Gene polymorphisms and serum levels of sVEGFR-1 in patients with systemic lupus erythematosus

Correlation between soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) concentration, VEGFR1 gene polymorphisms and systemic lupus erythematosus (SLE) risk remains unclear. The present case–control study comprised 254 SLE patients, 385 other rheumatic diseases patients and 390 healthy controls. Serum levels of sVEGFR-1 were detected by enzyme-linked immunosorbent assay. Seven VEGFR1 genetic variants (rs2296188, rs9943922, rs2296283, rs7324510, rs9554322, rs9582036, rs9554320) were genotyped by KASP. Serum levels of sVEGFR-1 were up-regulated in SLE and positively correlated with disease activity. Furthermore, serum sVEGFR-1 presented a distinctive elevation in SLE in comparison with other rheumatic diseases. Frequencies of allele T of rs2296283 and allele G of rs9554322 were significant lower in SLE patients (P = 0.003, P = 0.004). Frequencies of genotypes TT of rs2296188 and rs2296283 were declined in patients compared with healthy controls (P = 0.039, P = 0.033). CC genotype of rs7324510 and rs9582036 was negatively correlated with SLE risk (OR = 0.538, OR = 0.508). Distribution of GG, GC, GG + GC genotypes of rs9554322 were different between SLE patients and healthy controls (P = 0.027, P = 0.036, P = 0.010). Moreover, frequency of TC genotype of rs7324510 was higher in SLE patients with lupus headache (χ2 = 9.924, P = 0.039) and frequency of TC genotype of rs9943922 was lower in patients with cylindruriain (χ2 = 7.589, P = 0.026). Frequencies of allele C of rs7324510 and allele T of rs9943922 were decreased in SLE patients with cylindruria and hypocomplementemia, respectively (χ2 = 4.195, P = 0.041, χ2 = 3.971, P = 0.046). However, frequency of allele C of rs9554322 was increased in SLE patients with pyuria (χ2 = 11.702, P = 0.001). In addition, SLE patients carrying GG, GC, CC genotypes for rs9554322 had higher levels of serum sVEGFR-1. In conclusion, serum sVEGFR-1 was elevated in SLE patients and may be a disease marker. VEGFR1 gene polymorphisms related to risk of SLE in a Chinese Han population.


Scientific RepoRtS
| (2020) 10:15031 | https://doi.org/10.1038/s41598-020-72020-8 www.nature.com/scientificreports/ therapeutic target for VEGF-mediated pathological signaling. In systemic sclerosis (SSc) patients, lower serum level of sVEGFR-1 was detected in comparison with healthy controls 15 . In rheumatoid arthritis (RA) patients, expression of sVEGFR-1 was increased, correlating with VEGF concentration 16 . Serum level of sVEGFR-1 in patients with osteoarthritis (OA) was higher compared with that in non-arthritic controls 16 . VEGFR1 genetic variant associated with RA disease activity 17 . Collectively, sVEGFR-1 plays important roles in inflammatory and autoimmune diseases. To date, relationship of sVEGFR-1 and lupus is limited. What is the expression profile of sVEGFR-1 in lupus, if VEGFR1 gene polymorphisms relate to SLE risk needs to be discussed.

Results
Demographical and clinical characterization of study subjects. The Table 1). The Age of SLE group was matched among all control groups. For gender information about SLE group and controls, there was no gender difference between SLE patients and other study group excepting gout (P < 0.001) and AS (P < 0.001). In addition, the hypocomplementemia, proteinuria, arthritis and rash were the four dominant clinical characteristics in SLE patients (the proportion of 51.57%, 45.28%, 41.73% and 40.15%, respectively). Other clinical information was contained in Table 1.

