Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK:

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

rasa1-related arteriovenous malformation is driven by aberrant venous signalling.

Arteriovenous malformations (AVMs) develop where abnormal endothelial signalling allows direct connections between arteries and veins. Mutations in RASA1, a Ras GTPase activating protein, lead to AVMs in humans and, as we show, in zebrafish rasa1 mutants. rasa1 mutants develop cavernous AVMs that subsume part of the dorsal aorta and multiple veins in the caudal venous plexus (CVP) - a venous vascular bed. The AVMs progressively enlarge and fill with slow-flowing blood. We show that the AVM results in both higher minimum and maximum flow velocities, resulting in increased pulsatility in the aorta and decreased pulsatility in the vein. These hemodynamic changes correlate with reduced expression of the flow-responsive transcription factor klf2a. Remodelling of the CVP is impaired with an excess of intraluminal pillars, which is a sign of incomplete intussusceptive angiogenesis. Mechanistically, we show that the AVM arises from ectopic activation of MEK/ERK in the vein of rasa1 mutants, and that cell size is also increased in the vein. Blocking MEK/ERK signalling prevents AVM initiation in mutants. Alterations in venous MEK/ERK therefore drive the initiation of rasa1 AVMs.

Stroke-associated intergenic variants modulate a human FOXF2 transcriptional enhancer.

SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1, but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2, functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2, thereby modulating stroke risk.

SAM domain variants of EPHB4 associated with aberrant signaling are linked to lymphatic-related fetal hydrops and facial dysmorphology.

Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.

Vessel Metrics: A software tool for automated analysis of vascular structure in confocal imaging.

Images contain a wealth of information that is often under analyzed in biological studies. Developmental models of vascular disease are a powerful way to quantify developmentally regulated vessel phenotypes to identify the roots of the disease process. We present vessel Metrics, a software tool specifically designed to analyze developmental vascular microscopy images that will expedite the analysis of vascular images and provide consistency between research groups. We developed a segmentation algorithm that robustly quantifies different image types, developmental stages, organisms, and disease models at a similar accuracy level to a human observer. We validate the algorithm on confocal, lightsheet, and two photon microscopy data in a zebrafish model expressing fluorescent protein in the endothelial nuclei. The tool accurately segments data taken by multiple scientists on varying microscopes. We validate vascular parameters such as vessel density, network length, and diameter, across developmental stages, genetic mutations, and drug treatments, and show a favorable comparison to other freely available software tools. Additionally, we validate the tool in a mouse model. Vessel Metrics reduces the time to analyze experimental results, improves repeatability within and between institutions, and expands the percentage of a given vascular network analyzable in experiments.

Endothelial Semaphorin 3fb regulates Vegf pathway-mediated angiogenic sprouting.

Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling.

Development of vascular regulation in the zebrafish embryo.

The thin endothelial wall of a newly formed vessel is under enormous stress at the onset of blood flow, rapidly acquiring support from mural cells (pericytes and vascular smooth muscle cells; vSMCs) during development. Mural cells then develop vasoactivity (contraction and relaxation) but we have little information as to when this first develops or the extent to which pericytes and vSMCs contribute. For the first time, we determine the dynamic developmental acquisition of vasoactivity in vivo in the cerebral vasculature of zebrafish. We show that pericyte-covered vessels constrict in response to α1-adrenergic receptor agonists and dilate in response to nitric oxide donors at 4 days postfertilization (dpf) but have heterogeneous responses later, at 6 dpf. In contrast, vSMC-covered vessels constrict at 6 dpf, and dilate at both stages. Using genetic ablation, we demonstrate that vascular constriction and dilation is an active response. Our data suggest that both pericyte- and vSMC-covered vessels regulate their diameter in early development, and that their relative contributions change over developmental time.

MicroRNA26 attenuates vascular smooth muscle maturation via endothelial BMP signalling.

