Biomedical subjects
Publications and source records attributed to R Pili.
A modified procedure for the synthesis of Linomide is described. The synthesized drug was characterized and assessed for its in vivo antiangiogenic activity. In a murine angiogenesis assay Linomide treatment inhibited new blood vessel formation as documented by reduced microvessel area and blood volume.
Differentiation-inducing agents, such as retinoids and short-chain fatty acids, have an inhibitory effect on tumor cell proliferation and tumor growth in preclinical studies. Clinical trials involving these compounds as single agents have been suboptimal in terms of clinical benefit. Our study evaluated the combination of phenylbutyrate (PB) and 13-cis retinoic acid (CRA) as a differentiation and antiangiogenesis strategy for prostate cancer. On the basis of previous evidence, common signal transduction pathways and possible modulation of retinoid receptors and retinoid response elements by PB could be responsible for such activities. We assessed the effect of the combination of PB and CRA on human and rodent prostate carcinoma cell lines. The combination of PB and CRA inhibited cell proliferation and increased apoptosis in vitro in an additive fashion as compared with single agents (P < 0.014). Prostate tumor cells treated with both PB and CRA revealed an increased expression of a subtype of retinoic acid receptor (retinoic acid receptor-beta), suggesting a molecular mechanism for the biological additive effect. The combination of PB and CRA also inhibited prostate tumor growth in vivo (up to 82-92%) as compared with single agents (P < 0.025). Histological examination of tumor xenografts revealed decreased in vivo tumor cell proliferation, an increased apoptosis rate, and a reduced microvessel density in the animals treated with combined drugs, suggesting an antiangiogenesis effect of this combination. Thus, endothelial cell treatment with both PB and CRA resulted in reduced in vitro cell proliferation. In vivo testing using the Matrigel angiogenesis assay showed an additive inhibitory effect in the animals treated with a combination of PB + CRA (P < 0.004 versus single agents). In summary, this study showed an additive inhibitory effect of combination of differentiation agents PB and CRA on prostate tumor growth through a direct effect on both tumor and endothelial cells.
Gene transfer of angiogenic growth factors with replication-deficient recombinant adenovirus (Ad) vectors may provide a new approach to the treatment of ischemic diseases. To determine if Ad-infected cells could stimulate angiogenesis in vivo and to assess the tumorigenicity of cells infected with these vectors, NIH3T3 fibroblasts infected with Ad vectors coding for human acidic fibroblast growth factor (aFGF-1) were used in angiogenic and tumorigenic assays. Infected cells induced a strong angiogenic response in vivo, while cells infected with control virus did not. Stable 3T3 transfectants expressing the FGF-1 gene were also highly angiogenic and exhibited growth in soft agar, while Ad-infected cells did not. Ad-infected cells grew transiently in nude mice, whereas 3T3 transfectants formed large tumors which grew exponentially. Extrapolation of cell dose-response curves showed that a minimum of 1.5 x 10(4) infected cells were required for transient tumor cell growth in vivo. Ad-infected cells cultured in vitro for 30 days lost their invasive phenotype and the ability for transient cell growth in nude mice. Thus, phenotypic changes induced by Ad-mediated gene transfer of FGF-1 are transient both in vitro and in vivo, suggesting that these Ad vectors do not have tumorigenic potential. Stimulation of angiogenesis by Ad-infected cells may be useful for the evaluation of anti-angiogenic and anti-tumor agents.
