Variant V6 Research Articles

Versican V0 and V1 Guide Migratory Neural Crest Cells

We previously showed the selective expression of the chondroitin sulfate proteoglycans versican V0 and V1 in barrier tissues that impede the migration of neural crest cells during embryonic trunk development (Landolt, R. M., Vaughan, L., Winterhalter, K. H., and Zimmermann, D. R. (1995) Development 212, 2303-2312). To test for an active involvement of these isoforms in the guidance process, we have now established protocols to isolate intact versican V0 and V1 in quantities sufficient for functional experiments. Using stripe choice assays, we demonstrate that pure preparations of either a mixture of versican V0/V1 or V1 alone strongly inhibit the migration of multipotent Sox10/p75NTR double-positive early neural crest stem cells on fibronectin by interfering with cell-substrate adhesion. We show that this inhibition is largely core glycoprotein-dependent, as the complete removal of the glycosaminoglycan chains has only a minor effect on the inhibitory capacity. Our findings support the notion that versican variants V0 and V1 act, possibly in concert with other inhibitory molecules such as aggrecan and ephrins, in directing the migratory streams of neural crest cells to their appropriate target tissues.

Versican/PG-M Regulates Chondrogenesis as an Extracellular Matrix Molecule Crucial for Mesenchymal Condensation

Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Furthermore, treatment with beta-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. These results suggest that versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains.

Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.

Differential Expression of KCNQ4 in Inner Hair Cells and Sensory Neurons Is the Basis of Progressive High-Frequency Hearing Loss

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Short-term treatment with anti-CD44v7 antibody, but not CD44v4, restores the gut mucosa in established chronic dextran sulphate sodium (DSS)-induced colitis in mice

Increased expression of CD44 variant isoforms have been shown on the inflammatory infiltrates in human and mouse colitis and blockade or deletion of CD44 isoforms inhibit experimental colitis. The objective of this study was to find out if short-term treatment of CD44 antibodies specific to CD44v7, but not to other variant isoforms, suppresses leucocyte-endothelial interaction in chronic dextran sodium sulphate (DSS)-induced colitis in mice. Chronic colitis was induced by oral administration of four cycles of 5% DSS in BALB/c mice. Expression of CD44 was investigated on isolated mononuclear cells of the gut immune system. In established colitis, mice were treated with antibodies against CD44v7 or CD44v4 three times in 7 days. Intravital microscopy was used to study leucocyte-endothelial interactions and leucocyte extravasation. As a marker of inflammatory infiltrates myeloperoxidase was quantified in gut tissue. CD44-induced apoptosis was determined by fluorescence staining of hypodiploidic cell nuclei. In chronic DSS-induced colitis both CD44 variant isoforms, v4 and v7 were significantly up-regulated on mononuclear cells. However, whereas anti-CD44v7 antibody treatment induced a marked restoration of the gut mucosa and significantly reduced endothelial sticking and extravasation of circulating leucocyte in vivo (P < 0.01), application of anti-CD44v4 or an isotype control antibody had no anti-inflammatory effect. A significant reduction of myeloperoxidase activity was detected after blockade of CD44v7, but not v4. Short-term treatment with anti-CD44v7 antibody blocks T cell extravasation and recruitment to the intestinal mucosa and cures established experimental colitis.

Loss of Ep-CAM (CO17-1A) expression predicts survival in patients with gastric cancer.

Preoperative staging of gastric cancer is difficult and not optimal. The TNM stage is an important prognostic factor, but it can only be assessed reliably after surgery. Therefore, there is need for additional, reliable prognostic factors that can be determined preoperatively in order to select patients who might benefit from (neo) adjuvant treatment. Expression of immunohistochemical markers was demonstrated to be associated with tumour progression and metastasis. The expression of p53, CD44 (splice variants v5, v6 and v9), E-cadherin, Ep-CAM (CO17-1A antigen) and c-erB2/neu were investigated in tumour tissues of 300 patients from the Dutch Gastric Cancer Trial, investigating the value of extended lymphadenectomy compared to that of limited lymphadenectomy). The expression of tumour markers was analysed with respect to patient survival. Patients without loss of Ep-CAM-expression of tumour cells (19%) had a significantly better 10-year survival (P<0.0001) compared to patients with any loss: 42% (s.e.=7%) vs 22% (s.e.=3%). Patients with CD44v6 (VFF18) expression in more than 25% of the tumour cells (69% of the patients) also had a significantly better survival (P=0.01) compared to patients with expression in less than 25% of the tumour cells: 10 year survival rate of 29% (s.e.=3%) vs 19% (s.e.=4%). The prognostic value of both markers was stronger in stages I and II, and independent of the TNM stage. Ep-CAM and CD44v6-expression provides prognostic information additional to the TNM stage. Loss of Ep-CAM-expression identifies aggressive tumours especially in patients with stage I and II disease. This information may be helpful in selecting patients suitable for surgery or for additional treatment pre- or postoperatively.

