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PubMed2004

Modulation of ezrin and E-cadherin expression by IL-1beta and TGF-beta1 in human trophoblasts.

OBJECTIVES: The present study examines the effects of IL-1beta and TGF-beta1 in modulation of ezrin, E-cadherin, CD44 and beta-catenin expression in human trophoblast cells which may lead to their altered cytoskeleton dynamics during cell-to-cell and cell-to-matrix interactions. METHODS: Trophoblast (extravillous and villous) cells isolated and purified from early and term placentae and human choriocarcinoma cell line JEG-3 used in this study were challenged with either IL-1beta or TGF-beta1 (10 ng/ml) for 12 h following which RT-PCR was performed for ezrin, E-cadherin, CD44 and beta-catenin. Immunolocalization of these proteins was carried out in the chorionic villi as well as in the cultured cells stimulated by the cytokines. Western Blot was also performed to study the regulation of ezrin and E-cadherin in primary extravillous, villous and term trophoblast by these cytokines. Scanning electron microscopy (SEM) and Matrigel Invasion Assay was used to study the effect of these cytokines on cellular morphology and invasion. RESULTS: IL-1beta induced a down regulation in the expression of ezrin, E-cadherin and beta-catenin while upregulation of CD44 message in both primary trophoblast and JEG-3 cells. On the contrary, TGF-beta1 exhibited just an opposite effect, i.e. up regulation of ezrin, E-cadherin, beta-catenin, and down regulation of CD44. These observations were further corroborated with the immunolocalization findings of the above proteins in first trimester and term villous tissue, the former having predominance of IL-1beta and the latter of TGF-beta1 [Am. J. Reprod. Immunol. 48 (2002) 210]. Cellular morphology as observed through SEM revealed an enhanced cell-to-matrix adhesion with poor cell-cell interaction following IL-1beta challenge and a strong intercellular adhesion with weak cell-to-matrix interaction in presence of TGF-beta1. Crystal violet staining and Matrigel invasion revealed a higher invasion index following IL-1beta challenge and a low invasion index following TGF-beta1 challenge. CONCLUSION: IL-1beta mediated increased cell-to-matrix interaction with reduced cell-to-cell adhesion along with reduced ezrin and E-cadherin expression is associated with enhanced invasiveness while TGF-beta1 mediated up regulation of cell-to-cell adhesion with reduced cell-to-matrix interaction along with an increased ezrin and E-cadherin expression, is associated with reduced invasiveness, along with an altered cellular morphology. These facts therefore indicate the possible role of the two cytokines during cell motility and invasion through alteration of cell-matrix and cell-cell interaction.

Cadherins↗
PubMed2006

Integrin alpha5 is involved in fibronectin-induced human extravillous trophoblast invasion.

To identify the molecules involved in human extravillous trophoblast (EVT) invasion, we raised murine mAbs that react with EVTs and obtained one mAb (CHL3) that inhibited invasion of a human choriocarcinoma-derived cell line, BeWo cells. The N-terminal 22 aminoacid sequence of the CHL3 antigen (150kDa) purified from placental tissue completely matched that of integrin alpha5, which is known to interact with fibronectin. Double immunohistochemical staining and flow cytometry confirmed the reactivity of CHL3 with integrin alpha5 and its expression on the surface of BeWo cells and human EVTs isolated from villous explant cultures. CHL3 mAb inhibited the attachment of human EVTs and BeWo cells to fibronectin-coated dishes, but not to Matrigel dishes. In the Matrigel invasion assay supplemented with or without fibronectin, the invasion of isolated EVTs and BeWo cells was attenuated by treatment with CHL3 without affecting cell proliferation. During invasion assays, the production of matrix metalloproteases 2 and 9 was not changed by CHL3. These findings suggest that interaction with fibronectin through integrin alpha5 plays an important role in human extravillous trophoblast invasion.

Amino Acid Sequence↗
PubMed2005

An imbalance between vascular endothelial growth factor and its soluble receptor in placental villous explants of intrauterine growth-restricted pregnancies.

