Intra-hippocampal administration of the VEGF receptor blocker PTK787/ZK222584 impairs long-term memory.

A number of studies have established a role for vascular endothelial growth factor (VEGF) in angiogenesis. Recent reports have shown that VEGF overexpression in the hippocampus improves learning and memory and is associated with enhanced neurogenesis. PTK787/ZK222584 (PTK/ZK) is a reported inhibitor of VEGFR signaling that is currently being tested for its effects on lung and colon cancer. However, the influence of this drug on cognition has not been examined. In the present study, we questioned if post-training administration of PTK/ZK influences hippocampus-dependent memory. When administered to rats immediately following massed training in the Morris water maze, PTK/ZK impaired spatial memory retention tested 48 h later. This impairment was evidenced by increased latency to the hidden platform and fewer platform crossings. However, this impairment was not associated with a change in neurogenesis during this time frame. PTK/ZK infusion did not reduce VEGFR or AKT phosphorylation, but increased the phosphorylation of ERK. These studies suggest that VEGFR inhibitors such as PTK/ZK may negatively influence cognition.

Effect of VEGF treatment on the blood-spinal cord barrier permeability in experimental spinal cord injury: dynamic contrast-enhanced magnetic resonance imaging.

Compromised blood-spinal cord barrier (BSCB) is a factor in the outcome following traumatic spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and vascular permeability. The role of VEGF in SCI is controversial. Relatively little is known about the spatial and temporal changes in the BSCB permeability following administration of VEGF in experimental SCI. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) studies were performed to noninvasively follow spatial and temporal changes in the BSCB permeability following acute administration of VEGF in experimental SCI over a post-injury period of 56 days. The DCE-MRI data was analyzed using a two-compartment pharmacokinetic model. Animals were assessed for open field locomotion using the Basso-Beattie-Bresnahan score. These studies demonstrate that the BSCB permeability was greater at all time points in the VEGF-treated animals compared to saline controls, most significantly in the epicenter region of injury. Although a significant temporal reduction in the BSCB permeability was observed in the VEGF-treated animals, BSCB permeability remained elevated even during the chronic phase. VEGF treatment resulted in earlier improvement in locomotor ability during the chronic phase of SCI. This study suggests a beneficial role of acutely administered VEGF in hastening neurobehavioral recovery after SCI.

Enhanced in vivo angiogenesis within a model tissue engineered construct using biodegradable microspheres containing encapsulated VEGF

Introduction. Tissue engineering techniques offer a potential means to develop a tissue engineered construct (TEC) for the treatment of tissue and organ deficiencies. However, a lack of adequate vascularization is a limiting factor in the development of most viable engineered tissues. Vascular endothelial growth factor (VEGF) could aid in the development of a viable vascular network within TECs. The long-term goals of this research are to develop clinically relevant, appropriately vascularized TECs for use in humans. This project tested the hypothesis that the delivery of VEGF via controlled release from biodegradable microspheres would increase the vascular density and rate of angiogenesis within a model TEC. ^ Materials and methods. Biodegradable VEGF-encapsulated microspheres were manufactured using a novel method entitled the Solid Encapsulation/Single Emulsion/Solvent Extraction technique. Using a PLGA/PEG polymer blend, microspheres were manufactured and characterized in vitro. A model TEC using fibrin was designed for in vivo tissue engineering experimentation. At the appropriate timepoint, the TECs were explanted, and stained and quantified for CD31 using a novel semi-automated thresholding technique. ^ Results. In vitro results show the microspheres could be manufactured, stored, degrade, and release biologically active VEGF. The in vivo investigations revealed that skeletal muscle was the optimal implantation site as compared to dermis. In addition, the TECs containing fibrin with VEGF demonstrated significantly more angiogenesis than the controls. The TECs containing VEGF microspheres displayed a significant increase in vascular density by day 10. Furthermore, TECs containing VEGF microspheres had a significantly increased relative rate of angiogenesis from implantation day 5 to day 10. ^ Conclusions. A novel technique for producing microspheres loaded with biologically active proteins was developed. A defined concentration of microspheres can deliver a quantifiable level of VEGF with known release kinetics. A novel model TEC for in vivo tissue engineering investigations was developed. VEGF and VEGF microspheres stimulate angiogenesis within the model TEC. This investigation determined that biodegradable rhVEGF 165-encapsulated microspheres increased the vascular density and relative rate of angiogenesis within a model TEC. Future applications could include the incorporation of microvascular fragments into the model TEC and the incorporation of specific tissues, such as fat or bone. ^

A blood-borne PDGF/VEGF-like ligand initiates wound-induced epidermal cell migration in Drosophila larvae

