Cerebral microvascular smooth muscle in tissue culture

Summary Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco’s modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a “hill and valley” pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-³⁵]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, α-actin. The identity of α-actin is confirmed by analysis of NH₂-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of α-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation. This work was supported by Veteran’s Administration Research Funds, National Institutes of Health Grant HL 14230 (Arteriosclerosis Specialized Center of Research, sponsored by the National Heart, Lung, and Blood Institute), and Cardiovascular Research Program Project Grant from the National Institutes of Health to the University of Iowa (HL-14388). A. R. S. is a postdoctoral fellow of the National Institute of General Medical Sciences (GM-09020). P. A. R. is an established investigator of the American Heart Association.     

Tissue culture of human and canine thoracic duct endothelium

Summary Endothelial cells from the canine or human thoracic duct were harvested using 0.2% collagenase digestion and grown in Media 199, supplemented with fetal bovine serum. The canine endothelial cells grew to confluence (4.4 to 12×10⁴ cells/cm²) in 6 to 10 d; doubling times ranged from 1.5 to 2.8 d. There was a minimum critical density for cell growth between 500 and 10 000 cells/cm². The canine endothelial cells have been maintained in culture for periods up to 11 mo. The human thoracic duct endothelial cells are more difficult to grow and maintain. Endothelial cells were isolated from 5 out of 35 human thoracic ducts and grew for periods of up to 2 wk before degenerating. Both human and canine endothelial cells were Factor VIII positive. It has thus been demonstrated that it is possible to grow canine and, less easily, human thoracic duct endothelium in tissue culture.     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.     

Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

Summary Human myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. This investigation was supported, in part, by U.S. Public Health Service Grants 5-P50-HD11149 and 5-P01-AG00306.     

Cerebral microvessels and derived cells in tissue culture: Isolation and preliminary characterization

Summary Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 μm to 25 μm and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present. This work was supported in part by an Arteriosclerosis Specialized center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health (HL-14230) and Grant 584-127703 from the Veterans Administration.     

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity

Summary Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 μM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morpholgic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.     

Isolation of adult canine venous endothelium for tissue culture

Summary In order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact. This research was supported by National Heart, Lung and Blood Institute Contract NIH-NO1-HV-2054 and Grant HL23345.     

Growth of human muscle in tissue culture

Summary A method is described for the culture of normal and diseased human muscle cells. Cell outgrowth was obtained from 63/63 biopsies, and cells differentiated to form myotubes in 57/63 biopsies. The culture technique used readily permitted the growth of both normal and diseased human muscle cells. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and the Medical Research Council.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

An optimized culture medium for human vascular endothelial cells from umbilical cord veins

Various polypeptide growth factors, culture substrates, basal media, sera and further supplements were assayed for improvement of growth of human vascular endothelial cells from umbilical cord veins. The resulting optimized medium consisted of gelatinized culture substrates, a mixture (1:1) of Iscove's MDM and Ham's F12 basal media supplemented with 20% newborn calf serum, 500 ng/ml crude fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 g/ml transferrin, 5 g/ml insulin and 10 g/ml heparin. The medium allowed long term cultivation of HUVEC up to 45 generations with maximal cell densities of about 10⁵ cells per cm² and a minimal doubling time of about 14 hours at low cell densities.Abbreviations HUVEC Human Endothelial Cells From Umbilical Cord Veins - FGF Fibroblast growth factor - EGF Epidermal Growth Factor - FCS Fetal Calf Serum - NCS Newborn Calf Serum - HBS HEPES-Buffered Saline - ECM Extracellular Matrix - LHM Peptide PyroGlu-His-Ser-Phe-Thr-Ile-Lys-Ile-ThrCONH₂ - IF 1:1 mixture of Iscove's MDM and F12 basal media     

Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

Summary Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of a microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 μm diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 μm diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific α-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Laminin isoforms in endothelial and perivascular basement membranes

Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.     

Lymphangiogenesis in vitro: Formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells

Summary Lymphatic endothelial cells grown long term in culture form lymphatic capillarylike tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.     

Porcine aortic organ culture: A model to study the cellular response to vascular injury

Summary Organ cultures of porcine thoracic aorta were studied to define the characteristics of this system as a model to study the reaction of endothelial cells (ECs) and the underlying smooth muscle cells (SMCs) to injury. Both nonwounded and wounded cultures the latter having had part of the endothelial surface gently denuded with a scalpel blade, were studied over a 7 d period by scanning and transmission electron microscopy. The results showed that the nonwounded ECs underwent a shape change from elongated to polygonal within 24 h in culture. In both nonwounded and wounded explants there was cell proliferation beneath the nondenuded endothelium so that by 7 d several layers of cells were present showing features of the secretory type of SMCs. This proliferation, however, did not occur if the endothelium was totally removed from the aorta. There was also evidence of gaps between the surface ECs, and by 7 d lamellipodia of cells beneath the surface were present in these gaps. Occasionally, elongated cells were seen to be present on the surface of the endothelium. In the wounded organ culture, cell migration and proliferation occurred extending from the wound edge and producing a covering of cells on the denuded area. There were also multilayered cells beneath the surface similar to the nonwounded area. Occasional foam cells were seen in the depth of the multilayered proliferating cells. The results indicate that organ culture of porcine thoracic aorta is a good model to study the reaction of ECs and underlying SMCs to injury. This work was supported by a grant from the Ontario Heart Foundation.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture

Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Isolation and culture of vascular smooth muscle cells from rat placenta

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe₃O ₄) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the “hill and valley” morphology. PVSMCs were exposed to angiotensin II (1 μmol/L) and resulted in sharply increased intracellular Ca ²⁺ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.