OBJECTIVES: Human umbilical vascular endothelial cells (HUVECs), seeded on Matrigel (BD Biosciences, Bedford, UK), undergo an angiogenic-like process. We hypothesized that placental explants from normal pregnancies, maintained in cultures of different oxygen, would liberate factors that could be measured in this system. We further tested the angiogenic potential of placentae from intrauterine growth-restricted (IUGR) pregnancies and the effects of vascular endothelial growth factor (VEGF) blockade. METHODS: Placental villous explants were maintained in culture at 3% and 20% O2. The resultant media was added to HUVECs seeded on 80% Matrigel. Cells were incubated at 6% O2 in accordance with the natural placental environment. After 6 hours, cells were fixed and stained and the length and number of tubules measured by morphometric imaging. Finally, VEGF and soluble VEGF receptor (sVEGFR-1) were recorded in the explant conditioned media. RESULTS: Within the angiogenic assay, recombinant human VEGF significantly enhanced tubule outgrowth (branching and elongation) and this effect was blocked with neutralising antibody. Compared to 20% O2, media of placental explants conditioned at 3% O2 significantly encouraged tubule length and numbers. Again this affect was ablated by VEGF blockade. In cases of IUGR, conditioned media at 3% O2 showed a significant reduction in tubule growth. This was paralleled by a decline in available VEGF brought about an exaggeration in liberated sVEGFR-1. Notably, venous cord serum from IUGR pregnancies showed a similar elevation in sVEGFR-1. CONCLUSION: Under restricted oxygen, placental angiogenic potential is suppressed in IUGR pregnancies through the overproduction of placental sVEGFR-1. This reduction may discourage normal placental vascularization and impact on fetal development.

Cell Culture Techniques↗
PubMed2006

A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts.

BACKGROUND: Currently available vascular grafts have been limited by variable patency rates, material availability, and immunological rejection. The creation of a tissue-engineered vascular graft (TEVG) from autologous stem cells would potentially overcome these limitations. As a first step in creating a completely autologous TEVG, our objective was to develop a novel system for culturing undifferentiated mouse embryonic stem cells (mESC) in a three-dimensional (3D) configuration and under physiological pulsatile flow and pressure conditions. MATERIALS AND METHODS: A bioreactor was created to provide pulsatile conditions to a specially modified four-well Labtek Chamber-Slide culture system. Undifferentiated mESC were either suspended in a 3D Matrigel matrix or suspended only in cell-culture media within the culture system. Pulsatile conditions were applied to the suspended cells and visualized by video microscopy. RESULTS: Undifferentiated mESC were successfully embedded in a 3D Matrigel matrix and could withstand physiological pulsatile conditions. Video microscopy demonstrated that the mESC in the 3D matrix were constrained to the wells of the culture system, moved in unison with the applied flows, and were not washed downstream; this was in contrast to the mESC suspended in media alone. CONCLUSIONS: Undifferentiated mESC can be grown in 3D and under pulsatile conditions. We will use these results to study the effects of long-term pulsatile conditions on the differentiation of mESC into endothelial cells, smooth muscle cells, and fibroblast cells with the long-term goal of creating a completely autologous TEVG.

Animals↗
PubMed2005

The selective epidermal growth factor receptor tyrosine kinase inhibitor PD153035 suppresses expression of prometastasis phenotypes in malignant pleural mesothelioma cells in vitro.

OBJECTIVE: Malignant pleural mesothelioma is notoriously refractory to aggressive multimodality therapy. Epidermal growth factor receptor expression has been observed on malignant pleural mesothelioma cells. Epidermal growth factor receptor-mediated signaling promotes tumorigenesis and metastasis of cancer cells. The purpose of this study is to evaluate the ability of the epidermal growth factor receptor tyrosine kinase inhibitor PD153035 to abrogate the expression of prometastasis phenotypes in malignant pleural mesothelioma cells in vitro. METHODS: Epidermal growth factor receptor expression of malignant pleural mesothelioma cells and primary normal cells was quantitated by means of flow cytometry. PD153035-mediated growth inhibition was determined by means of 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan and clonogenic assays. Cell motility and invasion of extracellular matrix was evaluated with in vitro wound-healing and Matrigel invasion assays, respectively. Vascular epidermal growth factor levels in conditioned media were measured by using enzyme-linked immunosorbent assay. RESULTS: Epidermal growth factor receptor expression was detected on all 6 cultured malignant pleural mesothelioma cells, with 4 of 6 having normal receptor expression and 2 of 6 overexpressing the receptor. PD153035 suppressed cell motility and cell invasion through a Matrigel membrane, regardless of the baseline epidermal growth factor receptor expression. Decreased vascular epidermal growth factor production and significant inhibition of growth only occurred in malignant pleural mesothelioma cells that overexpress epidermal growth factor receptor. CONCLUSIONS: Epidermal growth factor receptor tyrosine kinase inhibitor PD153035 significantly inhibited motility and invasion in malignant pleural mesothelioma cells in vitro, regardless of their epidermal growth factor receptor expression levels. Inhibition of epidermal growth factor receptor-dependent signaling might be a useful strategy to diminish malignant pleural mesothelioma recurrence after aggressive cytoreductive surgery.

