Endothelial Transdifferentiation Research Articles

Transforming Growth Factor-β Receptor Internalization via Caveolae Is Regulated by Tubulin-β2 and Tubulin-β3 during Endothelial-Mesenchymal Transition

Fibrotic disorders, which are caused by long-term inflammation, are observed in numerous organs. These disorders are regulated mainly through transforming growth factor (TGF)-β family proteins by a fundamental cellular mechanism, known as the endothelial-mesenchymal transition. Therefore, there is a pressing need to identify the mechanisms and potential therapeutic targets that enable the inhibition of endothelial transdifferentiation. This study is the first to demonstrate that glycosylation of tubulin-β2 and tubulin-β3 in microtubules enhances sensitivity to TGF-β1 stimulation in human microvascular endothelial cells. We observed that the microtubules enriched in glycosylated tubulin-β2 and tubulin-β3 were necessary for caveolae-dependent TGF-β receptor internalization. Post-translational modulation is critical for the generation of myofibroblasts through endothelial-mesenchymal transition during fibrosis development. We suggest that microtubule glycosylation may become the target of new effective therapies for patients with recognized fibrotic diseases.

Identification of Macrophage Genotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome.

Background Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC. Methods and Results Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CACiv). Genes and pathways of interest were further evaluated using qPCR comparing CACiv versus CD14+ monocytic cells. The CACiv transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CACiv was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CACiv, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation. Conclusions CACiv are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation.

Erythropoietin (rhEPOa) promotes endothelial transdifferentiation of stem cells of the apical papilla (SCAP)

ObjectiveMesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair damaged tissue, immunosuppression, ability to differentiate into several cell types and their secretome. Earlier studies have demonstrated their angiogenic potential in vitro and in vivo. However, little is known regarding pro-angiogenic inducers of stable endothelial transdifferentiation of MSCs. Here, we employed human MSCs from the Apical Papilla (SCAP) and investigated whether recombinant human erythropoietin-alpha (rhEPOa) could act as such inducer. DesignCultured SCAP cells were exposed to rhEPOa and assessed for cell growth kinetics, viability and morphology, as well as their capacity to form capillary tubule structures in selected microenvironments. RT-PCR was used to monitor endothelial markers and activation of EPO/EPOR pathway signaling components; while gelatin zymographies to assess activation of MMP-2. ResultsrhEPOa treatment initially (48 h) accelerated cell proliferation and allowed SCAP to sprout micro-tubular structures. Morphological and biochemical differentiation was accompanied by activation of MMP-2 and upregulation of PECAM-1, VEGFR2, vWF and VE-cadherin/CDH5. SCAP expressed the cognate EPO-R, while rhEPOa-treated SCAP exhibited higher expression of molecules involved in EPO/EPOR pathway (EPOR and JAK2). ConclusionrhEPOa is capable of promoting endothelial transdifferentiation of SCAP which may be of clinical value in treating of ischemic disorders.

Endothelial trans-differentiation in glioblastoma recurring after radiotherapy

We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.

ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells

During normal tumor growth and in response to some therapies, tumor cells experience acute or chronic deprivation of nutrients and oxygen and induce tumor vascularization. While this occurs predominately through sprouting angiogenesis, tumor cells have also been shown to directly contribute to vessel formation through vascular mimicry (VM) and/or endothelial transdifferentiation. The extrinsic and intrinsic mechanisms underlying tumor cell adoption of endothelial phenotypes, however, are not well understood. Here we show that serum withdrawal induces mesenchymal breast cancer cells to undergo VM and that knockdown of the epithelial-to-mesenchymal transition (EMT) regulator, Zinc finger E-box binding homeobox 1 (ZEB1), or overexpression of the ZEB1-repressed microRNAs (miRNAs), miR-200c, miR-183, miR-96 and miR-182 inhibits this process. We find that secreted proteins Fibronectin 1 (FN1) and serine protease inhibitor (serpin) family E member 2 (SERPINE2) are essential for VM in this system. These secreted factors are upregulated in mesenchymal cells in response to serum withdrawal, and overexpression of VM-inhibiting miRNAs abrogates this upregulation. Intriguingly, the receptors for these secreted proteins, low-density lipoprotein receptor-related protein 1 (LRP1) and Integrin beta 1 (ITGB1), are also targets of the VM-inhibiting miRNAs, suggesting that autocrine signaling stimulating VM is regulated by ZEB1-repressed miRNA clusters. Together, these data provide mechanistic insight into the regulation of VM and suggest that miRNAs repressed during EMT, in addition to suppressing migratory and stem-like properties of tumor cells, also inhibit endothelial phenotypes of breast cancer cells adopted in response to a nutrient-deficient microenvironment.

