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,1
Departments of
* Oncology and
Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
Department of Nephrology, Rabin Medical Center, Petach-Tikva, Israel;
Department of Medical Biochemistry, University of Uppsala, The Biomedical Center, Uppsala, Sweden; and
|| Cancer and Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel
2Correspondence: Cancer and Vascular Biology Research Center, Faculty of Medicine, Technion, Haifa 31096, Israel. E-mail: vlodavsk{at}cc.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transgenic mice • mammary glands • hair growth • implantation • heparan sulfate • angiogenesis • body weight
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) chains are covalently O-linked (1
, 2)
. The ability of HSPGs to interact with various extracellular matrix (ECM) macromolecules and with different attachment sites on the cell membrane suggests a key role for this proteoglycan in the structural integrity, self-assembly, insolubility, and permeaselective properties of the basement membrane (BM) and ECM, as well as in cell adhesion and locomotion (1
2
3
4
5
6)
. Moreover, the HS chains, unique in their ability to bind a multitude of proteins, ensure that a wide variety of bioactive molecules bind to the cell surface and ECM and thereby function in the control of diverse normal and pathological processes (1
2
3
4
5
6
7
8)
. HSPGs are clearly the most common low-affinity receptors that function in the reception and modulation of various growth factors, morphogenes, and chemokines (1
, 7
, 8)
.
Although the majority of studies of cell interaction with the microenvironment focus on proteolytic enzymes (9
, 10)
, the involvement of glycosaminoglycan (e.g., heparan sulfate) -degrading enzymes (e.g., heparanase) has been underestimated, primarily due to a lack of appropriate molecular probes to explore their causative role in cell-ECM interactions and related effects. Long-term research on the biology of the heparanase enzyme led to the cloning of a human gene encoding HS-degrading endoglycosidase (heparanase) (11
12
13
14)
. The enzyme appears to play important roles in biological processes such as inflammation, vascularization, and cancer metastasis (14
15
16
17)
. Cloning of the same gene by several groups suggests that mammalian cells express primarily a single dominant functional heparanase, making it a promising target for basic and applied research on cell interaction with the microenvironment and for potential therapeutic applications in diverse disorders involving abnormal cell migration, invasion, and tissue remodeling (14
, 15)
.
The HS chains are cleaved by heparanase at only a few sites, resulting in HS fragments of appreciable size and biological activities (18
, 19)
. Heparanase is synthesized as a 65 kDa protein. The mature active 50 kDa enzyme, isolated from cells and tissues, has its amino terminus 157 amino acids downstream from the initiation codon, indicating post-translational proteolytic processing of a latent heparanase polypeptide (11
12
13
, 20)
. Heparanase has been identified in invasive normal and malignant cells, including cytotrophoblasts, keratinocytes, activated cells of the immune system, lymphoma, melanoma, and carcinoma cells (14
, 15
, 17
, 21)
. In a recent study, we demonstrated that the proangiogenic and prometastatic properties of heparanase are tightly regulated by its cellular localization and secretion (22)
. Moreover, cell surface expression of the enzyme was found to elicit a firm cell adhesion independent of its enzymatic activity (23)
, suggesting a possible involvement of heparanase in cell-ECM interaction, signal transduction, and cell survival.
Mice overexpressing HSPGs were generated and shown to exhibit altered feeding behavior (24)
and vascularization properties (25)
. On the other hand, mice homozygous for mutations in HS biosynthetic enzymes either die in the early neonatal period (26
, 27)
or exhibit bilateral renal agenesis together with milder phenotypic changes in their cytoskeleton and nervous system (28
, 29)
. To further elucidate the functional biological significance of HS and heparanase, we have generated and characterized homozygous transgenic mice overexpressing mammalian heparanase. To identify a broad spectrum of phenotypes, we applied the actin promoter to drive overexpression of the heparanase gene in all tissues. Western blot analysis, immunostaining, and measurements of heparanase enzymatic activity revealed expression of the heparanase protein in most tissues of the hpa-tg mice. Here, we describe characteristic features of the observed phenotypes, emphasizing the involvement of heparanase and HS in embryonic implantation, tissue morphogenesis, vascularization, and feeding behavior.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was modified to contain a unique EcoRI site at position 1719. An XbaI-EcoRI 1.7 kb fragment containing the entire open reading frame of heparanase was cloned into the compatible sites of the vector. Before injection, the plasmid pCAGGS-hpa was digested with SalI and PstI to isolate the expression cassette and eliminate bacterial DNA sequences. The resulting fragment contained the CMV-IE enhancer, chicken ß-actin promoter, and hpa cDNA, followed by a rabbit ß-globin poly adenylation site (Fig. 1
A). The DNA fragment containing the hpa expression cassette was injected into fertilized eggs derived from C57BL x Balb/c breed. Mice developed from the injected blastocytes were tested for the presence of the human hpa transgene in their genome.
