Evaluation of glycosaminoglycans and heparanase in placentas of women with preeclampsia

Clinica Chimica Acta 437 (2014) 155–160

Contents lists available at ScienceDirect

Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

Evaluation of glycosaminoglycans and heparanase in placentas of women with preeclampsia Eduardo Augusto Brosco Famá a,b, Renan Salvioni Souza b, Carina Mucciolo Melo c, Luciano Melo Pompei a, Maria Aparecida Silva Pinhal b,c,⁎ a b c

Obstetrics/Gynecology Department, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazil Biochemistry Department, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazil Biochemistry Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 16 January 2014 Received in revised form 17 July 2014 Accepted 18 July 2014 Available online 30 July 2014 Keywords: Preeclampsia Heparan sulfate Dermatan sulfate Hyaluronic acid Heparanase

a b s t r a c t Background: Preeclampsia is a multisystem disorder whose etiology remains unclear. It is already known that circulation of soluble fms-like tyrosine kinase-1 (sFlt-1) is directly involved in pre-eclampsia development. However, the molecular mechanisms involved with sFlt-1 shedding are still unidentified. We identified, quantified glycosaminoglycans and determined the enzymatic activity of heparanase in placentas of women with preeclampsia, in order to possibly explain if these compounds could be related to cellular processes involved with preeclampsia. Methods: A total of 45 samples collected from placentas, 15 samples from placentas of preeclampsia women and 30 samples from non-affected women. Heparan sulfate and dermatan sulfate were identified and quantified by agarose gel electrophoresis, whilst hyaluronic acid was quantified by an ELISA like assay. Heparanase activity was determined using biotynilated heparan sulfate as substrate. Results: The results showed that dermatan sulfate (P = 0.019), heparan sulfate levels (P = 0.015) and heparanase activity (P = 0.006) in preeclampsia were significantly higher than in the control group. There was no significant difference between the groups for hyaluronic acid expression in placentas (P = 0.110). The present study is the first to demonstrate directly the increase of heparan sulfate in human placentas from patients with preeclampsia, suggesting that endogenous heparan sulfate could be involved in the release of sFlt-1 from placenta, increasing the level of circulating sFlt-1. Conclusion: Alterations of extracellular matrix components in placentas with preeclampsia raise the possibility that heparan sulfate released by heparanase is involved in mechanisms of preeclampsia development. Published by Elsevier B.V.

1. Introduction Preeclampsia is characterized by the presence of arterial hypertension and significant proteinuria after 20 weeks of pregnancy [1]. It is a multifactorial, multisystem disorder whose etiology has not been fully elucidated [2–5]. Decreased vascular endothelial growth factor (VEGF) levels and increased levels of the soluble fms-like tyrosine kinase-1 (sFlt-1) have been implicated in the pathophysiology of preeclampsia [6–9]. Glycosaminoglycans (GAG) participate in several biological signaling processes connecting intracellular and extracellular environments [10]. Marked differences in GAG sulfation patterns were observed in placental preeclampsia [11]. It is already known that exogenous heparan sulfate (HS) binds to sFlt-1, and that heparin can compete with HS ⁎ Corresponding author at: Biochemistry Department UNIFESP, Rua Três de Maio, 1004th Floor, Vila Clementino, São Paulo, SP 04044-020, Brazil. Tel.: +55 11 55793175; fax: +55 11 55736407. E-mail address: [email protected] (M.A.S. Pinhal).

http://dx.doi.org/10.1016/j.cca.2014.07.023 0009-8981/Published by Elsevier B.V.

