Cytotoxicity and genotoxicity studies of two free-radical generators (AAPH and SIN-1) in human microvascular endothelial cells (HMEC-1) and human peripheral lymphocytes

Vascular endothelial cells, smooth muscle cells, macrophages and other cell types in the arterial wall may develop oxidative/nitrosative damage by generation of reactive oxygen/nitrogen species, which could alter endothelial cell function. These changes could play a key role in acute inflammatory processes, atherosclerosis and neurodegenerative pathogenesis. A human microvascular endothelial cell line (HMEC-1) and human peripheral lymphocytes were employed to investigate the cytotoxic and genotoxic effects induced by reactive peroxyl radicals and peroxynitrite generated from 2,2'-azo-bis-(2-amidinopropane)-dihydrochloride (AAPH) and 3-morpholinosydnonimine (SIN-1), respectively. The peroxides generated by AAPH were cytotoxic but not genotoxic in HMEC-1 cells and in peripheral lymphocytes (in separate culture and in whole blood). SIN-1 showed progressive cytotoxicity to HMEC-1 at doses of 10-75μM. In the same range of concentrations a significant increase in apoptotic cells and micronuclei was observed. DNA flow-cytometric analysis indicated that 100 and 200μM SIN-1 significantly increased the proportion of cells in G(2) phase compared with the control. SIN-1 decomposition products, NO and superoxide anion or peroxynitrite, induced greater cytotoxicity in lymphocyte cultures (separately and in whole blood) supplemented with HEPES - the organic buffer that is widely used to maintain stable physiological pH in cell cultures -, due to H(2)O(2) production, than in cultures without HEPES. In contrast, increased genotoxicity was observed in both lymphocyte cultures in the absence of HEPES due to the reduced cytotoxicity. In the cell systems employed in this study the genotoxic effect appears closely dependent on the nature of radical species generated by SIN-1.     

Morphology and transfection study of human microvascular endothelial cell angiogenesis: an in vitro three-dimensional model

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.     

Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS

Acute respiratory distress syndrome (ARDS) is a contemporary term incorporating the historic ‘acute lung injury’ and the colloquial term ‘shock lung’. ARDS remains a serious and enigmatic human disease, causing significant mortality. The mechanisms involved at the alveolar cell/capillary endothelial interface have been explored but to date we lack clarity on the role of intracellular calcium ([Ca²⁺]i) fluxes across this interface. To explore the mechanisms of Ca²⁺ induced inflammatory reaction in epithelial cells and pulmonary microvascular endothelial cells (HMVEC) located at the two sides of blood-air barrier, lung epithelial A549 and HMVEC cells were treated with LPS. Our results demonstrated that LPS evoked the increase of [Ca²⁺]i, TNF-α and IL-8 in both cells types. The [Ca²⁺]i increases involved intracellular but not extracellular Ca²⁺ sources in A549, but both intracellular and extracellular Ca²⁺ sources in HMVEC cells. The effects of LPS on both cells types were completely inhibited by the combination of LPS and CaSR-targeted siRNA. Furthermore, LPS-inhibited cell proliferations were significantly reversed by the combined treatment. Therefore, LPS induced different mechanisms of [Ca²⁺]i increase during the activation of CaSR in A549 and HMVEC cells, which translates into functional outputs related to ARDS.     

Secretory response of endothelin-1 in cultured human glomerular microvascular endothelial cells to shear stress

The shear-induced secretory response of endothelin-1 (ET-1) by human microvascular endothelial cells was studied using paired human glomerular microvascular endothelial cell (HGMEC) cultured monolayers exposed to steady-state laminar shear stress for up to 10 hours. The first cell monolayer was subjected to a shear stress of 0.65 N m-2 and the second, 1.3 N m-2. ET-1 secretion was determined by radioimmunoassay. Over 10 hours of shear, the total cumulative secretion of ET-1 was 237.4 pg/cm2 for the monolayer exposed to 1.3 N m-2 and 143.6 pg/cm2 for the monolayer exposed to 0.65 N m-2. The average ET-1 secretion rate was 20.90 +/- 2.15 and 12.45 +/- 1.05 pg/cm2.h at 0.65 N m-2 and 1.3 N m-2, respectively. The results showed that ET-1 secretion varied with the time of shear in a nonlinear fashion. Although the level of shear stress affected the absolute value of ET-1 cumulative secretion and secretion rate, the major secretion period for both monolayers occurred between 2.0 and 8.0 hours, with the peak secretion rate occurring at approximately 5 hours. Thus, the response of cultured human microvascular endothelial cells to shear stress differed from that of large vessel endothelial cell cultures in terms of ET-1 secretion. In addition to the level of shear stress, the time of shear was also an important determinant of ET-1 secretion. Consequently, the heterogeneity of vascular endothelial cells and the time of shear should both be considered in future research on the secretion of vascular endothelial cell cultures.     

Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function

There isincreasing evidence that sensory nerves may participate in cutaneousinflammatory responses by the release of neuropeptides such assubstance P (SP). We examined the direct effect of SP on human dermalmicrovascular endothelial cell (HDMEC) intercellular adhesion molecule1 (ICAM-1) expression and function. Our results indicated that,although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMECintracellular Ca²⁺ levels,primarily by the activation of NK-1R. Immunohistochemistry studieslikewise demonstrated that HDMEC predominantly expressed NK-1R. Theaddition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1mRNA levels, followed by a fivefold increase in ICAM-1 cell surfaceexpression. This functionally resulted in a threefold increase in⁵¹Cr-labeled binding of J-Ylymphoblastoid cells to HDMEC. In vivo studies demonstrated a markedincrease in microvascular ICAM-1 immunostaining 24 and 48 h afterapplication of capsaicin to the skin. These results indicate thatneuropeptides such as SP are capable of directly activating HDMEC toexpress increased levels of functional ICAM-1 and further support therole of the cutaneous neurological system in modulating inflammatoryprocesses in the skin.

