The plasma level of mCRP is linked to cardiovascular disease in antineutrophil cytoplasmic antibody-associated vasculitis

Background C-reactive protein (CRP) has two natural isomers: C-reactive protein pentamer (pCRP) and C-reactive protein monomer (mCRP). The levels of CRP are significantly elevated in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). mCRP not only activates the endothelial cells, platelets, leukocytes, and complements, but also has a proinflammatory structural subtype that can localize and deposit in inflammatory tissues. Thus, it regulates a variety of clinical diseases, such as ischemia/reperfusion (I/R) injury, Alzheimer’s disease, age-related macular degeneration, and cardiovascular disease. We hypothesized that plasma mCRP levels are related to cardiovascular disease in AAV. Methods In this cross-sectional study, 37 patients with AAV were assessed. Brain natriuretic peptide (BNP) and mCRP in plasma were assessed by enzyme-linked immunosorbent assay (ELISA). The acute ST-segment elevation myocardial infarction (STEMI) was diagnosed by coronary angiography, and the Gensini score calculated. Echocardiography evaluated the ejection fraction (EF%), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), and left ventricular mass index (LVMI). Estimated glomerular filtration rate (eGFR) was calculated based on serum creatinine, age, and gender. Results The plasma level of mCRP in AAV was significantly higher than that in healthy volunteers (P < 0.001). Then, mCRP and CRP levels were compared with and without STEMI complications in AAV. The plasma level of mCRP was higher, but that of CRP was lower in STEMI. The plasma level of mCRP was correlated with Birmingham vasculitis activity score (BVAS), eGFR, BNP, EF%, LVEDV, LVESV, LVMI, and STEMI complications’ Gensini score in AAV; however, CRP did not correlate with BNP, EF%, LVEDV, LVESV, LVMI, and Gensini score. Conclusions The plasma level of mCRP was related to cardiovascular diseases in AAV patients.

C-reactive protein (CRP) is an acute-phase protein that serves as an inflammatory response biomarker [10,11]. During activity and relapse of AAV disease, serum CRP levels increase rapidly, followed by a decrease during remission of the disease [12,13]. Human CRP in blood circulation consists of C-reactive protein pentamer (pCRP) and tissue-related C-reactive protein monomer (mCRP) [14]. In atherosclerosis-related experiments in mice, CRP bound to the Fc receptor CD32, while mCRP bound to another human Fc receptor subtype CD16 in neutrophils and performed function that was opposite to that of CRP [15]. Thus, FC receptor may be the key that links CRP and mCRP to AAV.
The most common pathogenesis of ST-segment elevation myocardial infarction (STEMI) is atherosclerotic fragile plaque rupture and thrombus-induced cardiac muscle cell necrosis. Also, inflammatory reactions are crucial responses [16,17]. CRP is involved in acute myocardial infarction. mCRP is obtained from the dissociation process of necrobiosis. Moreover, mCRP explains the function of CRP, while pCRP does not promote inflammation [18]. In vitro tests have shown that mCRP induces the synthesis of neutrophil-derived chemokines, stimulates the expression of monocyte integrin [19], and promotes neutrophil adhesion to vascular endothelial cells [20].
CRP is the classical acute-phase protein named for its ability to precipitate and bind to the pneumococcal Cpolysaccharide. Although the blood circulation concentration is low in healthy individuals, the level of CRP increases sharply when tissue damage and inflammation occur [21,22]. Currently, CRP is used not only as a marker of inflammation but also as a crucial and independent predictor of atherosclerotic thrombosis (including cardiovascular events) [22].
Active AAV patients have high levels of CRP [13,23]. Also, renal involvement is common in patients with AAV, including renal tubulointerstitial (TI) lesions [24,25]. mCRP is an acute-phase protein in tissues or cells, wherein it might act as an antigen in acute tubular interstitium (ATIN) [25]. Serum CRP is decomposed into mCRP and induces the generation of free oxygen radicals on the surface of apoptotic and necrotic cells, resulting in inflammatory reactions [26]. It was also found that mCRP is not only related to thrombosis [27] and atherosclerotic plaque rupture but can also be concentrated in localized areas after transient myocardial ischemia and related to acute myocardial infarction (AMI) [28]. Therefore, we hypothesized that plasma mCRP levels are related to cardiovascular disease in AAV.

