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As head-and-neck radiotherapy treatments become more complex and sophisticated, and the need to control and stabilise the positioning of intra-oral anatomy becomes more important, leading the increasing use of oral positioning stents during head-and-neck radiotherapy simulation and delivery. As an alternative to the established practice of creating oral positioning stents using wax, this study investigated the use of a 3D printing technique. An Ender 5 3D printer (Creality 3D, Shenzhen, China) was used, with PLA+ "food-safe" polylactic acid filament (3D Fillies, Dandenong South, Australia), to produce a low-density 3D printed duplicate of a conventional wax stent. The physical and dosimetric effects of the two stents were evaluated using radiochromic film in a solid head phantom that was modified to include flexible parts. The Varian Eclipse treatment planning system (Varian Medical Systems, Palo Alto, USA) was used to calculate the dose from two different head-and-neck treatment plans for the phantom with each of the two stents. Examination of the resulting four dose distributions showed that both stents effectively pushed sensitive oral tissues away from the treatment targets, even though most of the phantom was solid. Film measurements confirmed the accuracy of the dose calculations from the treatment planning system, despite the steep density gradients in the treated volume, and demonstrated that the 3D print could be a suitable replacement for the wax stent. This study demonstrated a useful method for dosimetrically testing novel oral positioning stents. We recommend the development of flexible phantoms for future studies.

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or CDK6 significantly overcomes the resistance of luminal breast cancer cells to AKT inhibitors in vitro and in vivo. Our study reports the involvement of BRD4/FOXO3a/CDK6 axis in AKTi resistance and provides potential therapeutic strategies for treating resistant breast cancer., The molecular mechanism underlying the resistance of AKT inhibitors in breast cancer is still elusive. Here, the authors demonstrate that BRD4/FOXO3a axis upregulates CDK6 promoter activity to promote resistance to AKT inhibition in breast cancer cells and that blocking the action of CDK6 re-sensitizes resistant cancer cells to growth inhibition.

Rationale: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30% to 40% of strokes. Inflammation has been suggested as a critical etiologic factor. However, there is lack of experimental evidence. Objective: In this study, we investigated inflammation-associated stroke using a mouse model that developed spontaneous stroke because of myeloid deficiency of TGF-β (transforming growth factor-β) signaling. Methods and Results: We report that mice with deletion of Tgfbr2 in myeloid cells ( Tgfbr2 Myeko ) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild-type mice via Tgfbr2 Myeko bone marrow transplant and can be rescued in Tgfbr2 Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation and cerebral endotheliopathy, characterized by elevated NF-κB (nuclear factor-κB) activation and TNF (tumor necrosis factor) production by myeloid cells. A high-fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence. Conclusions: Our studies show that TGF-β signaling in myeloid cells is required for maintenance of vascular health and provide insight into inflammation-mediated cerebrovascular disease and stroke.

Treatment of cancer metastasis has been largely ineffective. It is paramount to understand the mechanisms underlying the metastatic process, of which the tumor microenvironment is an indispensable participant. What are the critical cellular and molecular players at the primary tumor site where metastatic cascade initiates? How is tumor-associated inflammation regulated? How do altered vasculatures contribute to metastasis? What is the dynamic nature or heterogeneity of primary tumors and what are the challenges to catch a moving target? This review summarizes recent progress, mechanistic understanding, and options for metastasis-targeted therapy.

Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.

Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, subsets of myeloid cells have also been reported to have anti-tumor properties. It is not clear whether there is a transition between anti- and pro- tumor function of these myeloid cells, and if so, what are the underlying molecular mechanisms. Here we report platelet factor 4 (PF4), or CXCL4, but not the other family members CXCL9, 10, and 11, was produced at higher levels in the normal lung and early stage premetastatic lungs but decreased in later stage lungs. PF4 was mostly produced by Ly6G+CD11b+ myeloid cell subset. Although the number of Ly6G+CD11b+ cells was increased in the premetastatic lungs, the expression level of PF4 in these cells was decreased during the metastatic progression. Deletion of PF4 (PF4 knockout or KO mice) led an increased metastasis suggesting an inhibitory function of PF4. There were two underlying mechanisms: decreased blood vessel integrity in the premetastatic lungs and increased production of hematopoietic stem/progenitor cells (HSCs) and myeloid derived suppressor cells (MDSCs) in tumor-bearing PF4 KO mice. In cancer patients, PF4 expression levels were negatively correlated with tumor stage and positively correlated with patient survival. Our studies suggest that PF4 is a critical anti-tumor factor in the premetastatic site. Our finding of PF4 function in the tumor host provides new insight to the mechanistic understanding of tumor metastasis.

