Maternal plasma angiogenic and inflammatory factor profiling in foetal Down syndrome

Objective and design Angiogenic factors are proteins that are related to certain foetal chromosomal abnormalities. The aim of this study was to determine the concentration of 60 angiogenic factors in the plasma of women with offspring possessing trisomy 21/Down syndrome (DS). Method After analysing karyotyping results, we selected 20 patients with foetuses possessing DS, and for the control group, we selected 28 healthy patients with uncomplicated pregnancies who delivered healthy newborns at term (i.e., 15–18 weeks of gestation). To assess the concentration of proteins in the blood plasma, we used a protein macroarray which enabled simultaneous determination of 60 angiogenic factors per sample. Results We observed a statistically significant increase in the concentration of these five angiogenic and inflammatory factors: TGFb1 (p = 0.039), angiostatin (p = 0.0142), I-309 (p = 0.0476), TGFb3 (p = 0.0395), and VEGF-D (p = 0.0173)—compared to concentrations in patients with healthy foetuses. Conclusion Our findings suggest that angiogenic factors may play role in DS pathogenesis.


Introduction
Down syndrome (DS), the most frequent chromosomal aberration, is caused by an extra chromosome 21 or a fragment thereof [1]. Based  of Child Health and Human Development, the incidence of DS in the United States is estimated at between 1:800 and 1:1000 live births. DS occurs equally often across ethnic and social groups [2] and is a disease of congenital anomalies, which includes congenital heart defects, gastrointestinal anomalies, immune system defects, thyroid disease, bone defects, genitourinary system defects, strabismus, mental retardation, and many other conditions [3]. A pregnancy involving a DS foetus is accompanied by a great number of biochemical variations in the maternal plasma, likely induced by the additional chromosome 21. Our recent research in reference to other articles, shows that pregnancies with foetal chromosomal aberrations are strongly connected with an imbalance in chemokines and bioactive lipids, such as sphingolipids, which may suggest new potential pathomechanisms in foetal DS [4][5][6][7][8]. Additionally, the status of selected cytokines and angiogenic factors in amniotic fluid from chromosomally abnormal pregnancies has also already been described in our research [9][10][11].
In the scientific literature scientists in recent studies demonstrates that most of the differentially expressed genes in trisomy 21 play a role in angiogenesis, immune responses and inflammation processes [5]. The clinical observations suggest that individuals with DS have a significantly lower incidence of angiogenesis-dependent diseases, with scientists noting the strong endogenous angiogenesis inhibitors' activity. Current pharmacological cancer treatment is focused on the inhibition of angiogenic factors (or its receptors). Since individuals with DS present an antiangiogenic state due to increased activity of endogenous angiogenic inhibitors, one can speculate that the endogenous angiogenic inhibitors are potential candidates for future cancer therapy [12]. Moreover, Hattori et al. have found evidence for the hypothesis that disturbed concentrations of some chemokines, angiogenic factors and different circulating metabolites in the blood of pregnant women can stimulate a mother's immune response to produce auto-antibodies directed against these proteins [13]. Scientists have determined the impact of the reduced number and altered morphology of endothelial progenitor cells on the impaired process of angiogenesis and immune response in individuals with DS [5]. Therefore, measurement of the poorly tested angiogenic and inflammatory factors in pregnancies with foetal chromosomal abnormalities could lead to a better understanding of the influence of DS on such pregnancies.
The results of this research could enable a better understanding of abnormal foetal development processes and could direct future investigations for the modification of the developmental process of foetuses with trisomy 21. Since the pathology of the syndrome is extremely complicated, and the additional chromosome 21 causes myriad foetal pathologies with imbalance of angiogenic factors and they can induce the maternal and foetal immunology system, identifying abnormal signals and understanding the pathological mechanism(s) is highly desirable. Therefore major goal of this research is description of potential role of angiogenic factors in patomechanism of foetal DS and interaction between mother and foetal body.

