JRI Journal of Reproduction and Infertility 2228-5482 2251-676X Avicenna Research Institute JRI-13-131 Original Article RHAMM Expression in the Rat Endometrium during the Estrous Cycle and following Implantation Ozbilgin Kemal 1 * Boz Banu 1 Tuğyan Kazım 2 Inan Sevinç 1 Vatansever Seda 1 Department of Histology and Embryology, School of Medicine, Celal Bayar University, Manisa, Turkey Department of Histology and Embryology, School of Medicine, Dokuz Eylul University, Izmir, Turkey Corresponding Author: Kemal Ozbilgin, Department of Histology and Embryology, School of Medicine, Celal Bayar University, Manisa, Turkey. E-mail: kemalozbilgin@yahoo.com Jul-Sep 2012 13 3 131 137 15 01 2012 20 05 2012 Copyright © 2012 Avicenna Research Institute 2012

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Background

Receptor for hyaluronic acid mediated motility (RHAMM) has intracellular and extracellular functions. In this study, we focus on the expression of RHAMM in the rat uterus during estrous cycle and implantation period.

Methods

The female adult rats were divided into six groups following estrous cycle determination (n = 36). The utreri of rats were collected according to estrous cycle phases (menstruation group). For the implantation groups, uteri were obtained on D4, D5 and D6 (day of implantation) of pregnancy. The tissue samples were fixed and cut into 5 µm thick sections. RHAMM was investigated using immunohisto-chemical techniques and the intensity of RHAMM was evaluated by using the H-score technique. Comparisons between groups were performed using Kruskal-Wallis test.

Results

The RHAMM immunoreactivity of uterine antimesometrial epithelium (343.00±12.81), mesometrial subepithelium (285.00±27.26) and mesometrial stroma (270.00±36.00) were more prominent (p < 0.05) in the proestrus than estrus (275.00± 25.96; 220.00±14.48; 218.00±11.19) and diestrus (262.00±20.71; 192.50± 29.25; 216.00±12.97) groups, respectively. The most intense staining was seen in the epithelium on day four (275.50±30.06) and six (293.50±34.47) of pregnancy (p < 0.05). Strong RHAMM expressions were in both mature and predecidual cells on D5 (256.00±18.71), (247.50±22.14) and D6 (256.00±30.72), (265.00±14.87), respectively. RHAMM expression was prominent in the nondecidual region on D5 (270.00± 13.36).

Conclusion

Considering the role of RHAMM in cell proliferation, differentiation and angiogenesis, spatiotemporal expression of RHAMM in the uterus during estrous cycle and peri-implantation period is a means through which uterus becomes receptive for developing an embryo.

Estrous cycle Immunohistochemistry Implantation RHAMM Uterus

To cite this article: Ozbilgin K, Boz B, Tuğyan K, Inan S, Vatansever S. RHAMM Expression in the Rat Endometrium during the Estrous Cycle and following Implantation. J Reprod Infertil. 2012;13(3):131-137.

Introduction

Receptor for Hyaluronic Acid Mediated Motility (RHAMM, CD168) is a Hyaluronic acid (HA) receptor with many intracellular and extracellular functions (1). RHAMM regulates various cellular and dynamic processes, such as cell-to-cell adhesion, cell migration, morphogenesis, cell proliferation (including mitosis), cell signaling, regulation of gene expression, RNA splicing, cell differentiation, and metastasis (2). Savani et al. reported that anti–RHAMM anti-bodies block the migration of endothelial cells, which is an important key to the process of tissue injury and angiogenesis (3). It is known that over-expression of RHAMM correlates with an increase in the mitogen activated protein (MAP) kinase and progression of breast cancer (4), with the histological grade, invasion and metastasis of endometrial carcinoma (5), with adverse prognostic factors in colorectal cancer (6), and with gastric tumor progression (7).

Concerning reproductive tissues, several reports have described RHAMM-mediated promotion of cell growth and movement, sperm motility (8), angiogenesis (3) and embryonic development (9). Choudhary at al. showed, for the first time, that RHAMM is differentially expressed during all stages of preimplantation human embryos and human embryonic stem cells (hESC), and indicated that RHAMM knockdown results in down-regulation of several pluripotency markers in hESCs, induction of early extraembryonic lineage, loss of cell viability, and changes in hESC cycle (2).

