In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro- and anti-angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1-6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non-angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

Abstract. Soluble guanylyl cyclase (sGC) catalyzes the biosynthesis of cGMP in response to binding of L-arginine-derived nitric oxide (NO). Functionally, the NO-sGC-cGMP signaling pathway in kidney and liver has been associated with regional hemodynamics and the regulation of glomerular parameters. The distribution of the ubiquitous sGC isoform α1β1 sGC was studied with a novel, highly specific antibody against the β1 subunit. In parallel, the presence of mRNA encoding both subunits was investigated by using in situ hybridization and reverse transcription-PCR assays. The NO-induced, sGC-dependent accumulation of cGMP in cytosolic extracts of tissues and cells was measured in vitro. Renal glomerular arterioles, including the renin-producing granular cells, mesangium, and descending vasa recta, as well as cortical and medullary interstitial fibroblasts, expressed sGC. Stimulation of isolated mesangial cells, renal fibroblasts, and hepatic Ito cells with a NO donor resulted in markedly increased cytosolic cGMP levels. This assessment of sGC expression and activity in vascular and interstitial cells of kidney and liver may have implications for understanding the role of local cGMP signaling cascades.

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis.

No abstract