Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40.

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

Dobutamine stress cine-MRI of cardiac function in the hearts of adult cardiomyocyte-specific VEGF knockout mice.

A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

The role of vascular endothelial growth factor in angiogenesis.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenic inducer as well as a mediator of vascular permeability. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFr-1) and KDR (VEGFR-2). VEGF is essential for developmental angiogenesis and is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF in tumors and intraocular neovascular syndromes. Currently, several clinical trials are ongoing to test the hypothesis that inhibition of VEGF activity may be beneficial for these conditions.

Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor.

Growth of the human rhabdomyosarcoma A673 cell line in nude mice is substantially reduced but not completely suppressed after systemic administration of the antihuman vascular endothelial growth factor (VEGF) monoclonal antibody (Mab) A.4.6.1. Potentially, such escape might be attributable to incomplete local penetration of the antibody because of a diffusion barrier associated with tumor growth. Alternatively, it might reflect a compensatory up-regulation of murine VEGF, produced by the stroma of the host, or of other angiogenic factor genes. To test these potential mechanisms, systemic administration of Mab A.4.6.1, was performed in conjunction with intratumoral administration of an irrelevant antibody, an antihuman VEGF Fab or mFlt(1-3)-IgG that neutralizes both human and murine VEGF. Tumor growth in the systemic-plus-intratumoral anti-VEGF group was not different from that in the systemic anti-VEGF-plus-intratumoral-control antibody group, arguing against the possibility that bioavailability is the factor that limits the antitumor efficacy of Mab A.4.6.1. However, intratumoral mFlt(l-3)-IgG administration dramatically enhanced the activity of systemic anti-VEGF Mab and resulted in complete suppression of tumor growth, which indicated that host VEGF significantly contributes to tumor growth. Systemic administration of mFlt(1-3)-IgG alone replicated these findings. Histological analysis of residual tumor tissues revealed an almost complete absence of host-derived vasculature and massive tumor-cell necrosis in the mFlt(1-3)-IgG groups. Such extensive necrotic areas were not present in the other groups. Real-time reverse transcription-PCR analysis of total RNA derived from tumor tissues indicated strong up-regulation of both human and murine VEGF as well as other genes regulated by hypoxia. Our findings emphasize the need to completely block VEGF for maximal inhibition of tumor growth.

Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state.

VEGF-A has been implicated in regulating the initial angiogenic invasion events that are essential for endochondral bone formation. VEGF-A mRNA expression was indeed found in the sclerotome of the developing somite and in the limb-bud mesenchyme at E10.5 in mouse development but declined during chondrogenesis and became upregulated in hypertrophic chondrocytes prior to angiogenic invasion. To determine the functional importance of VEGF-A expression in the developing chondrogenic tissues, VEGF-A was conditionally inactivated during early embryonic development using Collagen2a1-Cre transgenic lines. Deletion of a single VEGF-A allele in Collagen2a1-Cre-expressing cells results in embryonic lethality around E10.5. This lethality is characterized by aberrant development of the dorsal aorta and intersomitic blood vessels, along with defects in the developing endocardial and myocardial layers of the heart. A small percentage of VEGF(Flox)/+, Collagen2a1-Cre fetuses survive until E17.5, show aberrant endochondral bone formation and develop a heart phenotype resembling a dilated form of ischemic cardiomyopathy. These results provide insights into the function of VEGF-A in heart and endochondral bone formation and underscore the importance of tightly controlled levels of VEGF-A during development.

Angiogenesis and bone growth.

Vascularization of the growth plate region represents a key mechanism for the coupling of two fundamental processes determining the rate of bone growth, chondrogenesis (cartilage production) and osteogenesis (bone formation). Precise coupling is crucial during periods of rapid bone growth or fracture repair in adults, and changes in the balance might induce pathologic conditions such as osteoarthritis and ectopic bone formation. During the formation of the growth plates of long bones, there is a close and dynamic interaction between developing vascular structures and the cartilage, which is one of the least vascular tissues in the body. Recent experimental findings provide an explanation why the close proximity of cartilage and vasculature is mutually exclusive: vascular invasion of cartilage is associated with chondrocyte apoptosis and consequently, inhibition of angiogenesis in the growth plate delays chondrocyte cell death, resulting in a massive expansion in the number of hypertrophic cartilage cells in the growth plate. The fundamental importance of chondrocytes in the growth, development and repair of the skeleton has led to intense investigation of the mechanisms that regulate chondrocyte maturation and apoptosis.

