Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.

The present study was undertaken to define the 5' and 3' regulatory sequences of human von Willebrand factor gene that confer tissue-specific expression in vivo. Transgenic mice were generated bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon fused in-frame to the Escherichia coli lacZ gene. In situ histochemical analyses in independent lines demonstrated that the von Willebrand factor promoter targeted expression of LacZ to a subpopulation of endothelial cells in the yolk sac and adult brain. LacZ activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart, and aorta, as well as in megakaryocytes. In contrast, in mice containing the lacZ gene targeted to the thrombomodulin locus, the 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside reaction product was detected throughout the vascular tree. These data highlight the existence of regional differences in endothelial cell gene regulation and suggest that the 733-bp von Willebrand factor promoter may be useful as a molecular marker to investigate endothelial cell diversity.

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Lisil oxidase e propriedades pró-tumorigênicas de pericitos

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com β-aminopropionitrile (βAPN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral

Avaliação na linhagem endotelial tEnd dos efeitos diretos da transferência gênica de IFNbeta e p19arf e efeitos parácrinos mediados pela linhagem B16 transduzida pelos mesmos vetores adenovirais

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.

Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva

A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P < 0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS). Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding. To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed. Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells. The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal toot for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.

Homocysteine and cardiovascular disease in renal disease

Elevated homocysteine (hyperhomocysteinaemia) in renal patients is a major concern for physicians. Although cause and effect between homocysteine and cardiovascular disease (CVD) has not been established in either the general population or renal patients, there is much evidence that this relationship does exist. Purported mechanisms that may explain this effect include increases in endothelial injury, smooth muscle cell proliferation, low-density lipoprotein oxidation and changes in haemostatic balance. Renal patients have a much greater incidence of hyperhomocysteinaemia and this may be explained by decreases in either the renal or extrarenal metabolism of the compound. We conclude that data from long-term placebo-controlled trials are urgently required to determine whether hyperhomocysteinaemia in renal patients is a cause of CVD events and requires therapeutic targeting.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Short-term peaks in glucose promote renal fibrogenesis independently of total glucose exposure

Postprandial hyperglycemia is implicated as a risk factor predisposing to vascular complications. This study was designed to assess recurrent short-term increases in glucose on markers of renal fibrogenesis. Human renal cortical fibroblasts were exposed to fluctuating short-term (2 h) increases to 15 mM D-glucose, three times a day over 72 h, on a background of 5 mM D-glucose. To determine whether observed changes were due to fluctuating osmolality, identical experiments were undertaken with cells exposed to L-glucose. Parallel experiments were performed in cells exposed to 5 mM D-glucose and constant exposure to either 15 or 7.5 mM D-glucose. Fluctuating D-glucose increased extracellular matrix, as measured by proline incorporation ( P < 0.05), collagen IV ( P < 0.005), and fibronectin production ( P < 0.001), in association with increased tissue inhibitor of matrix metalloproteinase (MMP) ( P < 0.05). Sustained exposure to 15 mM D-glucose increased fibronectin ( P < 0.001), in association with increased MMP-2 ( P = 0.01) and MMP-9 activity ( P < 0.05), suggestive of a protective effect on collagen matrix accumulation. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA was increased after short-term (90 min) exposure to 15 mM glucose (P < 0.05) and after 24-h exposure to 7.5 mM ? ( P < 0.05). Normalization of TGF-beta(1) secretion occurred within 48 h of constant exposure to an elevated glucose. Fluctuating L-glucose also induced TGF-beta(1) mRNA and a profibrotic profile, however, to a lesser extent than observed with exposure to fluctuating D-glucose. The results suggest that exposure to fluctuating glucose concentrations increases renal interstitial fibrosis compared with stable elevations in D-glucose. The effects are, in part, due to the inherent osmotic changes.

Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184135 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study. (c) 2005 Elsevier Inc. All rights reserved.

Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.