for sFlt-1 binding. Interestingly, Low Molecular Weight Heparin (LMWH) administered for coagulation prophylaxis to women at risk of coagulation disorders increased circulating sFlt-1 levels compared to normal untreated pregnant controls [12]. It has already been shown that decorin, a chondroitin sulfate and dermatan sulfate proteoglycan inhibits the activity of transforming growth factor beta (TGF-β). TGF-β produced in the fetal–maternal interface plays a crucial role in the control of trophoblast invasion in the uterus [13]. Consequently, both chondroitin sulfate and dermatan sulfate may modulate trophoblast invasion. Hyaluronan or hyaluronic acid (HA) is an extracellular matrix polysaccharide present at low concentrations in plasma. Normally, HA is rapidly eliminated from the blood by the liver. Increased concentration of circulating HA has been found in women with preeclampsia [14]. Histochemical analysis used to detect HA in placentas from uncomplicated pregnancies and patients with preeclampsia, showed enhanced staining in the stroma and blood vessel walls. HA was found within and on the surface of intervillous and perivillous fibrinoid deposits [15]. Since fibrinoid deposits of HA are increased in preeclampsia,

156

E.A.B. Famá et al. / Clinica Chimica Acta 437 (2014) 155–160

resulting from infarcted villi, this HA from fibrinoid tissue is able to reach maternal blood and may explain increased levels of circulating HA in the plasma of women with preeclampsia. Heparanase, an endo-β-glucuronidase, participates in the degradation of the heparan sulfate proteoglycan chains [16]. This enzyme presents two isoforms, a precursor with no apparent enzymatic activity (65 kDa) that undergoes proteolytic activity to form the mature active enzyme, a heterodimer containing a 50 kDa subunit associated with an 8 kDa. This posttranslational processing of heparanase is performed by a papain-like cysteine proteinase, called Cathepsin L [17]. Inhibition of heparanase with a neutralizing antibody resulted in a marked reduction in sFlt-1 secretion of normal and preeclampsia explants [18]. Moreover, the level of sFlt-1 in the serum of heparanaseoverexpressing transgenic mice was nearly double that of wild-type mice [12]. Although many studies have already shown a larger reservoir of sFtl-1 in the placenta and the role of heparanase and heparan sulfate in modulating the release of sFlt-1 into the circulation, until now, no study has examined GAG and heparanase activity in placental tissues from preeclampsia patients. 2. Methods

with the ethical principles of the Declaration of Helsinki. The samples were collected after informed consent had been granted. The study procedures were approved by the Ethics Committee of the Women's Hospital and Faculdade de Medicina ABC (number 259/2009). Preeclampsia was defined as high blood pressure, above 140/90 mm Hg, associated with proteinuria ≥ 300 mg/24-h urine or dipstick ≥ 1 + after 20 weeks of gestation [1]. Severe preeclampsia was defined following clinical and laboratory features, such as hypertension N 160/ 110 mm Hg, signs of imminent eclampsia, eclampsia, HELLP syndrome and proteinuria ≥ 5 g/24-h urine. It is important to point out that the severe preeclampsia group was also evaluated for early-onset disease (gestational age b 34 weeks) and late-onset disease (≥34 weeks). Included in the case group were pregnant women diagnosed with preeclampsia, that present singleton pregnancy, regardless of the type of delivery (vaginal or abdominal) or gestational age. The control group was composed of single pregnancy without preeclampsia from any type of delivery (vaginal or abdominal) or gestational age. We excluded patients from both groups who presented multiple gestations, chronic hypertension, diabetes mellitus, chronic kidney disease, thrombophilia, collagenosis and illicit drug use. The placental sample was obtained from a square measuring 5 × 5 cm, around the center of the umbilical cord insertion. The material was rinsed with 0.9% saline solution to remove blood and amniotic fluid.

2.1. Patients and tissue samples 2.2. Identification and quantification of sulfated glycosaminoglycans A case–control study was conducted and placentas of pregnant women with preeclampsia (n = 15) and without preeclampsia (n = 30) were collected. This study was conducted in accordance

Tissue samples were homogenized and kept in acetone for 24 h, changing the solution 4 times. The obtained dry powder tissue

Table 1 Features of preeclampsia and non-affected women.

Age (y)

Race

Family history of arterial hypertension

Family history of Diabetes mellitus

Previous abortion

Smoking habit

Number of gestations

Personal history of arterial hypertension

Proteinuria (mg/24 h)

a b

t-Student for independent samples. Fisher Exact Test or its extension.

