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Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor α (ERα) negativity. Here we explored the role of the second ER subtype, ERβ, and report expression in IBC tumors and its correlation with reduced metastasis. Ablation of ERβ in IBC cells promoted cell migration and activated gene networks that control actin reorganization, including G-protein–coupled receptors and downstream effectors that activate Rho GTPases. Analysis of preclinical mouse models of IBC revealed decreased metastasis of IBC tumors when ERβ was expressed or activated by chemical agonists. Our findings support a tumor-suppressive role of ERβ by demonstrating the ability of the receptor to inhibit dissemination of IBC cells and prevent metastasis. On the basis of these findings, we propose ERβ as a potentially novel biomarker and therapeutic target that can inhibit IBC metastasis and reduce its associated mortality.Significance:These findings demonstrate the capacity of ERβ to elicit antimetastatic effects in highly aggressive inflammatory breast cancer and propose ERβ and the identified associated genes as potential therapeutic targets in this disease.

Table with patient characteristics

Table with altered genes from microarray analysis

Table with patient characteristics

Contains supplementary methods

Supplementary figures 1-8. Supplementary Figure 1. ERβ expression is associated with clinical outcome in IBC patients. Supplementary Figure 2. Associations between ERβ expression and clinical outcome in patients with breast cancer. Supplementary Figure 3. Morphology of IBC cells with different ERβ levels. Supplementary Figure 4. Invasion and migration of IBC cells with different ERβ expression Supplementary Figure 5. ERβ affects metastasis of IBC tumors. Supplementary Figure 6. ERβ regulates genes that control actin cytoskeleton reorganization. Supplementary Figure 7. ERβ regulates genes that are involved in acting cytoskeleton remodeling. Supplementary Figure 8. RhoC and ERβ expression in IBC cells.

Ischemic heart disease is a leading cause of death in the world. Formation of new blood vessels is fundamental to cardiac reperfusion and repair after myocardial infarction (MI), and therapeutic promotion of angiogenesis, therefore, has emerged as a promising strategy for treating ischemic heart diseases. However, the therapeutic efficacy is small and transient in most of pro-angiogenic therapy. Here, we characterize unexpectedly prominent vessel aberrancy, albeit a robust angiogenesis, in MI-associated cardiac tissue by 3-dementional light sheet fluorescence imaging. Our single-cell transcriptome analysis of mouse MI tissues with Cdh5-Cre ; Rosa-LSL-tdTomato -mediated endothelial lineage tracing identifies a robust EC plasticity to acquire mesenchymal gene signature in MI-associated ECs, peaked at 1 week and partially retained at 4 and 8 weeks after MI. Moreover, cardiac ECs undergo transcriptional activation to drive mesenchymal phenotypes including over-proliferation, migration and increased permeability, which lead to vessel abnormality after exposure to in vitro ischemic microenvironment. Furthermore, our single-cell and bulk RNA-seq analysis uncovers a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, culminating in aberrant vascularization. Notably, EC-specific knockout of PDGFR-β abrogates mesenchymal gene expression in ECs, attenuates vascular abnormalities in the infarcted tissue, and improves cardiac repair after MI. Interestingly, our single-nuclei analysis uncovers that genetic ablation of PDGFR-β in ECs temporally reprograms cardiomyocyte metabolism and enhances myocyte survival and proliferation. Pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery normalizes vasculature and improves cardiac function recovery after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer exciting opportunities for normalizing vasculature and treating ischemic heart disease.

Myocardial infarction (MI) is a leading cause of death worldwide, largely because efficient interventions to restore cardiac function after MI are currently lacking. Here, we characterize vascular aberrancies induced by MI, and propose to target acquired endothelial cell (EC) changes to normalize vessels and promote cardiac repair after MI. Single-cell transcriptome analyses of MI-associated ECs indicates that ECs acquire mesenchymal gene signature that result in phenotypic and functional changes and lead to vessel abnormalities. We identify a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, altogether causing aberrant vascularization. EC-specific knockout of PDGFR-β, pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery in mice attenuates vascular abnormalities in the infarcted tissue and improves cardiac repair after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer opportunities for normalizing vasculature and treating ischemic heart diseases.