Gene transfer techniques may provide efficient treatment for a variety of malignant neoplasms. A replication-deficient adenovirus (Ad) vector which carries the cDNA for wild-type p53 (AdCMV.p53) was tested for its in vitro and in vivo effects on the growth of murine melanoma cell line B16-G3.26 and human melanoma cell line SK-MEL-24. The growth of B16-G3.26 cells infected with AdCMV.p53 was inhibited when compared to the uninfected cells or cells infected with the control vector AdCMV.NLS beta gal. Similarly, the growth of SK-MEL-24 cells infected with AdCMV.p53 was also below that of AdCMV.NLS beta gal-infected and uninfected controls. DNA laddering using agarose gel electrophoresis and in situ labeling of DNA fragmentation (TUNEL) showed that AdCMV.p53-infected murine and human melanoma cells underwent apoptosis. Nude mice injected s.c. either with B16-G3.26 cells or with SK-MEL-24 cells developed localized tumors. These tumors were subsequently infiltrated with either AdCMV.p53, AdCMV.NLS beta gal or saline alone. One week after infection, B16-G3.26 tumors exposed to AdCMV.p53 were 2.5 times smaller than control tumors and exhibited DNA fragmentation. A similar growth-inhibitory effect of AdCMV.p53 was observed with SK-MEL-24 tumors. Thus, Ad-mediated wild-type p53 overexpression resulted in melanoma cell apoptosis and inhibition of melanoma growth in vitro and in vivo. These gene therapy approaches may be useful in targeting rapidly growing, malignant melanomas in a clinical setting.
The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Experiments in vivo with castanospermine, an inhibitor of the glucosidases that convert protein N-linked high mannose carbohydrates to complex oligosaccharides, resulted in significant inhibition of tumor growth in nude mice. Angiogenesis to basic fibroblast growth factor in castanospermine-treated C57/BL mice was similarly reduced. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. In vitro differentiation models using Matrigel and postconfluent cultures of endothelial cells were used to study the effects of glycosidase inhibitors on endothelial cell behavior. FACS analysis of cell surface oligosaccharides using either Concanavalin A or L-phytohemagglutinin lectins confirmed an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-N-acetylglucosamine on mannose residues of Asn-linked oligosaccharides upon drug treatment. Castanospermine and the glucosidase inhibitor N-methyldeoxynojirimycin prevented the morphological differentiation of endothelial cells in vitro. These compounds did not alter the proliferation of cultured endothelial cells or their ability to attach to various extracellular matrix molecules. However, the cells showed a reduced ability to migrate and to invade basement membrane gels in vitro and an increased tendency to form aggregates that was inhibitable by D-mannose. These studies suggest that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth.
In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF1-154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF1-154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF1-154). AdCMV.NLS beta gal, an adenovirus vector coding for beta-galactosidase (beta-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF1-154 and with AdCMV.aFGF1-154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF1-154 than with AdCMV.aFGF1-154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 microliters of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 x 10(8) pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF1-154 and with AdCMV.sp+aFGF1-154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLS beta gal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF1-154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.
To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.
BACKGROUND: Cancer incidence increases with age, but the growth and spread of tumors is often slow and prolonged in the elderly. Transplantable murine tumors grow and spread less readily in older mice and can be used as models to study the effect of host age. Neovascularization is crucial for the growth of solid tumors, and alterations in the host vascular response may underlie the changes in tumor growth occurring with age. PURPOSE: We have used transplantable tumor cells and tumors, and tumor extracts, to better understand differences in the biology of tumor growth and vascularization as a function of host age. METHODS: Englebreth-Holm-Swarm (EHS) carcinoma and B16-F10 melanoma cells were injected into C57BL mice of different ages. Tumor growth, histology, and cellular DNA synthesis were compared. Vascularization was determined using basic fibroblast growth factor and an in vivo angiogenesis assay. EHS tumor extracts were assayed for biologic activity in vitro using human endothelial cells and in vivo using mouse EHS-BAM carcinoma and human TSU-Pr1 prostate carcinoma cells. RESULTS: EHS tumors formed larger tumors in young than in old C57BL mice. Rapid tumor growth resumed upon transfer of tumor tissue from old animals into young animals. The rate of DNA synthesis of tumor tissue from old animals in organ culture was lower than in tissue from young animals. Histologically, tumors grown in old animals exhibited a threefold higher ratio of extracellular matrix to tumor cells than those grown in young animals. Tumors from adult animals exhibited numerous small blood vessels; those from old animals contained fewer, much larger vessels. Similar results were observed in young mice fed a reduced-calorie diet. Young animals elicited a greater and more rapid angiogenic response than old animals. Extracts of tumors grown in old animals failed to support endothelial cell differentiation in culture. Tumor cells injected together with such old extracts showed reduced tumor growth in nude mice. CONCLUSIONS: The rate of growth and morphology of the EHS tumor were altered with age, partly due to a reduced capacity to vascularize the tumors because of a lack of angiogenic factors or the presence of host inhibitors. IMPLICATIONS: Alterations in host factors detected in this tumor model may underlie a variety of age-dependent changes that could influence tumor growth and the repair and regeneration of normal tissue. Reducing the vascularization of tumors represents a potential target to reduce their growth and progression.
