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.

Comparison of Prognostic Scores in Early Phase Clinical Trials: A 10-year Single Centre Australian Experience.

BACKGROUND/AIM: Early phase clinical trials (EPCTs) assess the tolerability of novel anti-cancer therapeutics in patients with advanced malignancy. Patient selection is important given the modest clinical benefit and time commitments for trials. Prognostic scores have been developed to facilitate identification of high-risk patients. This study aimed to compare five prognostic scores to predict survival for patients on an EPCT. PATIENTS AND METHODS: We performed a retrospective review of patients enrolled in EPCT at Liverpool Hospital, Sydney, from 2013 to 2023. Demographic, biochemical, and survival data were collected from electronic medical records. The score from five prognostic scoring systems (Royal Marsden hospital, MD Anderson Cancer centre, Gustave Roussy Immune, MD Anderson Immune Checkpoint Inhibitor and Princess Margaret Hospital Index) were calculated. Overall survival was measured using the Kaplan-Meier method and predictive discrimination was assessed using Harrell's c-index. RESULTS: A total of 218 patients across 36 EPCTs were included. The median overall survival was 9.8 months with 22% of patients dying in less than 90 days. Seventeen to thirty-four percent of patients were categorised as high-risk. The MDACC score obtained the highest predictability for overall survival for the whole cohort (c-index=0.67, 95%CI=0.62-0.72) and the immunotherapy-based cohort (c-index= 0.65, 95%CI=0.59-0.71). However, all scores performed similarly with a significant overlap in the confidence intervals. CONCLUSION: Our retrospective audit confirms the utility of prognostic scores to predict survival in an Australian EPCT cohort, with similar predictive discrimination across various scoring systems. Integration of these prognostic tools into EPCT screening processes may optimise benefits and reduce risks associated with EPCTs.

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.

A microtiter peg lid with ziggurat geometry for medium-throughput antibiotic testing and in situ imaging of biofilms.

Bacteria biofilm responses to disinfectants and antibiotics are quantified and observed using multiple methods, though microscopy, particularly confocal laser scanning microscopy (CLSM) is preferred due to speed, a reduction in user error, and in situ analysis. CLSM can resolve biological and spatial heterogeneity of biofilms in 3D with limited throughput. The microplate peg-lid-based assay, described in ASTM E2799-22, is a medium-throughput method for testing biofilms but does not permit in situ imaging. Breaking off the peg, as recommended by the manufacturer, risks sample damage, and is limited to easily accessible pegs. Here we report modifications to the peg optimized for in situ visualization and visualization of all pegs. We report similar antibiotic challenge recovery via colony formation following the ASTM E2799-22 protocol and in situ imaging. We report novel quantifiable effects of antibiotics on biofilm morphologies, specifically biofilm streamers. The new design bridges the MBEC® assays design that selects for biofilm phenotypes with in situ imaging needs.

Analysis of treatment cost differences in patients undergoing femoral versus radial access in outpatient diagnostic cerebral arteriograms.

BACKGROUND: Outpatient diagnostic cerebral arteriograms are the most common procedure in neuroendovascular surgery, and the use of transradial access for these studies is growing. Although transradial access has been associated with lower hospital costs for elective diagnostic and interventional neuroendovascular procedures, no study has compared transfemoral access and transradial access costs for a homogenous population of patients undergoing outpatient diagnostic cerebral arteriogram. METHODS: In this single-center retrospective study, the Value Driven Outcomes database was used to evaluate treatment costs for patients who underwent outpatient diagnostic cerebral arteriogram from January 2019 to December 2022. Propensity-score matching was performed to reduce confounders. Costs from each encounter were subcategorized into imaging, supplies, pharmacy, procedures, labs, and facility costs. RESULTS: After matching, 337 patients each for transradial access and transfemoral access were available for analysis. A total of 118,992 cost data points were associated with all encounters. Overall, per-visit costs were 15.2% cheaper for patients who underwent transradial access versus transfemoral access (p < 0.001). Most of the cost difference was due to supplies (35.2% cost difference, p < 0.001) and procedure costs (9.3% cost difference, p < 0.001). No statistical differences were observed between the two approaches in imaging, pharmacy, labs, and facility costs (all p > 0.05). CONCLUSIONS: Costs for outpatient diagnostic cerebral arteriogram were lower in patients who underwent transradial access versus transfemoral access because of supply and procedure costs. Understanding reasons for cost differences in common procedures is important for creating strategies to reduce overall healthcare costs. Additionally, addressing the cost differences of newer techniques may increase the likelihood that they are more readily implemented by hospitals and providers.