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with Sjӧgren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.

Interleukin-33 (IL-33) is one of the members of the IL-1 family of cytokines and a ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that is known to affect Th2 inflammatory response with partial effects on Th1 responses. This cytokine is released by epithelial and smooth muscle cells of the airway system during their injury by several environmental stimuli, such as allergens, viruses, helminths, and pollutants. IL-33 is an alarmin that acts as an endogenous danger signal, and it has been known to affect various types of cells, such as mast cells, basophils, eosinophils, T cells, and specific subsets of innate lymphoid cells (ILCs). In recent findings, this cytokine is believed to have a critical role in several types of cancers, such as lung cancer, liver cancer, and head and neck squamous cell cancer. The expression of IL-33/ST2 in cancer tissues shows a close association with tumor growth and tumor progression in several types of cancer, suggesting the IL-33/ST2 pathway as a potential target for therapy.

Decellularized tissue matrices are popular as scaffolding materials for tissue engineering application. However, it is unclear whether interspecies differences in tissue parameters influence the quality of tissue grafts that are engineered using human stem cells. In this study, decellularized cow and human bone scaffolds were compared for engineering bone grafts using human induced pluripotent stem cell-derived mesodermal progenitor cells and despite minor differences in architecture and mass composition, both scaffolds equally support cell viability and tissue mineralization. Decellularized cow bone scaffolds therefore represent a suitable and more affordable alternative for engineering human bone grafts for basic and applied research.

Myeloid-derived suppressor cells (MDSCs) are greatly expanded in cancer patients and tumor-bearing mice. They infiltrate into tumors and modulate the tumor microenvironment. In an effort to identify molecular mediators responsible for expansion and the tumor-promoting function of MDSCs, we discovered CCAAT/enhancer binding protein alpha (C/EBPα) expression was significantly reduced in MDSCs from tumor-bearing mice compared to non-tumor-bearing hosts. Tumor-conditioned medium down-regulated C/EBPα expression, suggesting tumor secreted factors inhibiting the gene expression. Consistent with the function of C/EBPα in regulating the balance between proliferation and growth arrest in hematopoietic progenitors, myeloid lineage specific deletion of C/EBPα resulted in significantly enhanced MDSC proliferation and expansion, as well as an increase of myeloid progenitors and a decrease of mature cells. In addition, deletion of C/EBPα in MDSCs enhanced the pro-angiogenic, immune suppressive and pro-tumorigenic behavior of these cells by upregulating the production of iNOS and arginase, as well as MMP-9 and VEGF. Accordingly, tumors growing in C/EBPα conditional null mice displayed greater MDSC infiltration, increased vascularization and accelerated tumor growth. Taken together, this study reveals dual negative roles of C/EBPα in the expansion as well as pro-angiogenic and immune suppressive functions in MDSCs.