As small regulatory transcripts, microRNAs (miRs) act as genetic 'fine tuners' of posttranscriptional events, and as genetic switches to promote phenotypic switching. The miR miR26a targets the BMP signalling effector, smad1. We show that loss of miR26a leads to hemorrhage (a loss of vascular stability) in vivo, suggesting altered vascular differentiation. Reduction in miR26a levels increases smad1 mRNA and phospho-Smad1 (pSmad1) levels. We show that increasing BMP signalling by overexpression of smad1 also leads to hemorrhage. Normalization of Smad1 levels through double knockdown of miR26a and smad1 rescues hemorrhage, suggesting a direct relationship between miR26a, smad1 and vascular stability. Using an in vivo BMP genetic reporter and pSmad1 staining, we show that the effect of miR26a on smooth muscle differentiation is non-autonomous; BMP signalling is active in embryonic endothelial cells, but not in smooth muscle cells. Nonetheless, increased BMP signalling due to loss of miR26a results in an increase in acta2-expressing smooth muscle cell numbers and promotes a differentiated smooth muscle morphology. Similarly, forced expression of smad1 in endothelial cells leads to an increase in smooth muscle cell number and coverage. Furthermore, smooth muscle phenotypes caused by inhibition of the BMP pathway are rescued by loss of miR26a. Taken together, our data suggest that miR26a modulates BMP signalling in endothelial cells and indirectly promotes a differentiated smooth muscle phenotype. Our data highlights how crosstalk from BMP-responsive endothelium to smooth muscle is important for smooth muscle differentiation.

Altered Expression of TMEM43 Causes Abnormal Cardiac Structure and Function in Zebrafish.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.

Psychosocial and lifestyle predictors of distress and well-being in people with mental illness during the COVID-19 pandemic.

OBJECTIVES: People with mental illness may be vulnerable to psychological distress and reduced well-being during the COVID-19 pandemic. The aim of this study was to assess psychosocial and lifestyle predictors of distress and well-being in people with mental illness during the pandemic. METHOD: People with mental illness who participated in an exercise programme prior to the pandemic were invited to complete surveys about mental health and lifestyle corresponding to before and during the pandemic. RESULTS: Social support reduced, alcohol intake increased, and sleep quality and diet worsened during the pandemic, contributing to distress. Psychological distress was associated with the two or more mental illnesses, and negatively associated with having a physical disease. Better diet appeared to protect against increases in distress; loneliness hindered improvements in well-being. CONCLUSIONS: Healthy lifestyle programmes designed to improve social connection may improve health for people with mental illnesses during and after the COVID-19 pandemic.

Preferences of people with mental illness for engaging in exercise programs under COVID-19 restrictions.

OBJECTIVES: People with mental illness may be vulnerable to decline in mental health and reduced physical activity because of the COVID-19 pandemic and associated restrictions. The aim of this study was to inform the design of physical activity interventions for implementation under these conditions to improve/maintain well-being and physical activity in this population. METHODS: People with mental illness who had participated in a physical activity program prior to the pandemic were invited to complete a survey about the impact of COVID-19 on mental health and physical activity and their preferences for engaging in a physical activity program under pandemic-related restrictions. RESULTS: More than half the 59 respondents reported worse mental health and lower physical activity during the pandemic. The preferred format for a physical activity program was one-on-one exercise instruction in-person in a park. Program components endorsed as helpful included incentivization, provision of exercise equipment and fitness devices, and daily exercise programs. About a third of the participants reported limitations in using technology for a physical activity program. CONCLUSIONS: In-person exercise support is preferred by people with mental illnesses during pandemic-related restrictions. Enablement strategies such as providing equipment and self-monitoring devices should be utilized; assistance may be needed to incorporate the use of technology in exercise programs.

foxc1 is required for embryonic head vascular smooth muscle differentiation in zebrafish.

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrß positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

Pericyte Biology in Zebrafish.

The zebrafish is an outstanding model for studying vascular biology in vivo. Pericytes and vascular smooth muscle cells can be imaged as they associate with vessels and provide stability and integrity to the vasculature. In zebrafish, pericytes associate with the cerebral and trunk vasculature on the second day of development, as assayed by pdgfrß and notch3 markers. In the head, cerebral pericytes are neural crest derived, except for the pericytes of the hindbrain vasculature, which are mesoderm derived. Similar to the hindbrain, pericytes on the trunk vasculature are also mesoderm derived. Regardless of their location, pericyte development depends on a complex interaction between blood flow and signalling pathways, such as Notch, SONIC HEDGEHOG and BMP signalling, all of which positively regulate pericyte numbers.Pericyte numbers rapidly increase as development proceeds in order to stabilize both the blood-brain barrier and the vasculature and hence, prevent haemorrhage. Consequently, compromised pericyte development results in compromised vascular integrity, which then evolves into detrimental pathologies. Some of these pathologies have been modelled in zebrafish by inducing mutations in the notch3, foxc1 and foxf2 genes. These zebrafish models provide insights into the mechanisms of disease as associated with pericyte biology. Going forward, these models may be key contributors in elucidating the role of vascular mural cells in regulating vessel diameter and hence, blood flow.

