WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

Cdc42 is required for cytoskeletal support of endothelial cell adhesion during blood vessel formation in mice.

The Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain unclear. Here, we assess the cellular mechanisms by which Cdc42 regulates mammalian vascular morphogenesis and maintenance. In vivo deletion of Cdc42 in embryonic ECs (Cdc42(Tie2KO)) results in blocked lumen formation and endothelial tearing, leading to lethality of mutant embryos by E9-10 due to failed blood circulation. Similarly, inducible deletion of Cdc42 (Cdc42(Cad5KO)) at mid-gestation blocks angiogenic tubulogenesis. By contrast, deletion of Cdc42 in postnatal retinal vessels leads to aberrant vascular remodeling and sprouting, as well as markedly reduced filopodia formation. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. We also identify Cdc42 effectors Pak2/4 and N-WASP, as well as the actomyosin machinery, to be crucial for EC actin organization. This work supports the notion of Cdc42 as a central regulator of the cellular machinery in ECs that drives blood vessel formation.

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II.

RATIONALE: Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells, apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 (Ras-interacting protein 1) is required for apical junction clearance, as well as for regulation of Rho GTPase (enzyme that hydrolyzes GTP) activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. OBJECTIVE: The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. METHODS AND RESULTS: Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of endothelial cell apical junctions during vascular tubulogenesis depends on Rasip1, as well as the GTPase Cdc42 (cell division control protein 42 homolog) and the kinase Pak4 (serine/threonine-protein kinase 4). Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all blocks endothelial cell tubulogenesis. By contrast, inactivation of RhoA (Ras homologue gene family member A) signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII (nonmuscle myosin II) either before, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. CONCLUSIONS: Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

Vascular development in the vertebrate pancreas.

The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium.

Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth.

Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular 'tubulogenesis' are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1(-/-) ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC-EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.

Saccadic Impairment Associated With Remote History of Mild Traumatic Brain Injury.

Evidence suggests that mild traumatic brain injury (TBI) is associated with long-term changes in brain function, but conventional neurocognitive tools are often insensitive to deficits after 90 days. Eye movements have been proposed as a means to identify more chronic forms of impairment. In this study, saccadic, manual, and conventional neuropsychological measures were compared between participants with remote mild TBI and well-matched control participants. Saccadic impairment was more frequent within the mild TBI group, and a history of multiple injuries or high symptom burden appeared to compound this risk. However, other neurocognitive measures did not differ by group, number of injuries, or symptom severity. These results suggest that saccadic impairment may reflect chronic effects of mild TBI that conventional measures are unable to detect.

Predictors of neurobehavioral symptoms in a university population: a multivariate approach using a postconcussive symptom questionnaire.

Several factors have been linked to severity of postconcussive-type (neurobehavioral) symptoms. In this study, predictors of neurobehavioral symptoms were examined using multivariate methods to determine the relative importance of each. Data regarding demographics, symptoms, current alcohol use, history of traumatic brain injury (TBI), orthopedic injuries, and psychiatric/developmental diagnoses were collected via questionnaire from 3027 university students. The most prominent predictors of symptoms were gender, history of depression or anxiety, history of attention-deficit/hyperactivity disorder or learning disability diagnosis, and frequency of alcohol use. Prior mild TBI was significantly related to overall symptoms, but this effect was small in comparison to other predictors. These results provide further evidence that neurobehavioral symptoms are multi-determined phenomena, and highlight the importance of psychiatric comorbidity, demographic factors, and health behaviors to neurobehavioral symptom presentation after mild TBI.

A comparison of long-term postconcussive symptoms between university students with and without a history of mild traumatic brain injury or orthopedic injury.

Mild traumatic brain injury (mild TBI) is often associated with postconcussive symptoms such as headache, memory problems, and irritability. However, high rates of similar symptoms in groups without a history of TBI raise questions about the clinical validity of the postconcussive syndrome. This study was conducted to address these issues through systematic examination of symptoms reported by those with and without a history of mild TBI or orthopedic injury. Responses to the Postconcussion Syndrome Checklist (PCSC), demographic information, and medical history were collected via online questionnaire from 3027 non-referred university students (2280 without a history of mild TBI or orthopedic injury, 491 with a history of orthopedic injury, and 256 with post-acute mild TBI). Although the mild TBI group reported higher mean levels of symptoms, confirmatory factor analyses demonstrated that symptoms clustered into parallel cognitive, somatic, affective, and sensory factors in all three groups. Despite modestly higher mean symptoms among those with a history of mild TBI, symptom clusters did not differ from non-TBI groups. These findings cast doubts about the clinical validity of the "postconcussive syndrome" and raise questions about pathways by which mild TBI and other factors may influence the expression of chronic symptoms.

