Clinica Chimica Acta 437 (2014) 155–160

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Evaluation of glycosaminoglycans and heparanase in placentas ofwomenwith 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 Obstetrics/Gynecology Department, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazilb Biochemistry Department, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazilc Biochemistry Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil

⁎ Corresponding author at: Biochemistry Departmen1004th Floor, Vila Clementino, São Paulo, SP 04044-020,fax: +55 11 55736407.

E-mail address: maspinhal@yahoo.com.br (M.A.S. Pinh

http://dx.doi.org/10.1016/j.cca.2014.07.0230009-8981/Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 16 January 2014Received in revised form 17 July 2014Accepted 18 July 2014Available online 30 July 2014

Keywords:PreeclampsiaHeparan sulfateDermatan sulfateHyaluronic acidHeparanase

Background: Preeclampsia is amultisystemdisorderwhose etiology remains unclear. It is already known that cir-culation of soluble fms-like tyrosine kinase-1 (sFlt-1) is directly involved in pre-eclampsia development. Howev-er, the molecular mechanisms involved with sFlt-1 shedding are still unidentified. We identified, quantifiedglycosaminoglycans and determined the enzymatic activity of heparanase in placentas of women with pre-eclampsia, in order to possibly explain if these compounds could be related to cellular processes involved withpreeclampsia.Methods: A total of 45 samples collected from placentas, 15 samples from placentas of preeclampsia women and30 samples from non-affected women. Heparan sulfate and dermatan sulfate were identified and quantified byagarose gel electrophoresis, whilst hyaluronic acid was quantified by an ELISA like assay. Heparanase activitywas 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. Therewas no significant difference between the groups for hyaluronic acid expression in placentas (P = 0.110). Thepresent study is thefirst to demonstrate directly the increase of heparan sulfate inhumanplacentas frompatientswith preeclampsia, suggesting that endogenous heparan sulfate could be involved in the release of sFlt-1 fromplacenta, increasing the level of circulating sFlt-1.Conclusion: Alterations of extracellular matrix components in placentas with preeclampsia raise the possibilitythat 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 hyperten-sion and significant proteinuria after 20 weeks of pregnancy [1]. It is amultifactorial, multisystem disorder whose etiology has not been fullyelucidated [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 signal-ing processes connecting intracellular and extracellular environments[10]. Marked differences in GAG sulfation patterns were observed inplacental preeclampsia [11]. It is already known that exogenous hepa-ran sulfate (HS) binds to sFlt-1, and that heparin can compete with HS

t UNIFESP, Rua Três de Maio,Brazil. Tel.: +55 11 55793175;

al).

for sFlt-1 binding. Interestingly, Low Molecular Weight Heparin(LMWH) administered for coagulation prophylaxis to women at riskof coagulation disorders increased circulating sFlt-1 levels comparedto normal untreated pregnant controls [12].

It has already been shown that decorin, a chondroitin sulfate anddermatan sulfate proteoglycan inhibits the activity of transforminggrowth factor beta (TGF-β). TGF-β produced in the fetal–maternalinterface plays a crucial role in the control of trophoblast invasion inthe uterus [13]. Consequently, both chondroitin sulfate and dermatansulfate may modulate trophoblast invasion.

Hyaluronan or hyaluronic acid (HA) is an extracellular matrix poly-saccharide present at low concentrations in plasma. Normally, HA israpidly eliminated from the blood by the liver. Increased concentrationof circulating HA has been found in women with preeclampsia [14].Histochemical analysis used to detect HA in placentas from uncompli-cated pregnancies and patients with preeclampsia, showed enhancedstaining in the stroma and blood vessel walls. HA was found withinand on the surface of intervillous and perivillous fibrinoid deposits[15]. Since fibrinoid deposits of HA are increased in preeclampsia,

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resulting from infarcted villi, this HA from fibrinoid tissue is able toreach maternal blood and may explain increased levels of circulatingHA in the plasma of women with preeclampsia.

