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Biomedical subjects

Michael S Pepper

Publications and source records attributed to Michael S Pepper.

At least 19 recordsLinked to original sources
PubMed2006

Bovine microvascular endothelial cells immortalized with human telomerase.

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.

Animals↗
PubMed2006

Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation.

Early revascularization of pancreatic islet cells after transplantation is crucial for engraftment, and it has been suggested that vascular endothelial growth factor-A (VEGF-A) plays a significant role in this process. Although VEGF gene therapy can improve angiogenesis, uncontrolled VEGF secretion can lead to vascular tumor formation. Here we have explored the role of temporal VEGF expression, controlled by a tetracycline (TC)-regulated promoter, on revascularization and engraftment of genetically modified beta cells following transplantation. To this end, we modified the CDM3D beta cell line using a lentiviral vector to promote secretion of VEGF-A either in a TC-regulated (TET cells) or a constitutive (PGK cells) manner. VEGF secretion, angiogenesis, cell proliferation, and stimulated insulin secretion were assessed in vitro. VEGF secretion was increased in TET and PGK cells, and VEGF delivery resulted in angiogenesis, whereas addition of TC inhibited these processes. Insulin secretion by the three cell types was similar. We used a syngeneic mouse model of transplantation to assess the effects of this controlled VEGF expression in vivo. Time to normoglycemia, intraperitoneal glucose tolerance test, graft vascular density, and cellular mass were evaluated. Increased expression of VEGF resulted in significantly better revascularization and engraftment after transplantation when compared to control cells. In vivo, there was a significant increase in vascular density in grafted TET and PGK cells versus control cells. Moreover, the time for diabetic mice to return to normoglycemia and the stimulated plasma glucose clearance were also significantly accelerated in mice transplanted with TET and PGK cells when compared to control cells. VEGF was only needed during the first 2-3 weeks after transplantation; when removed, normoglycemia and graft vascularization were maintained. TC-treated mice grafted with TC-treated cells failed to restore normoglycemia. This approach allowed us to switch off VEGF secretion when the desired effects had been achieved. TC-regulated temporal expression of VEGF using a gene therapy approach presents a novel way to improve early revascularization and engraftment after islet cell transplantation.

Animals↗
PubMed2005

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

Animals↗
PubMed2005

Lymphangiogenesis in human gynaecological cancers.

The metastatic spread of tumor cells is responsible for the majority of cancer deaths, and with few exceptions, all cancers can metastasize. Clinical findings have for a long time suggested that by providing a pathway for tumor cell dissemination, tumor-associated lymphatics act as key components of metastatic spread. This is believed to occur principally via pre-existing and possibly also newly formed lymphatics (lymphangiogenesis). Increased expression of vascular endothelial growth factor-C (VEGF-C) and VEGF-D in primary tumors correlates with increased dissemination of tumor cells to regional lymph nodes (LNs) in a variety of human carcinomas. Here we will review the mechanisms of lymphangiogenesis, particularly in the context of metastatic tumor spread, and will critically examine the role of VEGF-C and VEGF-D in this process in gynaecological cancers. Potential anti-lymphangiogenic strategies are also discussed.

Female↗
PubMed2005

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.

Animals↗
PubMed2005

Downregulation of vascular endothelial-cadherin expression is associated with an increase in vascular tumor growth and hemorrhagic complications.

The pathogenesis of vascular tumors such as angiosarcomas is poorly understood. Cadherin expression inversely correlates with tumor malignancy and the endothelial specific VE-cadherin is low or absent in angiosarcomas, suggesting an inhibitory role for this protein in tumor progression. In this paper we report that PmyT VE-cadherin null (VEC null) endothelial cells form larger vascular tumors in nude mice when injected subcutaneously as compared to isogenic VE-cadherin positive (VEC pos) cells. This effect requires the association of beta-catenin to VEcadherin, since a VE-cadherin mutant lacking the domain responsible for beta-catenin binding (Deltabetacat) cannot rescue the phenotype. In VEC null cells beta-catenin is phosphorylated and partly degraded. N-cadherin is increased and detected at junctions. VEC null cells also present an altered fibrinolytic activity with increases in tPA, uPA, uPAR and a strong reduction in PAI-1, which may be correlated to the high incidence of abrupt hemorrhages in VEC null tumors. Overall, these data strongly suggest that downregulation of VE-cadherin in endothelial tumors may have important consequences for tumor growth and bleeding complications.

