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Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.

Murine light chain 3 (LC3) exists as two isoforms, LC3alpha and beta: LC3beta is an RNA-binding protein that enhances fibronectin (FN) mRNA translation, and is also a marker of autophagy. We report embryonic expression patterns for LC3alpha and LC3beta, with some overlap but notable differences in the brain, and in tissues of non-neuronal origin. LC3beta knockout (-/-) mice develop normally without a compensatory increase in LC3alpha. LC3beta-/- embryonic fibroblasts (MEFs) exhibit reduced FN synthesis but maintain wild type (WT) levels of FN protein. No significant changes in proteins associated with FN turnover, i.e., caveolin-1, LRP-1, or matrix metalloproteinases were identified. Autophagosomes form in amino acid-starved LC3beta-/-MEFs, and Caesarean-delivered pups survive as long as WT pups without an increase in LC3-related proteins linked to autophagy. These results suggest novel compensatory mechanisms for loss of LC3beta, ensuring proper FN accumulation and autophagy during fetal and neonatal life.

Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (P<0.05). This correlated with increased NO production (P<0.05) and fibronectin synthesis (P<0.05). TNF-alpha induction of fibronectin synthesis was abrogated by the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (P<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (P<0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P<0.05). Northern blotting showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. TNF-alpha increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.

Ductus arteriosus intimal cushion formation is characterized by fibronectin-dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthesis in ductus SMC is regulated by the interaction of LC-3, a microtubule-associated protein, with an AU-rich element (ARE) in the 3'-untranslated region of fibronectin mRNA, facilitating its recruitment to polyribosomes for translation. Since nitric oxide (NO) is implicated in posttranscriptional gene regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis. NO production was sevenfold higher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronectin synthesis by approximately 45-50% (P<0.05), whereas the NO donor, SNAP, increased ductus fibronectin synthesis approximately onefold (P<0.05); neither agent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP increased and L-NMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to the fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production regulate the posttranscriptional increase in fibronectin synthesis required for SMC motility.

Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.

Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Progressive pulmonary hypertension is characterized by smooth muscle cell proliferation and migration leading to occlusive arterial lesions. Previously, using cultured smooth muscle cells, we demonstrated that epidermal growth factor (EGF)-dependent proliferation and migration are dependent on tenascin-C (Tn) and cellular fibronectin (Fn), respectively. In this study we applied immunohistochemistry to lung biopsy tissue from patients with congenital heart defects and pulmonary hypertension to determine how the distribution and intensity of Tn, EGF, proliferating cell nuclear antigen (PCNA), and Fn expression related to arterial abnormalities. With mildly increased wall thickness, minimal Tn, PCNA, and EGF was evident. With progressive hypertrophy, moderately intense foci of Tn were apparent in the adventitia, periendothelium, and occasionally the media but not consistently co-distributing with EGF and PCNA. With obstructive lesions, intense neointimal Tn expression co-localized with EGF and PCNA. Fn accumulation in the periendothelium increased with medial hypertrophy and became more widespread in a diffuse pattern with neointimal formation. The neointima was predominantly composed of alpha-smooth-muscle-actin-positive cells, occasional inflammatory cells with no evidence of apoptosis. These studies are consistent with Tn modulating EGF-dependent neointimal smooth muscle cell proliferation and Fn providing a gradient for smooth muscle cell migration from media to neointima.

We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL)-1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL-1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production.

Ultrastructural observations in lung tissue implicated an endogenous vascular elastase (EVE), in the pathobiology of pulmonary vascular disease. In experimental rats, increased activity of a 20 kDa serine proteinase related to adipsin precedes the development of sustained pulmonary hypertension and vascular abnormalities. A further increase in activity is related to malignant progression of the disease. A cause and effect relationship was suggested by studies in which elastase inhibitors successfully prevented or retarded progression of pulmonary hypertension. In vitro studies have shown that both serum and endothelial factors induce EVE via tyrosine kinase intracellular signalling. Induction of EVE can release basic fibroblast growth factor from the extracellular matrix in an active form stimulating smooth muscle cell proliferation. Elastase activity was also observed in the process of smooth muscle cell migration and neointimal formation in coronary arteries following experimental cardiac transplantation. An immune/inflammatory response is observed with increased production of cytokines, tumor necrosis factor-alpha and interleukin (IL)-1 beta, reciprocally up-regulating production of fibronectin, a glycoprotein which mediated smooth muscle cell migration. The action of IL-1 beta in inducing fibronectin is facilitated by the production of elastin peptides generated by increased activity of an elastase in the coronary arteries. Our studies suggest that ligation of the elastin binding protein by elastin peptides unmasks IL-1 receptors. Fibronectin also stimulates transendothelial migration of lymphocytes which perpetuates the inflammatory response leading to neointimal formation in this model. Masking integrins on T cells with a decoy synthetic CS-1 (fibronectin) peptide largely prevented transendothelial migration and coronary neointimal formation following cardiac transplant.

