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1.  Inhibition of c-Met reduces lymphatic metastasis in RIP-Tag2 transgenic mice 
Cancer research  2013;73(12):3692-3703.
Inhibition of vascular endothelial growth factor (VEGF) signaling can promote lymph node metastasis in preclinical models, but the mechanism is not fully understood, and successful methods of prevention have not been found. Signaling of hepatocyte growth factor (HGF) and its receptor c-Met can promote the growth of lymphatics and metastasis of some tumors. We sought to explore the contributions of c-Met signaling to lymph node metastasis after inhibition of VEGF signaling. In particular, we examined whether c-Met is upregulated in lymphatics in or near pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice and whether lymph node metastasis can be reduced by concurrent inhibition of VEGF and c-Met signaling. Inhibition of VEGF signaling by anti-VEGF antibody or sunitinib in mice from age 14 to 17 weeks was accompanied by more intratumoral lymphatics, more tumor cells inside lymphatics, and more lymph node metastases. Under these conditions, lymphatic endothelial cells - like tumor cells - had strong immunoreactivity for c-Met and phospho-c-Met. c-Met blockade by the selective inhibitor PF-04217903 significantly reduced metastasis to local lymph nodes. Together, these results indicate that inhibition of VEGF signaling in RIP-Tag2 mice upregulates c-Met expression in lymphatic endothelial cells, increases the number of intratumoral lymphatics and number of tumor cells within lymphatics, and promotes metastasis to local lymph nodes. Prevention of lymph node metastasis by PF-04217903 in this setting implicates c-Met signaling in tumor cell spread to lymph nodes.
PMCID: PMC3686901  PMID: 23576559
2.  Dynamics of Airway Blood Vessels and Lymphatics 
Blood vessels and lymphatic vessels in the respiratory tract play key roles in inflammation. By undergoing adaptive remodeling and growth, blood vessels undergo changes that enable the extravasation of plasma and leukocytes into inflamed tissues, and lymphatic vessels adjust to the increased fluid clearance and cell traffic involved in immune responses. Blood vessels and lymphatics in adult airways are strikingly different from those of late-stage embryos. Before birth, blood vessels in mouse airways make up a primitive plexus similar to that of the yolk sac. This plexus undergoes rapid and extensive remodeling at birth. In the early neonatal period, parts of the plexus regress. Capillaries then rapidly regrow, and with arterioles and venules form the characteristic adult vascular pattern. Lymphatic vessels of the airways also undergo rapid changes around birth, when lymphatic endothelial cells develop button-like intercellular junctions specialized for efficient fluid uptake. Among the mechanisms that underlie the onset of rapid vascular remodeling at birth, changes in tissue oxygen tension and mechanical forces associated with breathing are likely to be involved, along with growth factors that promote the growth and maturation of blood vessels and lymphatics. Whatever the mechanisms, the dynamic nature of airway blood vessels and lymphatics during perinatal development foretells the extraordinary vascular plasticity found in many diseases.
PMCID: PMC3359074  PMID: 22052927
angiogenesis; blood vessels; lymphatics; lymphangiogenesis; respiratory tract
3.  Cathepsin L Protects Mice from Mycoplasmal Infection and Is Essential for Airway Lymphangiogenesis 
Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis–infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl−/− and Ctsl+/+ mice. After infection, Ctsl−/− mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl+/+ mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl−/− mice than in Ctsl+/+ mice after infection for 3 days. Ctsl−/− mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl+/+ mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl+/+ mice, little lymphangiogenesis occurred in Ctsl−/− mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl+/+ mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.
PMCID: PMC3824055  PMID: 23600672
cathepsin L; mycoplasma; pneumonia; bronchitis; lymphangiogenesis
4.  IL-7 production in murine lymphatic endothelial cells and induction in the setting of peripheral lymphopenia 
International Immunology  2013;25(8):471-483.
IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.
PMCID: PMC3720065  PMID: 23657000
endothelium; HIV; interleukin-7; lymphatic; lymph node; lymphopenia; myeloid; stromal
5.  Suppression of tumor invasion and metastasis by concurrent inhibition of c-Met and VEGF signaling in pancreatic neuroendocrine tumors 
Cancer Discovery  2012;2(3):270-287.
