RESUMEN
Microvascular endothelial cells isolated from abdominal skin of diabetic and nondiabetic adults were maintained in culture by serial passage. Both cell types showed typical endothelial cell morphology, expressed factor VII-associated antigen, contained Weibel-Palade bodies, and produced an extensive subendothelial extracellular matrix containing type IV (basement membrane) procollagen. Biosynthetic studies using radioactive amino acids indicated that under the conditions of cell culture the matrix proteins newly synthesized by MEC of both cell types were similar in type and amount. Both cell types produced type IV procollagen, laminin, and fibronectin, which were deposited in the matrix. Electron microscopy showed that the matrices of both cell types had a similar multilayered, discontinuous, filamentous ultrastructure. Immunoperoxidase staining showed type IV collagen to be distributed similarly, in a fibrillar meshwork, in both matrices. The extractability of individual matrix macromolecules from both matrices was identical; 4 M urea or guanidine-HCl partially removed fibronectin and thrombospondin, but reducing agent was required to solubilize type IV procollagen. The results suggest that diabetic microangiopathy is not due to an inherent defect in the endothelium, and that this in vitro system may be useful for examining environmental factors possibly involved in its development.
Asunto(s)
Diabetes Mellitus/metabolismo , Endotelio/metabolismo , Proteínas de la Membrana/biosíntesis , Procolágeno/biosíntesis , Piel/irrigación sanguínea , Anciano , Anciano de 80 o más Años , Membrana Basal/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotelio/citología , Endotelio/ultraestructura , Fibronectinas/biosíntesis , Humanos , Técnicas para Inmunoenzimas , Laminina/biosíntesis , Microscopía Electrónica , Persona de Mediana Edad , Piel/metabolismo , Piel/ultraestructuraRESUMEN
Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.
Asunto(s)
Capilares/metabolismo , Colágeno/biosíntesis , Piel/irrigación sanguínea , Membrana Basal/metabolismo , Células Cultivadas , Endotelio/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Recién Nacido , Cinética , Masculino , Peso Molecular , Pepsina A , Procolágeno/biosíntesis , Procolágeno/aislamiento & purificaciónRESUMEN
Nearly one-third of all young homosexual men diagnosed as having acquired immune-deficiency syndrome (AIDS) develop a disseminated form of dermal Kaposi's sarcoma (KS). Although the histogenesis of KS cells is unclear, certain evidence suggests that the aberrant cells are of endothelial derivation. We have examined the presence and distribution of connective tissue-specific and basement membrane-specific macromolecules by indirect immunofluorescence and immunoperoxidase staining of frozen sections in early cutaneous lesions of KS from individuals with AIDS. The KS cells typically line the spaces between collagen bundles of the reticular dermis. When stained for the connective tissue-specific glycoprotein fibronectin, all Kaposi's sarcoma lesions showed an intense staining pattern, revealing a complex array of linear deposits of antigen that outlined the exterior surface of the collagen bundles. Antibodies to laminin and type IV collagen, both basement membrane-specific macromolecules, produced an intense staining pattern similar to that found with the anti-fibronectin antiserum, indicating that all 3 antigens are closely codistributed. In contrast, antibodies to type I collagen, the major collagen of the dermis, uniformly stained the collagen bundles in the KS lesions and in the normal control skin. Antiserum to factor VIII-associated antigen, an antigen specific to blood vascular endothelium, frequently stained the KS lesions but the staining pattern was diffuse and of variable intensity. The results suggest that KS cells are derived from the endothelium of the blood microvasculature and maintain their secretory phenotype of secreting basement membrane-specific macromolecules.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Colágeno/análisis , Fibronectinas/análisis , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Sarcoma de Kaposi/complicaciones , Especificidad de Anticuerpos , Antígenos/análisis , Membrana Basal , Factor VIII/análisis , Factor VIII/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Laminina/inmunología , Masculino , Sarcoma de Kaposi/análisis , Factor de von WillebrandRESUMEN
We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.