Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2013; 17: 874-878 Placental expression of vimentin, desmin and ultrastructural changes in the villi in patients with HELLP syndrome

M.E. SAK, E. DEVECI 1, A. TURGUT, S. SAK 2, M.S. EVSEN, T. GUL, S. KALKANLI 3

Department of Obstetrics and Gynecology, School of Medicine, Dicle University, Diyarbakir, Turkey 1Department of Histology and Embryology, School of Medicine, Dicle University, Diyarbakir, Turkey 2Department of Obstetrics and Gynecology, Diyarbakir Maternity and Children Hospital, Diyarbakir, Turkey 3Department of Immunology, School of Medicine, Dicle University, Diyarbakir, Turkey

Abstract. – OBJECTIVES : To examine placen - drome is categorized as a gestational hyperten - tal expression of vimentin and desmin in relation sive disorder, and seen as the more severe variant to ultrastructural changes within the placental 1,2 villi in cases of HELLP syndrome. of preeclampsia (PE) . STUDY DESIGN: Formaldehyde-fixed and paraf - Hemolysis, elevated liver enzymes, and low fin-embedded specimens of 15 healthy pregnant platelet count (HELLP) occur in 20% of severe PE and 13 Hemolysis, elevated liver enzymes, and cases. The pathogenesis of HELLP syndrome is low platelet count (HELLP) syndrome, placentas not clear and the obstetric approach with the in - were used for Harris hematoxylin staining, vi - duction of delivery is still the only specific therapy mentin and desmin immunohistochemistry, and transmission electron microscopy (TEM). in HELLP syndrome. Involvement of the coagula - RESULTS: Increased of fibrinoid necrosis in tion system is seen in HELLP patients, which is vascular wall and the periphery of villi were ob - not present in PE patients without HELLP 3. An - served in sections of the placentas with HELLP giogenic factors, cell adhesion proteins, immuno - syndrome. Increased expression of vimentin in logical factors, matrix metalloproteinases and their the intravillous area and increased expression of inhibitors are all anticipated to play a role in this desmin on blood vessel wall, were seen in placen - 4,5 tas of patients with HELLP syndrome when com - crucial process of spiral artery dilatation . pared to placentas of healthy pregnant. In order to characterise the differentiation of CONCLUSIONS: Augmentation of intermediate placental stromal cells in the human placenta, a filaments, desmin, vimentin may disturb normal variable layer of extravascular stromal cells lying movements of endothelial cells, and may display beneath the trophoblast expressed vimentin (V) placental dysfunction that is unable to compensate or vimentin and desmin (VD) were investigated the endothelial instability and the related hyperten - on placental tissue of different gestational age. sion in HELLP syndrome. Further studies are needed to get more definit results and also to com - The expression patterns of the cytoskeletal pro - pare HELLP syndrome with preeclampsia. teins vimentin, desmin, alpha- and gamma- smooth muscle actin, pan-actin, smooth muscle Key Words: myosin, and the monoclonal antibody GB 42, as HELLP Syndrome, Placental expression, Vimentin, Desmin. a marker of myofibroblasts and intermediate fila - ments were arranged as concentric layers with in - creasing stage of differentiation around the fetal stem vessels in placental villi 6. The placenta and Introduction the incomplete trophoblast invasion of spiral ar - teries have a central role in pathogenesis of HELLP syndrome is a form of preeclampsia HELLP syndrome, but especially in severe pre- during pregnancy that often progresses to a life- eclampsia and in the HELLP syndrome there is a threatening complication. HELLP usually occurs systemic endothelial activation and damage 7. in the later stages of pregnancy, or sometimes af - The aim of this study is to examine placental ter delivery. Despite reports describing HELLP expression of vimentin and desmin in relation to in patients with normal or minimally elevated ultrastructural changes within the placental villi blood pressure without proteinuria, HELLP syn - in cases of HELLP syndrome.