Invasion and metastasis increase after inhibition of vascular endothelial growth factor (VEGF) signaling in some preclinical tumor models. The present study asked whether selective VEGF inhibition is sufficient to increase invasion and metastasis and whether selective c-Met inhibition is sufficient to block this effect. Treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody reduced tumor burden but increased tumor hypoxia, HIF-1α, and c-Met activation, and also increased invasion and metastasis. However, invasion and metastasis were reduced by concurrent inhibition of c-Met by PF-04217903 or PF-02341066 (crizotinib). Similar benefit was found in orthotopic Panc-1 pancreatic carcinomas treated with sunitinib plus PF-04217903 and in RIP-Tag2 tumors treated with XL184 (cabozantinib), which simultaneously blocks VEGF and c-Met signaling. These findings document that invasion and metastasis are promoted by selective inhibition of VEGF signaling and can be reduced by concurrent inhibition of c-Met.
PMCID: PMC3354652  PMID: 22585997
Evasive resistance; pancreatic cancer; angiogenesis inhibitors
6.  VEGF and c-Met Blockade Amplify Angiogenesis Inhibition in Pancreatic Islet Cancer 
Cancer research  2011;71(14):4758-4768.
Angiogenesis inhibitors that block vascular endothelial growth factor receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTKs). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small molecule inhibitors that potently block multiple RTKs including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 to those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated ~ 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis.
PMCID: PMC3138890  PMID: 21613405
Angiogenesis inhibitors; c-Met; VEGF; receptor tyrosine kinases; XL880 (foretinib, GSK1363089); XL184 (cabozantinib); XL999; RIP-Tag2 mice
7.  Molecular Determinants of Lung Development 
Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
PMCID: PMC3955361  PMID: 23607856
lung development; lung cell fate; lung cell differentiation; tissue interaction; environmental impact
8.  Plasticity of Airway Lymphatics in Development and Disease 
The dynamic nature of lymphatic vessels is reflected by structural and functional modifications that coincide with changes in their environment. Lymphatics in the respiratory tract undergo rapid changes around birth, during adaptation to air breathing, when lymphatic endothelial cells develop button-like intercellular junctions specialized for efficient fluid uptake and transport. In inflammatory conditions, lymphatic vessels proliferate and undergo remodeling to accommodate greater plasma leakage and immune cell trafficking. However, the newly formed lymphatics are abnormal, and resolution of inflammation is not accompanied by complete reversal of the lymphatic vessel changes back to the baseline. As the understanding of lymphatic plasticity advances, approaches for eliminating the abnormal vessels and improving the functionality of those that remain move closer to reality. This chapter provides an overview of what is known about lymphatic vessel growth, remodeling, and other forms of plasticity that occur during development or inflammation, with an emphasis on the respiratory tract. Also addressed is the limited reversibility of changes in lymphatics during the resolution of inflammation.
PMCID: PMC3955364  PMID: 24276885
9.  Molecular Determinants of Lung Development 
Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
PMCID: PMC3955361  PMID: 23607856
lung development; lung cell fate; lung cell differentiation; tissue interaction; environmental impact
10.  Rapid remodeling of airway vascular architecture at birth 
Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder-like adult pattern. Hypoxia and HIF-1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation.
PMCID: PMC2997630  PMID: 20730909
Respiratory tract; blood vessels; angiogenesis; vascular regression; hypoxia; VEGF
11.  Identification of pY654-β-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia 
Oncogene  2012;32(42):10.1038/onc.2012.530.
Hypoxia is linked to epithelial mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of β-catenin to hypoxia-inducible factor-1α (HIF1α) signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-β-catenin and HIF1α. All pY654-β-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for β-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-β-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated β-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-β-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-β-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.
PMCID: PMC3871884  PMID: 23246962
hypoxia; signaling; transcription; tumor; epithelial
12.  Complementary Actions of Inhibitors of Angiopoietin-2 and VEGF on Tumor Angiogenesis and Growth 
Cancer research  2010;70(6):2213-2223.