The relative importance of bone marrow-derived cell populations to adult neovascularization is not clear. There are increasing evidences that myeloid cell lineage may play a role in neovascularization1–4. In a previous report, we demonstrated that Gr-1+CD11b+ myeloid cells improve both angiogenesis and vasculogenesis in tumor1. A recent report by Kim et al showed that Muscle-derived Gr1(dim)CD11b(+) myeloid cells enhance neovascularization in an ischemic hind limb model3. Similarly, a study with the same ischemic hindlimb model also verified the enhanced neovascularization properties uniquely associated with proangiogenic cells derived from common myeloid progenitors4. In the present study, we further investigated whether intravenous administration of Gr1+CD11b+ myeloid cells increases neovascularization and improves cardiac function after heart infarction. C57BL/6 mice were purchased from Jakson Lab. All animals received humane care and the study protocols were approved by Vanderbilt Medical University Animal Care and Use Committee. We sorted Gr-1+CD11b+ myeloid cells from the spleens of mice as previously described5. To investigate whether Gr-1+CD11b+ myeloid cells increase angiogenesis in vitro, we cultured Gr-1+CD11b+ myeloid cells with aortic ring 6. At the seventh day, we found that endothelial sprouts in Gr-1+CD11b+ myeloid cell treated group are significantly more than that in no cell treated group (23.2 ± 3.7 versus 13.4 ± 2.4 control, p < 0.01, n = 5 in each group, fig 1A and B), which indicated that Gr-1+CD11b+ myeloid cells increase angiogenesis in vitro. In order to see whether Gr-1+CD11b+ myeloid cells increase angiogenesis in vivo, we performed dorsal window model in mice7. We injected Gr-1+CD11b+ myeloid cells into tail vain of mice to see whether injected Gr-1+CD11b+ myeloid cells home to the site of window chamber. By tracing Gr-1+CD11b+ myeloid cells marked with PKH-26 (Fig 1C-a), we found that the Gr-1+CD11b+ myeloid cells home to the site of the window chamber after intravenous injection (Fig 1C-b). We further found the intravenous injection of Gr-1+CD11b+ myeloid cells obviously increase blood vessel branches at the site of window chamber, compared with saline treated groups, which suggested that the Gr-1+CD11b+ myeloid cells also increase angiogenesis in vivo (Fig 1D). Fig 1 Gr-1+CD11b+ myeloid cells increase angiogenesis in vitro and in vivo (A) Representive endothelial sprouts after 7 days of aortic ring sprouts assay. a, aorta ring culture with no cells; b, aorta ring culture with Gr-1+CD11b+ myeloid cells. Magnification ... Next, we injected Gr-1+CD11b+ myeloid cells via tail vain to see whether Gr-1+CD11b+ myeloid cells improve heart function after heart infarction with a model of LAD ligation in mice8. After 4 weeks of LAD ligation and myeloid cells injection, the cardiac function was evaluated by echocardiography. The measured index indicated that myeloid cells significantly improve heart FS (37.2 ± 7.8% versus 27.3 ± 7.2% control, p < 0.05, n = 5 in each group, Fig 2A and B). The hearts were harvested after 4 weeks, and Masson's trichrome staining was performed9. The results indicated that myeloid cells significantly decrease infarct size after LAD ligation (28.9 ± 7.4% versus 45.8 ± 5.0% control, p < 0.01, n = 5 in each group, Fig 2C and D). We further performed capillary density measurement and tracing myeloid cells marked with PKH-26 in the infarct hearts. The results showed that myeloid cells significantly increase capillary density in the board area of infarction (159.2 ± 13.3 versus 96.8 ± 9.0 control, p < 0.01, n = 5 in each group Fig 2E and F). By tracing the myeloid cells and immunostaining of endothelial cells with anti-CD31 antibody, we found a few Gr-1+CD11b+ myeloid cells incorporate into vasculature (Fig 2G). These indicated Gr-1+CD11b+ myeloid cells improve cardiac function after heart infarction via both angiogenesis and vasculogenesis. Fig 2 Intravenous injection of Gr-1+CD11b+ myeloid cells improves cardiac function and decreases cardiac infarct size after heart infarction via increasing neovascularization. (A) Representive echocardography of heart after 4 weeks of LAD ligation. a, Gr-1+CD11b+ ... In conclusion, the intravenous administration of Gr-1+CD11b+ myeloid cells increases neovascularization and improves cardiac function after heart infarction. The Gr-1+CD11b+ myeloid cells could be used as a potential cell source in the field of cell therapy for ischemic cardiovascular diseases.

Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as K m , V max , K i and K a , which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.—Boutté, A. M., Friedman, D. B., Bogyo, M., Min, Y., Yang, L., Lin, P. C. Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis.

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

IL-32 is a newly discovered protein found in human and certain primates, but absent in rodent. Various reports suggest its role as a proinflammatory mediator. Since vascular endothelium is critical in inflammation, we investigate IL-32 in endothelial cells. We found that the gene is expressed in human endothelial cells and Akt strongly induces its expression. Sequence analysis indicates IL-32 beta as the major isoform in endothelial cells. Surprisingly, we did not detect any secretion of IL-32 beta in human endothelial cells; instead we observed co-localization of IL-32 beta with endoplasmic reticulum, suggesting IL-32 beta is an intracellular protein in these cells. Promoter analysis identified a minimum required region for IL-32 transcription at -0.1 to +0.5 kb around the initially identified transcription start site. We also defined a transcriptional suppressor-binding site at -2.0 to -1.5 kb. Importantly, RNA ligase mediated rapid amplification of cDNA ends in endothelial cells determined the transcription start site at the 328 bp downstream from the original identified site. Finally, we found a positive correlation of IL-32 levels with human breast cancer and glioblastoma multiforme (GBM). These findings improve our understanding of IL-32 in vascular endothelium. IL-32 expression might be valuable as a biomarker for cancer.