874 Corresponding Author: Muhammet Erdal Sak, MD; e-mail: [email protected] Placental expression of vimentin, desmin and ultrastructural changes in HELLP syndrome

Materials and Methods Following incubation with primary antibody (mouse monoclonal anti-human vimentin; Santa- This study was performed at the Dicle Universi - Cruz, Santa Cruz, CA, USA) 1:20, and secondary ty, School of Medicine, Department of Obstetrics mouse monoclonal anti-human desmin (Dako, and Gynecology. The study included 13 pregnant Troy, MI, USA) 1:100, respectively, for 40 min at patients with HELLP syndrome(study group) and room temperature, the detection was performed us - 15 healthy pregnants at between 24 and 35 weeks ing DAB + peroxidase kit (Dako). Sections were gestational age (control group) . The study was ap - counterstained with hematoxylin. Simultaneous proved by the local instutional review board. Con - control experiments with the omission of either pri - trol group, healthy pregnancy was defined as preg - mary or secondary antibody gave negative results. nancy characterized by normal blood pressure val - ues (< 140/90 mmHg), negative proteinuria and Transmission Electron Microscopy normal values of laboratory findings . The HELLP The tissue samples were immediately placed in syndrome was defined as hemolysis (peripheral 2.5% glutaraldehyde, buffered for 4 h, then fixed blood smear findings and lactate dehydrogenase in OsO 4 for 2 h, dehydrated in graded ethanols, (LDH) > 600 U/L, or serum total bilirubin level > and embedded in araldite. Semi-thin 1µm-thick 1.2 mg/dL), decreased platelet count (< 100,000 sections were cut and stained with methylene cells/lL), and elevated liver enzymes [aspartate blue-azure II for light microscopic examination. aminotransferase (AST) > 70 U/L]. Thin sections of 70 nm were stained with lead cit - Gestational weeks of the women were deter - rate-uranyl acetate, and examined and pho - mined according to the last date of menstruation tographed under KARL Zeiss EVO LS10 STEM and/or ultrasonographic measurements. All pa - Electron microscope (Perbody, MA, USA). tients included in the study were monitored in the intensive care unit. In HELLP Group vital signs of Statistical Analysis all patients were monitored and magnesium thera - Statistical analyses were carried out using the py was initiated. Loading dose of magnesium (6 statistical package SPSS 15.0 for Windows g) was administered in 20 minutes, and mainte - (SPSS Inc., Chicago, IL, USA). The Mann-Whit - nance dose was given at a rate of 2 g per hour. ney U test was performed to compare data from On the first admission, variables such as blood control and HELLP patients. pressure, whole blood counts, ALT, AST, and LDH were evaluated. The decision to deliver was determined based on obstetric anamnesis, mater - Results nal and fetal status, and Bishop Scores. Vaginal delivery was preferred, however in the presence The characteristics of the patients studied are of unsuitable cervix, fetal distress, and in patients summarized in Table I. Histopathological com - with previous caesarian delivery, Cesarian sec - parison was made of placental morphology be - tion was performed. tween the two groups. Structural differences Immediately after delivery, normal and patho - were found between the normal placental termi - logical placentas were transported from the deliv - nal villi (Figure 1 a) and pathological forms of ery room to the laboratory and, after preliminary villi were observed in HELLP placenta. gross examination, two series of tissue samples Syncytial nodes and an increase in syncytial were obtained. The specimens were immersed in bridges, heavy bleeding foci were seen in the inter - 10% buffered formaldehyde, and 5 µm thick sec - villous space of the histopathological findings in tions were cut and made into slides. These were the HELLP group. Chorionic villous maturation, processed for Hematoxylen-Eosin and Masson’s vascular dilatation and progression of villous con - trichrome staining, carried out according to con - gestion were also increased in the connective tissue ventional procedures. (Figure 1 b,c). In another section obtained from the HELLP group, with the expansion of blood ves - Immunohistochemistry sels, fibrinoid necrosis becomes evident in the arte - The immunohistochemical detection of vimentin rial wall, and fibrous tissue was increased in stro - and desmin was performed as follows. After de - mal cells and connective tissue fibers. Mesenchy - paraffinization and antigen retrieval in a microwave mal cells are loosely arranged throughout the vil - oven, the endogenous peroxidase activity and the lous interior, and contain the intermediate filament non-specific antigen binding sites were blocked. (IF) proteins vimentin and desmin.