Collecting plasma samples
This cross-sectional study utilized plasma samples collected from 37 patients with AAV and 20 healthy volunteers. The AAV patients were diagnosed in the Department of Nephrology, Affiliated Hospital of Inner Mongolia Medical University, from October 2018 to November 2019. The patients met the criteria set at the Chapel Hill Consensus Meeting [29]. All the participants provided informed consent. Subjects with other autoimmune diseases, such as IgA nephropathy, rheumatoid arthritis, Graves' disease, and lupus nephritis, as well as secondary and various acute infectious vasculitis, were excluded. A volume of 5 mL venous blood from each patient was withdrawn and collected in EDTA anticoagulation tubes within 24 h of admission. The supernatant was separated by centrifugation at 3000 rpm, 10 min, and stored at − 80°C for subsequent use. Repeated freeze-thaw cycles are prohibited for this sample. The disease activity was based on the Birmingham vasculitis activity score (BVAS) in AAV patients [30]. We collected baseline patient data including status, age, gender, tobacco, alcohol, hypertension, diabetes, obesity, and biochemical indexes. These cardiovascular risk factors included triglyceride (TG), total cholesterol (Tch), lowdensity lipoprotein (LDL-C), high-density lipoprotein (HDL-C), CRP, and fibrinogen (Fib). The study was approved by the Ethics Committee of the Helsinki Declaration of Inner Mongolia Medical University.

Detection of mCRP by enzyme-linked immunosorbent assay (ELISA)
Plasma mCRP levels were tested by ELISA using a commercial kit (Kamaishu Biotechnology Co, Shanghai, China). The 96-well plate was coated with mCRP and blocked to reduce non-specific binding. Antibodies compete for binding to a plate or sample. This kit uses the double-antibody sandwich method to determine the level of human mCRP in the specimen. The microtiter plates were coated with purified human mCRP antibodies to make solid-phase antibodies. Then, mCRP was added to the microwells, followed by the addition of HRP-labeled mCRP antibodies to form an antibody-antigen complex, as assessed by ELISA. Subsequently, the substrate tetramethylbenzidine (TMB) was used to develop the color. The absorbance (OD value) of the colored complex was measured at 450 nm, and the human mCRP concentration in the sample was calculated by a standard curve [31].
Detection of brain natriuretic peptide (BNP) by ELISA BNP was detected by a double-antibody sandwich immunoenzyme method, using Unicel-TM-DXI800 (Beckman, CA, USA) full-automated immunoassay instrument. The ELISA was performed, as described previously [32].

Statistical analysis
All data were statistically analyzed using SPSS19.0. All grouped data were tested for normality before further analysis. The two groups of enumeration data that conformed to the normal distribution were compared using an independent t test. If any of the groups did not match, a non-parametric rank-sum test was used. If the data fulfilled the normal distribution, it would be expressed as means ( x ) ± standard deviation (SD). The Shapiro-Wilk test, median, and interquartile range (IQR) were used to check the degree of dispersion of the data. The correlation between the two groups of data was analyzed; if both groups met the normal distribution, Pearson's rank correlation was used, and if any group did not meet the normal distribution, Spearman's rank correlation was utilized. Cross-tabulation χ 2 test was used for gender comparison. P < 0.05 indicated statistical significance.