The uterus undergoes extensive remodeling during estrous cycle and embryo implantation (10).

In estrous cycle and on day 4 of pregnancy, the rat endometrial stroma has two morphologically distinct compartments, denominated supepithelium and deep stroma. The superficial stroma underlying the luminal epithelium is formed by four or five layers of round shaped and compactly arranged endometrial fibroblasts. The deep stroma, situated between the superficial stroma and the myometrium, is composed of elongated and loosely arranged endometrial fibroblasts.

On days 5 and 6 of pregnancy, rat endometrial stroma has five compartments. The first two compartments (supepithelium and deep stroma) are similar to those of the estrous cycle. The third compartment is peridecidua which contains fibroblasts that are in the process of redifferentiating into decidual cells. The mature decidua layer is formed by fully transformed decidual cells. The last compartment, the non-decidua compartment, is a layer of nontransformed fibroblasts situated close to the myometrium.

During both these processes, mitosis, cell pro-liferation, differentiation and migration of cells have been observed in the endometrium (11). It is known that RHAMM plays an important role in several cellular events, but the role of RHAMM during estrous cycle and embryo implantation has not been investigated much. In this study we aimed to investigate whether RAHMM is expressed by uterine cells in estrous cycle and implantation days.

Methods

The study was approved by the Animal Ethical Committee of the faculty of Medical Medicine affiliated to Dokuz Eylül University and was conducted in accordance with the recommendations outlined in Guidelines for Care and Use of Experimental Animals. A total of 36 female adult Wistar albino rats with body weights ranging from 200 − 230 gr were subjected to a constant cycle of 12 hr of light and 12 hr of darkness. The animals were maintained at a constant temperature of 22 °C in the Experimental Animal Unit of the Faculty. Daily vaginal cytology specimens were collected and prepared to establish the estrous cycle of each animal. The vaginal smears were obtained by cotton-tipped applicators and fixed on a slide by 5% alcohol. The smears were stained by Giemsa stain. Following three or more successive normal estrous cycles, the animals were divided into six groups:

Group I (n = 6): Estrous group, proestrus; Group II (n = 6): Estrous group, estrus; Group III (n = 6): Estrous group, diestrus; Group IV (n = 6): Implantation group, day 4; Group V (n = 6): Implantation group, day 5; Group VI (n = 6): Implantation group, day 6.

The first three groups of animals (proestrus, estrus, and diestrus) were humanely hilled according to the estrous cycle. Later, the rate in the implantation group were mated with proven fertile male rats at the proestrus period. Mating was confirmed by the presence of sperm in the vaginal smears. The day of mating was termed the 0.5th day of pregnancy. Pregnancy was confirmed by the presence of leukocytes and mucus in the vaginal smear. The implantation sites were identified by intravenous injection of 1% Chicago blue (Sigma) in 0.85% sodium chloride. The animals were sacrificed on D4 to D6 of implantation. The uterine horns of all animals were placed in fixative and were then cut along the antimesometrial border to expose their endometrial lining. Paraffin blocks of the tissue were cut in 5 µm sections and collected on poly-L-lysine coated slides (Sigma, St. Louis, MO, USA). Tissue sections were deparaffinized in xylene and rehydrated in a decreasing graded series of ethanol. For antigen retrieval, sections were boiled in a microwave oven in citrate buffer (10 mM, pH = 6.0) for 10 min and left to cool for 20 min. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol for 20 min. The sections were incubated with primary antibody as monoclonal rabbit anti-RHAMM (Boster Bio-tecnology, China) in a humidified chamber at room temperature for 60 min. The antigen–antibody complex was detected by using a biotin-labeled secondary antibody (Bulk Kit, Invitrogen Corp., Camarillo, CA, USA) and a streptavidin–peroxidase complex (LabVision), respectively, for 20 min. Each step was followed by three washes in phosphate buffered saline (PBS, pH = 7.4) unless otherwise stated. The resulting signal was developed by diaminobenzidine (DAB), (Spring Biosciene, Fremont, CA, USA). Sections were counterstained by Mayer's hematoxilen (Richord-Allan Scientific, CA, USA) and finally mounted in Entellan. Two histologists who had no knowledge of the groups examined all the immunostained sections. The proportion of epithetlial, subepithelial, predecidual, mature decidual and non-decidual cells in each selected field was determined.

