Fibrinogen induces endothelial cell permeability

Many cardiovascular and cerebrovascular disorders are accompanied by an increased blood content of fibrinogen (Fg), a high molecular weight plasma adhesion protein. Fg is a biomarker of inflammation and its degradation products have been associated with microvascular leakage. We tested the hypothesis that at pathologically high levels, Fg increases endothelial cell (EC) permeability through extracellular signal regulated kinase (ERK) signaling and by inducing F-actin formation. In cultured ECs, Fg binding to intercellular adhesion molecule-1 and to α₅β₁ integrin, caused phosphorylation of ERK. Subsequently, F-actin formation increased and coincided with formation of gaps between ECs, which corresponded with increased permeability of ECs to albumin. Our data suggest that formation of F-actin and gaps may be the mechanism for increased albumin leakage through the EC monolayer. The present study indicates that elevated un-degraded Fg may be a factor causing microvascular permeability that typically accompanies cardiovascular and cerebrovascular disorders.     

Recombinant expression of human transforming growth factor-β isoforms in Chinese hamster ovary cells

Transforming growth factor-βs (TGF-βs) are multi functional growth modulators implicated in several physiological processes which include embryogenesis, inflammation, immune-suppression, wound healing, carcinogenesis and cellular differentiation. For clinical use, recombinant expression of TGF-βs is the only practical source because of very low yields from natural sources. Here, we report the recombinant expression of human TGF-βl and TGF-β2 in a mammalian expression system using a high expression eukaryotic vector driven by a cytomegalovirus promoter. Expression levels are as high as 0.97 μg/ml of TGF-βl and 0.24 μg/ml of TGF-β2 in conditioned media, sufficient for purification without the need for amplification of the gene using methotrexate.     

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Summary This study reports the establishment ofα-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP₁) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP₁ produce low levels ofα-amylase and relatively high levels ofα-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP₁ are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation ofα-amylase in these cells.     

Establishment and characterization of immortalized cell lines from rat parotid glands

Summary This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV ₃ ^neo and pSV ₅ ^neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV ₅ ^neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV ₃ ^neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen,α-amylase mRNAs of 1176 and 702 bp, andα-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.     

Localization ofα 2-macroglobulin in human primary sarcomas and synthesis in established cell linesin human primary sarcomas and synthesis in established cell lines

Summary The presence ofα ₂-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α ₂M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection ofα ₂M in situ and in vitro an antiserum to tumor-associatedα ₂-macroglobulin was used. Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society.     

Assessment of the effect of candidate anti-inflammatory treatments on the interaction between meningococci and inflammatory cellsin vitro in a whole blood model

A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A). Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular activation was determined. Using elastase-α ₁-antitrypsin (elastase-α ₁-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α ₁-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α ₁-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min. Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release. This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory agents for evaluation in clinical trials.     

Natural secretory products of human neural and microvessel endothelial cells

Neurons, glia, and endothelial cells of the cerebral microvasculature co-exist in intimate proximity in nervous tissues, and their homeostatic interactions in health, as well as coordinated response to injury, have led to the concept that they form the basic elements of a functional neurovascular unit. During the course of normal cellular metabolism, growth, and development, each of these brain cell types secrete various species of potentially neurotoxic peptides and factors, events that increase in magnitude as brain cells age. This article reviews contemporary research on the secretory products of the three primary cell types that constitute the neurovascular unit in deep brain regions. We provide some novel in vitro data that illustrate potentially pathogenic paracrine effects within primary cells of the neurovascular unit. For example, the pro-inflammatory cytokine interleukin (IL)-1β was found to stimulate amyloid-β (Aβ) peptide release from human neural cells, and human brain microvessel endothelial cells exposed to transient hypoxia were found to secrete IL-1β at concentrations known to induce Aβ42 peptide release from human neural cells. Hypoxia and excessive IL-1β and Aβ42 abundance are typical pathogenic stress factors implicated in the initiation and development of common, chronic neurological disorders such as Alzheimer's disease. These data support the hypothesis that paracrine effects of stressed constituent cells of the neurovascular unit may contribute to “spreading effects” characteristic of progressive neurodegenerative disorders.     

miRNA-206 regulates human pulmonary microvascular endothelial cell apoptosis via targeting in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a leading cause of death due to tis high morbidity and mortality. microRNAs have emerged as new biomarkers for the prognosis and diagnosis of patients with COPD. In this study, we aimed to investigate the expression of microRNA-206 (miR-206) in lung tissues from COPD patients and to explore the regulatory role of miR-206 in the human pulmonary microvascular endothelial cells (HPMECs). Our results showed that cigarette smoke extract (CSE) promoted cell apoptosis, increased caspase-3 activity, and upregulated the expression of miR-206 in HPMECs, which was significantly reversed by the miR-206 knockdown. Transfection with miR-206 mimics led to cell apoptosis and was closely related to changes in the protein expression levels of caspase-3, caspase-9, and Bcl-2 in HPMECs. Further bioinformatics prediction analysis revealed that the 3′-untranslated region (3′UTR) of Notch3 and vascular endothelial growth factor-A (VEGFA) harbored miR-206-binding sites, and overexpression of miR-206 repressed the luciferase activity of the vectors containing Notch3 and VEGFA 3′UTR. Overexpression of either Notch3 or VEGFA attenuated miR-206-induced cell apoptosis in HPMECs. More importantly, miR-206 expression was upregulated in the lung tissues from COPD patients and was positively corrected with forced expiratory volume 1% predicted in COPD patients, while Notch3 and VEGFA mRNA levels were downregulated and were negatively correlated with the expression level of miR-206 in the lung tissues from COPD patients. In conclusion, our results showed that miR-206 was upregulated in COPD patients and CSE-treated HPMECs, promoted cell apoptosis via directly targeting Notch3 and VEGFA in HPMECs.     