We have examined the role of extracellular matrix (ECM), cell growth, and dihydrotestosterone on the expression of prostate-specific antigen (PSA) by human prostatic carcinoma cells LNCaP. ECM induced a transient decrease in PSA mRNA even in the presence of growth factors. PSA mRNA, but not actin mRNA, was down-regulated on ECM in a biphasic manner and was not detected up to 48 hr after culture, but was re-expressed after 3 days. Cycloheximide and actinomycin D pretreatment did not prevent ECM-induced down-regulation of PSA mRNA, while actinomycin D-treated cells on plastic maintained stable PSA mRNA levels. DNA synthesis and PSA glycoprotein secretion were also transiently suppressed on ECM. LNCaP growth inhibition correlated with decreased glyceraldehyde phosphate dehydrogenase mRNA levels. However, the transient growth suppression induced by ECM was not observed with primary endothelial cells on Matrigel. Down-regulation of PSA mRNA by culture on Matrigel was reversible upon transfer to a different matrix substrate. Re-expression was highest on heparan sulfate proteoglycan (4-fold) and fibronectin or collagen I (2-fold) compared to plastic or laminin. Our results indicate that the morphology and proliferation of LNCaP cells may be regulated by the ability of ECM to control cellular differentiation and proliferation.
BACKGROUND: Increases in both leukocyte and endothelial cytosolic free [Ca2+] may be involved in intercellular adhesion by regulating the affinity of surface adhesion molecules or by facilitating transendothelial leukocyte migration. The purpose of this study was to examine the effect of initial contact and subsequent adhesion of human neutrophils or monocytes on human aortic endothelial [Ca2+]. METHODS AND RESULTS: Endothelial monolayers were loaded with the fluorescent Ca2+ indicator indo 1 and exposed to isolated human peripheral blood neutrophils or to a cultured human monocyte cell line. A rapid, fourfold to fivefold increase in endothelial cytosolic [Ca2+] occurred within seconds of leukocyte contact. No increase in endothelial [Ca2+] occurred on contact of 18.25-microns inert microspheres, isolated red blood cells, or suspensions of cultured human aortic endothelial cells. In experiments performed on monolayers grown in 1-mm2 capillary flow tubes, the increase in endothelial cytosolic [Ca2+] on initial leukocyte contact was found to be related to the subsequent resistance to leukocyte detachment during exposure to arterial levels of shear stress (13.4 dyne.cm-2). The increase in endothelial cytosolic [Ca2+] during leukocyte contact was not inhibited in Ca(2+)-free buffer but was abolished by prior depletion of an endoplasmic reticulum Ca2+ store by thapsigargin. Pretreatment of neutrophils with R15.7, a specific monoclonal antibody to the adhesion protein CD-18, inhibited the increase in endothelial cytosolic [Ca2+] on neutrophil contact. CONCLUSIONS: Initial contact leading to subsequent adhesion of human leukocytes to human aortic endothelial cells releases an endothelial intracellular Ca2+ store. This may, in part, be mediated by specific adhesion proteins and may in turn regulate the affinity of surface adhesion molecules or facilitate transendothelial migration of leukocytes.