Proteoglycans and catabolic products of proteoglycans present in ligament.

The aim of the present study was to characterize the proteoglycans and catabolic products of proteoglycans present in the tensile region of ligament and explant cultures of this tissue, and to compare these with those observed in the tensile region of tendon. Approx. 90% of the total proteoglycans in fresh ligament was decorin, as estimated by N-terminal amino acid sequence analysis. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V1 and/or V2) and aggrecan. Approx. 23% of decorin detected in the matrix was degraded. Intact decorin and decorin fragments similar to those observed in the matrix that retained the N-terminus were also observed in the medium of ligament cultures. Intact biglycan core protein was detected in the matrix and medium of ligament cultures, and two fragments originating from the N-terminal region of biglycan were observed in the matrix of cultured ligament. Versican and versican fragments that retained the N-terminus of versican core protein were detected in fresh matrix and medium of tendon cultures. Approx. 42% of versican present in the fresh ligament was degraded. Aggrecan catabolites appearing in the culture medium were derived from aggrecanase cleavage of the core protein. An intact link protein and a degradation product from the N-terminal region of type XII collagen were also detected in the medium of the ligament explant.

Elevated expression of hyaluronan and its CD44 receptor in the duodenal mucosa of coeliac patients

Since hyaluronan (HA) metabolism is disturbed in some malignant tumours and in inflammatory diseases, we analysed HA and its receptor CD44 as well as the expression of the Ki67 nuclear protein, a marker of cell proliferation, in histological sections of duodenal biopsies of coeliac disease patients and controls. The study group consisted of 52 patients with coeliac disease in remission, 40 patients with newly diagnosed disease and 10 healthy control subjects. HA was detected with a specific biotinylated probe prepared from cartilage aggrecan and link protein, and CD44 with an antibody recognizing all forms of CD44 and another specific for its v6 variant. For the expression of the nuclear protein, monoclonal antibody MIB-1 was used. The percentage of HA-positive cells in surface epithelium was higher in newly diagnosed patients (13%) compared with patients in remission (11%) and controls (2%). In addition, HA intensity in the lamina propria was decreased in the newly diagnosed patients. In patients with active disease, 22-26% of the surface epithelium was CD44+, whereas the corresponding figure in patients in remission was 5%, and that of controls 1%. The more intensive MIB-1 labelling in the duodenal epithelium of coeliac patients without treatment was normalized after gluten-free diet. The HA-positive coat on surface epithelium seen even in patients in remission suggests persistent or even permanent changes in the epithelial permeability barrier in coeliac disease.

Prostate cancer invasion is influenced more by expression of a CD44 isoform including variant 9 than by Muc18

The standard form of cell adhesion glycoprotein CD44 is a metastasis suppressor in prostate cancer. However, we previously showed by RT-PCR and Western blotting that cancer overexpresses unique CD44 variant v7–v10 isoforms. Muc18 is another cell adhesion marker reportedly overexpressed by prostate cancer. Matched frozen section-confirmed tumor and benign tissues were harvested from 10 prostatectomy specimens and tumor was microdissected from two lymph node metastases. Tissues were homogenized for RNA preparations, and RT-PCR was performed for the CD44v7–v10 sequence. In cultured prostate cancer cells, we caused RNA interference against CD44v9 and/or Muc18. We used PC3M cells and a derivative cell line called Gsα, that constitutively expresses this G-protein and is more invasive. Lipofection was performed for a green fluorescent protein plasmid and for two 22-mer DNA fragments, cloned into a plasmid expression vector to generate hairpin, interfering dsRNA. Assays for invasion into Matrigel, a basement membrane matrix, were performed in 4–5 experiments. RT-PCR demonstrated expression of a 608 bp band representing CD44v7–v10 or a 638 bp band of CD44v6–v10 in prostate cancer tissues and metastases but not benign tissue. Cultured Gsα cells overexpressed CD44v9 by comparison with PC3M cells. At 90 h after 6-hour lipofection, protein silencing was evident by Western blots. Silencing the CD44v9 expression reduced invasiveness into Matrigel to 21.6±7.0% in PC3M cells (P<0.001) and 31.2±18.3% in Gsα cells (P=0.001), compared to cells exposed to transfection vehicle alone. Silencing Muc18 expression reduced invasiveness to 76.9±13.5% of the control value in PC3M cells (P<0.05) and 84.8±29.9% in Gsα cells (P=0.18). Prostate cancer invasion is facilitated more by its overexpression of CD44 variant 9 than by Muc18. Its relative overexpression by Gsα cells is a novel finding, suggesting a link between signal transduction and cell adhesion marker expression.