Circulating tumor cells (CTC) are known to be present in the blood of patients with glioblastoma (GBM). Here we report that GBM-derived CTC possess a cancer stem cell (CSC)-like phenotype and contribute to local tumorigenesis and recurrence by the process of self-seeding. Genetic probes showed that mouse GBM-derived CTC exhibited Sox2/ETn transcriptional activation and expressed glioma CSC markers, consistent with robust expression of stemness-associated genes including SOX2, OCT4, and NANOG in human GBM patient-derived samples containing CTC. A transgenic mouse model demonstrated that CTC returned to the primary tumor and generated new tumors with enhanced tumorigenic capacity. These CTCs were resistant to radiotherapy and chemotherapy and to circulation stress-induced cell apoptosis. Single-cell RNA-seq analysis revealed that Wnt activation induced stemness and chemoresistance in CTC. Collectively, these findings identify GBM-derived CTC as CSC-like cells and suggest that targeting Wnt may offer therapeutic opportunities for eliminating these treatment-refractory cells in GBM. Significance: These findings identify CTCs as an alternative source for in situ tumor invasion and recurrence through local micrometastasis, warranting eradication of systemic "out-of-tumor" CTCs as a promising new therapeutic opportunity for GBM.

Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis.

Therapeutic resistance remains a persistent challenge for patients with glioblastoma (GBM). Here, we report that endothelial cells (ECs) acquire transformation into mesenchymal stem cell (MSC)-like cells in GBM, driving tumor resistance to cytotoxic treatment. Single-cell transcriptome analysis identified an EC-derived cell population that robustly expresses mesenchymal genes and stem cell-associated markers, suggesting that ECs undergo mesenchymal transformation and stemness-like activation in GBM microenvironment. Furthermore, we identified a c-Met-mediated axis that induces β-catenin phosphorylation at Ser675 and Wnt signaling activation, inducing multidrug resistance-associated protein-1 (MRP-1) expression and leading to EC stemness-like activation and chemoresistance. Last, genetic ablation of β-catenin in ECs overcome GBM tumor resistance to temozolomide (TMZ) chemotherapy in vivo. Combination of Wnt inhibition and TMZ chemotherapy eliminated tumor-associated ECs, inhibited GBM growth, and increased mouse survival. These findings identified a cell plasticity-based, microenvironment-dependent mechanism that controls tumor chemoresistance, and suggest that targeting Wnt/β-catenin-mediated EC transformation and stemness activation may overcome therapeutic resistance in GBM.

Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor α (ERα) negativity. Here we explored the role of the second ER subtype, ERβ, and report expression in IBC tumors and its correlation with reduced metastasis. Ablation of ERβ in IBC cells promoted cell migration and activated gene networks that control actin reorganization, including G-protein–coupled receptors and downstream effectors that activate Rho GTPases. Analysis of preclinical mouse models of IBC revealed decreased metastasis of IBC tumors when ERβ was expressed or activated by chemical agonists. Our findings support a tumor-suppressive role of ERβ by demonstrating the ability of the receptor to inhibit dissemination of IBC cells and prevent metastasis. On the basis of these findings, we propose ERβ as a potentially novel biomarker and therapeutic target that can inhibit IBC metastasis and reduce its associated mortality. Significance: These findings demonstrate the capacity of ERβ to elicit antimetastatic effects in highly aggressive inflammatory breast cancer and propose ERβ and the identified associated genes as potential therapeutic targets in this disease.

