Vascular endothelial growth factor-A (VEGF) is required for endothelial cell differentiation and survival. To investigate the renoprotective properties of VEGF in diabetes an inducible Cre-loxP gene targeting system was used to excise VEGF from podocytes of adult mice (VEGFKO). Diabetes was induced by streptozotocin (STZ) at 2.5 weeks of age and VEGFKO was induced by doxycycline (dox) at 3-4 weeks of age. Blood and urine were collected weekly to monitor for hyperglycaemia and proteinuria, respectively. Mice were dissected 8 weeks after diabetes induction or earlier if morbidly ill; twenty percent of the mice in the DM+VEGFKO group died before the surrogate endpoint. Glomerular VEGF mRNA expression was decreased in VEGFKO mice compared to controls. However, DM+VEGFKO mice had significantly greater proteinuria, degrees of glomerular sclerosis, and glomerular cell apoptosis. These results confirm that VEGF is normally upregulated in diabetes but reducing VEGF expression in diabetes causes severe kidney injury.

Vascular endothelial growth factor-A (VEGF) is required for endothelial cell differentiation and survival. To investigate the renoprotective properties of VEGF in diabetes an inducible Cre-loxP gene targeting system was used to excise VEGF from podocytes of adult mice (VEGFKO). Diabetes was induced by streptozotocin (STZ) at 2.5 weeks of age and VEGFKO was induced by doxycycline (dox) at 3-4 weeks of age. Blood and urine were collected weekly to monitor for hyperglycaemia and proteinuria, respectively. Mice were dissected 8 weeks after diabetes induction or earlier if morbidly ill; twenty percent of the mice in the DM+VEGFKO group died before the surrogate endpoint. Glomerular VEGF mRNA expression was decreased in VEGFKO mice compared to controls. However, DM+VEGFKO mice had significantly greater proteinuria, degrees of glomerular sclerosis, and glomerular cell apoptosis. These results confirm that VEGF is normally upregulated in diabetes but reducing VEGF expression in diabetes causes severe kidney injury.

Bibliography: leaves 119-136 / ix, 136 leaves, [9] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2006" Vita. Includes bibliographical references.

Recreating organ-specific microenvironments of the extracellular matrix (ECM) in vitro has been an ongoing challenge in biofabrication. In this study, I present a biofunctional ECM-mimicking protein scaffold with tunable biochemical, mechanical and topographical properties. This scaffold, formed by microfibres, displays three favorable characteristics as a cell culture platform: high-loading of key ECM proteins, single-layered mesh membrane with controllable mesh size, and flexibility for supporting a range of cell culture configurations. Decellularized extracellular matrix (dECM) powder was used to fabricate this protein scaffold, as a close replicate of the chemical composition of physiological ECM. The highest dECM concentration in the solidified protein scaffold was 50 wt%, with gelatin consisting the rest. In practice, a high density of dECM-laden nano- to microfibres was directly patterned on a variety of substrates to form a single layer of mesh membrane, using the low-voltage electrospinning patterning (LEP) method. The smallest fibre diameter was measured at 450 nm, the smallest mesh size of the membrane was below 1 μm, and the thickness of the membrane was estimated to be less than 2 μm. This fabrication method demonstrated a good preservation of the key ECM proteins and growth factors, including collagen IV, laminin, fibronectin, VEGF and b-FGF. The integrated fibrous mesh exhibited robust mechanical properties, with tunable fibril Young’s modulus for over two orders of magnitude in the physiological range (depending on the dECM concentration). Combining this mesh membrane with 3D printing, a cell culture device was constructed. Co-culture of human glomerulus endothelial cells and podocytes was performed on this device, to simulate the blood-to-urine interface in vitro. Good cell attachment and viability were demonstrated, and specific cell differentiation and fibronectin secretion were observed. This dECM-laden protein scaffold sees the potential to be incorporated into a glomerulus-on-chip model, to further improve the physiological relevance of in vitro pathological models.