Quercetin Inhibits Pulmonary Arterial Endothelial Cell Transdifferentiation Possibly by Akt and Erk1/2 Pathways.

This study aimed to investigate the effects and mechanisms of quercetin on pulmonary arterial endothelial cell (PAEC) transdifferentiation into smooth muscle-like cells. TGF-β1-induced PAEC transdifferentiation models were applied to evaluate the pharmacological actions of quercetin. PAEC proliferation was detected with CCK8 method and BurdU immunocytochemistry. Meanwhile, the identification and transdifferentiation of PAECs were determined by FVIII immunofluorescence staining and α-SMA protein expression. The related mechanism was elucidated based on the levels of Akt and Erk1/2 signal pathways. As a result, quercetin effectively inhibited the TGF-β1-induced proliferation and transdifferentiation of the PAECs and activation of Akt/Erk1/2 cascade in the cells. In conclusion, quercetin is demonstrated to be effective for pulmonary arterial hypertension (PAH) probably by inhibiting endothelial transdifferentiation possibly via modulating Akt and Erk1/2 expressions.

Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.

Abstract A18: miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer

Abstract Tumor vascularization is a fundamental step in solid tumor progression and is orchestrated by different pathways of vasculogenesis. In malignant tumors, neoplastic cells can differentiate into endothelial-like cells acquiring the expression of endothelial markers (i.e. CD31 and CD34) and participating in the formation of vascular-like structures that functionally deliver oxygen and nutrients to the tumor site. We recently identified PDGFRβ as an important player of this process in triple negative breast cancer (TNBC). Interestingly, increasing evidence supported a connection between PDGFRβ and epithelial to mesenchymal transition (EMT), important step for the endothelial trans-differentiation process. PDGFR signaling arose as a promising target for TNBC; thus a better understanding of this new role of PDGFRβ could be relevant in the biology and treatment of TNBC. We then aimed at investigating microRNAs able to regulate tumor vascularization via PDGFRβ-mediated endothelial differentiation in TNBC, focusing on miR-9 and miR-200 family in light of their known relation with PDGFRβ and EMT. Tube formation assay showed that in MDA-MB-231 and MDA-MB-157 TNBC cell lines transfection of miR-9 and miR-200 family members (miR-200b and miR-200c) promoted or inhibited, respectively, the ability of cells to form vascular-like structures when seeded on matrigel. Stimulation of PDGFRβ with PDGF-BB ligand induced miR-9 expression and enhanced the loop formation ability of treated cells. This advantage was, however, almost completely abrogated by the concomitant inhibition of miR-9, thus demonstrating that miR-9 contributed to PDGFRβ-mediated vasculogenic properties of TNBC. Moreover, we found that silencing of STARD13, direct target of miR-9 validated by luciferase reporter assay, improved the vasculogenic potential. Ectopic expression of miR-200 members, instead, suppressed PDGFRβ at protein level in both TNBC cell lines. Furthermore, the silencing of ZEB1, known target of miR-200 family, induced downregulation of PDGFRβ at protein and mRNA level; interestingly, the mRNA levels of ZEB1 and PDGFRβ strongly correlated in the TNBC subset of the TGCA dataset. The in vivo effect of miR-9 and miR-200 family on vasculogenic properties of TNBC was then validated first through the generation of MDA-MB-231 clones for stable inhibition of miR-9 and restoration of miR-200c; the resulting miR-9 sponge and miR-200c xenografted tumors showed lower number of vascular lacunae than controls, identified through immunohistochemical (IHC) staining for CD31. These results were validated in orthotopic MDA-MB-231 breast tumors treated with miR-9 inhibitors or miR-200c mimics by peritumoral injection. We finally evaluated PDGFRβ, by IHC, and miR-9 and miR-200c, by Real-time PCR, in a cohort of TNBC. IHC analysis demonstrated that PDGFRβ staining identified tumor cells participating in vascular lacunae, data further confirmed by immunofluorescence co-localization of PDGFRβ and CD31. Interestingly, unlike miR-9, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFRβ. Finally, in situ hybridization analysis revealed that miR-200c was mainly expressed by tumor cells. In conclusion, our results suggest that miR-9 and miR-200 family play an opposite role in the regulation of the vasculogenic aptitude of TNBC. MiR-9 is induced by PDGFRβ and enhances the vasculogenic properties of tumor cells in part through the negative regulation of STARD13. MiR-200, instead, inhibits this aggressive phenotype indirectly suppressing PDGFRβ through targeting of ZEB1. Citation Format: Elvira D'Ippolito, Ilaria Plantamura, Lucia Bongiovanni, Patrizia Casalini, Sara Baroni, Claudia Piovan, Rosaria Orlandi, Ambra V. Gualeni, Annunziata Gloghini, Anna Rossini, Filippo De Braud, Elda Tagliabue, Claudio Tripodo, Marilena V. Iorio. miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A18.