Therapeutic resistance remains a persistent challenge for patients with malignant tumors. Here, we reveal that endothelial cells (ECs) acquire transformation into mesenchymal stem cell (MSC)–like cells in glioblastoma (GBM), driving tumor resistance to cytotoxic treatment. Transcriptome analysis by RNA sequencing (RNA-seq) revealed that ECs undergo mesenchymal transformation and stemness-like activation in GBM microenvironment. Furthermore, we identified a c-Met-mediated axis that induces β-catenin phosphorylation at Ser(675) and Wnt signaling activation, inducing multidrug resistance–associated protein-1(MRP-1) expression and leading to EC stemness-like activation and chemoresistance. Last, genetic ablation of β-catenin in ECs overcome GBM tumor resistance to temozolomide (TMZ) chemotherapy in vivo. Combination of Wnt inhibition and TMZ chemotherapy eliminated tumor-associated ECs, inhibited GBM growth, and increased mouse survival. These findings identified a cell plasticity–based, microenvironment-dependent mechanism that controls tumor chemoresistance, and suggest that targeting Wnt/β-catenin–mediated EC transformation and stemness activation may overcome therapeutic resistance in GBM.

Malignant solid tumors are characterized by aberrant vascularity that fuels the formation of an immune-hostile microenvironment and induces resistance to immunotherapy. Vascular abnormalities may be driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide screening of mesenchymal-like transcriptional activation in human glioblastoma (GBM)-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic reprogramming and aberrant vascularization. PAK4 knockout induces adhesion protein re-expression in ECs, reduces vascular abnormalities, improves T cell infiltration and inhibits GBM growth in mice. Moreover, PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to chimeric antigen receptor–T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer a unique opportunity to recondition the vascular microenvironment and strengthen cancer immunotherapy. Fan and colleagues report that inhibition of PAK4 normalizes the tumor vascular microenvironment and sensitizes glioblastomas to CAR-T cell immunotherapy.

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase–14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor–derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.

Angiogenesis is a hallmark of cancer. However, most malignant solid tumors exhibit robust resistance to current anti-angiogenic therapies that primarily target VEGF pathways. Here we report that endothelial-mesenchymal transformation induces glioblastoma (GBM) resistance to anti-angiogenic therapy by downregulating VEGFR-2 expression in tumor-associated endothelial cells (ECs). We show that VEGFR-2 expression is markedly reduced in human and mouse GBM ECs. Transcriptome analysis verifies reduced VEGFR-2 expression in ECs under GBM conditions and shows increased mesenchymal gene expression in these cells. Furthermore, we identify a PDGF/NF-κB/Snail axis that induces mesenchymal transformation and reduces VEGFR-2 expression in ECs. Finally, dual inhibition of VEGFR and PDGFR eliminates tumor-associated ECs and improves animal survival in GBM-bearing mice. Notably, EC-specific knockout of PDGFR-β sensitizes tumors to VEGF-neutralizing treatment. These findings reveal an endothelial plasticity-mediated mechanism that controls anti-angiogenic therapy resistance, and suggest that vascular de-transformation may offer promising opportunities for anti-vascular therapy in cancer., Resistance to anti-angiogenic therapies often occurs in patients. Here, the authors demonstrate the role of PDGF signaling in GBM resistance to anti-VEGF treatment via a mechanism that involves endothelial-mesenchymal transformation and transcriptional regulation of VEGFR-2.

Vascularization is a fundamental process in development, wound healing, and the progression of cardiovascular diseases. Formation of new blood vessels with functional and structural integrity is crucial for organ growth as well as post-injury tissue repair and regeneration. Although vascular sprouting and outgrowth, i.e. , angiogenesis, has been extensively studied, the underlying mechanisms for vessel formation remain largely unexplored. Here we reveal a critical role of phosphatidylinositol 4,5-bisphosphate (PIP2) for vessel formation in endothelial cells (ECs). Moreover, by using mass spectrometry, our identified Tbc1d2b as a novel PIP2-binding protein in vessel-formatting ECs. Considering its critical function in vessel formation, we name this protein “vesselin”. We showed that vesselin preferentially localizes to blood vessels in various human tissues, and that its siRNA-mediated knockdown inhibits EC tube formation, supporting its major role in vessel formation. Remarkably, injection of inhibitory vesselin morpholino into the embryos blocks blood vessel formation and zebrafish development. Vesselin contains PH (pleckstrin homology) and TBC (Tre2/Bub2/Cdc16) domains, which are known to bind phosphatidylinositols and display GTPase-activating protein (GAP) activities towards Rab GTPases, respectively. Consistently, we found by mass spectrometry that vesselin interacts with the small GTPase Rab13 and small GTPase Rac1. Finally, our further mechanistic studies showed that vesselin interacts with Rac1 and Rab13 during EC vessel formation, and regulates their activity in a PIP2-dependent manner. Thus, we uncover a previously unidentified regulatory system for EC vessel formation, providing molecular resolution of vascular morphogenesis. Targeting vesselin may provide new therapeutic strategies in cardiovascular diseases.