Performance Enhancement Assessment and Coaching in US Army Special Operations: Rapidly Enhancing Performance Through Targeted, Tailored Feedback.

BACKGROUND: Performance enhancement coaching poses significant benefits to individuals and organizations, such as improved job satisfaction and goal achievement. Given their training and experience in assessment and feedback, operational psychologists assigned to Special Operations units are uniquely positioned to provide performance enhancement coaching tailored to Operators and enablers. A preliminary program evaluation was conducted of the Performance Enhancement Assessment and Coaching (PEAC) Program. METHODS: A sample of 32 Operators and enablers assigned to a US Army Special Operations Forces (ARSOF) unit voluntarily participated in the PEAC Program and completed one 90-minute coaching session. Following their coaching session, Soldiers provided qualitative and quantitative feedback on their coaching experience. RESULTS: Soldiers overwhelmingly agreed that the PEAC Program was worth their time and helpful towards achieving their goals. Results indicate the PEAC Program enhanced Soldiers' perceived self-awareness, self-efficacy, and job performance. Results also suggest performance enhancement coaching may improve pass rates on interpersonally demanding Special Operations courses. CONCLUSION: Performance enhancement coaching delivers considerable value for Special Operations personnel and their organizations in relatively minimal time. Operational psychologist coaches (OPCs) assigned to Special Operations units can leverage their assessment skills to provide targeted, tailored performance enhancement coaching and increase value to their organizations.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Increased risk for age-related impairment in visual attention associated with mild traumatic brain injury: Evidence from saccadic response times.

It was hypothesized that risk for age-related impairment in attention would be greater among those with remote history of mild TBI than individuals without history of head injury. Twenty-seven adults with remote history of mild TBI and a well-matched comparison group of 54 uninjured controls completed a computerized test of visual attention while saccadic and manual response times were recorded. Within the mild TBI group only, older age was associated with slower saccadic responses and poorer saccadic inhibition. Saccadic slowing was mitigated in situations where the timing and location of attention targets was fully predictable. Mild TBI was not associated with age-related increases in risk for neuropsychological impairment or neurobehavioral symptoms. These results provide preliminary evidence that risk for age-related impairment in visual attention may be higher among those with a history of mild TBI. Saccadic measures may provide enhanced sensitivity to this subtle form of cognitive impairment.

Src- and Fyn-dependent apical membrane trafficking events control endothelial lumen formation during vascular tube morphogenesis.

Here we examine the question of how endothelial cells (ECs) develop their apical membrane surface domain during lumen and tube formation. We demonstrate marked apical membrane targeting of activated Src kinases to this apical domain during early and late stages of this process. Immunostaining for phosphotyrosine or phospho-Src reveals apical membrane staining in intracellular vacuoles initially. This is then followed by vacuole to vacuole fusion events to generate an apical luminal membrane, which is similarly decorated with activated phospho-Src kinases. Functional blockade of Src kinases completely blocks EC lumen and tube formation, whether this occurs during vasculogenic tube assembly or angiogenic sprouting events. Multiple Src kinases participate in this apical membrane formation process and siRNA suppression of Src, Fyn and Yes, but not Lyn, blocks EC lumen formation. We also demonstrate strong apical targeting of Src-GFP and Fyn-GFP fusion proteins and increasing their expression enhances lumen formation. Finally, we show that Src- and Fyn-associated vacuoles track and fuse along a subapically polarized microtubule cytoskeleton, which is highly acetylated. These vacuoles generate the apical luminal membrane in a stereotypically polarized, perinuclear position. Overall, our study identifies a critical role for Src kinases in creating and decorating the EC apical membrane surface during early and late stages of lumen and tube formation, a central event in the molecular control of vascular morphogenesis.

Assessment of Performance Validity Using Embedded Saccadic and Manual Indices on a Continuous Performance Test.

OBJECTIVE: In addition to manual (i.e., "button press") metrics, oculomotor metrics demonstrate considerable promise as tools for detecting invalid responding in neurocognitive assessment. This study was conducted to evaluate saccadic and manual metrics from a computerized continuous performance test as embedded indices of performance validity. METHOD: Receiver operating characteristic analyses, logistic regressions, and ANOVAs were performed to evaluate saccadic and manual metrics in classification of healthy adults instructed to feign deficits ("Fake Bad" group; n = 24), healthy adults instructed to perform their best ("Best Effort" group; n = 26), and adults with a history of mild traumatic brain injury (TBI) who passed a series of validity indices ("mTBI-Pass" group; n = 19). RESULTS: Several saccadic and manual metrics achieved outstanding classification accuracy between Fake Bad versus Best Effort and mTBI-Pass groups, including variability (consistency) of saccadic and manual response time (RT), saccadic commission errors, and manual omission errors. Very large effect sizes were obtained between Fake Bad and Best Effort groups (Cohen's d range: 1.89-2.90; r range: .75-.78) as well as between Fake Bad and mTBI-Pass groups (Cohen's d range: 1.32-2.21; r range: .69-.71). The Fake Bad group consistently had higher saccadic and manual RT variability, more saccadic commission errors, and more manual omission errors than the Best Effort and mTBI-Pass groups. CONCLUSIONS: These findings are the first to demonstrate that eye movements can be used to detect invalid responding in neurocognitive assessment. These results also provide compelling evidence that concurrently measured saccadic and manual metrics can detect invalid responding with high levels of sensitivity and specificity.