Patterning mechanisms of the sub-intestinal venous plexus in zebrafish.

Despite considerable interest in angiogenesis, organ-specific angiogenesis remains less well characterized. The vessels that absorb nutrients from the yolk and later provide blood supply to the developing digestive system are primarily venous in origin. In zebrafish, these are the vessels of the Sub-intestinal venous plexus (SIVP) and they represent a new candidate model to gain an insight into the mechanisms of venous angiogenesis. Unlike other vessel beds in zebrafish, the SIVP is not stereotypically patterned and lacks obvious sources of patterning information. However, by examining the area of vessel coverage, number of compartments, proliferation and migration speed we have identified common developmental steps in SIVP formation. We applied our analysis of SIVP development to obd mutants that have a mutation in the guidance receptor PlexinD1. obd mutants show dysregulation of nearly all parameters of SIVP formation. We show that the SIVP responds to a unique combination of pathways that control both arterial and venous growth in other systems. Blocking Shh, Notch and Pdgf signaling has no effect on SIVP growth. However Vegf promotes sprouting of the predominantly venous plexus and Bmp promotes outgrowth of the structure. We propose that the SIVP is a unique model to understand novel mechanisms utilized in organ-specific angiogenesis.

The LIM-homeodomain transcription factor Islet2a promotes angioblast migration.

Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion.

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. We know little about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. Here, we show that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. We propose a novel, tissue-autonomous mechanism of organ cohesion, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.

Quantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells.

Nanoparticle (NP) interactions with biological tissues are affected by the size, shape and surface chemistry of the NPs. Here we use in vivo (zebrafish) and in vitro (HUVEC) models to investigate association of quantum dots (QDs) with endothelial cells and the effect of fluid flow. After injection into the developing zebrafish, circulating QDs associate with endothelium and penetrate surrounding tissue parenchyma over time. Amino-functionalized QDs cluster, interact with cells, and clear more rapidly than carboxy-functionalized QDs in vivo, highlighting charge influences. QDs show stronger accumulation in slow-flowing, small caliber venous vessels than in fast-flowing high caliber arterial vessels. Parallel-plate flow experiments with HUVEC support these findings, showing reduced QD-EC association with increasing flow. In vivo, flow arrest after nanoparticle injection still results in venous accumulation at 18 h. Overall our results suggest that both QD charge and blood flow modulate particle-endothelial cell interactions.

Zac1 Regulates the Differentiation and Migration of Neocortical Neurons via Pac1.

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. Significance statement: Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader-Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.

Comparative analysis of genes regulated by Dzip1/iguana and hedgehog in zebrafish.

BACKGROUND: The zebrafish genetic mutant iguana (igu) has defects in the ciliary basal body protein Dzip1, causing improper cilia formation. Dzip1 also interacts with the downstream transcriptional activators of Hedgehog (Hh), the Gli proteins, and Hh signaling is disrupted in igu mutants. Hh governs a wide range of developmental processes, including stabilizing developing blood vessels to prevent hemorrhage. Using igu mutant embryos and embryos treated with the Hh pathway antagonist cyclopamine, we conducted a microarray to determine genes involved in Hh signaling mediating vascular stability. RESULTS: We identified 40 genes with significantly altered expression in both igu mutants and cyclopamine-treated embryos. For a subset of these, we used in situ hybridization to determine localization during embryonic development and confirm the expression changes seen on the array. CONCLUSIONS: Through comparing gene expression changes in a genetic model of vascular instability with a chemical inhibition of Hh signaling, we identified a set of 40 differentially expressed genes with potential roles in vascular stabilization.