Alteration of heat shock protein 20 expression in preeclamptic patients and its effect in vascular and coagulation function

Abstract

Preeclampsia (PE) is a pregnancy-specific, multi-system disorder and the leading cause of maternal and perinatal morbidity and mortality in obstetrics worldwide. Excessive vasoconstriction and dysregulated coagulation function are closely associated with PE. Heat shock protein 20 (HSP20) is ubiquitously expressed under normal physiological conditions and has important roles in vascular dilatation and suppression of platelet aggregation. However, the role of HSP20 in the pathogenesis of PE remains unclear. In this study, we collected chorionic plate resistance arteries (CPAs) and serum from 118 healthy pregnant women and 80 women with PE and detected the levels of HSP20 and its phosphorylated form. Both HSP20 and phosphorylated HSP20 were downregulated in CPAs from women with PE. Comparison of the vasodilative ability of CPAs from the two groups showed impaired relaxation responses to acetyl choline in preeclamptic vessels. In addition to the reduced HSP20 in serum from women with PE, the platelet distribution width and mean platelet volume were also decreased, and the activated partial thromboplastin time and thromboplastin time were elevated. With regard to the vital roles of HSP20 in mediating vasorelaxation and coagulation function, the decreased HSP20 might contribute to the pathogenesis of PE.

Keywords

preeclampsia / heat shock protein 20 / vascular relaxation / coagulation-fibrinolytic system

Introduction

Preeclampsia (PE) is characterized by new-onset hypertension (blood pressure consistently>140/90 mmHg) occurring in pregnant women after 20 weeks of gestation and is accompanied by evidence of organ injury (proteinuria, thrombocytopenia, renal or liver impairment, pulmonary edema, or cerebral/visual symptoms suggesting cerebral cortical dysfunction) [¹]. PE remains a complicated, multi-system disorder and a main cause of maternal and fetal morbidity and mortality worldwide [²]. Once established, the progression of this disease cannot be prevented, and delivery is the only cure [³]. Moreover, women with a history of PE have a high risk of acquiring cardiovascular diseases, and the infants born to PE women have an increased risk of neuropathy and bronchopulmonary dysplasia caused by preterm birth [⁴,⁵].

The mechanisms underlying PE remain elusive, but extensive studies suggest that abnormal trophoblast invasion and placentation and incomplete remodeling of spiral arteries are responsible for most of the pathology of PE [⁶,⁷]. Vascular disorder characterized by generalized endothelial damage and vasospasm, increased oxidative stress, derangements in platelets and alterations in coagulation also contribute to this disease [⁸]. Thus, understanding these complex pathophysiological changes would significantly advance our knowledge of PE.

Heat shock proteins (HSPs) are a diverse protein group ubiquitously expressed under normal physiological conditions to mediate diverse activities. According to their molecular weight, HSPs exist in six families: small heat shock proteins (sHSPs), HSP40, HSP60, HSP70, HSP90, and HSP100 [⁹]. Abnormalities in some HSPs have been reported for several disorders and have specifically been described in PE. For example, HSP70 and 90 are increased in placental endothelial cells from PE patients and are associated with endothelial cell viability [¹⁰]. Serum HSP70 is also increased in PE and reflects systemic inflammation, oxidative stress, and hepatocellular injury [¹¹,¹²]. HSP27 is a sHSP spatially distributed in human placenta and is selectively regulated during PE [¹³]. Similarly, HSP20 (17 kDa), which was identified in the mid-1990s as a byproduct of the purification of HSPB1 and HSPB5 [¹⁴], has been gradually confirmed important in physiology. For instance, HSP20 phosphorylation at ser16 (pHSP20) mediates smooth muscle relaxation, and its reduced expression is associated with impaired vascular relaxation [¹⁵,¹⁶]. These data indicate the importance of HSP20 and its phosphorylation in maintaining normal vascular function. HSP20 is also a potential regulator of platelet function [¹⁷]: HSP20 inhibits thrombin-induced aggregation of platelets at low concentrations [¹⁸], and this function may be achieved by blocking thrombin-induced calcium entrance from platelet exteriors [¹⁹]. HSP20 in human blood could restrains platelet activation, suggesting its potential influence on coagulation. In addition, the overexpression of HSP20 has cardioprotective and anti-apoptotic effects [²⁰,²¹]. Therefore, we hypothesized that HSP20 may be involved in the development of PE, but few reports have been found in this regard. To address this issue, we evaluated the vascular function of chorionic plate resistance arteries and identified HSP20 location and abundance in normal pregnant women and those with PE. We measured serum HSP20 and compared the coagulation factors to explore the relationship between HSP20 and PE.

