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1.  Enzastaurin, an inhibitor of PKCβ, Enhances Antiangiogenic Effects and Cytotoxicity of Radiation against Endothelial Cells1,2 
Translational Oncology  2008;1(4):195-201.
PURPOSE: Angiogenesis plays an important role in pancreas cancer pathobiology. Pancreatic tumor cells secrete vascular endothelial growth factor (VEGF), activating endothelial cell protein kinase C beta (PKCβ) that phosphorylates GSK3β to suppress apoptosis and promote endothelial cell proliferation and microvessel formation. We used Enzastaurin (Enz) to test the hypothesis that inhibition of PKCβ results in radiosensitization of endothelial cells in culture and in vivo. MATERIALS/METHODS: We measured PKCβ phosphorylation, VEGF pathway signaling, colony formation, and capillary sprout formation in primary human dermal microvessel endothelial cells (HDMECs) after Enz or radiation (RT) treatment. Microvessel density and tumor volume of human pancreatic cancer xenografts in nude mice were measured after treatment with Enz, RT, or both. RESULTS: Enz inhibited PKCβ and radiosensitized HDMEC with an enhancement ratio of 1.31 ± 0.05. Enz combined with RT reduced HDMEC capillary sprouting to a greater extent than either agent alone. Enz prevented radiation-induced GSK3β phosphorylation of serine 9 while having no direct effect on VEGFR phosphorylation. Treatment of xenografts with Enz and radiation produced greater reductions in microvessel density than either treatment alone. The reduction in microvessel density corresponded with increased tumor growth delay. CONCLUSIONS: Enz-induced PKCβ inhibition radiosensitizes human endothelial cells and enhances the antiangiogenic effects of RT. The combination of Enz and RT reduced microvessel density and resulted in increased growth delay in pancreatic cancer xenografts, without increase in toxicity. These results provide the rationale for combining PKCβ inhibition with radiation and further investigating such regimens in pancreatic cancer.
PMCID: PMC2582168  PMID: 19043530
2.  MicroRNA expression after ionizing radiation in human endothelial cells 
Endothelial cells (EC) in tumor and normal tissue constitute critical radiotherapy targets. MicroRNAs have emerged as master switchers of the cellular transcriptome. Here, we seek to investigate the role of miRNAs in primary human dermal microvascular endothelial cells (HDMEC) after ionizing radiation.
The microRNA status in HDMEC after 2 Gy radiation treatment was measured using oligo-microarrays covering 361 miRNAs. To functionally analyze the role of radiation-induced differentially regulated miRNAs, cells were transfected with miRNA precursor or inhibitor constructs. Clonogenic survival and proliferation assays were performed.
Radiation up-regulated miRNA expression levels included let-7g, miR-16, miR-20a, miR-21 and miR-29c, while miR-18a, miR-125a, miR-127, miR-148b, miR-189 and miR-503 were down-regulated. We found that overexpression or inhibition of let-7g, miR-189, and miR-20a markedly influenced clonogenic survival and cell proliferation per se. Notably, the radiosensitivity of HDMEC was significantly influenced by differential expression of miR-125a, -127, -189, and let-7g. While miR-125a and miR-189 had a radioprotective effect, miR-127 and let-7g enhanced radiosensitivity in human endothelial cells.
Our data show that ionizing radiation changes microRNA levels in human endothelial cells and, moreover, exerts biological effects on cell growth and clonogenicity as validated in functional assays. The data also suggest that the miRNAs which are differentially expressed after radiation modulate the intrinsic radiosensitivity of endothelial cells in subsequent irradiations. This indicates that miRNAs are part of the innate response mechanism of the endothelium to radiation.
PMCID: PMC2859352  PMID: 20346162
3.  Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling1 
Neoplasia (New York, N.Y.)  2008;10(2):131-139.
Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.
PMCID: PMC2244688  PMID: 18283335
4.  Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor. 
Journal of Clinical Investigation  1997;99(11):2691-2700.