Materials and methods
The study and control groups consisted of women who underwent routine amniocentesis between the 15 th and 18 th week of gestation at the Department of Reproduction and Gynecological Endocrinology of the Medical University of Bialystok, Poland. Recruitment occurred between September 2012 and March 2015. We recruited only non-febrile women without any chronic or acute disease, and we excluded women taking any hormonal or anti-inflammatory treatment, as well as those with vaginal or urinary tract symptoms that would suggest infection.
The study protocol was approved by the Local Ethical Committee of Medical University of Bialystok, Poland, and informed written consent was obtained from each participant (ethics committee approval no: R-I-002/36/2014). We obtained 10 mL of peripheral blood, which was collected for EDTA probes after amniocentesis, from the participants. The blood was then centrifuged, and the plasma was subsequently separated and frozen at −80˚C. After analysis of the karyotyping results, we chose 20 women with foetuses with trisomy 21, and for the control group, we selected 28 healthy patients with uncomplicated pregnancies who delivered healthy newborns at term.
To determine the concentration of angiogenic factors in the blood plasma, we used a multiplex method as previously detailed [8,10], which allows for simultaneous determination of 60 proteins per sample. Like a traditional sandwich-based ELISA, this method utilizes a pair of specific protein antibodies for detection. A capture antibody is first bound to the glass surface. After incubation with the sample, the target angiogenic factor is trapped on the solid surface. A second biotin-labelled detection antibody, which can recognize a different isotope of the target factor, is then added. The protein factor-antibody-biotin complex is then visualized through the addition of the streptavidin-labelled Cy3 equivalent dye using a laser scanner (GenePix 4100A) and the following two software programs: GenePix Pro7 and Q-Analyzer [11,[13][14][15].
Descriptive statistics including the mean concentration and standard error of the mean were calculated for selected factors, henceforth referred to as "features". To detect statistically significant differences between considered groups (i.e., DS group versus control group), either analysis of variance or a non-parametric method (e.g., Wilcoxon rank-sum test) was applied. The choice of an appropriate method was made upon fulfilling the normality and homogeneity of variance assumptions, and in the case of violation of at least one condition, a non-parametric approach was employed.
The normality of features distribution was checked with the Shapiro-Wilk test [16] and the homogeneity of variances using Levene's test [17]. A statistical significance level of p = 0.05 was applied for all statistical tests.

Results
Clinical characteristics of the patients are presented in Table 1. Patients from both groups were matched for maternal age, number of pregnancies, and gestational age at data collection to ensure that there were no statistically significant differences between the groups. Mean concentrations and standard errors of maternal plasma angiogenic factor concentrations in each study group are presented in Table 2.
In Table 3 we have divided significant factors for pro or anti-agiogenic proteins.

Discussion
Human chromosome 21 encodes the antiangiogenic factors as endostatin (ES), β-amyloid peptide (BAP) and Down Syndrome Critical Region 1 (DSCR-1) [18]. As in DS, there are three copies of the 21 chromosome; those genes are overexpressed. Therefore, individuals with DS were reported as a model for systemic antiangiogenic state due to overexpression of angiogenic factors. The novelty of our study is the increased expression of five angiogenic factors-TGFb1, TGFb3, angiostatin, VEGF-D, and I-309-found in maternal serum, encoded in the 19, 14, 6, X and 17 chromosomes, respectively. Transforming growth factor beta 1 and 3 (TGFb1), (TGFb3) are two subtypes of the same protein, which are part of a vast cytokine group. These proangiogenic factors are natural modulators of cell transformation, proliferation and stimulation involved in the immunological and vascular system [19]. In the scientific literature, Hattori et al. have demonstrated a correlation between TGFb1 and DS. For example, Hattori et al. [20] and van der Wal et al. [21] found high concentrations of TGFb1 in plaques in Alzheimer's disease and DS pathologies. In addition, TGFb1 is a factor in responses to neurodegeneration: TGFb1 mRNA and protein were recently shown to increase in animal brains that cause local differentiation or neuronal death (which occurs in Alzheimer's disease and DS) [22]. In our research, we found a statistically significant increase in the concentration of TGFb1 and TGFb3 in the plasma of women with foetal DS, which may confirm that these proteins are indeed involved in neurodegeneration processes in the foetus body. To our knowledge, there are no other studies that describe the level of TGFb proteins in the plasma of women with foetal DS. Furthermore, Dalgliesh et al.