875 M.E. Sak, E. Deveci, A. Turgut, S. Sak, M, S. Evsen, T. Gul, S. Kalkanli

Table I. Characteristics of control and HELLP groups.

Controls HELLP Characteristic (n = 15) (n = 13) p value

Age (year) 28.0 ± 4.0 29.9 ± 2.5 NS Parity 1.8 ± 6.7 1.7 ± 7.2 NS Gestational age at delivery (weeks) 31.2 ± 2.2 30.1 ± 1.7 NS Systolic blood pressure at registration (mmHg) 117.0 ± 10.3 156.1 ± 12.6 < .001 Diastolic blood pressure at registration (mmHg) 70.3 ± 8.1 91.5 ± 13.4 < .001 Placental weight (g) 432.6 ± 48.4 380.0 ± 22.7 < .001

Endothelial damage has caused extravasation of desmin-positive cells in cases with HELLP syn - blood components and the formation of fibrin in drome. The HELLP group showed positive ex - chorionic vessel wall as a result of shrinkage due pression of vimentin around blood vessels, con - to degenerative changes in endothelial cells in pla - nective tissue, and stromal cells (Figure 1 d,e). centas with HELLP syndrome (Figure 1 b). Tuni - The immunohistochemical detection of IF ca intima injury and endothelial dysfunction has desmin demonstrated noticeable differences be - been observed in a majority of chorionic villi ves - tween normal and HELLP placentas villi (Figure sels. Increase of ıntravillous nonvascular connec - 1 f,g). Desmin was confined to the bodies and tive tissue fibers and desmin, vimentin cytokeratin cytoplasmic projections of stromal cells, which protein expression around blood vessels has been had a close relationship to arteries. In transverse concentrated at the location of the vessel wall sections, desmin-positive cells were apposed to damage in other sections of chorionic villi, in pla - the outer surface of arteries; their desmin-posi - centas with HELLP syndrome (Figure 1 d). tive cytoplasmic projections were spread to the Fusiform-shaped view showed degenerative subtrophoblastic villous area, where arteries were changes in endothelial cells and also thinning of mainly located. In sections from the HELLP the endothelial basement membrane increased dis - group, smooth muscle cells showed intense ex - tribution of blood cells and fibrin formation in ass - pression of desmin. The increased of fibrinoid esment of the electron microscope (Figure 2 c). necrosis in vascular wall and the periphery of vil - Large vessels of the chorionic villi and stem vil - li were observed in sections of the placentas with li showed thick walls consisting of vimentin, with patients of HELLP syndrome (Figure 1 b,c). In -

Figure 1. Control group H-E Bar 20 µm. 1b, HELLP group: Sincistial nodes and an increase in syncytial bridges H-E Bar 100 µm. 1c, HELLP group: Fibrinoid necrosis and a increase fibrous tissue (Trichrom-Masson Bar) 50 µm. 1d, HELLP group: Vimentin expression in stem villous (Brown-black) Bar 50 µm. 1e, HELLP group: Around blood vessels, connective tissue, increased expression of vimentin Bar 10 µm. 1f, HELLP group: Desmin expresion in choronic villous Bar 100 µm. 1g, HELLP group: Desmin positive expression in smooth muscle cells Bar 50 µm. 2a, Control group (Uranyl-acetate X6800). 2b, HELLP group: Syncytial degeneration, reduction of microvilli (Uranyl-acetate X8600). 2c, HELLP group: An increase in col - lagen and hypertrophy in pericyte cells vacuolation was seen in some places (Uranyl-acetate X6800).