The presence of mast cells near capillary sprouting sites suggests an association between mast cells and angiogenesis. However, the role of mast cells in blood vessel development remains to be defined. In an attempt to elucidate this relationship, we investigated the effect of human mast cells (HMC-1) and their products on human dermal microvascular endothelial cell (HDMEC) tube formation. Coculture of HMC-1 with HDMEC led to a dose-response increase in the network area of vascular tube growth. Moreover, the extent of neovascularization was enhanced greatly when HMC-1 were degranulated in the presence of HDMEC. Further examination using antagonists to various mast cell products revealed a blunted response (73-88% decrease) in the area of vascular tube formation if specific inhibitors of tryptase were present. Tryptase (3 microg/ml) directly added to HDMEC caused a significant augmentation of capillary growth, which was suppressed by specific tryptase inhibitors. Tryptase also directly induced cell proliferation of HDMEC in a dose-dependent fashion (2 pM-2 nM). Our results suggest that mast cells act at sites of new vessel formation by secreting tryptase, which then functions as a potent and previously unrecognized angiogenic factor.
PMCID: PMC508115  PMID: 9169499
5.  Metronomic treatment of temozolomide increases anti-angiogenicity accompanied by down-regulated O6-methylguanine-DNA methyltransferase expression in endothelial cells 
Metronomic chemotherapy is a continuous low-dose administration of chemotherapeutic agents to minimize toxicity and target tumor-associated endothelial cells. This therapy is beneficial to anti-angiogenic efficacy which is linked to the inhibition of tumor growth. In the present study, we compared the anti-angiogenicity of temozolomide in human umbilical vein endothelial cells (HUVECs) between conventional and metronomic treatment. Metronomic treatment of temozolomide (TMZ) (6.25 and 12.5 μM) showed increased inhibition of the proliferation of HUVECs compared to an equivalent conventional treatment of TMZ. The differential effects between conventional and metronomic treatment of TMZ were also noted in cell migration and angiogenic tube formation. Notably, the expression level of O6-methylguanine-DNA methyltransferase (MGMT) was markedly reduced in the HUVECs treated with metronomic TMZ (12.5 and 25 μM) compared to cells treated with conventional treatment of TMZ. Accordingly, HUVECs treated with metronomic treatment of TMZ were more sensitive to TMZ treatment. Taken together, metronomic chemotherapy with TMZ enhances the inhibition of angiogenesis accompanied by the down-regulation of MGMT expression in endothelial cells when compared to conventional chemotherapy.
PMCID: PMC3440646  PMID: 22977508
metronomic chemotherapy; anti-angiogenicity; human umbilical vein endothelial cells; temozolomide; O6-methylguanine-DNA-methyltransferase
6.  Level of Endothelial Cell Apoptosis Required for a Significant Decrease in Microvessel Density 
Experimental cell research  2007;313(16):3645-3657.
Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF165) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.
PMCID: PMC2080879  PMID: 17720154
7.  Activation of iCaspase-9 in Neovessels Inhibits Oral Tumor Progression 
Journal of dental research  2006;85(5):436-441.
Tumors of the oral cavity are highly vascularized malignancies. Disruption of neovascular networks was shown to limit the access of nutrients and oxygen to tumor cells and inhibit tumor progression. Here, we evaluated the effect of the activation of an artificial death switch (iCaspase-9) expressed in neovascular endothelial cells on the progression of oral tumors. We used biodegradable scaffolds to co-implant human dermal microvascular endothelial cells stably expressing iCaspase-9 (HDMEC-iCasp9) with oral cancer cells expressing luciferase (OSCC3-luc or UM-SCC-17B-luc) in immunodeficient mice. Alternatively, untransduced HDMEC were co-implanted with oral cancer cells, and a transcriptionaly targeted adenovirus (Ad-VEGFR2-iCasp-9) was injected locally to deliver iCaspase-9 to neovascular endothelial cells. In vivo bioluminescence demonstrated that tumor progression was inhibited, and immunohisto-chemistry showed that microvessel density was decreased, when iCaspase-9 was activated in tumor-associated microvessels. We conclude that activation of iCaspase-9 in neovascular endothelial cells is sufficient to inhibit the progression of xenografted oral tumors.
PMCID: PMC2220154  PMID: 16632757
angiogenesis; neovascularization; apoptosis; suicide gene; bioluminescence
8.  Radio-sensitivities and angiogenic signaling pathways of irradiated normal endothelial cells derived from diverse human organs 
Journal of radiation research  2012;53(4):570-580.
The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and VEGFR-2, whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kinase-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure.