We previously reported infiltration of immune-inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)-1 beta. We now investigate, using a porcine endothelial-smooth muscle cell co-culture system, whether IL-1 beta-stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL-2, were characterized by fluorescence-activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL-1 beta. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking alpha 4 beta 1 and alpha 5 beta 1 integrin receptors on lymphocyte surfaces, respectively. A 55-70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium, we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine-mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface alpha 4 beta 1 and alpha 5 beta 1 integrins.

Background: The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury., Methods: Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility., Results: The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta., Conclusions: Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury.

Graft arteriopathy, a leading cause of cardiac allograft failure, is associated with increased intimal smooth muscle cells, inflammatory cells, and accumulation of extracellular matrix. We hypothesized that cellular fibronectin plays a pivotal role in the progression of the allograft arteriopathy by directing the transendothelial trafficking of inflammatory cells through interaction of the connecting segment-1 (CS1) motif with the very late antigen-4 (VLA-4) integrin, and tested this in vivo using a blocking peptide. Cholesterol-fed rabbits underwent heterotopic cardiac transplantation without immunosuppression. The treatment group (n = 7) received a synthetic CS1 peptide (1 mg/kg per d, subcutaneously), and the controls (n = 7) received an inactive peptide (1 mg/kg per d, subcutaneously). At 7-8 d after transplantation, hearts were harvested and sectioned for morphometric analysis and immunohistochemical studies. We observed a > 50% decrease in the incidence (P < 0.001) and severity (P < 0.001) of donor coronary artery intimal thickening in the CS1-treated compared with the control group. These findings correlated with reduced infiltration of T cells (P < 0.05), a trend toward decreased expression of adhesion molecules (P < 0.06), and less accumulation of fibronectin (P < 0.03). Our data suggest that the VLA-4-fibronectin interaction is critical to the progression of the allograft arteriopathy by perpetuating the immune-inflammatory response in the vessel wall.

We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-1 beta and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-1 beta was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-1 beta and tumor necrosis factor (TNF)-alpha has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-alpha expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-1 beta, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-alpha antibodies, and exogenous TNF-alpha upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-alpha-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-1 beta antibodies. Likewise, IL-1 beta-stimulated fibronectin synthesis was downregulated to control levels when TNF-alpha neutralizing antibodies were added. Combining TNF-alpha and IL-1 beta enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-1 beta and TNF-alpha activity and provide the first demonstration of a 'cytokine loop' modulating matrix production.

Background: Graft coronary arteriopathy has become the major complication observed in the late follow-up of cardiac transplant patients. We investigated this process experimentally in piglets after a heterotopic cardiac transplant and observed early changes in donor coronary arteries compatible with an immune-inflammatory process, ie, there is increased expression of interleukin-1 beta (IL-1 beta), fibronectin, and activated lymphocytes associated with intimal thickening., Methods and Results: In this study, we cultured porcine coronary artery endothelial cells from host and donor hearts and found similarities in morphology and uptake of acetylated low-density lipoprotein (LDL). As well, host and donor cells showed similar patterns of growth, protein, and glycosaminoglycan synthesis. Endothelial cell fibronectin synthesis was determined after radiolabeling with [35S]-methionine in serum-free medium, gelatin-sepharose extraction of the culture medium and resolution on 5% SDS-PAGE. Donor coronary artery endothelial cell fibronectin synthesis was up to fivefold higher than that of host but was not associated with comparable increased levels of fibronectin mRNA. IL-1 beta appeared to mediate this enhanced fibronectin production, since the IL-1 receptor antagonist caused a 50% decrease in this feature, a change not observed in host cells. Furthermore, donor endothelial cells produced twice the amount of IL-1 beta compared with host cells as judged by immunoprecipitation., Conclusions: Increased donor coronary artery endothelial cell fibronectin appears to be regulated at least partly by an autocrine mechanism involving IL-1 beta, and fibronectin may mediate lymphocyte trafficking and smooth muscle cell migration related to graft arteriopathy.