Two randomly selected areas were scored, and in sections where all the staining appeared intense, one random field was chosen. The proportion of epithelial, subepithelial, predecidual, mature decidual and non-decidual cells in each selected field was determined by counting them at a high magnification. At least 100 cells were scored per X40 field for each animal in all the groups. All sections were scored in a semiquantitative fashion, by considering both the intensity and percentage of cell staining. Intensities were classified as 0 (no staining), +1 (weak staining), +2 (distinct staining) and +3 (very strong staining). The staining of RHAMM was graded semiquantitatively and the H-score was calculated using the following equation: H-score = ΣPi (i + 1), where i = intensity of staining with a value of 1, 2 or 3 (weak, moderate or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100.

Statistical analysis

The data were summarized as median±Range. Comparisons between all groups were made using Kruskal-Wallis test. The p < 0.05 was considered significant. The statistical analysis was performed by SPSS, version 10 for Windows.

Results

Immunohistochemically, anti-RHAMM antibody positivity was seen in the membranous region of uterine cells. Concentration of RHAMM peaked (343.00±12.81) in the uterine antimezometrial epithelium of rats in the proestrus group compared with mezometrial region (275.00±27.89), and estrus (275.00±25.96) and diestrus groups (262.00± 20.71), (Table 1). RHAMM expression in the sub-epithelium of the proestrus group (285.00±27.26) was much stronger than estrus (220.00±14.48) and diestrus groups (192.50±29.25), (Table 1). Moreover, RHAMM immunoreactivity was very high in the stroma during proestrus (270.50±36.00) compared with the estrus (218.00±11.19) and diestreous groups (216.00±12.97), (Table 1 and Figure 1).

Immunohistochemistry of RHAMM in endometrium of Estrous groups: Expressions are strong in the epithetlial cells (E) of antimesometrial region, as well as sub-epithelial cells (SE) and deep stromal cells (DS) in the proestrus group (1a). Moderate RHAMM expressions are seen in the epithelium (E), subepithelium (SE) and deep stroma (DS) in both estrus (1b) and diestrus groups (1c). Negative control without primary antibody (1d). M: Myometrium; Me: Mesometrium. Original magnification 100×, insert 400×

H-score values of RHAMM immunoreactivities in different compartment of endometrium at estrous groups

RHAMM Proestrus Estrus Diestrus

Mesometrial Antimesometrial Mesometrial Antimesometrial Mesometrial Antimesometrial
Epithelium 275.00±27.89 343.00±12.81* 268.00±29.29 275.00±25.96 257.50±16.21 262.00±20.71
Subepithelium 285.00±27.26 * 255.00±32.04 220.00±14.48 278.00±17.31 192.50±29.25 268.00±17.17
Deep stroma 270.00±36.00 * 282.00±27.53 218.00±11.19 226.50±9.22 216.00±12.97 264.50±32.76

Values are medians±range of three independent experiments analysed by Kruskal-Wallis test compared with proestrus, estrus and diestrus.

p < 0.05

The most intense immunoreactivity of RHAMM was in the epithelium on D4 (275.50±30.06) and D6 (293.50±34.47) of pregnancy compared with D5 (243.33±17.04), (Table 2). Although there was no statistical difference in RHAMM expression of the subepithelial and deep stroma on D4, D5 or D6 but decidual region was different. RHAMM expressions were strong in both mature decidua and predecidua cells on D5 (256.00±18.71 and 247.00±22.14, respectively) and on D6 (256.00± 30.72 and 265.00±14.87, respectively), (Table 2 and Figure 2).

Immunohistochemistry of RHAMM in endometrium of implantation groups: RHAMM expression of epithelial cells (E) are strong in mesometrium on D4 (2a) and D6 (2c) compared with antimesometrium. There are no differences in RHAMM expression in the subepithelium (SE) among the implantation groups. Strong RHAMM expressions are seen in the mesometrial part of predecidual (PD) and mature decidual (MD) regions on D5 (2b) and D6 compared with the antimesometrial region. In the non-decidual regions (ND), RHAMM expressions are strong in the mesometrium on D4 and D5. Negative control without primary antibody (2d). M: Myometrium; Me: Mesometrium. Original magnification 100×, insert 400×

H-score values of RHAMM immunoreactivities in different compartments of endometrium on day 4 of implantation groups