PMCID: PMC3393354  PMID: 22843622
ionizing radiation; normal endothelial cells; angiogenesis
9.  Pharmacokinetic-Directed Dosing of Vandetanib and Docetaxel in a Mouse Model of Human Squamous Cell Carcinoma 
Molecular cancer therapeutics  2008;7(9):3006-3017.
Docetaxel (DTX), usually administered according to maximum tolerated dosing (MTD), can inhibit endothelial cell (EC) proliferation at low nM concentrations. DTX may exert antiangiogenic effects if dosed so plasma levels are maintained at low nM concentrations over a prolonged time. We evaluated metronomic and MTD-based dosing of DTX with and without vandetanib (VAN), a VEGFR-2 and EGFR tyrosine kinase inhibitor with antiangiogenic and anti-tumor activity, in a head and neck xenograft model.
Experimental Design
A murine physiologically-based pharmacokinetic model was modified to predict DTX distribution following intraperitoneal administration to design dosing regimens that target pre-specified plasma concentrations, for anti-endothelial effects (metronomic), or exposure, to mimic 30mg/m2 (weekly/MTD) DTX in humans. Animals were treated for 28 days with 1mg/kg/day (DTX1) or 6mg/kg q4d (DTX6) DTX with or without VAN (15 mg/kg/day p.o.) in mice bearing UMSCC2 tumor xenografts.
The DTX1 dosing scheme was adjusted to treatment for 10 days followed by 9 days off due to severe GI toxicity. All treatment groups significantly reduced tumor volume, tumor proliferation (Ki-67) and tumor EC proliferation (Ki-67/vonWillebrand factor) compared with control. Addition of VAN to DTX treatment significantly enhanced tumor growth inhibition over single agent therapy.
A positive correlation of tumor EC proliferation with tumor growth rates demonstrates VAN and DTX antiangiogenic effects. Due to the morbidity observed with DTX1 treatment it is difficult to clearly ascertain if metronomic schedules will be effective for treatment. DTX with VAN is effective in treating UMSCC2 xenografts at concentrations relevant to exposures in humans.
PMCID: PMC2673509  PMID: 18790781
pharmacokinetic modeling; docetaxel; tyrosine kinase inhibitor; head and neck cancer; xenograft
10.  Anti-tumor and anti-angiogenic effects of metronomic dosing of BH3-mimetics 
Cancer research  2011;72(3):716-725.
Recent studies have shown that Bcl-2 functions as a pro-angiogenic signaling molecule in addition to its well-known effect as an inhibitor of apoptosis. The discovery of AT101, a BH3-mimetic drug that is effective and well tolerated when administered orally, suggested the possibility of using a molecularly targeted drug in a metronomic regimen. Here, we generated xenograft squamous cell carcinomas (SCC) with humanized vasculature in immunodeficient mice. Mice received taxotere in combination with either daily 10 mg/kg AT101 (metronomic regimen) or weekly 70 mg/kg AT101 (bolus regimen). The effect of single drug AT101 on angiogenesis, and combination AT101/taxotere on the survival of endothelial cells and SCC cells, were also evaluated in vitro. Metronomic AT101 increased mouse survival (p=0.02), decreased tumor mitotic index (p=0.0009), and decreased tumor microvessel density (p=0.0052), as compared to bolus delivery of AT101. Notably, the substantial potentiation of the anti-tumor effect observed in the metronomic AT101 group was achieved using the same amount of drug and without significant changes in systemic toxicities. In vitro, combination of AT101 and taxotere showed additive toxicity for endothelial cells and synergistic or additive toxicity for tumor cells (SCC). Interestingly, low-dose (sub-apoptotic) concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, these data unveiled the benefit of metronomic delivery of a molecularly targeted drug, and suggested that patients with squamous cell carcinomas might benefit from continuous administration of low dose BH3-mimetic drugs.
PMCID: PMC3748951  PMID: 22158856
Developmental therapeutics; targeted therapy; angiogenesis; Bcl-2; squamous cell carcinoma
11.  Enhanced anti-tumor and anti-angiogenic effects of metronomic cyclophosphamide combined with Endostar in a xenograft model of human lung cancer 
Oncology Reports  2012;28(2):439-445.