Microlens topography in conjunction with vascular endothelial growth factor induces endothelial transdifferentiation of human mesenchymal stem cells into vasculogenic progenitors.

Microlens topography in conjunction with vascular endothelial growth factor induces endothelial transdifferentiation of human mesenchymal stem cells into vasculogenic progenitors.

Endothelial Transdifferentiation of Tumor Cells Triggered by the Twist1-Jagged1-KLF4 Axis: Relationship between Cancer Stemness and Angiogenesis.

Tumor hypoxia is associated with malignant biological phenotype including enhanced angiogenesis and metastasis. Hypoxia increases the expression of vascular endothelial cell growth factor (VEGF), which directly participates in angiogenesis by recruiting endothelial cells into hypoxic area and stimulating their proliferation, for increasing vascular density. Recent research in tumor biology has focused on the model in which tumor-derived endothelial cells arise from tumor stem-like cells, but the detailed mechanism is not clear. Twist1, an important regulator of epithelial-mesenchymal transition (EMT), has been shown to mediate tumor metastasis and induce tumor angiogenesis. Notch signaling has been demonstrated to be an important player in vascular development and tumor angiogenesis. KLF4 (Krüppel-like factor 4) is a factor commonly used for the generation of induced pluripotent stem (iPS) cells. KLF4 also plays an important role in the differentiation of endothelial cells. Although Twist1 is known as a master regulator of mesoderm development, it is unknown whether Twist1 could be involved in endothelial transdifferentiation of tumor-derived cells. This review focuses on the role of Twist1-Jagged1/Notch-KLF4 axis on tumor-derived endothelial transdifferentiation, tumorigenesis, metastasis, and cancer stemness.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

A twist tale of cancer metastasis and tumor angiogenesis.

Twist1 is an evolutionally conserved transcription factor. Originally identified in Drosophila as a key regulator for mesoderm development, it was later implicated in many human diseases, including Saethre-Chotzen syndrome and cancer. Twist1's involvement in cancer has been well recognized. Driven by hypoxia-induced factor-1 (HIF-1), Twist1 has been considered as a proto-oncogene and its overexpression has been observed in a wide variety of human cancers. High expression level of Twist1 is closely related to tumor aggressiveness and metastatic potential. In cancer cells, Twist1 has been shown to function as a key regulator of epithelial-mesenchymal transition (EMT), a critical process for metastasis initiation. Twist1 has also been implicated in maintaining cancer stemness for self-renewal and chemoresistance. This review first summarizes the roles of Twist1 in embryo development and Saethre-Chotzen syndrome followed by a discussion of Twist1's critical functions in cancer. In particular, the review focuses on the recent discovery of Twist1's capability to promote endothelial transdifferentiation of cancer cells beyond EMT.