Multimodal assessment of visual attention using the Bethesda Eye & Attention Measure (BEAM).

INTRODUCTION: Computerized cognitive tests measuring manual response time (RT) and errors are often used in the assessment of visual attention. Evidence suggests that saccadic RT and errors may also provide valuable information about attention. This study was conducted to examine a novel approach to multimodal assessment of visual attention incorporating concurrent measurements of saccadic eye movements and manual responses. METHOD: A computerized cognitive task, the Bethesda Eye & Attention Measure (BEAM) v.34, was designed to evaluate key attention networks through concurrent measurement of saccadic and manual RT and inhibition errors. Results from a community sample of n = 54 adults were analyzed to examine effects of BEAM attention cues on manual and saccadic RT and inhibition errors, internal reliability of BEAM metrics, relationships between parallel saccadic and manual metrics, and relationships of BEAM metrics to demographic characteristics. RESULTS: Effects of BEAM attention cues (alerting, orienting, interference, gap, and no-go signals) were consistent with previous literature examining key attention processes. However, corresponding saccadic and manual measurements were weakly related to each other, and only manual measurements were related to estimated verbal intelligence or years of education. CONCLUSIONS: This study provides preliminary support for the feasibility of multimodal assessment of visual attention using the BEAM. Results suggest that BEAM saccadic and manual metrics provide divergent measurements. Additional research will be needed to obtain comprehensive normative data, to cross-validate BEAM measurements with other indicators of neural and cognitive function, and to evaluate the utility of these metrics within clinical populations of interest.

Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.

A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKß, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2ß1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network assembly. Overall, this work defines novel key regulators and their functional roles during human EC tubulogenesis.

Asthma prevalence in European, Maori, and Pacific children in New Zealand: ISAAC study.

The International Study of Asthma and Allergies in Childhood (ISAAC) demonstrated that the highest prevalence of asthma in the world is in English-speaking countries, including New Zealand. In this paper, we compare asthma symptom prevalence in the three major ethnic groups (Maori, Pacific, and European) in the six participating centers in New Zealand. Hospital admission rates for asthma are higher among Maori and Pacific children compared to European children. The working hypothesis was that there were important differences in prevalence of asthma symptoms or diagnosis between ethnic groups which might explain these observed differences in asthma morbidity. In each center in 1992-1993, we sampled approximately 3000 children at each of the age brackets 6-7 years and 13-14 years. There were 37592 participants. Maori children had higher rates of diagnosed asthma and reported asthma symptoms than Pacific children in both age groups (diagnosed asthma in 6-7-year-olds: Maori, 31.7%; Pacific, 21.2%; 95% confidence interval on difference (CID), 7.2, 13.8; P < 0.001; 13-14-year-olds: Maori, 24.7%; Pacific, 19.2%; CID 2.5, 8.5; P < 0.001; recent wheeze in 6-7-year-olds: Maori, 27.6%; Pacific, 22.0%; CID, 2.6, 8.6; P < 0.001; 13-14-year-olds: Maori, 30.8%; Pacific, 21.1%; CID, 4.8, 14.5; P < 0.001;). European children had rates intermediate between those of Maori and Pacific children (6-7-year-olds) or similar to those of Maori children (13-14-year-olds), but had the lowest prevalence of night waking with wheeze in both age groups (e.g., 6-7-year-olds: European, 2.6%; Maori, 5.8%; Pacific, 5.7%; European-Maori CID: -4.2, -2.2, P < 0.001; European-Pacific CID: -4.7, -1.7, P < 0.001; Maori-Pacific CID: -1.7, 1.8, P = 1.0). The pattern of differences closely resembled that in a 1985 Auckland study, despite a 1.5-1.7-fold overall increase in prevalence. In conclusion, there are important differences in asthma prevalence among Maori, Pacific, and European children. These differences are small compared to worldwide variation, but their pattern is stable over time. The higher rate of severe asthma symptoms that Maori and Pacific children report may be one reason for the increased asthma morbidity in these groups. Further studies are needed to determine the reasons for these apparent differences in asthma severity.