Autocrine Hyaluronan Influences Sprouting and Lumen Formation During HUVEC Tubulogenesis In Vitro.

Although many studies have focused on a role for hyaluronan (HA) of interstitial extracellular matrix (presumably produced by non-vascular "stromal" cells) in regulating vascular growth, we herein examine the influence of "autocrine HA" produced by vascular endothelial cells themselves on tubulogenesis, using human umbilical vein endothelial cells (HUVECs) in angiogenic and vasculogenic three-dimensional collagen gel cultures. Relative to unstimulated controls, tubulogenic HUVECs upregulated HAS2 mRNA and increased the synthesis of cell-associated HA (but not HA secreted into media). Confocal microscopy/immunofluorescence on cultures fixed with neutral-buffered 10% formalin (NBF) revealed cytoplasmic HAS2 in HUVEC cords and tubes. Cultures fixed with NBF (with cetylpyridinium chloride added to retain HA), stained for HA using "affinity fluorescence" (biotinylated HA-binding protein with streptavidin-fluor), and viewed by confocal microscopy showed HA throughout tube lumens, but little/no HA on the abluminal sides of the tubes or in the surrounding collagen gel. Lumen formation in angiogenic and vasculogenic cultures was strongly suppressed by metabolic inhibitors of HA synthesis (mannose and 4-methylumbelliferone). Hyaluronidase strongly inhibited lumen formation in angiogenic cultures, but not in vasculogenic cultures (where developing lumens are not open to culture medium). Collectively, our results point to a role for autocrine, luminal HA in microvascular sprouting and lumen development. (J Histochem Cytochem 69: 415-428, 2021).

Modulation of hyaluronan synthases and involvement of T cell-derived hyaluronan in autoimmune responses to transplanted islets.

The extracellular matrix glycosaminoglycan hyaluronan (HA) accumulates in human and mouse islets during the onset of autoimmune type 1 diabetes (T1D). HA plays a critical role in T1D pathogenesis, as spontaneous disease is blocked in mice fed the HA synthesis inhibitor 4-methylumbelliferone (4MU). The present study demonstrates the involvement of HA in T cell-mediated autoimmune responses to transplanted islets and in in vivo and in vitro T cell activation. Scaffolded islet implants (SIs) loaded with RIP-mOVA mouse islets expressing chicken ovalbumin (OVA) on their ß cells were grafted into T and B cell-deficient RIP-mOVA mice, which subsequently received CD4+ T cells from DO11.10 transgenic mice bearing OVA peptide-specific T cell receptors (TcRs), followed by injection of OVA peptide to induce an immune response to the OVA-expressing islets. By affinity histochemistry (AHC), HA was greatly increased in grafted islets with T cell infiltrates (compared to islets grafted into mice lacking T cells) and a portion of this HA co-localized with the infiltrating T cells. Transferred T cells underwent HA synthase (HAS) isoform switching - T cells isolated from the SI grafts strongly upregulated HAS1 and HAS2 mRNAs and downregulated HAS3 mRNA, in contrast to T cells from graft-draining mesenteric lymph nodes, which expressed HAS3 mRNA only. Expression of HAS1 and HAS2 proteins by T cells in SI infiltrates was confirmed by immunohistochemistry (IHC). DO11.10 mice fed 4MU had suppressed in vivo T cell immune priming (measured as a reduced recall response to OVA peptide) compared to T cells from control mice fed a normal diet. In co-cultures of naïve DO11.10 T cells and OVA peptide-loaded antigen-presenting cells (APCs), pre-exposure of the T cells (but not pre-exposure of APCs) to 4MU inhibited early T cell activation (CD69 expression). In addition, T cells exposed to 4MU during activation in vitro with anti-CD3/CD28 antibodies had inhibited phosphorylation of the CD3ζ subunit of the TcR, a very early event in TcR signaling. Collectively, our results demonstrate that T cell-derived HA plays a significant role in T cell immune responses, and that expression of T cell HAS isoforms changes in a locale-specific manner during in vivo priming and functional phases of the T cell response.

