Actin cytoskeletal isoforms in human endothelial cellsin vitro: Alteration with cell passage

Summary The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but notβ-actin orα-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction ofα-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behavior occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that inprolonged passage, bothα-smooth muscle actin andβ-actin were expressed, whereas HPMEC at P4 showed a lower level ofβ-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirement for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons ofin vitro andin vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.     

Culture and characterization of bovine mesenteric lymphatic endothelium

Summary Lymphatic endothelial cells isolated from bovine mesenteric lymphatic vessels were cultured and characterized. Lymphatic endothelial cells grew as a monolayer displaying an elongated morphology in preconfluent primary cultures. When confluent, the cells exhibited a polygonal morphology to form a “cobblestone” pattern previously described for cultured vascular endothelium. All culture lymphatic endothelial cells expressed Factor VIII-related antigen and boundUlex europaeus I lectin. Ultrastructurally, cultured lymphatic endothelium was characterized by the presence of Weibel-Palade bodies as well as the usual cytoplasmic organelles.     

Fucosterol decreases angiotensin converting enzyme levels with reduction of glucocorticoid receptors in endothelial cells

The modulation of angiotensin converting enzyme (ACE) levels was studied using fucosterol, one of phytosterols, in cultured bovine carotid endothelial cells. Addition of fucosterol to the culture medium resulted in the decrease of ACE activity of endothelial cells; however, fucosterol did not directly inhibit ACE activity. Dexamethasone elevated the levels of ACE in normal cells, but this effect was not seen in the fucosterol-treated cells. Receptor assays showed that the amount of glucocorticoid receptors in fucosterol-treated cells decreased to an undetectable level. These results indicate that fucosterol lowers the ACE levels on the endothelial cells by inhibiting the synthesis of glucocorticoid receptors involved in the regulation of ACE levels.     

Elevation of angiotensin converting enzyme in bovine endothelial cells quantitated by an ELISA.

Levels of angiotensin converting enzyme (ACE) in cultured bovine pulmonary artery endothelial cells treated with dexamethasone, aldosterone, 3,3',5'-triiodo-L-thyronine, Ca2+ ionophore, 3-isobutyl-1-methylxanthine, dibutyryl cAMP and forskolin were quantitated by an enzyme linked immunosorbent assay (ELISA). The configuration for the ELISA consisted of purified bovine lung ACE adsorbed to a solid phase competing with endothelial cellular ACE for a limited amount of anti-ACE immunoglobulin. ACE-IgG complex on the solid phase was detected by goat anti-rabbit IgG-alkaline phosphatase conjugate with enzymatic activity measured by p-nitrophenylphosphate as substrate. This ELISA detected ACE with a sensitivity of 32 ng/ml cellular ACE. Elevation in cellular ACE catalytic activity as measured by fluorescent assay of detergent extracts from bovine endothelial cells corresponded well with an increase in ACE protein as determined by the ELISA. These results provide direct evidence that increases in catalytic activity of ACE produced in endothelial cells by a variety of agents result from enhancement of the synthesis of ACE protein.     

Regulation of angiotensin I-converting enzyme activity in serially cultivated bovine endothelial cells

Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.     

Macro- and microvascular endothelial cells in vitro: Maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

Summary Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of³⁵S-methionine-labeled whole cells. Two-dimensional gel analysis of³⁵S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or “latent” heterogeneity in the ability of these different cells to respond to “developmental signals” (i.e. mitogenic medium) in vitro. This work was supported in part by a grant from the American Heart Association (860929), NIH (GM29127), and a Biomedical Research Grant from the University of Massachusetts.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Effect of dephosphorylated 2′,5′-trioligoadenylate on the entry of sodium ions into human neuroblastoma cells