Association of VEGFR1 gene polymorphisms with clinical and laboratory features in SLE.
Given that SLE patients had diverse phenotypes and genetic predisposition, we explored association between VEGFR1 polymorphisms and clinical, laboratory features. The genotype and allele frequencies of VEGFR1 polymorphisms in SLE patients with different clinical manifestations were listed in Table 6. As the results shown, there was an increased frequency of TC genotype in patients with lupus headache in contrast to patients without this feature for rs7324510 (χ 2 = 9.924, P = 0.039). For rs9943922, TC genotype was different in patients with cylindruria as compared with patients without this feature (χ 2 = 7.589, P = 0.026). Distribution of CC, CA, AA genotypes of rs9582036 and rs9554320 was different between SLE patients with and without pyuria (χ 2 = 14.437, P = 0.003; χ 2 = 15.074, P = 0.001). In addition, C allele frequency of rs7324510 was lower in SLE patients with hypocomplementemia comparing to those without (χ 2 = 4.195, P = 0.041). T allele frequency of rs9943922 polymorphism was different between SLE patients with cylindruria and those without (χ 2 = 3.971, P = 0.046). It was found that the C allele frequency of rs9554322 was increased in SLE patients with pyuria    Table 1).
Variation of VEGFR1 and concentration of serum sVEGFR-1. To discuss possible significance of VEGFR1 SNPs on serum sVEGFR-1 concentration, serum sVEGFR-1 levels were compared according to genotypes of individual SNP. Analysis indicated that serum levels of sVEGFR-1 were significantly different among SLE patients carrying GG, GC, CC genotypes for rs9554322 (P = 0.027, Fig. 4A). The other SNPs were not related to serum levels of sVEGFR-1 (Fig. 4B-G).

