Margaret E. Schelling

Characterization of a functional angiotensin IV receptor on coronary microvascular endothelial cells

A new class of angiotensin receptors has recently been identified that exhibits both high specificity and affinity for the hexapeptide (3-8) fragment of angiotensin II, angiotensin IV (AngIV). Here, utilizing radioligand binding, we fully characterize AngIV binding at the AT4 receptor on cultured bovine coronary venular endothelial cells (CVEC), and report that when AngIV and bFGF are presented simultaneously an enhancement of DNA synthesis results that is significantly greater than that produced by bFGF alone. The level of DNA synthesis was determined by the incorporation of [3H]thymidine into quiescent CVEC monolayers following exposure to 10 nM AngIV and 10 ng/ml bFGF for 1, 3, 5, 7, 9, or 11 days. A significant enhancement of DNA synthesis (P < 0.01) was seen following 3, 5, 7, 9 and 11 days exposure. In addition, AngIV does not bind to bFGF or heparin, and conversely, bFGF is unable to compete for AngIV binding which suggests that this synergistic response is mediated by independent receptors for these ligands. Results of this study indicate that microvascular endothelial cells are significantly more responsive to bFGF in the presence of nanomolar concentrations of AngIV.

Magnetic bead purification as a rapid and efficient method for enhanced antibody specificity for plant sample immunoblotting and immunolocalization

Molecular analysis of plant tissues with antibodies has traditionally been hindered by the presence of high non-specific binding by plant cell walls and other components along with significant contaminants within sera that retard identification of specific plant tissue targets. Methods which rely on immobile solid supports conjugated with high-affinity molecular entities, have been used to purify sera. Despite their wide use, traditional antibody purification methods can result in low yields or activity and can produce significant levels of secondary contaminants, resulting in high non-specific background and dilution of tissue-specific signals. Mobile support matrixes like magnetic beads conjugated with high-affinity antisera have recently become an efficient alternative method for isolating and identifying diverse molecular targets. In this study, rabbit anti-calreticulin (CRT) immunoglobulin G (IgG) was isolated from whole anti-CRT sera with magnetic beads and tested by Western blot and immunocytochemistry for CRT localization in Pistia stratiotes plant tissues. IgG protein quantitation and purity was compared between purified and non-purified pre-immune and anti-CRT sera using spectrophotometric, reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorescence staining followed by quantitative densitometry analysis. Anti-CRT IgG binding specificity after purification was determined by Western blot of total soluble protein extract. Purified and non-purified pre-immune and anti-CRT samples were subsequently utilized for CRT immunogold localization in Pistia tissue sections and visualized with confocal microscopy. The results demonstrate that magnetic bead purified anti-CRT IgG from whole serum shows enhanced specificity and reduced background. The ease of use and speed of this IgG purification technique should find widespread use in the plant biology field.

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.

Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

Endothelial cell properties may be studied by either detecting their functional activity or immunologic reactivity. Antibodies against the fibroblast growth factor (FGF) receptor may help delineate signal transduction pathways involved in the proliferation of endothelial cells and angiogenesis. This paper describes membrane fragment preparation from cultured coronary venular endothelial cells for obtaining intact FGF receptors for the assay of the anti-receptor antibodies.

Detection of multiple fibronectin isoforms in fetal fibroblast monolayer culture: a novel method for the qualitative and quantitative detection of multiple antigens.

Abstract Analyzing the expression of multiple distinct antigens within a single monolayer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of multiple antigens within a single monolayer. By generating an immunohistochemical grid which divides a monolayer in a standard tissue culture dish into 20 distinct areas, we were able to detect and quantify four individual fibronectin (FN) isoforms within a single fibroblast monolayer culture. Quantification of each isoform was performed using a modified enzyme-linked immunoassay. In addition, within the same monolayer, each FN isoform was detected using standard immunohistochemical detection with DAB visualization. Using this novel approach to immunohistochemical analysis we determined that within the first 4 days of culture, the quantity of all FN isoforms increases faster than the number of cells. However, upon reaching confluency, the quantity of FN/cell drops dramatically. After reaching confluency, the amount of FN/cell levels off and remains constant within the postconfluent monolayer. Statistical analysis of the quantity of FN/cell indicates that a significant reduction in the amount of FN/cell occurs in the 2 days prior to reaching confluency. The distribution of all the FN isoforms, with the exception of B-FN, was found along the length of the cell body. In contrast, the distribution of B-FN was altered in postconfluent monolayers where it was detected only in distinct locations within the monolayer.

Expression and assembly of the angiogenic marker B-fibronectin by endothelial cells in vitro: Regulation by confluency

Recent studies have demonstrated the association of the rare fibronectin (FN) isoform B-FN with newly formed blood vessels. Although B-FN is a unique angiogenesis marker, it is not clear whether B-FN associated with the neovasculature is expressed by the endothelial cells (EC) or obtained from the microenvironment. Here we report on a study analyzing the expression and assembly of B-FN by bovine microvascular EC (BMEC) in vitro. We determined that BMEC express B-FN and assemble it into fibrils during monolayer culture. Furthermore, in addition to assembling endogenous B-FN, subconfluent BMEC can assemble exogenous B-FN provided by a non-EC source. Upon reaching confluency the assembly and expression of B-FN by BMEC is inhibited and the previously assembled B-FN is eventually eliminated from postconfluent EC monolayers. Indeed, confluent BMEC assemble neither endogenous nor exogenous B-FN, while they continue to assemble other FN isoforms. We conclude that BMEC in vitro express B-FN and assemble B-FN fibrils using endogenous as well as exogenous B-FN. The expression and assembly of B-FN are tightly regulated by confluency. Our observations in vitro are a plausible explanation for the absence of B-FN in established blood vessels in vivo, and the subsequent reappearance of B-FN in angiogenic vessels. Furthermore, since our observations of B-FN assembly in BMEC monolayers correspond to previously published observations in vivo, B-FN may be utilized as an appropriate marker for angiogenic behavior of EC in vitro.