Using a radioisotope technique, we studied the effect of dephosphorylated 2′,5′-trioligoadenylate (2′,5′ ApApA) on the entry of sodium ions into cultured human neuroblastoma cells (IMR 32 strain). Short-term (nearly 1 h) action of 2′,5′ ApApA did not influence the entry of sodium ions through voltage-operated sodium channels in the absence of neurotoxins modulating the characteristics of these channels (toxin of a scorpion, Leiurus quinquestriatus, + veratrine). At the same time, 2′,5′ ApApA weakened in a dose-dependent manner the entry of sodium ions through sodium channels opened upon the action of the above neurotoxins. In cells cultured for 22 h in a medium containing 5 · 10⁻⁶ M 2′,5′ ApApA, the entry of sodium ions in the absence of neurotoxins was 25% greater, while in the presence of neurotoxins it was 24% smaller than that in the control cells. Tetrodotoxin (TTX, 4 · 10⁻⁷ M) blocked completely sodium entry through sodium channels in cells cultured in the absence of 2′,5′ ApApA, while in cells cultured in the presence of this adenylate TTX decreased the entry by 64%. It is hypothesized that long-lasting action of 2′,5′ ApApA results in the appearance of voltage-operated TTX-insensitive sodium channels in the plasma membrane of IMR 32 cells. Our data show that 2′,5′ ApApA is capable of modulating to a considerable extent the functioning of mechanisms controlling transport of sodium ions in cells of human neuroblastoma cells of the IMR 32 strain. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 3–8, January–February, 2008.     

Effects of culture conditions on the synthesis of human chorionic gonadotropin by placental organ cultures

Summary Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix infuence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [¹²⁵Iiododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% CO₂ (approximately 20% O₂), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3′5′ dibutyryl cyclic AMP. Presented in part at the meeting of the American Association for Cancer Research, April 1978. This work is being submitted in partial fulfillment of the Ph.D. requirements in the Department of Biology, Catholic University of America.     

Identification of trophoblast-specific binding sites for GATA-2 that are essential for rat placental lactogen-I gene expression

We identified a 3.4-kb 5′-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) −2,487 and −2,310 in the 5′-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp −2,443 to −2,415 and −2,374 to −2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue. Gon-Sup Kim and Yeoung-Gyu Ko are contributed equally to this work.     

Thromboxane modulates endothelial permeability

The study tests the role of thromboxane in modulating microvascular permeability in vitro. Cultured monolayers of bovine aortic endothelial cells were challenged with the thromboxane (Tx) mimic U46619. This led to disassembly of actin microfilaments, cell rounding, border retraction and interendotheHal gap formation. Pretreatment with the Tx receptor antagonist SQ 29,548 prevented the Tx mimic-induced cytoskeletal changes. The Tx mimic also altered endothelial cell barrier function. Increased permeability was indicated by the increased passage of labelled albumin across monolayers cultured on microcarriers, relative to untreated endothelial cells (p < 0.05). Furthermore, electron microscopy of endothelial cells cultured on the basement membrane of human placental amnion indicated increased permeability based on wide, interendotheHal gap formation and transit of the tracer horseradish peroxidase. Quantification of interendothelial gaps revealed an eleven-fold increase with the Tx mimic relative to untreated endothial cells (p < 0.05) and prevention by pretreatment with the Tx receptor antagonist (p < 0.05). These data indicate that Tx directly modulates the permeability of endothelial cell in vitro.     

Elevation of angiotensin converting enzyme by 3-isobutyl-1-methylxanthine in cultured endothelial cells: a possible role for calmodulin

Incubation of cultured bovine pulmonary artery endothelial cells with 200 microM of 3-isobutyl-1-methylxanthine (IBMX) for 24 hr produced a five- to tenfold increase in cellular angiotensin converting enzyme activity (ACE) above that of untreated control cells. A lesser increase was observed in medium ACE. Other methylxanthines produced a similar, but less marked, effect. The elevation of ACE seemed to require de novo protein synthesis since it was reduced by 0.1 microgram/ml cycloheximide. Elevation of cellular cAMP was detected at 30 min after introduction of IBMX, then rapidly returned to control levels at 1 hour, while elevation in cellular ACE at 24 hr required contact with IBMX for at least 2 hr. Hence, the transient elevation in cAMP is unlikely to be the cause of the elevation of ACE. Phorbol ester and synthetic diacyl glycerol OAG, activators of protein kinase C, did not elevate ACE. Indomethacin, at a concentration known to inhibit cyclooxygenase activity, had no effect on the elevation of ACE. The elevation of ACE by IBMX was not affected by the calcium channel blocker verapamil or the calcium chelator EGTA. In contrast, the effect of IBMX was totally abolished by the calmodulin inhibitors trifluoperazine and calmidazolium. The data show that IBMX elevates endothelial cell ACE and suggest that the elevation is mediated by a calcium-calmodulin complex. The studies demonstrate a novel effect of methylxanthines on endothelial cells in culture.     