Heparanase, an endo-β-glucuronidase, participates in the degrada-tion of the heparan sulfate proteoglycan chains [16]. This enzyme pre-sents two isoforms, a precursor with no apparent enzymatic activity(65 kDa) that undergoes proteolytic activity to form the mature activeenzyme, a heterodimer containing a 50 kDa subunit associated withan 8 kDa. This posttranslational processing of heparanase is performedby a papain-like cysteine proteinase, called Cathepsin L [17].

Inhibition of heparanase with a neutralizing antibody resulted in amarked reduction in sFlt-1 secretion of normal and preeclampsia ex-plants [18]. Moreover, the level of sFlt-1 in the serum of heparanase-overexpressing transgenic mice was nearly double that of wild-typemice [12].

Althoughmany studies have already shown a larger reservoir of sFtl-1in the placenta and the role of heparanase and heparan sulfate in modu-lating the release of sFlt-1 into the circulation, until now, no study hasexamined GAG and heparanase activity in placental tissues frompreeclampsia patients.

2. Methods

2.1. Patients and tissue samples

A case–control study was conducted and placentas of pregnantwomen with preeclampsia (n = 15) and without preeclampsia(n = 30) were collected. This study was conducted in accordance

Table 1Features of preeclampsia and non-affected women.

Age (y) NMeanMedianMinimumMaximumStandard deviation

Race CaucasianBlackOthersTotal

Family history of arterial hypertension PositiveNegativeTotal

Family history of Diabetes mellitus PositiveNegativeTotal

Previous abortion PositiveNegativeTotal

Smoking habit PositiveNegativeTotal

Number of gestations 12346Total

Personal history of arterial hypertension PositiveNegativeTotal

Proteinuria (mg/24 h) NMeanMedianMinimumMaximumStandard deviation

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

with the ethical principles of the Declaration of Helsinki. The sampleswere collected after informed consent had been granted. The studyprocedures were approved by the Ethics Committee of the Women'sHospital and Faculdade de Medicina ABC (number 259/2009). Pre-eclampsia 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 fol-lowing clinical and laboratory features, such as hypertension N 160/110 mm Hg, signs of imminent eclampsia, eclampsia, HELLP syndromeand proteinuria≥ 5 g/24-h urine. It is important to point out that the se-vere preeclampsia group was also evaluated for early-onset disease(gestational age b 34 weeks) and late-onset disease (≥34 weeks). In-cluded in the case group were pregnant women diagnosed with pre-eclampsia, that present singleton pregnancy, regardless of the type ofdelivery (vaginal or abdominal) or gestational age. The control groupwas composed of single pregnancy without preeclampsia from anytype of delivery (vaginal or abdominal) or gestational age.We excludedpatients from both groups who presented multiple gestations, chronichypertension, diabetes mellitus, chronic kidney disease, thrombophilia,collagenosis and illicit drug use. The placental sample was obtainedfrom a square measuring 5 × 5 cm, around the center of the umbilicalcord insertion. The material was rinsed with 0.9% saline solution toremove blood and amniotic fluid.