Day 4 Day 5 Day 6

Mesometrial Antimesometrial Mesometrial Antimesometrial Mesometrial Antimesometrial
Epithelium
 Implantation 275.50±30.06 * 235.00±20.33 243.00±17.04 268.50±37.08 293.50±34.47 * 252.50±12.14
 Interimplantation 215.00±22.48 243.00±44.74 252.50±33.56 276.00±18.64 189.00±23.23 214.50±17.87
Subepitelium
 Implantation 217.50±5.19 223.00±30.43 224.00±69.25 207.50±10.18 264.50±70.14 223.00±9.93
 Interimplantation 222.50±21.65 226.00±21.68 259.00±9.50 254.00±9.57 238.00±3.08 195.00±48.17
Deep stroma
 Implantation 210.00±31.17 202.00±49.54 217.50±38.20 231.50±27.06 209.00±28.65 201.00±18.06
 Interimplantation 250.00±9.07 211.00±11.77 262.00±19.46 245.50±37.43 238.00±10.78 223.50±23.18
Predecidua
 Implantation -- -- 256.00±18.71 * 220.00±11.64 256.00±30.72 * 250.50±13.36
 Interimplantation -- -- 248.50±11.72 198.50±5.55 260.50±11.51 233.50±8.41
Mature deciduas
 Implantation -- -- 247.50±22.14 * 232.50±23.70 265.00±14.87 * 207.00±14.61
 Interimplantation -- -- 273.50±29.47 252.50±29.60 258.00±17.42 247.50±19.51
Non-decidual cells
 Implantation -- -- 270.00±13.36 * 248.00±12.37 211.00±10.67 219.00±8.91
 Interimplantation -- -- 255.50±14.67 258.50±12.29 258.00±18.69 241.00±17.04

Values are medians±range of three independent experiments analysed by Kruskal-Wallis test compared on days 4, 5 and 6.

p < 0.05

Discussion

RHAMM stimulates cell migration and locomotion via activation of a signal transduction cascade upon HA binding (2). This article demonstrates, for the first time, that RHAMM undergoes substantial variation within the estrous cycle and during implantation days of rat endometrium. Peaks of RHAMM expression coincide both phases of profound cellular proliferation of estrous cycle and embryo implantation in rat, where RHAMM may contribute endometrial renewal and embryo implantation.

It is well known that the functionalis layer of endometrium sloughs off during menses and parturition, and the putative endometrial stem cells residing in the basal layer regenerate the functionalis layer (12). The basal layer is believed to behave as a germinal compartment from which various types of endometrial cells proliferate and differentiate. We observed that the RHAMM immunoreactivity of uterine epithelial cells and sub-epithelial cells increased in proestrus compared with estrus and diestrus cycles (Table 1). It is known that uterine epithelium undergoes renewal during the proestrus of estrous cycle (11) and we hypothesized that the increases in RHAMM immunoreactivity in the epithelium and subepithelium may be related to mitosis of the uterine stem cells which are found in both the epithelial and stromal compartments of the endometrium (13).

It is known that RHAMM is highly expressed in the G2/M phase of the cell cycle, thus, controlling mitosis (14). RHAMM is a centrosomal protein that localizes to interphase microtubules, spindle poles, the anaphase midbody and the telophase midzone microtubules. RHAMM contains a centrosome targeting carboxy-terminal basic leucine zipper and, like Xklp2, interacts with the dynein motor complex (14). Overexpression of RHAMM was also detected in high mitotic index in malignancies (4).

Because of the fundamental role for RHAMM in mediating and HA-induced cell migratory response, the strong RHAMM immunoreactivity in both uterine epithelium and subepithelium of the proestrus cycle may be related to stem cell motility. We thought that RHAMM played an important role in stem cell motility of the subepithelium toward epithelium and stroma. RHAMM is believed to increase cellular motility through direct interaction with the cytoskeleton and may have a role in the separation and migration of daughter cells following mitosis (15). It has been reported that certain anti-RHAMM antibodies block the HA-dependent migration of a variety of cell types (16).