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Genotyping of transgenic mice
Genomic DNA was extracted from tail tips of the mice and the human hpa transgene sequence was amplified using human hpa specific PCR primers. Tail fragments were incubated overnight at 55°C in lysis buffer (8 M urea, 0.2 M Tris-HCl, 0.4 M NaCl, 20 mM EDTA, 1% N-laurylsarcosine, 10 µg/mL proteinase K) (31)
. The dissolved tissue underwent phenol extraction and ethanol precipitation to obtain highly purified genomic DNA.
Integration of the human heparanase cDNA in the mouse genome was verified by PCR using two sets of primers. The first set was designed to amplify the 5' region of the transgene. It included a ß-actin promoter specific primer (5'-ATAGGCAGCTGACCTGA-3'; designated 5'-pCAGGs) and human hpa specific primer (5'-TGACTTGAGATTGCCAGTAACTTC-3'; designated Hpa-300). The second set of primers was designed to amplify the 3' region of the transgene and included a human hpa specific primer (5'-CTGTCCAACTCAATGGTCTAACTC-3'; designated Hpa-830) and a primer specific to the plasmid derived 3'-untranslated region (5'-TCTAGAGCCTCTGCTAACCA-3'; designated 3'-pCAGGS). PCR conditions were: 2 min at 95°C, followed by 33 cycles of 15 s at 95°C, 1 min at 58°C and 1 min at 72°C.
Analysis of heparan sulfate
Newborn control and hpa-tg mice were injected subcutaneously with Na2[35S]O4 (Amersham Bioscience, Buckinghamshire, UK; 50 µCi/2 µL) and incubated for 2 h. The mice were killed and the whole body was homogenized in Tris-HCl buffer (50 mM, pH 7.4) containing 4 M urea and 1% (v/w) Triton X-100. Homogenates were incubated at 4°C overnight and the supernatant was applied on a 1 mL DEAE-Sephacel column equilibrated with extraction buffer. The column was eluted with 50 mM acetic acid, pH 4, containing 1.5 M NaCl and 4 M urea, and the released material was desalted on a PD-10 column in 10% ethanol. The radiolabeled material was treated (overnight, 37°C) with chondroitinase ABC (1 U/mL) in a total volume of 200 µL, followed by treatment with 0.5 M NaOH for 16 h at 4°C. The free HS chains were recovered by purification on 1 mL DEAE-Sephacel column and analyzed by gel filtration on a Superose-12 column in Tris-HCl buffer (50 mM, pH 7.4) containing 0.1% Triton X-100 and 0.5 M NaCl (32)
. Size-defined standards of hyaluronan (120 kDa) and heparin (3, 13 and 35 kDa) were kindly provided by Dr. P. Heldin and Dr. I. Bjork (University of Uppsala, Uppsala, Sweden), respectively.
Preparation of ECM coated dishes
Bovine corneal endothelial cells were established and cultured as described (33)
. Subendothelial ECM was exposed by dissolving the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH4OH, followed by four washes with PBS (33)
. The ECM remained intact, free of cellular debris and firmly attached to the entire area of the tissue culture dish (33)
. For preparation of sulfate-labeled ECM, corneal endothelial cells were cultured in the presence of Na2[35S]O4 (Amersham Bioscience) added (25 µCi/mL) on days 1 and 5 after seeding (11
, 33)
. Seven to 10 days later, the cell monolayer was dissolved and the ECM exposed, as described (11
, 33)
.