Antineoplastic Agents↗
PubMed2006

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus).

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10-12 month lactation period, female fur seals suckle pups on shore for 2-3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 microm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (alphaS2-casein, beta-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell-matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Animals↗
PubMed2006

Transcriptional profiling implicates TGFbeta/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts.

Bile duct morphogenesis involves sequential induction of biliary specific gene expression, bilayer generation, cell proliferation, remodeling and apoptosis. HBC-3 cells are a model system to study differentiation of hepatoblasts along the hepatocytic or bile ductular lineage in vitro and in vivo. We used microarray to define molecular pathways during ductular differentiation in response to Matrigel. The temporal pattern of expression of marker genes induced was similar to that observed during bile duct formation in vivo. Notch, HNF1beta, Polycystic kidney disease 2, Bicaudal C 1 and beta-catenin were up regulated during the time course. Functional clustering analysis revealed significant up regulation of clusters of genes involved in extracellular matrix remodeling, ion transport, vacuoles, lytic vacuoles, pro-apoptotic and anti-apoptotic genes, transcription factors and negative regulators of the cell proliferation, while genes involved in the cell cycle were significantly down regulated. Notch signaling pathway was activated by treatment with Matrigel. In addition, TGFbeta/BMP signaling pathway members including the type I TGFbeta receptor and Smads 3, 4 and 5 were significantly up regulated, as were several TGFbeta/BMP responsive genes including Hey 1, a regulator of Notch pathway signaling. SMADS 3, 4 and 5 were present in the nuclear fraction of HBC-3 cells during ductular differentiation in vitro, but not during hepatocyte differentiation. SMAD 5 was preferentially expressed in hepatoblasts undergoing bile duct morphogenesis in the fetal liver, while the TGFbeta/BMP signaling antagonist chordin, was expressed throughout the liver suggesting a mechanism by which TGFbeta/BMP signaling is limited to hepatoblasts that contact portal mesenchyme in vivo.

Animals↗
PubMedDate not supplied

TNFalpha-mediated induction of PAI-1 restricts invasion of HTR-8/SVneo trophoblast cells.

The pro-inflammatory cytokine TNFalpha has numerous effects on placental trophoblasts. Here, we investigated the effects of the cytokine on gene expression and function of the extravillous trophoblast cell line HTR-8/SVneo. Wound healing and Matrigel invasion assays demonstrate that TNFalpha impairs motility and invasiveness. In contrast, counting of cumulative cell numbers and FACS analyses revealed that the cytokine did neither affect proliferation nor distribution of cell cycle phases. Immunocytochemistry of the cytokeratin 18 neo-epitope suggests that TNFalpha did not induce apoptosis in HTR-8/SVneo cells. Gelatine zymography and enzyme activity assays of supernatants of TNFalpha-treated cells demonstrate elevation of the pro- and active form of MMP-9 suggesting that increased expression of the protease cannot overcome the TNFalpha-inhibitory effect on cell invasion. Semi-quantitative RT-PCR analyses suggest that the cytokine may not alter mRNA levels of uPA and tPA. However, elevated expression of PAI-1 was detected by RT-PCR, as well as by Northern and Western blot analyses. Supplementation of PAI-1-blocking antibodies restored invasion of TNF-alpha-incubated HTR-8/SVneo cells through Matrigel-coated transwells. In addition, immunocytochemistry revealed nuclear accumulation of the p65 subunit of NFkappaB in the presence of the cytokine. EMSA indicated TNFalpha-induced binding of the inflammatory transcription factor to an NFkappaB consensus sequence and to the NFkappaB recognition site located in the PAI-1 promoter. The data suggest that TNFalpha restricts trophoblast invasion mainly by increasing the expression of PAI-1. Induction of the inhibitor may involve TNFalpha-stimulated activation of NFkappaB.