2.2. Identification and quantification of sulfated glycosaminoglycans

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

Control group Preeclampsia P

30 15 NSa

24.2 27.325.0 2.016.0 16.035.0 38.05.5 7.320 71.4% 12 80.0% NSb

2 3.6% 1 6.7%8 25.0% 2 13.3%30 100.0% 15 100.0%10 34.5% 3 20.0% NSb

20 65.5% 12 80.0%30 100.0% 15 100.0%6 20.7% 1 6.7% NSb

24 79.3% 14 93.3%30 100.0% 15 100.0%1 3.4% – – NSb

29 96.6% 15 100.0%30 100.0% 15 100.0%3 10.3% – – NSb

27 89.7% 15 100.0%30 100.0% 15 100.0%11 35.7% 6 40.0% NSa

10 32.1% 4 26.7%7 25.0% 5 33.3%1 3.6% – –

1 3.6% – –

30 100.0% 15 100.0%– – 3 20.0% 0.034b

30 100.0% 12 80.0%30 100.0% 15 100.0%– 7 –

– 554.3– 340.0– 300.0– 1400.0– 421.0

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(100 mg) was submitted to proteolysis in alcalase (4 mg/ml in Tris-chloridric acid 0.05 mol/l, pH 8, containing 1.5 mmol/l sodium chlo-ride) at a ratio of 1:4 for 72 h at 60 °C. Trichloroacetic acid (10% finalconcentration) was added to the mixture, maintained at 4 °C for15 min. The supernatant containing glycosaminoglycans was obtainedafter centrifugation (10 min, 3500 ×g, 4 °C). Sulfated glycosaminogly-cans 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 inwater andmaintained frozen for furtheranalysis. Sulfated glycosaminoglycans (heparan sulfate, dermatan sulfateand chondroitin sulfate) were identified and quantified by agarosegel electrophoresis in 0.05 mol/l 1,3-diaminopropane-acetatebuffer, pH 9.0. After electrophoresis, for 1 h at 100V, glycosaminoglycanswere precipitated in agarose gel using 0.1% cetyl-trimethylammonium-bromide (Sigma-Aldrich) for 2 h at room temperature. The gel wasdried and stained with toluidine blue (0.1% in acetic acid:ethanol:water; 0.1:5:4.9, v:v:v). Glycosaminoglycan quantification was carriedout by densitometry at 530 nm. The extinction coefficients of the glycos-aminoglycans were calculated using standards of chondroitin 4-sulfatefrom whale cartilage (Seikagaku Kogyo Co.), dermatan sulfate (frompig skin) and heparan sulfate (from bovine pancreas). The agarose gel

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

Controlgroup

Preeclampsia P

Delivery Cesarean 6 14 b0.001b

Vaginal 22 1Forceps 2 –

Total 30 15Newborn's gender Male 12 6 NSc

Female 18 9Total 30 15

One-minute APGARscore

N 30 15 NSd

Mean 8.5 8.3Median 9.0 9.0Minimum 6.0 6.0Maximum 10.0 9.0Standarddeviation

0.7 1.0

Five-minute APGARscore

N 30 15 NSd

Mean 9.4 9.4Median 9.0 9.0Minimum 8.0 8.0Maximum 10.0 10.0Standarddeviation

0.6 0.6

Newborn's weight (g) N 30 15 NSa

Mean 3333.3 2972.7Median 3340.0 3140.0Minimum 2506.0 1196.0Maximum 3954.0 4056.0Standarddeviation

319.6 858.0

Time of collection ofplacenta postpartum(hours)

N 30 15 NSa

Mean 1.53 1.65Median 1.00 1.00Minimum 0.17 0.33Maximum 5.00 7.00Standarddeviation

1.40 1.77

Gestational age (weeks) N 30 15 0.028a

Mean 39.0 36.9Median 39.1 38.4Minimum 37.3 29.3Maximum 41.3 40.1Standarddeviation

0.8 3.3

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.

electrophoresis method error was approximately 5%. Identification ofthe sulfated glycosaminoglycans was based on the migration of thecompounds compared with that of the standards. The identity ofthe different sulfated glycosaminoglycans was confirmed by degradationwith 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 acidwasquantified after proteolysis of the tissue samplesas previously described. For the dosage of hyaluronic acid, a fluorimetricmethod was used. First, link protein was coated (1 mg protein/ml), in a96-well plate, and kept overnight at 4 °C. Link protein was isolated frombovine nasal cartilage purified by affinity chromatography. It containsthe globular region G1 aggrecan that binds specifically to hyaluronicacid. The plates were washed 3 times with wash buffer (0.05 mol/lTris-chloridric acid, pH 8) and then 200 μl of blocking solution wasadded to each well. The assay was performed using 100 μl of each sam-ple in triplicate that had undergone proteolysis. The plates were incu-bated overnight at 4 °C, washed 3 times with wash buffer andincubated with 100 μl of biotinylated link protein (1 mg/ml), dilutedin blocking buffer (1:5000), and kept under stirring at room tempera-ture for 90min. The plates were again washed 3 times with wash bufferand incubated with streptavidin conjugated to Europium, diluted1:10,000 in blocking solution under stirring for 30min at room temper-ature. Then the plates were washed 3 times with wash buffer. Afterwashing, aliquots of 200 μl of developing solution were added to thewells and the plates were kept under stirring for 10 min. The fluores-cence quantification was performed using a Victor 2 ELISA reader(PerkinElmer, Life Sciences). This method detects hyaluronic acid be-tween 0.2 and 500 μg/l. The values were processed automatically bythe program MultCalc® (PerkinElmer, Life Sciences, Turku, Finland).