Heparanase activity
Fresh tissues derived from hpa-tg and control mice were homogenized in buffer containing 10 mM phosphate-citrate, pH 6.0, 150 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, and 0.5% Nonidet P-40. The supernatant fractions were analyzed for heparanase activity, as described (11
, 22
, 34)
. Briefly, samples containing equal amount of protein were incubated (16 h, 37°C) on sulfate-labeled ECM coated 35 mm dishes in 1 mL of heparanase reaction buffer (20 mM phosphate-citrate, pH 5.8, 1 mM dithiothreitol, 1 mM CaCl2, 50 mM NaCl). The incubation medium containing sulfate-labeled degradation fragments released from ECM was subjected to gel filtration on a Sepharose CL-6B column. Intact HSPGs were eluted just after the void volume (Kav<0.2, fractions 1-10); HS degradation fragments were eluted toward the Vt of the column (0.5<Kav<0.8, fractions 15-35) (11
, 22
, 34)
.
Antibodies
Monoclonal mouse anti-human heparanase antibodies (mAb 130) directed against the carboxyl terminus of the 50 kDa active enzyme were produced as described previously (11)
. These antibodies do not recognize the mouse heparanase. Polyclonal antibodies (Ab-p9), recognizing both human and mouse enzymes were directed against a synthetic peptide corresponding to residues 335-353 of human heparanase. Both antibodies were kindly provided by InSight Ltd. (Rehovot, Israel).
Western blot analysis
For immunoblot analysis, aliquots of tissue extracts were mixed with heparin-Sepharose Fast Flow beads (Amersham Biosciences). The beads were then washed with PBS (x3) and boiled in Laemmli buffer. Proteins were separated by electrophoresis in 10% SDS-polyacrylamide gel (PAGE) and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA). Heparanase was detected by anti-human heparanase monoclonal antibodies (mAb130) (11)
, followed by HRP-conjugated anti-mouse antibodies (Jackson Laboratories, Bar Harbor, ME, USA) and enhanced chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions (11
, 35)
. Fuji medical X-ray film Super RX was used.
Histology
Tissue samples were fixed with 4% formaldehyde in PBS, embedded in paraffin, and sectioned (5 µm sections). After deparaffinization and rehydration, sections were washed (3x) with PBS and stained with hematoxyline/eosine or Mason-Trichrom, as described (22
, 36)
. Tissue sections were then washed, mounted with 90% glycerol in PBS, and visualized with a Zeiss axioscope microscope.
Immunohistochemistry
Immunohistochemistry was performed as described with minor modifications (11
, 36)
. Briefly, 5 µm sections were deparaffinized and rehydrated. The tissue was then denatured for 3 min in a microwave oven in citrate buffer (0.01 M, pH 6.0). Blocking steps included successive incubations in 3% H2O2 in methanol and 5% goat serum. Tissue sections were incubated with anti-human heparanase antibodies (mAb 130; Ab-p9) or DMEM supplemented with 3.3% horse serum as control, followed by incubation with the respective HRP conjugated goat anti-mouse or goat anti-rabbit antibodies (Jackson Laboratories). Color was developed using Zymed AEC substrate kit (Zymed, South San-Francisco, CA, USA) for 10 min, followed by counterstaining with Mayer’s hematoxylin (11
, 36)
. Vascular endothelial cells were stained with anti-Von Willibrand polyclonal antibodies (DAKO, Glostrup, Denmark), as described (37)
.
Electron microscopy
Kidneys were placed on ice and slices were fixed in 0.5% glutaraldehyde/4% formaldehyde in PBS, pH 7.4. Tissue blocks (1 mm3) were postfixed with 1% OsO4 in Veronal-acetate buffer, pH 7.4, for 1 h at 4°C, dehydrated in ethanol and propylene oxide, and embedded in araldite (Polysciences, Washington, PA, USA). Ultrathin araldite sections were mounted on naked 400 mesh grids, stained with uranyl acetate and lead citrate, and coated with carbon. Examination of sections was carried out using a JEOL-100B electron microscope at 80 KV (38)
.
Embryo implantation
Hpa-tg and control female mice (8-wk-old, 15 mice/group) were caged with males overnight and checked for the presence of vaginal plugs the following day. The day on which the plug was found was designated as the first day of pregnancy. Seven days later, the female mice were killed and their uteri removed and analyzed for the number of implanted embryos.
Mammary gland whole mounts
Whole-mount mammary glands, prepared as described (39)
, were fixed in Tellys fixative (100 mL EtOH 70%, 5 mL Formalin, 5 mL glacial acetic acid), rehydrated, and stained with hematoxylin for 3 h. After staining, the glands were washed in tap water (1 h), dehydrated, and stored in methyl salicylate. Microvessel density was determined in areas with the highest vascularization. Individual vessels were counted by a blinded observer on 400x microscopic field (0.196 mm2). A total of 12 fields/mammary gland (4
sections x 3 fields) were analyzed and the mean value ± SD was determined, as described (22)
.
