Angiogenic Models Research Articles

Notch1 signaling regulates Sox9 and VEGFA expression and governs BMP2-induced endochondral ossification of mesenchymal stem cells

Although bone morphogenetic protein 2 (BMP2) can induce chondrogenic differentiation of mesenchymal stem cells (MSCs), its induction of endochondral ossification limits the application of BMP2-based cartilage regeneration. Here, we clarified the mechanisms of BMP2-induced endochondral ossification of MSCs. In vitro and in vivo chondrogenic, osteogenic, and angiogenic differentiation models of MSCs were constructed. The expression of target genes was identified at both protein and mRNA levels. RNA sequencing, molecular docking, co-immunoprecipitation, and chromatin immunoprecipitation followed by sequencing were applied to investigate the molecular mechanisms. We found that BMP2 up-regulated the expression of Notch receptors and ligands in MSCs. Notch1 signaling activation was related to inhibition of chondrogenic differentiation, promotion of osteogenic and angiogenic differentiation. In vivo ectopic stem cell implantation identified that Notch1 signaling activation blocked BMP2-induced chondrogenesis and facilitated endochondral ossification of MSCs. Mechanistically, we elucidated Notch1 intracellular domain (NICD1)-RBPjk complex binding to SRY-box transcription factor 9 (Sox9) and vascular endothelial growth factor A (VEGFA) promoters to decrease Sox9 expression and increase VEGFA expression. These findings suggest that Notch1 signaling can regulate BMP2-induced endochondral ossification by promoting RBPjk-mediated Sox9 inactivation and VEGFA expression. It is conceivable that targeting Notch1 signaling mediated endochondral ossification would benefit BMP2-based cartilage regeneration.

Single-cell and Bulk RNA-Seq reveal angiogenic heterogeneity and microenvironmental features to evaluate prognosis and therapeutic response in lung adenocarcinoma.

Angiogenesis stands as a pivotal hallmark in lung adenocarcinoma (LUAD), intricately shaping the tumor microenvironment (TME) and influencing LUAD progression. It emerges as a promising therapeutic target for LUAD, affecting patients' prognosis. However, its role in TME, LUAD prognosis, and its clinical applicability remain shrouded in mystery. We employed integrated single-cell and bulk transcriptome sequencing to unravel the heterogeneity of angiogenesis within LUAD cells. Through "consensus clustering", we delineated distinct angiogenic clusters and deciphered their TME features. "Monocle2" was used to unravel divergent trajectories within malignant cell subpopulations of LUAD. Additionally, regulon submodules and specific cellular communication patterns of cells in different angiogenic states were analyzed by "pyscenic" and "Cellchat" algorithms. The "univariate Cox" and "LASSO" algorithms were applied to build angiogenic prognostic models. Immunohistochemistry (IHC) on clinical samples validated the role of model factors in LUAD angiogenesis. We utilized CTRP 2.0 and PRISM databases for pinpointing sensitive drugs against lung adenocarcinoma. Two clusters for the activation of angiogenesis were identified, with Cluster 1 showing a poor prognosis and a pro-cancerous TME. Three differentiated states of malignant epithelial LUAD cells were identified, which had different degrees of angiogenic activation, were regulated by three different regulon submodules, and had completely different crosstalk from other cells in TME. The experiments validate that SLC2A1 promotes angiogenesis in LUAD. ARS (Angiogenesis related score) had a high prognostic value; low ARSs showed immunotherapy benefits, whereas high ARSs were sensitive to 15 chemotherapeutic agents. The assessment of angiogenic clusters helps to determine the prognostic and TME characteristics of LUAD. Angiogenic prognostic models can be used to assess the prognosis, immunotherapeutic response, and chemotherapeutic drug sensitivity of LUAD.

Phytochemicals Showing Antiangiogenic Effect in Pre-clinical Models and their Potential as an Alternative to Existing Therapeutics.