Ischemic heart disease is the most common cause of death in the Western world, largely due to myocardial infarction (MI). After MI, formation of new blood vessels, i.e. , neovascularization, is crucial for ischemic tissue reperfusion, repair and regeneration. However, the newly formed vasculatures in infarcted tissue are characterized by functional and structural abnormalities, which compromise vascular delivery function and impede cardiac recovery after MI. Likewise, aberrant non-productive neovascularization, albeit previously under-appreciated, represents a promising therapeutic target for post-MI treatment. Here we reveal that mesenchymal transformation-mediated endothelial cell (EC) plasticity induces aberrant post-ischemic neovascularization. In contrast to the old concept implicating that ECs undergo endothelial mesenchymal transitions to generate fibroblasts de novo in infarcted cardiac tissue, we suggest that ECs acquire mesenchymal phenotypes including high proliferation and motility to generate excessive abnormal vasculatures after MI. By utilizing genetic EC lineage tracing and single-cell RNAseq technologies with a mouse MI model induced by ligation of left anterior descending coronary artery, our transcriptome analysis uncovers that ECs undergo mesenchymal transformation in infarcted tissues. Moreover, exposure of cardiac ECs to ischemic microenvironment in vitro induces EC expression of mesenchymal genes including S100A4, ACTA2, and CDH2, and interestingly, EC functions including tube formation and uptake of ac-LDL are retained, suggesting EC mesenchymal transformation without lineage transition. Furthermore, we identify a PDGF/Snail-mediated axis that controls EC transformation under hypoxia. Notably, genetic ablation of PDGF receptor-β in ECs promotes blood perfusion and tissue repair after hindlimb ischemia and MI in mice. These findings identify a novel cellular mechanism controlling non-productive neovascularization after ischemia, and suggest that targeting EC plasticity may offer promising therapeutic opportunities for normalization of aberrant neovasculature and improvement of tissue repair in ischemic heart diseases.

Spatiotemporal regulation of tumor immunity remains largely unexplored. Here we identify a vascular niche that controls alternative macrophage activation in glioblastoma (GBM). We show that tumor-promoting macrophages are spatially proximate to GBM-associated endothelial cells (ECs), permissive for angiocrine-induced macrophage polarization. We identify ECs as one of the major sources for interleukin-6 (IL-6) expression in GBM microenvironment. Furthermore, we reveal that colony-stimulating factor-1 and angiocrine IL-6 induce robust arginase-1 expression and macrophage alternative activation, mediated through peroxisome proliferator-activated receptor-γ-dependent transcriptional activation of hypoxia-inducible factor-2α. Finally, utilizing a genetic murine GBM model, we show that EC-specific knockout of IL-6 inhibits macrophage alternative activation and improves survival in the GBM-bearing mice. These findings illustrate a vascular niche-dependent mechanism for alternative macrophage activation and cancer progression, and suggest that targeting endothelial IL-6 may offer a selective and efficient therapeutic strategy for GBM, and possibly other solid malignant tumors., Macrophages in the tumour microenvironment (TME) acquire tumour-promoting functions upon M2 polarization. Here the authors show, in a mouse model of glioblastoma, that endothelial cells in the TME induce macrophage M2 polarization via IL-6 and that depletion of endothelial IL-6 improves survival.

