Two-dimensional peptide mapping of fibronectins from bovine aortic endothelial cells and bovine plasma

We have made a comparison between plasma and endothelial cell fibronectin, since these cells are in intimate contact with plasma $\text{in vivo}$. Cellular and secreted fibronectins were purified from cloned lines of adult bovine aortic endothelial cells, and compared to purified bovine plasma fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional peptide mapping. When unreduced, all three fibronectins migrated on gels as single bands with M_r 440,000. After reduction, cellular and secreted fibronectins migrated on gels as single bands with M_r 220,000, but plasma fibronectin migrated as two bands with M_r 220,000 and 210,000. All three fibronectins, including the two subunits of plasma fibronectin, had identical structures by peptide mapping analysis.     

Surface modification of polystyrene Petri dishes by plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine for enhanced culturing and migration of bovine aortic endothelial cells

Abstract

Plasma surface modification is an effective method for changing material properties to control cell behavior on a surface. This study investigates the efficiency of a plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine (ppTTDDA) film coated on a polystyrene (PS) Petri dish, which is a biocompatible surface with carbon- and oxygen-based chemical species. The adhesion, proliferation, and migration properties of bovine aortic endothelial cells (BAECs) were profoundly enhanced in the ppTTDDA-coated PS Petri dishes without extracellular matrix (ECM) proteins, when compared with the uncoated PS Petri dishes. These observations indicate that ppTTDDA-coated PS Petri dishes can directly interact with cells, regardless of cell adhesion molecules. The increased cell affinity was attributed to the high concentration of carboxyl group on the surface of the ppTTDDA film. Such a carboxyl surface showed an excellent ability to promote culturing of BAECs. Plasma surface modification techniques are effective in improving biocompatibility and provide a surface environment for cell culture.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Spontaneous transformation of a cloned cell line of normal diploid bovine vascular endothelial cells

Summary We report here the spontaneous transformation of a normal diploid bovine fetal aortic endothelial cell line. This cell line showed a period of rapid proliferation, followed by a period of declining proliferative activity, as judged by both a decline in the number of population doublings achieved from seeding to subcultivation and a decrease in the fraction of cells incorporating [³H]thymidine. During the decline in proliferation, foci of small cells appeared amid a background of larger senescent-appearing cells. The cultures then regained proliferative activity and have been maintained in our laboratory for more than 22 months. The transformants are characterized by (a) an indefinite life span, (b) a morphology that is more spindle shaped as compared to pretransformants, and (c) heteroploidy with chromosome translocations. This work was supported by the U.S. National Institute of Health (NIH) Grant AG-00378. S. D. G. is a predoctoral trainee supported by U.S. Public Health Service Grant CA-09191-06, E. H. is supported under NIH Grant AG0-2100, and W. W. N. is the S. Emlen Stokes Professor of Genetics at the Institute for Medical Research.     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Primary culture of microvascular endothelial cells from bovine retina

Summary To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type, which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels were plated on tissue culture grade plastic in fetal bovine serum. Supported by Research Grants 5R01-EY03772 and 5R01-ES02380 from the U.S. Public Health Service (G. W. G.) and Established Investigator Award 31-107 from the American Heart Association (A. L. B.).     

Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells

Summary Endothelial and smooth muscle cells were isolated from human adult large blood vessels to compare their proliferative response to hormones and growth factors. Neural extracts and the medium from differentiated hepatoma cells were used as concentrated sources of required hormones and growth factors that supported both cell types. Active hormones and growth factors were identified from the neural extracts and hepatoma medium by substitution or direct isolation and biochemical characterization. Epidermal growth factor, lipoproteins, and heparin-binding growth factors elicited growth-stimulatory effects on both endothelial and smooth muscle cells. Both types of human vascular cells displayed 7600 to 8600 specific heparin-binding growth factor receptors per cell with a similar apparent dissociation constant (Kd) of 200 to 250 pM. Heparin modified the response of both endothelial and smooth muscle cells to heparin-binding growth factors dependent on the type of heparin-binding growth factor and amount of heparinlike material present. In addition, heparin exerted a growth factor-independent inhibition of smooth muscle cell proliferation. Platelet-derived growth factor, insulinlike growth factors, and glucocorticoid specifically supported proliferation of smooth muscle cells with no apparent effect on endothelial cell proliferation. Growth-factorlike proteinase inhibitors had an impact specifically on endothelial cell proliferation. Transforming growth factor beta was a specific inhibitor of endothelial cells, but had a positive effect on smooth muscle cell proliferation. The results provide a framework for differential control of the two vascular cell types at normal and atherosclerotic blood vessel sites by the balance among positive and negative effectors of endocrine, paracrine and autocrine origin. This research was supported by NIH grants CA37589, HL33847, and AM35310 from the National Institutes of Health, Bethesda, MD; grant 1718 from the Council for Tobacco Research; and a grant from RJR/Nabisco, Inc.     

Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

Sumamry A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1×10⁻⁹ M), isobutyl methylxanthine (3.3×10⁻⁵ M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies. This research was supported by grant AG 01312 from the U.S. Public Health Service, Washington, D.C.     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness

Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good ‘cobblestone-like’ morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.