Bruce M. Pratt

Endothelial cell-matrix interactions: in vitro models of angiogenesis.

Introduction The endothelium is composed of a number of heterogeneous cell populations from a variety of vascular beds. Endothelial cells from these diverse beds share some common structures and functions but also exhibit a wide range of diversity in their morphological appearance, function, and response to injury (2,6,22). One factor that is thought to play key, if not pivotal, roles in the modulation of endothelial cell behavior is the extracellular matrix (21,24,2 5). A great deal of morphological evidence (at light and electron microscopic levels) supports the notion that the nature of the subendothelial matrix underlying endothelial cells varies depending on such criteria as size and type of artery and the particular microvascular bed examined (15,36,37). These differences in matrix, although still incompletely documented biochemically and immunochemicaliy, have led to the hypothesis that the subendothehal matrix is a determining factor in modulating the behavior of the overlying endothelial cell population. In this review we will discuss data, accrued from in vitro systems, that support this notion. Both large vessel (arterial) and microvascular (capillary) endothelial cell culture systems will be discussed. Because of the limited length of this review, its content is selective and will focus on the data of only several of the many laboratories actively working in this area.

Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH). rhTSH was expressed by co-transfecting a human α-subunit cDNA with a human β-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be >99% by SDS polyacryl-amide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

Endothelial Cell-Extracellular Matrix Interactions

The role of extracellular matrix as a modulator of cell behavior is widely accepted and is currently under intensive study by a number of investigators using diverse cell, tissue, and organ culture systems.10,34 Several general findings have emerged from this work, namely, that cell behavior is dramatically different when cells are grown and maintained on extracellular matrix as compared to tissue culture plastic or glass; and that cell behavior can be modulated, depending on the composition and organization of the matrix component(s) or tissue used.35 For example, in recent studies Lwebuga-Mukasa et al. have demonstrated that cultured type II pneumocytes exhibit variable behavior patterns, depending on the nature of the underlying matrix on which they are cultured. When cultured on the stromal aspect of the acellular amnionic membrane, these cells appear flattened, having few cytoplasmic lamellar bodies, basolateral junctional complexes, and apical microvilli. In contrast, when cultured on the basement membrane surface of the amnion, they maintain their cuboidal morphology and have abundant microvilli, basolateral junctional complexes, and cytoplasmic lamellar bodies.21,22 Additionally, in both instances the cultured cells produce a basal lamina-like structure. Conversely, when cultured and maintained on an acellular pulmonary basement membrane, these cells do not synthesize a basal lamina. With time they become flattened and attenuated, losing their cytoplasmic lamellar bodies and apical microvilli but maintaining their basolateral junctional complexes, suggesting a “differentiation” into type I pneumocytes.22 As a further example, Ingber et al. have studied the effects of extracellular matrix on the behavior of rat pancreatic adenocarcinoma cells. This tumor exhibits cytodifferentiation in vivo when closely associated with vascular or peritoneal connective tissue, suggesting the possibility

Differences in the biological activities of transforming growth factor-beta and platelet-derived growth factor in vivo.

Transforming growth factor-beta 1 (TGF-beta 1 and recombinant platelet-derived growth factor-BB (rPDGF-BB) promoted an extensive, dose-dependent development of fibrous connective tissue when continuously delivered for 8 days by mini-osmotic pumps implanted subcutaneously in adult guinea pigs. Biochemical analyses demonstrated that TGF-beta 1 and rPDGF-BB stimulated dose-dependent increases in the dry weight, and protein, DNA, collagen, and glycosaminoglycan (GAG) contents of the fibrous connective tissue capsule that enveloped the pumps. The GAG/DNA mass ratio was markedly elevated by TGF-beta 1, but the collagen/DNA, protein/DNA, and collagen/protein ratios were not significantly increased. In contrast, rPDGF-BB generally decreased these mass ratios. Histological analyses suggested that this was due to the fact that rPDGF-BB induced a very cellular response with a marked influx of neutrophils and fibroblasts. TGF-beta 1 induced significantly less cellular response, which consisted primarily fibroblasts and macrophages. These results indicated that rPDGF-BB and TGF-beta 1 induced connective tissue deposition in vivo in a dose-dependent fashion, although the cellular nature of the responses as well as the structural composition of the extracellular matrices were clearly distinguishable between the two growth factors.

Transforming Growth Factors-βl and β2 Induce Synthesis and Accumulation of Hyaluronate and Chondroitin Sulfate In Vivo

Subcutaneous implantation in rats of partially purified transforming growth factor-beta (TGF-beta) derived from bovine bone induced extensive development of connective tissue with associated edema. Subcutaneous injection of pure TGF-beta 1 or TGF-beta 2 also induced connective tissue deposition in mice and guinea pigs. Sustained release of TGF-beta 1 from mini-osmotic pumps implanted subcutaneously in mature guinea pigs promoted connective tissue deposition that encapsulated the pumps. Biochemical analyses of the connective tissue capsule demonstrated that TGF-beta 1 induced a dose-dependent accumulation of glycosaminoglycans (GAGs). The GAG/DNA ratio also increased as a function of the rate of TGF-beta 1 released, suggesting that the factor increased production of GAGs per cell. Cellulose acetate gel electrophoresis of the GAGs and hydrolysis with specific glycosidases revealed that the majority of GAGs consisted of hyaluronate and chondroitin sulfate. These results demonstrate that TGF-beta 1 and TGF-beta 2 stimulate the production of not only collagenous extracellular matrix components, but also dramatically increase the in vivo synthesis of hyaluronate and chondroitin sulfate.