Regeneration of elastic fibers by three-dimensional culture on a collagen scaffold and the addition of latent TGF-ß binding protein 4 to improve elastic matrix deposition.

The objective of this study was to investigate the effects of latent TGF-ß binding protein 4 (LTBP-4) on elastic fiber regeneration in three-dimensional cultures of human dermal fibroblasts (HDFs). Appropriate collagen scaffold for elastic fiber regeneration was also examined. Collagen sponges cross-linked at 120 °C and composed of small pores (25 µm on average) was favorable for elastic fiber regeneration by HDFs. Addition of LTBP-4, followed by culture for 21 days, accelerated elastic fiber accumulation within the scaffolds. Conditioned scaffolds containing either HDFs or LTBP-4-built mature elastic fibers were implanted between the dermis and the cutaneous muscle of mice. The combined use of HDFs and LTBP-4 resulted in thicker tissues containing elastic fibers. These results indicate that weakly cross-linked collagen sponges can be used as scaffolds for regenerating elastic fibers both in vitro and in vivo, and that the addition of LTBP-4 accelerates the deposition of both elastin and fibrillin-1, and increases cell proliferation. These techniques may be useful for generating cutaneous or cardiovascular tissue equivalents; furthermore, they may serve as a useful method for the three-dimensional analyses of drugs used to treat skin diseases or to examine the microstructure of elastin networks.

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor ß (TGF-ß), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-ß, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9-14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.

Local injection of latency-associated peptide, a linker propeptide specific for active form of transforming growth factor-beta1, inhibits dermal sclerosis in bleomycin-induced murine scleroderma.

Transforming growth factor-ß1 (TGF-ß1) has been thought to play a key role in the pathogenesis of scleroderma; however, therapeutic approaches targeting TGF-ß1 and/or related molecules have provided inconsistent results. In this study, we demonstrate the antifibrotic effects of local administration of latency-associated peptide (LAP), a linker propeptide that specifically converts the active form of TGF-ß1 to the inactive from, in the bleomycin (BLM)-induced scleroderma mouse model. Histologically, co-injection of BLM and LAP into the dorsal skin prevented proinflammatory and later sclerotic responses, features seen in mice injected with BLM alone or together with PBS as control. In addition, the skin sites co-injected with BLM and LAP showed a marked decrease in mast cell infiltration. Isoform-specific ELISA and real-time RT-PCR revealed transient decreases in connective tissue growth factor and collagen α1(I) mRNA expression 2 weeks after the co-injection, preceded by a decrease in active TGF-ß1 protein production. In contrast, the baseline expression of TGF-ß1 mRNA remained unchanged. By contrast, after induction of scleroderma by BLM, the inhibitory effects of LAP did not occur, suggesting time course-dependent TGF-ß1 regulation. Our data may have novel therapeutic implications regarding in vivo TGF-ß1 inactivation in human scleroderma, and the post-transcriptional interrelationship between major fibrogenic cytokines in the autoimmune aspects of the disease.

Therapeutic benefit of treatment with anti-thymocyte globulin and latent TGF-beta1 in the MRL/lpr lupus mouse model.

The pathogenesis of systemic lupus erythematosus is believed to involve defects in regulatory T cell (Treg) activity and abnormal activation of B and T lymphocytes. The purpose of this study was to test the therapeutic potential of rabbit anti-mouse thymocyte globulin (ATG), a lymphocyte-depleting agent, in conjunction with transforming growth factor (TGF)-beta1, a factor involved in the induction and expansion of Tregs. MRL/lpr mice with active disease were treated with ATG followed by a 12-day course of latent TGF-beta1 during the period of lymphocyte repopulation. Treatment with ATG + latent TGF-beta1 synergistically inhibited the progression of proteinuria and albuminuria and provided a significant improvement in long-term survival. This therapeutic benefit correlated histologically with reduced glomerular pathology and protein cast formation. The mechanism of action did not involve suppression of autoantibody formation but may involve the activity of CD4+CD25+FoxP3+ Tregs, which were found to be induced by ATG + TGF-beta1 treatment in vitro.