Wound healing is a conserved survival response whose function is to restore the integrity of the tissue after physical trauma. Despite numerous studies in the wound healing field, the signals and pathways that orchestrate and control the wound healing program are still not entirely known. To identify additional signals and pathways that regulate epidermal wound repair in Drosophila larvae, we performed a pilot in vivo RNAi screen using a live reporter for epidermal morphology and a wounding assay. From our pilot screen we identified Pvr, the Drosophila homolog of the vertebrate PDGF/VEGF receptors, and six other genes as epidermal wound healing genes. Morphological analysis of wound-edge cells lacking Pvr or the Drosophila Jun N-terminal Kinase (JNK), previously implicated in larval wound closure, suggest that Pvr signaling leads to cell process extension into the wound site while JNK mediates transient dedifferentiation of wound-edge epidermal cells. Furthermore, we found that one of the three known Pvr ligands, Pvf1, is also required for epidermal wound closure. Through tissue-specific knock down and rescue experiments, we propose a model in which epidermally-produced Pvf1 may be sequestered into the hemolymph (blood) and that tissue damage locally exposes blood-borne Pvf1 to Pvr receptors on epidermal cells at the wound edge, thus initiating epidermal cell process extension and migration into the wound gap. Together, our data suggest that the Pvr and JNK signaling pathways act in parallel to control different aspects of wound closure and that PDGF/VEGF ligands and receptors may have a conserved autocrine role in epidermal wound closure. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model by Ricardo H. Alvarez Several mechanisms of cell resistance are often accountable for unsuccessful chemotherapy against cancer. Another reason, which has received increased attention, is the inefficient transport of anticancer drugs into tumor tissue. These impaired transports of chemotherapy into the tumor have been attributed to abnormal microvasculature and to pathologically increased tumor hypertension also called: interstitial fluid pressure (IFP). The pathophysiological processes leading to elevated tumor IFP are poorly understood. Here, in a preclinical breast cancer model, it is argued that a condition of raised IFP is a major factor in preventing optimal access of systemically administered chemotherapy agents. In our experimental model, we used a GILM2 human breast cancer in xenografts; mice were treated with different doses of paclitaxel –a widely used antimicrotubular agent, and bevacizumab –monoclonal antibody against vascular endothelial growth factor (VEGF). The proposed research project is designed to test the hypothesis that paclitaxel in combination with bevacizumab decreases the tumor IPF by restoring tumor permeability and increasing chemotherapy delivery. We demonstrated that the combination of paclitaxel and bevacizumab produced greater tumor control than either agent given alone and this combination reduced the IFP, producing an increment of 75% of apoptosis compared with the control arm. In addition, the intra-tumor paclitaxel quantification by liquid chromatography/Mass Spectrometry (LC/MS) demonstrated that lower dose of both agents showed a synergistic effect compared with high dose of treatment, where there is no significantly increase of paclitaxel into the tumor. These preclinical results are likely to have broad implications for the utility of anti-angiogenic therapies alone and in combination with chemotherapeutic agents.

NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis.

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis.

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

The Role of Cancer-Associated Fibroblasts in Lung Tumorigenesis

The extracellular milieu is rich in growth factors that drive tumor progression,but the mechanisms that govern tumor cell sensitivity to those ligands have notbeen fully defined. In this study, we address this question in mice that developmetastatic lung adenocarcinomas through the suppression of the microRNA-200 (miR-200) family. Cancer-associated fibroblasts (CAF) enhance tumorgrowth and invasion by secreting VEGF-A that binds to VEGFR1, a processrequired for tumor growth and metastasis in mice and correlated with a poorprognosis in lung adenocarcinoma patients. In this study, we discovered thatmiR-200 blocked CAF-induced tumor cell invasion by directly targetingVEGFR1 in tumor cells. In the context of previous studies, our findings suggestthat the miR-200 family is a point of convergence for diverse biologic processesthat regulate tumor cell proliferation, invasion, and metastasis; its target genesixdrive epithelial-to-mesenchymal transition (ZEB1 and ZEB2) and promotesensitivity to a potent tumor growth factor emanating from the microenvironment(VEGFR1). Clinical trials should focus not only on the role of VEGFR1 inangiogenesis but also on the expression and activation of VEGFR1 in tumorcells by stromal sources of VEGF-A in the tumor microenvironment as a targetfor metastasis prevention.

Dual Targeting of Tumor Angiogenesis and Chemotherapy by Endostatin- Cytosine Deaminase-Uracil PhosphoribosylTransferase

Antiangiogenesis is a promising anti-tumor strategy through inhibition tumor vascularformation to suppress tumor growth. Targeting specific VEGF/R has been showntherapeutic benefits in many cancer types and become a first approvedantiangiogenic modalities by Food and Drug Administration (FDA) in United States.However, interruption of homeostasis in normal tissues that is likely due to theinhibition of VEGF/R signaling pathway induces unfavorable side effects. Moreover,cytostatic nature of antiangiogenic drugs frequently causes less tumor cell specifickilling activity, and cancer cells escaped from cell death induced by these drugseven gain more malignant phenotypes, resulting in tumor invasion and metastasis.To overcome these issues, we developed a novel anti-tumor therapeutic EndoCDfusion protein which linked endostatin (Endo) to cytosine deaminase-uracilvphosphoribosyl transferase (CD). Endo targets unique tumor endothelial cells toprovide tumor-specific antiangiogenesis activity and also carries CD to the localtumor area, where it serves nontoxic prodrug 5-fluorocytosine (5-FC) enzymaticconversion reaction to anti-metabolite chemotherapy drug 5-fluorouracil (5-FU). Wedemonstrated that 5-FU concentration was highly increased in tumor sites, resultingin high level of endothelial cells and tumor cells cytotoxic efficacy. Furthermore,EndoCD/5-FC therapy decreased tumor growth and colorectal liver metastasisincident compared with bevacizumab/5-FU treatment in human breast and colorectalliver metastasis orthotropic animal models. In cardiotoxicity safety profile,EndoCD/5-FC is a contrast to bevacizumab/5-FU; lower risk of cardiotoxicityinduction or heart function failure was found in EndoCD/5-FC treatment thanbevacizumab/5-FU does in mice. EndoCD/5-FC showed more potent therapeuticefficacy with high safety profile and provided stronger tumor invasion or metastasisinhibition than antiangiogenic drugs. Together, EndoCD fusion protein with 5-FCshowed dual tumor targeting activities including antiangiogenesis and tumor localchemotherapy, and it could serve as an alternative option for antiangiogenic therapy.

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^