Characterization of the Pall Celeris system as a point-of-care device for therapeutic angiogenesis

Background aimsThe Pall Celeris system is a filtration-based point-of-care device designed to obtain a high concentrate of peripheral blood total nucleated cells (PB-TNCs). We have characterized the Pall Celeris–derived TNCs for their in vitro and in vivo angiogenic potency. MethodsPB-TNCs isolated from healthy donors were characterized through the use of flow cytometry and functional assays, aiming to assess migratory capacity, ability to form capillary-like structures, endothelial trans-differentiation and paracrine factor secretion. In a hind limb ischemia mouse model, we evaluated perfusion immediately and 7 days after surgery, along with capillary, arteriole and regenerative fiber density and local bio-distribution. ResultsHuman PB-TNCs isolated by use of the Pall Celeris filtration system were shown to secrete a panel of angiogenic factors and migrate in response to vascular endothelial growth factor and stromal-derived factor-1 stimuli. Moreover, after injection in a mouse model of hind limb ischemia, PB-TNCs induced neovascularization by increasing capillary, arteriole and regenerative fiber numbers, with human cells detected in murine tissue up to 7 days after ischemia. ConclusionsThe Pall Celeris system may represent a novel, effective and reliable point-of-care device to obtain a PB-derived cell product with adequate potency for therapeutic angiogenesis.

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

BackgroundMesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo.MethodsMesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).ResultshWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.ConclusionThese results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

Cultured Human Bone Marrow–Derived CD31+ Cells Are Effective for Cardiac and Vascular Repair Through Enhanced Angiogenic, Adhesion, and Anti-Inflammatory Effects

BackgroundCell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31+ cells are a promising cell source owing to their high angiovasculogenic and paracrine activities. ObjectivesThis study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31+ cells, and to determine whether these cultured CD31+ cells are effective for cardiac and vascular repair. MethodsCD31+ cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms. ResultsThe CD31+ cells cultured in endothelial cell medium (EC-CD31+ cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31+ cells. When implanted into mouse MI or HLI models, EC-CD31+ cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31+ cells. Histologically, injected EC-CD31+ cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year. ConclusionsShort-term cultured EC-CD31+ cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31+ cells may be a promising source for treating ischemic cardiovascular diseases.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings

BackgroundTumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.MethodsTo characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts.ResultsAs expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated.ConclusionsTaken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

Abstract 360: IL-10-inhibits Pressure Overload-induced Homing, Proliferation And Differentiation Of Non-resident Fibroblast Progenitors And Improve Heart Function.

Background: Recently we have shown that IL-10, an anti-inflammatory cytokine, markedly inhibited the pressure overload-induced cardiac fibrosis, however, antifibrotic mechanisms of IL-10 are largely unknown. In most of organs, including heart, extracellular matrix (ECM) remodeling is primarily mediated by excessive proliferation of activated fibroblasts and myofibroblasts. Here we hypothesized that IL-10 inhibits stress-induced homing, proliferation and differentiation of nonresident bone marrow-derived fibroblast progenitor cells and therefore, attenuates cardiac remodeling and improves of heart function. Methods and Results: Cardiac hypertrophy was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice compared to WT. TAC significantly increased TGF-β, collagen Iα and IIIα genes expression. Systemic recombinant mouse IL-10 administration markedly improved LV function, inhibited TAC-induced cardiac fibrosis and fibrosis associated genes expression. To identify the role of fibroblast progenitor cells (FPCs), we measured the mobilization of FPCs (Prominin1 positive cells) from bone marrow to heart by FACs. Exacerbated mobilization of FPCs in peripheral blood and heart in IL-10 KO mice were found 3 and 7 days after aortic constriction. Bone marrow transplantation experiments were performed where WT-GFP positive marrow was transplanted in BM depleted IL-10 KO mice. TAC-induced mobilization was significantly reduced in WT-transplanted marrow as compare to TAC-IL-10 KO mice. To identify the role IL-10 on TGFβ-induced endothelial cells trans-differentiation to myofibroblasts, we treated aortic endothelial cells with IL-10 and TGFβ2 for 96 hrs. Both Immunocytochemistry and Western blot analysis results suggested that TGF-β2-induced EndMT was significantly inhibited by IL-10 treatment. To understand the mechanisms, we found that TGF-β2-induced Notch1 signaling was reduced by IL-10. Conclusion: Taken together our observations suggest that the anti-fibrotic effects of IL-10 treatment are mediated by reduced proliferation and differentiation of non-resident myofibroblasts.