Group II-Pregnancies with Down Syndrome (n = 20)
Maternal age (median ± SD) 37.5 ± 6.83 36 ± 6.61 Number of pregnancies (median ± SD)   found increased levels of activin and inhibin in the plasma and placenta of women with foetal DS; these proteins belong to the same family as TGFb, ostensibly confirming our results [23].

Group I-Pregnancies without Down syndrome (n = 28) Group II-Pregnancies with Down syndrome (n = 20) P-value Angiogenic factors concentration (pg/ml) Mean ± SEM Group I-Group II
Galambos et al. previously demonstrated the increased lung expression of antiangiogenic factors, including as COL18A1, COL4A3, TIMP3 and APP. Here, it was assumed that the increase of antiangiogenic factors' expression leads to impaired lung vascular growth. Lung histology from patients with DS confirmed impaired lung vascular and alveolar development [18,24]. In the present study, we found elevated levels of another antiangiogenic protein, angiostatin, in maternal plasma. Despite the fact that there are no other peer-reviewed studies about the levels of this protein in pregnant women with foetal DS, this protein is notable in the context of impaired angiogenesis and cardiovascular disease, which often occurs in individuals with DS [1]. Proteins such as metalloproteinases (MMPs), elastase and prostate-specific antigen (PSA), release angiostatin from plasminogen during enzymatic division [25]. Hopkins et al. observed reduced levels of plasminogen activator inhibitor-1 (PAI-I) in the blood of adults with DS, consequently resulting in increased plasminogen level activity [26]. The elevated levels and activity of plasminogen can be a potential source of angiostatin. In addition, Lambert-Messerlian et al. revealed increased concentrations of PSA in maternal serum from pregnancies affected by foetal DS [27]. As previously mentioned, PSA is one of the major activators of angiostatin synthesis.  Table 3. Significant proteins-pro/anti angiogenic function.

VEGF-D proangiogenic
Vascular endothelial growth factor D (VEGF-D) was also shown to be elevated in our participants. Generally, VEGF plays a major role in the growth of blood and lymphatic vessels. VEGF-D through enzymatic processes has conformation that fits and activates the VEGFR-2 and VEGFR-3 receptors [28]. Like the above-described proteins, VEGF-D has not been previously studied in DS, but VEGF has been tested in this disease model. Yao et al. [29] showed that VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells, which results in the inhibition of angiogenesis and tumour growth [29]. Conversely, in our experiment, VEGF was not statistically significant. The mechanism and function of VEGF-D in foetal DS remains unclear, but this remains a notable protein requiring further research. The presence of three copies of chromosome 21 results in overexpression of its resident genes. Fuentes et al. presented the overexpression of the DSCR1, the product of the 21 chromosome gene, which acts as an inhibitor of calcineurin-mediated signalling pathways [30]. DSCR1 encodes a protein that inhibits VEGF-mediated angiogenic signalling by the calcineurin pathway, and its overexpression was confirmed by Baek et al. The diminution of calcineurin activity is reached by the overexpression of another 21 chromosome gene, Dyrk 1a. Scientists believe that DSCR1 and DYRK1A could be candidates for potential cancer therapy target [31].
I-309 (CCL1) is the last protein that was present at a higher concentration in the plasma of women with foetal DS. It is difficult to explain the elevated levels of I-309 in maternal plasma because of the small amount of research on processes involving this protein. Bernardini et al. Showed that I-309 is a new proangiogenic protein that connects to umbilical vein cells (HUVECs) and induces their chemotaxis, invasion, and differentiation [32]. Such a process could suggest that the protein is transplanted from the placenta into the mother's bloodstream.
In this publication, we found that selected angiogenic factors could be predictive factors for Down syndrome pregnancies and may play a role in the pathology of trisomy of chromosome 21. Currently, in the international literature, there is no relevant research focused on the roles of these factors in the pathogenesis of DS. Therefore, it is difficult to form definite conclusions about variations in the levels of angiogenic factors. However, due to the complexity of the pathomechanism responsible for DS, additional functional experiments should be performed.