Holger Weber

Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.

Angiogenesis is essential in bone fracture healing for restoring blood flow to the fracture site. Vascular endothelial growth factor (VEGF) and its receptor have been implicated in this process. Despite the importance of angiogenesis for the healing processes of damaged bones, the role of VEGF signaling in modulation of osteogenic differentiation in human mesenchymal stem cells has not been investigated in great detail. We examined the expression of VEGF‐A and VEGFR‐1 in human adult mesenchymal stem cells derived from trabecular bone (hTBCs). VEGF‐A was found to be secreted in a differentiation dependent manner during osteogenesis. Transcripts for VEGF‐A were also seen to be elevated during osteogenesis. In addition, transcripts for VEGF‐A and the corresponding receptor VEGFR‐1 were upregulated under hypoxic conditions in undifferentiated hTBCs. To investigate the signaling of VEGF‐A on osteogenesis recombinant hTBCs were generated. High expression of VEGF‐A stimulated mineralization, whereas high expression of sFLT‐1, an antagonist to VEGF‐A, reduced mineralization suggesting that VEGF‐A acts as autocrine factor for osteoblast differentiation. In addition, VEGF‐A secreted by hTBCs promotes sprouting of endothelial cells (HUVE) demonstrating a paracrine role in blood vessel formation. In summary, an in vitro analysis of transgene effects on cellular behavior can be used to predict an effective ex vivo gene therapy.

Spheroid-based engineering of a human vasculature in mice.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Spheroid-based human endothelial cell microvessel formation in vivo

The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versatility of cellular assays with the complexity of readouts of in vivo experimentation, we have developed an endothelial transplantation assay. This assay is based on grafting ex vivo generated EC spheroids (2 d) in a suitable matrix in immunocompromised mice, to give rise to a 3D network of capillaries (20 d). This vasculature connects to the mouse vasculature, is perfused and matures by recruiting mouse mural cells. Here we describe the detailed protocol for this assay, including generation of spheroids, injection into mice, excision and processing of resulting plugs, and quantification by immunohistochemical analysis of the resulting vasculature.

Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro.

Given the need for robust and cost‐efficient in vitro models to study angiogenesis and reproducibly analyze potential pro‐ and antiangiogenic compounds in preclinical studies, we developed a 3‐dimensional in vitro angiogenesis assay that is based on collagen gel‐embedded, size‐defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time‐lapse video microscopy, we show that tip cells lead the formation of capillary‐like and partially lumenized sprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165. Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis‐related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC50 values of individual compounds, the sprouting results were partially affected by the HUVEC passage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid‐based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro‐ and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.—Heiss, M., Hellström, M., Kalén, M., May, T., Weber, H., Hecker, M., Augustin, H. G., Korff, T. Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro. FASEB J. 29, 3076‐3084 (2015). www.fasebj.org

Differential Endothelial Transcriptomics Identifies Semaphorin 3G as a Vascular Class 3 Semaphorin

Objective—To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results—Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Conclusion—Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell–expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

The BTB-Kelch Protein KLEIP Controls Endothelial Migration and Sprouting Angiogenesis

Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1&agr;. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF- and basic fibroblast growth factor–induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF- or basic fibroblast growth factor–mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases.

Gamma-Secretase Inhibitor Treatment Promotes VEGF-A-Driven Blood Vessel Growth and Vascular Leakage but Disrupts Neovascular Perfusion

The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimers disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.

Cell Lines Expressing Recombinant Transmembrane Domain–Activated Receptor Kinases as Tools for Drug Discovery

Many receptor tyrosine kinases (RTKs) represent bona fide drug targets in oncology. Effective compounds are available, but treatment invariably leads to resistance, often due to RTK mutations. The discovery of second-generation inhibitors requires cellular models of resistant RTKs. An approach using artificial transmembrane domains (TMDs) to activate RTKs was explored for the rapid generation of simple, ligand-independent cellular RTK assays, including resistance mutants. The RTKs epidermal growth factor receptor (EGFR), MET, and KIT were chosen in a proof-of-concept study. Their intracellular domains were inserted into a series of expression vectors encoding artificial TMDs, and they were tested for autophosphorylation activity in transient transfection assays. Active constructs could be identified for MET and EGFR, but not for KIT. Rat1 cell pools were generated expressing the MET or EGFR constructs, and their sensitivity to reference tool compounds was compared to that of MKN-45 or A431 cells. A good correlation between natural and recombinant cells led us to build a panel of clinically relevant MET mutant cell pools, based on the wild-type construct, which were then profiled via MET autophosphorylation and soft agar assays. In summary, a platform was established that allows for the rapid generation of cellular models for RTKs and their resistance mutants.

The Potency of Refined Mouse Models: Implications for Clinical Trials

Many promising anti-cancer compounds fail in the clinical phase despite extensive testing in animal models. Reasons for this failure are varied, ranging from inadequate mouse models to dosing schemes that cannot be applied in humans. The apparent shortcomings of exisiting pre-clinical studies suggests a need for improved mouse models. Fur- thermore, the development of an increasing number of targeted drugs requires that biomarkers and/or the in vivo imaging of the tumor or its environment need to be established, ideally before the clinical candidate is selected. In this review, we focus on recent progress in mouse models, emphasizing imaging techniques, biomarker development, the tumor microen- vironment and orthotopic metastatic tumor models. We discuss the application of the latter models to drug testing, making comparisons with classical subcutaneous xenograft models, and also models utilizing transgenic animals. Imaging modali- ties used to detect orthotopically implanted cells in the mouse, e.g. fluorescence or luciferase, which allow relatively high throughput measurements, will be compared to PET, CT, or MRI, which are closer to the clinical application. Examples for a combination of the latter imaging systems with luciferase or fluorescence in the mouse to develop or improve clini- cal imaging will be presented. Finally, the value of biomarkers in mouse models and strategies to identify novel target- specific biomarkers in mouse models will be discussed.