Angiogenesis, the formation of new blood vessels from a pre-existing vascular network, is an important hallmark of several pathological conditions, such as tumor growth and metastasis, proliferative retinopathies, including proliferative diabetic retinopathy and retinopathy of prematurity, age-related macular degeneration, rheumatoid arthritis, psoriasis, and endometriosis. Putting a halt to pathology-driven angiogenesis is considered an important therapeutic strategy to slow down or reduce the severity of pathological disorders. Considering the attrition rate of synthetic antiangiogenic compounds from the lab to reaching the market due to severe side effects, several compounds of natural origin are being explored for their antiangiogenic properties. Employing pre-clinical models for the evaluation of novel antiangiogenic compounds is a promising strategy for rapid screening of antiangiogenic compounds. These studies use a spectrum of angiogenic model systems that include HUVEC two-dimensional culture, nude mice, chick chorioallantoic membrane, transgenic zebrafish, and dorsal aorta from rats and chicks, depending upon available resources. The present article emphasizes the antiangiogenic activity of the phytochemicals shown to exhibit antiangiogenic behavior in these well-defined existing angiogenic models and highlights key molecular targets. Different models help to get a quick understanding of the efficacy and therapeutics mechanism of emerging lead molecules. The inherent variability in assays and corresponding different phytochemicals tested in each study prevent their immediate utilization in clinical studies. This review will discuss phytochemicals discovered using suitable preclinical antiangiogenic models, along with a special mention of leads that have entered clinical evaluation.

574 Three-dimensional culture of dermal papilla cells improves their in vitro angiogenic activity

Tissue-engineered skin constructs correct vascularization remains a major challenge. We have shown that the presence of dermal papilla cells (DPCs) in these constructs favors the vascularization process, resulting in a better wound healing and graft take. To culture DPC as spheres increases the expression of angiogenic genes such as VEGF, angiogenin and FGF. Since angiogenesis in scaffolds is essential for grafts to survive and integrate with existing host tissue, our aim was to compare the ability of monolayer and sphere cultures of DPC to favor endothelial cell migration and neovessel development by using two angiogenic study models.

LRP-1 Matricellular Receptor Involvement in Triple Negative Breast Cancer Tumor Angiogenesis.

Background: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression. Methods: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs’ angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways. Results: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs’ angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-β signaling and plasminogen/plasmin system. Conclusions: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

336 In vitro models to study angiogenic effects of dermal papilla cells as cellular component of skin substitutes

Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries. However, its correct vascularization remains a major challenge. We have shown that the presence of dermal papilla cells (DPCs) in these constructs favors the vascularization process, resulting in a better wound healing and graft take. We also have seen DPC-spheres culture increase the expression of angiogenic genes as VEGF, angiogenin and FGF. Since angiogenesis in scaffolds is essential for grafts to survive and integrate with existing host tissue, our aim was to develop two in vitro models to quantify angiogenesis stimulation of monolayer and spheres [G1] cultures of DPC. The first model was a migration assay, in which DPC or dermal fibroblasts (DF) were co-cultured as monolayers with Human Umbilical Vein Endothelial Cells (HUVEC) in a transwell system. We observed more migrated HUVEC in the coculture with DPC than in the DF or medium controls. The second model was an endothelial cell tube formation assay where HUVEC, resuspended in DPC-conditioned medium and seeded on Matrigel, showed an increase in the total tubule’s length and in the number of segments and joints, compared to the medium control. To be able to compare the inductive molecules secretion between monolayer and spheres [G2] -DPC cultures, we looked for the condition in which both systems contained the same amount of metabolically active cells. In our culture conditions, 55 spheres [G3] were metabolically equivalent to 104 cells/cm2 monolayer seeded cells. Using this equivalence, we are evaluating the effects that sphere culture conditions have on the angiogenic abilities of DPCs. We conclude that these two angiogenic models are useful to measure, in vitro, the stimulation of angiogenesis of cell cultures to be used as dermal components in skin substitutes. In this way, the use of DPC in skin substitutes could favor the vascularization of the grafts, and thus the closure of the wound and the graft take.

Reversine suppresses osteosarcoma cell growth through targeting BMP-Smad1/5/8-mediated angiogenesis

Reversine, or 2-(4-morpholinoanilino)-6cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule exhibits tumor-suppressive activities through different molecular mechanisms. In this study, in vitro and in vivo angiogenic models were used to elucidate the effect of Reversine on angiogenesis in the tumor suppression. Firstly, we grafted osteosarcoma-derived MNNG/HOS cell aggregates onto chick embryonic chorioallantoic membrane (CAM) to examine the vascularization of these grafts following Reversine treatment. Following culture, it was determined that Reversine inhibited MNNG/HOS grafts growth, and decreased the density of blood vessels in the chick CAM. We then used CAM and chick embryonic yolk-sac membrane (YSM) to investigate the effects of Reversine on angiogenesis. The results revealed Reversine inhibited the proliferation of endothelial cells, where cells were mainly arrested at G1/S phase of the cell cycle. Scratch-wound assay with HUVECs revealed that Reversine suppressed cell migration in vitro. Furthermore, endothelial cells tube formation assay and chick aortic arch sprouting assay demonstrated Reversine inhibited the sprouting, migration of endothelial cells. Lastly, qPCR and western blot analyses showed BMP-associated Smad1/5/8 signaling expressions were up-regulated by Reversine treatment. Our results showed that Reversine could suppress tumor growth by inhibiting angiogenesis through BMP signaling, and suggests a potential use of Reversine as an anti-tumor therapy.