MicroRNAs (miRNAs) are a class of non-coding, regulatory small RNAs of ∼22 nt. It was implicated that these small RNAs play critical roles in various important biological processes. During development, some miRNAs are specifically expressed in individual tissues and at particular developmental stages. Many miRNAs show distinct expression patterns in the development of central nervous system, including spinal cord. In this study, we first reported the miRNAs expression in the development of mouse spinal cord. Differentially expressed miRNAs in embryonic (day 13.5) and neonatal mice spinal cords were identified. The predicted target genes of the differentially expressed miRNAs were subject to gene ontology and KEGG pathway analysis, and several nervous development-related pathways were enriched, implying that these miRNAs may be involved in these pathways that regulate mouse spinal cord development.

Cancer stem cells (CSCs) - known to be resistant to genotoxic radiation and chemotherapy - are fundamental to therapy failure and cancer relapse. Here, we reveal that glioma CSCs are hypersensitive to radiation, but a temporal DNA repair mechanism converts the intrinsic sensitivity to genomic instability and treatment resistance. Transcriptome analysis identifies DNA-dependent protein kinase (DNA-PK) as a predominant DNA repair enzyme in CSCs. Notably, DNA-PK activity is suppressed after irradiation when ROS induce the dissociation of DNA-PKcs with Ku70/80, resulting in delayed DNA repair and radiosensitivity; subsequently, after ROS clearance, the accumulated DNA damage and robust activation of DNA-PK induce genomic instability, facilitated by Rad50-mediated cell-cycle arrest, leading to enhanced malignancy, CSC overgrowth, and radioresistance. Finally, we show a requisite in vivo role for DNA-PK in CSC-mediated radioresistance and glioma progression. These findings identify a time-sensitive mechanism controlling CSC resistance to DNA-damaging treatments and suggest DNA-PK/Rad50 as promising targets for CSC eradication.

Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo . Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.

Lipoprotein(a) [Lp(a)] resembles low-density lipoprotein (LDL), with an LDL lipid core and apolipoprotein B (apoB), but contains a unique apolipoprotein, apo(a). Elevated Lp(a) is an independent risk factor for coronary and peripheral vascular diseases. The size and concentration of plasma Lp(a) are related to the synthetic rate, not the catabolic rate, and are highly variable with small isoforms associated with high concentrations and pathogenic risk. Apo(a) is synthesized in the liver, although assembly of apo(a) and LDL may occur in the hepatocytes or plasma. While the uptake and clearance site of Lp(a) is poorly delineated, the kidney is the site of apo(a) fragment excretion. The structure of apo(a) has high homology to plasminogen, the zymogen for plasmin and the primary clot lysis enzyme. Apo(a) interferes with plasminogen binding to C-terminal lysines of cell surface and extracellular matrix proteins. Lp(a) and apo(a) inhibit fibrinolysis and accumulate in the vascular wall in atherosclerotic lesions. The pathogenic role of Lp(a) is not known. Small isoforms and high concentrations of Lp(a) are found in healthy octogenarians that suggest Lp(a) may also have a physiological role. Studies of Lp(a) function have been limited since it is not found in commonly studied small mammals. An important aspect of Lp(a) metabolism is the modification of circulating Lp(a), which has the potential to alter the functions of Lp(a). There are no therapeutic drugs that selectively target elevated Lp(a), but a number of possible agents are being considered. Recently, new modifiers of apo(a) synthesis have been identified. This review reports the regulation of Lp(a) metabolism and potential sites for therapeutic targets.

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.

