Browsing by Subject "Angiogenesis"

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

Pathological angiogenesis is a widespread biological phenomenon that influences the progression of various diseases, including autoimmune conditions, cancers, and microbial infections. One infection in particular, tuberculosis, is associated with the induction of a potent pro-angiogenic signaling cascade that facilitates bacterial growth and accelerates disease progression. A synthesis of early studies on bacterial factors that drive host angiogenesis with modern genetic findings identified the mycobacterial glycolipid trehalose 6-6'-dimycolate (TDM) as a critical factor driving vascular endothelial growth factor (VEGFA) production and angiogenesis during mycobacterial infection. Despite these recent findings, many of the underlying host response mechanisms remain unknown. The introductory chapter will serve to introduce the reader to the major concepts addressed in this work: Mycobacterium tuberculosis and the disease it causes, the role of macrophages in health and disease, the function of pattern recognition receptors in detecting microbial ligands, the specific downstream intracellular signaling pathway of interest for this work (mediated by the transcription factor, nuclear factor of activated T cells, NFAT), the contributions of angiogenesis to diverse contexts and pathologies, and the promise of host-directed therapies to overcome challenges associated with traditional treatment approaches in infectious disease. Chapter 2 describes the new and existing methodological approaches that were required to complete this work. This work utilizes the zebrafish-Mycobacterium marinum model of tuberculosis infection to facilitate in depth in vivo observation and quantitation of these phenomena. Using this model in tandem with human macrophage cell culture, I was able to model major aspects of the host-pathogen interface, enabling me to identify a critical role for a macrophage-C-type lectin receptor-NFATC2-VEGFA signaling axis required for the angiogenic response to mycobacterial infection and TDM, findings that comprise the core of this work and are detailed at length in Chapter 3. The analysis of the large amounts of data generated in this work required creative approaches to data processing and analysis. To this end, I have developed a set of novel processing modalities in Python and R that are capable of the rapid and reproducible processing of images as well as certain aspects of automated data collection therefrom. These macros, many written for the FIJI/ImageJ programming environment, serve as the infrastructure on which the rest of this work has been built. These will be detailed in Chapter 4. Finally, this body of work leaves many questions as yet unanswered. While it is clear that NFAT signaling is required for VEGFA production, the precise mechanism by which this may work is unclear and could be mediated by either direct DNA binding or indirect activation or cooperative binding with some other transcriptional activator. There also exist a variety of other potential NFAT- and angiogenesis-related phenotypes worthy of exploring using the tools and approaches I have developed. It is my hope that the findings herein stimulate further study on the contributions of NFAT signaling to the host immune response to mycobacterial infection and evaluation of the potential of NFAT inhibition as host-directed therapy to tuberculosis.

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.

Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.

The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Efforts to create a reliable, long–term implantable glucose sensor have been stymied by the effects of the foreign body response and wound healing that introduce delayed response times as well as unpredictable sensor performance. Loss of vascularization from fibrotic encapsulation around implanted sensors is purported as a key contributor to sensor failure, as glucose and oxygen transport to the sensor becomes impeded. Improving sensor performance by increasing angiogenesis and/or reducing capsule thickness using tissue-modifying textured coatings is attractive because texturing is not dependent upon a depletable drug reservoir. A significant range of materials and pore sizes are capable of promoting angiogenesis and reducing capsule thickness, provided pores have open-architecture with dimensions sufficiently large enough to allow inflammatory cell infiltration.

Poly–L–lactic acid was gas foamed/salt leached with ammonium bicarbonate to produce porous coatings for Medtronic MiniMed SOF–sensor glucose sensors. Coating properties included 30μm pore diameters, 90% porosity, and 50μm wall thickness. Cytotoxicity, degradation, and sensor response time studies were performed to ensure the porous coatings were non–toxic and negligibly retarded glucose diffusion prior to in vivo testing. Histology was used to evaluate angiogenesis and collagen deposition adjacent to porous coated and bare (i.e. smooth, uncoated) non–functional sensor strips after three weeks in the rat dorsal subcutis. Functional Medtronic glucose sensors, with and without porous coatings, were percutaneously implanted in the rat dorsum to assess if the angiogenic–inducing properties observed around the non–functional porous coated sensor strips translated into stable, non–attenuated sensor signals over two and three weeks. MiniLinkTM transmitters were attached to the rats, permitting continuous glucose monitoring. Vessel counts and collagen deposition adjacent to sensors were determined from histological analysis. A one–sided dorsal window model was developed to further evaluate the interplay between vascularization and sensor performance Sensors were inserted beneath the windows, allowing visualization of microvascular changes adjacent to sensor surfaces, with simultaneous evaluation of how vascular changes impacted interstitial glucose monitoring.