Correction to: Angiogenic Marker Prognostic Models in Pregnant Women With Hypertension.

Correction to: Angiogenic Marker Prognostic Models in Pregnant Women With Hypertension.

Microfluidics for Angiogenesis Research.

Angiogenesis is a natural and vital phenomenon of neovascularization that occurs from pre-existing vasculature, being present in many physiological processes, namely in development, reproduction and regeneration. Being a highly dynamic and tightly regulated process, its abnormal expression can be on the basis of several pathologies. For that reason, angiogenesis has been a subject of major interest among the scientific community, being transverse to different areas and founding particular attention in tissue engineering and cancer research fields. Microfluidics has emerged as a powerful tool for modelling this phenomenon, thereby surpassing the limitations associated to conventional angiogenic models. Holding a tremendous flexibility in terms of experimental design towards a specific goal, microfluidic systems can offer an unlimited number of opportunities for investigating angiogenesis in many relevant scenarios, namely from its fundamental comprehension in normal physiological processes to the identification and testing of new therapeutic targets involved on pathological angiogenesis. Additionally, microvascular 3D in vitro models are now opening up new prospects in different fields, being used for investigating and establishing guidelines for the development of next generation of 3D functional vascularized grafts. The promising applications of this emerging technology in angiogenesis studies are herein overviewed, encompassing fundamental and applied research.

Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/β-arr1 (β-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

Hypoxia activates placental growth factor expression in lymphatic endothelial cells.

Placental growth factor (PlGF), a proangiogenic member of vascular endothelial growth family, is active during pathological conditions like cancer, metastasis formation and hind limb ischemia and in wound healing. Endothelial cells express PlGF and hypoxia positively modulates in vitro its expression. To verify whether hypoxia modulates PlGF expression in different cellular contexts and in vivo, we first analyzed five human and five mouse cancer cell lines showing that in eight of them hypoxia positively modulates PlGF. Next, we analyzed xenograft colorectal cancer tumors showing that human cancer cells were able to express PlGF in hypoxic area of the tumor. Surprisingly, we did not visualize mouse PlGF in CD31 positive tumor vessels, but in low CD31 positive vessels, a characteristic of lymphatic vessels. We found that hypoxia effectively activates PlGF expression in lymphatic endothelial cells as well as in LYVE1 positive tumor vessels. We also investigated two additional mouse angiogenic models, hind limb ischemia and wound healing, and we confirmed that lymphatic vessels of both ischemic muscles and skin express PlGF. These results show for the first time that hypoxia activates PlGF expression in lymphatic endothelial cells, which have to be considered an additional source for PlGF production in pathological contexts.

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy

Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

BP-1T, an antiangiogenic benzophenone-thiazole pharmacophore, counteracts HIF-1 signalling through p53/MDM2-mediated HIF-1α proteasomal degradation.

Hypoxia is a feature of all solid tumours, contributing to tumour progression. Activation of HIF-1α plays a critical role in promoting tumour angiogenesis and metastasis. Since its expression is positively correlated with poor prognosis for cancer patients, HIF-1α is one of the most convincing anticancer targets. BP-1T is a novel antiproliferative agent with promising antiangiogenic effects. In the present study, the molecular mechanism underlying cytotoxic/antiangiogenic effects of BP-1T on tumour/non-tumour angiogenesis was evaluated. Evidences show that BP-1T exhibits potent cytotoxicity with prolonged activity and effectively regressed neovessel formation both in reliable non-tumour and tumour angiogenic models. The expression of CoCl2-induced HIF-1α was inhibited by BP-1T in various p53 (WT)-expressing cancer cells, including A549, MCF-7 and DLA, but not in mutant p53-expressing SCC-9 cells. Mechanistically, BP-1T mediates the HIF-1α proteasomal degradation by activating p53/MDM2 pathway and thereby downregulated HIF-1α-dependent angiogenic genes such as VEGF-A, Flt-1, MMP-2 and MMP-9 under hypoxic condition of in vitro and in vivo solid tumour, eventually leading to abolition of migration and invasion. Based on these observations, we conclude that BP-1T acts on HIF-1α degradation through p53/MDM2 proteasome pathway.

