Search

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

Refereed/Peer-reviewed Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

BACKGROUND: Wnt/β-catenin signaling is a highly conserved signaling pathway that regulates the transcription factor PROX1. The role of β-catenin and PROX1 in pancreatic cancer is ambiguous, as some studies have associated their expression with tumor regression and some with tumor progression. OBJECTIVE: We have investigated their expression in surgically treated pancreatic cancer patients receiving neoadjuvant therapy (NAT), and patients treated upfront with surgery (US). We furthermore compared the expression of β-catenin and PROX1 between patients who had a good or poor response to NAT. METHODS: We evaluated β-catenin and PROX1 expression through immunohistochemistry in 88 neoadjuvant and 144 upfront surgery patients by scoring the intensity of the immunopositivity as 0–3, corresponding to negative, weak, moderate, or strong. We developed a six-tier grading scheme for the neoadjuvant responses by analyzing the remaining tumor cells in surgical specimen histological sections. RESULTS: Strong β-catenin immunopositivity associated with improved survival in the patients with good NAT-response (≤10% residual tumor cells) (Hazard ratio [HR] 0.26 95%, confidence interval [CI] 0.07–0.88 p = 0.030). Additionally, the combined moderate β-catenin and PROX1 expression associated with improved survival (HR 0.20 95% CI 0.05–0–76 p = 0.018) among the good responders. Among the patients with a poor NAT-response (> 10% residual tumor cells), both strong β-catenin immunopositivity and strong combined β-catenin and PROX1 associated with shorter survival (HR 2.03 95% CI 1.16–3.55 p = 0.013, and HR 3.1 95% CI 1.08–8.94 p = 0.03, respectively). PROX1 alone was not associated with survival. CONCLUSIONS: Strong β-catenin immunopositivity and combined strong or moderate β-catenin and PROX1 immunopositivity associated with improved survival among the good NAT-responders and worse survival among the poor NAT-responders.

Supplementary Table 1 and Fig 1-8 including legends. Table S1: Observed and expected numbers of Pf4-Cre;Pdgfbfl/fl embryos at different developmental stages. Figure S1: Platelet-specific PDGFB knockout embryos display no obvious abnormalities. Figure S2: Platelet counts and proportion in peripheral blood from WT and pl-PDGFB KO mice. Figure S3: Generation of RIP1-Tag2 mice lacking PDGFB in platelets. Figure S4: Lack of platelet PDGFB does not affect the angiogenic switch or tumor growth in RT2 mice. Figure S5: Tumor vessels in MC38 colon carcinoma in mice with platelet-specific PDGFB ablation have impaired function and decreased pericyte coverage. Figure S6: Increased spontaneous lung metastasis in mice with platelet-specific PDGFB ablation in the HCmel12 melanoma model. Figure S7: Reduced pericyte coverage already in pre-tumorigenic tissue in mice lacking PDGFB in platelets. Figure S8: Expression of Cxcl3, Cxcl5, Cxcl7 and Cxcl15 in tumor tissue from Pf4-Cre- and Pf4-Cre+ mice.

Supplementary Figure S4

Abnormal vasculature in tumors leads to poor tissue perfusion and cytostatic drug delivery. Although drugs inducing vascular normalization, for example, angiopoietin-2 (Ang2)-blocking antibodies, have shown promising results in preclinical tumor models, clinical studies have so far shown only little efficacy. Because Ang2 is known to play a protective role in stressed endothelial cells, we tested here whether Ang2 blocking could enhance radiation-induced tumor vascular damage. Tumor-bearing mice were treated with anti-Ang2 antibodies every 3 or 4 days starting 3 days before 3 × 2 Gy or 4 × 0.5 Gy whole-body or tumor-focused radiation. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal tumors. Single-cell RNA-sequencing revealed that Ang2 blocking rescued radiation-induced decreases in T cells and cells of the monocyte/macrophage lineage. In addition, anti-Ang2 enhanced radiation-induced apoptosis in cultured endothelial cells. In vivo, combination treatment decreased tumor vasculature and increased tumor necrosis in comparison with tumors treated with monotherapies. These results suggest that a combination of Ang2-blocking antibodies with radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice.Significance:These findings offer a preclinical rationale for further testing of the use of radiation in combination with Ang2-blocking antibodies to improve the overall outcome of cancer treatment.

Antibody concentration and blocking conditions

Data from endothelial cell qPCR array.

