Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

<p>Adherens junctions are calcium-dependent cell-cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl family of tyrosine kinases have been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins.</p><p>Here we use pharmacological inhibition and RNA interference to identify the Abl family kinases as critical mediators of cadherin-mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl-dependent regulation of the Rho-ROCK-myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto-myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain-of-function promotes adherens junction formation through a Crk-dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation.</p><p>Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl-Crk-Rac.</p><p>Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover.</p> / Dissertation

Nerve autografts are widely used clinically to repair nerve grafts. However, nerve grafts have many limitations, such as, availability of donor nerve grafts, and loss of function at donor site. To overcome these problems, we have used a tissue engineering approach to design three-dimensional (3D) agarose scaffolds containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) to mimic in vivo conditions to promote nerve regeneration in rats. To determine the effect of LN-1 gradients on neurite extension in vitro, dorsal root ganglia (DRG) from chick embryos were cultured in 3D hydrogels. A gradient of LN-1 molecules in agarose gels was made by diffusion technique. LN-1 was then immobilized to the agarose hydrogels using a photo-crosslinker, Sulfo-SANPAH (Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate). Anisotropic scaffolds with three different slopes of LN-1 gradients were used. Isotropic scaffolds with uniform concentrations of LN-1, at various levels, were used as a positive control. DRG cultured in anisotropic scaffolds with optimal slope of LN-1 gradient extended neurites twice as fast as DRG in optimal concentration in isotropic scaffolds. Also, in the anisotropic scaffolds the faster growing neurites were aligned along the direction of LN-1 gradient. To promote nerve regeneration in vivo, tubular polysulfone guidance channels containing agarose hydrogels with gradients of LN-1 and NGF (anisotropic scaffolds) were used to bridge 20-mm nerve gaps in rats. Nerve autografts were used as positive controls and isotropic scaffolds, with uniform concentration of LN-1 and NGF, were used as negative controls. After 4-months, the rats were sacrificed and nerve histology was done to test for nerve regeneration. Only anisotropic scaffolds and nerve autografts contained evidence of axonal regeneration. Both groups had similar numbers of myelinated axons and similar axonal-diameter distribution. However, nerve graft group performed better in functional outcome as measured by relative gastrocnemius muscle weight (RGMW) and electrophysiology. Optimization of performance of anisotropic scaffolds by varying the LN-1 and NGF concentration gradients might lead to development of scaffolds that can perform as well as nerve auotgrafts for nerve regeneration over long nerve gaps.

In pigs and other mammals, embryonic losses often occur during implantation when the conceptus (embryo plus its extra-embryonic membranes) attaches to the maternal uterine epithelium. Mechanisms controlling this process are not completely understood. Integrins and growth factors are among many molecules likely involved in controlling implantation. Numerous integrins (ITG), including subunits ITGAV (alpha v), ITGB1 (beta 1), ITGB3 (beta 3), and ITGB5 (beta 5), and transforming growth factor betas (TGFBs), in both latent and active forms, are present at the porcine conceptus-maternal interface. TGFBs are released as latent precursors which cannot interact with TGFBRs prior to their activation. Latency associated peptide (LAP), part of the TGFB latent complex, contains an amino acid sequence Arg-Gly-Asp (RGD) that is found in other extracellular matrix molecules and may interact with and signal through integrins. We hypothesize that LAP will bind to and activate ITGAV-containing heterodimers at the conceptus-maternal interface and that these interactions are a functional component of implantation. We also hypothesize that TGFB acting via TGFBRs has critical roles during peri-implantation, and such roles may include promoting conceptus development, survival, and adhesion. Immunofluorescence was used to colocalize TGFB, LAP, and integrins in porcine peri-implantation uterus and conceptus; immunohistochemistry of phosphorylated SMAD2/3 provided evidence of TGFB activity. Affinity chromatography identified cell surface integrins on porcine trophectoderm that are capable of binding LAP. In vivo, intrauterine infusions of LAP with its native RGD site (LAP-RGD) resulted in inhibition of conceptus elongation; LAP-RGE infusions yielded normal-appearing filamentous conceptuses at d13 of pregnancy. At d24, allantois length and fetal weights were greater in gilts which received LAP-RGE infusions compared to controls which received vehicle only. Results provide evidence for 1) active and latent TGFB in porcine conceptus and uterus; 2) receptor-ligand interactions of integrins and LAP; 3) integrin aggregation and potential focal adhesion formation at the conceptus-maternal interface; and 4) TGFB- and/or integrin-associated mechanisms which regulate conceptus elongation and placental and fetal size. Collectively, results suggest that TGFB and integrins are extensively involved in communication at the porcine conceptus-maternal interface, particularly regulating conceptus development, adhesion, and placental and fetal development.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Genetic factors playing an important role in disease susceptibility have long been suggested. Transforming growth factor-beta (TGF-beta) regulates differentiation and proliferation of T cells. Therefore, it could be a candidate gene for the development of systemic lupus erythematosus. Allelic polymorphisms in TGF-beta promoter region (-988, -800, -509) and in exon 1 (codon 10 and codon 25) have been suggested to associate with SLE susceptibility. Allelic polymorphisms at positions -988, -800, -509, codon 10 and codon 25 on TGF-beta gene in 138 SLE patients and 182 healthy controls were analyzed in this study. TGF-beta polymorphisms were determined by PCR amplification and sequencing. With the previous polymorphic data of interleukin-4 (IL-4) -590 and interleukin-10 (IL-10) -819, associations of cytokine genotyoe and allele frequencies were analyzed. Results showed that there were differences in the genotype distribution of TGF-beta promoter region at position -509 and in the signal sequence at codon 10 (Leu¡÷Pro) between case and control groups in this study. However, no significant differences were found for all the TGF-beta polymorphisms. Allele frequency of IL-10 -819 was significantly associated with the susceptibility of SLE (p = 0.011). No significant associations were found between lupus nephritis with all the cytokine polymorphisms, but CNS involvement and lung involvement were associated with the polymorphisms studied in this research.

No description available.

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NF£eB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, I£eB and it¡¦s subunit IKK£\, IKK£], IKK£^ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1£\ in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and £],£^-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

Lübeck, Med. Universität, Habilitation, 2000.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xii, 118 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 102-115). Also available on the World Wide Web.