Inhibition of angiopoietin-2 (Ang2) can slow tumor growth, but the underlying mechanism is not fully understood. Because Ang2 is expressed in growing blood vessels and promotes angiogenesis driven by vascular endothelial growth factor (VEGF), we asked whether the anti-tumor effect of Ang2 inhibition results from reduced sprouting angiogenesis and whether the effect is augmented by inhibition of VEGF from tumor cells. Using Colo205 human colon carcinomas in nude mice as a model, we found that selective inhibition of Ang2 by the peptide-Fc fusion protein L1-7(N) reduced the number of vascular sprouts by 46% and tumor growth by 62% over 26 days. Strikingly, when the Ang2 inhibitor was combined with a function-blocking anti-VEGF antibody, the number of sprouts was reduced by 82%, tumor vascularity was reduced by 67%, and tumor growth slowed by 91% compared to controls. The reduction in tumor growth was accompanied by decreased cell proliferation and increased apoptosis. We conclude that inhibition of Ang2 slows tumor growth by limiting the expansion of the tumor vasculature by sprouting angiogenesis, in a manner that is complemented by concurrent inhibition of VEGF and leads to reduced proliferation and increased apoptosis of tumor cells.
PMCID: PMC2840050  PMID: 20197469
angiogenesis; angiopoietin-2; VEGF; Colo205 tumors
13.  Functionally specialized junctions between endothelial cells of lymphatic vessels 
The Journal of Experimental Medicine  2007;204(10):2349-2362.
Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946–953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf–shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction–associated proteins, including occludin, claudin-5, zonula occludens–1, junctional adhesion molecule–A, and endothelial cell–selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule–1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics.
PMCID: PMC2118470  PMID: 17846148
14.  Permeability to Macromolecular Contrast Media Quantified by Dynamic MRI Correlates with Tumor Tissue Assays of Vascular Endothelial Growth Factor (VEGF) 
European Journal of Radiology  2011;81(5):891-896.
To correlate dynamic MRI assays of macromolecular endothelial permeability with microscopic area-density measurements of vascular endothelial growth factor (VEGF) in tumors.
Methods and Material
This study compared tumor xenografts from two different human cancer cell lines, MDA-MB-231 tumors (n=5), and MDA-MB-435 (n=8), reported to express respectively higher and lower levels of VEGF. Dynamic MRI was enhanced by a prototype macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)35. Quantitative estimates of tumor microvascular permeability (KPS; μl/min·100cm3), obtained using a two-compartment kinetic model, were correlated with immunohistochemical measurements of VEGF in each tumor.
Mean KPS was 2.4 times greater in MDA-MB-231 tumors (KPS=58±30.9μl/min·100cm3) than in MDA-MB-435 tumors (KPS=24±8.4μl/min·100cm) (p<0.05). Correspondingly, the area-density of VEGF in MDA-MB-231 tumors was 2.6 times greater (27.3±2.2%, p<0.05) than in MDA-MB-435 cancers (10.5±0.5%, p<0.05). Considering all tumors without regard to cell type, a significant positive correlation (r=0.67, p<0.05) was observed between MRI-estimated endothelial permeability and VEGF immunoreactivity.
Correlation of MRI assays of endothelial permeability to a MMCM and VEGF immunoreactivity of tumors support the hypothesis that VEGF is a major contributor to increased macromolecular permeability in cancers. When applied clinically, the MMCM-enhanced MRI approach could help to optimize the appropriate application of VEGF-inhibiting therapy on an individual patient basis.
PMCID: PMC3242157  PMID: 21889860
DCE-MRI; macromolecular contrast media; endothelial permeability; immunohistochemical tumor VEGF levels; correlation
15.  Rapid vascular regrowth in tumors after reversal of VEGF inhibition 
Journal of Clinical Investigation  2006;116(10):2610-2621.
Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%–60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less α–SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.