     

Effects of extracorporeal shock waves (ESW) on human bone marrow cell cultures]

INTRODUCTION: Extracorporeal shockwave therapy (ESWT) is a new method of treating insertion tendopathy and pseudo-arthrosis, the clinical importance of which cannot yet be definitively assessed, and the underlying mechanisms of which are still unclear. AIM: To develop an experimental set-up enabling the standardised application of ESWT to human bone marrow cell culture and the determination of the effect of ESWT. MATERIAL AND METHODS: After 14 days incubation, human bone marrow cell cultures were subjected to ESWT using 200/500 pulses at an energy flux densities ED + of 0.03, 0.04, 0.07, 0.11 and 0.25 millijoule/mm2. Samples were obtained for LDH measurement 15 minutes, 4 h and 18 h after ESWT. Transmission light microscopy was carried out before and after ESWT to determine cell numbers and for morphological analysis. RESULTS: Gaps in the cellular tissue first appear at an energy of 0.01 millijoule/mm2. At energies of 0.25 millijoule/mm2, morphologically altered cells, thinned out cellular tissue with a cell-free focal zone are found. At low energy levels, defects have been repaired ca. 1 week after ESWT. No significant increase in LDH was detected at any of the energy levels applied. CONCLUSION: Increasing energy and higher pulse frequency is associated with an increase in the size and number of holes in cellular tissue and in cell separation. Regeneration capability (regrowth, sprouting, normal cell form) decreases as the energy level increases. Changes can be detected even at the lowest energy flux densities, which up until now had been assumed to have no effect on cell morphology or number. The standardised application of ESWT to human bone marrow cell cultures provides reproducible results that can be controlled by a placebo ESWT application.     

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing

sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O?) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O? concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3'-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3'-end processing.     

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.     

Role of nitric oxide in human pulmonary microvascular endothelial cell adhesion

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.     

Identification of a glialblastoma cell differentiation factor-related gene mRNA in human microvascular endothelial cells

Vascular endothelial cells (VEC) transduce mitogenic and chemoattractant signals in response to erythropoietin (Epo). An analysis of changes in gene expression in VEC would be helpful to understanding the molecular nature of mitogenic signals. An effective method for analysis of gene expression is through differential display. Using this approach, we obtained from Epo-treated human microvascular endothelial cells (HMVEC) a cDNA fragment with characteristics of the 3'end of mRNA. Using the cDNA fragment, we then isolated a full-length clone from a HMVEC cDNA library. The cDNA of interest encodes a protein consisting of 404 amino acids with a carboxy-terminal end sequence identical to glialblastoma cell differentiation factor-related protein (GBDR1). Northern blot analysis showed that GBDR1 mRNA was ubiquitously expressed in human tissues. In Southern blot analysis, GBDR1 cDNA identified a single gene on chromosome 9. Since analysis of the amino acid sequence revealed several putative phosphorylation sites for different protein kinases, the GBDR1 protein was expressed and purified from bacterial extracts and, as predicted, casein kinase II phosphorylated GBDR1 in vitro. Immunofluorescence and biochemical data revealed that the GBDR1 protein is not entirely localized in the cytosolic fraction, suggesting that it may interact with another protein(s). These findings demonstrate that GBDR1 is an intracellular signaling molecule that may play a role in the regulation of endothelial cell growth.     

Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.     

Lung endothelial cells strengthen,but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model

The blood–air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood–air barrier.     

Phenotypic comparison between mesothelial and microvascular endothelial cell lineages using conventional endothelial cell markers, cytoskeletal protein markers and in vitro assays of angiogenic potential

Endothelial and mesothelial cells are mesodermally derived simple squamous epithelial cells. A controversy concerning the ontogenetic origin of neoplasms derived from these cell types, commonly cited in the literature, is whether Kaposi's sarcoma is a mesothelioma or an angioma. To assess the similarities and differences between these cell types, pulmonary microvascular endothelial cells (PMVEC) and pericardial mesothelial cells (PMC) were cultured in vitro. PMVEC and PMC were found to be difficult to distinguish from one another by histological criteria alone. Both cell types formed contact-inhibited, and 'cobblestone', monolayers typical of simple epithelial cells. PMVEC and PMC demonstrated positive immunoreactivity to Factor VIII-related antigen and angiotensin-converting enzyme (ACE) antigen. They also showed uptake of 1,1'-dioctacecyl-1,3,3,3',3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) in 4 h. Both PMVEC and PMC expressed low ACE activities when compared to macrovessel endothelial cells. PMVEC and PMC shared similar isoform profiles for vimentin and actin. Both cell types expressed the simple epithelial keratins, cytokeratins 8 and 19, though PMC contained 50% more cytokeratins than PMVEC. Additionally, PMC contained cytokeratin 18, an intermediate filament protein not detectable in PMVEC. PMC formed 15 times as many epithelial ringlets or "stomata" as PMVEC. PMVEC but not PMC could be induced in vitro to differentiate into branching tube-like structures in response to their culture environment. Reorganization of PMVEC into vessel-like structures was more rapid and complete than PMC when embedded in three-dimensional collagen I lattices, cultured on Matrigel or exposed to a shaped-pulsed electromagnetic field. The angiogenic response of PMVEC to specialized culture conditions in vitro may reflect their phenotypic differentiation state characterized by anastomosing vascular structures in vivo, whereas PMC remain differentiated into monolayer sheet-like structures.