Excessive and abnormal vasculature characterizes the microenvironment that fuels the progression of malignant tumors including glioblastoma multiforme (GBM), the grade IV glioma. GBM is among the most lethal human malignancies, distinguished by extensive microvascular hyperplasia due to intravessel endothelial cell (EC) proliferation with unknown etiology. Here, we show that c-Met phosphorylation induces endothelial-mesenchymal transition (Endo-MT), contributing significantly to vascular abnormality, GBM progression, and therapeutic resistance. Utilizing murine orthotropic GBM models induced by transplantation of GL26 glioma cells and RCAS/tv-a-mediated somatic gene transfer of platelet-derived growth factor (PDGF) in Ink-4a/Arf−/-PTEN−/- mice, we reveal robust Endo-MT in tumor vasculature, characterized by expression of the fibroblast-specific marker FSP-1 in 60% of CD31+ or CD105+ ECs, and the co-expression level correlates with the aggressiveness of glioma in humans. Cell fate analysis by lineage tracing Tie2+ ECs shows over half of FSP-1+ fibroblasts are of EC origin in the mouse GBM models. Tumor-conditioned medium induces in vitro EC acquisition of mesenchymal gene signature including FSP-1 and N-cadherin expression, confirming the microenvironment-dependent Endo-MT. Furthermore, proteomic analysis identifies a critical role of c-Met in Endo-MT, which is required for the increased EC proliferation and migration and vessel hyperpermeability in the GBM microenvironment. EC-specific c-Met phosphorylation induces MMP-14 expression, leading to Endo-MT and vascular abnormality. Finally, pharmacological inhibition of c-Met phosphorylation normalizes blood vessels, reduces vascular density and Endo-MT, blocks tumor progression, and sensitizes tumor to temozolomide treatment; all of which improves overall survival in the GBM-bearing mice. These findings reveal a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting c-Met phosphorylation and Endo-MT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors.

Background Reprogrammed cells, including induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cells (NT-ESCs), are similar in many respects to natural embryonic stem cells (ESCs). However, previous studies have demonstrated that iPSCs retain a gene expression signature that is unique from that of ESCs, including differences in microRNA (miRNA) expression, while NT-ESCs are more faithfully reprogrammed cells and have better developmental potential compared with iPSCs. Results We focused on miRNA expression and explored the difference between ESCs and reprogrammed cells, especially ESCs and NT-ESCs. We also compared the distinct expression patterns among iPSCs, NT-ESCs and NT-iPSCs. The results demonstrated that reprogrammed cells (iPSCs and NT-ESCs) have unique miRNA expression patterns compared with ESCs. The comparison of differently reprogrammed cells (NT-ESCs, NT-iPSCs and iPSCs) suggests that several miRNAs have key roles in the distinct developmental potential of reprogrammed cells. Conclusions Our data suggest that miRNAs play a part in the difference between ESCs and reprogrammed cells, as well as between MEFs and pluripotent cells. The variation of miRNA expression in reprogrammed cells derived using different reprogramming strategies suggests different characteristics induced by nuclear transfer and iPSC generation, as well as different developmental potential among NT-ESCs, iPSCs and NT-iPSCs. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-488) contains supplementary material, which is available to authorized users.

Establishing the pattern of expression of transmitters and peptides as well as their receptors in different neuronal types is crucial for understanding the circuitry in various regions of the brain. Previous studies have demonstrated that the transmitter and peptide phenotypes in mouse dorsal spinal cord neurons are determined by the transcription factors Tlx1/3 and Ptf1a. Here we show that these transcription factors also determine the expression of two distinct sets of transmitter and peptide receptor genes in this region. We have screened the expression of 78 receptor genes in the spinal dorsal horn by in situ hybridization. We found that receptor genes Gabra1, Gabra5, Gabrb2, Gria3, Grin3a, Grin3b, Galr1, and Npy1r were preferentially expressed in Tlx3-expressing glutamatergic neurons and their derivatives, and deletion of Tlx1 and Tlx3 resulted in the loss of expression of these receptor genes. Furthermore, we obtained genetic evidence that Tlx3 uses distinct pathways to control the expression of receptor genes. We also found that receptor genes Grm3, Grm4, Grm5, Grik1, Grik2, Grik3, and Sstr2 were mainly expressed in Pax2-expressing GABAergic neurons in the spinal dorsal horn, and their expression in this region was abolished or markedly reduced in Ptf1a and Pax2 deletion mutant mice. Together, our studies indicate that Tlx1/3 and Ptf1a, the key transcription factors for fate determination of glutamatergic and GABAergic neurons in the dorsal spinal cord, are also responsible for controlling the expression of two distinct sets of transmitter and peptide receptor genes.