Versican splice variant messenger RNA expression in normal human Achilles tendon and tendinopathies.

Versican is the principal large proteoglycan expressed in mid-tendon, but its role in tendon pathology is unknown. Our objective was to define the expression of versican isoform splice variant messenger ribonucleic acid (mRNA) in normal Achilles tendons, in chronic painful tendinopathy and in ruptured tendons. Total RNA isolated from frozen tendon samples (normal n = 14; chronic painful tendinopathy n = 10; ruptured n = 8) was assayed by relative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for total versican, versican variants V0, V1, V2, V3 and type I collagen alpha1 mRNA, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Differences between sample groups were tested by Wilcoxon statistics. Painful and ruptured tendons showed a significant decrease (median 2-fold) in the expression of versican mRNA, in contrast to an increased expression (median 8-fold) of type I collagen alpha1 mRNA in painful tendons. Versican splice variants V0 and V1 mRNA were readily detected in normal samples, V3 levels were substantially lower, and V2 levels were more variable. Each of V1, V2 and V3 mRNA showed significant decreases in expression in painful and ruptured tendons, but V0 was not significantly changed. Changes in versican expression relative to that of collagen, and alterations in the balance of versican splice variants, may contribute to changes in matrix structure and function in tendinopathies.

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, ∼80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V0 and/or V1) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K171 and D180 were observed in the matrix and one core protein with an amino-terminal sequence commencing at G189 was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E373–A374, E1480–G1481 and E1771–A1772. The analysis of versican catabolites (splice variants V0 and/or V1) also showed evidence of degradation of the core protein by aggrecanase within the GAG-β subdomain, as well as cleavage by other proteinase(s) within the GAG-α and GAG-β subdomains of versican (variants V0 and/or V2). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

Graded phenotypic response to partial and complete deficiency of a brain-specific transcript variant of the winged helix transcription factor RFX4.

One line of mice harboring a cardiac-specific epoxygenase transgene developed head swelling and rapid neurological decline in young adulthood, and had marked hydrocephalus of the lateral and third ventricles. The transgene was found to be inserted into an intron in the mouse Rfx4 locus. This insertion apparently prevented expression of a novel variant transcript of RFX4 (RFX4_v3), a member of the regulatory factor X family of winged helix transcription factors. Interruption of two alleles resulted in profound failure of dorsal midline brain structure formation and perinatal death, presumably by interfering with expression of downstream genes. Interruption of a single allele prevented formation of the subcommissural organ, a structure important for cerebrospinal fluid flow through the aqueduct of Sylvius, and resulted in congenital hydrocephalus. These data implicate the RFX4_v3 variant transcript as being crucial for early brain development, as well as for the genesis of the subcommissural organ. These findings may be relevant to human congenital hydrocephalus, a birth defect that affects approximately 0.6 per 1000 newborns.

Differential expression of versican isoforms is a component of the human melanoma cell differentiation process

Versican is a large chondroitin sulfate proteoglycan produced by human melanoma cell lines and malignant melanocytic lesions. In the present work, we have analyzed the expression of versican spliced variants V0, V1, V2 and V3 in human melanoma cell lines at several differentiation degrees. The isoform expression pattern depends on the degree of cell differentiation. Differentiated cell lines do not produce any of the versican isoforms as analyzed by Western blot, Northern blot and RT-PCR. All cell lines with an early or intermediate degree of differentiation (AX3, SK-mel-37, Rider, SK-mel-1.36-1–5 and SK-mel-3.44) expressed V0 and V1 transcripts, whereas V2 and V3 expression was shown only by the undifferentiated cell lines SK-mel-1.36-1–5 and Rider. Furthermore, we have analyzed the expression of versican isoforms in SK-mel-3.44 and SK-mel-1.36-1–5 cells induced to differentiate by TPA treatment. The expression of the large V0, V1 and V2 isoforms practically disappears in differentiated cells, whereas V3 remains detectable by RT-PCR analysis.