The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium, but its function has not been established. We investigated whether functional ryanodine-sensitive Ca2+ stores are present in cultured endothelial cells from rat aorta (RAECs), human aorta (HAECs), human umbilical vein (HUVECs), and bovine pulmonary artery (BPAECs) and what role these may play in intracellular Ca2+ regulation. Under resting conditions, HAECs, BPAECs, and HUVECs demonstrated a slow increase in intracellular Ca2+ (indexed by indo 1 fluorescence) on exposure to 5 mumol/L ryanodine, whereas RAECs did not. However, after an initial bradykinin exposure in RAECs, ryanodine markedly blunted the rapid increase in Ca2+ on a second exposure to bradykinin. In HUVECs, ryanodine in buffer with 1.5 mmol/L Ca2+ did not inhibit the agonist-sensitive Ca2+ increase, whereas it blunted the rapid increase in Ca2+ on histamine exposure in buffer with 5 mmol/L Ca2+, suggesting that increasing [Ca2+] enhances the binding of ryanodine to its receptor. Thus, functional ryanodine-sensitive Ca2+ stores are present in vascular endothelial cells. These appear to be involved in regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments.
Circulating immune complexes (CIC) have been detected in several autoimmune diseases, and studies have also suggested that CIC provide a useful tool as tumor markers. In order to identify differences or similarities in antigenic composition, CIC from 23 patients with gastrointestinal (GI) tumors, from 20 patients with stage III and IV melanoma and from 6 patients with inflammatory bowel disease (IBD) were studied. Serum from all GI, melanoma and IBD patients showed higher levels of CIC than controls. SDS/PAGE electrophoresis under reducing conditions revealed some differences between cancer and IBD patients as far as the CIC protein composition was concerned. In melanoma patients, two fast-migrating bands, in the regions of 71-74 and 30-49 kD, were found, consistent with previously isolated and characterized antigens described in the literature.
The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.
Coagulation disorders are frequently detected in patients affected by different tumours even though clinical symptoms occur in a very small percentage of such subjects. Coagulation processes are probably involved in the mechanism of metastatic spread. We assayed the plasma levels of thrombin-antithrombin III (TAT) complexes in a group of 276 patients with several tumours in different stages in order to achieve a better understanding of the complex interactions between coagulation disorders and either tumour growth or metastatic spread. High levels of TAT complexes were found in 51% of localized, 66.3% of metastatic and 58.3% of patients with no evidence of disease; a statistically significant difference was observed comparing metastatic cancer either with localized (p < 0.00015) or with free-of-disease (p < 0.004) groups. Gastrointestinal tract neoplasms showed higher levels of TAT complexes in the metastatic than in the localized group. No difference was seen between small-cell and non-small-cell lung-localized cancer. Our results confirm the frequent coexistence of cancer and subclinical blood coagulation disorders. The evidence of higher levels of TAT complexes in metastatic cancer than in the other groups could be related to the mechanisms involved in tumour spread.
BACKGROUND: Blood vessel growth is necessary for normal tissue homeostatis and contributes to solid tumor growth. Methods to quantitate neovascularization should be useful in testing biological factors and drugs that regulate angiogenesis or to induce a vascular supply to promote wound healing. EXPERIMENTAL DESIGN: An extract of basement membrane proteins (Matrigel) was found to reconstitute into a gel when injected subcutaneously into C57/BL mice and to support an intense vascular response when supplemented with angiogenic factors. RESULTS: New vessels and von Willebrand factor antigen staining were apparent in the gel 2-3 days after injection, reaching a maximum after 3-5 days. Hemoglobin content of the gels was found to parallel the increase in vessels in the gel allowing ready quantitation. Angiogenesis was obtained with both acidic and basic fibroblast growth factors and was enhanced by heparin. Several substances were tested for angiostatic activity in this assay by coinjection in Matrigel with fibroblast growth factor and heparin. Platelet-derived growth factor BB, interleukin 1-beta, interleukin-6, and transforming growth factor-beta were potent inhibitors of neovascularization induced by fibroblast growth factor. Tumor necrosis factor-alpha did not alter the response but was alone a potent inducer of neovascularization when coinjected with Matrigel and heparin. Consistent with the previously demonstrated importance of collagenase in mediating endothelial cell invasion, a tissue inhibitor of metalloproteinases that also inhibits collagenases was found to be a potent inhibitor of fibroblast growth factor-induced angiogenesis. CONCLUSIONS: Our assay allows the ready quantitative assessment of angiogenic and anti-angiogenic factors and should be useful in the isolation of endothelial cells from the capillaries that penetrate into the gel.