N Mean Median Minimum Maximum Standard deviation Caucasian Black Others Total Positive Negative Total Positive Negative Total Positive Negative Total Positive Negative Total 1 2 3 4 6 Total Positive Negative Total N Mean Median Minimum Maximum Standard deviation

Control group

Preeclampsia

P

30 24.2 25.0 16.0 35.0 5.5 20 2 8 30 10 20 30 6 24 30 1 29 30 3 27 30 11 10 7 1 1 30 – 30 30 – – – – – –

15 27.3 2.0 16.0 38.0 7.3 12 1 2 15 3 12 15 1 14 15 – 15 15 – 15 15 6 4 5 – – 15 3 12 15 7 554.3 340.0 300.0 1400.0 421.0

NSa

71.4% 3.6% 25.0% 100.0% 34.5% 65.5% 100.0% 20.7% 79.3% 100.0% 3.4% 96.6% 100.0% 10.3% 89.7% 100.0% 35.7% 32.1% 25.0% 3.6% 3.6% 100.0% – 100.0% 100.0%

80.0% 6.7% 13.3% 100.0% 20.0% 80.0% 100.0% 6.7% 93.3% 100.0% – 100.0% 100.0% – 100.0% 100.0% 40.0% 26.7% 33.3% – – 100.0% 20.0% 80.0% 100.0%

NSb

NSb

NSb

NSb

NSb

NSa

0.034b

–

E.A.B. Famá et al. / Clinica Chimica Acta 437 (2014) 155–160

(100 mg) was submitted to proteolysis in alcalase (4 mg/ml in Trischloridric acid 0.05 mol/l, pH 8, containing 1.5 mmol/l sodium chloride) at a ratio of 1:4 for 72 h at 60 °C. Trichloroacetic acid (10% final concentration) was added to the mixture, maintained at 4 °C for 15 min. The supernatant containing glycosaminoglycans was obtained after centrifugation (10 min, 3500 ×g, 4 °C). Sulfated glycosaminoglycans were precipitated by adding two volumes of methanol (24 h, 20 °C). The precipitate was collected by centrifugation (20 min, 3500 ×g, 4 °C), dried, dissolved in water and maintained frozen for further analysis. Sulfated glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin sulfate) were identified and quantified by agarose gel electrophoresis in 0.05 mol/l 1,3-diaminopropane-acetate buffer, pH 9.0. After electrophoresis, for 1 h at 100 V, glycosaminoglycans were precipitated in agarose gel using 0.1% cetyl-trimethylammoniumbromide (Sigma-Aldrich) for 2 h at room temperature. The gel was dried and stained with toluidine blue (0.1% in acetic acid:ethanol: water; 0.1:5:4.9, v:v:v). Glycosaminoglycan quantification was carried out by densitometry at 530 nm. The extinction coefficients of the glycosaminoglycans were calculated using standards of chondroitin 4-sulfate from whale cartilage (Seikagaku Kogyo Co.), dermatan sulfate (from pig skin) and heparan sulfate (from bovine pancreas). The agarose gel

Table 2 Clinical characteristics of non-affected women and preeclampsia patients. Control group Delivery

Newborn's gender

One-minute APGAR score

Five-minute APGAR score

Newborn's weight (g)

Time of collection of placenta postpartum (hours)

Gestational age (weeks)

Cesarean Vaginal Forceps Total Male Female Total N Mean Median Minimum Maximum Standard deviation N Mean Median Minimum Maximum Standard deviation N Mean Median Minimum Maximum Standard deviation N Mean Median Minimum Maximum Standard deviation N Mean Median Minimum Maximum Standard deviation

N, number of samples. a t-Student for independent samples. b Fisher Exact Test or its extension. c Pearson's chi-square test. d Mann–Whitney.