Objective— The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. Approach and Results— Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S 473 phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα −/− monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR) −/− mice reconstituted with IKKα −/− hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR −/− mice transplanted with wild-type cells. Conclusions— Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can assess tumor perfusion, microvascular vessel wall permeability and extravascular-extracellular volume fraction. Analysis of DCE-MRI data is usually based on indicator dilution theory that requires knowledge of the concentration of the contrast agent in the blood plasma, the arterial input function (AIF). A method is presented that compares the tissues of interest (TOI) curve shape to that of a reference region (RR), thereby eliminating the need for direct AIF measurement. By assigning literature values for Ktrans (the blood perfusion-vessel permeability product) and v(e) (extravascular-extracellular volume fraction) in a reference tissue, it is possible to extract the Ktrans and v(e) values for a TOI without knowledge of the AIF. The operational RR equation for DCE-MRI analysis is derived, and its sensitivity to noise and incorrect assignment of the RR parameters is tested via simulations. The method is robust at noise levels of 10%, returning accurate (+/-20% in the worst case) and precise (+/-15% in the worst case) values. Errors in the TOI Ktrans and v(e) values scale approximately linearly with the errors in the assigned RR Ktrans and v(e) values. The methodology is then applied to a Lewis Lung Carcinoma mouse tumor model. A slowly enhancing TOI yielded Ktrans=0.039+/-0.002 min-1 and v(e)=0.46+/-0.01, while a rapidly enhancing region yielded Ktrans=0.35+/-0.05 min-1 and v(e)=0.31+/-0.01. Parametric Ktrans and v(e) mappings manifested a tumor periphery with elevated Ktrans (>0.30 min-1) and v(e) (>0.30) values. The main advantage of the RR approach is that it allows for quantitative assessment of tissue properties without having to obtain high temporal resolution images to characterize an AIF. This allows for acquiring images with higher spatial resolution and/or SNR, and therefore, increased ability to probe tissue heterogeneity.

We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage–displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzyme's substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST–anti-Akt1–MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser473 Akt and GSK-3α/β phosphorylation, suggesting that Ser473 is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited in vitro by the anti-Akt1 fusion protein. Furthermore, GST–anti-Akt1–MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST–anti-Akt1–MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both in vitro and in vivo.

We demonstrate a novel tumor-promoting role of myeloid immune suppressor Gr+CD11b+ cells, which are evident in cancer patients and tumor-bearing animals. These cells constitute approximately 5% of total cells in tumors. Tumors coinjected with Gr+CD11b+ cells exhibited increased vascular density, vascular maturation, and decreased necrosis. These immune cells produce high levels of MMP9. Deletion of MMP9 in these cells completely abolishes their tumor-promoting ability. Gr+CD11b+ cells were also found to directly incorporate into tumor endothelium. Consistent with this observation, Gr+CD11b+ cells acquire endothelial cell (EC) properties in tumor microenvironment and proangiogenic culture conditions. Our data provide evidence that Gr+CD11b+ cells of immune origin induced by tumors directly contribute to tumor growth and vascularization by producing MMP9 and differentiating into ECs.

Objective Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium-specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis. Methods Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor α (TNFα)–induced Tie2 and Ang1 expression. Results Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen-induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNFα-induced angiogenesis in a mouse cornea assay. In addition, we observed that TNFα can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNFα up-regulates Tie2 in endothelial cells through nuclear factor κB, and it up-regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes. Conclusion This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNFα to pathologic angiogenesis.

Inflammation is a beneficial response that can remove pathogens, repair injured tissue and restore homeostasis to damaged tissues and organs. However, increasing evidence indicate that chronic inflammation plays a pivotal role in tumor development, as well as progression, metastasis, and resistance to chemotherapy. We will review the current knowledge regarding the contribution of inflammation to epithelial mesenchymal transition. We will also provide some perspectives on the relationship between ER-stress signals and metabolism, and the role of these processes in the development of inflammation.

δ-catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis.

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.

Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr-1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr-1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear-punch injury in diabetic or non-diabetic mice. The Gr-1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr-1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr-1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF-1 axis plays an important role in the homing of Gr-1+CD11b+ myeloid cells to the site of injury. In addition, Gr-1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr-1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing.

