We developed an agent-based model of endothelial sprout initiations based on time-lapse confocal imaging in vitro that outperforms Monte Carlo simulations, suggesting that sprout location and frequency are not purely stochastic behaviors.
Blood vessel formation is essential for vertebrate development and is primarily achieved by angiogenesis - endothelial cell sprouting from pre-existing vessels. Vessel networks expand when sprouts form new connections, a process whose regulation is poorly understood. Here, we show that vessel anastomosis is spatially regulated by Flt1 (VEGFR1), a VEGFA receptor that acts as a decoy receptor. In vivo, expanding vessel networks favor interactions with Flt1 mutant mouse endothelial cells. Live imaging in human endothelial cells in vitro revealed that stable connections are preceded by transient contacts from extending sprouts, suggesting sampling of potential target sites, and lowered Flt1 levels reduced transient contacts and increased VEGFA signaling. Endothelial cells at target sites with reduced Flt1 and/or elevated protrusive activity were more likely to form stable connections with incoming sprouts. Target cells with reduced membrane-localized Flt1 (mFlt1), but not soluble Flt1, recapitulated the bias towards stable connections, suggesting that relative mFlt1 expression spatially influences the selection of stable connections. Thus, sprout anastomosis parameters are regulated by VEGFA signaling, and stable connections are spatially regulated by endothelial cell-intrinsic modulation of mFlt1, suggesting new ways to manipulate vessel network formation.
Soluble VEGF receptor 1 (sFlt1) modulates the guidance cues provided to sprouting blood vessels by vascular endothelial growth factor (VEGF). Based on experimental observations in developing vasculature, we hypothesize that a local reduction in sFlt1 expression can control angiogenesis by increasing local VEGF receptor 2 (Flk1) signaling. To quantify sFlt1 modulation of VEGF signaling, we developed an experimentally-based computational model describing spatial transport of VEGF and its receptors. Our model represents the local environment of a single blood vessel and nearby tissue: parenchymal cells secrete VEGF, which diffuses and binds extracellular matrix and sFlt1; VEGF binds endothelial cells via membrane-bound Flt1 and Flk1; and endothelial cells secrete sFlt1. The model allows us to quantify the relative contributions of VEGF sequestration by sFlt1 in the interstitial space and sFlt1 heterodimerization with surface receptors, both of which can decrease VEGF receptor signaling. Our simulations predict that when a sprout-leading tip cell secretes less sFlt1 than neighboring cells, local sFlt1 sequestration of VEGF is decreased, thus increasing VEGF-Flk1 levels on the surface of the low-sFlt1 secreting tip cell. We also show that the proximity of neighboring tip cells may alter VEGF receptor binding. Supported by NIH R00HL093219 (FMG), R01HL43174 (VLB), F32HL95359, T32CA9156 & AHA 0826082E (JCC)
Define a role for perivascular cells during developmental retinal angiogenesis in the context of EC Notch1-DLL4 signaling at the multicellular network level.The retinal vasculature is highly sensitive to growth factor-mediated intercellular signaling. Although EC signaling has been explored in detail, it remains unclear how PC function to modulate these signals that lead to a diverse set of vascular network patterns in health and disease. We have developed an ABM of retinal angiogenesis that incorporates both ECs and PCs to investigate the formation of vascular network patterns as a function of pericyte coverage. We use our model to test the hypothesis that PC modulate Notch1-DLL4 signaling in endothelial cell-endothelial cell interactions.Agent-based model (ABM) simulations that include PCs more accurately predict experimentally observed vascular network morphologies than simulations that lack PCs, suggesting that PCs may influence sprouting behaviors through physical blockade of endothelial intercellular connections.This study supports a role for PCs as a physical buffer to signal propagation during vascular network formation-a barrier that may be important for generating healthy microvascular network patterns.
Abstract Therapeutic strategies in which recombinant growth factors are injected to stimulate arteriogenesis in patients suffering from occlusive vascular disease stand to benefit from improved targeting, less invasiveness, better growth‐factor stability, and more sustained growth‐factor release. A microbubble contrast‐agent‐based system facilitates nanoparticle deposition in tissues that are targeted by 1‐MHz ultrasound. This system can then be used to deliver poly( D , L ‐lactic‐co‐glycolic acid) nanoparticles containing fibroblast growth factor‐2 to mouse adductor muscles in a model of hind‐limb arterial insufficiency. Two weeks after treatment, significant increases in both the caliber and total number of collateral arterioles are observed, indicating that the delivery of nanoparticles bearing fibroblast growth factor‐2 by ultrasonic microbubble destruction may represent an effective and minimally invasive strategy for the targeted stimulation of therapeutic arteriogenesis.
Capillaries within the microcirculation are essential for oxygen delivery and nutrient/waste exchange, among other critical functions. Microvascular bioengineering approaches have sought to recapitulate many key features of these capillary networks, with an increasing appreciation for the necessity of incorporating vascular pericytes. Here, we briefly review established and more recent insights into important aspects of pericyte identification and function within the microvasculature. We then consider the importance of including vascular pericytes in various bioengineered microvessel platforms including 3D culturing and microfluidic systems. We also discuss how vascular pericytes are a vital component in the construction of computational models that simulate microcirculation phenomena including angiogenesis, microvascular biomechanics, and kinetics of exchange across the vessel wall. In reviewing these topics, we highlight the notion that incorporating pericytes into microvascular bioengineering applications will increase their utility and accelerate the translation of basic discoveries to clinical solutions for vascular-related pathologies.