Antibodies, Blocking↗
PubMed2006

Platelet-activating factor (PAF) is the effector of IFN gamma-stimulated invasiveness and motility in a B16 melanoma line.

In this study, we investigated whether PAF synthesized by F10-M3 cells (a clone of B16-F10 melanoma line) mediates the increased capacity of these cells to penetrate into Matrigel upon stimulation with IFN gamma. The determination of PAF synthesized by IFN gamma-stimulated tumor cells revealed that 70% of newly synthesized PAF was released into growth media, while the remaining 30% was associated with the cell bodies. An experimental protocol based on the use of WEB 2086, a PAF receptorial antagonist, was designed to explore which of the two fractions of PAF synthesized by IFN gamma-stimulated F10-M3 cells (released into the growth medium or associated with the cell bodies) is essential to their capacity to migrate through Matrigel. We found that the PAF secreted into growth medium is the fraction responsible for the enhanced invasiveness of melanoma cells stimulated with IFN gamma. We also investigated whether motility of melanoma cells is stimulated by IFN gamma, and, if so, whether PAF is involved in this effect. We found that WEB 2086 prevented the remodeling of stress fibers, examined as an index of cell motility, that we observed in F10-M3 cells stimulated with IFN gamma. Furthermore, the observation that PAF receptor is expressed in IFN gamma-stimulated melanoma cells suggests that the invasive phenotype (e.g. migration through a reconstituted basement membrane and motility) promoted by PAF is based on an autocrine mechanism. On the whole, these results might indicate that PAF contributes to the expression of properties typical of an invasive phenotype in tumor cells stimulated with cytokines.

Animals↗
PubMed2004

Chronic exposure of transforming growth factor beta 1 confers a more aggressive tumor phenotype through downregulation of p21(WAF1/CIP1) in conditionally immortalized pancreatic epithelial cells.

BACKGROUND: Recent studies have demonstrated that transforming growth factor beta 1 (TGF-beta1) expression is markedly enhanced in invasive ductal pancreatic adenocarcinomas, although the precise role of TGF-beta1 in pancreatic carcinogenesis remains unclear. We analyzed TGF-beta1 expression in pancreatic intraepithelial neoplasias (PanINs) and the effects of chronic TGF-beta1 exposure on conditionally immortalized pancreatic epithelial (IMPE) cells. METHODS: Sixty-one PanIN lesions were immunohistochemically stained with a polyclonal rabbit antibody against human TGF-beta1. Growth-inhibitory effects of short-term exposure to TGF-beta1 were examined in IMPE cells. IMPE cells resistant to TGF-beta1 (IMPE-Tr cells) were generated by continuous exposure to 1 ng/mL of TGF-beta1 for more than 50 days. Phenotypic alterations of IMPE-Tr cells were examined by soft agar and Matrigel assay and Western blot analysis. IMPE and IMPE-Tr cells were injected subcutaneously into nude mice for an in vivo tumorigenicity assay. RESULTS: Forty-six percent of PanINs (28/61) were positive for TGF-beta1 expression, whereas all the epithelia of normal pancreatic ducts were negative. TGF-beta1 treatment showed the marked growth-inhibitory effects (>75%) in IMPE cells, whereas its effects were not observed in IMPE-Tr cells. IMPE-Tr cells were more spindle shaped compared with IMPE cells. In soft agar and Matrigel, formations of many colonies were observed in IMPE-Tr cells, but not in IMPE cells. Interestingly, the expression of p21(WAF1/CIP1) was induced by short-term exposure to TGF-beta1 in IMPE cells, whereas the induction was decreased in IMPE-Tr cells. All of the IMPE-Tr cell-injected mice (5/5) had subcutaneous tumors, although no tumor was found in the IMPE cell-injected mice. CONCLUSIONS: TGF-beta1 expression in PanINs and neoplastic transformation of IMPE cells by long-term exposure to TGF-beta1 suggest that TGF-beta1 may act as a tumor promoter in the early stage of pancreatic carcinogenesis.