876 Placental expression of vimentin, desmin and ultrastructural changes in HELLP syndrome creased expression of vimentin in the intravillous the fetal stem vessels in term placental villi they area and increased expression of desmin on were arranged as concentric layers with increas - blood vessel wall, supports the pathological find - ing stage of differentiation. A variable layer of ings by light microscopy in patients with HELLP extravascular stromal cells lying beneath the tro - syndrome (Figure 1 e,g). Ultrastructural exami - phoblast expressed vimentin or vimentin and nation, syncytial degeneration, and reduction of desmin. They were mitotically active. Significant microvilli vacuolation were seen in some places. expression of intermediate filament proteins, In Figure 2 a is healthy placenta and we observed desmin and vimentin, in the inter-villous area of an increase in the collagen component of villous connective tissue and vessel wall were shown stroma (Figure 2 b) in cases with HELLP syn - that cytokeratin mechanism may play an active drome . Degenerative changes of heterochroma - role in HELLP syndrome. Desmin and vimentin tine large nuclei in syncytial bridges as a result of thought to play a role in villous contractility and junction of the sincityotrofoblast cytoplasmic modulating by ımpact on maternal and fetal pla - cells and cisternal expansion with an increased cental circulation. Ultrastructurally, V cells re - vacuoles in endoplasmic reticulum were ob - sembled typical mesenchymal cells and VD cells served in ultrastructural section of the HELLP corresponded to fibroblasts 6. In our study we group placentas. Edema located in the sub syncy - showed positive and strongly increased im - tial area due to syncytial cells changes, and also munoreactivity of vimentin in vessel lumen and loss of the cristae in mitochondria of citotro - the stromal cells around the vessel wall. foblast cells were observed (Figure 2 b). Vinnars et al. showed that in addition to Another section from the HELLP group retroplacental hematoma, placental infarction showed collagen fibers, pericytes hypertrophy and decidual arteriopathy are also significantly and increase in villus (Figure 2 c). In addition, more frequent in preeclampsia and HELLP syn - there was thinning of the endothelial cell mem - drome 10 . Some researchers reported that desmin brane, which showed deterioration in places. occurs in different forms with the commence - Platelets was observed in blood vessels that ment of the development of decidua. This is due showed the electron dense (Figure 2 c). to increased synthesis of cell shape and or - ganelle distribution of activity associated with ra pid transformation 10-13 . In the present work we Discussion showed vimentin and desmin expression in the stromal blood vessels and connective tissue areas HELLP syndrome is a systemic obstetric com - of the region in the tunica media layer, combined plication and pathogenesis is not completely un - with increased mesenchymal cell density in pla - derstood. Fischer et al 8 investigated vascular re - centas with HELLP syndrome. activity in both PE and HELLP patients, and The basis of the pathophysiology of pre- showed that vascular resistance during reactive eclampsia and HELLP syndrome is vasospasm. hyperemia was increased in women with PE Hemolysis is one of the major features of compared with both the HELLP group and con - HELLP syndrome. Red blood cell fragmentation trol subjects. They concluded that vasodilatory caused by more blood flow formed along the reactivity is reduced in PE but not in HELLP damaged endothel, leads to tunica intima injury, syndrome, suggesting different pathogenetic endothelial dysfunction and the formation of fib - mechanisms in the two populations, and probably rin deposition. Vascular hemorrhage and fibrin a more pathological vascular development in PE deposition changes due to endothelial damage than in HELLP. Augmentation of cytoskeletons was observed significantly in HELLP syndrome in fetal capillary endothelial cells is one of the also in our study . Increased fibrin deposition and well known pathologic changes in toxemic pla - the distribution of the free form of the blood centae. There are few studies on these filaments, compounds in placental sections, supports the especially on their ontogeny. pathophysiological involvement of HELLP syn - Vimentin and desmin are intermediate fila - drome. Subendotelial fibrin storage takes place in ment proteins found in various mesenchymal and the vessel wall. This result was significantly ob - skeletal muscle cells, respectively 9. Different served by us. subpopulations of extravascular stromal cells Degenerative changes of heterochromatine were distinguished according to typical co-ex - large nuclei in syncytial bridges as a result of pression patterns of cytoskeletal proteins. Around junction of the sinsityotrofoblast cytoplasmic