Hair growth
Backskin hair of 2-month-old hpa-tg and control female mice (10 mice/group) at the end of the first hair cycle was clipped with electric clipper. Hair growth rate was examined every other day for 25 days. Heparanase expression (immunostaining) and vascular density were evaluated in skin samples taken from 8 and 16 day postnatal hpa-tg and control mice.
Biochemical analysis
Urine and blood samples were collected from hpa-tg and control mice and subjected to biochemical analysis. Urine samples were tested for total protein and creatinine content, applying the automated Kodak 250 system. Blood samples were examined for creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) content, using an automated Kodak 950 system (40)
.
Cell surface binding of bFGF
Fibroblasts were isolated from embryos of hpa-tg and control mice, 15 days postgestation. Cells were cultured in DMEM/RPMI/F-12 (1:1:1) medium supplemented with 10% FCS. Confluent cells were incubated (1 h, 4°C) with increasing concentrations (1–10 ng/mL) of 125I-bFGF (2.3 µCi/ng, Amersham Bioscience) in triplicate 35 mm dishes in 1 mL serum-free RPMI medium containing 0.5% BSA. The cells were washed (x3), trypsinized, and the radioactivity of the cell pellets was counted in a 
-counter (41)
.
Statistics
Student’s t test was used for statistical analysis of the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was microinjected to fertilized eggs of C57BL/6 x Balb/c origin to produce transgenic mice overexpressing the hpa cDNA. Forty-two pups were born and 4 positive human heparanase founder transgenic mice were identified by tail tip genotyping. Each of these founder mice bears a different copy number of the human hpa cDNA in their genome, as revealed by a semiquantitative PCR reaction specific for the human hpa cDNA (Fig. 1B
). Founder mice were mated with C57BL/6 mice to create F1 mice and those were mated among themselves to create F2 mice. Homozygous F2 mice from each of the four founder lines termed G1–G4 were identified by Southern blot analysis and quantitative PCR (data not shown). Homozygosity was verified by mating with wild-type C57BL/6 mice, where all the pups were expected to be heterozygous. F2 negative mice from the same littermate served as controls in all the experiments.
Heparanase expression pattern and activity
Expression of the heparanase protein in the four founder lines was verified by Western blot analysis of tissues derived from F2 homozygous hpa-tg mice. Homogenized tissue samples, partially purified on heparin-Sepharose, were subjected to 10% SDS-PAGE. After protein transfer to Immobilon P membrane, heparanase was identified with mAb130 specifically directed against the human enzyme. Figure 2
A demonstrates human heparanase expression in various tissues of G1 mice. The processed 50 kDa human enzyme was overexpressed in all tissues examined. Line G3 expressed the human heparanase in all tissues examined, but to a lesser extent than G1 (not shown). Line G2 revealed an expression pattern similar to G3, except that heparanase was not detected in the kidney whereas line G4 expressed the human enzyme mainly in the heart and lung, with little or no expression in other tissues (not shown). All tissues expressed the 50 kDa processed enzyme, suggesting a proper processing of the full-length proenzyme. In some tissues (i.e., gut, heart), both latent (65 kDa) and active (50 kDa) forms of the enzyme were detected (Fig. 2A
). On the basis of Western blot analysis, the G1 line expressing the highest amount of heparanase was chosen as the preferred line for further investigation. The results were, however, confirmed with G3 mice to rule out a positional effect.
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Immunostaining of the hpa-tg tissues with mAb 130 (Fig. 2B
) confirmed the expression pattern revealed by the Western blot analysis. The human heparanase was mostly localized in cytoplasmic granules. Sections of liver (Fig. 2B
, top) and colon (Fig. 2B
, bottom) are shown, but a similar staining pattern was observed in other tissues. Extracts of heart, lung, and brain tissues were evaluated for heparanase activity. The tissues were homogenized in heparanase reaction buffer and equal amounts of protein were incubated with sulfate-labeled ECM. As shown in Fig. 2C
, tissues extracted from hpa-tg mice exhibited 6- to 30-fold higher heparanase activity than endogenous heparanase activity determined in the corresponding tissues of control mice.