Summary Pericytes (PCs), cells that extend along capillaries to contribute stability and other critical functions to established vasculature, are attracting attention from various fields involving vascular-related pathologies. Here, we demonstrate primary evidence of PC communication with endothelial cells (ECs) prior to tube coalescence. Observations of apparent PCs during early embryogenesis urged development of a mouse embryonic stem cell line (DR-ESCs), enabling unique dual-reporter investigations into earliest PC-EC interactions. Live imaging of differentiating DR-ESCs corroborated emergence of a PC lineage, which preceded EC differentiation, and further revealed highly dynamic PC-EC interactions during coordinated vessel formation. We show direct PC-EC communication via cell microinjection and dye-transfer, and RNA-seq analysis indicates a PC-EC coupling mechanism via gap junction Connexin43 (Cx43), exclusively up-regulated throughout DR-ESC differentiation. High resolution imaging of embryonic and postnatal mouse vasculature substantiates Cx43 plaques at PC-EC borders. These findings indicate a new role for PCs during vasculogenesis via Cx43-mediated communication with ECs.
Objective— We have previously shown that, under certain conditions, ultrasonic microbubble destruction creates arteriogenesis and angiogenesis in skeletal muscle. Here, we tested whether this neovascularization response enhances hyperemia in a rat model of arterial insufficiency and is dependent on the recruitment of bone marrow–derived cells (BMDCs) to treated tissues via a β2 integrin (CD18)-dependent mechanism. Methods and Results— Sprague-Dawley rats, C57BL/6 wild-type mice, and C57BL/6 chimeric mice engrafted with BMDCs from either GFP + or CD18 −/− mice received bilateral femoral artery ligations. Microbubbles (MBs) were intravenously injected, and one gracilis muscle was exposed to pulsed 1 MHz ultrasound (US). Rat hindlimbs exhibited significant increases in adenosine-induced hyperemia and arteriogenesis compared to contralateral controls at 14 and 28 days posttreatment. US-MB–treated wild-type C57BL/6 mice exhibited significant arteriogenesis, angiogenesis, and CD11b + monocyte recruitment; however, these responses were all completely blocked in CD18 −/− chimeric mice. The number of BMDCs increased in US-MB–treated muscles of GFP + chimeric mice; however, GFP + BMDCs did not incorporate into microvessels as vascular cells. Conclusion— In skeletal muscle affected by arterial occlusion, arteriogenesis and hyperemia can be significantly enhanced by ultrasonic MB destruction. This response depends on the recruitment, but not vascular incorporation, of BMDCs via a CD18-dependent mechanism.
Introduction During blood vessel development, cross‐talk between the vascular endothelial growth factor (VEGF) and Notch pathways, among others, coordinates the sprouting of endothelial “tip” cells from existing vessels. These tip cells in turn secrete platelet‐derived growth factor‐BB (PDGF‐BB) to recruit pericytes for the stabilization of nascent vessel branches1. We therefore hypothesize that PDGF‐BB production from endothelial cells must be tightly regulated such that pericyte recruitment closely matches the rate of endothelial tip cell formation. To test this hypothesis, we expanded our recently published model of angiogenesis2 in the developing mouse retina to include endothelial cell production of, and pericyte responsiveness to, PDGF‐BB. Methods Our previous agent‐based model (ABM) was implemented in NetLogo and captures the effect of pericytes on endothelial tip cell formation at the angiogenic front of the developing mouse retina2. Here, we incorporated additional rules to govern (i) PDGF‐BB secretion from endothelial cells, with tip cells increasing their production, (ii) pericyte migration, and (iii) pericyte movement towards regions with the highest PDGF‐BB concentrations. In addition to monitoring output levels of each molecular species, the ABM endpoints included the spatial position of each cellular agent, yielding insight into relative locations of endothelial cells, tip cells, and migrating pericytes. Results Incorporation of the additional rules governing PDGF‐BB and pericytes did not cause any abnormal changes to the previously validated cell behaviors within by our published model. One important parameter that we explored in initial simulations was the rate of PDGF‐BB secretion from endothelial tip cells. Interestingly, if the tip cell PDGF‐BB secretion rate was relatively high, neighboring pericytes would ensheath tip cells such that outward migration of these endothelial cells was limited, or completely blocked, even though they retained other tip cell characteristics, such as higher expression of VEGF receptor 2. In addition, the number of endothelial tip cells did not change across a range from low to high PDGF‐BB production rates. Discussion Using ABM to simulate the key growth factor signaling pathways (VEGF, Notch, PDGF‐BB) and cell behaviors, including pericyte‐endothelial cell interactions, that contribute to retinal vascular development has led to one intriguing new hypothesis that can be tested experimentally: if endothelial cell migration rate outward from an existing vessel outpaces the tip cell production rate of PDGF‐BB, pericyte migration along newly formed branches will be impaired, which could adversely affect neo‐vessel stability/longevity. Testing this, and other related hypotheses generated by the ABM, will likely yield new insights into basic principles for normal blood vessel formation, as well as into pathological settings in which high levels of angiogenic growth factors fail to generate functional new blood vessels. Additionally, this computational model will inspire the generation of new experimental tools and models to validate the findings from these simulations. Support or Funding Information University of Virginia‐VIrginia Tech Carilion Seed Fund (to J.C. and S.P.), NIH R56HL133826 (to J.C.) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