A Role for HAPLN1 During Phenotypic Modulation of Human Lung Fibroblasts In Vitro.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-ß1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling.

Controlled release of monoclonal antibodies from poly-l-lysine-coated alginate spheres within a scaffolded implant mitigates autoimmune responses to transplanted islets and limits systemic antibody toxicity.

Immunomodulatory monoclonal antibodies (IM-mAbs) are a cornerstone of modern immunotherapy; however, when administered systemically (i.e., via injection), these agents can generate a variety of negative side effects. For many diseases, systemic delivery of IM-mAbs is the most effective mode of treatment, but in instances where the cellular target occupies a limited, well-defined space (e.g., solid tumors or cellularized implants) local, controlled release of IM-mAbs might be desirable. Antibodies are highly sensitive to a variety of environmental conditions, which limit the kinds of polymers suitable for antibody retention and controlled release. The present study evaluates the release of antibodies from biocompatible, 2-mm diameter alginate spheres coated with poly-l-lysine and a thin outer layer of alginate (APA spheres). In vitro, rates of antibody release (including IM-mAbs) could be incrementally decreased and made linear by incrementally increasing the quantity of poly-l-lysine deposited on the alginate, with linear release lasting in one scenario for at least 46 days. To evaluate the bioactivity in vivo of IM-mAbs, APA spheres loaded with either anti-CD3ε or anti-CD95 mAb were incorporated into scaffolded islet implant (SI) test-beds and the SIs implanted into a mouse model of autoimmune (type 1) diabetes. Release of mAbs within the implanted SIs resulted in reduced autoimmune responses to both transplanted and native islets. Notably, mice implanted with APA spheres loaded with quantities of anti-CD95 mAb that would be lethal if given systemically showed immunomodulation with no toxic side effects. Collectively, our results indicate that APA spheres are a relatively simple means to evaluate the effects of local, controlled release of IM-mAbs in a way that preserves mAb function and limits systemic toxicity.

Local, Controlled Release In Vivo of Vascular Endothelial Growth Factor Within a Subcutaneous Scaffolded Islet Implant Reduces Early Islet Necrosis and Improves Performance of the Graft.

Islet transplantation remains the only alternative to daily insulin therapy for control of type 1 diabetes (T1D) in humans. To avoid the drawbacks of intrahepatic islet transplantation, we are developing a scaffolded islet implant to transplant islets into nonhepatic sites. The implant test bed, sized for mice, consists of a limited (2-mm) thickness, large-pore polymeric sponge scaffold perforated with peripheral cavities that contain islets suspended in a collagen hydrogel. A central cavity in the scaffold holds a 2-mm diameter alginate sphere for controlled release of the angiogenic cytokine vascular endothelial growth factor ( VEGF). Host microvessels readily penetrate the scaffold and collagen gel to vascularize the islets. Here, we evaluate the performance of the implant in a subcutaneous (SC) graft site. Implants incorporating 500 syngeneic islets reversed streptozotocin-induced diabetes in mice approximately 30 d after SC placement. Controlled release of a modest quantity (20 ng) of VEGF within the implant significantly reduced the time to normoglycemia compared to control implants lacking VEGF. Investigation of underlying causes for this effect revealed that inclusion of 20 ng of VEGF in the implants significantly reduced central necrosis of islets 24 h after grafting and increased implant vascularization (measured 12 d after grafting). Collectively, our results demonstrate (1) that the scaffolded islet implant design can reverse diabetes in SC sites in the absence of prevascularization of the graft site and (2) that relatively low quantities of VEGF, delivered by controlled release within the implant, can be a useful approach to limit islet stress after grafting.

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.

Native fibrillar collagen membranes of micron-scale and submicron thicknesses for cell support and perfusion.

Fibrillar type I collagen is nontoxic, biocompatible, and possesses considerable strength and stability. In a study of scaffolds for use in laminated tissue substitutes, we examined the properties of membranes made from air-dried hydrogels of collagen fibrils that were polymerized from native, monomeric collagen. Planar collagen membranes (CMs) of 0.1-5.3 microm dry thickness were made by variation of the collagen concentration and/or the volume of the hydrogel. The planar CMs, which were comprised of a dense feltwork of long collagen fibrils 70-100 nm in diameter, showed considerable resistance to rupture and retained their membranous character after 6 weeks in tissue culture medium at 37 degrees C. CMs that were relatively thick when dry exhibited a greater proportional increase in rehydrated thickness and a greater diffusivity (when rehydrated) to 4.3 kDa dextran than did CMs that were relatively thin when dry. Hollow, tubular CMs of several configurations were prepared by embedment of solid, removable forms into collagen hydrogels prior to drying. By use of special fixtures, a planar CM that incorporated multiple, parallel tubes was fabricated. In summary, hydrogels of fibrillar collagen can be transformed into membranous structures suitable for tissue engineering applications.

Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment.

For several years, microgrooved substrates have been evaluated as a means to orient cells in engineered tissues. Recently, we fabricated thin (0.1-5.3 microm) planar and tubular collagen membranes (CMs) from air-dried hydrogels of native, fibrillar type I collagen (Vernon et al., Biomaterials 2004;26:1109-17). The CMs were strong, stable, and permeable and, hence, of potential use as scaffolds for tissue engineering. In the present study, planar CMs supported a robust attachment, spreading, and proliferation of human dermal fibroblasts (HDFs) and human umbilical artery smooth muscle cells (HUASMCs). Collagen hydrogels were air-dried onto microgrooved templates and subsequently removed in the form of grooved CMs with the potential to align cells. The grooved CMs were highly effective at inducing HDFs and HUASMCs to elongate and align, as revealed by scanning electron microscopy and by assays of f-actin and nuclear orientation. Alignment of cells was maintained at high cell densities. CMs with grooves of substantially different widths and depths were similarly effective in causing cell alignment; however, cells aligned poorly on CMs that had grooves less than 1 microm in depth. Grooved CMs with the capability to align cells might be of considerable use in the fabrication of tissue substitutes.

Elongation and secretion of tissue inhibitor of metalloproteinases 1 by human microvascular endothelial cells cultured in collagen gels is stimulated by mitomycin c.

During angiogenesis, interactions between endothelial cells (ECs) and the surrounding extracellular matrix are influenced by matrix metalloproteinases (MMPs) and their cognate inhibitors, the TIMPs. The authors discovered that the secretion of TIMP-1 by human microvascular ECs (hmECs) cultured within gels of native, fibrillar collagen was increased robustly by mitomycin C (MMC), an inhibitor of cell proliferation. In contrast, hmECs cultured on plastic coated with gelatin or with native fibrillar collagen exhibited nil (on gelatin) or very modest (on native collagen) increases in TIMP-1 upon exposure to MMC. Notably, none of the cultures altered the secretion of TIMP-2, or MMP-1 and -2, in response to MMC. hmECs cultured within collagen gels elongated significantly after exposure to MMC, a response the authors concluded was mediated by TIMP-1, because elongation could be inhibited completely with a function-blocking antibody to TIMP-1. Moreover, substitution of purified human TIMP-1 for MMC induced a similar elongation by hmECs. hmECs cultured within collagen gels did not proliferate under the conditions used in this study; therefore, inhibited proliferation was not a factor in the altered cell shape and TIMP-1 secretion elicited by MMC. These results illustrate that antiproliferative compounds should be used with caution in studies of MMP regulation by ECs.

Overexpression of decorin by rat arterial smooth muscle cells enhances contraction of type I collagen in vitro.