Discussion
In the present study, we revealed that sVEGFR-1 serum levels associated with severity of SLE as well as VEGFR1 genetic variants existed in Chinese Han population. These findings added evidence that serum sVEGFR-1 and VEGFR1 gene were implicated in SLE pathogenesis. To our knowledge, this is the first study describing the network of serum sVEGFR-1, VEGFR1 gene and SLE risk. According to the findings, we observed that SLE patients showed higher serum levels of sVEGFR-1 which positively correlated with disease activity. Furthermore, genotype TT of rs2296188, TT of rs9943922, TT, TT + TC of rs2296283, CC of rs7324510, GG, GC, GG + GC of rs9554322 and CC of rs9582036 related to genetic susceptibility of SLE. SLE patients carrying rs2296283 G allele, rs9554322 T allele had decreased risk of SLE. Moreover, we found that VEGFR1 rs9554322 polymorphism may be a genetic factor for regulating sVEGFR-1 expression. SLE is an autoimmune disease with a panel of clinical manifestations such as lupus nephritis that results from a variety of immunological and vascular abnormalities. Relationship between sVEGFR-1 and SLE is not clearly elucidated to date. VEGF is a crucial factor in circulating angiogenesis. It is able to modulate proliferation of endothelial cells, regulate chemotaxis, and capillary hyperpermeability in angiogenesis 18 . Studies indicated that sVEGFR-1 may be involved in SLE by affecting VEGF-mediated activation of angiogenesis. Several studies found that serum concentration of VEGF was elevated in SLE patients [19][20][21] . Renal glomerular microvasculature  www.nature.com/scientificreports/ is susceptible to local VEGF-A 22 . VEGF mediates the glomerular endothelial cell proliferation and survival in damaged glomerular capillaries 23 . The dysregulation of VEGF is involved in initiation of glomerular injury 24 . Therefore, regulation of VEGF is important in lupus nephritis development. sVEGFR-1 was encoded by a specifically spliced form of VEGFR-1 mRNA. It is comprised of 656 N-terminal residues of the receptor, with a specific 30 amino acid tail at C-terminus. In SLE patients, plasma levels of sVEGFR-1 were found higher in patients with active lupus nephritis 25 . Similarly, in our study, we found that serum sVEGFR-1 was up-regulated in SLE and positively correlated with disease activity. In the inflammatory circumstance, sVEGFR-1 was secreted by activated monocytes 25 . High concentration of sVEGFR-1 antagonized the protective effect of VEGF, aggravating endothelial cell damage 26 . Therefore, it is hypothesized that sVEGFR-1 was up-regulated in the inflammatory circumstance of SLE, which bound to VEGF, leading to endothelial cell damage, further promoting inflammation in SLE.
Concerning VEGFR1 gene polymorphisms, our findings found that six SNPs (rs2296188, rs9943922, rs2296283, rs7324510, rs9554322, rs9582036) correlated with risk of SLE. It is worthy that rs9554322 and rs9582036 was the first time found to relation with SLE. Genotypes of GG, GC and GG + GC in rs9554322 were significantly lower in patients than in healthy controls, suggesting that rs9554322 polymorphisms may negatively correlate with the risk of SLE in Chinese Han population (OR = 0.417, OR = 0.694, OR = 0.649). Genotype CC of rs9582036 was related to SLE risk (OR = 0.508). It is possible that regulatory, structural or quantitative polymorphisms at the VEGFR1 locus may affect VEGF signaling pathway and enhance susceptibility to some www.nature.com/scientificreports/ angiogenic conditions. In our study, rs2296283 located in the functional 3′-UTR region and other three SNPs (rs2296188, rs9943922 and rs7324510) were within the introns. In a Polish study, there was no statistic difference in allele or genotypes frequencies of rs2296283 between RA patients and healthy controls 17 . On the contrary, we obtained a statistical difference in the frequencies of genotype TT and TT + TC for rs2296283 in SLE patients (OR = 0.513, OR = 0.588). Compared with the Polish study, we further observed statistic differences in genotype TT of rs2296188, TT of rs9943922 and CC of rs7324510 (OR = 0.578, OR = 0.619, OR = 0.538), which were encoded in the introns. Therefore, rs2296283 located in the functional region of VEGFR1 gene may influence the mRNA translation and stability through regulating polyadenylation, miRNA-mRNA and protein-mRNA interactions. However, further study needs to verify the effect of rs2296283 within the functional region. The significance of SNPs located in non-coding region of VEGFR1 (introns) were not clear. Several studies showed that polymorphisms in non-coding region of VEGFR1 possibly silenced or enhanced the transcriptional activity of sVEGFR-1 27,28 . Thus, it is postulated that polymorphisms in the non-coding region may implicate in selective splicing of RNA and promote the transcriptional activity of target protein. It is well known that SNP is the predominant pattern in the genomic DNA sequence variation. The majority of SNPs affected the gene expression instead of the protein composition. In agreement with previous studies, our results exhibited that six of seven SNPs (including rs2296188, rs9943922, rs2296283, rs7324510, rs9582036, rs9554320) were not related with sVEGFR-1 levels. Several GWAS studies indicated that although most of SNPs did not possess the direct ability to change the gene expression, these SNPs sometimes served for the functional counterparts which regulated gene expression and protein assemble 29,30 . Thus, we hypothesized that the six SNPs selected in this study may not directly influence sVEGFR-1 concentration in SLE patients. On the other side, serum levels of sVEGFR-1 were different among rs9554322 genotypes (P = 0.029). SLE patients with rs9554322 GG genotype had higher sVEGFR-1 protein levels, suggesting that VEGFR1 rs9554322 G/C genetic variant may contribute to abnormal sVEGFR-1 serum levels. However, what is the exact role of polymorphisms in VEGFR1 gene, sVEGFR-1 protein expression needs to be discussed and whether the selected SNPs in the current study can affect VEGFR1 gene, sVEGFR-1 protein expression needs to be conducted by functional study in the future. There are several limitations in this study. First, the sample size in our study is relatively limited, where cases were recruited from two hospitals. Considering the large number of Chinese Han population, larger scale and multi-center studies are needed in the future. Second, the clear mechanism of sVEGFR-1 involves in SLE pathogenesis needs to discuss.
In summary, the present study showed that serum levels of sVEGFR-1 were elevated in SLE patients, may be a disease marker, and VEGFR1 gene polymorphisms related to risk of SLE in a Chinese Han population.

Materials and methods
Study subjects. Case-control studies were conducted for sVEGFR-1 serum levels and gene polymorphisms.   sVEGFR-1 protein measurement. sVEGFR-1 protein levels of SLE patients and control groups were assessed by enzyme-linked immunosorbent assay (ELISA) kits (Cusabio, Houston, USA) in accordance with the manufacturer's protocol. All samples were measured in duplicates and plates were read automatically at an absorbance of 450 nm using LT-4000MS reader (Labtech International Ltd, East Sussex, UK). Concentration was calculated on the basis of a linear standard curve. The detection limit was 0.039 ng/ml. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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