Preeclampsia P b0.001b

6 22 2 30 12 18 30 30 8.5 9.0 6.0 10.0 0.7

14 1 – 15 6 9 15 15 8.3 9.0 6.0 9.0 1.0

30 9.4 9.0 8.0 10.0 0.6

15 9.4 9.0 8.0 10.0 0.6

NSd

15 2972.7 3140.0 1196.0 4056.0 858.0

NSa

30 1.53 1.00 0.17 5.00 1.40

15 1.65 1.00 0.33 7.00 1.77

NSa

30 39.0 39.1 37.3 41.3 0.8

15 36.9 38.4 29.3 40.1 3.3

0.028a

30 3333.3 3340.0 2506.0 3954.0 319.6

NSc

157

electrophoresis method error was approximately 5%. Identification of the sulfated glycosaminoglycans was based on the migration of the compounds compared with that of the standards. The identity of the different sulfated glycosaminoglycans was confirmed by degradation with specific lyases: chondroitinases AC and ABC (Seikagaku Kogyo Co.) and heparatinases obtained by Nader et al. as previously described [19]. 2.3. Dosage of hyaluronic acid Hyaluronic acid was quantified after proteolysis of the tissue samples as previously described. For the dosage of hyaluronic acid, a fluorimetric method was used. First, link protein was coated (1 mg protein/ml), in a 96-well plate, and kept overnight at 4 °C. Link protein was isolated from bovine nasal cartilage purified by affinity chromatography. It contains the globular region G1 aggrecan that binds specifically to hyaluronic acid. The plates were washed 3 times with wash buffer (0.05 mol/l Tris-chloridric acid, pH 8) and then 200 μl of blocking solution was added to each well. The assay was performed using 100 μl of each sample in triplicate that had undergone proteolysis. The plates were incubated overnight at 4 °C, washed 3 times with wash buffer and incubated with 100 μl of biotinylated link protein (1 mg/ml), diluted in blocking buffer (1:5000), and kept under stirring at room temperature for 90 min. The plates were again washed 3 times with wash buffer and incubated with streptavidin conjugated to Europium, diluted 1:10,000 in blocking solution under stirring for 30 min at room temperature. Then the plates were washed 3 times with wash buffer. After washing, aliquots of 200 μl of developing solution were added to the wells and the plates were kept under stirring for 10 min. The fluorescence quantification was performed using a Victor 2 ELISA reader (PerkinElmer, Life Sciences). This method detects hyaluronic acid between 0.2 and 500 μg/l. The values were processed automatically by the program MultCalc® (PerkinElmer, Life Sciences, Turku, Finland).

NSd

2.4. Heparanase activity Heparanase activity was measured by an ELISA-like method using 15% biotinylated heparan sulfate immobilized in poly-L-lysine multiwell [20]. All solutions were prepared in sodium acetate buffer 25 mmol/l, pH 5.5. Tissue samples were homogenized in inhibitory protease cocktail P-8340 (Sigma-Aldrich) and incubated on pre-coated plate for 18 h at 37 °C. After several washings, non-degraded biotinylated heparan sulfate was detected by incubation with europium-conjugated streptavidin

Fig. 1. Gel electrophoresis. 1. Standard of sulfated glycosaminoglycans, 5 μg of heparan sulfate (HS), 5 μg of dermatan sulfate (DS), 5 μg of chondroitin sulfate (CS). 2. Sample obtained from placenta of preeclampsia patients. 3 and 4. Samples obtained from placentas of non-affected women (control).

158

E.A.B. Famá et al. / Clinica Chimica Acta 437 (2014) 155–160

(1:1,000) for 40 min at 25 °C. The plate was washed with acetate buffer to remove unbound streptavidin. Finally, 100 μl of enhancement solution (PerkinElmer Life Sciences-Wallac Oy) was added to each well under stirring for 3 min at room temperature. This procedure releases the europium bound to streptavidin. A time-resolved fluorometer was used to measure free europium and the data (counts/s) were processed automatically using MultCalc software (PerkinElmer Life Sciences-Wallac Oy). A standard curve of different concentrations of biotinylated heparan sulfate was performed in order to convert the values of counts/s obtained to percentage of heparanase activity based on heparan sulfate degradation. 2.5. Statistical analysis The results were expressed as means and SD or SE. The statistical difference was confirmed by Student's t-test, Mann–Whitney test, ANOVA with a fixed factor, and Kruskal–Wallis test with a statistical significance of P b 0.05. The significant correlations between variables were assessed by Pearson correlation coefficient. Statistical analyses were performed by the Statistical Package for the Social Sciences (SPSS) ver 19.0 for Windows. 3. Results The samples in this study consisted of 45 placental tissues, 30 samples (66.7%) obtained from healthy women and 15 samples (33.3%) from patients with preeclampsia. The groups presented no significant difference in ages (P = 0.115) as shown in Table 1. The control group had an average age of 24.2 y, ranging from 16 to 35 y with an SD of 5.5 y. The preeclampsia group had a mean age of 27.3 y, ranging from 16 to 38 y, with an SD of 7.3 y.