OBJECTIVE: Overexpression of decorin reduces neointimal thickening in balloon-injured carotid arteries of rats by decreasing the volume of neointimal extracellular matrix (ECM). We examined the hypothesis that decorin regulates ECM volume by stimulating cell-mediated contraction of collagen-rich ECMs. METHODS AND RESULTS: Rat arterial smooth muscle cells (ASMCs) transduced with bovine decorin cDNA by retroviral transfection (LDSN) exhibited enhanced contraction of collagen gels in vitro when compared with vector-only transduced (LXSN) cells. Addition of recombinant decorin to LXSN or LDSN cells did not stimulate contraction of collagen gels. Enhanced contraction of collagen by LDSN cells was unaffected by the metalloproteinase inhibitor GM6001. LDSN cells exhibited increased expression of type I collagen mRNA when compared with that of LXSN cells. Correspondingly, collagen gel contraction by LDSN cells was reduced by inhibition of collagen synthesis by 3,4-l-dehydroproline (L-DHP). Antibodies to alpha1beta1-integrin, but not to alpha2beta1-integrin, blocked collagen contraction by both LXSN and LDSN cells. However, LXSN and LDSN cells expressed similar levels of alpha1- and beta1-integrin mRNAs. CONCLUSIONS: Decorin synthesized de novo by ASMCs increases type I collagen synthesis and enhances contraction of collagen gels. Regulated synthesis of decorin may be a useful therapeutic approach to reduce ECM volume in vascular disease.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

New technologies in vitro for analysis of cell movement on or within collagen gels.

The movement of cells through extracellular matrix (ECM) is a critical component of many normal and pathological processes in vivo. Consequently, efforts to characterize motility-associated interactions between cells and ECM have led to the development of methods to observe and quantify (assay) the movement of cells under simplified conditions in vitro. In this report, we describe a novel method (the bullseye assay) and apparatus for the concentration of cells into small, precisely sized and shaped circular disks (bullseyes) that serve as starting points for migration of cells within ECM. The same apparatus is used to form the bullseyes and position them at the center of flat disks (windows) of gelled collagen that are supported at the edges by rings of nylon mesh. Complete assemblies, each consisting of a bullseye, collagen window and nylon mesh ring, are transferred to tissue culture wells for assay of cell migration either within or on top of the collagen window. Studies of the migratory responses of three different cell types to specific cytokines demonstrated that the bullseye assay was sensitive, rapid to set up, and easy to use. In conjunction with the bullseye assay, we developed a novel annular grayscale method for quantification of cell migration from digital images. The method is easily mastered, is derived from a measurement program in the public domain, is not subjective and is more discriminative than other techniques of measurement.

Inhibited angiogenesis in aging: a role for TIMP-2.

Factors responsible for age-associated impairment of angiogenesis are poorly understood. We observed that in aged mice, new fibrovascular tissue within subcutaneous polyvinyl alcohol sponges expressed more tissue inhibitor of metalloproteinases (TIMP)-2 than did corresponding tissue from young mice. In complementary studies in vitro, we utilized young and aged human microvascular endothelial cell lines (hmEC36 and hmEC90, respectively) and compared their morphogenetic capacity within three-dimensional collagen. HmEC90 exhibited poor formation of tubular, capillary-like structures in vitro, diminished expression of active matrix metalloproteinase (MMP)-2, and similar or lesser amounts of MT1-MMP relative to hmEC36. Correspondingly, the MMP inhibitor GM6001 decreased tubulogenesis by hmEC36 to levels observed for hmEC90. In vitro, hmEC90 expressed similar quantities of TIMP-1, but more TIMP-2 than did hmEC36. Accordingly, purified TIMP-2 inhibited tubulogenesis by hmEC36. Collectively, our studies indicate that elevated levels of TIMP-2 modulate decreased angiogenesis in aged tissues, most likely via TIMP-2-mediated inhibition of MMP-2 and MT1-MMP.

An improved method for the collagen gel contraction assay.