Materials and methods

Subjects

We enrolled 80 women with PE and 118 healthy women in their third trimester of pregnancy. All subjects were admitted to Tongji Hospital from 2014 to 2016 and consented to cesarean delivery. Among the 80 women with PE, 50 were diagnosed with severe PE and 30 with mild PE. Exclusion criteria included diabetes or gestational diabetes, chronic hypertension, infectious diseases recognized in pregnancy, premature rupture of membranes, intrahepatic cholestasis of pregnancy, hyper- or hypothyroidism, or cardiac diseases. Controls had no signs of gestational complications or fetal distress and all gave birth to healthy neonates of appropriate size for gestational age.

PE was diagnosed according to the definition of the America College of Obstetricians and Gynecologists using two criteria [¹]. Mild PE was defined as a systolic blood pressure greater than or equal to 140 mmHg or a diastolic blood pressure greater than or equal to 90 mmHg on two occasions at least 4 h apart after 20 weeks of gestation in a woman with a previously normal blood pressure. The other criterion is proteinuria greater than or equal to 300 mg per 24 h urine collection or protein/creatinine ratio greater than or equal to 0.3 and a dipstick reading of 1+ . In the absence of proteinuria, hypertension onset was considered diagnostic of PE if accompanied by thrombocytopenia, renal insufficiency, impaired liver function, pulmonary edema, and cerebral or visual symptoms. Severe PE was defined with the presence of any of the following conditions: systolic blood pressure of 160 mmHg or higher, diastolic blood pressure of 110 mmHg or higher on two occasions at least 4 h apart when the patient was on bed rest, thrombocytopenia (platelet count<100 000/mL), liver function indicated by abnormally elevated liver enzymes (up to twice the normal value), severe persistent right upper quadrant or epigastric pain unresponsive to medication and not accounted for by alternative diagnoses or both, progressive renal insufficiency (serum creatinine concentration>1.1 mg/dL or doubled serum creatinine in the absence of preexisting renal diseases), pulmonary edema, and onset of cerebral or visual disturbances. The study was approved by the local ethics committee, and patient consent was obtained prior to delivery.

Sample collection

In brief, 2 mL fasting blood sample was drawn before caesarean section and collected into a vacuum tube (purple cap) containing 2.0 mg/mL EDTA-2K and preserved at 37 °C for platelet analysis. Another 2 mL fasting blood sample for coagulation factors and serum HSP20 measurement was collected into a vacuum tube (blue cap) containing sodium citrate (32.06 mg/mL, final concentration 3.8%) in a 9:1 volume ratio. Serum was centrifuged at 3000×g for 10 min, and supernatant was separated and divided into two aliquots: one was maintained at room temperature for D-dimer measurement and the remainder was frozen at −80 °C until later analysis.

Chorionic plate resistance arteries were dissected immediately after removing the placenta from the uterus during caesarean section. Vessels were carefully freed of connective tissues and washed in sterile PSS. A small section of vessels was fixed with paraformaldehyde for immunohistochemistry. Afterward, a 3 mm vessel ring was excised to measure the myogenic tone, and the rest was refrigerated at −80 °C until subsequent protein analysis.

Immunohistochemical analysis

Chorionic plate resistance arteries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunohistochemistry was performed on 5 mm-thick sections of paraffin-embedded samples. Sections were dewaxed, rehydrated, and incubated with 3% H₂O₂ for 25 min, blocked with 3% BSA for 30 min, and incubated overnight with a rabbit anti-HSP20 and -pHSP20 poly antibody (1:200, Abcam, UK). Samples were subsequently incubated for 50 min with secondary antibodies and labeled with biotin at optimal concentrations for 30 min at 37 °C. Protein expression was measured using horseradish peroxidase-conjugated streptavidin–avidin complex whereas sections incubated solely with secondary antibody were negative controls.

Western blot

Chorionic plate resistance arteries were homogenized in cold RIPA buffer (50 mmol/L Tris·HCl [pH 7.4], 0.15 mmol/L NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, and 1 mmol/L EDTA) enriched with 1 mmol/L cocktail. Homogenate was separated by centrifugation at 12 000×g for 20 min at 4 °C. The supernatant was kept in ice. Total protein was measured with a protein assay kit (Bio-Rad, USA). Protein samples were loaded equally and separated using 12% SDS-PAGE and then transferred to a polyvinylidenedifluoride membrane (Millipore Biosciences, Singapore). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature, probed with anti-HSP20 (1:2000) or anti-pHSP20 (1:2000), and kept overnight at 4 °C. After washing (three times for 5 min each) in TBST, the blots were incubated with anti-mouse or anti-rabbit secondary antibodies (1:2000; Santa Cruz Biotechnology, USA) at room temperature for 1 h and then washed (four times for 5 min each) in TBST. Immunoblotting signals were measured using ECL (Amersham Biosciences, USA) and exposed to X-ray film (Kodak, USA). Densitometry analysis was performed using ImageLab.