Animals↗
PubMed2004

Lymphatic vessel density and vascular endothelial growth factor-C expression correlate with malignant behavior in human pancreatic endocrine tumors.

Metastatic dissemination of tumor cells to regional lymph nodes is a common early feature of many human cancers including pancreatic adenocarcinoma. In contrast, lymph node metastasis is more variably observed in pancreatic endocrine tumors. The objective of this study was to assess the lymphatic system of human pancreatic endocrine tumors and correlate this to clinical behavior. Immunohistochemistry was performed using antibodies to two recently identified markers of lymphatic endothelium, namely, LYVE-1 and podoplanin, and to the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C. As has been reported previously, we observed that in the normal pancreas, islets of Langerhans are devoid of intra-islet lymphatics, but that lymphatics are present in connective tissue in association with ducts and blood vessels. We found that both benign and malignant pancreatic endocrine tumors contain intratumoral lymphatic vessels. Lymphatic vessel density was related to the size of the tumor in benign tumors and to the presence of liver metastasis but not to lymph node metastasis in malignant tumors. VEGF-C was expressed in tumor cells: 4 of 19 (21%) benign tumors were positive, whereas 6 of 9 (67%) borderline tumors and 9 of 11 (82%) carcinomas were positive. These findings strongly suggest that lymphangiogenesis occurs in pancreatic endocrine tumors and that lymphatic invasion and the development of metastases are associated with VEGF-C expression.

Adolescent↗
PubMed2004

Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C.

In this report we utilize a novel antagonist antibody to the human VEGFR-3 to elucidate the role of this receptor in in vitro tubular morphogenesis of bovine and human endothelial cells (EC cells) induced by VEGF-C. The antibody hF4-3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4-3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). Binding of hF4-3C5 shows that about half of bovine aortic endothelial (BAE) cells express VEGFR-3 and cells in this subpopulation are primarily responsible for the chemotactic response to the mature form of VEGF-C (VEGF-C(DeltaNDeltaC)). This response was strongly inhibited by the addition of hF4-3C5. In vitro tube formation by BAE cells induced by VEGF-C(DeltaNDeltaC) was reduced by greater than 60% by hF4-3C5 whereas the response to VEGF(165) was unaffected. Addition of hF4-3C5 together with an antagonist antibody to VEGFR-2 completely abolished the response to VEGF-C(DeltaNDeltaC). Similar results were obtained with HUVECs. Together, these findings point to a role for VEGFR-3 in vascular tubular morphogenesis and highlight the utility of hF4-3C5 as a tool for the investigation of the biology of VEGFR-3.

Animals↗
PubMed2004

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo.

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.

Allantois↗
PubMed2004

Haemangiomas are formed by cells expressing high levels of alphavbeta3 integrin and lacking acetylated LDL uptake.

Haemangiomas are benign tumours occurring in up to 12% of Caucasians, particularly in infancy and childhood. In the present study, two variant cell lines were isolated from murine endothelioma cells. One variant, named t.End.1V(high), represented 16.9% of the parental cell population and was selected by virtue of high expression levels of integrin alphavbeta3 and reduced capacity to endocytose acetylated low-density lipoproteins (Ac-LDLs). A second variant, named t.End.1V(low), represented 38.8% of the parental endothelioma cell line, expressed low levels of alphavbeta3 integrin, and was able to endocytose Ac-LDL. These phenotypic modifications were stable and correlated with specific morphological and functional properties of the two variant cell lines. While the t.End.1V(high) cells induced the formation of large haemangiomas when injected subcutaneously into mice, the t.End.1V(low) cells formed haemangiocytomas. When compared with t.End.1V(low) cells, the t.End.1V(high) cells showed increased migratory capacity, lacked an inflammatory response, and formed cord-like structures in fibrin gels. In contrast, the t.End.1V(low) cells organized into cysts with a lumen in fibrin gels. They rarely formed blood-filled haemangiomas in vivo and recruited host smooth muscle cells, a phenomenon typical for vessel wall maturation of resting cells. These data suggest that Ac-LDL uptake and the level of alphavbeta3 integrin expression are linked to the ability of endothelial cells to form large haemangiomas in vivo.