The collagen gel contraction (CGC) assay is used frequently to study the cell-mediated reorganization of the extracellular natrix. In a typical CGC assay, cells embedded in a disk-shaped lattice (gel) of native type I collagen fibers compress the fibers and, consequently, reduce the diameter of the collagen disk within h or d. The degree to which the collagen is contracted is usually quantified by measurement of the diameter or the area of the disk. During CCC assays, friction or adhesion (or both) between gels and their culture containers can cause gels to be incompletely contracted or to acquire distorted shapes. Such occurrences degrade the reproducibility and reliability of measurements of gel dimensions. To address these problems, we developed an oil-supported collagen retraction (OSCR) assay that creates an environment of low friction and adhesion around the contracting collagen gel. The OSCR assay is accomplished with simple equipment and is easily performed, sensitive, and consistently yields fully contracted gels with minimal distortion.

ADAMTS-4 and biglycan are expressed at high levels and co-localize to podosomes during endothelial cell tubulogenesis in vitro.

Proteolysis of the extracellular matrix influences vascular growth. We examined the expression of ADAMTS-1, -4, and -5 metalloproteinases and their proteoglycan substrates versican, decorin, and biglycan as human umbilical vein endothelial cells (HUVECs) formed tubes within type I collagen gels in vitro. Tubulogenic and control HUVEC cultures expressed low levels of ADAMTS-1 and -5 mRNAs, but ADAMTS-4 mRNA was relatively abundant and was significantly elevated (as was ADAMTS-4 protein) in tubulogenic cultures versus controls. Immunocytochemistry revealed ADAMTS-4 in f-actin- and cortactin-positive podosome-like puncta in single cells and mature tubes. Tubulogenic and control cultures expressed low levels of versican and decorin mRNAs; however, peak levels of biglycan mRNA were 400- and 16,000-fold that of versican and decorin, respectively. Biglycan mRNA was highest at 3 hr, declined steadily through day 7 and, at 12 hr and beyond, was significantly lower in tubulogenic cultures than in controls. Western blots of extracellular matrix from tubulogenic cultures contained bands corresponding to biglycan and its cleavage products. By immunocytochemistry, biglycan was found in the pericellular matrix surrounding endothelial tubes and in cell-associated puncta that co-localized with ADAMTS-4 and cortactin. Collectively, our results suggest that ADAMTS-4 and its substrate biglycan are involved in tubulogenesis by endothelial cells.

IL-10 induction from implants delivering pancreatic islets and hyaluronan.

Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

Reversal of diabetes in mice with a bioengineered islet implant incorporating a type I collagen hydrogel and sustained release of vascular endothelial growth factor.

We have developed a bioengineered implant (BI) to evaluate strategies to promote graft survival and function in models of islet transplantation in mice. The BI, sized for implantation within a fold of intestinal mesentery, consists of a disk-shaped, polyvinyl alcohol sponge infused with a type I collagen hydrogel that contains dispersed donor islets. To promote islet vascularization, the BI incorporates a spherical alginate hydrogel for sustained release of vascular endothelial growth factor (VEGF). BIs that contained 450-500 islets from syngeneic (C57Bl/6) donors and 20 ng of VEGF reversed streptozotocin (STZ)-induced diabetes in 100% of mice (8/8), whereas BIs that contained an equivalent number of islets, but which lacked VEGF, reversed STZ-induced diabetes in only 62.5% of mice (5/8). Between these "+VEGF" and "-VEGF" groups, the time to achieve normoglycemia (8-18 days after implantation) did not differ statistically; however, transitory, postoperative hypoglycemia was markedly reduced in the +VEGF group relative to the -VEGF group. Notably, none of the mice that achieved normoglycemia in these two groups required exogenous insulin therapy once the BIs began to fully regulate levels of blood glucose. Moreover, the transplanted mice responded to glucose challenge in a near-normal manner, as compared to the responses of healthy, nondiabetic (control) mice that had not received STZ. In future studies, the BIs described here will serve as platforms to evaluate the capability of immunomodulatory compounds, delivered locally within the BI, to prevent or reverse diabetes in the setting of autoimmune (type 1) diabetes.

Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contraction.

Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of (35)S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM.