Cyclic mechanical strain guides capillary-like morphology in vitro

Introduction Stretching of tissue stimulates angiogenesis but increased motion at a fracture site hinders revascularisation. In vitro studies have indicated that mechanical stimuli promote angiogenic responses in endothelial cells, but can either inhibit or enhance responses when applied directly to angiogenesis assays. We anticipated that cyclic tension applied during endothelial network assembly would increase vascular structure formation up to a certain threshold. Methods Fibroblast/HUVEC co-cultures were subjected to cyclic equibiaxial strain (1 Hz; 6 h/day; 7 days) using the FlexerCell FX-4000T system and limiting rings for simultaneous application of multiple strain magnitudes (0–13%). Cells were labelled using anti-PECAM-1, and image analysis provided measures of endothelial network length and numbers of junctions. Results Cyclic stretching had no significant effect on the total length of endothelial networks (P > 0.2) but resulted in a strain-dependent decrease in branching and localised alignments of endothelial structures, which were in turn aligned with the supporting fibroblastic construct. Conclusion The organisation of endothelial networks under cyclic strain is dominated by structural adaptation to the supporting construct. It may be that, in fracture healing, the formation and integrity of the granulation tissue and callus is ultimately critical in revascularisation and its failure under severe strain conditions.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

"Candidatus Similichlamydia laticola", a novel Chlamydia-like agent of epitheliocystis in seven consecutive cohorts of farmed Australian barramundi, lates calcarifer (Bloch)

Six consecutively hatched cohorts and one cohort of pre-hatch eggs of farmed barramundi (Lates calcarifer) from south Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To identify and characterise the bacteria, 59 gill samples and three pre-hatch egg samples were processed for histology, in situ hybridisation and 16S rRNA amplification, sequencing and comprehensive phylogenetic analysis. Cases of epitheliocystis were observed microscopically and characterised by membrane-enclosed basophilic cysts filled with a granular material that caused hypertrophy of the epithelial cells. In situ hybridisation with a Chlamydiales-specific probe lead to specific labelling of the epitheliocystis inclusions within the gill epithelium. Two distinct but closely related 16S rRNA chlamydial sequences were amplified from gill DNA across the seven cohorts, including from pre-hatch eggs. These genotype sequences were found to be novel, sharing 97.1 - 97.5% similarity to the next closest 16S rRNA sequence, Ca. Similichlamydia latridicola, from Australian striped trumpeter. Comprehensive phylogenetic analysis of these genotype sequences against representative members of the Chlamydiales order and against other epitheliocystis agents revealed these Chlamydia-like organisms to be novel and taxonomically placed them within the recently proposed genus Ca. Similichlamydia. Following Fredricks and Relman's molecular postulates and based on these observations, we propose the epitheliocystis agents of barramundi to be known as "Candidatus Similichlamydia laticola" (sp. nov.).

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.

Collagen-induced activation of the Mr. 72,000 type IV collagenase in normal and malignant human fibroblastoid cells

Although the Mr. 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of Mr 59,000 and/or Mr 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor β or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analy-sis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.

Cells Tissues Organs : preface

This volume stems from the 1st International Conference on Epithelial-Mesenchymal Transitions (EMT), which was convened by the editors on October 5–8, 2003 in the beautiful setting of Port Douglas, Queensland, Australia. EMT, the name given to the transformation of cells arranged in a coherent layer – epithelial cells – to more individualistic and potential motile cells – mesenchymal cells – was recognized decades ago by Prof. Elisabeth (Betty) Hay (Harvard Medical School, Boston, Mass., USA) as a primary mechanism in embryogenesis for remodelling tissues. More recently EMT has been seen as crucial to the spread and invasion of carcinoma, and more recently still, EMT-like changes have been detected in various pathologies marked by fi brosis. Despite the basic and clinical importance of EMT, this extremely rapidly growing fi eld had never previously had a conference devoted to it, and indeed the disciplines of developmental biology, cancer and pathology rarely interact although they have much to share. The chapters assembled for this volume encompass these three major themes of the meeting, development, pathology and cancer, and further highlight the commonality in terms of mechanisms and outcomes among them...

Invasive activity and chemotactic response to growth factors by Kaposi's sarcoma cells

Kaposi's sarcoma (KS) is a relatively low grade neoplasm, classically occurring in the skin of elderly men. A more virulent and invasive form of Kaposi's sarcoma has been described in patients with acquired immune deficiency syndrome (AIDS). The origin and identification of the tumor cells in these lesions is controversial. Here we have studied the behavior of cells derived from KS lesions in an in vitro assay which measures the ability of cells to invade through a reconstituted basement membrane. In agreement with previous work, KS cells obtained under selective culture conditions were invasive showing activity comparable to that of malignant tumor cells. Normal fibroblasts, smooth muscle cells, and endothelial cells did not demonstrate invasive behavior under the same experimental conditions. To characterize further the nature of the KS cells we tested the chemotactic response of cells from the most invasive line to a variety of growth factors and compared their response to those of fibroblasts, smooth muscle, and endothelial cells. These studies suggest that normal cells respond to a unique repertoire of chemotactic factors. The chemotactic response of the KS cells most closely resembled that of smooth muscle cells and was quite distinct from endothelial cells. These results indicate that the KS-derived cultures contain invasive cells with a smooth muscle cell-like phenotype.

Endothelial precursor cells home to a vascularized tissue engineering chamber by application of the angiogenic chemokine CXCl12

Tissue engineering of vascularized constructs has great utility in reconstructive surgery. While we have been successful in generating vascularized granulation-like tissue and adipose tissue in an in vivo tissue engineering chamber, production of other differentiated tissues in a stable construct remains a challenge. One approach is to utilize potent differentiation factors, which can influence the base tissue. Endothelial precursor cells (EPCs) have the ability to both carry differentiation factors and home to developing vasculature. In this study, proof-of-principle experiments demonstrate that such cells can be recruited from the circulation into an in vivo tissue engineering chamber. CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 was infused into the chamber through Alzet osmotic pumps and chamber cannulation between days 0 and 7, and facilitated recruitment of systemically inoculated exogenous human EPCs injected on day 6. CXCL12 infusion resulted in an eightfold increase in EPC recruitment, 2 (p = 0.03) and 7 days postinfusion (p = 0.008). Delivery of chemotactic/proliferation and/or differentiation factors and appropriately timed introduction of effective cells may allow us to better exploit the regenerative potential of the established chamber construct. © Copyright 2009, Mary Ann Liebert, Inc. 2009.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.