Porous coating did have angiogenic–inducing effects on the surrounding tissue. When fully implanted in the rat dorsum, sensor strips with porous coatings induced three–fold more vessels within 100μm2 of the sensor strip surface after three weeks and two-fold more cumulative vessel lengths within 1mm2 after two weeks, compared to bare surfaces. In contrast, when percutaneously implanted in the rat dorsum, porous coated and bare sensors were equally highly vascularized, with two–fold more vessels than fully implanted bare sensors.

Despite increased angiogenesis adjacent to percutaneous sensors, sensor signal attenuation occurred over 14 days, suggesting that angiogenesis plays a secondary role in maintaining sensor function. Percutaneously implanted porous coated sensors had greater reductions in baseline current (20 to 50+%) over two weeks than bare sensors (10 to 30%). Mechanical stresses imposed by percutaneous tethering may override the beneficial effects of porous coatings. Furthermore, integration of the porous coating with the surrounding tissue may have increased tissue tearing at the porous coating–tissue, increasing inflammation and collagen deposition resulting in greater signal attenuation compared with bare sensors. Future investigations of the role mechanical irritation has on wound healing around percutaneous glucose sensors are warranted.

A functioning vasculature is critical for the supply of nutrients to other systems as well as a host of physiologic and pathologic processes. Vascular development and maintenance are tightly regulated by a number of signaling processes, among which the Tie proteins are two functioning receptors. Although they have been shown to exhibit essential roles in endothelial cell sprouting and quiescence, the mechanistic details of Tie interactions and the effects of their associations with the Angiopoietins have not been elucidated. Studies in this thesis investigated the effects of Ang2 on Tie1 activation, signaling, and cellular responses within the context of both native and immortalized endothelial cells. Additionally, we investigated the role of Ang2 in the cellular reorganization and subsequent downregulation of Tie1. We observed that Ang2, but not Ang1, induces phosphorylation of Tie1 in endothelial cells and that the extracellular domain of Tie2 is required for Ang2-mediated activation of Tie1. Furthermore, we demonstrated that Tie1 activation leads to signaling through the Akt axis, and the consequent stimulation of anti-apoptotic and pro-proliferative cellular effects. Additionally, we demonstrated that Ang2 induces a concentration and time dependent downregulation of Tie1, and that Tie2's role in the process appears to be recruitment of the ligand to the multimeric Tie complexes. Interestingly, although Ang2 stimulation is necessary, we demonstrated that Ang2 activation of Tie1 receptor complexes is not required for ligand induced downregulation of the receptor. Finally, we characterized the modulatory role of Tie1 with regards to Angiopoietin signaling through Tie2, and observed that removal of Tie1 from the surface of endothelial cells induces Ang2 activation of Tie2 leading to increases in cell survival signaling cascades. Taken together, these data shed new light on Angiopoietin signaling through the Tie receptors, further characterize the interactions between Tie1 and Tie2, suggest novel forms of endothelial cell regulation within developing and mature vasculature, and may have implications in signaling within a host of physiologic and pathologic states.

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its activity as a kinase. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1-mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

The Transforming Growth Factor-β (TGF-β) superfamily of ligands and receptors play critical roles in angiogenesis, evidenced by their essential role in physiologic angiogenesis and their perturbation in pathologic angiogenesis. In development, loss or mutation of Activin receptor-Like Kinase 1 (ALK1), an endothelium- specific TGF-β superfamily receptor kinase, leads to disordered angiogenesis and results in the hereditary vascular disease, Hereditary Hemorrhagic Telangiectasia (HHT). A co-receptor in the TGF-β superfamily, the type III Transforming Growth Factor-β Receptor (TβRIII), is known to be important for the vasculature, with genetic deletion of TβRIII resulting in embryonic lethality in mice characterized by severe defects in cardiac and hepatic vasculogenesis. Despite these observations, how TβRIII regulates the vasculature remains unknown. The aim of this study is to elucidate how TβRIII regulates ALK1 signaling and endothelial behavior.

Here, we establish that vascular endothelial cells express relatively high levels of TβRIII, alongside the canonical endothelium-restricted co-receptor, endoglin. Moreover, using biophysical and biochemical techniques, we have established that TβRIII forms stable complexes with ALK1. Through genetic knockout approaches using CRISPR, we demonstrate that loss of TβRIII impairs ALK1-induced Smad phosphorylation as well as endothelial angiogenic potential, including the ability to form capillary tubes.

We have uncovered a novel TβRIII/ALK1 interaction, the role of TβRIII in ALK1-mediated signaling and TβRIII’s role in functional endothelial biology. Understanding TβRIII’s function in the developing endothelium may lead to the development of innovative pharmacological treatments aimed at normalizing angiogenesis and improve our ability to anticipate the potential adverse effects of therapies targeting TGF-β superfamily receptors.