Towards retrievable vascularized bioartificial pancreas: induction and long-lasting stability of polymeric mesh implant vascularized with the help of acidic and basic fibroblast growth factors and hydrogel coating.

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

Expression of capillary basement membrane components during sequential phases of wound angiogenesis.

Before capillaries sprout to form new vessels in a wound, the endothelial cells are sequestered from the surrounding stromal or provisional matrix by a well organized protein envelope called the basement membrane (BM). After breaching the BM, endothelial cells are exposed to the wound provisional matrix and begin to migrate and proliferate. Endothelial derived basement membrane proteins and molecules of the provisional matrix are mutually accessible to the endothelial cell surface during migration. Eventually, new capillaries again segregate in a formed envelope of basement membrane and resume a tubular morphology. Endothelial cell recognition of the architecture and concentration of basement membrane ligands appears to be an important determinant of capillary morphology during angiogenesis. In this study, we characterized the molecular composition, expression and steady-state transcript levels of BM proteins during sequential stages of wound angiogenesis. Specific analysis of rat capillary BM transcripts was achieved by employing a space-filling wound model which did not have non-capillary BM. Invading capillaries appeared between days 3 and 5 and matured by day 12. Occasionally, vessels larger than capillaries were observed to form by a process resembling vasculogenesis. Steady-state transcript levels for subunits of all major BM proteins studied were readily measured by day 3, and laminin and type IV collagen immunostaining were evident at the periphery of all vessels studied. From vessel initiation to regression, the transcript levels most changed were the alpha 1 and alpha 2 transcripts of type IV collagen; after an early peak, they exhibited a sharp three-fold decline as the response progressed. Conversely, entactin and laminin subunits did not decline as the response progressed, suggesting an increasing ratio of expression relative to type IV collagen. Perlecan expression was inconsistent, but it appeared to decline during the late phase of the response. Laminin beta 1 and gamma 1, but not alpha 1 or beta 2, transcripts were expressed by forming capillaries, providing evidence that the laminin 1, 3, 4 and 7 isotypes are not expressed by growing capillaries. These results also demonstrate that the steady-state ratios of type IV collagen transcripts to laminin and entactin transcripts are greatest during the early proliferative-migratory phase of angiogenesis but decrease significantly in later phases, when vessel maturation and tube formation predominate.

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth.

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.

A laminin-pepsin fragment with cell attachment and neurite outgrowth activity at distinct sites.

Laminin is a large basement membrane glycoprotein which influences the behavior and morphology of a variety of cells. We have found that laminin and a pepsin fragment of laminin (P-lam) contain distinct sites for HT-1080 human fibrosarcoma cell attachment and for neurite outgrowth activity of PC12 and NG108-15 cell lines. Reduction and alkylation of laminin and P-lam fragment disulfide bonds, in the absence of denaturing agents, markedly reduced the cell attachment activity without reducing the neurite outgrowth response. The P-lam fragment (approximately 375 kDa) was found to contain part of the cross region of laminin and a portion of the long arm, on the basis of recognition by antisera against laminin synthetic peptides and fusion proteins. Modification of arginine residues by cyclohexanedione also had no effect on neurite outgrowth but reduced HT-1080 cell adhesion. Modification of lysine residues by succinic and citraconic anhydride, however, abolished laminin neurite outgrowth but not cell attachment activity. Neurite outgrowth activity was recovered by reversing the lysine modification. These data support the existence on laminin of separate sites for cell attachment and for neurite outgrowth.

A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth.

Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19-mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior.

Heterogeneity of elastin expression in cutis laxa fibroblast strains.

Cutis laxa is a genetically heterogeneous connective tissue disease that occurs in both inherited and acquired forms. The most apparent defect is loose, redundant, nonresilient skin, but systemic connective tissue abnormalities exist, especially in conjunction with the early onset or autosomal recessive variety. The elastic fiber shows morphologic alterations. We studied dermal skin biopsies and cultured skin fibroblasts from 6 patients with congenital forms of cutis laxa in an effort to correlate alterations in elastin morphology and metabolism. In general, ultrastructural analysis revealed occasional variance in collagen fiber diameter, whereas elastic tissue varied in content, appearance, and the proportion and manner by which elastin and microfibrillar component associated. Fibroblast cell lines comprised of normal donors from a similar age group produced an average of 35 +/- 10 X 10(3) tropoelastin molecular equivalents per cell per hour, as measured by an ELISA. Three of six cutis laxa cell strains were markedly (5-20-fold) reduced in tropoelastin production. Two of these cell strains had specifically reduced levels of tropoelastin production relative to total protein synthesis. Analysis of elastin specific messenger RNA levels indicated this reduced expression of tropoelastin was regulated at a pretranslational level. In other strains, diminished production of elastin did not appear to be the primary defect, underscoring the heterogeneous nature of cutis laxa at both the biochemical and ultrastructural levels.

Laminin A chain synthetic peptide which supports neurite outgrowth.

Neurons from peripheral and central nervous tissue as well as from established cell lines respond to low concentrations of laminin with rapid extension of axon-like processes. Two sites on laminin have been identified which stimulate neurite outgrowth, the major site residing at the end of the long arm of laminin. Recently laminin has been cloned and sequenced allowing for synthetic peptides to be prepared and tested for biological activity. We report here that antisera against synthetic peptides corresponding to A and to B1 chain sequences at the end of the long arm can partially inhibit laminin-mediated neurite outgrowth. Further, we show that a 19 amino acid synthetic peptide (CSRARKQAASIKVAVSADR) from the long arm of the laminin A chain is capable of stimulating neuronal-like process formation to almost the same extent as laminin and competes with laminin for stimulation of neurite outgrowth.

Laminin neural activity and binding proteins.