Standard chemotherapy for advanced NSCLC has reached a therapeutic plateau. More effective strategies must be explored. The purpose of this study was to evaluate the role of metronomic chemotherapy combined with an angiogenesis inhibitor in non-small cell lung cancer (NSCLC). A total of 114 BALB/c nude mice were inoculated subcutaneously with human NSCLC cells (A549), and when xenograft tumors were palpable, mice were randomly injected with saline as controls (Ctrl), or treated with metronomic cyclophosphamide (MET CPA), recombinant human endostatin, Endostar (Endo), MET CPA combined with Endostar (MET CPA + Endo) or maximum tolerance dose of CPA (MTD CPA), respectively. The growth of xenograft tumors and mouse survival were monitored. The frequency of peripheral blood circulating endothelial cells (CECs), microvessel density (MVD) and pericyte coverage was determined using flow cytometry and immunofluorescence staining. In comparison with the controls, treatment with either drug significantly inhibited the growth of xenograft tumors in mice. Treatment with MET CPA or Endostar, but not with MTD CPA, significantly reduced the frequency of peripheral blood total and viable CECs and the value of MVD. Endostar also considerably reduced pericyte coverage in xenograft tumors. Moreover, MET CPA combined with Endostar further reduced the frequency of peripheral blood CECs, the value of MVD, and pericyte coverage, with concomitant delay in tumor growth and extension of mouse survival. Our results indicate that MET CPA combined with Endostar results in enhanced anti-tumor and anti-angiogenic effects in a xenograft model of human lung cancer. Combined therapy with metronomic chemotherapy and an angiogenesis inhibitor may serve as a promising treatment strategy for patients with advanced NSCLC.
PMCID: PMC3583573  PMID: 22641525
non-small cell lung cancer; metronomic chemotherapy; cyclophosphamide; Endostar; angiogenesis
12.  Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells 
The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear.
Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls.
Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC.
The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds.
PMCID: PMC3653709  PMID: 23514369
13.  Inhibition of tumor angiogenesis by oral etoposide 
The chemotherapeutic agent etoposide is a topoisomerase II inhibitor widely used for cancer therapy. Low-dose oral etoposide, administered at close regular intervals, has potent anti-tumor activity in patients who are refractory to intravenous etoposide; however, the mechanism remains unclear. Since endothelial cells may be more sensitive than tumor cells to chemotherapy agents, we determined the effects of etoposide alone and in combination with oral cyclooxygenase-2 inhibitors and peroxisome-proliferator activated receptor γ ligands on angiogenesis and tumor growth in xenograft tumor models. Optimal anti-angiogenic (metronomic) and anti-tumor doses of etoposide on angiogenesis, primary tumor growth and metastasis were established alone and in combination therapy. Etoposide inhibited endothelial and tumor cell proliferation, decreased vascular endothelial growth factor (VEGF) production by tumor cells and suppressed endothelial tube formation at non-cytotoxic concentrations. In our in vivo studies, oral etoposide inhibited fibroblast growth factor 2 and VEGF-induced corneal neovascularization, VEGF-induced vascular permeability and increased levels of the endogenous angiogenesis inhibitor endostatin in mice. In addition, etoposide inhibited Lewis lung carcinoma (LLC) and human glioblastoma (U87) primary tumor growth as well as spontaneous lung metastasis in a LLC resection model. Furthermore, etoposide had synergistic anti-tumor activity in combination with celecoxib and rosiglitazone, which are also oral anti-angiogenic and anti-tumor agents. Etoposide inhibits angiogenesis in vitro and in vivo by indirect and direct mechanisms of action. Combining etoposide with celecoxib and rosiglitazone increases its efficacy and merits further investigation in future clinical trials to determine the potential usefulness of etoposide in combinatory anti-angiogenic chemotherapy.
PMCID: PMC3445956  PMID: 22993597
etoposide; tumor angiogenesis; metronomic chemotherapy; oral combination therapy; peroxisome-proliferator activated receptor
14.  Metronomic S-1 Chemotherapy and Vandetanib: An Efficacious and Nontoxic Treatment for Hepatocellular Carcinoma1 
Neoplasia (New York, N.Y.)  2011;13(3):187-197.
Metronomic chemotherapy involves frequent, regular administration of cytotoxic drugs at nontoxic doses, usually without prolonged breaks. We investigated the therapeutic efficacies of metronomic S-1, an oral 5-fluorouracil prodrug, and vandetanib, an epidermal growth factor receptor and vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, in models of hepatocellular carcinoma (HCC).