Abstract Basement membranes (BM) underlie epithelia and endothelia and surround many tissues. In cutaneous BM epithelial cells are attached to the stroma via multiprotein complexes called hemidesmosomes (HD). Collagen XVII and integrin α6β4 are components of HD and they bind to laminin 332, a component of anchoring filaments, extracellularly. The main interest of this study is the function of collagen XVII and its interactions with these proteins. What is known about the function of collagen XVII is mostly derived from its role as an adhesive component in cutaneous HD. Here we demonstrate for the first time that collagen XVII is expressed by podocytes in the human and murine glomerulus and that mutant mice lacking collagen XVII in addition to small size, blisters and diffuse hair loss, also have deficient glomerular development and a high mortality rate. We also show for the first time at the protein level that collagen XVII is expressed, and probably has a functional interaction with laminin 332, in normal colon epithelia. We demonstrate that collagen XVII is expressed by the invasive cells of human colorectal carcinoma (CRC) samples and its immunostaining is increased in metastasis in CRC. The higher proportion of collagen XVII positive tumor cells correlates with decreased disease-free survival and cancer-specific survival times and we also suggest a functional interaction between collagen XVII and laminin 332 in CRC. Previous studies have suggested that collagen XVII participates in keratinocyte migration by affecting the correlation of HD disassembly and assembly, its expression is increased in squamous cell carcinoma (SCC) and it may have a role in cell adhesion and migration in SCC carcinogenesis. Here we demonstrate upregulated collagen XVII, integrin β4 and laminin γ2 expression in actinic keratosis, Bowen’s disease and SCC. The expression of collagen XVII was increased with a high degree of variation, especially in samples taken from areas where SCC is particularly invasive. We also demonstrate in the SCC-25 cell line that lack of collagen XVII or integrin β4 severely disrupts the adhesion, migration and invasivity of these cells. Taken together, in this study we show that collagen XVII is needed for normal glomerular development, is expressed in normal colon epithelia and participates in CRC and SCC carcinogenesis together with laminin 332 and integrin β4. / Tiivistelmä Tyvikalvot sijaitsevat epiteelin ja endoteelin alla ja ympäröivät monia kudoksia. Ihon tyvikalvossa epiteelisoluja alla olevaan verinahkaan kiinnittää rakenne, jota kutsutaan hemidesmosomiksi (HD). Kollageeni XVII ja integriin α6β4 ovat HD:n rakenneproteiineja. Ne kiinnittyvät solun ulkopuolella laminiin 332 nimiseen proteiiniin, joka muodostaa ankkurifilamentit. Kollageeni XVII ilmentyminen ja toiminta yhdessä näiden kahden proteiinin kanssa on tämän tutkimuksen keskeisin kohde. Valtaosa tutkimuksista, jotka käsittelevät kollageeni XVII:ää, koskevat sen toimintaa ihon keratinosyyteissä. Tässä tutkimuksessa osoitimme ensi kertaa, että hiiren ja ihmisen munuaiskerästen podosyyttisolut ilmentävät kollageeni XVII. Geenimanipuloidut hiiret, joilta kollageeni XVII oli poistettu, olivat pieniä, kehittivät rakkuloita ja karvattomuutta, niillä oli korkea kuolleisuus ja niiden munuaiskerästen kehitys oli häiriintynyt. Kollageeni XVII esiintymistä proteiinitasolla, sekä mahdollista toiminnallista yhteyttä laminiin 332:een, ei aiemmin ole osoitettu paksusuolen epiteelissä. Havaitsimme, että paksu- ja peräsuolen adenokarsinooman (CRC) invasiivinen solukko ilmentää kollageeni XVII:ää, kollageeni XVII esiintyminen on merkittävän voimakasta CRC:n metastasoinnin yhteydessä ja lisääntynyt kollageeni XVII esiintyminen lyhentää syöpävapaata aikaa ja heikentää syöpäspesifistä selviytymistä. Myös CRC:ssä kollageeni XVII toiminta voi liittyä laminiini 332:een. Aiempien tutkimusten mukaan kollageeni XVII osallistuu keratinosyyttien migratioon vaikuttamalla toimivien HD:ien määrään. Sen määrän on havaittu olevan korkeampi okasolusyövässä (SCC) ja sen on ehdotettu osallistuvan syöpäsolujen adheesioon ja migraatioon SCC:n kehittyessä. Me osoitimme kohonneen kollageeni XVII, integriini β4 ja laminiini γ2 ilmenemisen aktiinisessa keratoosissa, Bowenin taudissa sekä SCC:ssä. Kollageeni XVII määrä oli korkea, mutta vaihteli paljon, sekä hiiren että ihmisen invasiivisilla SCC alueilla. Havaitsimme myös SCC-25 solulinjalla, että kollageeni XVII tai integriini β4 puutos häiritsee vakavasti solujen adheesiota, migraatiota ja invaasiota. Yhteenvetona tässä työssä osoitimme, että kollageeni XVII:ää tarvitaan munuaiskerästen kehittymisessä, sitä esiintyy paksusuolen epiteelissä, ja että kollageeni XVII osallistuu CRC:n ja SCC:n kehittymiseen yhdessä integriini β4:n ja laminiini 332:n kanssa.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.

No description available.

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