John C. Chappell

Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1

Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed misguided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sflt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus, we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF-A. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.

Influence of injection site, microvascular pressureand ultrasound variables on microbubble-mediated delivery of microspheres to muscle ☆

OBJECTIVES Our objective was to test the hypothesis that the ultrasound pulsing interval (PI), microbubble injection site and microvascular pressure significantly influence the transport of 100-nm microspheres to muscle through extravasation sites created by the destruction of microbubbles with ultrasound. BACKGROUND Microbubbles show promise as targeted drug and gene delivery agents; however, designing optimal microbubble-based therapies will require an understanding of the factors that influence the transport of microbubble-delivered, gene-bearing vehicles to tissue. METHODS Ultrasound at 1 MHz, with a peak negative pressure amplitude of 0.75 MPa, was applied to microbubbles and 100-nm microspheres in exteriorized rat spinotrapezius muscle. Ultrasound PIs of 1, 3, 5 and 10 s, arterial microsphere injection times of 10 or 40 s and arterial versus venous injection sites were tested. RESULTS Extravasation point creation and microsphere delivery were greatest when the ultrasound PI was 5 or 10 s. No significant differences in extravasation point creation or microsphere delivery were observed with arterial versus venous microbubble injection, but a trend toward increased microsphere delivery with arterial injection may exist. Decreasing the arterial injection time from 40 to 10 s increased microvascular pressure, which, in turn, substantially enhanced microsphere transport to tissue, without a concomitant increase in the number of extravasation points. CONCLUSIONS The ultrasound PI and microvascular pressure significantly influence the creation of extravasation points and the transport of microspheres to tissue. These factors may be important in designing and optimizing contrast ultrasound-based therapies.

Targeted Delivery of Nanoparticles Bearing Fibroblast Growth Factor-2 by Ultrasonic Microbubble Destruction for Therapeutic Arteriogenesis

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.

Vascular Development: Genetic Mechanisms and Links to Vascular Disease

Vertebrate development depends on the formation of intricate vascular networks at numerous sites and in precise patterns; these vascular networks supply oxygen and nutrients to the rapidly expanding tissues of the embryo. Embryonic blood vessels are composed of endothelial cells and pericytes that organize and expand into highly branched conduits. Proper development of the vasculature requires heterogeneity in the response of endothelial cells to angiogenic cues provided by other tissues and organs. The pathogenesis of vascular diseases results from genetic mutations in pathways that provide these cues and in signals that coordinate endothelial heterogeneity during blood vessel formation. Here we provide a brief overview of different aspects of blood vessel formation and then discuss three essential signaling pathways that help establish vessel networks and maintain endothelial phenotypic heterogeneity during vascular development: the vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), and the Notch/Delta/Jagged pathways. The VEGF pathway is critical for the initiation and spatial coordination of angiogenic sprouting and endothelial proliferation, BMP signaling appears to act in a context-dependent manner to promote angiogenic expansion and remodeling, and the Notch pathway is a critical integrator of endothelial cell phenotypes and heterogeneity. We also discuss human genetic mutations that affect these pathways and the resulting pathological conditions.

Ultrasonic Microbubble Destruction Stimulates Therapeutic Arteriogenesis Via the CD18-Dependent Recruitment of Bone Marrow–Derived Cells

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.

Regulation of blood vessel sprouting.

Blood vessels are essential conduits of nutrients and oxygen throughout the body. The formation of these vessels involves angiogenic sprouting, a complex process entailing highly integrated cell behaviors and signaling pathways. In this review, we discuss how endothelial cells initiate a vessel sprout through interactions with their environment and with one another, particularly through lateral inhibition. We review the composition of the local environment, which contains an initial set of guidance cues to facilitate the proper outward migration of the sprout as it emerges from a parent vessel. The long-range guidance and sprout stability cues provided by soluble molecules, extracellular matrix components, and interactions with other cell types are also discussed. We also examine emerging evidence for mechanisms that govern sprout fusion with its target and lumen formation.

Computational modeling of interacting VEGF and soluble VEGF receptor concentration gradients

Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF–Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF–Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis.

Targeted Therapeutic Applications of Acoustically Active Microspheres in the Microcirculation

The targeted delivery of intravascular drugs and genes across the endothelial barrier with only minimal side effects remains a significant obstacle in establishing effective therapies for many pathological conditions. Recent investigations have shown that contrast agent microbubbles, which are typically used for image enhancement in diagnostic ultrasound, may also be promising tools in emergent, ultrasound–based therapies. Explorations of the bioeffects generated by ultrasound–microbubble interactions indicate that these phenomena may be exploited for clinical utility such as in the targeted revascularization of flow‐deficient tissues. Moreover, development of this treatment modality may also include using ultrasound–microbubble interactions to deliver therapeutic material to tissues, and reporter genes and therapeutic agents have been successfully transferred from the microcirculation to tissue in various animal models of normal and pathological function. This article reviews the recent studies aimed at using interactions between ultrasound and contrast agent microbubbles in the microcirculation for therapeutic purposes. Furthermore, the authors present investigations involving microspheres that are of a different design compared to current microbubble contrast agents, yet are acoustically active and demonstrate potential as tools for targeted delivery. Future directions necessary to address current challenges and advance these techniques to clinical practicality are also discussed.

Flt-1 (Vascular Endothelial Growth Factor Receptor-1) Is Essential for the Vascular Endothelial Growth Factor–Notch Feedback Loop During Angiogenesis

Objective—Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. Approach and Results—Wild-type and flt-1−/− mouse embryonic stem cell–derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1−/− embryonic stem cell–derived vessel branching dysmorphogenesis and endothelial hyperproliferation, and rescue of flt-1−/− vessels was accompanied by a reduction in elevated Notch targets. Surprisingly, wild-type vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in wild-type endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels but not caudal vein plexuses. In contrast, exogenous VEGF caused wild-type embryonic stem cell–derived vessel and zebrafish intersegmental vessel dysmorphogenesis that was rescued by Notch blockade. Conclusions—Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1−/− blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.

How blood vessel networks are made and measured.

Tissue and organ viability depends on the proper systemic distribution of cells, nutrients, and oxygen through blood vessel networks. These networks arise in part via angiogenic sprouting. Vessel sprouting involves the precise coordination of several endothelial cell processes including cell-cell communication, cell migration, and proliferation. In this review, we discuss zebrafish and mammalian models of blood vessel sprouting and the quantification methods used to assess vessel sprouting and network formation in these models. We also review the mechanisms involved in angiogenic sprouting, and we propose that the process consists of distinct stages. Sprout initiation involves endothelial cell interactions with neighboring cells and the environment to establish a specialized tip cell responsible for leading the emerging sprout. Furthermore, local sprout guidance cues that spatially regulate this outward migration are discussed. We also examine subsequent events, such as sprout fusion and lumenization, that lead to maturation of a nascent sprout into a patent blood vessel.