We compared anti-HCC effects and toxicity in the six treatment groups: control (untreated), maximum tolerated dose (MTD) S-1, metronomic S-1, vandetanib, MTD S-1 with vandetanib, and metronomic S-1 with vandetanib. Tumor microvessel density (MVD) and tumor apoptosis were evaluated by immunohistochemistry. The expression of VEGF and thrombospondin-1, an endogenous inhibitor of angiogenesis, was analyzed by Western blot.
Metronomic S-1 significantly inhibited tumor growth, which was enhanced by combination with vandetanib. With respect to toxicities, MTD S-1 caused severe body weight loss and myelosuppression, whereas metronomic S-1 did not cause any overt toxicities. Moreover, metronomic S-1 or metronomic S-1 with vandetanib prolonged survival, the latter treatment providing the greatest benefit. Metronomic S-1 and metronomic S-1 with vandetanib decreased MVDs and increased apoptosis in tumor tissues. The expression of VEGF in tumor tissues was upregulated by vandetanib and metronomic S-1 with vandetanib, whereas the expression of thrombospondin-1 was upregulated by metronomic S-1 and metronomic S-1 with vandetanib.
Metronomic S-1 with an antiangiogenic agent seems to be an effective and safe therapeutic strategy for HCC.
PMCID: PMC3050862  PMID: 21390182
15.  Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells. 
Journal of Clinical Investigation  1997;100(10):2521-2529.
We have compared the adhesion of Plasmodium falciparum-infected erythrocytes to human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) and have assessed the relative roles of the receptors CD36 and intercellular adhesion molecule-1 (ICAM-1). HUVEC (a cell line that expresses high levels of ICAM-1 but no CD36) mediate low levels of adhesion, whereas HDMEC (which constitutively express CD36) mediate high levels of adhesion even before ICAM-1 induction ICAM-1 expression leads to yet greater levels of adhesion, which are inhibited both by anti-ICAM-1 and CD36 mAbs, despite no increase in the expression of CD36. The results indicate the presence of a substantial population of infected cells that require the presence of both receptors to establish adhesion. Synergy between these receptors could be demonstrated using a number of parasite lines, but it could not be predicted from the binding of these same parasite lines to purified ICAM-1 and CD36. This phenomenon could not be reproduced using either purified receptors presented on plastic, or formalin-fixed HDMEC, suggesting that receptor mobility is important. This is the first study to demonstrate receptor synergy in malaria cytoadherence to human endothelial cells, a phenomenon necessary for parasite survival and associated with disease severity.
PMCID: PMC508452  PMID: 9366566
16.  Antigen Presentation by Human Microvascular Endothelial Cells Triggers ICAM-1-dependent Transendothelial Protrusion by and Fractalkine-dependent Transendothelial Migration of Effector Memory CD4+ T Cells 
TCR engagement on adherent human effector memory (EM) CD4+ T cells by TNF-treated human umbilical vein endothelial cells (HUVEC) under flow induces formation of a transendothelial protrusion (TEP) by the T cell but fails to induce transendothelial migration (TEM). In contrast, TCR engagement of the same T cell populations by TNF-treated human dermal microvascular cells (HDMEC) not only induces TEP formation, but triggers TEM at or near the interendothelial cell junctions via a process in which TEP formation appears to be the first step. Transduction of adhesion molecules in unactivated HDMEC and use of blocking antibodies as conducted with TNF-activated HDMEC indicate that ICAM-1 plays a nonredundant role in TCR-driven TEP formation and TEM, and that TCR-driven TEM is also dependent upon fractalkine. TEP formation, dependence on ICAM-1 and dependence on fractalkine distinguish TCR-induced TEM from IP-10 induced TEM. These in vitro observations suggest that presentation of antigen by human microvascular EC to circulating CD4+ EM T cells may function to initiate recall responses in peripheral tissues.
PMCID: PMC2435282  PMID: 18523306
Endothelial cells; T cells; T cell receptors; chemokines; superantigens
17.  Modulation of the Antitumor Activity of Metronomic Cyclophosphamide by the Angiogenesis Inhibitor Axitinib 
Molecular cancer therapeutics  2008;7(1):79-89.