Fig. 2. Profile of sulfated glycosaminoglycans extracted from placentas. Thirty samples were collected from placentas of pregnant women without preeclampsia (control) and 15 samples from placentas of preeclampsia patients (preeclampsia). The black lines indicate the mean values obtained in each group by quantification of dermatan sulfate and heparan sulfate, respectively. The values were obtained by quantification of agarose gel electrophoresis as described in the Methods and expressed as μg/mg of tissue. A. Dermatan sulfate. *P = 0.019. B. Heparan sulfate. *P = 0.015.

There were no statistically significant differences between the group of healthy pregnant women with preeclampsia group considering race, family history of hypertension, diabetes mellitus, previous abortion, smoking habit, number of previous pregnancies, newborn's gender, newborn's weight, or APGAR score evaluation in the first and fifth minutes. Similarly, there was no difference between the groups considering the time to collect placenta samples after delivery. We found a higher incidence of previous personal history of hypertension in preeclampsia patients (P = 0.034), higher incidence of cesarean delivery (P b 0.001) and lower gestational age at delivery (P = 0.028) compared to the healthy pregnant women (Tables 1 and 2). Gel electrophoresis analysis demonstrated a significant increase of sulfated glycosaminoglycans (GAG) in the preeclampsia samples compared to the control group (Fig. 1). Sulfated galactosaminoglycans in the placental samples was completely degraded after digestion with chondroitinase ABC, confirming the presence of iduronic acid residues and, consequently, the identification of dermatan sulfate as the galactosaminoglycan in the tissue samples (data not shown). The mean value of dermatan sulfate obtained from the placentas of healthy pregnant women was 0.102 ± 0.055 μg/mg tissue, ranging from 0.043 to 0.244 μg/mg tissue, whilst in the samples of preeclampsia patients the average was 0.144 ± 0.074 μg/mg tissue, ranging from 0.048 to 0.322 μg/mg tissue. A significant increase was therefore observed in the dermatan sulfate level of preeclampsia tissues compared to the healthy placentas (P = 0.019), as shown in Fig. 2A. The average for heparan sulfate was 0.078 ± 0.041 μg/mg tissue, ranging from 0.034 to 0.190 μg/mg tissue and 0.113 ± 0.063 μg/mg tissue, ranging from 0.041 to 0.272 μg/mg tissue (P = 0.015), for nonaffected placental tissues and preeclampsia placental tissues, respectively (Fig. 2B). There was no difference in the hyaluronic acid determined in samples obtained from patients with preeclampsia (3.076 ± 5.103 μg/mg tissue, ranging from 0.040 to 18.785 μg/mg tissue), compared to the values obtained from non-affected patients (1.322 ± 1.876 μg/mg tissue, ranging from 0.002 to 8.852 μg/mg tissue; P = 0.110), as demonstrated in Fig. 3. Fig. 4 shows that there was a significant difference in heparanase activity between tissues obtained from the placentas of non-affected women compared to the preeclampsia patients. The mean and SD of heparanase activity were 0.61 ± 0.19 μg of degraded HS/g tissue and 0.83 ± 0.28 μg of degraded HS/g tissue (P = 0.006), for non-affected placental and preeclampsia placental tissues, respectively. We were also interested in investigating the Pearson correlation coefficient between some patient's characteristics and the levels of GAG and heparanase activity. The analysis showed an association between a higher age of pregnant women with preeclampsia and lower levels of dermatan sulfate (P = 0.001), heparan sulfate (P = 0.002) and

Fig. 3. Hyaluronic acid levels from placentas. Thirty samples were collected from placentas of pregnant women without preeclampsia (control) and 15 samples from placentas of preeclampsia patients (preeclampsia). The black lines indicate the mean values obtained in each group by quantification of hyaluronic acid by ELISA-like assay. The assay was performed three times and the values are expressed as μg/mg of tissue. *P = 0.110.