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 μm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r ² = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1α (IL-1α) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1α/β neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1α secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines. Received: 15 October 1998 / Accepted: 17 February 1999     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Characterization of Esterases in a Brazilian Population of Zaprionus Indianus (Diptera: Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using α- and β-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as β-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as α-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.     

The hsa-miR-5739 modulates the endoglin network in endothelial cells derived from human embryonic stem cells

MicroRNAs are small, noncoding RNAs that bind to seed sequences on the 3′ untranslated regions of their target genes and then negatively regulate gene expressions via the RISC complex. The novel miRNA, hsa-miR-5739, was cloned and characterized its function and cellular expression in current study. The hsa-miR-5739 downregulated endothelial cells that were derived from human ES cells significantly suppressed the translational level of endoglin. This study showed that characterized hsa-miR-5739 expression by performing expression during endothelial differentiation and demonstrate potential roles of hsa-miR-5739 in human endothelial cell differentiation.     

Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis

Human microvascular endothelial cell-1 (HMEC-1) generated by transfection with SV40 large T antigen has been the prevailing model for in vitro studies on endothelium. However, the transduction of SV40 may lead to unwanted cell behaviors which are absent in primary cells. Thus, establishing a new microvascular endothelial cell line, which is capable of maintaining inherent features of primary endothelial cells, appears to be extremely important. Here, we immortalized primary human microvascular endothelial cells (pHMECs) by engineering the human telomerase catalytic protein (hTERT) into the cells. Endothelial cell-specific markers were examined and the angiogenic responses were characterized in these cells (termed as HMVECs, for human microvascular endothelial cells). We found that VEGF receptor 2 (Flk-1/KDR), tie1, and tie2 expression is preserved in HMVEC, whereas Flk-1/KDR is absent in HMEC-1. In addition, HMVEC showed similar angiogenic responses to VEGF as HMEC-1. Furthermore, the HMVEC line was found to generate a prominent angiogenic response to periostin, a potent angiogenic factor identified recently. The data indicate that HMVEC may serve as a suitable in vitro endothelium model.     

The common bile duct ligation in rat: a relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing α-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.     

Immunohistochemical localization of estrogen receptors ERα and ERβ in the spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary during postnatal development

This study was designed to determine the expression pattern of estrogen receptor (ER) subtypes in the Acomys cahirinus ovarian cells during its postnatal development. Immunohistochemical studies revealed the presence of ERα and ERβ in germinal epithelium cells and interstitial tissue. Both these ER subtypes were also seen in granulosa cells and oocytes of growing follicles, however, the level of ERβ expression was higher in comparison with ERα. In contrast to ERβ, ERα protein was also present in theca cells. The expression of ERs increased with animals’ age, but it decreased during follicular maturation. Moreover, the immunolocalization of ER subtypes in luteal cells showed that not ERβ, but ERα expression is up-regulated throughout corpus luteum development. These immunohistochemical studies demonstrate, for the first time, that ERα is also expressed in the mouse granulosa cells and it may be a mediator of estrogen action in granulosa cells proliferation and differentiation.     

Towards a Complex, Plural and Dynamic Approach to Diversity: Rejoinder to Myers and Patil, Podani, and Sarkar

This reply paper includes two brief remarks in rejoinder to the commentary papers of Myers and Patil, Podani, and Sarkar. The first observation concerns the fundamental nature of ecological diversity measures, while the second one specifically addresses some interesting mathematical connections between α- and β-diversity.     

Paradoxical effects of hypoxia-mimicking divalent cobalt ions in human endothelial cells in vitro

Divalent cobalt ions (Co²⁺) induce the expression of hypoxia responsive genes and are often used in cell biology to mimic hypoxia. In this in vitro study we compared the effects of hypoxia and Co²⁺ on human endothelial cells and examined processes that are stimulated in hypoxia in vivo (proliferation and angiogenesis). We analyzed the expression of the hypoxia-inducible factor-1 (HIF-1) under different hypoxic conditions (3% and nearly 0% O₂) and Co²⁺-concentrations (0.01–0.7 mM). As in hypoxia, the amount of HIF-1 protein was enhanced by exposure to Co²⁺ (did not correlate with mRNA amount). However, contrary to the results of hypoxia, in vitro-angiogenesis was inhibited after exposure to even low Co²⁺-concentrations (0.01 mM). This led to the conclusion that although hypoxia signaling after Co²⁺-exposure took place, further yet unknown Co²⁺-induced event(s) must have occurred. (Mol Cell Biochem 270: 157–166, 2005)