Spinal cord injury results in tissue necrosis in and around the lesion site, commonly leading to the formation of a fluid-filled cyst. This pathological end point represents a physical gap that impedes axonal regeneration. To overcome the obstacle of the cavity, we have explored the extent to which axonal substrates can be bioengineered through electrospinning, a process that uses an electrical field to produce fine fibres of synthetic or biological molecules. Recently, we demonstrated the potential of electrospinning to generate an aligned matrix that can influence the directionality and growth of axons. Here, we show that this matrix can be supplemented with nerve growth factor and chondroitinase ABC to provide trophic support and neutralize glial-derived inhibitory proteins. Moreover, we show how air-gap electrospinning can be used to generate a cylindrical matrix that matches the shape of the cord. Upon implantation in a completely transected rat spinal cord, matrices supplemented with NGF and chondroitinase ABC promote significant functional recovery. An examination of these matrices post-implantation shows that electrospun aligned monofilaments induce a more robust cellular infiltration than unaligned monofilaments. Further, a vascular network is generated in these matrices, with some endothelial cells using the electrospun fibres as a growth substrate. The presence of axons within these implanted matrices demonstrates that they facilitate axon regeneration following spinal cord injury. Collectively, these results demonstrate the potential of electrospinning to generate an aligned substrate that can provide trophic support, directional guidance cues and regeneration-inhibitory neutralizing compounds to regenerating axons following spinal cord injury. Copyright © 2016 John WileySons, Ltd.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive function on immune response. In this review, we discuss recent studies about mechanisms of expansion and suppressive function of MDSCs during inflammation, infection and autoimmune diseases, as well as pro-angiogenic and pro-metastatic functions of these cells in tumor development. Further, we focus on novel studies of MDSCs and therapeutic approaches to eliminate these cells in cancer.

Background Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization. Methodology/Principal Findings In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2−/− tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1. Conclusion Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production.

Expansion of Gr-1+/CD11b+ myeloid derived suppressor cells (MDSCs) is governed by the presence of increasingly metastatic, malignant primary tumors. Metastasis, not the primary tumor, is often the cause of mortality. This study sought to fully characterize the MDSC proteome in response to metastatic and non-metastatic mammary tumors using label-free mass spectrometry shotgun proteomics in a mouse model with tumor cell lines, 67NR and 4T1, derived from the same tumor. 67NR cells form only primary mammary tumors, whereas 4T1 cells readily metastasize to the lungs, lymph nodes, and blood. Overall analysis identified a total of 2825 protein groups with a 0.78% false discovery rate. Of the 2814 true identifications, 43 proteins were exclusive to the 67NR group, 153 were exclusive to the 4T1 group, and 2618 were shared. Among the shared cohort, 26 proteins were increased and 31 were decreased in the metastatic 4T1 cohort compared to non-metastatic 67NR controls after filtering. MDSCs selectively express proteins involved in the γ-glutamyl transferase, glutathione synthase pathways, CREB transcription factor signaling, and other pathways involved in platelet aggregation, as well as lipid and amino acid metabolism, in response to highly metastatic 4T1 tumors. Cell cycle regulation dominated protein pathways and ontological groups of the 67NR non-metastatic group. Not only does this study provide a starting point to identify potential biomarkers of metastasis expressed by MDSCs; it identifies critical pathways that are unique to non-metastatic and metastatic conditions. Therapeutic interventions aimed at these pathways in MDSC may offer a new route to control malignancy and metastasis.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFRβ, showing that two PDGF-B protomers clamp PDGFRβ at their dimerization seam. The PDGF-B:PDGFRβ interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFRβ. Calorimetric data also shows that the membrane-proximal homotypic PDGFRα interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.

Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable and are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared with normal and benign breast tumors and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclasts and Tie2 is present in a certain population of these cells, we isolated Tie2+ and Tie2− myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2− compared with Tie2+ CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclasts and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 as a therapeutic target for controlling not only tumor angiogenesis but also osteolytic bone metastasis in breast cancer. Cancer Res; 70(7); 2819–28

Background Inflammation is associated with most diseases, which makes understanding the mechanisms of inflammation vitally important. Methodology/Principal Findings Here, we demonstrate a critical function of interleukin-32β (IL-32β) in vascular inflammation. IL-32β is present in tissues from humans, but is absent in rodents. We found that the gene is highly expressed in endothelial cells. Three isoforms of IL-32, named IL-32α, β, and ε, were cloned from human endothelial cells, with IL-32β being the major isoform. Pro-inflammatory cytokines (TNFα and IL-1β) induced IL-32β expression through NF-κB. Conversely, IL-32β propagated vascular inflammation via induction of vascular cell adhesion molecules and inflammatory cytokines. Accordingly, IL-32β increased adhesion of inflammatory cells to activated endothelial cells, a paramount process in inflammation. These results illustrate a positive feedback regulation that intensifies and prolongs inflammation. Importantly, endothelial/hematopoietic expression of IL-32β in transgenic mice elevated inflammation and worsened sepsis. This was demonstrated by significant elevation of leukocyte infiltration and serum levels of TNFα and IL-1β, increased vascular permeability and lung damage, and accelerated animal death. Together, our results reveal an important function of IL-32 in vascular inflammation and sepsis development. Conclusions/Significance Our results reveal an important function of IL-32 in vascular inflammation and sepsis development.

The transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in many types of cancers and has been implicated in gene expression important for angiogenesis, tumor growth, progression, and metastasis. Here, we show that the NF-kappaB activator, IkappaB kinase-alpha (IKKalpha), but not IKKbeta, promotes endothelial cell motility and tumor angiogenesis. IKKalpha is elevated in tumor vasculature compared with normal endothelium. Overexpression of IKKalpha in endothelial cells promoted cell motility and vascular tubule formation in a three-dimensional culture assay, and conversely, knockdown of IKKalpha in endothelial cells inhibited cell motility, compared with controls. Interestingly, blocking NF-kappaB activation totally abolished IKKalpha-induced angiogenic function. Furthermore, using a tumor and endothelial cell cotransplantation model, we show that overexpression of IKKalpha in endothelial cells significantly increased tumor vascular formation compared with controls, which contributed to increased tumor growth and tumor cell proliferation, and decreased tumor cell apoptosis. Collectively, these findings have identified a new function for IKKalpha through the canonical NF-kappaB pathway in tumor angiogenesis.

Models have been developed for the analysis of dynamic contrast-enhanced MRI (DCE-MRI) data that do not require direct measurements of the arterial input function; such methods are referred to as reference region models. These models typically return estimates of the volume transfer constant (K(trans)) and the extravascular extracellular volume fraction (v(e)). To date such models have assumed a linear relationship between the measured R(1) ( identical with 1/T(1)) and the concentration of contrast agent, a transformation referred to as the fast exchange limit, but this assumption is not valid for all concentrations of an agent. A theory for DCE-MRI reference region models which accounts for water exchange is presented, evaluated in simulations, and applied in tumor-bearing mice. Using reasonable parameter values, simulations show that the assumption of fast exchange can underestimate K(trans) and v(e) by up to 82% and 46%, respectively. By analyzing a large region of interest and a single voxel the new model can return parameters within approximately +/-10% and +/-25%, respectively, of their true values. Analysis of experimental data shows that the new approach returns K(trans) and v(e) values that are up to 90% and 73%, respectively, greater than conventional fast exchange analyses.

Nanotechnology has advanced greatly in recent years and antiangiogenic therapy is becoming a promising approach for cancer treatment. Therefore, a combination of nanotechnology with antiangiogenic therapy should significantly enhance our ability to treat this deadly disease. αvβ3-integrin has often been used to target ‘angiogenically active’ tumor endothelium and many nanoparticles have been developed by combining this molecule with other agents for imaging and/or therapy. Development of improved and multifunctional nanoparticles is in progress for the enhancement of imaging, targeting, delivery and other processes. Modification for clinical use has been challenging. Concerns remain regarding safety, stability and tissue targeting of the particles, yet significant progress has been made. In this review, studies targeting ‘angiogenically active’ tumor endothelium using nanoparticles are discussed. The number of studies using multifunctional nanotechnology in tumor angiogenesis is still limited; however, significant advancement in this area is expected to come in the near future.

Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.

Purpose To test the repeatability of a reference region (RR) model for the analysis of dynamic contrast-enhanced MRI (DCE-MRI) in a mouse model of cancer at high field. Materials and Methods Seven mice were injected with 106 4T1 mammary carcinoma cells and imaged eight to 10 days later on a Varian 7.0T scanner. Two DCE-MRI studies were performed for each mouse (separated by 2.5 hours). The RR model was used to analyze the data, and returned estimates on the perfusion-permeability index (Ktrans) for the RR and the tissue of interest (TOI), as well as the extravascular extracellular volume fraction (ve) for the TOI. Results When the first injection was compared with the second injection, all parameters tested were highly correlated (r2 = 0.90, 0.62, 0.82 for the RR Ktrans, TOI Ktrans, and TOI ve, respectively, with P < 0.001 for all). To observe a statistically significant change (at the 5% level) in a treatment study with seven animals in each group, log10 changes of 0.084 and 0.077 in the tumor Ktrans and ve, respectively, are required. Conclusion If a reliable arterial input function (AIF) is unavailable, the RR model is a reasonable alternative to measuring MRI contrast-agent (CA) kinetics in mouse models of cancer at high field. J. Magn. Reson. Imaging 2006. © 2006 Wiley-Liss, Inc.