PMCID: PMC1578604  PMID: 17016557
16.  In situ force mapping of mammary gland transformation 
Tumor progression is characterized by an incremental stiffening of the tissue. The importance of tissue rigidity to cancer is appreciated, yet the contribution of specific tissue elements to tumor stiffening and their physiological significance remains unclear. We performed high-resolution atomic force microscopy indentation in live and snap-frozen fluorescently labeled mammary tissues to explore the origin of the tissue stiffening associated with mammary tumor development in PyMT mice. The tumor epithelium, the tumor-associated vasculature and the extracellular matrix all contributed to mammary gland stiffening as it transitioned from normal to invasive carcinoma. Consistent with the concept that extracellular matrix stiffness modifies cell tension, we found that isolated transformed mammary epithelial cells were intrinsically stiffer than their normal counterparts but that the malignant epithelium in situ was far stiffer than isolated breast tumor cells. Moreover, using an in situ vitrification approach, we determined that the extracellular matrix adjacent to the epithelium progressively stiffened as tissue evolved from normal through benign to an invasive state. Importantly, we also noted that there was significant mechanical heterogeneity within the transformed tissue both in the epithelium and the tumor-associated neovasculature. The vascular bed within the tumor core was substantially stiffer than the large patent vessels at the invasive front that are surrounded by the stiffest extracellular matrix. These findings clarify the contribution of individual mammary gland tissue elements to the altered biomechanical landscape of cancerous tissues and emphasize the importance of studying cancer cell evolution under conditions that preserve native interactions.
PMCID: PMC3564969  PMID: 21842067
17.  Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation 
Journal of Clinical Investigation  2005;115(2):247-257.
Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases.
PMCID: PMC544601  PMID: 15668734
18.  Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung 
Nature medicine  2004;10(10):1095-1103.
Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF165 transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (TH2)-mediated inflammation. In these mice, VEGF induced, through IL-13–dependent and –independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and TH2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by TH2 versus TH1 cells. In this setting, it had a critical role in TH2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive TH2 inflammation. VEGF regulation may be therapeutic in asthma and other TH2 disorders.
PMCID: PMC3434232  PMID: 15378055
19.  Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors 
Cancer research  2011;71(5):1573-1583.
The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin which only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts anti-angiogenic and anti-tumoral effects that are even more efficacious when combined with a VEGFR antagonist.
PMCID: PMC3077087  PMID: 21363918
mTOR; VEGF; angiogenesis; vascular regrowth; RIP-Tag2 tumors
20.  New antibody to stop tumor angiogenesis and lymphatic spread by blocking receptor partnering 
Cancer cell  2010;18(6):541-543.
Tvorogov et al. (2010) describe in this issue of Cancer Cell an antibody that inhibits homodimerization of vascular endothelial growth factor receptor-3 (VEGFR-3) and its heterodimerization with VEGFR-2, but not ligand binding. The work provides mechanistic insights into receptor dimerization and an approach to suppress both angiogenesis and lymphangiogenesis.
PMCID: PMC3031147  PMID: 21156277
21.  MR Signal Characteristics of Viable and Apoptotic Human Mesenchymal Stem Cells in MASI for Treatment of Osteoarthritis 
Investigative radiology  2010;45(10):634-640.
To compare MR signal characteristics of contrast agent-labeled apoptotic and viable human mesenchymal stem cells (hMSCs) in matrix associated stem cell implants (MASI).
hMSCs were labeled with FDA approved ferumoxides nanoparticles. One group (A) remained untreated while a second group (B) underwent Mitomycin C-induced apoptosis induction. Viability of group A and apoptosis of group B was confirmed by Caspase-assays and TUNEL stains. Labeled viable hMSCs, unlabeled viable hMSCs, labeled apoptotic hMSCs and unlabeled apoptotic hMSCs (n=7 samples each) in an agarose scaffold were implanted into cartilage defects of porcine patellae specimens and underwent MR imaging at 7T, using T1 weighted SE sequences, T2-weighted SE sequences and T2*-weighted GE sequences. Signal to noise ratios (SNR) of the implants were calculated and compared between different experimental groups using linear mixed regression models (LMM).
Ferumoxides-labeled hMSCs provided a strong negative T2 and T2*-enhancement. Corresponding SNR data of labeled hMSCs were significantly lower compared to unlabeled controls (p<0.05). Apoptosis induction resulted in a significant signal decline of ferumoxides-labeled hMSC transplants on short TE T2-weighted sequences. SNR data of labeled apoptotic hMSCs were significantly lower compared to labeled viable hMSCs (p<0.05).