HA is a naturally existing molecule in the female reproductive tract. It is present in the human endometrium (17) and its concentrations have been shown to increase dramatically on the day of implantation in mice (18). One of the main signaling receptors for HA is RHAMM (1) which regulates various cellular and dynamic processes, such as cell-to-cell adhesions. Although there were no differences in antimesometrial epithelia for RHAMM immunoreactivity among the implantation days, the mesometrial RHAMM immunoreactivity increased in the implantation group, especially on days 4 and 6. We thought that RHAMM may be involved in the adhesion of embryo during implantation. Several studies have been performed in order to evaluate the ability of HA in promoting embryo implantation. Although the major biological functions of HA are still unknown, various mechanisms can be proposed for its beneficial effect on implantation. However, the results are still conflicting. Considering the presence of HA increases during implantation days, HA improves embryo implantation; these effects may be mediated by RHAMM, as well as CD44, its major cell surface receptor (19).

Decidualization is the transformation from small spindle-shape cells to large plump decidual cells, which is essential for normal implantation of blastocyst. It is known that rodent decidualization occurs after normal entrance into pregnancy following cervical stimulation and insemination on about day 4.5; it does not begin naturally during the estrous cycle (20). Initially, decidualization occurs in several cell layers of the endometrial stroma immediately adjacent to the implanting conceptus. This area is known as the primary decidual zone and is located adjacent to the anti-mesometrial chamber of the uterine lumen that surrounds the conceptus. In this study, we observed that RHAMM immunoreactivity increased on D4 and D5 in non-decidual, cells and on D5 to D6 in mature and predecidual cells. The increases of RHAMM in non-decidual area during decidualization period may be related to the start of decidualization and also the increases in pre- and mature decidual areas related to RHAMM.

RHAMM mRNA and protein are poorly expressed in most normal human tissues (21) but RHAMM expression increases in pathologic conditions such as wound repair in response to hypoxia and fibrogenic factors (TGFβ1) (22). Tong et al. reported that in wound repair RHAMM acts as a fibrogenic factor required for temporal and spatial regulation of granulation tissue formation and resolution. An underlying signaling defect associated with Rh−/− wounds is deregulated ERK1, 2 activation, which promotes fibroblast migration, as well as mesenchymal cell differentiation (23).

An angiogenic network is also formed in the uterine stromal bed, critically supporting the early development of the embryo. Endothelial cells which are in close proximity to decidual cells pro-liferate to form a new dense vascular network in pregnant uterus (24). Decidual angiogenesis and maintenance of vasculature in the early postimplantation period is an absolute requirement for normal pregnancy development. VEGF/VEGFR-2 pathway is a key regulator of decidual angiogenesis (25). We observed that RHAMM expression increased in stromal cells during D5 and D6, and we thought that RHAMM also had a function during angiogenesis. Matou-Nasri S et al. reported, for the first time, that CD44 and RHAMM were both involved in oligosaccharides of hyaluronan-induced endothelial tube formation in Matrigel, mediating distinct angiogenic signaling pathway (26). In addition, several other studies have demonstrated an association between HA and tumor vascularization. In some invasive tumors, revascularization occurrs adjacent to a region of desmoplasia rich in HA. HA concentrations increased dramatically in the ECM of human breast tissue during carcinoma infiltration (27).

In conclusion, RHAMM may have an important role in uterus both estrous cycle, and invasion and implantation period via promotes cell proliferation, differentiation and angiogenesis.

Conclusion

Considering the role of RHAMM in cell proliferation, differentiation and angiogenesis, it seems that spatiotemporal expression of RHAMM in the uterus during estrous cycle and peri-implantation period is a means through which uterus becomes receptive for developing an embryo.

Acknowledgement

The study was supported by Celal Bayar University Scientific Research Projects Commission (FEF: 2008/111).

Conflict of Interest

Authors declare no conflict of interest.