877 M.E. Sak, E. Deveci, A. Turgut, S. Sak, M, S. Evsen, T. Gul, S. Kalkanli cells and cisternal expansion with an increased 2) BAXTER JK, W EINSTEIN L. Hellp syndrome: the state vacuoles in endoplasmic reticulum is defined as a of the art. Obstet Gynecol Surv 2004; 59: 838- sign of moving towards to cell apoptosis (Figure 845. 2 b). The degenerative change in the area of ın - 3) CURTIN WM, W EINSTEIN L. A review of HELLP syn - drome. J Perinatol 1999; 19: 138-143. travillous syncytial nodes, subsyncytial edema, 4) PIJNENBORG R, V ERCRUYSSE L, H ANSSENS M. The uter - increase of collagen fiber and loss of mitochon - ine spiral arteries in human pregnancy: facts and drial cristae in sitotrofoblast; induce apoptosis as controversies. Placenta 2006; 27: 939-958. well as significantly induced endothelial damage 5) REDMAN CW, S ARGENT IL . Latest advances in under - were observed. standing preeclampsia. Science 2005; 308: 1592- Increase of connective tissue fibers and protein 1594. expression of vimentin, desmin around blood ves - 6) KOHNEN G, K ERTSCHANSKA S, D EMIR R, K AUFMANN P. sels especially concentrate at the location of the Placental villous stroma as a model system for damage vessel wall for the repair of vessel wall myofibroblast differentiation. Histochem Cell Biol and the fluidity of the blood. This report can be 1996; 105: 6, 415-429. 7) LANDI B, T RANQUILLI AL . HELLP syndrome and pla - support by advanced techniques such as western cental inflammatory pathology. Minerva Ginecol blot, immune fluorescent assay in future studies. 2008; 60: 389-398. 8) FISCHER T, S CHNEIDER MP, S CHOBEL HP, H EUSSER K, L AN - GENFELD M, S CHMIEDER RE . Vascular reactivity in pa - Conclusions tients with preeclampsia and Hellp (hemolysis, el - evated liver enzymes, and low platelet count) syn - We observed that increased expression of vi - drome. Am J Obstet Gynecol 2000; 183: 1489- 1494. mentin in the intravillous area and increased ex - pression of desmin on blood vessel wall in pa - 9) ISE H, K OBAYASHI S, G OTO M, S ATO T, K AWAKUBO M, TAKAHASHI M, I KEDA U, A KAIKE T. Vimentin and tients with HELLP syndrome when compared to desmin possess GlcNAc-binding lectin-like prop - placentas of healthy pregnant. Augmentation of erties on cell surfaces. Glycobiology 2010; 20: intermediate filaments, desmin, vimentin may 843-864. disturb normal movements of endothelial cells, 10) VINNARS MT, W IJNAENDTS LC, W ESTGREN M, B OLTE and may display placental dysfunction that is un - AC, P APADOGIANNAKIS N, N ASIELL J. Severe able to compensate the endothelial instability and preeclampsia with, without HELLP differ with re - gard to placental pathology. Hypertension 2008; the related hypertension in HELLP syndrome. 51: 1295-1299. Further studies are needed to compare HELLP 11) ABRAHAMSON PA, Z ORN TM. Implantation and decid - syndrome with preeclampsia. ualization in Rodents. J Exp Zool 1993; 266: 603- 628. 12) GLASSER RS, J ULIAN J. Intermediate filament protein References as a marker of uterine stromal cell decidualiza - tion. Biol Reprod 1986; 35: 463-474. 1) SIBAI BM . The Hellp syndrome (hemolysis, elevat - 13) GLASSER RS, L AMPELO S, M UNIR IM, J ULIAN JA. Expres - ed liver enzymes, and low platelets): much ado sion of desmin, laminin and fibronectin during in about nothing? Am J Obstet Gynecol 1990; 162: situ differentiation (decidualization) of rat uterine 311-316. stromal cells. Differentiation 1987; 35: 132-142.

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