Objective In a previous study, we demonstrated that Tie2 regulates angiogenesis in arthritis. The current study was performed to determine whether systemic delivery of a soluble Tie2 receptor (ExTek) using an adenoviral vector (AdExTek) as a Tie2 inhibitor affects arthritis development and progression in an animal model. Methods We used a collagen-induced arthritis (CIA) mouse model to study the outcome of treatment with either AdExTek or a control vector. The onset, incidence, and severity of arthritis were quantified. Immunohistologic analysis of endothelium obtained from the paws was performed. Bone destruction in paws was analyzed using phase-contrast radiography. Results The data showed that systemic delivery of ExTek before disease development significantly inhibited the onset, incidence, and severity of arthritis. When AdExTek was given after disease onset, the severity of disease in mice treated with AdExTek was significantly lower than that in the control group at 35 days postimmunization, which correlated with significantly diminished angiogenesis in mouse paws. Strikingly, AdExTek treatment protected bone from erosion in the CIA model and reduced levels of RANKL. No differences in collagen-specific antibodies were detected between these 2 groups. Conclusion We demonstrated that blocking Tie2 receptor activation inhibits angiogenesis and arthritis development and protects against bone destruction in a CIA mouse model. These findings identify Tie2 as a therapeutic target for arthritis treatment and imply that interventions designed to target the Tie2 pathway could be clinically beneficial.

Inflammation and angiogenesis are associated with pathological disorders. TNF-alpha is a major inflammatory cytokine that also regulates angiogenesis. TNF-alpha has been shown to regulate Tie-2 and angiopoietin (Ang) expression, but the functional significance is less clear. In this study, we showed that TNF-alpha induced a weak angiogenic response in a mouse cornea assay. Systemic overexpression of Ang-1 or Ang-2 dramatically increased corneal angiogenesis induced by TNF-alpha. In the absence of TNF-alpha, neither Ang-1 nor Ang-2 promoted corneal angiogenesis. Low doses (0-25 ng/ml) of TNF-alpha increased vascular branch formation of cultured endothelial cells. Overexpression of Ang-1 or Ang-2 enhanced the effects of TNF-alpha. These data suggest that Tie-2 signaling synergistically amplifies and participates in TNF-alpha-mediated angiogenesis. In addition, high doses (/=50 ng/ml) of TNF-alpha induced apoptosis in endothelial cells, but addition of Ang-1 or Ang-2 significantly reduced cell death. Enhanced endothelial cell survival was correlated with Akt phosphorylation. Collectively, our data reveal dual functional roles of Tie-2: low doses enhance TNF-alpha-induced angiogenesis, and high doses attenuate TNF-alpha-induced cell death. The study provides evidence supporting a role for Tie-2 in inflammatory angiogenesis.

Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To survive and function, transplanted islets must revascularize because islet isolation severs arterial and venous connections; the current paradigm is that islet revascularization originates from the transplant recipient. Because isolated islets retain intraislet endothelial cells, we determined whether these endothelial cells contribute to the revascularization using a murine model with tagged endothelial cells (lacZ knock-in to Flk-1/VEGFR2 gene) and using transplanted human islets. At 3–5 weeks after transplantation beneath the renal capsule, we found that islets were revascularized and that the transplant recipient vasculature indeed contributed to the revascularization process. Using the lacZ-tagged endothelial cell model, we found that intraislet endothelial cells not only survived after transplantation but became a functional part of revascularized islet graft. A similar contribution of intraislet endothelial cells was also seen with human islets transplanted into an immunodeficient mouse model. In the murine model, individual blood vessels within the islet graft consisted of donor or recipient endothelial cells or were a chimera of donor and recipient endothelial cells, indicating that both sources of endothelial cells contribute to the new vasculature. These observations suggest that interventions to activate, amplify, or sustain intraislet endothelial cells before and after transplantation may facilitate islet revascularization, enhance islet survival, and improve islet transplantation.

