Release of endothelial cell associated VEGFR2 during TGF-ß modulated angiogenesis in vitro.

BACKGROUND: Sprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-ß) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-ß1 under low serum conditions. RESULTS: TGF-ß1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-ß co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-ß1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes. CONCLUSIONS: Taken together, our results suggest that endothelial cells exposed to TGF-ß1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Ex Vivo Mouse Aortic Ring Angiogenesis Assay.

The ex vivo aortic ring assay is one of the most widely used protocols to study sprouting angiogenesis. It is a highly adaptable method that can be utilized to investigate the effects of different growth factors, small-molecule drugs, and genetic modifications on vascular sprouting in a physiologically relevant setting. In this chapter we describe a simple and optimized protocol for investigating vascular sprouting in the mouse aortic ring model. The protocol describes the harvesting and embedding of the aortic rings in a collagen matrix, treatment of the rings with agents of interest, and the visualization and quantification of the vascular sprouts.

Filamin A pre-mRNA editing modulates vascularization and tumor growth.

Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are interpreted as guanosines by the translational machinery. Consequently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the consequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A editing critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly by altering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its interaction with the extracellular matrix. We therefore identify filamin A editing as a critical component for angiogenesis, tumor growth, and metastasis formation.

Recapitulating the Angiogenic Switch in a Hydrogel-Based 3D In Vitro Tumor-Stroma Model.

To ensure nutrient and oxygen supply, tumors beyond a size of 1-2 mm3 need a connection to the vascular system. Thus, tumor cells modify physiological tissue homeostasis by secreting inflammatory and angiogenic cytokines. This leads to the activation of the tumor microenvironment and the turning of the angiogenic switch, resulting in tumor vascularization and growth. To inhibit tumor growth by developing efficient anti-angiogenic therapies, an in depth understanding of the molecular mechanism initiating angiogenesis is essential. Yet so far, predominantly 2D cell cultures or animal models have been used to clarify the interactions within the tumor stroma, resulting in poor transferability of the data obtained to the in vivo situation. Consequently, there is an abundant need for complex, humanized, 3D models in vitro. We established a dextran-hydrogel-based 3D organotypic in vitro model containing microtumor spheroids, macrophages, neutrophils, fibroblasts and endothelial cells, allowing for the analysis of tumor-stroma interactions in a controlled and modifiable environment. During the cultivation period of 21 days, the microtumor spheroids in the model grew in size and endothelial cells formed elongated tubular structures resembling capillary vessels, that appeared to extend towards the tumor spheroids. The tubular structures exhibited complex bifurcations and expanded without adding external angiogenic factors such as VEGF to the culture. To allow high-throughput screening of therapeutic candidates, the 3D cell culture model was successfully miniaturized to a 96-well format, while still maintaining the same level of tumor spheroid growth and vascular sprouting. The quantification of VEGF in the conditioned medium of these cultures showed a continuous increase during the cultivation period, suggesting the contribution of endogenous VEGF to the induction of the angiogenic switch and vascular sprouting. Thus, this model is highly suitable as a testing platform for novel anticancer therapeutics targeting the tumor as well as the vascular compartment.

FOXF1 Mediates Endothelial Progenitor Functions and Regulates Vascular Sprouting.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

Knockdown of HIF-1α impairs post-ischemic vascular reconstruction in the brain via deficient homing and sprouting bmEPCs.

Although the critical role of hypoxia inducible factor-1α (HIF-1α) in cerebral neovascularization after stroke has been well characterized, the details regarding the regulation of endothelial progenitor cell (EPC)-dependent neovascularization by HIF-1α are not completely understood. Using lentiviral shRNA to knockdown HIF-1α, we showed that HIF-1α plays a central role in bone marrow-derived EPC (bmEPC) homing and sprouting in the post-acute stage of ischemic Sprague Dawley (SD) rat brains. First, knockdown of HIF-1α decreased the homing of both endogenous and exogenous bmEPCs to the ischemic brain. Additionally, the knockdown impaired the incorporation and sprouting of bmEPCs in the ischemic brain. In vitro, knockdown of HIF-1α inhibited the spheroid sprouting and tube formation of bmEPCs. Mechanically, the HIF-1α-dependent recruitment of bmEPCs to the ischemic brain was relative to the CXCL12/CXCR4 axis and HMGB1, which were relative to astrocytes. In addition, the loss of HIF-1α resulted in deficient expression levels of VEGF-A, Flk-1, NRP1, and Dll4 in the ischemic brains, bmEPCs, and astrocytes. These findings suggested that HIF-1α implicates in bmEPC homing via CXCL12/CXCR4 and HMGB1 and that it promotes bmEPC sprouting via VEGF-A/flk1-NRP1/Dll4.

Inhibition of the transcriptional repressor complex Bcl-6/BCoR induces endothelial sprouting but does not promote tumor growth.

The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors.

Therapeutic angiogenesis: angiogenic growth factors for ischemic heart disease.

Stem cells encode vascular endothelial growth factors (VEGFs), fibroblastic growth factors (FGFs), stem cell factor, stromal cell-derived factor, platelet growth factor and angiopoietin that can contribute to myocardial vascularization. VEGFs and FGFs are the most investigated growth factors. VEGFs regulate angiogenesis and vasculogenesis. FGFs stimulate vessel cell proliferation and differentiation and are regulators of endothelial cell migration, proliferation and survival. Clinical trials of VEGF or FGF for myocardial angiogenesis have produced disparate results. The efficacy of therapeutic angiogenesis can be improved by: (1) identifying the most optimal patients; (2) increased knowledge of angiogenic factor pharmacokinetics and proper dose; (3) prolonging contact of angiogenic factors with the myocardium; (4) increasing the efficiency of VEGF or FGF gene transduction; and (5) utilizing PET or MRI to measure myocardial perfusion and perfusion reserve.

Podosomes and invadopodia: tools to breach vascular basement membrane.

The vascular basement membrane (BM) is a thin and dense cross-linked extracellular matrix layer that covers and protects blood vessels. Understanding how cells cross the physical barrier of the vascular BM will provide greater insight into the potentially critical role of vascular BM breaching in cancer extravasation, leukocyte trafficking and angiogenic sprouting. In the last year, new evidence has mechanistically linked the breaching of vascular BM with the formation of specific cellular micro-domains known as podosomes and invadopodia. These structures are specialized cell-matrix contacts with an inherent ability to degrade the extracellular matrix. Specifically, the formation of podosomes or invadopodia was shown as an important step in vascular sprouting and tumor cell extravasation, respectively. Here, we review and comment on these recent findings and explore the functions of podosomes and invadopodia within the context of pathological processes such as tumor dissemination and tumor angiogenesis.