2.4. Heparanase activity

Heparanase activity was measured by an ELISA-like method using15% biotinylated heparan sulfate immobilized in poly-L-lysinemultiwell[20]. All solutions were prepared in sodium acetate buffer 25 mmol/l,pH 5.5. Tissue samples were homogenized in inhibitory protease cock-tail P-8340 (Sigma-Aldrich) and incubated on pre-coated plate for 18 hat 37 °C. After several washings, non-degraded biotinylated heparan sul-fate was detected by incubation with europium-conjugated streptavidin

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

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(1:1,000) for 40 min at 25 °C. The plate was washed with acetate bufferto remove unbound streptavidin. Finally, 100 μl of enhancement solution(PerkinElmer Life Sciences-Wallac Oy) was added to each well understirring for 3min at room temperature. This procedure releases the euro-pium bound to streptavidin. A time-resolved fluorometer was usedto measure free europium and the data (counts/s) were processed auto-matically using MultCalc software (PerkinElmer Life Sciences-WallacOy). A standard curve of different concentrations of biotinylated heparansulfate was performed in order to convert the values of counts/s obtain-ed to percentage of heparanase activity based on heparan sulfatedegradation.

2.5. Statistical analysis

The resultswere expressed asmeans and SD or SE. The statistical dif-ference was confirmed by Student's t-test, Mann–Whitney test, ANOVAwith a fixed factor, and Kruskal–Wallis testwith a statistical significanceof P b 0.05. The significant correlations between variableswere assessedby Pearson correlation coefficient. Statistical analyses were performedby the Statistical Package for the Social Sciences (SPSS) ver 19.0 forWindows.

3. Results

The samples in this study consisted of 45 placental tissues, 30 sam-ples (66.7%) obtained from healthy women and 15 samples (33.3%)from patients with preeclampsia. The groups presented no significantdifference in ages (P = 0.115) as shown in Table 1. The control grouphad an average age of 24.2 y, ranging from 16 to 35 y with an SD of5.5 y. The preeclampsia group had a mean age of 27.3 y, ranging from16 to 38 y, with an SD of 7.3 y.

Fig. 2. Profile of sulfated glycosaminoglycans extracted from placentas. Thirty sampleswere collected from placentas of pregnant women without preeclampsia (control) and15 samples from placentas of preeclampsia patients (preeclampsia). The black lines indi-cate the mean values obtained in each group by quantification of dermatan sulfate andheparan sulfate, respectively. The values were obtained by quantification of agarose gelelectrophoresis as described in theMethods and expressed as μg/mg of tissue. A.Dermatansulfate. *P = 0.019. B. Heparan sulfate. *P = 0.015.

Therewere no statistically significant differences between the groupof 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 mi-nutes. Similarly, there was no difference between the groups consider-ing the time to collect placenta samples after delivery. We found ahigher incidence of previous personal history of hypertension in pre-eclampsia patients (P = 0.034), higher incidence of cesarean delivery(P b 0.001) and lower gestational age at delivery (P= 0.028) comparedto the healthy pregnant women (Tables 1 and 2). Gel electrophoresisanalysis demonstrated a significant increase of sulfated glycosaminogly-cans (GAG) in the preeclampsia samples compared to the control group(Fig. 1).

Sulfated galactosaminoglycans in the placental samples wascompletely degraded after digestionwith chondroitinaseABC, confirmingthe presence of iduronic acid residues and, consequently, the identifica-tion of dermatan sulfate as the galactosaminoglycan in the tissue samples(data not shown).

