Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when ...cells are allowed to establish cell-cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell-cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.
The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting ...outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC.
Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification.
Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05).
Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.
The angiopoietin (Ang)–Tie ligand–receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous ...diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1–Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel–Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang–Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.
The Tie receptors with their Angiopoietin ligands act as regulators of angiogenesis and vessel maturation. Tie2 exerts its functions through its supposed endothelial-specific expression. Yet, Tie2 is ...also expressed at lower levels by pericytes and it has not been unravelled through which mechanisms pericyte Angiopoietin/Tie signalling affects angiogenesis. Here we show that human and murine pericytes express functional Tie2 receptor. Silencing of Tie2 in pericytes results in a pro-migratory phenotype. Pericyte Tie2 controls sprouting angiogenesis in in vitro sprouting and in vivo spheroid assays. Tie2 downstream signalling in pericytes involves Calpain, Akt and FOXO3A. Ng2-Cre-driven deletion of pericyte-expressed Tie2 in mice transiently delays postnatal retinal angiogenesis. Yet, Tie2 deletion in pericytes results in a pronounced pro-angiogenic effect leading to enhanced tumour growth. Together, the data expand and revise the current concepts on vascular Angiopoietin/Tie signalling and propose a bidirectional, reciprocal EC-pericyte model of Tie2 signalling.
During angiogenesis, anastomosing capillary sprouts align to form complex three-dimensional networks of new blood vessels. Using an endothelial cell spheroid model that was developed to study ...endothelial cell differentiation processes, we have devised a novel collagen gel-based three-dimensional in vitro angiogenesis assay. In this assay, cell number-defined, gel-embedded endothelial cell spheroids act as a cellular delivery device, which serves as a focal starting point for the sprouting of lumenized capillary-like structures that can be induced to form complex anastomosing networks. Formation of capillary anastomoses is associated with tensional remodeling of the collagen matrix and directional sprouting of outgrowing capillaries towards each other. To analyze whether directional sprouting is dependent on cytokine gradients or on endothelial cell-derived tractional forces transduced through the extracellular matrix, we designed a matrix tension generator that enables the application of defined tensional forces on the extracellular matrix. Using this matrix tension generator, causal evidence is presented that tensional forces on a fibrillar extracellular matrix such as type I collagen, but not fibrin, are sufficient to guide directional outgrowth of endothelial cells. RGD peptides but not control RAD peptides disrupted the integrity of sprouting capillary-like structures and induced detachment of outgrowing endothelial cells cultured on top of collagen gels, but did not inhibit primary outgrowth of endothelial cells. The data establish the endothelial cell spheroid-based three-dimensional angiogenesis technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.
Microvessel density (MVD) counting techniques have been widely used to assess the vasculature in tumors. MVD counts assess the presence of blood vessels but do not give an indication of the degree of ...angiogenesis and the functional status of the tumor neovasculature. To analyze angiogenesis and the functional status of the tumor vascular bed, we have quantitated endothelial cell proliferation and the recruitment of pericytes in human tumors glioblastomas (n = 30), renal cell carcinomas (n = 22), colon carcinomas (n = 18), mammary carcinomas (n = 24), lung carcinomas (n = 15), and prostate carcinomas (n = 19). These findings were compared to the physiological angiogenesis in the cyclic bovine ovarian corpus luteum. Tissue sections were examined applying double-labeling immunohistochemical techniques to detect proliferating endothelial cells and to colocalize endothelial cells and pericytes. The following parameters were quantitated: (a) MVD count; (b) proliferating capillary index (PCI); (c) proliferating tumor versus endothelial cell index; and (d) microvessel pericyte coverage index (MPI). Based on endothelial cell proliferation, angiogenesis was found to be present in all tumors with characteristic and significant differences between the tumor types (glioblastomas, PCI = 9.6 +/- 6.1%; renal cell carcinomas, PCI = 9.4 +/- 5.2%; colon carcinomas, PCI = 7.8 +/- 5.2%; mammary carcinomas, PCI = 5.0 +/- 4.8%; lung carcinomas, PCI = 2.6 +/- 2.5%; prostate carcinomas, PCI = 2.0 +/- 1.4%). There was a considerable degree of heterogeneity in the intensity of angiogenesis within each tumor group, as indicated by large standard deviations. Even in the most angiogenic tumors, angiogenesis was found to be 4 to 20 times less intense as compared with the physiological angiogenesis in the growing ovarian corpus rubrum (PCI = 40.6 +/- 6.2%). Varying degrees of pericyte recruitment to the tumor microvasculature were determined in the different tumor types (glioblastomas, MPI = 12.7 +/- 7.9%; renal cell carcinomas, MPI = 17.9 +/- 7.8%; colon carcinomas, MPI = 65.4 +/- 10.5%; mammary carcinomas, MPI = 67.3 +/- 14.2%; lung carcinomas, MPI = 40.8 +/- 14.5%; prostate carcinomas, MPI = 29.6 +/- 9.5%). The data demonstrate distinct quantitative variations in the intensity of angiogenesis in malignant human tumors. Furthermore, the varying degrees of pericyte recruitment indicate differences in the functional status of the tumor vasculature in different tumors that may reflect varying degrees of maturation of the tumor vascular bed.