Supplementary Figure 1 from Suppression of Prostate Cancer Nodal and Systemic Metastasis by Blockade of the Lymphangiogenic Axis

Supplementary Figure 1 from Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth

PDF file - 93K, No effect on tumor growth in the Bmx-deleted male mice in the chemically induced skin carcinogenesis model or RIP1-Tag2 pancreatic insulinomas

Severe ascites is a hallmark of advanced ovarian cancer (OVCA), yet the underlying mechanism that creates an imbalance between peritoneal vascular leakage and lymphatic drainage is unknown. Here, we identified and characterized peritoneal lymphatic vessels in OVCA mice, a model generated by implantation of human OVCA cells into athymic nude mice. The OVCA mice displayed substantial lymphangiogenesis and lymphatic remodeling, massive infiltration of CD11b+/LYVE-1+ macrophages and disseminated carcinomatosis in the mesentery and diaphragm, and progressive chylous ascites formation. Functional assays indicated that the abnormally abundant lymphatic vessels in the diaphragm were not conductive in peritoneal fluid drainage. Moreover, lipid absorbed from the gut leaked out from the aberrant mesenteric lymphatic vessels. Our results indicate that vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF-A from CD11b+ macrophages are responsible for producing OVCA-induced dysfunctional lymphangiogenesis, although other cell types contribute to the increased ascites formation. Accordingly, the combined blockade of VEGF-C/D and VEGF-A signaling with soluble VEGF receptor-3 and VEGF-Trap, respectively, markedly inhibited chylous ascites formation. These findings provide additional therapeutic targets to ameliorate chylous ascites formation in patients with advanced OVCA. [Cancer Res 2008;68(4):1100–9]

Supplementary Methods, Figures 1-9, Tables 1-2 from Profound but Dysfunctional Lymphangiogenesis via Vascular Endothelial Growth Factor Ligands from CD11b+ Macrophages in Advanced Ovarian Cancer

Supplementary Figure 6 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Figure 7 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Figure 2 from Loss of Neural Cell Adhesion Molecule Induces Tumor Metastasis by Up-regulating Lymphangiogenesis

Supplementary Figure 2 from Angiopoietin-1 Overexpression Modulates Vascular Endothelium to Facilitate Tumor Cell Dissemination and Metastasis Establishment

PDF file - 196K, Normal postnatal development of retinal vasculature in Bmx deficient mice

Supplementary Figure 1 from Angiopoietin-1 Overexpression Modulates Vascular Endothelium to Facilitate Tumor Cell Dissemination and Metastasis Establishment

PDF file - 198K

Supplementary Legends for Figures 1-8, Tables 1-3, Methods from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Bmx (Bone marrow kinase in chromosome X), also known as Etk, is a member of the Tec family of nonreceptor tyrosine kinases. Bmx is expressed mainly in arterial endothelia and in myeloid hematopoietic cells. Bmx regulates ischemia-mediated arteriogenesis and lymphangiogenesis, but its role in tumor angiogenesis is not known. In this study, we characterized the function of Bmx in tumor growth using both Bmx knockout and transgenic mice. Isogenic colon, lung, and melanoma tumor xenotransplants showed reductions in growth and tumor angiogenesis in Bmx gene–deleted (−/−) mice, whereas developmental angiogenesis was not affected. In addition, growth of transgenic pancreatic islet carcinomas and intestinal adenomas was also slower in Bmx−/− mice. Knockout mice showed high levels of Bmx expression in endothelial cells of tumor-associated and peritumoral arteries. Moreover, endothelial cells lacking Bmx showed impaired phosphorylation of extracellular signal–regulated kinase (Erk) upon VEGF stimulation, indicating that Bmx contributes to the transduction of vascular endothelial growth factor signals. In transgenic mice overexpressing Bmx in epidermal keratinocytes, tumors induced by a two-stage chemical skin carcinogenesis treatment showed increased growth and angiogenesis. Our findings therefore indicate that Bmx activity contributes to tumor angiogenesis and growth. Cancer Res; 72(14); 3512–21. ©2012 AACR.

PDF file - 1.8MB, Effect of homozygous or haploid Bmx deletion on tumor growth in female mice in the chemical skin carcinogenesis model

Supplementary Tables 1-3 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Tables S1-S4. MMPs which are significantly over-expressed in melanoma cell lines compared to the mean expression of all cancer cell lines in the datasets with over 15 melanoma cell lines (S1); Outlier analysis of MMP16 copy number gain or over-expression using DNA and mRNA datasets containing more than 20 clinical melanoma samples (S2); MMP mRNA expression, LVI and clinicopathological data of human melanoma tumors (S3); S100B staining coverage, lymphatic and blood vessel densities of human melanoma tumors (S4)

Supplementary Table 1, Figures 1-7 from CXCR4 Signaling Regulates Metastasis of Chemoresistant Melanoma Cells by a Lymphatic Metastatic Niche

Supplementary Data. Supplementary Materials and Methods and Supplementary Figure Legends

Lymph node involvement denotes a poor outcome for patients with prostate cancer. Our group, along with others, has shown that initial tumor cell dissemination to regional lymph nodes via lymphatics also promotes systemic metastasis in mouse models. The aim of this study was to investigate the efficacy of suppressive therapies targeting either the angiogenic or lymphangiogenic axis in inhibiting regional lymph node and systemic metastasis in subcutaneous and orthotopic prostate tumor xenografts. Both androgen-dependent and more aggressive androgen-independent prostate tumors were used in our investigations. Interestingly, we observed that the threshold for dissemination is lower in the vascular-rich prostatic microenvironment compared with subcutaneously grafted tumors. Both vascular endothelial growth factor-C (VEGF-C) ligand trap (sVEGFR-3) and antibody directed against VEGFR-3 (mF4-31C1) significantly reduced tumor lymphangiogenesis and metastasis to regional lymph nodes and distal vital organs without influencing tumor growth. Conversely, angiogenic blockade by short hairpin RNA against VEGF or anti–VEGFR-2 antibody (DC101) reduced tumor blood vessel density, significantly delayed tumor growth, and reduced systemic metastasis, although it was ineffective in reducing lymphangiogenesis or nodal metastasis. Collectively, these data clarify the utility of vascular therapeutics in prostate tumor growth and metastasis, particularly in the context of the prostate microenvironment. Our findings highlight the importance of lymphangiogenic therapies in the control of regional lymph node and systemic metastasis. [Cancer Res 2008;68(19):7828–37] [Cancer Res 2008;68(19):7828–37]