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.

VEGF is required for growth and survival in neonatal mice.

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.

Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.

Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3'-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.

Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro.

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.

Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.

We examined the role of vascular endothelial growth factor (VEGF) in preventing apoptosis in primary human umbilical vein endothelial (HUVE) cells. VEGF was capable of preventing serum starvation-induced apoptosis at concentrations between 10 and 100 ng/ml. The addition of VEGF to serum-starved HUVE cells led to a 5. 2-fold induction of Bcl-2 after 36 h and to a transient, 2.4-fold induction of A1 after a 7-h incubation, as quantitated by real time reverse transcriptase-polymerase chain reaction analysis. Western blot analysis demonstrated a 2-3-fold induction of Bcl-2 protein after 18-36 h of exposure to VEGF and a transient induction of A1 after 7 h of VEGF stimulation. Moreover, overexpression of Bcl-2 by means of transient biolistic transfection experiments of HUVE cells was sufficient to prevent endothelial cells from apoptotic cell death in the absence of VEGF. These findings indicate that Bcl-2 plays an important role in mediating the survival activity of VEGF on endothelial cells.

Vascular endothelial growth factor is essential for corpus luteum angiogenesis.

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.

Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia.

Vascular endothelial growth factor (VEGF) and its two endothelial cell-specific receptor tyrosine kinases, Flk-1/KDR and Flt-1, play a key role in physiological and pathological angiogenesis. Hypoxia has been shown to be a major mechanism for up-regulation of VEGF and its receptors in vivo. When we exposed human umbilical vein endothelial cells to hypoxic conditions in vitro, we observed increased levels of Flt-1 expression. In contrast, Flk-1/KDR mRNA levels were unchanged or slightly repressed. These findings suggest a differential transcriptional regulation of the two receptors by hypoxia. To identify regulatory elements involved in the hypoxic response, promoter regions of the mouse Flt-1 and Flk-1/KDR genes were isolated and tested in conjunction with luciferase reporter gene. In transient transfection assays, hypoxia led to strong transcriptional activation of the Flt-1 promoter, whereas Flk-1/KDR transcription was essentially unchanged. Promoter deletion analysis demonstrated a 430-bp region of the Flt-1 promoter to be required for transcriptional activation in response to hypoxia. This region includes a heptamer sequence matching the hypoxia-inducible factor-1 (HIF) consensus binding site previously found in other hypoxia-inducible genes such as the VEGF gene and erythropoietin gene. We further narrowed down the element mediating the hypoxia response to a 40-base pair sequence including the putative HIF binding site. We show that this element acts like an enhancer, since it activated transcription irrespective of its location or orientation in the construct. Furthermore, mutations within the putative HIF consensus binding site lead to impaired transcriptional activation by hypoxia. These findings indicate that, unlike the KDR/Flk-1 gene, the Flt-1 receptor gene is directly up-regulated by hypoxia via a hypoxia-inducible enhancer element located at positions -976 to -937 of the Flt-1 promoter.

RNA polymerase II C-terminal domain required for enhancer-driven transcription.

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.

Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

Transcriptional activation modulated by homopolymeric glutamine and proline stretches.

Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

C-terminal domain (CTD) of RNA-polymerase II and N-terminal segment of the human TATA binding protein (TBP) can mediate remote and proximal transcriptional activation, respectively.

Activation domains of mammalian transcription factors can be subdivided into at least two functional classes. One, exemplified by the glutamine-rich activation domains of Oct and Sp1 factors, mediates transcriptional activation only from a proximal promoter position, and in response to an enhancer. The other, exemplified by the 'acidic' domain of the viral activator VP16, has the ability to activate from remote enhancer as well as from proximal promoter positions. Here we report that two proteins of the basal transcription apparatus also contain activation domains whose stimulatory effect can be detected in fusion proteins containing the GAL4 DNA binding domain. The human TATA-binding protein (TBP) contains at its N-terminus a domain with typical 'promoter' activity. We propose that the TBP N-terminal region acts as an auxiliary activation domain which reinforces the activity of other promoter-bound factors. The largest subunit of RNA polymerase II contains at its C-terminus a conserved heptad repeat structure (CTD). Both natural and synthetic CTD consensus repeats fused to GAL4 can activate transcription from remote positions like a typical enhancer-active domain. Accordingly we propose that the RNA polymerase II large subunit contains a 'portable' domain for transcriptional activation which may synergize with the activation domains of enhancer-bound transcription factors.