Endothelial transdifferentiation in hepatocellular carcinoma: loss of Stabilin‐2 expression in peri‐tumourous liver correlates with increased survival

Hepatocellular carcinoma (HCC) is a malignant tumour that is characterized by extensive vascular remodelling and responsiveness to treatment with the anti-angiogenic multikinase inhibitor sorafenib. The aim was to study endothelial remodelling in HCC. The murine inducible albumin-SV40-large T-antigen model and two tissue microarrays (TMA) with 295 tumourous and 83 peri-tumourous samples of 296 patients with HCC were analysed for expression of liver sinusoidal endothelial cell (LSEC)-specific marker proteins, stabilin-1 and stabilin-2, LYVE-1 and CD32b. LSEC marker proteins were sequentially lost during HCC progression in the murine HCC model being absent from tumour nodules larger than 800μm in diameter. Similarly, the TMA analysis of human HCCs revealed loss of all four marker proteins in the majority of tumourous tissue samples. Preservation of LYVE-1 expression showed a significant correlation with low grading (G1). In corresponding peri-tumourous liver tissue, loss of all marker proteins was seen in a minor proportion of cases (34%) while the majority of cases retained expression of at least one of the marker proteins. Loss of stabilin-2 expression in peri-tumourous liver tissue of patients with HCC was significantly less likely to occur (38%) than loss of the other marker proteins (63-95%) and it was associated with significantly longer tumour-specific (P=0.0523) and overall (P=0.0338) survival. Loss of stabilin-2 may enhance survival in HCC by preventing endothelial-tumour cell adhesive interactions and microvascular invasion. In summary, endothelial transdifferentiation is a major pathogenic event in HCC development indicating a switch from vessel co-option/intussusceptive angiogenesis to sprouting angiogenesis.

Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities

BackgroundChemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model.MethodsSCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel.ResultsHUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression.ConclusionsChemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

TNFα Accelerates Monocyte to Endothelial Transdifferentiation in Tumors by the Induction of Integrin α5 Expression and Adhesion to Fibronectin

Tumor-associated myeloid cells are believed to promote tumor development by stimulating tumor growth, angiogenesis, invasion, and metastasis. Tumor-associated myeloid cells that coexpress endothelial and myeloid markers represent a proangiogenic subpopulation known as vascular leukocytes. Recently, we and others had shown that tumor-derived TNFα promotes local tumor growth and vascularity. Our data suggested that tumor growth is in part due to TNFα-mediated increased numbers of tumor-associated vascular leukocytes (i.e., myeloid-endothelial biphenotypic cells). The work detailed herein explored the mechanism by which TNFα mediates endothelial differentiation of myeloid cells. Our studies showed that fibronectin is a robust facilitator of endothelial differentiation of blood mononuclear cells in vitro. We have found that TNFα treatment of monocytes significantly increased expression of α(5)β(1) integrin, a major fibronectin receptor enriched on endothelial cells, leading to a consequent fourfold increase in fibronectin adhesion. Furthermore, TNFα-treated monocytes upregulated expression of endothelial markers, flk-1(VEGFR2/KDR) and VE-cadherin. Integrin α(5) subunit inhibitory antibodies blocked adhesion to fibronectin as well as consequent upregulation of flk-1 and VE-cadherin transcripts, implying a role for outside-in signaling by the α(5)β(1) integrin after binding fibronectin. Finally, treatment of mouse tumors with anti-α(5) antibodies reduced accumulation of tumor vascular leukocytes in vivo. Our studies suggest that tumor cell-derived TNFα constitutes a tumor microenvironment signal that promotes differentiation of tumor-associated monocytes toward a proangiogenic/provasculogenic myeloid-endothelial phenotype via upregulation of the fibronectin receptor α(5)β(1).