Measurement of myogenic tone

Each vessel ring was mounted onto an individual myograph bath chamber (Catamount myotechnology, Catamount R&D, UK). Each myograph had four chambers filled with 15 mL of PSS solution heated to 37 °C and gassed with 95% O₂ and 5% CO₂. Vessels were mounted onto two inverted L-shaped steel wires (100 mm each), immersed in PSS, and normalized to 1 g initial tension. Vessels were allowed to equilibrate for 1 h, during which the liquid was changed every 20 min, and the tension maintained at 1 g. Relaxation of preconstricted vessels was assessed using incremental doses of acetyl choline (ACh) from 0.1 nmol/L to 10 mmol/L applied at 5 min intervals.

ELISA for coagulation factors

Platelet indices were measured using an automatic quantitative hematology analyzer (Sysmex, Japan). Coagulation factors were measured using an automatic blood coagulation analyzer (Stago, China). Human specific Imu-clone ELISA (American Diagnostica Inc., USA) and human Heat Shock Protein 20 Kit (Human Elisa kit 1169, China) were used for D-dimer and HSP20 measurements.

Data analysis

Results were expressed as mean±SD. One-way ANOVA was used to compare differences in normally distributed variables. P<0.05 indicated statistical significance. Figures were processed with GraphPad Prism 5.0 (GraphPad software, Inc., La Jolla, CA).

Results

No major differences existed in variables as detailed in Table 1.

HSP20 and pHSP20 were mainly located in the cytoplasm and on the cell surface of smooth muscle and endothelium. The average optical density (AOD) was significantly lower in PE than in the controls, indicating the decreased expression of HSP20/pHSP20 protein. No significant AOD difference was observed between mild and severe PE (Fig. 1).

In accordance with the immunohistochemical results, Western blot revealed the downregulated protein expressions of HSP20 and pHSP20 in PE groups (P<0.05), and no difference was found in the protein expressions between mild and severe PE (P>0.05; Fig. 2).

Chorionic plate resistance arteries were preconstricted with 60 mmol/L KCl and ACh produced a slight relaxation at the maximal dose in mild and severe PE (11.1% and 8.7%, respectively); however, the diastolic pressure was 26.2% at the same dose in the normal pregnancy group. The reduced relaxation was recorded at each dose of ACh from 10⁻⁹ mol/L to 10⁻⁵mol/L in PE groups compared with those in the normal pregnancy group (Fig. 3; P<0.05).

Platelet distribution width (PDW) and mean platelet volume (MPV) were significantly increased in PE, and no difference in platelet counts and platelet large cell ratios (PLCR) was found between the groups (Fig. 4B–4E). PLCR reflects the platelet function to a certain extent, and coagulation was assessed using prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and D-dimer. APTT and TT were prolonged in PE, but PT was comparable among the groups (Fig. 4F–4H). D-dimer was increased in severe PE compared with that in normal pregnancy and mild PE (Fig. 4I).

Discussion

Alterations in HSP20 in chorionic plate resistance arteries and maternal serum during the third trimester of healthy pregnancy are associated with PE. HSP20 phosphorylation is associated with smooth muscle relaxation [¹⁶]. HSP20 has three phosphorylation sites: Ser16, Ser59, and Ser157 [²⁰]. Given that Ser157 phosphorylation site does not exist in human HSP20 and the physiological role of serine 59 phosphorylation is unclear [²⁰], we measured the expression of pHSP20 on Ser16. The protein expression levels of HSP20 and pHSP20 were significantly decreased in chorionic plate resistance arteries in PE compared with those in normal pregnancy. These data were confirmed with immunohistochemistry and Western blot (Figs. 1 and 2). Given that placental vessels lack innervations and respond poorly to potent vasoactive agents from systemic circulation [²²,²³], local relaxing factors are necessary for the regulation of vascular function. Nitric oxide (NO) and prostacyclin are the most frequently-studied factors for their vasodilatory effects but both are deficient in women with PE. Our data describing the impairment of vasodilatation of placental arteries in PE predicted that systemic vasospasm is characteristic of PE. Although NO and endothelium-dependent hyperpolarizing factors contribute substantially in mediating the vasodilation of placental vessels, the mechanism is poorly understood. Reduced human placental artery relaxation has been suggested to be associated with low expression of HSP20 [²⁴]. Additionally, strong evidence was found that HSP20 could bind to actin in vivo and in vitro. The association of HSP20 with actin depends on its phosphorylation state, indicating its role in stabilizing the cytoskeleton and regulating vascular function. NO can phosphorylate HSP20 at Ser16 by activating guanylylcyclase (cyclic GMP) protein kinase G pathways [²⁵], and the increased HSP20 phosphorylation modulates NO released by the endothelium [¹⁴]. Thus, HSP20/pHSP20 may significantly contribute to the pathway of NO-mediated vasodilatation, and the downregulation of HSP20/pHSP20 in chorionic plate resistance arteries may be involved in the etiology and/or progression of PE.