Heparan sulfate molecular size and bFGF binding capacity
Overexpression of heparanase is expected to alter the amount and size of HS chains in the hpa-tg mice. Metabolic labeling (Na235SO4) and direct biochemical analysis (gel chromatography on Superose-12 column) of total HS extracted from newborn mice revealed a significant reduction from 
35 kDa to 12 kDa (peak elution positions) of metabolically 35S-labeled HS chains derived from control vs. hpa-tg mice (Fig. 3
A). A similar difference in elution profiles was obtained with samples extracted from adult tissues (not shown).
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Heparan sulfates function as low-affinity, high-capacity receptors for bFGF (41
42
43)
. To examine the effect of heparanase overexpression on cell surface HS, we determined the bFGF binding capacity of primary cultures derived from hpa-tg vs. control embryos. Fibroblasts 15 days postgestation were incubated (1 h, 4°C) with increasing concentrations of 125I-bFGF and their ability to bind bFGF was evaluated by measuring the radioactivity associated with the cells. As demonstrated in Fig. 3B
, binding of 125I-bFGF to fibroblasts derived from G1 embryos reached saturation at a lower bFGF concentration compared with control fibroblasts. Under these experimental conditions, bFGF binds primarily to HS, which function as low-affinity, high-capacity receptors for bFGF (42
, 43)
. This observation suggests that overexpression of heparanase reduces the amount and/or size of HS available for bFGF binding on the cell surface.
Biochemical parameters and feeding behavior
Serum and urine samples were collected from 13 transgenic and 11 control mice in order to evaluate effects on kidney function. Biochemical analysis included determination of creatinine concentration in urine and serum as well as urinary protein concentration. Analysis of these parameters revealed a marked increase (2-fold, P=0.016) in urinary protein content in hpa-tg (0.277 g/L±0.036) compared with control (0.155 g/L±0.028) mice (P=0.016). There was also a significant (P=0.024) difference in urinary protein to creatinine (p/c) ratio between hpa-tg (p/c=0.151) vs. control (p/c=0.033) mice. Creatinine concentration was elevated in the serum of hpa-tg (21.79 µmol/L±1.76) vs. control (20.45 µmol/L±1.03) mice (P=0.062). These data suggest that the hpa-tg mice suffered from mild renal failure, as indicated by electron microscopy examination of the kidney tissue. Analysis of thin araldite sections showed in the control mice normal podocyte architecture with numerous foot processes (Fig. 4
, bottom). However, in hpa-tg mice the glomeruli exhibited alterations in podocyte structure, as seen by a loss of podocyte architecture with flattening and effacement of foot processes (Fig. 4
, top). Similar alterations in kidney histology were described in human and experimental diseases associated with proteinuria (44)
.
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Differences between hpa-tg and control mice in AST, ALT, and ALP activities (not shown) were not significant, suggesting normal liver function, in spite of a pronounced heparanase overexpression observed in the liver tissue (Fig. 2B
).
G1 hpa-tg males (15 mice) fed a regular chow diet ate (at 6 months of age) 4.6 ± 0.1 g/day; control males (15 mice) ate 5.5 ± 0.2 g/day (Fig. 5A
; P<0.007). Body weight of the hpa-tg males was 33.4 ± 1.0 g/mouse vs. 39.5 ± 1.7 g/mouse of control mice (Fig. 5B
, P <0.015). A similar effect was obtained in female mice (not shown). Overexpression of syndecan 1 in the hypothalamus of transgenic mice was shown to be associated with an increase in food uptake and weight gain (24)
. The decrease in food consumption and body weight observed (Fig. 5)
may therefore be attributed to overexpression of heparanase in the brain of hpa-tg mice.
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Embryonic implantation and mammary gland morphogenesis
Homozygous hpa-tg mice were fertile and had a normal litter size and life span. However, when pregnant mice at day 7 were scarified, there was a significant increase in the number of transgenic embryos implanted in their uteri compared with that of control embryos (13±3 vs. 9±2, respectively, P=0.023, n=10) (Fig. 6
). This observation suggests a role for heparanase in embryonic implantation.