Animals↗
PubMed2004

Growth factors and nonparenchymal cell conditioned media induce mitogenic responses in stable long-term adult rat hepatocyte cultures.

Most prior studies have characterized hepatocyte proliferative responses in culture systems that do not express a stable differentiated phenotype. We investigated the DNA synthetic response of long-term stable hepatocyte cultures to growth factor stimulation as well as conditioning with nonparenchymal cells (NPCs). Primary rat hepatocytes were cultured on a single layer of collagen (h/C) or Matrigel (h/M), or in a collagen sandwich (C/h/C) or collagen-Matrigel sandwich (M/h/C). Hepatocytes were cultured for 7 days to allow phenotypic stabilization before growth factor addition, except for h/C cultures, which are unstable, where growth factors were added 1 day after seeding. Culture medium was supplemented with a mixture of hepatocyte, epidermal, and vascular endothelial growth factors and interleukin-6, either directly or after conditioning with NPCs for 24 h. Growth factors alone induced hepatocyte DNA synthesis, as measured via [3H]thymidine uptake, in the h/C, C/h/C, and M/h/C configurations. h/M exhibited very low levels of DNA synthesis. In the C/h/C and M/h/C configurations, the greatest stimulation was obtained using NPC-conditioned growth factors. This response was sustained for several days and without decreasing albumin or urea synthesis. These results suggest that hepatocyte mitogens and NPC-derived factors can stimulate DNA synthesis in stable and differentiated hepatocyte cultures.

Albumins↗
PubMed2005

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26.

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca(2+) signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the plasminogen 2epsilon-induced Ca(2+) signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel by 1-LN cells is blocked. Angiostatin 2epsilon is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2epsilon may also directly block tumor metastasis.

Angiostatins↗
PubMed2005

The effect of extracellular matrix on embryonic stem cell-derived cardiomyocytes.

Extracellular matrix (ECM) components significantly influence the growth characteristics of cardiomyocytes, development of spontaneous contractile activity and morphologic differentiation. The present study involves characterization of ES cell-derived cardiomyocytes cultured on different ECMs. We hypothesized that cardiogel, which is a naturally occurring ECM containing a complex mixture of laminin, fibronectin may better support ES cell-derived cardiomyocytes maturation in terms of chronotropic characteristics and subcellular structure development. Spontaneously differentiated cardiomyocytes from 7-day embryoid bodies (EBs) derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a fibroblast-derived ECM (cardiogel), and control (without ECM) for 7 + 21 days. The growth characteristics of cardiomyocytes were assessed by immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR), positive and negative chronotropic drugs and transmission electron microscopy (TEM). The spontaneously beating cells expressed markers characteristic of cardiomyocytes including alpha-actinin, desmin, troponin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin-43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF) and myosin light chain isoform-2V (MLC-2V). In addition, spontaneous beating of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel, by carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine and/or Bay-K. Ultrastructure analysis showed myofibrillar bundle organization and exhibited intercalated discs, T-tubules, Z-, A-, I-, H- and M-bands and the showed earlier maturation of the cardiomyocytes on cardiogel. We conclude that substructurally matured cardiomyocytes can be produced from ES cells and the cardiac fibroblast-derived ECM (cardiogel) supported earlier maturation of ES-derived cardiomyocytes, in terms of chronotropic characteristics and subcellular structure development.

Animals↗
PubMed2006

Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo.

