A Soluble Platelet-Derived Growth Factor Receptor-ß Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRß) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRß (sPDGFRß) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRß isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRß in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion-critical processes underlying neuronal health and function, and in turn, memory and cognition.

A Soluble Platelet-Derived Growth Factor Receptor-ß Originates via Pre-mRNA Splicing in the Healthy Brain and is Differentially Regulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRß) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRß (sPDGFRß) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRß isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRß in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.

Connexin 43 across the Vasculature: Gap Junctions and Beyond.

Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds.

Evaluating cell viability, capillary perfusion, and collateral tortuosity in an ex vivo mouse intestine fluidics model.

Numerous disease conditions involve the sudden or progressive loss of blood flow. Perfusion restoration is vital for returning affected organs to full health. While a range of clinical interventions can successfully restore flow to downstream tissues, the microvascular responses after a loss-of-flow event can vary over time and may involve substantial microvessel instability. Increased insight into perfusion-mediated capillary stability and access-to-flow is therefore essential for advancing therapeutic reperfusion strategies and improving patient outcomes. To that end, we developed a tissue-based microvascular fluidics model to better understand (i) microvascular stability and access-to-flow over an acute time course post-ischemia, and (ii) collateral flow in vessels neighboring an occlusion site. We utilized murine intestinal tissue regions by catheterizing a feeder artery and introducing perfusate at physiologically comparable flow-rates. The cannulated vessel as well as a portion of the downstream vessels and associated intestinal tissue were cultured while constant perfusion conditions were maintained. An occlusion was introduced in a selected arterial segment, and changes in perfusion within areas receiving varying degrees of collateral flow were observed over time. To observe the microvascular response to perfusion changes, we incorporated (i) tissues harboring cell-reporter constructs, specifically Ng2-DsRed labeling of intestinal pericytes, and (ii) different types of fluorescent perfusates to quantify capillary access-to-flow at discrete time points. In our model, we found that perfusion tracers could enter capillaries within regions downstream of an occlusion upon the initial introduction of perfusion, but at 24 h tissue perfusion was severely decreased. However, live/dead cell discrimination revealed that the tissue overall did not experience significant cell death, including that of microvascular pericytes, even after 48 h. Our findings suggest that altered flow conditions may rapidly initiate cellular responses that reduce capillary access-to-flow, even in the absence of cellular deterioration or hypoxia. Overall, this ex vivo tissue-based microfluidics model may serve as a platform upon which a variety of follow-on studies may be conducted. It will thus enhance our understanding of microvessel stability and access-to-flow during an occlusive event and the role of collateral flow during normal and disrupted perfusion.

Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43.

BACKGROUND: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. METHODS: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. RESULTS: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte-endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. CONCLUSIONS: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.

Pericyte heterogeneity identified by 3D ultrastructural analysis of the microvessel wall.

Confident identification of pericytes (PCs) remains an obstacle in the field, as a single molecular marker for these unique perivascular cells remains elusive. Adding to this challenge is the recent appreciation that PC populations may be heterogeneous, displaying a range of morphologies within capillary networks. We found additional support on the ultrastructural level for the classification of these PC subtypes-"thin-strand" (TSP), mesh (MP), and ensheathing (EP)-based on distinct morphological characteristics. Interestingly, we also found several examples of another cell type, likely a vascular smooth muscle cell, in a medial layer between endothelial cells (ECs) and pericytes (PCs) harboring characteristics of the ensheathing type. A conserved feature across the different PC subtypes was the presence of extracellular matrix (ECM) surrounding the vascular unit and distributed in between neighboring cells. The thickness of this vascular basement membrane was remarkably consistent depending on its location, but never strayed beyond a range of 150-300 nm unless thinned to facilitate closer proximity of neighboring cells (suggesting direct contact). The density of PC-EC contact points ("peg-and-socket" structures) was another distinguishing feature across the different PC subtypes, as were the apparent contact locations between vascular cells and brain parenchymal cells. In addition to this thinning, the extracellular matrix (ECM) surrounding EPs displayed another unique configuration in the form of extensions that emitted out radially into the surrounding parenchyma. Knowledge of the origin and function of these structures is still emerging, but their appearance suggests the potential for being mechanical elements and/or perhaps signaling nodes via embedded molecular cues. Overall, this unique ultrastructural perspective provides new insights into PC heterogeneity and the presence of medial cells within the microvessel wall, the consideration of extracellular matrix (ECM) coverage as another PC identification criteria, and unique extracellular matrix (ECM) configurations (i.e., radial extensions) that may reveal additional aspects of PC heterogeneity.

Pericyte migration and proliferation are tightly synchronized to endothelial cell sprouting dynamics.

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.

The cerebral microvasculature: Basic and clinical perspectives on stroke and glioma.