Abundant data now demonstrate that the growth of new blood vessels, termed angiogenesis, plays both pathological and beneficial roles in human disease. Based on these data, a tremendous effort has been undertaken to understand the molecular mechanisms that drive blood vessel growth in adult tissues. Tie2 recently was identified as a receptor tyrosine kinase expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature, suggesting a role in blood vessel maturation and maintenance. Based on these studies, we undertook a series of studies to probe the function of Tie2 in adult vasculature that will form the focus of this chapter. Consistent with a role in blood vessel growth in adult vasculature, Tie2 was upregulated and activated in the endothelium of rat ovary and in healing rat skin wounds, both areas of active angiogenesis. Moreover, Tie2 was upregulated in the endothelium of vascular "hot spots" in human breast cancer specimens. Surprisingly, Tie2 also was expressed and activated in the endothelium of all normal rat tissues examined, suggesting a role in maintenance of adult vasculature. To determine the functional role of Tie2 in tumor vasculature, a soluble Tie2 extracellular domain (ExTek) was designed that blocked the activation of Tie2 by its activating ligand, angiopoietin 1 (Ang1). Administration of recombinant ExTek protein or an ExTek adenovirus inhibited tumor growth and metastasis in rodent tumor models, demonstrating a functional role for Tie2 in pathological angiogenesis in adult tissues. To begin to understand the endothelial signaling pathways and cellular responses that mediate Tie2 function, we identified signaling molecules that are recruited to the activated, autophosphorylated Tie2 kinase domain. Two of these molecules, SHP2 and GRB2, are part of the pathway upstream of mitogen-activated protein kinase (MAPK) activation, a pathway that may be responsible for morphogenetic effects of Tie2 on endothelial cells. Another signaling molecule, p85, is responsible for recruitment of phosphatidylinositol 3 kinase (PI3-K) and activation of the Akt/PI3-K pathway. Akt/PI3-K has emerged as a critical pathway downstream of Tie2 that is necessary for cell survival effects as well as for chemotaxis, activation of endothelial nitric oxide synthase, and perhaps for anti-inflammatory effects of Tie2 activation. Taken together, these studies and many others demonstrate that the Tie2 pathway has important functions in adult tissues, in both quiescent vasculature and during angiogenesis, and help to validate the Tie2 pathway as a therapeutic target.

Tumor angiogenesis is essential for tumor growth and progression. Therefore, targeting tumor blood vessels is a promising approach for cancer therapy. Angiogenesis, the formation of blood vessels, is a multistep process, and strongly influenced by the microenvironment. There are no in vitro assays that can resemble this dynamic process in vivo. For this reason, animal models and imaging technologies are critical for studying tumor angiogenesis, identifying therapeutic targets as well as validating the targets. Non-invasive molecular imaging in animal models presents an unprecedented opportunity and ability for us to perform repetitive observations and analysis of the biological processes underlying tumor angiogenesis and tumor progression in living animals in real time. As we gain a better understanding of the fundamental molecular nature of cancer, these techniques will be an important adjunct in translating the knowledge into clinical practice. This important information may elucidate how the tumor blood vessels behave and respond to certain treatments and therapies. © 2003 Wiley-Liss, Inc.

Vascular and neuronal networks share a similar branching morphology, and emerging evidence implicates common mechanisms in the formation of both systems. delta-Catenin is considered a neuronal catenin regulating neuron cell-cell adhesion and cell motility. Here, we report expression of delta-catenin in vascular endothelium, and show that deletion of only one allele of delta-catenin is sufficient to impair endothelial cell motility and vascular assembly in vitro and pathological angiogenesis in vivo, thereby inhibiting tumor growth and wound healing. In contrast, deletion of one or both allele of delta-catenin had no effects on hormone-induced physiological angiogenesis in the uterus. Molecular analysis confirmed a gene dosage effect of delta-catenin on Rho GTPase activity. Moreover, we show that inflammatory cytokines, but not angiogenic factors, regulate delta-catenin expression, and the levels of delta-catenin positively correlate to human lung cancers. Collectively, our data suggest that inflammation, commonly associated with disease conditions, induces delta-catenin expression that specifically regulates pathological, and not physiological, angiogenesis. Because only pathological angiogenesis is sensitive to decreased levels of delta-catenin, this may provide a good target for antiangiogenic therapy.

Mounting evidence demonstrates that the formation of new blood vessels, termed angiogenesis, plays critical roles in human disease development and progression. Based on these findings, there has been a tremendous effort to investigate the molecular mechanisms that drive blood vessel growth in adult tissues. Compared to physiological angiogenesis, inflammation is often accompanied with pathological angiogenesis and often is the underlying causes of many diseases such as cancer, arthritis, atherosclerosis, and others. Inflammation induces angiogenesis and reciprocally, angiogenesis facilitate inflammation. A study of the interaction between angiogenesis and inflammation will enhance our understanding of the mechanisms of diseases. It may generate novel approaches for therapy. Tie2 was recently identified as a receptor tyrosine kinase expressed principally on vascular endothelium, making it an attractive molecular target for angiogenic therapy. This review discusses the regulation of Tie2 and its angiopoietin ligand family in inflammation-associated angiogenesis focusing on cancer, arthritis, and atherosclerosis. The complexity of angiogenesis and context-dependent regulation of angiopoietin/Tie2 signaling in angiogenesis requires further studies.