In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many ...Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35's primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.
Cell autonomous behaviors such as migration and orchestration of cell polarity programs are required for physiological tissue formation. Micropatterns are cell-adhesive shapes that confine cell(s) to ...a user defined geometry. This biophysical confinement allows researchers to standardize the cell shape, and in doing so, stereotype organelle and cytoskeletal systems that can have an arbitrary organization. Thus, micropatterning can be a powerful tool in interrogation of polarity programs by enforcing a homogenous cell shape and cytoskeletal organization. A major drawback of this approach is the equipment and reagent costs associated with fabrication. Here, we provide a characterization of a compound called Lipidure (2-Methacryloyloxy ethyl phosphorylcholine) that is up to 40X less expensive than other cell repulsive coating agents. We found that Lipidure is an effective cell-repulsive agent for photolithography-based micropattern fabrication. Our results demonstrate that Lipidure is sensitive to deep UV irradiation for photolithography masking, stable in both benchtop and aqueous environments, non-toxic in prolonged culture, and effective at constraining cell geometry for quantification of cytoskeletal systems.
Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely studied, area of ...endothelial cell regulation is vesicular trafficking, also termed sorting. After moving through the Golgi apparatus, proteins are shuttled to organelles, plugged into membranes, recycled, or degraded depending on the internal and extrinsic cues. A snapshot of these protein-sorting systems can be viewed as a trafficking signature that is not only unique to endothelial tissue, but critically important for blood vessel form and function. In this review, we will cover how vesicular trafficking impacts various aspects of angiogenesis, such as sprouting, lumen formation, vessel stabilization, and secretion, emphasizing the role of Rab GTPase family members and their various effectors.
Objectives
Vesicular trafficking dictates protein localization, functional activity, and half‐life, providing a critically important regulatory step in tissue development; however, there is little ...information detailing endothelial‐specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3‐dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events.
Methods
Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed‐cell microscopy.
Results
Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture.
Conclusions
Our results establish that the fibrin bead sprouting assay is well‐suited for imaging of trafficking events during angiogenic growth.
Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to ...induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation.
The formation of the vascular system is essential for embryonic development and homeostasis. However, transcriptional control of this process is not fully understood. Here we report an evolutionarily ...conserved role for the transcription factor CASZ1 (CASTOR) in blood vessel assembly and morphogenesis. In the absence of CASZ1, Xenopus embryos fail to develop a branched and lumenized vascular system, and CASZ1-depleted human endothelial cells display dramatic alterations in adhesion, morphology, and sprouting. Mechanistically, we show that CASZ1 directly regulates Epidermal Growth Factor-Like Domain 7 (Egfl7). We further demonstrate that defects of CASZ1- or EGFL7-depleted cells are in part due to diminished RhoA expression and impaired focal adhesion localization. Moreover, these abnormal endothelial cell behaviors in CASZ1-depleted cells can be rescued by restoration of Egfl7. Collectively, these studies show that CASZ1 is required to directly regulate an EGFL7/RhoA-mediated pathway to promote vertebrate vascular development.
► CASZ1 is a conserved transcription factor required for vascular patterning ► CASZ1 controls endothelial cell adhesion, contractility, and proliferation ► This control is exerted through the regulation of target genes Egfl7 and miR-126 ► RhoA functions downstream of CASZ1/EGFL7 to mediate endothelial cell behavior
Mutations in CASZ1 are associated with hypertension; however, the vascular function of this transcription factor has been unclear. Charpentier et al. identify Egfl7 as a direct target of CASZ1 in endothelial cells. These factors promote RhoA GTPase signaling to modulate cell behaviors that control vascular patterning and lumen formation.
Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G ...protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm, gene; AM, protein)—a mitogenic peptide hormone required for normal cardiovascular development—is tightly controlled by CXCR7. To this end, Cxcr7−/− mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes that can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7−/− mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development.
Display omitted
•Adrenomedullin (AM) is a biological ligand for CXCR7•CXCR7 scavenges AM peptide and dampens AM-mediated pERK in vitro and in vivo•Dynamic expression of CXCR7 is required for proper lymphangiogenesis•Genetic reduction of AM reverses lymphatic and cardiac hyperplasia of Cxcr7−/− mice
Atypical 7-transmembrane receptors, often called decoy receptors, act as molecular sinks to regulate ligand bioavailability and temper the signaling of canonical GPCR pathways. Here, Klein et al. identify a peptide hormone adrenomedullin as a biological ligand for one such decoy receptor, CXCR7, and discover a role for CXCR7 during lymphangiogenesis.
Abstract
Pericytes are critical for microvascular stability and maintenance, among other important physiological functions, yet their involvement in vessel formation processes remains poorly ...understood. To gain insight into pericyte behaviors during vascular remodeling, we developed two complementary tissue explant models utilizing ‘double reporter’ animals with fluorescently-labeled pericytes and endothelial cells (via Ng2:DsRed and Flk-1:eGFP genes, respectively). Time-lapse confocal imaging of active vessel remodeling within adult connective tissues and embryonic skin revealed a subset of pericytes detaching and migrating away from the vessel wall. Vessel-associated pericytes displayed rapid filopodial sampling near sprouting endothelial cells that emerged from parent vessels to form nascent branches. Pericytes near angiogenic sprouts were also more migratory, initiating persistent and directional movement along newly forming vessels. Pericyte cell divisions coincided more frequently with elongating endothelial sprouts, rather than sprout initiation sites, an observation confirmed with in vivo data from the developing mouse brain. Taken together, these data suggest that (i) pericyte detachment from the vessel wall may represent an important physiological process to enhance endothelial cell plasticity during vascular remodeling, and (ii) pericyte migration and proliferation are highly synchronized with endothelial cell behaviors during the coordinated expansion of a vascular network.
Endocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy ...and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin-mediated and clathrin-independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis.
Primary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently-tagged constructs for live-cell imaging.
We found that Arf6 localized to both filamentous actin and sites of endocytosis in two-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence.
Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation.