Angiogenesis In Normal Tissue Research Articles

NSD1 promotes esophageal cancer tumorigenesis via HIF1α signaling.

Unlike angiogenesis in normal tissues, tumor angiogenesis is typically dysregulated, during which the HIF1/VEGFA signaling pathway plays a pivotal role. Solid tumors generate immature vessels, which promote tumor progression and treatment resistance. NSD1 can di-methylate histone 3 lysine 36 and regulate transcription factors binding to the promoters of various genes. However, the role of NSD1 in tumorigenesis remains elusive. Here, we evaluated the relationship between NSD1 signaling and HIF1 signaling. It was found that NSD1 transcriptionally regulates HIF1α expression by recruiting STAT3 molecule into the HIF1α promoter. In vivo xenograft experiments further confirmed that HIF1α and STAT3 maintenance is essential for NSD1-mediated tumor progression and angiogenesis. Therefore, the NSD1/STAT3/HIF1α signaling pathway may be a novel and effective treatment target for ESCA.

A Pilot/Safety Study of sEphB4-Hsa in Combination with a Hypomethylating Agent for Patients with Relapsed or Refractory Myelodysplastic Syndrome and Acute Myeloid Leukemia Previously Treated with a Hypomethylating Agent

Background: Myelodysplastic syndrome (MDS) is a clonal hematopoietic neoplasm that results in bone marrow failure and frequently leads to acute myeloid leukemia (AML). Hypomethylating agents (HMA) are the only FDA-approved treatment for MDS and among few options for chemotherapy-ineligible patients with AML. There are limited options for patients in whom HMA therapy fails and who are not candidates for allogeneic stem cell transplant, so therapies that complement or restore sensitivity to HMAs are needed. Studies have shown increases in bone marrow microvessel density (MVD) and angiogenic markers in patients with MDS and AML. A decrease in MVD has been shown to correlate with response to hypomethylating agents in MDS. A receptor-ligand interaction, comprised of receptor EphB4 and membrane localized ligand EphrinB2, mediates angiogenesis in normal tissue and appears to be a target unique to many cancer types. We have previously shown EphB4 to be highly expressed and a driver of leukemic cell survival in a subset of AML patients. Our group has developed a human fusion protein, sEphB4-HSA, that blocks bidirectional signaling induced by EphB4-EphrinB2 interaction to inhibit tumor cell proliferation and angiogenesis. In phase I clinical trials of sEphB4-HSA in various tumor types, there were no myelosuppressive effects and minimal toxicity. Given its safety in phase I and potential to inhibit leukemic cell proliferation and angiogenesis, we proposed a pilot trial to evaluate the safety of sEphB4-HSA in combination with HMAs in MDS and AML patients who have failed treatment with HMAs. Methods: This pilot study was designed to enroll 6 patients with relapsed/refractory intermediate or high-risk MDS and 6 patients with AML refractory to or relapsed to HMA treatment and who are deemed unfit for chemotherapy. Treatment consisted of sEphB4-HSA 15 mg/kg IV every 2 weeks in combination with the FDA-approved HMA most recently or currently being used for treatment (decitabine 20mg/m2 IV/1hr on days 1 to 5 every 28 days or azacitidine 75mg/m2 SC or IV on days 1 to 7 every 28 days). Patients were treated for as long as they were receiving clinical benefit up to 12 months. The primary endpoint was toxicity and tolerability of sEphB4-HSA in combination with HMA. Toxicity was assessed and graded after each cycle according to the CTCAE version 4. Tolerability was defined as the ability to complete two cycles of treatment without the occurrence of dose-limiting toxicity. A secondary efficacy endpoint was to assess best overall response, based on the IWG Working Group Criteria for MDS and AML, during the first two cycles of treatment. Enrollment was stopped after 7 patients due to expiration of funding. Results: Three patients with intermediate-risk MDS were treated for a median duration of 6 cycles (2-12) and 4 patients with AML were treated for 2 cycles. Median age was 75.5 years (67.9-84.8) and 57.1% were male. HMA included azacitadine in 6 patients and decitabine in one patient. There were no dose-limiting toxicities. There were 8 grade 3/4 events attributed to HMA, which included: neutropenia (2), thrombocytopenia (3), and leukopenia (3). There were 3 grade 3/4 events attributed to sEphB4, which included: febrile neutropenia (1), leukopenia (1), and hypertension (1). Of the MDS patients, 2 had stable disease, 1 patient after 2 cycles and 1 patient after 4 cycles. One patient achieved a hematologic improvement-erythroid after 6 cycles. AML patients had no disease response. Reasons for treatment discontinuation were death (1), disease progression (2), patient's decision (1), physician's decision (2), and hospice (1). Notably, a comparison of bone marrow biopsies at baseline and after 8 weeks of treatment demonstrated a decrease in MVD (Figure 1). Discussion: This pilot study found sEphB4 in combination with HMAs to be tolerable with no significant toxicity beyond that expected with HMA therapy and associated with potential clinical benefit in MDS patients. Improvement in abnormal bone marrow MVD may indicate a potential for sEphB4-HSA plus HMA therapy to alter the malignant microenvironment in MDS/AML. Disclosures No relevant conflicts of interest to declare.

EGCG Suppresses Melanoma Tumor Angiogenesis and Growth without Affecting Angiogenesis and VEGF Expression in the Heart and Skeletal Muscles in Mice

Melanoma is a highly malignant cancer with a potent capacity to metastasize distantly and has a higher mortality. There is no effective therapy for high risk melanoma patients to prevent relapse or distant metastasis. Therefore effective chemoprevention strategies are needed. The present study mainly evaluates the effects of EGCG on melanoma angiogenesis, growth, and capillary density (CD) in the heart and skeletal muscles of mice. 5 x 10^5 B16F10 cells were inoculated into the right proximal dorsal of the back in the eight week old male mice (n=12). Then, 6 mice received EGCG at 50-100 mg/kg/d in drinking water for 4 weeks and 6 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, hearts, and limb muscles were collected and measured for VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. Compared to the control, EGCG treatment significantly reduced tumor weight (2.9±0.5 vs. 5.9±1.1 g; P<0.01; n=6), melanoma CD (117±9 vs. 167±23; P<0.01), and melanoma VEGF expression (32±1.5 vs. 42±2 pg/mg; P < 0.01), respectively. Also EGCG had no effects on body weight, heart weight, angiogenesis or VEGF expression in the heart and skeletal muscle of mice. EGCG (20-50 µg/ml) significantly inhibited the proliferation, migration, VEGF expression, and the activation of HIF-1α and NFαB in cultured B16F10 cells, respectively. These findings support the hypothesis that EGCG, a major green tea polyphenol, directly targets tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of melanoma, and that the down-regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1α and NFkB activation. EGCG has great potential as a chemopreventive agent because it has no effect on angiogenesis in normal tissue and has low toxicity.

Distinct roles of DKK1 and DKK2 in tumor angiogenesis

Tumor angiogenesis is essential for tumor invasive growth and metastasis, and generates abnormal vascular structures unlike developmental neovessel formation. To reduce tumor vascular abnormalities such as leakage and perivascular cell coverage deficiency that limit cancer therapy effectiveness, novel therapeutic approaches focus on vessel normalization. We have previously shown that Dickkopf-1 (DKK1), a Wnt antagonist, inhibits and its homolog DKK2 enhances, angiogenesis in normal tissues. In the present study, we investigated the effects of DKK1 and DKK2 on tumor growth and angiogenesis. Treatment of B16F10 melanoma-bearing mice with adenovirus expressing DKK1 significantly reduced tumor growth but DKK2 increased growth compared with controls. Similar pattern of tumor growth was observed in endothelial-specific DKK1 and DKK2 transgenic mice. Interestingly, tumor vascular density and perfusion were significantly decreased by DKK1 but increased by DKK2. Moreover, coverage of blood vessels by pericytes was reduced by DKK1, while DKK2 increased it. We further observed that DKK1 diminished retinal vessel density and increased avascular area in an in vivo murine model of oxygen-induced retinopathy, whereas DKK2 showed opposite results. These findings demonstrate that DKK1 and DKK2 have differential roles in normalization and functionality of tumor blood vessels, in addition to angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-013-9390-5) contains supplementary material, which is available to authorized users.

Sp/KLF Family and Tumor Angiogenesis in Pancreatic Cancer

Tumor angiogenesis play a significant role in genesis, development and metastasis of pancreatic cancer though the process is different from angiogenesis in normal tissues. VEGF, one of the most important angiogenesis factors, is modulated by inflammation factors as well as transcriptional factors, such as members of Sp/KLF family. Recent research showed that VEGF related inflammation factors and Sp/KLF family members form a complex network structure, which causes genesis and development of tumor. Conceivably, fully understanding the mechanism linking VEGF related inflammation factors and Sp/KLF family members would promote the concept of tumor angiogenesis. Furthermore, it could also help to design effective strategies to target the key components of the network and control the development and progression of tumor.

New pathways and mechanisms regulating and responding to Delta-like ligand 4–Notch signalling in tumour angiogenesis

Notch signalling is a key pathway controlling angiogenesis in normal tissues and tumours. This has become a major focus of development of anticancer therapy, but to develop this appropriately, we need further understanding of the mechanisms of regulation of Dll4 (Delta-like ligand 4), a key endothelial Notch ligand. Dll4 and VEGF (vascular endothelial growth factor) cross-talk, with VEGF up-regulation of Dll4 and Dll4 down-regulating VEGFR (VEGF receptor) signalling. Both are essential for normal angiogenesis, and blockade of one may produce compensatory changes in the other. The present review considers recent developments in the regulation of Dll4 expression and functions, its role as a mechanism of resistance to anti-angiogenic therapy, and methods needed to develop effective therapy against this target.

Urokinase Gene Transfer Augments Angiogenesis in Ischemic Skeletal and Myocardial Muscle

Urokinase plasminogen activator (uPA) is required for both endogenous and vascular endothelial growth factor (VEGF)-augmented angiogenesis in normal tissues, leading us to hypothesize that uPA augmentation by gene transfer might promote angiogenesis in ischemic tissues. Overexpression of uPA was studied in rat myocardial infarction (MI) and mouse hind limb ischemia models and compared with VEGF overexpression effects. Animals were divided into control and three experimental groups (n = 6), receiving intramuscular injections of plasmids as follows: (i) control (empty vector or expressing beta-galactosidase); (ii) uPA; (iii) VEGF(165); (iv) a 1:1 mixture of uPA and VEGF(165). The capillary densities in both ischemic models were greater (P < 0.05) in tissues treated with uPA, VEGF, or a combination of both than in controls. Infarct size was reduced in hearts from uPA and VEGF experimental groups compared with controls (P < 0.05). Local overexpression of uPA induced a marked increase in the number of macrophages and myofibroblasts present within infarcts. Hind limb blood flow was greater in all experimental groups by day 10 (P < 0.05). Overall, the effects of uPA and VEGF were uniformly comparable. Additional analysis revealed association of local edema with VEGF but not with uPA treatment. This study established that uPA gene therapy effectively induces functionally significant angiogenesis in models of acute MI and hind limb ischemia.

Mammalian Target of Rapamycin (mTOR) Inhibitors as Anti-Cancer Agents

Highly specific signal transduction inhibitors are being developed as anti-cancer agents against an array of molecular targets, with the promise of increased selectivity and lower toxicity than classic cytotoxic chemotherapy agents. Rapamycin and its analogues are a promising class of novel therapeutics that specifically inhibit signaling from the serine-threonine kinase, mammalian target of rapamycin (mTOR). mTOR is a key intermediary in multiple mitogenic signaling pathways and plays a central role in modulating proliferation and angiogenesis in normal tissues and neoplastic processes. Rapamycin potently inhibits T-cell proliferation, and is approved for clinical use as an immuno-suppressant following kidney transplantation. Hyperactivation of mTOR signaling has been implicated in tumorigenesis, and promising pre-clinical studies in several tumor types suggest that the anti-proliferative and anti-angiogenic properties of rapamycin may be useful in cancer therapy. These studies have led to several clinical trials evaluating the safety and efficacy of rapamycin analogs in cancer therapy. The goal of this article is to review the mechanism of action of rapamycin as an anti-cancer agent, and to review the clinical experience with rapamycin and rapamycin analogs as immunosuppressive and anti-neoplastic therapeutic agents.

Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis.