The mean value of dermatan sulfate obtained from the placentas ofhealthy pregnant women was 0.102 ± 0.055 μg/mg tissue, rangingfrom 0.043 to 0.244 μg/mg tissue, whilst in the samples of preeclampsiapatients the average was 0.144 ± 0.074 μg/mg tissue, ranging from0.048 to 0.322 μg/mg tissue. A significant increase was therefore ob-served in the dermatan sulfate level of preeclampsia tissues comparedto 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/mgtissue, ranging from 0.041 to 0.272 μg/mg tissue (P = 0.015), for non-affected placental tissues and preeclampsia placental tissues, respec-tively (Fig. 2B).

There was no difference in the hyaluronic acid determined in sam-ples obtained from patients with preeclampsia (3.076 ± 5.103 μg/mgtissue, ranging from 0.040 to 18.785 μg/mg tissue), compared to thevalues obtained from non-affected patients (1.322 ± 1.876 μg/mgtissue, ranging from0.002 to 8.852 μg/mg tissue; P= 0.110), as demon-strated in Fig. 3.

Fig. 4 shows that therewas a significant difference in heparanase ac-tivity between tissues obtained from the placentas of non-affectedwomen compared to the preeclampsia patients. The mean and SD ofheparanase activity were 0.61 ± 0.19 μg of degraded HS/g tissue and0.83 ± 0.28 μg of degraded HS/g tissue (P = 0.006), for non-affectedplacental and preeclampsia placental tissues, respectively.

We were also interested in investigating the Pearson correlation co-efficient between some patient's characteristics and the levels of GAGand heparanase activity. The analysis showed an association betweena higher age of pregnant women with preeclampsia and lower levelsof dermatan sulfate (P = 0.001), heparan sulfate (P = 0.002) and

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

Fig. 4. Heparanase activity. Enzymatic activity was determined in 30 samples obtainedfrom control placentas (pregnantwomenwithout preeclampsia) and 15 samples collectedfrom preeclampsia patients (preeclampsia). The black lines represent means. The assaywas performed in triplicate and expressed as μg of degraded heparan sulfate/g tissue *P= 0.006.

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hyaluronic acid (P = 0.012). Older preeclampsia patients also present-ed a decrease in heparanase activity (P = 0.003) as shown in Table 3.However, no association was observed between GAG profile andheparanase activity to the newborn's weight and gestational age com-paring control group and preeclampsia patients (Table 3).

The results prompted us to evaluate whether the glycosaminogly-cans and heparanase may be related to the severity of the disease. Con-sequently, preeclampsia women were classified into three differentsubgroups; severe preeclampsia with gestational age b 34 weeks,severe preeclampsia with gestational age ≥ 34 weeks and mildpreeclampsia (gestational age N 34 weeks). The early-onset group(b34 weeks) presented significantly higher levels of DS and HS com-paredwith others group (Suppl. Fig. 1A and B). Furthermore, a tendencyof enhanced heparanase activity was also detected in the severe early-onset group (Suppl. Fig. 1C).

4. Discussion

A number of studies have found a higher concentration of glycos-aminoglycans in preeclampsia patients [11,21,22]. However, identifica-tion and quantification of glycosaminoglycans and heparanase activityhave not been investigated in placental tissues until now. The presentstudy showed that there is a significant increase in sulfated glycosami-noglycan levels (heparan sulfate and dermatan sulfate) and heparanaseactivity in the placentas of patients with preeclampsia.

In the present studywe foundan increase of dermatan sulfate in pre-eclampsia group. One plausible explanation is that dermatan sulfatestrongly binds to TGF-β, expressed in placenta, inhibiting its activi-ty [13]. Therefore, high levels of dermatan sulfate may be involved

Table 3Pearson's correlation between clinical features and levels of glycosaminoglycans and heparana

Dermatan sulfate Heparan sulfate

ra pb nc ra pb

AgeControl group 0.126 0.515 30 0.124 0.522Preeclampsia −0.766 0.001 15 −0.723 0.002

Newborn's weightControl group −0.034 0.860 30 −0.125 0.520Preeclampsia −0.282 0.308 15 −0.327 0.235

Gestational ageControl group 0.131 0.497 30 0.043 0.825Preeclampsia −0.210 0.451 15 −0.193 0.490

a Pearson correlation coefficient.b Descriptive level.c Number of involved pairs.

with possible modulation of trophoblastic invasion in the uterus ofpatients with preeclampsia.