E.A.B. Famá et al. / Clinica Chimica Acta 437 (2014) 155–160

Fig. 4. Heparanase activity. Enzymatic activity was determined in 30 samples obtained from control placentas (pregnant women without preeclampsia) and 15 samples collected from preeclampsia patients (preeclampsia). The black lines represent means. The assay was performed in triplicate and expressed as μg of degraded heparan sulfate/g tissue *P = 0.006.

hyaluronic acid (P = 0.012). Older preeclampsia patients also presented a decrease in heparanase activity (P = 0.003) as shown in Table 3. However, no association was observed between GAG profile and heparanase activity to the newborn's weight and gestational age comparing control group and preeclampsia patients (Table 3). The results prompted us to evaluate whether the glycosaminoglycans and heparanase may be related to the severity of the disease. Consequently, preeclampsia women were classified into three different subgroups; severe preeclampsia with gestational age b 34 weeks, severe preeclampsia with gestational age ≥ 34 weeks and mild preeclampsia (gestational age N 34 weeks). The early-onset group (b34 weeks) presented significantly higher levels of DS and HS compared with others group (Suppl. Fig. 1A and B). Furthermore, a tendency of enhanced heparanase activity was also detected in the severe earlyonset group (Suppl. Fig. 1C).

4. Discussion A number of studies have found a higher concentration of glycosaminoglycans in preeclampsia patients [11,21,22]. However, identification and quantification of glycosaminoglycans and heparanase activity have not been investigated in placental tissues until now. The present study showed that there is a significant increase in sulfated glycosaminoglycan levels (heparan sulfate and dermatan sulfate) and heparanase activity in the placentas of patients with preeclampsia. In the present study we found an increase of dermatan sulfate in preeclampsia group. One plausible explanation is that dermatan sulfate strongly binds to TGF-β, expressed in placenta, inhibiting its activity [13]. Therefore, high levels of dermatan sulfate may be involved

159

with possible modulation of trophoblastic invasion in the uterus of patients with preeclampsia. Vascular endothelial growth factor (VEGF) is necessary for tissue angiogenesis and its expression has been found to be lower in preeclampsia. The VEGF is regulated by heparan sulfate, thus, a probable explanation for the increased expression of heparan sulfate found in placentas with preeclampsia might interfere in the regulation of VEGF by reducing its expression and inducing the onset of preeclampsia [22,23]. Furthermore, placental growth factor induces angiogenesis by exclusively binding to the membrane receptor Flt-1 in the endothelial cells, resulting in the stretching and winding of the pre-formed vessels [24]. Soluble Flt-1 in its free form acts as an antagonist of the angiogenesis by binding to vascular endothelial growth factor and placental growth factor, blocking their biological activity [6]. It is well known that heparan sulfate and heparin are structurally similar and can bind to sFlt-1, enabling this receptor to attach to blood vessels and placenta [18]. Therefore, increased levels of heparan sulfate in placenta of patients with preeclampsia may be related to elevated levels of sFlt-1, which has been implicated in the development of preeclampsia mechanisms. This enhanced level of heparan sulfate in preeclampsia placenta corroborates with previous studies described in the literature that showed increased levels of circulating sFlt-1 after treatment of pregnant women with heparin [12]. It was observed that pericellular retention of sFlt-1 is mediated through its high affinity binding to heparan sulfate and its release controlled, at least in part, by heparanase [25,26]. Heparanase expression was shown to be progressively upregulated during pregnancy, with progressive accumulation of sFlt-1 in the circulation. A comparison of heparanase protein expression levels between normal and preeclampsia placenta revealed a significant increase in preeclampsia [18,27]. Furthermore, it has been described in the literature that the inhibition of Cathepsin L reduced the secretion of sFlt-1, since this protease activates heparanase precursor [17,18]. The significant increase in heparanase activity of preeclampsia placental tissues observed in the present study confirms that heparanase is important to the pathophysiology of preeclampsia and is possibly involved with sFlt-1 shedding. In light of that observed, it could also be hypothesized that the mechanism of insertion and cellular trafficking for the sFlt1 receptor in the plasma membrane, may be dependent on endogenous heparan sulfate. Finally, tissue retention versus systemic release of sFlt-1 mediated by heparan sulfate might be regulated by heparanase activity (Fig. 5). The fact that heparanase and heparan sulfate have the ability to interfere in the concentration of sFlt1 suggests that possible manipulation of the system using heparan sulfate/heparin or heparanase could reduce the undue release of sFlt1. There was no significant difference in hyaluronic levels between the average of preeclampsia group and control group demonstrated. However, we verified some outlier values of HA in preeclampsia group,