Microvascular networks are vital components of the cardiovascular system, performing many key roles in maintaining the health and homeostasis of the tissues and organs in which they develop. As discussed in this review, the molecular and cellular components within the microcirculation orchestrate critical processes to establish functional capillary beds, including organization of endothelial cell (EC) polarity, guiding investment of vascular pericytes (PCs), and building the specialized extracellular matrix (ECM) that comprises the vascular basement membrane (vBM). Herein, we further discuss the unique features of the microvasculature in the central nervous system (CNS), focusing on the cells contributing to the neurovascular unit (NVU) that form and maintain the blood-brain barrier (BBB). With a focus on vascular PCs, we offer basic and clinical perspectives on neurovascular-related pathologies that involve defects within the cerebral microvasculature. Specifically, we present microvascular anomalies associated with glioblastoma multiforme (GBM) including defects in vascular-immune cell interactions and associated clinical therapies targeting microvessels (ie, vascular-disrupting/anti-angiogenic agents and focused ultrasound). We also discuss the involvement of the microcirculation in stroke responses and potential therapeutic approaches. Our goal was to compare the cellular and molecular changes that occur in the microvasculature and NVU, and to provide a commentary on factors driving disease progression in GBM and stroke. We conclude with a forward-looking perspective on the importance of microcirculation research in developing clinical treatments for these devastating conditions.

Pericytes in Vascular Development.

PURPOSE OF REVIEW: Pericytes are essential components of capillaries in many tissues and organs, contributing to vessel stability and integrity, with additional contributions to microvascular function still being discovered. We review current and foundational studies identifying pericyte differentiation mechanics and their roles in the earliest stages of vessel formation. RECENT FINDINGS: Recent advances in pericyte-focused tools and models have illuminated critical aspects of pericyte biology including their roles in vascular development.Pericytes likely collaborate with endothelial cells undergoing vasculogenesis, initiating direct interactions during sprouting and intussusceptive angiogenesis. Pericytes also provide important regulation of vascular growth including mechanisms underlying vessel pruning, rarefaction, and subsequent regrowth.

Blood Vessel Patterning on Retinal Astrocytes Requires Endothelial Flt-1 (VEGFR-1).

Feedback mechanisms are critical components of many pro-angiogenic signaling pathways that keep vessel growth within a functional range. The Vascular Endothelial Growth Factor-A (VEGF-A) pathway utilizes the decoy VEGF-A receptor Flt-1 to provide negative feedback regulation of VEGF-A signaling. In this study, we investigated how the genetic loss of flt-1 differentially affects the branching complexity of vascular networks in tissues despite similar effects on endothelial sprouting. We selectively ablated flt-1 in the post-natal retina and found that maximum induction of flt-1 loss resulted in alterations in endothelial sprouting and filopodial extension, ultimately yielding hyper-branched networks in the absence of changes in retinal astrocyte architecture. The mosaic deletion of flt-1 revealed that sprouting endothelial cells flanked by flt-1-/- regions of vasculature more extensively associated with underlying astrocytes and exhibited aberrant sprouting, independent of the tip cell genotype. Overall, our data support a model in which tissue patterning features, such as retinal astrocytes, integrate with flt-1-regulated angiogenic molecular and cellular mechanisms to yield optimal vessel patterning for a given tissue.

Applications of Ultrasound to Stimulate Therapeutic Revascularization.

Many pathological conditions are characterized or caused by the presence of an insufficient or aberrant local vasculature. Thus, therapeutic approaches aimed at modulating the caliber and/or density of the vasculature by controlling angiogenesis and arteriogenesis have been under development for many years. As our understanding of the underlying cellular and molecular mechanisms of these vascular growth processes continues to grow, so too do the available targets for therapeutic intervention. Nonetheless, the tools needed to implement such therapies have often had inherent weaknesses (i.e., invasiveness, expense, poor targeting, and control) that preclude successful outcomes. Approximately 20 years ago, the potential for using ultrasound as a new tool for therapeutically manipulating angiogenesis and arteriogenesis began to emerge. Indeed, the ability of ultrasound, especially when used in combination with contrast agent microbubbles, to mechanically manipulate the microvasculature has opened several doors for exploration. In turn, multiple studies on the influence of ultrasound-mediated bioeffects on vascular growth and the use of ultrasound for the targeted stimulation of blood vessel growth via drug and gene delivery have been performed and published over the years. In this review article, we first discuss the basic principles of therapeutic ultrasound for stimulating angiogenesis and arteriogenesis. We then follow this with a comprehensive cataloging of studies that have used ultrasound for stimulating revascularization to date. Finally, we offer a brief perspective on the future of such approaches, in the context of both further research development and possible clinical translation.

The pericyte microenvironment during vascular development.

Vascular pericytes provide critical contributions to the formation and integrity of the blood vessel wall within the microcirculation. Pericytes maintain vascular stability and homeostasis by promoting endothelial cell junctions and depositing extracellular matrix (ECM) components within the vascular basement membrane, among other vital functions. As their importance in sustaining microvessel health within various tissues and organs continues to emerge, so does their role in a number of pathological conditions including cancer, diabetic retinopathy, and neurological disorders. Here, we review vascular pericyte contributions to the development and remodeling of the microcirculation, with a focus on the local microenvironment during these processes. We discuss observations of their earliest involvement in vascular development and essential cues for their recruitment to the remodeling endothelium. Pericyte involvement in the angiogenic sprouting context is also considered with specific attention to crosstalk with endothelial cells such as through signaling regulation and ECM deposition. We also address specific aspects of the collective cell migration and dynamic interactions between pericytes and endothelial cells during angiogenic sprouting. Lastly, we discuss pericyte contributions to mechanisms underlying the transition from active vessel remodeling to the maturation and quiescence phase of vascular development.