Supplementary Figures S1-S9. MMP14 which is over-expressed in melanoma cell lines was not associated with patient survival (S1); Vasculature and morphology of human melanoma tumors (S2); MMP16 expression in WM852 cells and its correlation with tumor weight in melanoma xenografts (S3); No significant difference in the blood and lymphatic vessel densities was observed between shScr and shMMP16 tumors (S4); MMP16-silencing does not affect mouse collagen type I in xenografts (S5); Endogenous or exogenous MMP16 regulates collagen invasion and cell-cell contacts in WM852 and Bowes melanoma cells (S6); Endogenous or recombinant MMP16 regulates LEC spheroid intravasation of WM852 and Bowes melanoma cells (S7); Silencing of L1CAM cleaved by MMP16 rescued the transmigration across BECs but not the cell junction disassembly of shMMP16 cells (S8); L1CAM immunohistochemistry of xenografts and human samples (S9).

Supplementary Figure 5 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

The angiopoietin-1 (Ang1)/Tie2 signaling pathway is known to play an important role in the regulation of vascular maturation and maintenance of vessel integrity. In this study, we have investigated the effect of systemic Tie2 activation or inhibition on tumor growth and metastasis. We found that treatment with Ang1 delivered via an adenoviral vector promoted s.c. implanted tumor metastasis to the lungs. Ang1 treatment did not significantly increase vascular density in the tumors but induced enlargement of blood vessels in both the tumor and normal tissues, which increased tumor cell dissemination into the blood circulation. Ang1 also enhanced the formation of metastatic foci in the lungs when tumor cells were injected into the circulation via the tail vein. The effect of Ang1 on metastasis was validated by a simultaneous treatment with a soluble form of Tie2 (sTie2), which led to the suppression of Ang1-induced increase of tumor metastasis. Furthermore, using a highly metastatic tumor model, we confirmed that systemic treatment with sTie2 suppressed tumor metastasis to the lungs and lymph nodes, whereas tumor-associated angiogenesis and lymphangiogenesis were not significantly affected. This suggests that the Ang1/Tie2 signals contribute to tumor progression by increasing vascular entry and exit of tumor cells to facilitate tumor dissemination and establishment of metastases. [Cancer Res 2009;69(11):4656–64]

Supplementary Figure 2 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Table 1 from Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth

Supplementary Methods from Loss of Neural Cell Adhesion Molecule Induces Tumor Metastasis by Up-regulating Lymphangiogenesis

Supplementary Figure 3 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Figure Legends 1-2 from Angiopoietin-1 Overexpression Modulates Vascular Endothelium to Facilitate Tumor Cell Dissemination and Metastasis Establishment

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3–treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3–treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets. [Cancer Res 2007;67(2):593–9]

Supplementary Figure 4 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Figure 1 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Supplementary Figure 1 from Loss of Neural Cell Adhesion Molecule Induces Tumor Metastasis by Up-regulating Lymphangiogenesis

Supplementary Figure 8 from Fibroblast Growth Factor Receptor 4 Regulates Tumor Invasion by Coupling Fibroblast Growth Factor Signaling to Extracellular Matrix Degradation

Reduced expression of neural cell adhesion molecule (NCAM) has been implicated in the progression to tumor malignancy in cancer patients. Previously, we have shown that the loss of NCAM function causes the formation of lymph node metastasis in a transgenic mouse model of pancreatic β cell carcinogenesis (Rip1Tag2). Here we show that tumors of NCAM-deficient Rip1Tag2 transgenic mice exhibit up-regulated expression of the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and -D (17% in wild-type versus 60% in NCAM-deficient Rip1Tag2 mice) and, with it, increased lymphangiogenesis (0% in wild-type versus 19% in NCAM-deficient Rip1Tag2 mice). Repression of VEGF-C and -D function by adenoviral expression of a soluble form of their cognate receptor, VEGF receptor-3, results in reduced tumor lymphangiogenesis (56% versus 28% in control versus treated mice) and lymph node metastasis (36% versus 8% in control versus treated mice). The results indicate that the loss of NCAM function causes lymph node metastasis via VEGF-C- and VEGF-D-mediated lymphangiogenesis. These results also establish Rip1Tag2;NCAM-deficient mice as a unique model for stochastic, endogenous tumor lymphangiogenesis and lymph node metastasis in immunocompetent mice.