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Tissue sections from hpa-tg mice were processed for histological evaluation in order to detect morphological changes associated with overexpression of heparanase. Hematoxylin/eosin staining of most tissues revealed no readily detectable histological differences between the transgenic and control mice. Few phenotypic alterations were, however, clearly noted. Mammary glands of 3-month-old virgin homozygous hpa-tg mice were excised and whole-mount preparations were prepared for morphological examination. The transgenic mammary glands showed abnormal abundant side branches and precocious alveolar structures, typical of pregnant mice (Fig. 7
, top right). The diameter of many of the mammary ducts was larger in the hpa-tg compared with control mice. overbranching, hyperplasia, and widening of glands were best demonstrated in 6- to 12-month-old hpa-tg virgin mice (not shown). The same phenotype of extra side branches and wider ducts was noted in heterozygous transgenic mice, but to a lesser extent. The effect of heparanase overexpression on the mammary gland architecture was enhanced during pregnancy (Fig. 7
, lower panels). The alveoli were abnormally clustered and more abundant than in mammary glands of control pregnant mice. On day 7 of pregnancy the ducts were about fivefold wider than those of control mammary glands.
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The histology of the mammary glands was evaluated in tissue sections. Sections of mammary glands excised from control and hpa-tg mice were stained for heparanase, using polyclonal anti-heparanase antibodies (Ab-p9) recognizing both human and mouse heparinase. Heparanase was expressed in mammary glands of control pregnant mice to a much lesser extent than in glands removed from the hpa-tg mice (Fig. 8
A). Disruption of the BM surrounding the mammary ducts and alveoli of virgin and 7 day pregnant hpa-tg mice was visualized by Masson-Trichrom staining, primarily in areas of side branching (Fig. 8B
). It is conceivable that an increased heparanase activity in the epithelium of the hpa-tg mammary gland is associated with an increased digestion of HS in the BM and ECM, resulting in excess branching and abnormal formation of alveolar structures, as well as in widening of the ducts. These effects were enhanced during pregnancy. We have noted a marked increase in vascularization (mean vascular count) along the ducts and in between fat cells in the hpa-tg vs. control mice (12.5±3 vs. 4±1.5 vessels/microscopic field, respectively, P=0.0009) (Fig. 8C
). These results reflect a pronounced effect of heparanase overexpression on tissue morphogenesis and vascularization pattern.
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Hair growth
Backskin hair of 1-month-old hpa-tg and control female mice at the end of the first hair cycle was clipped and the rate of hair regrowth was compared. A significant difference in hair growth was noted as early as 5 days after clipping (not shown). Ten to 12 days later, the clipped area of the hpa-tg mice was fully covered with hairs (Fig. 9
A, right) compared with a sparse coverage seen in control mice (Fig. 9A
, left). The control mice exhibited almost complete hair coverage about a week later, reflecting an accelerated rate of hair regrowth in the hpa-tg mice. Histological examination of skin tissue sections derived from hpa-tg and control mice revealed no difference in the number of hair follicles per microscopic field. Follicle size was slightly higher in the hpa-tg mice, but the difference was not significant. Immunostaining with anti-heparanase antibodies revealed increased levels of heparanase in hair follicles of hpa-tg vs. control mice. Heparanase expression in control mice was confined solely to the outer root sheath (ORS) keratinocytes. In contrast, hpa-tg mice exhibited a high heparanase expression that was not restricted to the ORS, but rather occurred in all epithelial compartments of the follicle (Fig. 9B
). Improved follicle vascularization promotes hair growth and increases hair follicle and hair shaft size (45
, 46)
. To examine whether the accelerated hair growth seen in the hpa-tg mice was accompanied by increased vascularization of hair follicles, skin samples from 8 and 16 day postnatal hpa-tg and control mice were stained for blood vessel endothelial cells, using anti-Von Willebrand factor antibodies. As shown in Fig. 9C
, vascular density was higher in the skin of hpa-tg compared with control mice (7.5±1.1 vs. 3.5±1.3 microvessels/field, respectively, P=0.031), suggesting that increased vascularization may, among other effects, accelerate hair growth in the hpa-tg mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7
8)
. Enzymatic degradation of HS is therefore involved in processes such as cell migration, proliferation, and lipoprotein retention, associated (among other aspects) with cancer metastasis, inflammation, restenosis, angiogenesis, and lipid metabolism (1
, 3
4
5
6
7
8
, 14
, 15
, 47)
. Cloning of human heparanase, the predominant functional HS-degrading endoglycosidase in mammals, led us to produce transgenic mice overexpressing the human heparanase and characterize the enzyme involvement in tissue architecture, vascularization, and metabolism.