Within the phenotypically and functionally heterogeneous group of circulating progenitor cells (CPC), a subclass of cells with vascular repair potential have been identified. These CPC are detected and isolated based on single or combined expression of CD34, CD133 and VEGFR-2, and referred to as endothelial progenitor cells. Here we asked whether CPC subsets defined by single expression of these markers exhibit functional heterogeneity. As functional parameters, we chose the capacity of CPC to differentiate into endothelial cells. Moreover, we studied their role in remodeling by recruitment of inflammatory cells, an aspect that has been little explored. We established an in vivo model in which the intrinsic functional capacity of these human CPC subsets was studied. Human CD34+ CPC, but not CD133+ or VEGFR-2+ CPC, seeded in Matrigel pellets and transplanted subcutaneously in a nude mouse host, contributed little to donor-derived neovascularization. However, host angiogenesis in the Matrigel implant, as demonstrated by the presence of capillaries containing erythrocytes and expressing mouse CD31, was strong in response to implantation of human CD34+ CPC and significantly lower in response to the other two CPC subsets. Moreover, the CD34+ CPC subset was significantly superior to CD133+ CPC and VEGFR-2+ CPC in the recruitment of host monocytes/macrophages. These three CPC populations were further dissected into seven discrete subsets, based on three-parameter flow cytometry analysis of combined expression patterns of CD34, CD133 and VEGFR-2. In conclusion, in our system, CD34+ CPC contribute marginally to neovascularization by differentiation but are potent regulators of the host angiogenic and pro-inflammatory response, suggesting a possible role for these cells in the remodeling of vascular lesions.

AC133 Antigen↗
PubMed2001

Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Cell Division↗
PubMed2001

A synthetic tissue kallikrein inhibitor suppresses cancer cell invasiveness.

Serine proteinases modulate the interaction of tumor cells with extracellular matrix components during extravasation and metastasis. The serine proteinase tissue kallikrein has been previously demonstrated in several human adenocarcinomas, and we presently report the localization of immunoreactive kallikrein and its mRNA in pancreatic adenocarcinoma. In addition, a synthetic peptide-based inhibitor specific for tissue kallikrein (FE999024) was used in our studies to explore a possible role for kallikrein in cancer cell invasiveness. Matrigel invasion assays were performed with a human breast-cancer cell line, MDA-MB-231, which expresses tissue kallikrein in culture. In the presence of FE999024 invasion through Matrigel was inhibited in a dose-dependent manner to a maximum of 39%. We also developed a novel ex vivo assay in which breast cancer cells are infused into the pulmonary circulation of artificially ventilated explanted rat lungs. At intervals up to 6 hours after infusion pulmonary invasion was quantified by bronchial alveolar lavage to recover human cancer cells from the airspace. Invading cells in the lung interstitium were also quantified after immunohistochemistry with a monoclonal antibody specific for human cytokeratin 18. The synthetic kallikrein inhibitor attenuates breast cancer cell invasion into the airspace by 33% when quantified by lavage recovery and up to 34% as quantified in the lung interstitium by cytokeratin 18 immunostaining. Our results indicate tissue kallikrein may participate in the invasion and metastasis of human adenocarcinomas. The newly developed explanted rodent lung assay should be useful for the study of cancer cells, neutrophils, or other extravasating cells.

Adenocarcinoma↗
PubMed2003

Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.

Animals↗
PubMed2002

CD44 deficiency leads to enhanced neutrophil migration and lung injury in Escherichia coli pneumonia in mice.

CD44 is a major cell-surface receptor for hyaluronic acid (HA), a glycosaminoglycan component of extracellular matrix. HA-CD44 interactions have been implicated in leukocyte extravasation into an inflammatory site. This study examined the role of CD44 in acute inflammatory responses during pneumonias induced by Escherichia coli and Streptococcus pneumoniae using CD44-deficient mice. In E. coli-induced pneumonia, neutrophil accumulation in the lungs and edema formation was increased by 84% and 88%, respectively, in CD44-deficient mice compared to wild-type mice. In contrast, no difference was observed between these genotypes in S. pneumoniae-induced pneumonia, and the HA content in the lungs decreased after instillation of S. pneumoniae, but not E. coli, in both genotypes. Studies to determine the mechanisms for this enhanced response showed that: 1) neutrophil apoptosis was not different between these two genotypes in either type of pneumonia; 2) CD44 deficiency resulted in enhanced mRNA expression of several inflammatory genes; and 3) CD44-deficient neutrophils migrated through Matrigel in response to chemoattractants faster and in greater numbers than wild-type neutrophils in vitro and this increase was in part dependent on HA content in the Matrigel. These data demonstrate that CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a previously unrecognized role for CD44 in limiting the inflammatory response to E. coli.

Animals↗