Progressive modulation of endothelial phenotype during in vitro blood vessel formation.

"Sprouting" vascular endothelial cells were used as an in vitro model system to study the progressive morphologic and biosynthetic changes associated with the formation of tubular structures. In vitro, sprouting endothelial cells formed spontaneously without the addition of any exogenous factors from cultures of cloned endothelium exhibiting a polygonal/cobblestone phenotype. These phenotypically variant endothelial cells differentiated to form associated cell networks or nodules which gradually reorganized into tubular structures. Concomitant with these morphologic changes, the biosynthesis of extracellular matrix proteins was modulated, as determined by Northern blot analysis, metabolic labeling, and immunocytochemistry. The initial sprouting phase was characterized by the induction of type I collagen synthesis and the appearance of fibronectin containing the ED-A domain, in comparison to their absence in cloned cultures displaying a stable polygonal/cobblestone phenotype. The organizational stage, where the sprouting endothelial cells assembled into tubular structures, was additionally characterized by the expression of type IV collagen. These studies demonstrate that the progression from polygonal/cobblestone to sprouting cultures, and subsequent tubular organization, involves major alterations in extracellular matrix protein expression. This developmental phenomenon, although not completely analogous to blood vessel formation in vivo, nevertheless may be helpful in understanding the role of matrix macromolecules in the angiogenic process.     

Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.     

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.     

Effects of biaxial deformation on pulmonary artery endothelial cells

An apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (Mitrathane). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation-producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec-1 and a duty cycle of 0.5 sec-1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.     

An ultrastructural study of adhesive junctions in reaggregates of unincubated chick embryos.

Cell suspensions obtained by the dissociation of unincubated chick embryo blastoderms were allowed to reaggregate on a gyratory shaker for 24-48 hours. The reaggregates which form during this period consist of an inner phase of tightly packed cohesive cells surrounded by an external phase of loosely packed cells. This sorted out arrangement achieves its definitive form between 24 and 48 hours of rotation culture. It was determined that the external phase consists of primitive ectoderm and that the internal phase consists of primitive endoderm. Both 24- and 48-hour reaggregates were examined in the electron microscope and observations were directed to areas of close membrane apposition between cells. In 48-hour reaggregates, primitive endoderm cells were joined by many specialized junctions (desmosomes). The formation of desmosomes in reaggregates of dissociated unincubated chick embryo cells was correlated with the sorting out process.     

Comparison of the intracellular lipids of cultured vascular endothelial cells and fibroblasts

Summary The lipids of human umbilical vein endothelial cells, calf aortic endothelial cells and foreskin fibroblasts have been compared. Cell cultures were established, and, upon confluency, the lipids were extracted and analyzed with respect to total lipid content, classes of lipids and total lipid fatty acid composition. The total quantity of lipid per milligram protein found in both human umbilical vein endothelium and calf aorta endothelium was similar to that found in fibroblasts grown in similar medium. Both types of endothelium contained the same major neutral lipid classes as fibroblasts, although they contained more phospholipid than did fibroblasts. The fatty acid composition of the three cells examined was influenced by cell type as well as the type of serum in the culture medium. This work was supported by PHS Grants HL16058, HL19638, AM14626 and HL18827.