Growth and differentiation of the cultured secretory cells of the cow oviduct on reconstituted basement membrane

Isolated bovine oviduct epithelial cells were cultured on plastic precoated with matrigel. The epithelial cells seeded on 10 mg/ml matrigel often organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. This is the first report of describing the spontaneous tube formation of oviduct epithelial cells in vitro. The epithelial cells growing on this substratum became fully differentiated with the formation of junctional complexes and the production of secretory vesicles which migrated to apical plasmalemma. Epithelial cells seeded on 2 mg/ml matrigel (dry film) formed a subconfluent monolayer in 15-20 days after seeding. The histoarchitecture of the secretory cells growing on the matrigel dry film closely resembled that of intact epithelial cells. Occasional ciliated cells containing large numbers of mitochondria were observed in cell colonies growing on 2 mg/ml matrigel but they possessed very few intact cilia. The monolayer linearly incorporated 35S-methionine into proteins up to 8 hr in presence of estradiol or progesterone. The fluorography of the newly synthesized proteins indicated that the cell extracts of estradiol-stimulated cells contained an additional protein of approximate molecular weight of 60 kd as compared to the extracts of cells incubated without steroids or incubated with progesterone.     

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

Capillary endothelial cell cultures: phenotypic modulation by matrix components

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.     

Soluble epithin/PRSS14 secreted from cancer cells contains active angiogenic potential

Epithin (PRSS14/matriptase/ST14), a type II membrane protein, is involved in progression of epithelial cancers and metastasis as well as in the normal epidermal barrier function. When activated, it translocates into the cell-cell contacts and sheds into media. In order to understand the specific mechanism during tumor progression, we tested the angiogenic potential of secreted form of epithin. Epithin produced from the cancer cells shed more in hypoxia and induced motility of endothelial cells. Epithin enhanced the migration and invasion of mouse and bovine endothelial cells without cell proliferation. Furthermore, soluble epithin induced endothelial differentiation in the assay of the human endothelial microvessel-like tube formation and in that of the chicken chorioallantoic membrane. The knock-down of epithin in the 427 thymoma cell line abolished the protease activity of secreted epithin fraction, reduced the invasion of endothelial cells through matrigel, and tube formation activity. Only specific antibodies abolished the migration of endothelial cell and the vessel morphogenesis, suggesting that epithin specifically functions in these systems. Therefore, we propose that the secreted epithin in the hypoxic cancer microenvironment plays a role as a proangiogenic factor, and can be modulated with specific antibodies.     

Effect of an epoxy derivative of 2′5′-trioligoadenylate on human neuroblastoma cells

We studied the effect of an epoxy derivative of dephosphorylated 2′,5′-trioligoadenylate (5′,5′ApApAepoxy) resistive to the action of cellular phosphodiesterase on cells of human neuroblastoma IMR 32 cultured in vitro. Twenty-two hours after the addition of 5·10⁻⁶ M 2′,5′ApApAepoxy to the culture medium, the number of cells decreased by 20% (P < 0.05), while the content of protein in these cells increased, on average, by 52% (P < 0.01), as compared with the control. The activities of Na⁺,K⁺-and Ca²⁺, Mg²⁺-ATPases in a microsomal fraction obtained from cells cultured in the presence of 2′, 5′ ApApAepoxy decreased by 50% (P < 0.001) as compared with those in the control cells. Our data indicate that 2′,5′ApApAepoxy possess antiproliferative activity. According to our findings, the antiproliferative effect of 2′,5′ ApApAepoxy can, to a great extent, be explained by the fact that this oligoadenylate derivative significantly modulates the activities of Na⁺,K⁺-and Ca²⁺,Mg²⁺-ATPases. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 97–102, March–April, 2006.