We have generated four lines of transgenic mice ubiquitously overexpressing the human heparanase. The transgenic construct used to generate these mice encodes for the full-length 65 kDa pro-heparanase enzyme. Western blot analysis of tissue extracts revealed primarily the 50 kDa active enzyme, as also evident by measurements of heparanase activity, suggesting that the transgenic human proenzyme is being properly processed to produce the fully active human heparanase. All tissues expressing the human protein exhibited a high heparanase activity compared with the endogenous level of activity. It is conceivable that the basal activity determined in control tissue homogenates is due primarily to the presence of residual heparanase expressing blood cells such as platelets and neutrophils (21)
and, hence, may not necessarily represent heparanase activity expressed by the tissue cells themselves.
Direct biochemical analysis of HS isolated from newborn mice and adult tissues revealed a pronounced decrease (from 35 kDa to 12 kDa, corresponding to 140 and 50 sugar units, respectively) in the size of HS chains derived from control vs. hpa-tg mice, again reflecting an enhanced heparanase activity in most tissues. Despite this change, the mice appeared normal, were fertile, and exhibited a normal life span. Overexpression of heparanase resulted in a decrease in bFGF binding capacity of embryonic fibroblasts derived from hpa-tg vs. control embryos. HS function as low-affinity accessory receptors for heparin binding growth factors, including bFGF (41
42
43
, 48)
. Since the binding assay was not restricted to high-affinity receptor sites, amounting in most cells to <10% of the total binding, this result further indicates a lower content and/or size of HS available for bFGF binding on the surface of hpa-tg cells, due to enhanced degradation and shift in steady-state content and/or length of HS side chains. The effect on bFGF binding was, however, relatively small, taking into account that HS is cleaved by heparanase at specific intra-chain sites (18
, 19)
, leaving on the proteoglycan core protein HS moieties that can still bind bFGF. The bound bFGF and possibly other heparin binding factors may function in promoting vascularization, as indicated by an increased tissue vascularization observed in the mammary glands and skin of the hpa-tg mice.
Implantation
Among the phenotypic differences between the hpa-tg and control mice is the higher number of embryos observed in the uterus of transgenic mice during the first trimester of pregnancy. The following considerations suggest that embryonic implantation may in fact be attributed to overexpression of heparanase. Heparanase has been shown to promote neovascularization in wound healing and tumor models (16
, 22)
. Increased tissue vascularization was noted in response to a wound made in the skin of hpa-tg vs. control mice (our unpublished observations). Heparanase overexpression may thus promote excess vascularization and thereby ovulation in hpa-tg females. High levels of heparanase were previously reported in cytotrophoblasts (11
, 17
, 49)
. In the implantation site, heparanase may play a dual role. First, the heparanase molecule has been shown to mediate cell adhesion independent of its enzymatic activity (23)
. Its overexpression may therefore facilitate adhesive interactions at the very early stage of embryonic implantation. Heparanase may then support blastocyte implantation via recruitment of blood vessels, an activity that has been attributed to release of HS-bound angiogenic factors sequestered in the tissue as a complex with HS (16)
. A similar role was ascribed to MMP-9 in mediating the angiogenic switch during tumor progression (50)
. The same litter size in hpa-tg and control mice may indicate a higher miscarriage rate during pregnancy of hpa-tg females and/or a higher embryonic lethality rate. We could not distinguish between the two possibilities. Miscarriage and embryonic lethality may be due to heparanase mediated abnormal cell migration and tissue damage, causing instability and ontogenic death. In fact, HS-deficient mice die at the gastrula stage, indicating an absolute requirement for HS during progressive embryonic growth and morphogenesis (29)
. Cleavage of HS due to overexpression of heparanase may yield a similar effect, albeit to a much lower extent.
Feeding behavior
Transgenic expression of syndecan-1 in the hypothalamus results in obese hyperphagic mice, indicating that HS affects the regulation of energy homeostasis (24)
. It was suggested that HS indirectly interfere with the interaction between MC4-R and its endogenous agonist 
-MSH that normally results in reduced food intake and increased energy expenditure (51)
. HS bind the agouti-related protein (an endogenous antagonist of MC4-R), potentiate its interaction with MC4-R and thereby induce hyperphagia and increased body weight in the syndecan-1 overexpressing mice (24)
. The hpa-tg mice, characterized by reduced content and size of HS, appear to exhibit a mirror image of the syndecan-1 transgenic mice. The marked decrease in the size of HS observed in the hpa-tg mice may affect its binding to the agouti-related protein, resulting in reduced food consumption and body weight. An unrelated disorder in the digestive function of the hpa-tg mice could not be ruled out.