Essentials Mechanism of thrombin-induced inflammation is not fully understood. Thrombin induced monocyte adhesion and barrier loss require Angiopoietin-2 (Ang-2). Ang-2 mediates vessel leakage and ...monocyte adhesion through SHP-2/p38MAPK pathway. Calcium dependent SHP2/p38MAPK activation regulates Ang-2 expression through a feedback loop.
Background Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. Objective The aim of this study was to determine whether Angiopoietin-2 (Ang-2) has a role, if any, in regulating inflammatory signals initiated by thrombin. Methods Assessment of vascular leakage by Miles assay was performed by intra-dermal injection on the foot paw. Surface levels of intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry. Overexpression, knockdown and phosphorylation of proteins were determined by Western blotting. Results In time-course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein-derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin-induced monocyte adhesion and ICAM-1 expression. In addition, Ang-2(-/-) mice displayed defective vascular leakage when treated with thrombin. Introducing Ang-2 protein in Ang-2(-/-) mice failed to recover a wild-type phenotype. Mechanistically, Ang-2 appears to regulate the thrombin-activated calcium spike that is required for tyrosine phosphatase SHP2 and p38 MAPK activation. Further, down-regulation of SHP2 attenuated both thrombin-induced Ang-2 expression and monocyte adhesion. Down-regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as overexpression of the Gab1 mutant incapable of interacting with SHP2 (YFGab1), inhibited thrombin-mediated effects, including downstream activation of p38 MAPK, which in turn was required for Ang-2 expression. Conclusions The data establish an essential role of the Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage.
ABSTRACT
Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular ...remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3‐dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calciumdependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down‐regulation of PDGF‐B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation;inability to form capillary‐like sprouts in a VEGF‐dependent manner in a 3‐dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang‐2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang‐2‐mediated vessel destabilization resulting in VEGF responsiveness. Ang‐2 on its own was able to stimulate endothelial cells in the absence of Ang‐1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell‐mediated quiescence effects on endothelial cells.—Korff, T., Kimmina, S., Martiny‐Baron, G., Augustin, H. G. Blood vessel maturation in a 3‐dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 15, 447‐457 (2001)
We study the influence of the large-scale atmospheric contribution to the dynamics of the convective boundary layer (CBL) in a situation observed during the Boundary Layer Late Afternoon and Sunset ...Turbulence (BLLAST) field campaign. We employ two modeling approaches, the mixed-layer theory and large-eddy simulation (LES), with a complete data set of surface and upper-air atmospheric observations, to quantify the contributions of the advection of heat and moisture, and subsidence. We find that by only taking surface and entrainment fluxes into account, the boundary-layer height is overestimated by 70%. Constrained by surface and upper-air observations, we infer the large-scale vertical motions and horizontal advection of heat and moisture. Our findings show that subsidence has a clear diurnal pattern. Supported by the presence of a nearby mountain range, this pattern suggests that not only synoptic scales exert their influence on the boundary layer, but also mesoscale circulations. LES results show a satisfactory correspondence of the vertical structure of turbulent variables with observations. We also find that when large-scale advection and subsidence are included in the simulation, the values for turbulent kinetic energy are lower than without these large-scale forcings. We conclude that the prototypical CBL is a valid representation of the boundary-layer dynamics near regions characterized by complex topography and small-scale surface heterogeneity, provided that surface- and large-scale forcings are representative for the local boundary layer.
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