Vascular endothelial growth factor (VEGF) is necessary for tissueangiogenesis and its expression has been found to be lower in pre-eclampsia. The VEGF is regulated by heparan sulfate, thus, a probableexplanation for the increased expression of heparan sulfate found inplacentas with preeclampsia might interfere in the regulation of VEGFby reducing its expression and inducing the onset of preeclampsia [22,23].

Furthermore, placental growth factor induces angiogenesis by exclu-sively 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 angiogenesisby binding to vascular endothelial growth factor and placental growthfactor, blocking their biological activity [6]. It is well known that hepa-ran 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 patientswith preeclampsia may be related to elevated levels of sFlt-1, whichhas been implicated in the development of preeclampsia mechanisms.This enhanced level of heparan sulfate in preeclampsia placenta corrob-orates with previous studies described in the literature that showed in-creased levels of circulating sFlt-1 after treatment of pregnant womenwith heparin [12].

It was observed that pericellular retention of sFlt-1 is mediatedthrough its high affinity binding to heparan sulfate and its release con-trolled, at least in part, by heparanase [25,26]. Heparanase expressionwas shown to be progressively upregulated during pregnancy, withprogressive accumulation of sFlt-1 in the circulation. A comparison ofheparanase protein expression levels between normal and preeclamp-sia placenta revealed a significant increase in preeclampsia [18,27]. Fur-thermore, it has been described in the literature that the inhibition ofCathepsin L reduced the secretion of sFlt-1, since this protease activatesheparanase precursor [17,18]. The significant increase in heparanase ac-tivity of preeclampsia placental tissues observed in the present studyconfirms that heparanase is important to the pathophysiology of pre-eclampsia and is possibly involved with sFlt-1 shedding.

In light of that observed, it could also be hypothesized that themechanism of insertion and cellular trafficking for the sFlt1 receptorin the plasma membrane, may be dependent on endogenous heparansulfate. Finally, tissue retention versus systemic release of sFlt-1mediat-ed by heparan sulfatemight be regulated by heparanase activity (Fig. 5).The fact that heparanase and heparan sulfate have the ability to inter-fere in the concentration of sFlt1 suggests that possible manipulationof the systemusing heparan sulfate/heparin or heparanase could reducethe undue release of sFlt1.

There was no significant difference in hyaluronic levels between theaverage of preeclampsia group and control group demonstrated. How-ever, we verified some outlier values of HA in preeclampsia group,

se activity.

Hyaluronic acid Heparanase activity

nc ra pb nc ra pb nc

30 −0.167 0.388 30 0.093 0.645 3015 −0.628 0.012 15 −0.708 0.003 15

30 0.015 0.938 30 0.063 0.755 3015 −0.026 0.926 15 0.211 0.451 15

30 0.027 0.891 30 −0.016 0.936 3015 0.050 0.861 15 0.170 0.545 15

Fig. 5. Hypothetical scheme to explain preeclampsia. Both representative schemes explain differences between non-affected placenta (normal pregnancies) and placenta from patientwith preeclampsia (preeclamptic pregnancies). Heparan sulfate was increased in preeclampsia tissue. Heparan sulfate chain from proteoglycans is cleaved by heparanase and releasessFlt-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.

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suggesting that possibly high level of HAmay be an indicative of a largerarea of infarcted villi, as proposed by other authors [14].

These combined results confirmed that less than 34 weeks of deliv-ery (early-onset) presented a significant increased in dermatan sulfateand heparan sulfate. Hence, these biomarkers could be useful for classi-fying the severity of preeclampsia.

5. Conclusion

The present study has shown, for the first time a direct correlationbetween increased level of heparan sulfate and heparanase activity inthe placentas of women with preeclampsia, which can explain themechanisms of how these compounds are involved in the release ofsFlt-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

Weexpress gratitude for thefinancial support provided by FundaçãodeAmparo à Pesquisa do Estadode São Paulo (FAPESP) (2009/50061-0),Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)and Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq).

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