The process of neurite extension is complex and mechanisms involved probably vary depending on the local microenvironment (tissue site, extracellular matrix, neighboring cells, humoral factors, and developmental stage) of the developing or repairing neuron. Laminin contains at least one and perhaps more sites capable of stimulating process formation in a variety of neuronal cell types. This site contains a lysine and is likely located near a complex sugar residue. A complex of laminin and heparan sulfate proteoglycan appears to form a unique site with comparable activity, which is defined by monoclonal antibody recognition. Cell receptor complexes capable of recognizing the fibronectin cell attachment peptide RGD also bind laminin, while antisera to these complexes inhibit neurite outgrowth in some instances. The identification of laminin active sites and corresponding cell receptors could open new approaches to improving nerve regeneration in both the peripheral and central nervous system.

Increased elastin production by progeria skin fibroblasts is controlled by the steady-state levels of elastin mRNA.

Hutchinson-Gilford progeria is a unique, rare disease with markedly accelerated aging. The average lifespan of affected individuals is 12 years. Although the biochemical basis of the syndrome is unknown, its influence appears to be primarily upon mesodermal tissues. Characteristics such as the altered appearance of the skin and the extensive and fatal involvement of the cardiovascular system led us to study elastin production in cultured skin fibroblasts from three progeroid individuals. We found tropoelastin production by progeroid cells was elevated six- to nine-fold at the protein and mRNA levels, while relative collagen synthesis was similar to control strains. There was little difference between progeroid and normal cells in expression of total protein or in total cellular mRNA content. Western blot analysis of tropoelastin from progeroid fibroblasts confirmed increased production of elastin but revealed no gross changes in the molecular mass. The significant increase in tropoelastin expression lends support to the concept that progeria results from a mesenchymal dysplasia, and offers a possible biochemical marker for the phenotype.

Use of extracellular matrix components for cell culture.

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.

Developmental initiation of elastin gene expression by human fetal skin fibroblasts.

Elastin synthesis is initiated in many organs during the latter part of fetal development. By birth, accumulation of elastin in elastic fibers accounts in large part for the integrity and resilience of skin, blood vessels, and lungs. Developmental studies in several connective tissues of nonhuman vertebrates indicate that elastin synthesis is rapidly initiated during fetal life and that its expression is largely controlled by the abundance of its mRNA. Previous evidence for elastin synthesis in the developing human fetus has been derived from either histologic inference or studies of net accumulation. We now report that the developmental induction of cutaneous elastin synthesis appears to be stably reflected in cell culture. Production of elastin by human skin fibroblasts increased 7- to 14-fold between 17 and 19 weeks of gestation, reaching the levels found in neonatal skin fibroblasts. Consistent with other developmental studies, elastin synthesis was found to be under pretranslational control with relative mRNA levels increasing 6- to 15-fold by 19 weeks of gestation. Under the same circumstances, collagen expression and total protein synthesis were relatively constant among all strains examined. Human skin fibroblasts may thus be a useful system for examining developmentally regulated elastin gene expression.

Elastin production in human skin fibroblast cultures and its decline with age.

Recent studies have established that cultured human skin fibroblasts secrete the soluble precursor of elastin, tropoelastin (TE). The present studies evaluate, by an enzyme-linked immunosorbent assay, the stability of the TE phenotype and the effect of culture conditions and donor age on TE accumulation by human skin fibroblasts. Tropoelastin was maximally produced by 2 control fibroblast strains at early confluency (32-49 X 10(3) molecules/cell/h), and its serum-dependent accumulation in the medium was linear for at least 72 h. Inhibition of cross-linking had no effect on the rate of elastin production. Optimum serum concentrations for TE production differed for fibroblast cell strains derived from foreskin and trunk skin fibroblasts. Production of TE by human skin fibroblasts was stable through nearly 30 population doublings after which there was a greater than 2-fold decline in the rate of accumulation. In a cohort of donor strains, TE production appeared to decline at donor ages greater than or equal to 70 years. Under standard culture conditions, cell strains from normal donors of various ages produced TE at rates ranging from 25-69 X 10(3) molecules/cell/h. Rates of TE accumulation in medium were not significantly altered by degradation of TE, as a variety of cell strains tested exhibited minimal cell-associated elastolytic activity. Based on the demonstration of a stable elastin phenotype, skin fibroblast cultures provide a new system for studying regulation of elastin biosynthesis and evaluating potential defects in elastin metabolism associated with certain connective tissue disorders.

Demonstration of elastin gene expression in human skin fibroblast cultures and reduced tropoelastin production by cells from a patient with atrophoderma.

Atrophoderma is a rare dermal disorder characterized by a patchy distribution of areas apparently devoid of elastic fibers. Skin fibroblast cultures were established from the normal and affected dermis of a patient with this disorder. Human tropoelastin was identified in culture medium by use of electroblotting and anti-elastin antisera. An enzyme-linked immunosorbent assay was used to establish that significantly less elastin accumulated in the media of cultured cells from lesional fibroblasts over a 3-d period. Since elastin biosynthesis in most tissues is under pretranslational control, molecular hybridization to a nick-translated genomic elastin probe was performed; however, elastin messenger RNA levels were equivalent in both cell strains. Both strains produced less elastin than did normal skin fibroblasts. Extracellular proteolysis of elastin was evaluated as a possible mechanism. Elastase activity was increased and porcine tropoelastin was degraded four times faster, on a per-cell basis, in lesional fibroblast cultures than in cells derived from an unaffected site. The two cell strains exhibited no significant differences in collagen production or collagenase activity. These results are the first demonstration of elastin production by cultured human skin fibroblasts, and they suggest that the primary defect in atrophoderma may be a result of enhanced degradation of newly synthesized elastin precursors.