Animals↗
PubMed2004

Active-site inactivated FVIIa decreases thrombosis and necrosis in a random skin flap model of acute ischemia.

BACKGROUND: Previous studies have emphasized the role of ischemia in inducing vascular thrombosis. MATERIALS AND METHODS: Using a skin flap model of acute ischemia in the rat, we studied the effect of active-site inactivated factor VIIa (FVIIai), an inhibitor of tissue factor (TF), on tissue survival during acute ischemia. RESULTS: Ribonuclease protection analysis revealed an increase in TF in ischemic parts of the flap, and in situ hybridization localized this increase mainly to perivascular cells. A decrease in vascular thrombosis, as determined by fibrin immunostaining, was observed in FVIIai-treated animals. Intravenous administration of FVIIai had a positive impact on survival of the flap. Laser Doppler flowmetry revealed an increase in blood flow in the FVIIai-treated group. In treated animals, prothrombin time (PT) was increased (P < 0.01), whereas partial thromboplastin time (APTT) was unaltered; no significant impairment in systemic hemostasis (peri- and postoperative bleeding) was observed. CONCLUSIONS: These findings demonstrate that TF expression is increased in perivascular cells in ischemic skin flaps and that FVIIai, by inhibiting TF, increases flap survival.

Acute Disease↗
PubMed2004

Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span.

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.

Biomarkers↗
PubMed2004

Visualizing function in the tumor-associated lymphatic system.

The recent surge of interest in the lymphatic system can be attributed to two factors: the discovery of cytokines that induce the growth of new lymphatic capillaries and the identification of lymphatic endothelial-specific markers. In contrast to the above, there is a paucity of techniques for studying lymphatic function in vivo. This article reviews imaging and other techniques that allow the assessment of lymphatic function, particularly in the tumor microenvironment, and proposes novel solutions for probing the same in vivo.

Animals↗
PubMed2004

Lymphangiogenesis and tumour metastasis.

The lymphatic system serves to collect and transport interstitial fluid (lymph) within tissues, and plays an important role in the immune response. The lymphatic system also constitutes one of most important pathways of tumour dissemination. In several human cancers, increased expression in primary tumours of a new member of the vascular endothelial growth factor (VEGF) family, namely VEGF-C, is correlated with regional lymph node metastasis. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumour cells into immunodeficient mice have demonstrated a role for VEGF-C in tumour lymphangiogenesis and the subsequent formation of lymph node metastasis. However, there is at present very little evidence for lymphangiogenesis in human tumours, which is at variance with the data obtained in animal models. Nonetheless, the striking correlation between levels of VEGF-C in primary tumours and lymph node metastases exists. This suggests that VEGF-C may activate pre-existing lymphatics which then become actively involved in tumour cell chemotaxis, intralymphatic intravasation and distal dissemination. The role of VEGF-C in human tumour metastasis is therefore likely to involve lymphangiogenesis as well as its capacity to induce activation of pre-existing lymphatic endothelium.

Animals↗
PubMed2003

VEGF is a chemoattractant for FGF-2-stimulated neural progenitors.

Migration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.

Animals↗
PubMed2003

Lymphatic endothelium: morphological, molecular and functional properties.

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins, and is an important point of entry for leukocytes and tumor cells. The traditional view that lymphatic capillaries are passive participants in these tasks is currently being challenged. This overview highlights recent advances in our understanding of the molecular mechanisms underlying the formation and function of lymphatic vessels.

Animals↗
PubMed2003

Lymphangiogenesis and tumor metastasis.

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.

Animals↗