Antonino Passaniti

VEGF165 Expressed by a Replication-Deficient Recombinant Adenovirus Vector Induces Angiogenesis In Vivo

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.

A functional ryanodine-sensitive intracellular Ca2+ store is present in vascular endothelial cells.

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.

Adenovirus-mediated wild-type p53 overexpression inhibits endothelial cell differentiation in vitro and angiogenesis in vivo

Gene therapy with the tumor suppressor gene p53 induces cancer cell apoptosis in vitro and in vivo and inhibits tumor growth in nude mice. We hypothesized that, in addition to cancer cell apoptosis, a replication-deficient adenovirus vector which carries the cDNA for human wild-type p53 (AdCMV.p53) may also modulate endothelial cell function and inhibit angiogenesis. Human umbilical vein endothelial cells (HUVEC) were infected at different multiplicities of infection (MOI) with either AdCMV.p53, the control vector AdCMV.null or were not infected. Western blot analysis showed p53 overexpression up to 7 days after infection with AdCMV.p53. HUVEC proliferation was either not affected (20 and 50 MOI) or inhibited to comparable levels (100 MOI; P < 0.05) in adcmv.p53- and adcmv.null-infected versus uninfected cells. huvec differentiation into capillary-like structures on reconstituted basement membrane proteins (matrigel) was assessed 48 h after infection (100 moi). after 18 h on matrigel the capillary-like network formed by adcmv.p53-infected huvec was less extensive than that formed by both adcmv.null-infected and uninfected control cells (P < 0.05 versus either control). in contrast, conditioned medium from adcmv.p53-infected huvec did not modulate endothelial cell differentiation on matrigel. the effect of adcmv.p53 on angiogenesis in vivo was assessed by injecting this vector subcutaneously in mice; 3 days later matrigel containing basic fibroblast growth factor (bfgf) was injected at the same site. in other experiments adcmv.p53 was injected simultaneously with an ad vector coding for vascular endothelial growth factor (adcmv.vegf165) into the rat perirenal fat tissue. AdCMV.p53 significantly inhibited neovascularization induced by bFGF within the Matrigel plugs (P < 0.05) or by adcmv.vegf165 in the fat tissue (P < 0.05). thus, the anti-angiogenic effect of ad-mediated wild-type p53 overexpression may contribute to the ability of this viral vector to inhibit tumor growth.

Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store.

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.

Extracellular matrix inhibits apoptosis and enhances endothelial cell differentiation by a NFκB‐dependent mechanism

Hormonal and environmental factors that control the growth, differentiation, and regression of the vasculature are of fundamental importance in tumorigenesis and in the choice of therapeutic strategies. To test the hypothesis that estradiol (E2) and basement membrane proteins would affect the survival of vascular endothelial cells (EC), immortalized human umbilical vein endothelial cells (ECV304) were examined for their response to the chemotherapeutic drugs taxol and etoposide. ECV cell apoptosis was inhibited by E2 (taxol only) or attachment to extracellular matrix (ECM) (taxol or etoposide). E2 increased ECV growth, while ECM binding resulted in growth arrest and differentiation. Apoptosis was associated with decreased levels of Bcl‐2 and p21 proteins. E2 prevented down‐regulation of p21 and Bcl‐2 induced by taxol but did not prevent the down‐regulation of p21 induced by etoposide, consistent with the failure of E2 to inhibit etoposide‐induced cell death. However, ECM prevented p21 and Bcl‐2 down‐regulation induced by taxol or etoposide. Persistent activation of NFκB occurred after attachment of ECV cells to ECM, suggesting a role in survival or differentiation. IκBα levels were not affected by taxol but were reduced by etoposide treatment, while IκBβ levels did not change with drug treatment. E2 did not alter the levels of IκBα or IκBβ. Interestingly, levels of IκBα and IκBβ declined in etoposide‐treated ECV cells on ECM concomitant with the elevation of NFκB, suggesting that in these cells degradation of IκB may be responsible for NFκB activation. In agreement with these data, anti‐sense NFκB treatment of ECV cells inhibited differentiation on ECM, but did not affect cell survival. In conclusion, culture of ECV cells on ECM or treatment with E2 inhibited apoptosis. NFκB activation by ECM was necessary for cellular differentiation, rather than inhibition, of apoptosis. J. Cell. Biochem. 73:321–331, 1999.

Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors

During development of the embryonic limb, differentiation of mesenchymal progenitor cells into chondrocytes is regulated by cell shape, extracellular matrix, and growth and differentiation factors. In this study, reconstituted basement membrane (Matrigel) prepared from mouse Englebreth-Holm-Swarm tumor tissue was found to stimulate mesenchymal cell chondrogenesis in vitro and the production of cartilage at ectopic sites in athymic mice. The rate of chondrogenesis of mesenchymal cells from chick limb bud was increased four-fold by the addition of 400 micrograms/ml Matrigel to the media of micromass cultures, and this activity was not blocked by neutralizing antibodies to transforming growth factor-beta (TGF-beta) or fibroblast growth factor (FGF). Mesenchymal cells cultured on Matrigel, but not laminin or collagen type I or IV, formed spheres of condensed cells which stained with Alcian blue. Chick limb-bud mesenchymal cells suspended in Matrigel prepared from tumors grown in C57 mice aged 3, 12, or 26 months formed disks of hyaline cartilage within 2 weeks with wet weights of 59.1 mg, 35.7 mg, and 21.4 mg, indicating that the Matrigel from the old animals was less biologically active. In agreement with the in vivo data, Alcian blue staining of proteoglycan was over two-fold higher in micromass cultures supplemented with the Matrigel from young animals than in cultures treated with the Matrigel from old mice. A high-salt wash preparation of Matrigel from tumors grown in old mice increased the rate of chondrogenesis and cartilage production, suggesting that an inhibitor of chondrogenesis is produced by the old host. Thus, Matrigel contains chondrogenic activity distinct from TGF-beta or FGF. The aged host may produce factors that are inhibitory to mesenchymal cell differentiation and adversely affect cartilage formation and repair.