We previously described in piglets after heterotopic cardiac transplantation the early development of a coronary arteriopathy characterized by increased immunostaining for fibronectin and interleukin-1 beta (IL-1 beta) in the vessel wall. The objective of this study was to culture smooth muscle cells from donor and host coronary arteries in these piglets to determine whether donor cells produce more fibronectin than host cells as judged by increased protein and mRNA levels, and whether IL-1 beta may be regulating this increase by an autocrine mechanism involving increased production of the cytokine. We documented increased donor coronary artery smooth muscle cell fibronectin protein synthesis and mRNA compared to host. By using neutralizing antibodies to IL-1 beta, fibronectin protein synthesis and mRNA levels were reduced in donor cells to the levels observed in the host cells and a similar reduction in synthesis was observed with the IL-1 receptor antagonist. Immunoprecipitation of newly synthesized IL-1 beta revealed increased endogenous levels in donor compared to host cells. We therefore suggest in the coronary arteriopathy a pathophysiologic mechanism whereby IL-1 beta-mediated increased fibronectin synthesis may promote lymphocyte trapping and migration of medial smooth muscle cells leading to progressive intimal thickening associated with the post-cardiac transplant coronary arteriopathy.

The mechanism causing intimal thickening in the postcardiac transplant coronary arteriopathy (PCTCA) is associated with interactions between inflammatory cells and vascular cells. Our previous studies related intimal thickening to fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, and others have shown that cytokines, eg, interleukin (IL)-1 beta, up-regulate SMC fibronectin synthesis. In this study, we identified, in piglets, features compatible with early development of the PCTCA. Ultrastructure revealed increased SMC and inflammatory cells in the subendothelium. Immunohistochemistry showed major histocompatibility complex II presentation in the endothelium and adventitia, associated with infiltration of different subsets of inflammatory cells; increased IL-1 beta, particularly in the endothelium; and fibronectin, in the subendothelium and inner media, the latter confirmed by quantitative immunoelectron microscopy. In the PCTCA, increases in IL-1 beta and fibronectin could mediate adherence, transendothelial migration and trapping of inflammatory cells, and SMC migration into the subendothelium.

Background: In previous studies we established that there are developmentally regulated increases in endothelial hyaluronan (HA) and heparan sulfate (HS), and smooth muscle cell fibronectin (FN) related to the formation of intimal cushions, structures essential to the postnatal closure of the ductus arteriosus (DA). In this report, we investigated the mechanisms underlying these features to ascertain whether they were independently or coordinately regulated., Experimental Design: We determined by assessing HA polymer size and by pulse labeling with [3H]glucosamine whether the increased glycosaminoglycans (GAGs) incorporated in DA compared with aorta (Ao) endothelial cell matrices reflected increased synthesis of HA and HS rather than decreased degradation. We assessed whether transforming growth factor-beta (TGF-beta) may be responsible for the increased DA endothelial GAGs and smooth muscle FN production by confirming the presence of TGF-beta in DA tissue using immunohistochemistry and by assessing the effect of adding neutralizing antibodies to the cell cultures. We next determined whether the level of regulation of the increase in FN in DA smooth muscle cells was transcriptional or post-transcriptional by relating protein synthesis to steady state mRNA levels and to mRNA levels after serum stimulation. Using northern blot analyses with specific probes, we also explored the possibility that the FN produced by the DA and Ao was qualitatively different in the proportion of isoforms containing the V95+ region associated with secretion or the EIIIB+ region that has been related to migration., Results: We observed that HA polymer size produced by DA and Ao endothelial cells was similar and we further verified using pulse labeling that the increase in DA compared with Ao endothelial GAGs reflected increased synthesis of HA and HS rather than decreased degradation. There was increased immunostaining for TGF-beta in DA compared with Ao tissue and we showed that TGF-beta neutralizing antibodies reduced synthesis of GAGs by the DA endothelial cells to the level of that seen in the Ao cells, but did not reduce DA smooth muscle cell FN synthesis. The increase in FN synthesis by DA compared to Ao smooth muscle cells was not associated with increased levels of steady-state mRNA for FN. Furthermore, following serum-stimulated increases in FN mRNA, the DA yielded greater amounts of FN protein compared to Ao smooth muscle cells. The increased FN production by the DA smooth muscle cells could not be attributed to a relative lack of degradation of FN protein or mRNA, or to qualitative differences which might influence secretion, as both cell types contained similar proportions of EIIIB+ and V95+ isoforms of FN., Conclusions: These results would suggest that, in contrast to the TGF-beta dependent increase in DA endothelial GAG synthesis, the increase in DA smooth muscle FN synthesis arises through differences in post-transcriptional regulation that are likely independent of TGF-beta.

"Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo.