Ralf Erber

Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms

Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR‐2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR‐2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang‐1/Tie2 pathway. In contrast, targeting VEGFR‐2 plus the plate‐let‐derived growth factor receptor (PDGFR)‐β system (PDGFR‐β signaling (by SU6668) rapidly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR‐2 plus PDGFR‐β targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte‐endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti‐angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte‐associated pathways involved in vascular stabilization and maturation exert potent anti‐vascular effects.

Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2

Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (<< 1 mm(3)) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin‐1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non‐neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non‐neoplastic angiogenesis in general. This implies that both neoplastic and non‐neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand‐receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte‐free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia‐induced vascular remodeling using the pericyte reporter X‐LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up‐regulation of angiopoietin‐1 and PDGF‐B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie‐2 function either by soluble Tie‐2 or by a sulindac analog, an inhibitor of Tie‐2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie‐2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Compression-dependent Up-regulation of Ephrin-A2 in PDL Fibroblasts Attenuates Osteogenesis

Members of the ephrin/Eph family have recently been shown to be involved in the regulation of bone homeostasis in a murine model. The activation of the EphB4 receptor on osteoblasts by its ligand ephrin-B2 led to stimulation of osteoblastogenesis and therefore to bone formation. The activation of ephrin-A2-EphA2 signaling on osteoblasts inhibited the activation of osteoblast-specific gene expression, leading to bone resorption. Fibroblasts within the periodontal ligament periodontal ligament may be one of the first responders to orthodontic forces. Periodontal ligament fibroblasts (PDLF) are mechanoresponsive. Members of the ephrin/Eph family might link mechanical forces received by PDLF with the regulation of osteoblastogenesis on osteoblasts of the alveolar bone. To study whether ephrin-A2 is modulated upon compression, we subjected human primary PDLF to static compressive forces (30.3 g/cm2). Static compressive forces significantly induced the expression of ephrin-A2, while the expression of ephrin-B2 was significantly down-regulated. Moreover, osteoblasts of the alveolar bone stimulated with ephrin-A2 in vitro significantly suppressed their osteoblastogenic gene expression (RUNX2, ALPL) and decreased signs of osteoblastic differentiation, as demonstrated by a significantly reduced ALP activity. Together, these findings establish a role for this ligand/receptor system linking mechanical forces with the regulation of osteogenesis during orthodontic tooth movement.

Strain-dependent Up-regulation of Ephrin-B2 Protein in Periodontal Ligament Fibroblasts Contributes to Osteogenesis during Tooth Movement

Background: Regulation of bone remodeling during orthodontic tooth movement. Results: Tensile stress induces ephrin-B2 expression in PDL fibroblasts, and ephrin-B2-EphB4 interactions induce osteoblastogenesis in osteoblasts of the alveolar bone. Conclusion: Ephrin-B2-EphB4 signaling between PDLF and osteoblasts of the alveolar bone might contribute to bone remodeling during orthodontic tooth movement. Significance: Understanding the regulation of bone remodeling during orthodontic tooth movement is a prerequisite for pharmacological interventions. During orthodontic tooth movement, the application of adequate orthodontic forces allows teeth to be moved through the alveolar bone. These forces are transmitted through the periodontal ligaments (PDL) to the supporting alveolar bone and lead to deposition or resorption of bone, depending on whether the tissues are exposed to a tensile or compressive mechanical strain. Fibroblasts within the PDL (PDLF) are considered to be mechanoresponsive. The transduction mechanisms from mechanical loading of the PDLF to the initiation of bone remodeling are not clearly understood. Recently, members of the ephrin/Eph family have been shown to be involved in the regulation of bone homeostasis. For the first time, we demonstrate that PDLF exposed to tensile strain induce the expression of ephrin-B2 via a FAK-, Ras-, ERK1/2-, and SP1-dependent pathway. Osteoblasts of the alveolar bone stimulated with ephrin-B2 increased their osteoblastogenic gene expression and showed functional signs of osteoblastic differentiation. In a physiological setting, ephrin-B2-EphB4 signaling between PDLF and osteoblasts of the alveolar bone might contribute to osteogenesis at tension sites during orthodontic tooth movement.

In vitro labeling of glioma cells with gadofluorine M enhances T1 visibility without affecting glioma cell growth or motility

Gadofluorine is a novel macrocyclic, amphiphilic gadolinium‐based contrast agent. We found that malignant glioma cells could be labeled in vitro using Gadofluorine without the need for transfection agents or any other additional means. Labeling with Gadofluorine enhanced the visualization of glioma cells in T1‐weighted sequences, even if the cells had been cultured in medium without Gadofluorine over several days. The intracellular uptake of Gadofluorine was measured and the loss of relevant amounts of Gadofluorine into the cell culture medium was ruled out by MRI. Confocal laser fluorescence microscopy revealed Cy‐5‐labeled Gadofluorine in the perinuclear cytoplasmic region, but neither within the nucleus nor bound to the cell membrane. Adverse effects of cellular Gadofluorine uptake were ruled out by proliferation and migration assays. Finally, in vivo analyses provided good visibility of labeled glioma cells in T1‐weighted sequences after intracerebral injection in mice for more than 2 weeks. We thus conclude that Gadofluorine can easily be used to label glioma cells in vitro without affecting glioma cell biology. Gadofluorine provides an interesting alternative for cellular labeling if iron oxide particles are incorporated insufficiently by target cells or if the vicinity of susceptibility artifacts prohibits the use of signal‐decreasing contrast agents. Magn Reson Med 59:1014–1020, 2008.

Human primary cementoblasts respond to combined IL-1β stimulation and compression with an impaired BSP and CEMP-1 expression.

Cementoblasts are cells that produce, secrete and direct the production of cementum. Resorption lacunae occur in over 90% of teeth at the compression side of the periodontal ligament and might result in an irreversible loss of the original root length. We isolated and cultivated human primary cementoblasts and investigated their expression pattern concerning markers of the osteoblastogenic differentiation (RUNX2, OCN, ALP, and BSP) and CEMP-1. Compared to osteoblasts, cementoblasts displayed an expression pattern comparable to osteoblasts in an early stage of osteoblastogenic differentiation. Next, the human primary cementoblasts were stimulated with IL-1β (1 and 10ng/ml) for 24 and 96h and subsequently subjected to compressive forces (30.3g/cm(2)) for 1 and 6h. Our in vitro data demonstrated that BSP and CEMP-1 expression significantly decreased when stimulation was accompanied by compression, while compression or stimulation alone led to increased levels of BSP and decreased levels of CEMP-1. We concluded that human primary cementoblasts subjected to compression and IL-1β stimulation impeded BSP and CEMP-1 expression, proteins that are associated with cementogenesis.

Microvascular biodistribution of L19-SIP in angiogenesis targeting strategies

INTRODUCTION Various strategies using L19-mediated fibronectin targeting have become useful clinical tools in anti-tumour therapy and diagnostics. The aim of our study was to characterise the microvascular biodistribution and binding process during tumour angiogenesis and after anti-angiogenic therapy. MATERIALS AND METHODS SF126 glioma and F9 teratocarcinoma cells were implanted into dorsal skin fold chambers (SF126: n = 4; F9: n = 6). Using fluorescence and confocal intravital microscopy the biodistribution process was assessed at t = 0 h, t = 4 h and t = 24 h after intravenous application of Cy3-L19-SIP. Sunitinib treatment was applied for six days and microscopy was performed 2 and 6 days after treatment initiation. Analysed parameters included: vascular and interstitial binding, preferential binding sites of L19-SIP, microvascular blood flow rate, microvascular permeability. Histological analysis included CD31 and DAPI. RESULTS L19-SIP showed a specific and time-dependent neovascular binding with a secondary extravasation process reaching optimal vascular/interstitial binding ratio 4 hours after iv administration (F9: L19-SIP: vascular binding: 74.6 ± 14.5; interstitial binding: 46.8 ± 12.1; control vascular: 22,2 ± 16.6). Angiogenic sprouts were preferred binding sites (F9: L19-SIP: 188 ± 15.5; RTV: 90.6 ± 13.5). Anti-angiogenic therapy increased microvascular hemodynamics (SF126: Su: 106.6 ± 13.3 μl/sec; Untreated: 19.7 ± 9.1 μl/sec) and induced increased L19-SIP accumulation (SF 126: t24; Su: 92.6 ± 2.7; Untreated: 71.9 ± 5.9) in therapy resistant tumour vessels. CONCLUSION L19-SIP shows a time and blood-flow dependent microvascular biodistribution process with angiogenic sprouts as preferential binding sites followed by secondary extravasation of the antibody. Microvascular biodistribution is enhanced in anti-angiogenic-therapy resistant tumour vessels.

Compression Induces Ephrin-A2 in PDL Fibroblasts via c-fos

Ephrin-A2–EphA2 and ephrin-B2–EphB4 interactions have been implicated in the regulation of bone remodeling. We previously demonstrated a potential role for members of the Eph-ephrin family of receptor tyrosine kinases for bone remodeling during orthodontic tooth movement: compression-dependent upregulation of ephrin-A2 in fibroblasts of the periodontal ligament (PDL) attenuated osteogenesis in osteoblasts of the alveolar bone. However, factors affecting the regulation of ephrin-A2 expression upon the application of compressive forces remained unclear. Here, we report a mechano-dependent pathway of ephrin-A2 induction in PDL fibroblasts (PDLFs) involving extracellular signal–regulated kinases (ERK) 1/2 and c-fos. PDLF subjected to compressive forces (30.3 g/cm2) upregulated c-fos and ephrin-A2 mRNA and protein expression and displayed increased ERK1/2 phosphorylation. Inhibition of the MAP kinase kinase (MEK)/ERK1/2 pathway using the specific MEK inhibitor U0126 significantly reduced ephrin-A2 messenger RNA upregulation upon compression. Silencing of c-fos using a small interfering RNA approach led to a significant inhibition of ephrin-A2 induction upon the application of compressive forces. Interestingly, ephrin-A2 stimulation of PDLF induced c-fos expression and led also to the induction of ephrin-A2 expression. Using a reporter gene construct in murine 3T3 cells, we found that ephrin-A2 was able to stimulate serum response element (SRE)–dependent luciferase activity. As the regulation of c-fos is SRE dependent, ephrin-A2 might induce c-fos via SRE activation. Taken together, we provide evidence for an ERK1/2- and c-fos–dependent regulation of ephrin-A2 in compressed PDLF and suggest a novel pathway for ephrin-A2 induction emanating from ephrin-A2 itself. We showed previously that ephrin-A2 at compression sites might contribute to tooth movement by inhibiting osteogenic differentiation. The regulatory pathway of ephrin-A2 induction during tooth movement identified in this study might be accessible for pharmacological interventions.