Phi-Values beyond the ribosomally encoded amino acids: kinetic and thermodynamic consequences of incorporating trifluoromethyl amino acids in a globular protein.

The consequences of the substitution of 4,4,4-trifluorovaline for valine on the folding kinetics and thermodynamics of a globular protein are presented. Variants of the N-terminal domain of L9, a small alpha-beta protein, were prepared in which V3 or V21 was replaced by trifluorovaline. CD and NMR demonstrate that the structure is not perturbed. Both are more stable, the V3 variant by 0.8 kcal mol-1 and the V21 variant by 1.4 kcal mol-1. The increase of stability is significantly larger than that observed in coiled-coils on a per trifluoromethyl group basis. Folding is two-state, and the variants both fold faster than the wild type. The Phi-values are 0.16 and 0.11, respectively.

Phytoplasma host range and symptom expression in the pasture legume Stylosanthes

A field trial of 23 stylo taxa was assessed for susceptibility to stylosanthes little leaf (SLL) disease. The trial included Stylosanthes scabra cvv. Seca, Siran, and Fitzroy, S. guianensis var. guianensis cv. Cook, S. hamata cvv. Amiga and Verano and “ Stylosanthes seabrana” cvv. Unica and Primar, as well as seven unreleased cultivars from a CSIRO anthracnose resistance breeding program, and four unreleased introduced lines. Plants that had SLL symptoms were sampled on six occasions from March to August 1999 and tested for phytoplasmas, the presumed causal organism of this disease, using the polymerase chain reaction (PCR). All taxa were found to be susceptible to SLL disease except S. hamata cv. Verano and “ S. seabrana” cv. Unica. Varieties were ranked on disease incidence, with S. scabra varieties bred for anthracnose resistance having the highest incidence of the disease. These varieties also had significantly higher SLL incidence than that of the original introductions from which they were derived. Before testing, symptom profiles were recorded noting the presence of chlorosis, spike elongation, reddening of leaf tips and floral parts, and reduced leaf size (little leaf). Statistical analysis of the association of these characteristics with SLL disease showed that little leaf was the best disease indicator and that chlorosis and excessive reddening of leaf tips and floral parts were also associated with the disease. The presence of floral abnormalities was also recorded in the symptom profiles. At no time were normal flowers found on samples that had SLL, making this symptom a good indicator of SLL. However, not all plants with floral symptoms tested positive for phytoplasmas by PCR, probably because these symptoms are difficult to diagnose visually. Virescence and phyllody were the most common floral abnormalities and were found to be associated specifically with the phytoplasmas pigeon pea little leaf and sweet potato little leaf variant V4, respectively. An additional floral abnormality, termed abortion, was also observed in association with this disease.

Concentration of soluble CD44 standard and soluble CD44 variant V5 in the serum of patients with malignant bone tumors.

Concentration of soluble CD44 standard and soluble CD44 variant V5 in the serum of patients with malignant bone tumors.

Influence of epitopes CD44v3 and CD44v6 in the invasive behavior of fibroblast-like synoviocytes derived from rheumatoid arthritic joints.

To investigate the functional implications of CD44 splice variant expression in fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). FLS were isolated from synovial tissue obtained from both diseased and nondiseased joints. The expression of splice variants containing exons v3 and v6 was analyzed using immunocytochemistry with exon-specific antibodies and reverse transcription-polymerase chain reaction followed by Southern blotting. The invasive capacity of the cells was studied in a transwell invasion assay. FLS obtained from RA joints expressed various CD44 splicing combinations containing the variant exons v3 and/or v6. These cells were highly invasive, whereas cells from normal tissues, which lacked expression of CD44 splice variants, were not. Variant exons CD44v3 and CD44v6 were instrumental in matrix invasion in vitro, with cells enriched for CD44v3 and v6 exhibiting greater invasion and antibodies that specifically recognize CD44v3 and v6 abrogating this capacity to invade. Invasive cells showed a reduced expression of CD44v7/8, and antibodies against this epitope had no significant effect on cellular infiltration of the matrix. The antibodies had no effect on cell migration into the porous section of the transwell. FLS obtained from patients with RA express CD44 splice variants and are highly invasive, whereas cells obtained from healthy tissue do not express these variants and are not invasive. Expression of the epitopes CD44v3 and CD44v6 is instrumental in the invasive capacity but not in cell migration. This finding highlights a functional implication for the expression of CD44 splice variants at the level of matrix degradation.