The promising but still limited efficacy of angiogenesis inhibitors as monotherapies for cancer treatment indicates a need to integrate these agents into existing therapeutic regimens. Presently, we investigate the anti-tumor activity of the small molecule angiogenesis inhibitor axitinib (AG-013736) and its potential for combination with metronomic cyclophosphamide (CPA). Axitinib significantly inhibited angiogenesis in rat 9L tumors grown s.c. in scid mice, but only moderately delayed tumor growth. Combination of axitinib with metronomic CPA fully blocked 9L tumor growth upon initiation of drug treatment. In contrast, metronomic CPA alone required multiple treatment cycles to halt tumor growth. However, in contrast to the substantial tumor regression that is ultimately induced by metronomic CPA, the axitinib/CPA combination was tumor growth static. Axitinib did not inhibit hepatic activation of CPA or export of its activated metabolite, 4-OH-CPA, to extrahepatic tissues; rather, axitinib selectively decreased 9L tumor uptake of 4-OH-CPA by 30–40%. The reduced tumor penetration of 4-OH-CPA was associated with a decrease in CPA-induced tumor cell apoptosis and a block in the induction of the endogenous angiogenesis inhibitor TSP-1 in tumor-associated host cells, which may contribute to the absence of tumor regression with the axitinib/CPA combination. Finally, axitinib transiently increased 9L tumor cell apoptosis, indicating that its effects are not limited to the endothelial cell population. These findings highlight the multiple effects that may characterize anti-angiogenic agent–metronomic chemotherapy combinations, and suggest that careful optimization of drug scheduling and dosages will be required to maximize anti-tumor responses.
PMCID: PMC2390754  PMID: 18202011
anti-angiogenesis; axitinib (AG-013736); metronomic cyclophosphamide; combination therapy
18.  Overexpression of Laminin-8 in Human Dermal Microvascular Endothelial Cells Promotes Angiogenesis-Related Functions 
This study examined the effects of endogenous overexpression of laminin-8 on angiogenesis and wound healing in primary human dermal microvascular endothelial cells (HDMECs). HDMECs expressed laminin-8 and laminin-10, but no other laminins, as determined by radioimmunoprecipitation assay using a panel of antibodies to individual laminin chains. To study laminin-8 function, full-length human laminin α4 cDNA was retrovirally transferred to HDMEC, and specific overexpression of laminin-8 was verified by Western blot. Laminin-8 overexpression promoted endothelial cell spreading and migration in scratch assays and accelerated angiogenic tubule formation in collagen gel overlay assays. Strong inhibitory effect of β1 integrin and weak inhibition by αvβ3 integrin antibodies were observed in laminin-8-stimulated cell migration, but only β1 integrin antibodies affected tubule formation. These studies suggest that laminin-8 overexpression may prove to be a useful method to engineer HDMECs to promote angiogenesis and wound repair.
PMCID: PMC2535788  PMID: 16374451
19.  VEGF Is Involved in the Increase of Dermal Microvascular Permeability Induced by Tryptase 
ISRN Dermatology  2012;2012:941465.
Tryptases are predominantly mast cell-specific serine proteases with pleiotropic biological activities and play a critical role in skin allergic reactions, which are manifested with rapid edema and increases of vascular permeability. The exact mechanisms of mast cell tryptase promoting vascular permeability, however, are unclear and, therefore, we investigated the effect and mechanism of tryptase or human mast cells (HMC-1) supernatant on the permeability of human dermal microvascular endothelial cells (HDMECs). Both tryptase and HMC-1 supernatant increased permeability of HDMECs significantly, which was resisted by tryptase inhibitor APC366 and partially reversed by anti-VEGF antibody and SU5614 (catalytic inhibitor of VEGFR). Furthermore, addition of tryptase to HDMECs caused a significant increase of mRNA and protein levels of VEGF and its receptors (Flt-1 and Flk-1) by Real-time RT-PCR and Western blot, respectively. These results strongly suggest an important role of VEGF on the permeability enhancement induced by tryptase, which may lead to novel means of controlling allergic reaction in skin.
PMCID: PMC3361195  PMID: 22666607
20.  Sirtinol Treatment Reduces Inflammation in Human Dermal Microvascular Endothelial Cells 
PLoS ONE  2011;6(9):e24307.
Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.
PMCID: PMC3171404  PMID: 21931678
21.  Low-dose metronomic oral dosing of a prodrug of Gemcitabine (LY2334737) causes anti-tumor effects in the absence of inhibition of systemic vasculogenesis 
Molecular cancer therapeutics  2011;11(3):680-689.
Metronomic chemotherapy refers to the close, regular administration of conventional chemotherapy drugs at relatively low minimally toxic doses with no prolonged break periods; it is now showing encouraging results in various phase II clinical trials, and is currently undergoing phase III trial evaluation. It is thought to cause anti-tumor effects primarily by antiangiogenic mechanisms, both locally by targeting endothelial cells of the tumor neovasculature and systemically by effects on bone marrow derived cells, including circulating endothelial progenitor cells (CEPs). Previous studies have shown reduction of CEPs by metronomic administration of a number of different chemotherapeutic drugs, including vinblastine, cyclophosphamide, paclitaxel, topotecan, and tegafur plus uracil (UFT). However in addition to, or even instead of, anti angiogenic effects, metronomic chemotherapy may cause suppression of tumor growth by other mechanisms such as stimulating cytotoxic T cell responses, or by direct anti-tumor effects. Here we report results evaluating the properties of metronomic administration of an oral prodrug of gemcitabine LY2334737 (LY) in non tumor-bearing mice, and in preclinical models of human ovarian (SKOV3-13) and breast cancer (LM2-4) xenografts. Through daily gavage (at 6mg/kg/day) the schedules tested were devoid of toxicity and caused anti-tumor effects; however, a suppressive effect on CEPs was not detected. Unexpectedly metronomic LY administration caused increased blood flow in luciferase-tagged LM2-4 tumor xenografts; and this effect coincided with a relative increase in tumor bioluminescence. These results highlight the possibility of significant anti-tumor effects mediated by metronomic administration of some chemotherapy drugs without a concomitant inhibition of systemic angiogenesis.
PMCID: PMC3409688  PMID: 22188817
endothelial progenitor cells; blood flow; angiogenesis
22.  Low-Dose Metronomic Cyclophosphamide Combined with Vascular Disrupting Therapy Induces Potent Anti-Tumor Activity in Preclinical Human Tumor Xenograft Models 
Molecular cancer therapeutics  2009;8(10):2872-2881.
Vascular disrupting agents (VDAs) preferentially target the established but abnormal tumor vasculature, resulting in extensive intratumoral hypoxia and cell death. However, a rim of viable tumor tissue remains from which angiogenesis-dependent regrowth can occur, in part via mobilization and tumor colonization of circulating endothelial progenitor cells (CEPs). Co-treatment with an agent that blocks CEPs, such as VEGF-pathway targeting biologic antiangiogenic drugs, results in enhanced anti-tumor efficacy. We asked whether an alternative therapeutic modality – low-dose metronomic (LDM) chemotherapy could achieve the same result, given its CEP targeting effects.
Experimental Design
We studied the combination of the VDA OXi-4503 with daily administration of CEP-inhibiting, low-dose metronomic (LDM) cyclophosphamide to treat primary orthotopic tumors using the 231/LM2-4 breast cancer cell line and MeWo melanoma cell line. In addition, CEP mobilization and various tumor characteristics were assessed.
We found that daily oral LDM cyclophosphamide was capable of preventing the CEP spike and tumor colonization induced by OXi-4503; this was associated with a decrease in the tumor rim and marked suppression of primary 231/LM2-4 growth in nude as well as SCID mice. Similar results were found in MeWo bearing nude mice. The delay in tumor growth was accompanied by significant decreases in micro-vessel density, perfusion and proliferation, and a significant increase in tumor cell apoptosis. No overt toxicity was observed.
The combination of OXi-4503 and metronomic chemotherapy results in prolonged tumor control, thereby expanding the list of therapeutic agents that can be successfully integrated with metronomic low-dose chemotherapy.
PMCID: PMC2782518  PMID: 19825805
OXi-4503; low-dose cyclophosphamide; breast cancer; melanoma; endothelial progenitor cells
23.  Collaboration between Hepatic and Intratumoral Prodrug Activation in a P450 Prodrug-activation Gene Therapy Model for Cancer Treatment 
Molecular cancer therapeutics  2007;6(11):2879-2890.