Kidney structure and function
Intact structure of the glomerular BM is essential for proper kidney function. Elevated levels of protein and protein to creatinine ratio were found in the urine of hpa-tg mice, suggesting that overexpression of heparanase disrupts the filtration barrier and reabsorption functions of the kidney. The observed proteinuria may be due to a loss of the characteristic podocyte architecture and foot processes seen in hpa-tg compared with control mice. Similar alterations were reported in diseases associated with proteinuria (44)
. In fact, it has been demonstrated that heparanase is up-regulated and activated in glomeruli from rats with proteinuria and that the enzyme may be involved in the loss of glomerular charge seen in proteinuria (52)
. We recently suggested that overexpression of heparanase in diabetic patients may disrupt the permeaselective properties of the glomerular BM and that urinary heparanase is an early sign of kidney disfunction involved in the pathogenesis of diabetic nephropathy (53)
.
Mammary gland morphogenesis
The mammary gland of normal virgin mice is essentially quiescent. It remains dormant until pregnancy, when the BM surrounding the mammary ducts is degraded, enabling the mammary epithelial cells to form the alveolar structure (54)
. In contrast, mammary glands of virgin hpa-tg mice showed precocious alveolar development and maturation with primary and secondary ducts, similar to a normal gland at 9–12 days of pregnancy. Even more pronounced development was observed during pregnancy, where abundant secondary alveolar structures were observed in the transgenic vs. normal mammary glands. We propose that overexpression of heparanase may facilitate BM disruption and thereby enable overbranching, hyperplasia, and widening of ducts. Moreover, cleavage of HS by heparanase releases HS-bound growth factors stored in the BM and ECM (7
, 16)
, resulting in stimulation of cell proliferation, differentiation, and preneoplastic transformation, in a manner similar to that observed with transgenic mice overexpressing stromelysin (55)
. Excessive disruption of the BM near the budding sites of hpa-tg alveoli was clearly noted by Masson-Trichrom staining of collagen. This was associated with an increased number of blood vessels along the ducts and in between fat cells of the mammary gland tissue.
Hair growth
The growing hair follicle is surrounded by blood vessels that arise from the deep dermal vascular plexus (45
, 46)
. Transgenic overexpression of VEGF in ORS keratinocytes of hair follicles was found to induce perifollicular vascularization, resulting in accelerated hair regrowth after depilation, and increased size of hair follicles and hair shafts (45)
. overexpression of heparanase may accelerate hair growth by promoting vascularization and maturation of the hair follicle, as well as enhancing keratinocyte cell migration toward the basal and outer zones of the hair follicle.
Altogether, our results emphasize the involvement of heparanase in normal and pathological tissue morphogenesis, remodeling, and vascularization. Apart from actual structural disruption of tissue barriers and acceleration of cell migration, we attribute the above-described phenotypes to the ability of HS to sequester multiple bioactive molecules made bioavailable, in a given time and location, when HS is degraded by heparanase. In view of the increasing structural and functional significance of HS in cells and tissues, generation of an in vivo model for overexpression of heparanase provides an attractive experimental system to study the involvement of HS in a multitude of developmental, metabolic, and physiological processes, as well as in pathological situations such as cancer progression, inflammation, and diabetes. Whereas the hpa-tg mice appear normal, unique features are detected in response to perturbations such as wounding, pregnancy, and malignancy. We attribute the observed phenotypes to a change in cell adhesion and migration as well as to alterations in tissue microenvironment, resulting in an improved bioavailability of ECM-resi-dent factors promoting survival, growth, and vascularization of cells and tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 8, 2003. Accepted for publication October 8, 2003.
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REFERENCES |
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) and hpa-tg (
) mice, were incubated (16 h, 37°C pH 6.0) on 35S-ECM. Degradation products released into the incubation medium were subjected to gel filtration on CL-6B Sepharose columns, as described in Materials and Methods. Left: lungs; middle: brain; right: heart
) vs. control (•) mice was noticed, reflecting a marked reduction in the length of the HS side chains. B) Binding of 125I-bFGF. Primary cultures of embryonic fibroblasts were prepared from control (•) and hpa-tg (