The relationship between cancer and coagulation disorders is widely accepted. Such disorders can contribute to the metastatic spreading of the primary tumor. Aim of our study was to evaluate the alterations of thrombin-antithrombin III complex (TAT) measured by an ELISA plasma assay in a population of 78 patients suffering from various gastrointestinal tumors. We found high levels of TAT in 68.6% of the patients. Our data show that assay of TAT plasma levels may be a useful test in detecting early coagulation disorders in cancer patients.
Twenty-six institutionalized elderly subjects, selected as healthy according to the SENIEUR protocol, were compared to adult controls to establish correlations between affective disorders and immune abnormalities and to investigate underlying neuroendocrine mechanisms. After an extensive psychodiagnostic examination, 35% of the aged subjects were classified as depressed. Cutaneous delayed hypersensitivity tests showed reduced responses in the aged, but no correlation was found with the psychological status. Examination of the peripheral blood lymphocyte subsets revealed no imbalance in the percentages of CD3+, CD4+, CD8+ cells in the aged. A slight reduction in the CD4+/CD8+ cell ratio could however be detected in the non-depressed aged, as compared to adult controls. The CD4+/CD45R+ cell subset was reduced in non-depressed aged. The percentage of B lymphocytes was reduced in the aged, mostly in the non-depressed subjects. No changes were detected in the percent of OKDR+ cells. The percentage of CD16+ cells was found unchanged, while that of Leu7+ cells was significantly higher in the aged than in the adults and in the non-depressed than in the depressed aged. Leu7+ cell levels were negatively correlated with the depression score. On double labelling, the percent of CD16+/Leu7+ cells appears increased in the subgroup of depressed aged and positively correlated with age. Plasmatic and urinary cortisol levels were both positively correlated with depression score. Urinary cortisol level was higher in the depressed aged. These parameters, as well as plasmatic ACTH, beta-endorphin and urinary catecholamines, were not correlated with immune responses. Based on these findings, we recommend that the neuroendocrinological conditions should be taken into account when healthy subjects are examined in studies of immune senescence.
Lymphocyte activities were determined in a population of 26 institutionalized aged subjects, selected as healthy according to the SENIEUR protocol and previously reported to display immunological and endocrinological abnormalities correlated with depressive disorders. The lymphocyte mitotic response to PHA, which was reduced in aged as compared to adult subjects, was found to be significantly lower and negatively correlated with the depression score in the elderly subjects. In supernatants of PHA-stimulated lymphocyte culture from aged subjects, IL-2, IL-4 and gamma-IFN levels were very low and more severely affected in the depressed aged group. Each cytokine production was negatively correlated with age and depression score. NK activity was lower in the aged and it could be augmented by the addition of IL-2 or alpha-IFN, even though to a lesser extent than in the adult subjects. The nondepressed aged displayed higher levels of IL-2 inducible NK activity than the depressed aged subjects. IL-2 and alpha-IFN stimulated NK activities were negatively correlated with depression score. The present work indicates that the psychological status could affect lymphocyte reactivity in the aged. Given the relatively high frequency of affective disorders in these subjects, the psychological status should be considered in studies of immune senescence.