Victor B. Hatcher

Human brain-derived acidic and basic fibroblast growth factors: Amino terminal sequences and specific mitogenic activities

Extended amino terminal sequence determinations, made on both acidic and basic fibroblast growth factors from human brain, showed extensive homology with each other and with their respective bovine counterparts. Both human growth factors in the presence of heparin have equivalent specific mitogenic activities on human umbilical vein endothelial cells in culture whereas in the absence of heparin, the acidic mitogen is less than 1% as active as the basic growth factor.

LONG-TERM CULTURE OF HUMAN ENDOTHELIAL CELLS

SummaryHuman umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (103/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.

The complete amino acid sequence of human brain-derived acidic fibroblast growth factor

Acidic fibroblast growth factor is a potent mitogen for a variety of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of human brain-derived acidic fibroblast growth factor has been determined from amino terminal sequence analysis and carboxypeptidase A digestion of the whole protein and sequence analyses of peptides generated by tryptic, Staphylococcus aureus V8 protease and cyanogen bromide cleavages. A potential Asn-Gly-Ser glycosylation sequence is present in the human protein. The complete amino acid sequence is compared to that of the equivalent protein purified from bovine brain.

The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway

Summary We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma‐like multimers, and a non‐secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two‐dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro‐vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells—a LMW form of pro‐vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.

Protease activity of normal and PHA stimulated human lymphocytes

Abstract An assay measuring the release of TCA soluble radioactive peptides from 3 H acetylated casein or hemoglobin has been used to demonstrate that human peripheral blood lymphocytes contain a number of proteases, including cathepsin D, a neutral serine protease(s) inhibited by DFP and TLCK and probably a thiol protease(s) as well. We have also found a neutral protease activity bound to the surface of the lymphocyte, but not secreted into the medium which is not inhibited by TLCK. TLCK inhibits blast transformation to PHA under conditions that do not profoundly affect protein synthesis and inhibits the total extractable proteolytic activity of lymphocytes by approximately 25%. Lymphocytes contain one or more proteases that may play a role in blast transformation and other lymphocyte functions.

Relationship between cell surface protease activity and doubling time in various normal and transformed cells

A sensitive method for measuring cell surface and secreted protease activity utilizing 3H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloracetic acid soluble 3H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3--1 times more proteolytic activity than the normal cells. Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.

Staphylococcus aureus-human endothelial cell interactions

Staphylococcus aureus is an invasive pathogen capable of causing life-threatening disease. A major component of this pathogens virulence is its ability to invade normal endovascular tissue. We examined the interaction of S. aureus with cultured human endothelial cells and with human and rabbit endovascular tissue. Our ultrastructural study demonstrated a sequence of steps which occurred with staphylococcal invasion of human endothelial cells; adhesion, endocytosis, and intracellular replication. Ultimately, this resulted in cell disruption and death. Cytochemical staining of lysosomes demonstrated lysosomal fusion with both viable and killed intracellular bacteria without evidence of staphylococcal degradation. Quantitative studies using an in vitro infection assay demonstrated comparable rates of adhesion by viable and ultraviolet-killed bacteria, phagocytosis at a slower rate, and intracellular replication. The present study demonstrates an active role for the endothelial cell in the development and spread of endovascular staphylococcal infections. It also supports the use of this in vitro tissue culture system as a model for the study of bacterial invasion of the endothelium.

The isolation and purification of two anionic endothelial cell growth factors from human brain

Two anionic polypeptides which stimulate the proliferation of human umbilical vein endothelial cells have been isolated and purified to homogeneity from human brain using heparin affinity chromatography. The molecular weights of the polypeptides are 18,500 and 19,300; the isoelectric point for both polypeptides is at pH 5.2. The purified polypeptides differed in their ability to stimulate human endothelial cell growth. The half maximal activities observed for the 18.5 and 19.3 kilodalton polypeptides were at concentrations of 10.0 ng/ml and 0.9 ng/ml respectively.

Alterations in intracellular calcium following infection of human endothelial cells with Trypanosoma cruzi

Trypanosoma cruzi infection in cultured human umbilical vein endothelial cells increased basal cellular calcium levels from 55 to 110 nM, as monitored with the fluorescent probe, fura-2. It also influenced intracellular calcium such that consistently higher total levels were observed in response to bradykinin, angiotensin II and norepinephrine, as compared to similarly treated uninfected cells. However, bradykinin and angiotensin II-dependent increases in calcium, when considered as the absolute increment or fold elevation over basal, were significantly lower in infected endothelial cells. Infection also influenced changes in calcium levels due to agents that operate independently of plasma membrane receptors. In the presence of ionomycin, the magnitude and rate of rise of intracellular calcium were decreased; additionally the calcium peak was delayed and the subsequent decline slowed. Similar to the results with bradykinin and angiotensin II, infection decreased both the increment in and fold stimulation of intracellular calcium in response to ionomycin. In contrast, infection altered only the total calcium stimulated in response to oligomycin; neither the fold stimulation of, nor increment in intracellular calcium was affected. These results indicate that (1) infection by T. cruzi alters calcium homeostasis in endothelial cells under basal and stimulated conditions; (2) both receptor-dependent and receptor-independent mechanisms are affected by infection. The possible contribution of altered calcium homeostasis induced by T. cruzi in the pathogenesis of chagasic cardiomyopathy is considered.

Characterization of a chemotactic and cytotoxic proteinase from human skin

A proteinase (EC 3.4.-.-) active at physiological pH has been isolated from human skin utilizing gel filtration and affinity chromatography techniques. The proteinase has a molecular weight of approx. 28 000 and it is inhibited by alpha 2-macroglobulin, alpha 1-antitrypsin, C-1 inactivatory, soybean trypsin inhibitor and diisopropyl fluorophosphate. 2njection of 1 ng of purified proteinase into rabbit skin induces polymorphonuclear leukocyte infiltration of the cutis. Inhibition of enzyme activity with diisopropyl fluorophosphate inhibits the chemotactic effect. Addition of 0.2 microgram/ml of purified proteinase to fibroblast cultures kills the cells within minutes. This proteinase may play a significant role in modulating the inflammatory response after cellular injury.