Microvascular bioengineering: a focus on pericytes.

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.

Hypoxia, angiogenesis, and metabolism in the hereditary kidney cancers.

The field of hereditary kidney cancer has begun to mature following the identification of several germline syndromes that define genetic and molecular features of this cancer. Molecular defects within these hereditary syndromes demonstrate consistent deficits in angiogenesis and metabolic signaling, largely driven by altered hypoxia signaling. The classical mutation, loss of function of the von Hippel-Lindau (VHL) tumor suppressor, provides a human pathogenesis model for critical aspects of pseudohypoxia. These features are mimicked in a less common hereditary renal tumor syndrome, known as hereditary leiomyomatosis and renal cell carcinoma. Here, we review renal tumor angiogenesis and metabolism from a HIF-centric perspective, considering alterations in the hypoxic landscape, and molecular deviations resulting from high levels of HIF family members. Mutations underlying HIF deregulation drive multifactorial aberrations in angiogenic signals and metabolism. The mechanisms by which these defects drive tumor growth are still emerging. However, the distinctive patterns of angiogenesis and glycolysis-/glutamine-dependent bioenergetics provide insight into the cellular environment of these cancers. The result is a scenario permissive for aggressive tumorigenesis especially within the proximal renal tubule. These features of tumorigenesis have been highly actionable in kidney cancer treatments, and will likely continue as central tenets of kidney cancer therapeutics.

Excess vascular endothelial growth factor-A disrupts pericyte recruitment during blood vessel formation.

Pericyte investment into new blood vessels is essential for vascular development such that mis-regulation within this phase of vessel formation can contribute to numerous pathologies including arteriovenous and cerebrovascular malformations. It is critical therefore to illuminate how angiogenic signaling pathways intersect to regulate pericyte migration and investment. Here, we disrupted vascular endothelial growth factor-A (VEGF-A) signaling in ex vivo and in vitro models of sprouting angiogenesis, and found pericyte coverage to be compromised during VEGF-A perturbations. Pericytes had little to no expression of VEGF receptors, suggesting VEGF-A signaling defects affect endothelial cells directly but pericytes indirectly. Live imaging of ex vivo angiogenesis in mouse embryonic skin revealed limited pericyte migration during exposure to exogenous VEGF-A. During VEGF-A gain-of-function conditions, pericytes and endothelial cells displayed abnormal transcriptional changes within the platelet-derived growth factor-B (PDGF-B) and Notch pathways. To further test potential crosstalk between these pathways in pericytes, we stimulated embryonic pericytes with Notch ligands Delta-like 4 (Dll4) and Jagged-1 (Jag1) and found induction of Notch pathway activity but no changes in PDGF Receptor-ß (Pdgfrß) expression. In contrast, PDGFRß protein levels decreased with mis-regulated VEGF-A activity, observed in the effects on full-length PDGFRß and a truncated PDGFRß isoform generated by proteolytic cleavage or potentially by mRNA splicing. Overall, these observations support a model in which, during the initial stages of vascular development, pericyte distribution and coverage are indirectly affected by endothelial cell VEGF-A signaling and the downstream regulation of PDGF-B-PDGFRß dynamics, without substantial involvement of pericyte Notch signaling during these early stages.

Establishment and characterization of an embryonic pericyte cell line.

OBJECTIVE: Pericytes are specialized perivascular cells embedded within the basement membrane. These cells envelope the abluminal surface of endothelial cells and promote microvessel homeostasis. Recent discoveries of unique pericyte functions, particularly in neural tissues, underscore the need for overcoming existing challenges in establishing a functionally validated pericyte cell line. Here, we present methodologies for addressing these challenges as well as an embryonic pericyte cell line for use with in vitro and ex vivo experimental models. METHODS: We isolated an enriched population of NG2:DsRed+ pericytes from E12.5 mice. This pericyte cell line was compared to MEFs with respect to gene expression, cell morphology and migration, and engagement with endothelial cells during junction stabilization and angiogenesis. RESULTS: NG2+ pericytes displayed gene expression patterns, cell morphology, and 2D migration behaviors distinct from MEFs. In three different vessel formation models, pericytes from this line migrated to and incorporated into developing vessels. When co-cultured with HUVECs, these pericytes stimulated more robust VE-Cadherin junctions between HUVECs as compared to MEFs, as well as contributed to HUVEC organization into primitive vascular structures. CONCLUSIONS: Our data support use of this pericyte cell line in a broad range of models to further understand pericyte functionality during normal and pathological conditions.

Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch.

Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome.

Agent-based computational model of retinal angiogenesis simulates microvascular network morphology as a function of pericyte coverage.

OBJECTIVE: Define a role for perivascular cells during developmental retinal angiogenesis in the context of EC Notch1-DLL4 signaling at the multicellular network level. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis.

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.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS: In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS: Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS: These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.