Table 3 Pearson's correlation between clinical features and levels of glycosaminoglycans and heparanase activity. Dermatan sulfate a

Heparan sulfate b

r

p

Age Control group Preeclampsia

0.126 −0.766

0.515 0.001

30 15

Newborn's weight Control group Preeclampsia

−0.034 −0.282

0.860 0.308

Gestational age Control group Preeclampsia

0.131 −0.210

0.497 0.451

a b c

Pearson correlation coefficient. Descriptive level. Number of involved pairs.

n

c

r

a

Hyaluronic acid b

c

r

a

Heparanase activity ra

pb

nc

30 15

0.093 −0.708

0.645 0.003

30 15

0.938 0.926

30 15

0.063 0.211

0.755 0.451

30 15

0.891 0.861

30 15

−0.016 0.170

0.936 0.545

30 15

p

b

p

n

0.124 −0.723

0.522 0.002

30 15

−0.167 −0.628

0.388 0.012

30 15

−0.125 −0.327

0.520 0.235

30 15

0.015 −0.026

30 15

0.043 −0.193

0.825 0.490

30 15

0.027 0.050

n

c

160

E.A.B. Famá et al. / Clinica Chimica Acta 437 (2014) 155–160

Fig. 5. Hypothetical scheme to explain preeclampsia. Both representative schemes explain differences between non-affected placenta (normal pregnancies) and placenta from patient with preeclampsia (preeclamptic pregnancies). Heparan sulfate was increased in preeclampsia tissue. Heparan sulfate chain from proteoglycans is cleaved by heparanase and releases sFlt-1. Heparanase activity is higher in preeclamptic placenta, enhancing sFlt-1 shedding. HS, heparan sulfate; HPSE, heparanase; sFlt-1, soluble fms-like tyrosine kinase-1.

suggesting that possibly high level of HA may be an indicative of a larger area of infarcted villi, as proposed by other authors [14]. These combined results confirmed that less than 34 weeks of delivery (early-onset) presented a significant increased in dermatan sulfate and heparan sulfate. Hence, these biomarkers could be useful for classifying the severity of preeclampsia. 5. Conclusion The present study has shown, for the first time a direct correlation between increased level of heparan sulfate and heparanase activity in the placentas of women with preeclampsia, which can explain the mechanisms of how these compounds are involved in the release of sFlt-1 to the circulation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cca.2014.07.023. Acknowledgments We express gratitude for the financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2009/50061-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] Report of the National High Blood Pressure Education Program Working Group on high blood pressure in pregnancy. Am J Obstet Gynecol 2000;183:S1–S22. [2] Serrano NC. Immunology and genetic of preeclampsia. Clin Dev Immunol 2006; 13:197–201. [3] Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005;365:785–99. [4] Facca TA, Kirsztajn GM, Pereira AR, et al. Renal evaluation in women with preeclampsia. Nephron Extra 2012;2:125–32. [5] Sibai BM. Diagnosis and management of gestational hypertension and preeclampsia. Obstet Gynecol 2003;102:181–92. [6] Woolcock J, Hennessy A, Xu B, et al. Soluble Flt-1 as a diagnostic marker of preeclampsia. Aust N Z J Obstet Gynaecol 2008;48:64–70. [7] Carty DM, Delles C, Dominiczak AF. Novel biomarkers for predicting preeclampsia. Trends Cardiovasc Med 2008;18:186–94. [8] Molvarec A, Szarka A, Walentin S, Szucs E, Nagy B, Rigo Jr J. Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia. Hypertens Res 2010;33:892–8.

