). 37790, VCAM-1, ICAM-1, and PAI-1 ranged from −0.90 ~ −0.83, and were positively correlated (r=0.85), suggesting that those two molecules play a common role in AS-associated inflammation. Moreover, VCAM-1 levels displayed a significant positive correlation with ESR (r=0.79) and CRP (r=0.84), confirming the contributory role of this molecule in inflammatory activity. In like manner, r value for ICAM-1 with ESR (r=0.77) and CRP (r=0.82) further demonstrates that rising serum levels of ICAM-1 correlate with the severity of inflammation. In addition, a strong correlation (family = no; biplot ordinate) was observed between ESR and CRP (r=0.90), indicating the two to be representative inflammatory markers in patients with AS. These findings show the proximity between adhesion molecules and conventional markers of inflammation, potentially revealing their influence on assessing disease severity and evolution.
Ankylosing spondylitis (AS) is a chronic inflammatory condition. During its progression, common pathological changes include inflammation and bone destruction. As the disease advances, inflammation affects cartilaginous and synovial joints, as well as the attachment sites of ligaments and tendons to bone, leading to fibrous and bony ankylosis. In later stages, the spine undergoes a “bamboo-like” transformation, which can result in severe disability and loss of work capacity, imposing a significant burden on both the family and society [19]. According to epidemiological surveys [20], ankylosing spondylitis (AS) is more prevalent in men than in women, typically manifesting between the ages of 15 and 30. It is strongly associated with HLA-B27 expression. The progression of AS is significantly influenced by inflammation, making inflammation control essential for slowing disease progression [21]. The result in Table 1 has shown the significant elevation of ESR and CRP levels in AS in comparison with healthy control subjects (p < 0.001). Indeed, the mean ESR in the AS group (27.29 ± 18.19 mm/h) had a statistically significant higher than that in the control group (4.06 ± 2.36 mm/h), indicating that AS is associated with chronic inflammatory process. AS patients also had significantly higher levels of CRP (19.33 ± 4.25 mg/L) than controls (1.12 ± 1.98 mg/L), reinforcing the notion of systemic inflammation. These results are in accordance with previous studies by [22], which also indicated higher ESR and CRP in AS patients, confirming their status as the most important inflammatory markers in disease development. However, a few studies, like [23], show that ESR and CRP levels do not always correspond to disease severity in patients with non-radiographic axial SpA. This difference could be due to differences in duration of disease, genetic susceptibility, or individual immune response. In addition, more potent biomarkers, including IL-6 and TNF-α, are suggested to be more accurate markers of AS activity under some conditions. Overall, the literature overwhelmingly supports elevated ESR and CRP as a marked indication of AS and efficacy as markers of disease activity and treatment response despite a few studies that have conflicting findings [24]. As presented in Table 2, both VCAM-1 and ICAM-1 levels were significantly higher in AS patients compared to healthy controls (p < 0.001), indicating that endothelial dysfunction may have a close relationship with AS pathophysiology. The average level of VCAM-1 (2888.2 ± 722.15 ng/mL) determined in AS cases was markedly higher when contrasted with the control groups (1014.3 ± 136.5 ng/mL) reflecting organ damage determined by vascular inflammation and endothelial activation. Additionally, the reported levels of ICAM-1 were significantly increased (2.6 ± 0.39 ng/mL in AS patient’s vs 1.7 ± 0.12 ng/mL in controls), suggesting that immune-mediated vascular involvement may also play an important role in AS disease. Similar results were reported by others including [25], which showed that expression of adhesion molecules in AS begets atherosclerosis at the heart of cardiovascular risk. But other studies like the one by [26], showed no significant correlation between higher adhesion molecule levels and higher AS disease activity supporting the idea that endothelial dysfunction might be influenced by genetic and environmental factors. Although studies vary in their definitions and categorisation of AS severity, as well as the specific methods employed in their investigations, the overall literature certainly supports the concept that increased levels of VCAM-1 and ICAM-1 in AS patients adds to their increased cardiovascular risk and may even serve as markers to monitor disease progression and vascular involvement in AS [27]. Table 3 displays well-correlated relationship between inflammatory markers (ESR and CRP) with adhesion molecules (VCAM-1 and ICAM-1) ergodicity, implication of close association of systemic inflammation with endothelial dysfunction in AS patients. VCAM-1 strongly correlated with ICAM-1 (r = 0.85), suggesting that both adhesion molecules could be co-regulated in response to a chronic inflammatory state. Moreover, ESR and CRP are well correlated to each other (r = 0.90), which were suggestive of major inflammatory markers in AS. This ̈indexdescribes the correlation of VCAM-1 (r = 0.84) or ICAM-1 (r = 0.82) against CRP increased levels which may be associated with endothelial activation & contributes to vascular involvement in AS. Similar findings were also reported in studies including [28,29], showing a direct correlation between the degree of systemic inflammation and the expression levels of adhesion molecules in patients with AS and their proposed involvement in cardiovascular comorbidities. However, a handful of studies, such as [30], identified lower correlations, indicating that additional covariates such as genetic predisposition and disease duration may affect these associations. In summary, although differences were noted, the existing evidence do endorse the hypothesis that chronic inflammation in AS leads to endothelial dysfunction that could promote atherosclerosis and cardiovascular disease [31].
This research demonstrated that using nano-calcium extracted from eggshells possesses a significantly higher anti-inflammatory effect [32]. A marked decrease in the levels of the adhesion molecules VCAM-1 and ICAM-1 was observed, along with reductions in CRP and ESR, indicating an improvement in the inflammatory state. The efficacy of Nano-Ca is attributed to its small size, which allows for greater absorption and easier entry into cells, thus enabling it to regulate the immune response and reduce endothelial activation, a key factor in the elevation of these inflammatory molecules [33].
The study results showed that the use of calcium nanoparticles (CaNPs) led to a significant decrease in the levels of the inflammatory adhesion molecules VCAM-1 and ICAM-1 compared to both untreated and treated serum. This decrease is attributed to CaNPs’ high cellular uptake and their direct effect on endothelial cell activation, which reduces the inflammatory response. These results suggest that calcium nanoparticles possess a more potent anti-inflammatory effect and may represent a promising therapeutic option for managing chronic inflammation in patients with ankylosing spondylitis by reducing endothelial dysfunction markers and improving the inflammatory environment associated with disease progression [34].

CONCLUSION
The results of this study showed a significant increase in the levels of the inflammatory markers VCAM-1, ICAM-1, CRP, and ESR in patients with ankylosing spondylitis compared to healthy individuals, confirming the role of these molecules as diagnostic and prognostic factors associated with disease severity and endothelial dysfunction. Furthermore, experimental treatment with calcium nanoparticles demonstrated a significant decrease in adhesion molecules compared to untreated serum, indicating the anti-inflammatory efficacy of calcium nanoparticles and their ability to enhance the inflammatory response. Therefore, calcium nanoparticles represent a promising therapeutic option that warrants further investigation in future clinical and laboratory trials to develop innovative treatment strategies for patients with ankylosing spondylitis.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.