Abdominal Aortic Aneurysm (AAA) is a lethal disorder found primarily in adults over 65. Unlike coronary heart disease, the incidence of AAA is rapidly increasing, and currently there is no nonsurgical treatment. AAA is a chronic inflammatory disease that includes early leukocyte infiltration, destruction of the elastic lamina and extracellular matrix, expansion of the aorta, and eventually fatal aorta rupture. Components of the plasminogen (Plg) and MMP pathways that mediate leukocyte infiltration are prominent in AAA. The goal of this study is to investigate regulators of these pathways in AAA, namely the inhibition Plg by apo(a). In AAA patients, the association of elevated Lp(a) and progression of AAA is controversial. Apolipoprotein(a) [apo(a)], a component of lipoprotein(a) [Lp(a)], has a high homology to Plg, and interferes with Plg functions in vitro. In our previous studies apo(a), independent of Plg, inhibited neutrophil recruitment in a peritonitis model of inflammation, and in Plg deficient mice macrophage infiltration was impaired in CaCl2 induced AAA formation. Our hypothesis is that apo(a) inhibits AAA formation by reducing neutrophil or macrophage infiltration by interfering with Plg. To test our hypothesis, the CaCl2 induced AAA model was performed in 5 strains of mice: WT; Plg-/-; apo(a)tg; apo(a)tg:Plg-/-; and Lp(a)tg mice (6-14 mice analyzed per genotype). Three weeks after injury, the WT aorta diameter increased from 0.54 to 0.88mm (62%). The diameter changed in WT 0.31±0.11mm, apo(a)tg 0.06±0.02mm, apo(a):Plg -/- 0.39±0.07mm and Lp(a) 0.08±0.07mm. The dilation was significantly lower in apo(a) and Lp(a) mice compared to WT (P-/- injured aorta, accompanied by thining of the media layer and elastic lamellae fragmentation. In contrast, the aorta of apo(a) mice remained preserved. Neutrophils and macrophages were decreased in apo(a) mice compared to WT(P-/- mice, only neutrophil infiltration was decreased not macrophages. These results suggest that inhibition of neutrophils in apo(a) mice is independent of Plg, but reduced macrophage infiltration in apo(a) mice is Plg-dependent during AAA formation.

The involvement of microRNAs (miRNAs) in the development of the neural crest (NC) cells and other neuronal differentiation is still poorly understood. Here, we investigated the global function of miRNAs in embryonic development by examining the Wnt1-cre-mediated Dicer knockout mice. Dicer ablation resulted in malformation of the midbrain and cerebellum and failure of NC and dopaminergic differentiation. First, the Dicer mutant fetuses exhibited dramatic malformation of the tectum and cerebellum and the eyelids were open. Second, the skeletal structures that are derived from the cranial NC were lost or mostly ablated in Dicer mutant mice. Third, deletion of Dicer in the NC cells resulted in the malformation of the dorsal root ganglia, enteric nervous system and sympathetic ganglia. Interestingly, the expression of neuropeptide Y and its potential regulators TrkA, AP-2 alpha and AP-2 beta was largely abolished in sympathetic neurons of Dicer mutant mice. Fourth, in situ hybridization data revealed that the expression of miR-9, miR-124 and miR-218 in the midbrain and rostral hindbrain area was mostly eliminated in the Dicer mutant mice. We then demonstrated that the development of dopaminergic neurons was impaired in Dicer-deleted mice. Our studies therefore suggest that miRNAs contribute to the embryonic development in multiple locations.