References Hardwick C Hoare K Owens R Hohn HP Hook M Moore D Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility J Cell Biol. 1992 117 6 1343 50 Choudhary M Zhang X Stojkovic P Hyslop L Anyfantis G Herbert M Putative role of hya-luronan and its related genes, HAS2 and RHAMM, in human early preimplantation embryogenesis and embryonic stem cell characterization Stem Cells. 2007 25 12 3045 57 Savani RC Cao G Pooler PM Zaman A Zhou Z DeLisser HM Differential involvement of the hya-luronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis J Biol Chem. 2001 276 39 36770 8 Wang C Thor AD Moore DH 2nd Zhao Y Kerschmann R Stern R The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression Clin Cancer Res. 1998 4 3 567 76 Rein DT Roehrig K Schöndorf T Lazar A Fleisch M Niederacher D Expression of the hyaluronan receptor RHAMM in endometrial carcinomas suggests a role in tumour progression and metastasis J Cancer Res Clin Oncol. 2003 129 3 161 4 Lugli A Zlobec I Günthert U Minoo P Baker K Tornillo L Overexpression of the receptor for hyaluronic acid mediated motility is an independent adverse prognostic factor in colorectal cancer Mod Pathol. 2006 19 10 1302 9 Li H Guo L Li JW Liu N Qi R Liu J Expression of hyaluronan receptors CD44 and RHAMM in stomach cancers: relevance with tumor progression Int J Oncol. 2000 17 5 927 32 Kornovski BS McCoshen J Kredentser J Turley E The regulation of sperm motility by a novel hyaluronan receptor Fertil Steril. 1994 61 5 935 40 Stojkovic M Krebs O Kölle S Prelle K Assmann V Zakhartchenko V Developmental regulation of hyaluronan-binding protein (RHAMM/IHABP) expression in early bovine embryos Biol Reprod. 2003 68 1 60 6 Li S Davis B Evaluating rodent vaginal and uterine histology in toxicity studies Birth Defects Res B Dev Reprod Toxicol. 2007 80 3 246 52 Westwood FR The female rat reproductive cycle: a practical histological guide to staging Toxicol Pathol. 2008 36 3 375 84 Maruyama T Masuda H Ono M Kajitani T Yoshimura Y Human uterine stem/progenitor cells: their possible role in uterine physiology and pathology Reproduction. 2010 140 1 11 22 Taylor HS Endometrial cells derived from donor stem cells in bone marrow transplant recipients JAMA. 2004 292 1 81 5 Maxwell CA Keats JJ Crainie M Sun X Yen T Shibuya E RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability Mol Biol Cell. 2003 14 6 2262 76 Assmann V Jenkinson D Marshall JF Hart IR The intracellular hyaluronan receptor RHAMM/ IHABP interacts with microtubules and actin filaments J Cell Sci. 1999 112 Pt 22 3943 54 Turley EA Austen L Moore D Hoare K Ras-transformed cells express both CD44 and RHAMM hyaluronan receptors: only RHAMM is essential for hyaluronan-promoted locomotion Exp Cell Res. 1993 207 2 277 82 Salamonsen LA Shuster S Stern R Distribution of hyaluronan in human endometrium across the menstrual cycle. Implications for implantation and menstruation Cell Tissue Res. 2001 306 2 335 40 Carson DD Dutt A Tang JP Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function Dev Biol. 1987 120 1 228 35 Teixeira Gomes RC Verna C Nader HB dos Santos Simões R Dreyfuss JL Martins JR Concentration and distribution of hyaluronic acid in mouse uterus throughout the estrous cycle Fertil Steril. 2009 92 2 785 92 Gellersen B Brosens IA Brosens JJ Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives Semin Reprod Med. 2007 25 6 445 53 Evanko SP Tammi MI Tammi RH Wight TN Hyaluronan-dependent pericellular matrix Adv Drug Deliv Rev. 2007 59 13 1351 65 Samuel SK Hurta RA Spearman MA Wright JA Turley EA Greenberg AH TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan J Cell Biol. 1993 123 3 749 58 Tolg C Hamilton SR Nakrieko KA Kooshesh F Walton P McCarthy JB Rhamm-/- fibro-blasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair J Cell Biol. 2006 175 6 1017 28 Chakraborty I Das SK Dey SK Differential expression of vascular endothelial growth factor and its receptor mRNAs in the mouse uterus around the time of implantation J Endocrinol. 1995 147 2 339 52 Douglas NC Tang H Gomez R Pytowski B Hicklin DJ Sauer CM Vascular endothelial growth factor receptor 2 (VEGFR-2) functions to promote uterine decidual angiogenesis during early pregnancy in the mouse Endocrinology. 2009 150 8 3845 54 Matou-Nasri S Gaffney J Kumar S Slevin M Oligosaccharides of hyaluronan induce angiogenesis through distinct CD44 and RHAMM-mediated signalling pathways involving Cdc2 and gamma-adducin Int J Oncol. 2009 35 4 761 73 Losa GA Alini M Sulfated proteoglycans in the extracellular matrix of human breast tissues with infiltrating carcinoma Int J Cancer. 1993 54 4 552 7