HSP20 possesses anti-platelet potential and can restrain thrombin-induced platelet aggregation in a dose-dependent manner. HSP20 was significantly decreased in an in vivo hamster model of carotid artery endothelial injury. HSP20 may be secreted from the vascular wall after endothelial injury to regulate platelet function in the circulation [²⁵]. PE is described as a disease possessing wide-ranging endothelial cell damage [⁷]. Interestingly, we observed that serum HSP20 was decreased in PE compared with normal pregnancy, which can be explained two ways. First, an extensive reduction of HSP20 protein synthesis in systemic blood vessels of PE patients may occur. Endothelial injury is an early pathological change prior to onset of clinical symptoms. PE patients undergo physiological changes, and HSP20 expression would be affected by these changes over the course of PE [²⁶]. Furthermore, other sources of serum HSP20 may exist besides smooth muscle, which are presumably predominant in normal pregnant women but absent in PE for unknown reasons. Detailed information about intracellular distribution of HSP20 is scarce and intracellular HSP20 is strictly regulated — the mechanism may differ by tissue type [²⁷]. Nevertheless, decreased serum HSP20 in PE could not adequately regulate platelet function.

Blood platelets are required for hemostasis and thrombosis, so we measured coagulation factors to evaluate functional differences in platelets in PE and normal pregnancies. PE women whose condition was aggravated by HELLP syndrome were excluded, explaining a lack of differences in platelet counts and platelet large cell ratios between normal pregnancy and PE groups. PDW and MPV are potential markers of platelet activation, and both were increased in PE compared with normal pregnancies, indicating platelet dysfunction in PE, and may partly be attributed to the decreased level of serum HSP20.

Normal human pregnancy is associated with significant hemodynamic changes characterized by a protective hypercoagulable state due to alterations in coagulation and fibrinolytic systems [²⁷,²⁸]. This alteration reduces the risk of postpartum hemorrhage and prevents complications [²⁹]. However, during PE, a super-hypercoagulation state exists, increasing susceptibility to venous thromboembolism and disseminating intravascular coagulation [³⁰]. However, the cause and the mechanism of this phenomenon remain unclear. The generalization of endothelial dysfunction often leads to alterations of local anticoagulant properties in PE, and pathological activation of blood coagulation pathways in PE was chiefly attributed to abnormal platelet activation and endothelial cell dysfunction. We measured the coagulation factors and confirmed that APTT in severe PE patients was greater than that in normal pregnancy, but no significant difference was found in PT. These data agreed with reports by Han et al.[²⁸], implying that the function of endogenous coagulation pathways was, to a certain degree, impaired in severe PE whereas exogenous coagulation pathways were not significantly affected. TT and D-dimer indirectly reflect fibrinolytic system activity and both were increased and associated with PE severity, suggesting progressive hyperfibrinolysis in PE. Collectively, these results revealed complex changes in coagulation during PE. From previous reports about the potent inhibitive effect of HSP20 on platelet aggregation and activation [¹⁷], and because platelet activation is key to the inception of the coagulation pathway, we surmised that decreased serum HSP20 might contribute to a disturbed coagulation-fibrinolytic mechanism in PE. However, more work is required to confirm that coagulation pathways are modulated by HSP20 during PE.

Given the limitations of this cross-sectional study, we failed to identify a causal relationship between decreased HSP20 and PE. Longitudinal estimations of HSP20 throughout gestation would better define a role for this during PE, and may indicate that HSP20 is a marker for predicting PE onset or severity. Thus, HSP20 (pHSP20) protein was decreased in chorionic plate resistance arteries and in serum of PE women and vascular function and the endogenous coagulation and fibrinolytic systems were impaired to some degree in PE, suggesting that downregulation of HSP20 might be involved in PE pathophysiology.

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