Results
General data from AAV patients and healthy volunteers (normal control) All the 37 AAV patients, including 17 (45.9%) females and 20 (54.1%) males, harbored MPO-ANCA. Among the 4 AAV patients with STEMI complications, 2 (50.0%) were males and 2 (50.0%) were females, with average age 59.5 ± 14.3 years, while that of the 37 AAV patients was 58.5 ± 10.6 years. The healthy volunteers consisted of 10 (50.0%) males and 10 (50.0%) females, aged 55.3 ± 10.7 years. The comparison of age and sex between 37 AAV patient groups and healthy volunteers (n = 20, P = 0.266; n = 20, P = 0.788) did not show significant differences. Next, we used the same method to compare the age and gender of 4 patients with STEMI complications and 33 patients without STEMI complications in the AAV group. Therefore, both the AAV patient group and the normal control, as well as patients with and without STEMI complications in the AAV group, showed no significant difference in the age and sex (P > 0.05). Also, the relevant biochemical indicators were analyzed in AAV patients (Table 1).

Correlation between mCRP and cardiovascular disease risk factors
The risk factors of cardiovascular diseases include status, age, gender, tobacco, alcohol, hypertension, diabetes, and obesity. Moreover, the correlation between clinical indicators of cardiovascular risk factors (lipid-and inflammation-related factors) and mCRP were compared. We found that mCRP was negatively correlated with total cholesterol (TG) and CRP (r = − 0.592, P < 0.001; r = − 0.340, P = 0.040, respectively), (Table 2).
Plasma levels of mCRP were higher in AAV patients than in normal controls The plasma level of mCRP in AAV patients was 244.12 (226.12, 331.725) μg/mL and that in the normal control was 170.0 (135.7, 199.3) μg/mL. Next, we compared the plasma levels of mCRP in AAV patients and healthy volunteers (P < 0.001) and found significant differences (Fig. 1).
Plasma levels of mCRP were highest in patients with than in those without STEMI complications The AAV patients were divided into groups with (n = 4) and without STEMI complications (n = 33) groups (Fig. 2a).

Discussion
Accumulating evidence has shown that CRP is pathogenic in atherosclerosis, acute myocardial infarction, cerebral infarction, and AAV disease [37]. CRP is one of the biomarkers for assessing AAV disease activity. The serum CRP concentration increased during the AAV active phase and decreased rapidly with the remission of the disease [13,38]. mCRP can bind with complement factor H (CFH) and is more effective than pCRP in inhibiting alternative complement [39] and plays a critical role in the development of AAV [40,41]. Xu et al.