Expression of CD44v5 and -v6 in Barrett's Carcinoma Is Not Increased Compared to That in Nondysplastic Barrett's Mucosa

Barrett's adenocarcinoma currently shows the highest increase in the incidence of all malignant tumors. Reliable molecular markers to identify Barrett's patients at risk are still missing. Our own results demonstrate that the expression of CD44v6 correlates with the development of dysplasia in colorectal neoplasms. Therefore, we examined the expression of CD44 variants v5 and v6 in normal esophageal mucosa, nondysplastic Barrett's mucosa, and Barrett's carcinoma. mRNA from biopsy specimens of patients with Barrett's esophagus ( n = 19) or Barrett's carcinoma ( n = 15) and patients without esophageal diseases (controls; n = 9) were extracted and used as templates for cDNA synthesis. CD44 variants were detected by RT–PCR with primers hybridizing with CD44 sequences up- and downstream of variable exons. CD44v6 expression was found in 36 of 56 biopsy specimens (64%) of nondysplastic Barrett's mucosa, in 100% of squamous epithelium, and in none of the gastric mucosa specimens. Eleven of 15 specimens (73%) of Barrett's carcinoma tested positive for v6 expression. The identification of v5 expression did not give additional information. There was no correlation between CD44v5 or -v6 expression and staging or grading of the tumors. Expression of CD44v5 and -v6 seems to be independent of the development of cancer in Barrett's mucosa.

Retrovirally mediated overexpression of versican v3 by arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury.

Versican is an extracellular matrix (ECM) proteoglycan that is synthesized as multiple splice variants. In a recent study, we demonstrated that retroviral-mediated overexpression of the variant V3, which lacks chondroitin sulfate (CS) chains, altered arterial smooth muscle cell (ASMC) phenotype in short-term cell culture. We now report that V3-overexpressing ASMCs exhibit significantly increased expression of tropoelastin and increased formation of elastic fibers in long-term cell cultures. In addition, V3-overexpressing ASMCs seeded into ballooned rat carotid arteries continued to overexpress V3 and, at 4 weeks after seeding, produced a highly structured neointima significantly enriched in elastic fiber lamellae. In contrast to the hydrated, myxoid neointima produced by rounded or stellate vector-alone--transduced cells, V3-expressing cells produced a compact and highly ordered neointima, which contained elongated ASMCs that were arranged in parallel arrays and separated by densely packed collagen bundles and elastic fibers. These results indicate that a variant of versican is involved in elastic fiber assembly and may represent a novel therapeutic approach to facilitate the formation of elastic fibers.

Expression of CD44s and CD44 splice variants in human melanoma

The ability of tumor cells to adhere and detach from extracellular matrix and endothelial cells, is a crucial step in the metastatic process and may alter the clinical prognosis of some human tumors such as melanomas. CD44, the major cell surface receptor for hyaluronate, has been implicated in cell adhesion and in tumor progression. We studied the expression of standard CD44 molecule (CD44s) and its variants v3 and v6 in 57 human primary melanoma biopsies, without previous treatment. We analyzed the association between CD44 expression and the principal clinicopathological features, including survival. Fifty-six of 57 tumors expressed CD44s, associated to the cytoplasmic membrane. No expression of CD44v3 or CD44v6 was detected. No association between CD44s expression and prognostic factors such as tumor thickness, growth type, stage or anatomic site of the lesion was found. However, a positive correlation between CD44s expression and Clark level (Spearman, p<0.001) was found. While only 33.3% of melanomas Clark I + II showed high expression of CD44s (more than 50% of positive cells), 82.6% of melanomas Clark IV + V did so. Kaplan-Meier analysis revelead that patients whose melanomas had high expression of CD44s showed a reduced relapse free survival (RFS) rate, though without statistical significance. No difference between the level of CD44 expression and overall survival (OS) was found. We conclude that melanomas only expressed CD44s, and that its level was associated with Clark's stage. CD44s seems not to be useful as a tumor marker, because it does not predict either RFS or OS.