[8] Glycosyltransferase probes

Publisher Summary Glycosyltransferases catalyze the transfer of a monosaccharide from an activated sugar nucleotide donor to a specific acceptor such as monosaccharides, oligosaccharides, lipids, or amino acid residues. The chapter outlines various methods and presents examples applicable to the use of virtually any soluble or membrane-bound glycosyltransferase as a probe of oligosaccharide structure. An advantage of using glycosyltransferases as surface probes is that they selectively detect accessible substrates that may be important in extracellular interactions with other cells or ligands. Therefore, the exogenous resialylation of these cells appears to be efficient, suggesting that enzyme specificity and not steric hindrance is the major factor in determining the extent of reglycosylation. Many of the known glycosyltransferases are membrane-bound enzymes that must be purified using nonionic detergents. However, to use these transferases as probes of living cells, it is necessary to remove these detergents.

Hypomethylation of an exon I estrogen receptor CpG island in spontaneous and carcinogen-induced mammary tumorigenesis in the rat

Loss of methylation at a CpG island in exon I of the rat ER gene was observed in 48% of the spontaneous mammary tumors in old female Wistar rats and 22% of the contralateral normal mammary tissues. The majority of the methylation losses were total. Similarly, 50% of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in young Sprague-Dawley rats exhibited a partial or total loss of methylation at this site, while all normal mammary tissues in young rats were fully methylated. Loss of ER methylation also increased with age in normal mammary tissues of tumor-free rats approaching 12.5% in middle-aged and 43% in old rats. In addition, 66% of mammary glands obtained from young rats that are subsequently at an increased risk to develop breast cancer due to manipulation of in utero dietary fat intake, exhibited methylation loss while no methylation changes were observed in rats at no increased risk for breast cancer. Therefore, the loss of ER methylation is more extensive in mammary glands of rats at high than low breast cancer risk, in old than young, and in mammary tumors than in normal tissues. The data suggest that hypomethylation of a growth-associated ER gene may be a common event in mammary tumorigenesis in the rat and may be of predictive value as a marker of increased breast cancer risk in aged individuals.

Regulation of urokinase plasminogen activator (uPA) activity by E-cadherin and hormones in mammary epithelial cells.

Urokinase plasminogen activator (uPA) is involved in proteolysis of extracellular matrix during development and tumor cell invasion. In the present study, we examined the regulation of uPA in hormone‐responsive, noninvasive mammary epithelial cells by using fibrinolytic and caseinolytic enzyme activity assays. Urokinase PA expression was activated after contact with fibrin and initiation of cell–cell interactions that were mediated by E‐cadherin. Fibrinolysis occurred in zones surrounding cellular aggregates. Stromal matrix proteins that disrupted aggregation or anti‐E‐cadherin antibodies that inhibited cellular compaction inhibited fibrinolysis perhaps by increasing cell–matrix adhesion or preventing E‐cadherin signaling, respectively. Aggregation required the presence of divalent cations and was inhibited by serum and ethylene diaminetetraacetic acid, whereas serine protease inhibitors reduced uPA activity without affecting aggregation. Inhibitors of PA (type 2; PAI‐2) and a specific antisense uPA oligonucleotide also reduced enzymatic activity, suggesting that fibrinolysis depends on translational regulation of uPA. In addition, the activation of plasmin from plasminogen was inhibited by anti‐E‐cadherin antibodies and PAI‐2, consistent with a role for uPA. The data also support a role for transcriptional regulation of uPA activity because treatment of cells with progesterone, hydrocortisone, or dexamethasone inhibited uPA activation on fibrin without affecting cellular aggregation. Estradiol and insulin did not alter, whereas human chorionic gonadotropin and prolactin increased uPA activity. The expression of the 55‐kDa uPA activity was consistent with specific hormone action and correlated with protein expression by immunoblotting. Therefore, the alteration of downstream signaling events by hormones may affect uPA production. These results indicate that uPA is an enzyme that may be important in the degradation of extracellular matrix during development and that specific E‐cadherin interactions and hormones can regulate its activity. Investigation of the regulation of uPA in these cells may be useful in understanding and manipulating mammary gland remodeling. J. Cell. Physiol. 181:1–13, 1999. Published 1999 Wiley‐Liss, Inc.

Adenovirus-mediated gene transfer of fibroblast growth factor-1: Angiogenesis and tumorigenicity in nude mice

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 × 104 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. Int. J. Cancer 73:258–263, 1997. Published 1997 Wiley‐Liss, Inc.