Table 2 Comparison of the relationship between clinical indicators of cardiovascular risk factors in AAV
Abbreviations: TG triglyceride, Tch total cholesterol, LDL-C low-density lipoprotein, HDL-C high-density lipoprotein, CRP C-reactive protein, Fib fibrinogen showed that MPO inhibits the binding between CFH and mCRP, thereby inhibiting the regulatory activation of alternative complements. PR3 does not bind to either pCRP or mCRP, while MPO can bind to mCRP, which blocks the binding between mCRP and CFH [41]. Therefore, the mechanism of mCRP and AAV disease is indistinguishable.
CRP can be decomposed into mCRP at high temperature, urea, or acidic microenvironment [42,43]. mCRP activates platelets [20], monocytes [44], and endothelial cells [45]. It is a key substance that promotes inflammatory response [21,46]. The mechanism underlying the inflammatory response of endothelial cells is caused by the interaction of mCRP with neutrophils, macrophages, and platelets [46,47]. Furthermore, mCRP binds to CFH and directs it to the damaged part of the cell [48,49], inactivates C3b, and limits the progression of inflammation [39]. Therefore, we speculated that the role of mCRP in the pathogenesis of AAV disease might be related to CFH.
In patients with lupus, the level of anti-mCRP autoantibodies is related to the degree of renal interstitial lesions [50], and mCRP has been reported as an autoantigen in interstitial nephritis-associated uveitis (TINU) syndrome [51]. In AAV patients, anti-mCRP antibodies may be the cause of severe TI lesions [25].
Serum CRP levels increase due to acute infection, trauma, and inflammation [52]. Thus, CRP is commonly used for the risk stratification of cardiovascular disease [53,54], as well as a marker of generalized atherosclerosis [54,55]. Fig. 2 Plasma levels of mCRP were highest in STEMI complications in AAV than without STEMI complications in AAV and normal controls. a Thirty-seven AAV patients were divided into two groups: with STEMI complications (n = 4) and without STEMI complications (n = 33). b Plasma levels of mCRP in STEMI complications were highest in the three groups (with STEMI complications in AAV, without STEMI complications, normal). c Comparison of BVAS in groups with and without STEMI complications Fig. 3 Plasma levels of mCRP were compared with BVAS and eGFR in AAV. a Correlation between mCRP and BVAS. The plasma levels of mCRP were positively correlated with BVAS (r = 0.404, P = 0.013). BVAS, Birmingham vasculitis activity score. b Correlation between mCRP and eGFR. The plasma levels of mCRP were negatively correlated with eGFR (r = − 0.341, P = 0.039). eGFR, estimated glomerular filtration rate CRP levels are significantly increased in cardiovascular patients without any symptoms, obvious cardiovascular disease, unstable angina pectoris, myocardial infarction, and other diseases [22,56]. Also, it is directly involved in the response process of acute myocardial infarction [18]. Thielle et al. demonstrated that mCRP stimulates inflammation, while pCRP has no proinflammatory effect [45]. In addition, Diehl et al. detected mCRP deposition in human  [57]. Similarly, studies on cardiovascular disease have found that mCRP is present in endothelial cells cultured in vitro and can promote inflammatory responses [20]. In the current study, 37 patients with AAV were collected and 4 patients with STEMI complications were found. mCRP has lectin-like properties and can bind galactosecontaining residues [12,58,59]. ANCA-induced NETs activate the platelets and then promote mCRP formation on these platelets. Subsequently, the newly generated mCRP can further enhance thrombosis and inflammatory response during platelet activation. mCRP may be a potential link between thrombosis and inflammation in AAV. Therefore, we suspected that the occurrence of STEMI complications in patients was related to mCRP [12]. AAV exhibits various echocardiographic characteristics, such as decreased systolic function, increased diastolic dysfunction, and pulmonary hypertension at the time of diagnosis [60]. The current study focused on cardiovascular disease in patients with AAV, which reflected the clinical examination of cardiovascular function by echocardiography. EF%, LVEDV, and LVESV are indicators of heart function. LVMI is associated with the mortality of patients with chronic kidney disease (CKD) and is a high-risk factor of heart failure [61]. Therefore, we analyzed the correlation between EF%, LVEDV, LVESV, LVMI, and mCRP. In this study, we proved that circulating blood levels of CRP were significantly higher in AAV patients than in healthy volunteers. Also, mCRP levels, but not CRP levels, were significantly increased in patients with STEMI complications. Next, we analyzed the correlation between mCRP levels and the Gensini score of STEMI complications and that between mCRP levels, BNP, and EF% in patients with AAV and found that the level of mCRP was positively correlated with the Gensini score and BNP and negatively correlated with EF%.
Nevertheless, the present study had some limitations. First, it was not a longitudinal study because while collecting cardiovascular clinical indicators, a large amount of experimental data were collected first, followed by comparison analysis. Finally, it was found that the mCRP level was related to the different parameters mentioned in this study. However, AAV patients have low morbidity but high mortality [12]. Most AAV patients visit our department for treatment due to abnormal renal function. The patients we collected in this study were MPO-ANCA because it is a predominant condition in Chinese AAV patients [14,17]. Therefore, follow-up studies are imperative.

Conclusions
The level of mCRP is related not only to AAV disease but also to the occurrence of cardiovascular disease in patients with AAV. Therefore, mCRP can be used as a plasma biomarker of cardiovascular diseases in AAV.  Table 3 Comparison of mCRP and CRP with BNP, EF%, LVEDV, LVESV, LVMI, and Gensini score with STEMI complications Abbreviations: BNP brain natriuretic peptide, EF% echocardiography: ejection fraction, LVEDV left ventricular end-diastolic volume, LVESV left ventricular end-systolic volume, LVMI left ventricular mass index