Apoptosis of transplanted ferumoxides-labeled stem cells in cartilage defects can be visualized non-invasively by a significant signal decline on T2-weighted MR images. The described MR signal characteristics may serve as a non-invasive outcome measure for the assessment of MASI therapies in clinical practice. Additional studies are needed to further enhance the observed differences between viable and apoptotic cells, e.g. by further optimizing the applied MR pulse sequence parameters or by determining more robust T2-relaxation times.
PMCID: PMC2943569  PMID: 20808236
MR Imaging; molecular imaging; apoptosis; stem cells; cartilage
22.  Relaxation effects of Ferucarbotran-labeled mesenchymal stem cells at 1.5T and 3T: Discrimination of viable from lysed cells 
Human mesenchymal stem cells (hMSC) were labeled with Ferucarbotran by simple incubation and cultured for up to 14 days. Iron content was determined by spectrometry and the intracellular localization of the contrast agent uptake was studied by electron and confocal microscopy. At various time points after labeling, reaching from 1 to 14 days, samples with viable or lysed labeled hMSCs, as well as non-labeled controls underwent MR imaging. SE- and GE-sequences with multiple TRs and TEs were used at 1.5T and 3T on a clinical scanner. Spectrometry showed an initial iron oxide uptake of 7.08 pg per cell. Microscopy studies revealed lysosomal compartmentalization. Contrast agent effects of hMSC were persistent for up to 14 days after labeling. A marked difference in the T2-effect of compartmentalized iron oxides compared to free iron oxides was found on T2-weighted sequences, but not on T2*-sequences. The observed differences may be explained by the loss of compartmentalization of iron oxide particles, the uniformity of distribution and the subsequent increase in dephasing of protons on SE images. These results show that viable cells with compartmentalized iron oxides may – in principle – be distinguished from lysed cells or released iron oxides.
PMCID: PMC2931823  PMID: 19353670
Cell Labeling; Mesenchymal Stem Cells; MR Imaging; Ferucarbotran
23.  Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning 
Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Gαi-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.
PMCID: PMC2812554  PMID: 20026661
24.  Cellular source and amount of VEGF and PDGF in tumors determine response to angiogenesis inhibitors 
Cancer research  2009;69(10):4527-4536.
Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their receptors are important targets in cancer therapy based on angiogenesis inhibition. However, it is unclear whether inhibition of VEGF and PDGF together is more effective than inhibition of either one alone. Here, we used two contrasting tumor models to compare the effects of inhibiting VEGF or PDGF alone, by adenovirally-generated soluble receptors, to the effects of inhibiting both together. In RIP-Tag2 tumors, VEGF and PDGF inhibition together reduced tumor vascularity and abundance of pericytes. However, VEGF inhibition reduced tumor vascularity without decreasing pericyte density, and PDGF inhibition reduced pericytes without reducing tumor vascularity. By contrast, in Lewis lung carcinomas (LLC), inhibition of VEGF or PDGF reduced blood vessels and pericytes to the same extent as inhibition of both together. Similar results were obtained using tyrosine kinase inhibitors AG-013736 and Imatinib. In LLC, VEGF expression was largely restricted to pericytes, and PDGF was largely restricted to endothelial cells, but in RIP-Tag2 tumors expression of both growth factors was more widespread and significantly greater than in LLC. These findings suggest that inhibition of PDGF in LLC reduced pericytes, and then tumor vessels regressed because pericytes were the main source of VEGF. The vasculature of RIP-Tag2 tumors, where most VEGF is from tumor cells, was more resistant to PDGF inhibition. The findings emphasize the interdependence of pericytes and endothelial cells in tumors and the importance of tumor phenotype in determining the cellular effects of VEGF and PDGF inhibitors on tumor vessels.
PMCID: PMC2759816  PMID: 19401451
25.  TNF-α drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice 
The Journal of Clinical Investigation  2009;119(10):2954-2964.
Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-α drives the changes in blood vessels and lymphatics in M. pulmonis–infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-α expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-α signaling was inhibited by a blocking antibody or was silenced in Tnfr1–/– mice. When administered after infection was established, the TNF-α–specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-α on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-α is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.
PMCID: PMC2752063  PMID: 19759514

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