Overgrown, abnormal vasculature characterizes the microenvironment that fuels cancer progression and induces therapeutic resistance. Here we reveal that endothelial plasticity drives excessive and abnormal tumor angiogenesis. Glioblastoma multiforme (GBM) is among the most lethal of human malignancies, distinguished by prominent vascularity and extreme vascular abnormality. We identify endothelial fibro-transformation (Endo-FT) in GBM, contributing significantly to aberrant vascularization, tumor progression, and therapeutic resistance. Utilizing human GBM specimens and allograft and genetic murine GBM models driven by RCAS/Ntv-a-mediated gene transfer of PDGF in Ink4a-Arf-/-PTEN-/- mice, we reveal robust Endo-FT in the tumor vasculature, characterized by endothelial cell (EC) expression of the fibroblast and mesenchymal cell markers including FSP-1, α-smooth muscle actin, N-cadherin. Moreover, genetic lineage tracing shows a prominent population of GBM-associated fibroblast-like cells with EC origin. Furthermore, glioma-conditioned medium induces EC in vitro acquisition of the fibroblast signature with decreased expression of EC-specific proteins including CD31, VE-cadherin, Tie2, VEGFR2, leading to enhanced cell proliferation, motility, and invasiveness, and vessel permeability. Interestingly, the fibroblast-like ECs retain the key EC-specific functions including Ac-LDL absorption and tube formation, suggesting Endo-FT without complete EC transition to fibroblasts. Proteomic analysis identifies c-Met is a regulator of Endo-FT, requisite for the vascular abnormality. Pharmacological inhibition and siRNA-mediated knockdown of c-Met or neutralization with a HGF antibody remarkably abrogate tumor-conditioned medium-induced Endo-FT and EC malfunctions, suggesting a critical role of HGF/c-Met in Endo-FT. Furthermore, c-Met activation induces ETS-1-dependent matrix metalloproteinase (MMP)-14 expression, leading to VE-cadherin cleavage and Endo-FT. Finally, we test the in vivo role of c-Met-mediated Endo-FT in GBM progression by using mice with Tie2-Cre-driven, EC-specific Met knockout. Met deletion in ECs significantly improves mouse survival and inhibits GBM growth after temozolomide (TMZ) treatment. Moreover, Met deletion in ECs abolishes Endo-FT and vascular abnormality, as shown by remarkable decreases in CD31+FSP-1+ cell population and intratumor hypoxia, and increases in vasculature-mediated perfusion and vascular pericyte coverage, suggesting that c-Met-mediated Endo-FT is required for vascular abnormality and GBM progression and therapeutic resistance. In sum, these findings illustrate a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting Endo-FT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors. Citation Format: Menggui Huang, Yi Fan. Endothelial plasticity generates aberrant angiogenesis and therapy resistance in glioblastoma. [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 5222. doi:10.1158/1538-7445.AM2015-5222

Inhibitory neurons in the dorsal horn synthesize a variety of neurotransmitters, including GABA, glycine and a set of peptides. Here we show that three transcription factors, Ptf1a, Pax2, and Lbx1, which have been reported to promote a GABAergic cell fate, also specify glycinergic and peptidergic transmitter phenotypes. First, Ptf1a appears to be a master regulator, as indicated by a requirement of Ptf1a for the expression of glycinergic marker GlyT2 and a set of peptides, including neuropeptide Y (NPY), nociceptin/orphanin FQ (N/OFQ), somatostatin (SOM), enkephalin (ENK), dynorphin (DYN) and galanin (GAL). Second, Pax2 is a downstream target of Ptf1a and controls subsets of transmitter phenotypes, including the expression of GlyT2, NPY, N/OFQ, DYN, and GAL, but is dispensable for SOM or ENK expression. Third, for Lbx1, due to neuronal cell loss at late stages, our analyses focused on early embryonic stages, and we found that Lbx1 is required for the expression of GlyT2, NPY, N/OFQ and is partially responsible for SOM expression. Our studies therefore suggest a coordinated and hierarchical specification of a variety of neurotransmitters in dorsal spinal inhibitory neurons.