[9] Molvarec A, Ito M, Shima T, et al. Decreased proportion of peripheral blood vascular endothelial growth factor-expressing T and natural killer cells in preeclampsia. Am J Obstet Gynecol 2010;203:567 [e561-568]. [10] Souza RS, Pinhal MAS. Interactions in physiological processes: the importance dynamics between extracellular matrix and proteoglycans. Arq Bras Cienc Saúde 2011;36:48–54. [11] Warda M, Zhang F, Radwan M, et al. Is human placenta proteoglycan remodeling involved in pre-eclampsia? Glycoconj J 2008;25:441–50. [12] Rosenberg VA, Buhimschi IA, Lockwood CJ, et al. Heparin elevates circulating soluble fms-like tyrosine kinase-1 immunoreactivity in pregnant women receiving anticoagulation therapy. Circulation 2011;124:2543–53. [13] Lysiak JJ, Hunt J, Pringle GA, Lala PK. Localization of transforming growth factor beta and its natural inhibitor decorin in the human placenta and decidua throughout gestation. Placenta 1995;16:221–31. [14] Berg S, Engman A, Holmgren S, Lundahl T, Laurent TC. Increased plasma hyaluronan in severe pre-eclampsia and eclampsia. Scand J Clin Lab Invest 2001;61:131–7. [15] Matejevic D, Neudeck H, Graf R, Muller T, Dietl J. Localization of hyaluronan with a hyaluronan-specific hyaluronic acid binding protein in the placenta in preeclampsia. Gynecol Obstet Investig 2001;52:257–9. [16] Vlodavsky I, Friedmann Y, Elkin M, et al. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med 1999; 5:793–802. [17] Abboud-Jarrous G, Atzmon R, Peretz T, et al. Cathepsin L is responsible for processing and activation of proheparanase through multiple cleavages of a linker segment. J Biol Chem 2008;283:18167–76. [18] Sela S, Natanson-Yaron S, Zcharia E, Vlodavsky I, Yagel S, Keshet E. Local retention versus systemic release of soluble VEGF receptor-1 are mediated by heparinbinding and regulated by heparanase. Circ Res 2011;108:1063–70. [19] Nader HB, Porcionatto MA, Tersariol IL, et al. Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy. J Biol Chem 1990;265:16807–13. [20] Boucas RI, Trindade ES, Tersariol IL, Dietrich CP, Nader HB. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells. Anal Chim Acta 2008;618:218–26. [21] Gogiel T, Bankowski E, Jaworski S. Pre-eclampsia-associated differential expression of proteoglycans in the umbilical cord arteries. Pathobiology 2001;69:212–8. [22] Chui A, Murthi P, Brennecke SP, Ignjatovic V, Monagle PT, Said JM. The expression of placental proteoglycans in pre-eclampsia. Gynecol Obstet Investig 2012;73:277–84. [23] Murthi P, Faisal FA, Rajaraman G, et al. Placental biglycan expression is decreased in human idiopathic fetal growth restriction. Placenta 2010;31:712–7. [24] Powe CE, Levine RJ, Karumanchi SA. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation 2011;123:2856–69. [25] Haimov-Kochman R, Friedmann Y, Prus D, et al. Localization of heparanase in normal and pathological human placenta. Mol Hum Reprod 2002;8:566–73. [26] Yagel S. Angiogenesis in gestational vascular complications. Thromb Res 2011; 127(Suppl. 3):S64–6. [27] Wang A, Holston AM, Yu KF, et al. Circulating anti-angiogenic factors during hypertensive pregnancy and increased risk of respiratory distress syndrome in preterm neonates. J Matern Fetal Neonatal Med 2012;25:1447–52.