Evaluation of Antiangiogenic Efficacy of Emilia sonchifolia (L.) DC on Tumor-Specific Neovessel Formation by Regulating MMPs, VEGF, and Proinflammatory Cytokines.

Formation of new blood vessels from preexisting vasculature is an indispensable process in tumor initiation, invasion, and metastasis. Novel therapeutic approaches target endothelial cells involved in the process of angiogenesis, due to their genetic stability relative to the rapidly mutating drug-resistant cancer cells. In the present study, we investigated the effect of an active fraction from Emilia sonchifolia, belonging to the family Asteraceae, a plant well known for its anti-inflammatory and antitumor effects, on the inhibition of tumor-specific angiogenesis. Administration of the active fraction from E sonchifolia (AFES; 5 mg/kg, body weight, intraperitoneally) containing the major compound γ-humulene significantly inhibited B16F10 melanoma-induced capillary formation in C57BL/6 mice. The level of serum vascular endothelial growth factor and serum proinflammatory cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor were also reduced significantly. At the same time, administration of AFES significantly enhanced the production of antiangiogenic factors such as tissue inhibitor of matrix metalloproteinase-1. Dose-dependent reduction can be seen in the budding and expansion of microvessels from rat thoracic aorta by AFES treatment. Inhibition of the activation of proenzyme to active enzyme of matrix metalloproteinase along with a successful reduction of proliferation, invasion, and migration of human umbilical vein endothelial cells demonstrated the antiangiogenic effect of AFES in vitro. To date, no study has examined the antiangiogenic activity of this plant with already well-known anti-inflammatory and antitumor effects. Results obtained in the present study by using both in vivo and in vitro angiogenic models altogether proved the inhibitory effect of AFES on tumor-specific neovessel formation.

Abstract 4133: ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic cancer models

Abstract Genomic alterations in fibroblast growth factor receptors (FGFR) and upregulation of vascular endothelial growth factor receptors (VEGFR) are often found in the same cancer types such as gastric, lung and breast and these alterations correlate with patient survival and disease progression. In addition, both FGFR and VEGFR signaling promote tumor angiogenesis. Activation of FGFR signaling has also been described to function as a compensatory angiogenic signal following development of resistance to VEGF inhibition. ODM-203 is a novel and selective inhibitor of the FGFR and VEGFR families exhibiting equal potency to both receptors both in vitro and in vivo. In recombinant kinase assays, ODM-203 inhibits both the FGFR and VEGFR family kinases in the low nanomolar range (IC50 6-35 nM). In cells, ODM-203 suppresses FGFR and VEGFR signaling with similar potency. It also inhibits VEGF-induced endothelial cell (HUVEC) proliferation (IC50 33 nM) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nM). In vivo, ODM-203 shows strong anti-tumor activity in FGFR-dependent xenograft models and in angiogenic xenograft models with similar well tolerated doses. In addition ODM-203 inhibits metastatic tumor growth in angiogenic xenograft models. Lack of activity in a FGFR and VEGFR independent tumor models further supports the selective mode of action of ODM-203. Oral administration of ODM-203 was well tolerated with only FGFR or VEGFR inhibition related physiological events observed in vivo in rat and dog. Based on our findings, ODM-203 has a unique profile with equal potency for both FGFR and VEGFR kinase families which correlates with with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic tumor models. Therefore, a clinical trial with ODM-203 is ongoing in patients with solid tumors (NCT 02264418). Citation Format: Tim Holmström, Anu Moilanen, Mari Björkman, Tero Linnanen, Gerd Wohlfahrt, Stefan Karlsson, Riikka Oksala, Timo Korjamo, Susanta Samajdar, Srinivasan Rajagopalan, Shekar Chelur, Kishore Narayan, Raghuveer Ramachandra, Thomas Anthony, Samiulla DS, Murali Ramachandra, Pekka Kallio. ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong activity on primary and metastatic tumor growth in FGFR-dependent and angiogenic cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2015-4133

Intermedin restricts vessel sprouting by inhibiting the loosening of endothelial junction

Vessel sprouting from pre-existing vasculature is a key step for the formation of a functional vasculature. The low level of vascular endothelial growth factor (VEGF) induces normal and stable angiogenesis, whereas high level of VEGF causes irregular and over sprouted vasculature. Intermedin (IMD) is a novel member of calcitonin family, and was found to be able to restrict the excessive vessel sprouting. However, the underlying mechanism had not been elucidated. In this study, using in vitro and in vivo angiogenic models, we found that the loosening of endothelial junction could significantly increase the ability of low-dose VEGF to induce vessel sprouting. IMD inhibited the junction dissociation-induced vessel sprouting by re-establishing the complex of vascular endothelial cadherin on the cell–cell contact. Our findings suggested a novel mechanism through which IMD could restrict the excessive vessel sprouting by preventing the endothelial junction from dissociation, and provide new insight into the understanding of the regulation of sprouting angiogenesis.

Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice.

BackgroundThe capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma.MethodsPC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed.ResultsGLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed.ConclusionsCPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.

Pro‐angiogenic potential of human chorion‐derived stem cells: in vitro and in vivo evaluation

Human chorion-derived stem cells (hCDSC) were previously shown to demonstrate multipotent properties with promising angiogenic characteristics in monolayer-cell culture system. In our study, we investigated the angiogenic capability of hCDSC in 3-dimensional (3D) in vitro and in vivo angiogenic models for the purpose of future application in the treatment of ischaemic diseases. Human CDSC were evaluated for angiogenic and endogenic genes expressions by quantitative PCR. Growth factors secretions were quantified using ELISA. In vitro and in vivo vascular formations were evaluated by histological analysis and confocal microscopic imaging. PECAM-1+ and vWF+ vascular-like structures were observed in both in vitro and in vivo angiogenesis models. High secretions of VEGF and bFGF by hCDSC with increased expressions of angiogenic and endogenic genes suggested the possible angiogenic promoting mechanisms by hCDSC. The cooperation of hCDSC with HUVECS to generate vessel-like structures in our systems is an indication that there will be positive interactions of hCDSC with existing endothelial cells when injected into ischaemic tissues. Hence, hCDSC is suggested as the novel approach in the future treatment of ischaemic diseases.

In-Vitro and Ex-Vivo Investigations of the Microtubule Binding Drug Targetin on Angiogenesis.

BackgroundIntervention aimed at disrupting or inhibiting newly formed vascular network is highly desired to attenuate the progression of angiogenesis-dependent diseases. In cancer, this is tightly associated with the generation of VEGF by hypoxia inducible factor-1α following its activation by hypoxia. In light of the multiple cellular roles played by microtubules and their involvement in the processing of the hypoxia inducible factor-1α transcript, modulation of microtubule dynamics is emerging as a logical approach to suppress tumor reliance on angiogenesis. Targetin is a novel noscapinoid that interferes with microtubule dynamicity and inhibits the growth of cell lines from many types of cancers.Methods and ResultsUtilizing in-vitro and ex-vivo angiogenic models, we discovered the vascular disrupting and anti-angiogenic properties of Targetin. Targetin disrupted pre-assembled capillary-like networks of human endothelial cells by severing cell-cell junctions, inhibiting endothelial cell proliferation and metabolic activity in the presence and absence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Furthermore, we show that Targetin significantly inhibits the formation of neovasculature network sprouting from rat aortic explants stimulated with proangiogenic stimuli, namely VEGF or bFGF.ConclusionWe conclude that Targetin is a potential clinically promising anti-angiogenic agent for the treatment of many diseases including cancers.

Intermedin

Intermedin (IMD), a member of calcitonin family, was suggested to play a role in angiogenesis and cancer. The aim of this study was to investigate the role of IMD in the angiogenic process and the underlying mechanism, and the possibility for it to be used as a target for angiogenesis-based anticancer therapies. Using in vivo and in vitro 3-dimensional angiogenic models, we found that IMD induced a well-ordered vasculature with hierarchical structure and had a synergistic effect with vascular endothelial growth factor. Using RNA interference, real-time polymerase chain reaction, and Western blot analysis, we found that IMD alleviated the undesirable effects of vascular endothelial growth factor by restricting the excessive vessel sprouting and uneven lumen formation through the regulation of vascular endothelial-cadherin and identified its receptor on the endothelial cells. Both mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt activation were involved in the effects. Furthermore, using experimental tumor models, we demonstrated that IMD was involved in tumor angiogenesis, and the blockade of IMD severely impaired blood supply and eventually inhibited tumor growth. We demonstrated that IMD played a critical role in the vascular remodeling process and tumor angiogenesis and may serve as a novel target for the development of angiogenesis-based anticancer therapies.