Physiologically, angiogenesis is tightly regulated, or otherwise it leads to pathological processes, such as tumors, inflammatory diseases, gynecological diseases and diabetic retinopathy. The vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis. The VEGF gene expression is regulated by a variety of stimuli. Hypoxia is one of the most potent inducers of the VEGF expression. The hypoxia inducible factor 1 (HIF-1) plays as a key transcription factor in hypoxia-mediated VEGF gene upregulation. Nitric oxide (NO) as well as hypoxia is reported to upregulate the VEGF gene by enhancing HIF-1 activity. The Akt/protein kinase B (PKB) pathway may be involved in NO-mediated HIF-1 activation in limited cell lines. There are some reports of negative effects of NO on HIF-1 and VEGF activity. These conflicting data of NO effects may be attributed mainly to the amount of released NO. Indeed, NO can be a positive or negative modulator of the VEGF gene under the same conditions simply by changing its amounts. The VEGF-mediated angiogenesis requires NO production from activated endothelial NO synthase (eNOS). Activation of eNOS by VEGF involves several pathways including Akt/PKB, Ca(2+)/calmodulin, and protein kinase C. The NO-mediated VEGF expression can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity, and the VEGF-mediated NO production by eNOS can be also modulated by HIF-1 and HO-1 activity, depending upon the amount of produced NO. These reciprocal relations between NO and VEGF may contribute to regulated angiogenesis in normal tissues.

Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis.

Physiologically, angiogenesis is tightly regulated, or otherwise it leads to pathological processes, such as tumors, inflammatory diseases, gynecological diseases and diabetic retinopathy. The vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis. The VEGF gene expression is regulated by a variety of stimuli. Hypoxia is one of the most potent inducers of the VEGF expression. The hypoxia inducible factor 1 (HIF-1) plays as a key transcription factor in hypoxia-mediated VEGF gene upregulation. Nitric oxide (NO) as well as hypoxia is reported to upregulate the VEGF gene by enhancing HIF-1 activity. The Akt/protein kinase B (PKB) pathway may be involved in NO-mediated HIF-1 activation in limited cell lines. There are some reports of negative effects of NO on HIF-1 and VEGF activity. These conflicting data of NO effects may be attributed mainly to the amount of released NO. Indeed, NO can be a positive or negative modulator of the VEGF gene under the same conditions simply by changing its amounts. The VEGF-mediated angiogenesis requires NO production from activated endothelial NO synthase (eNOS). Activation of eNOS by VEGF involves several pathways including Akt/PKB, Ca(2+)/calmodulin, and protein kinase C. The NO-mediated VEGF expression can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity, and the VEGF-mediated NO production by eNOS can be also modulated by HIF-1 and HO-1 activity, depending upon the amount of produced NO. These reciprocal relations between NO and VEGF may contribute to regulated angiogenesis in normal tissues.

Angiogenesis in normal tissue adjacent to colon cancer.

Angiogenesis in malignant neoplasms, as measured by microvessel density, has been shown to correlate with survival or stage in some studies of breast, gastric, and colorectal cancer. We hypothesized that aggressive cancers promote angiogenesis in normal tissue adjacent to the invading neoplasm. To test this hypothesis, 36 specimens of colon adenocarcinoma curatively resected between 1986 and 1990 were sectioned and stained for factor VIII-related antigen, vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8). Microvessel density was measured within the colon cancer and in adjacent, histologically normal tissue. Clinical/pathological variables were examined using multivariate analysis and Student t-test. Microvessel density was higher in the neoplasms (26.0+/-1.66/ 0.25 mm2) than in the surrounding normal tissue (22.3+/-1.88/0.25 mm2) (P=0.03). The difference was primarily due to smaller neoplasms (T1 and T2) which had vessel counts of 10.6+/-0.74/0.25 mm2 in the adjacent normal tissue compared to vessel counts of 18.9+/-3.02/0.25 mm2 within these tumors (P=0.02). T3 and T4 neoplasms had equivalent amounts of angiogenesis within the lesion (26.9+/-1.81/0.25 mm2) and in the histologically normal margin (24.2+/-1.98/0.25 mm2) (P=0.12). VEGF was present in the tumor microenvironment in 100% and IL-8 in 45% of specimens stained for these angiogenic cytokines. Microvessel density did not correlate with 5-year survival. Our data suggest that colon cancers that invade through the muscularis propria may have a greater ability to induce angiogenesis in adjacent normal tissue.