Presently, we investigate the mechanisms whereby intratumoral expression of a cyclophosphamide (CPA)-activating hepatic cytochrome P450 gene enhances therapeutic activity when CPA is given on an every 6-day (metronomic) schedule. In P450-deficient 9L gliosarcomas grown in scid mice, metronomic CPA substantially decreased tumor microvessel density and induced a ~70% loss of endothelial cells that began after the second CPA treatment. These responses were accompanied by increased expression of the endogenous angiogenesis inhibitor thrombospondin-1 in tumor-associated host cells but by decreased expression in 9L tumor cells. These anti-angiogenic responses preceded tumor regression and are likely key to the therapeutic activity of metronomic CPA. Unexpectedly, 9L/2B11 tumors, grown from 9L cells infected with retrovirus encoding the CPA-activating P450 2B11, exhibited anti-angiogenic responses very similar to 9L tumors. This indicates that the tumor endothelial cell population is well exposed to liver-activated CPA metabolites and that intratumoral P450 confers limited additional anti-endothelial cell bystander activity. In contrast, TUNEL assay revealed an increase in apoptosis that preceded the anti-angiogenic response and was substantially enhanced by intratumoral P450 2B11 expression. 9L/2B11 tumor regression was accompanied by an overall loss of tumor cellularity and by substantial enlargement of remaining P450-immunoreactive tumor cells as the number of P450-positive tumor cell decreased and the P450 protein content declined with CPA treatment. We conclude that metronomic CPA regresses P450-expressing tumors by two independent but complementary mechanisms: increased tumor cell killing via intratumoral P450-catalyzed prodrug activation, coupled with strong anti-angiogenic activity, which is primarily associated with hepatic prodrug activation.
PMCID: PMC2613795  PMID: 17989319
Cyclophosphamide; Anti-angiogenesis; Gene-Directed Enzyme-Prodrug Therapy
24.  Antiangiogenic and anticolorectal cancer effects of metronomic irinotecan chemotherapy alone and in combination with semaxinib 
British Journal of Cancer  2008;98(10):1619-1629.
Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.
PMCID: PMC2391121  PMID: 18443598
metronomic chemotherapy; angiogenesis; irinotecan; semaxinib; colon cancer
25.  Low-dosage metronomic chemotherapy and angiogenesis: topoisomerase inhibitors irinotecan and mitoxantrone stimulate VEGF-A-mediated angiogenesis 
Apmis  2012;120(2):147-156.
Albertsson P, Lennernäs B, Norrby K. Low-dosage metronomic chemotherapy and angiogenesis: topoisomerase inhibitors irinotecan and mitoxantrone stimulate VEGF-A-mediated angiogenesis. APMIS 2011.
Metronomic chemotherapy with cytotoxic agents has been shown to inhibit angiogenesis and, consequently, tumor growth by targeting vascular endothelial cells (ECs). In these regimens, anti-tumor activities additional to anti-angiogenesis may operate. Moreover, chemotherapy typically generates reactive oxygen species in targeted ECs, which can affect angiogenesis. The aim of the present study was to assess the systemic effect of low-dosage metronomic treatment with either irinotecan or mitoxantrone on angiogenesis induced by VEGF-A. Angiogenesis was induced in normal adult rat mesentery by intraperitoneal injection of a low dosage of VEGF-A. Thereafter, irinotecan and mitoxantrone were infused separately continuously at minimally toxic dosages for 14 consecutive days via a subcutaneous osmotic minipump. Angiogenesis was assessed in terms of objective and quantitative variables using morphologic and computerized image analyses. Irinotecan or mitoxantrone significantly stimulated angiogenesis, with ironotecan increasing angiogenesis by 104%, when compared with the vehicle-treated animals. Low-dosage metronomic chemotherapy with irinotecan or mitoxantrone stimulates angiogenesis in the normal mesentery of rats, probably by inducing low-level oxidative stress in the targeted ECs. Whether or not this pertains to tumor angiogenesis may be difficult to confirm, as several anti-tumor modes may operate during low-dosage metronomic chemotherapy.
PMCID: PMC3321228  PMID: 22229270
Angiogenesis; VEGF; metronomic chemotherapy; irinotecan; mitoxantrone; rat; reactive oxygen species

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