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.

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.

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.

Hnrnpul1 controls transcription, splicing, and modulates skeletal and limb development in vivo.

Mutations in RNA-binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb, and neurological symptoms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription, and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced body and fin growth and missing bones. Defects in craniofacial tendon growth and adult-onset caudal scoliosis are also seen. We demonstrate a role for Hnrnpul1 in alternative splicing and transcriptional regulation using RNA-sequencing, particularly of genes involved in translation, ubiquitination, and DNA damage. Given its cross-species conservation and role in splicing, it would not be surprising if it had a role in human development. Whole-exome sequencing detected a homozygous frameshift variant in HNRNPUL1 in 2 siblings with congenital limb malformations, which is a candidate gene for their limb malformations. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring the potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.

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.

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.

Semaphorin 3fa Controls Ocular Vascularization From the Embryo Through to the Adult.

Purpose: Pathological blood vessel growth in the eye is implicated in several diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. The limits of current disease therapies have created the need to identify and characterize new antiangiogenic drugs. Here, we identify the secreted chemorepellent semaphorin-3fa (Sema3fa) as an endogenous anti-angiogenic in the eye. Methods: We generated a CRISPR/Cas9 sema3fa zebrafish mutant line, sema3faca304/304. We assessed the retinal and choroidal vasculature in both larval and adult wild-type and sema3fa mutant zebrafish. Results: We find sema3fa mRNA is expressed by the ciliary marginal zone, neural retina, and retinal pigment epithelium of zebrafish larvae as choroidal vascularization emerges and the hyaloid/retinal vasculature is remodeled. The hyaloid vessels of sema3fa mutants develop appropriately but fail to remodel during the larval period, with adult mutants exhibiting a denser network of capillaries in the retinal periphery than seen in wild-type. The choroid vasculature is also defective in that it develops precociously, and aberrant, leaky sprouts are present in the normally avascular outer retina of both sema3faca304/304 larvae and adult fish. Conclusions: Sema3fa is a key endogenous signal for maintaining an avascular retina and preventing pathologic vascularization. Furthermore, we provide a new experimentally accessible model for studying choroid neovascularization (CNV) resulting from primary changes in the retinal environment that lead to downstream vessel infiltration.

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.

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.

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.

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.

Mutations in ILK, encoding integrin-linked kinase, are associated with arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy is a genetic heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes leading to life-threatening ventricular arrhythmias, heart failure, and sudden cardiac death. Mutations in genes encoding cardiac junctional proteins are known to cause about half of cases, while remaining genetic causes are unknown. Using exome sequencing, we identified 2 missense variants (p.H33N and p.H77Y) that were predicted to be damaging in the integrin-linked kinase (ILK) gene in 2 unrelated families. The p.H33N variant was found to be de novo. ILK links integrins and the actin cytoskeleton, and is essential for the maintenance of normal cardiac function. Both of the new variants are located in the ILK ankyrin repeat domain, which binds to the first LIM domain of the adaptor proteins PINCH1 and PINCH2. In silico binding studies proposed that the human variants disrupt the ILK-PINCH complex. Recombinant mutant ILK expressed in H9c2 rat myoblast cells shows aberrant prominent cytoplasmic localization compared to the wild-type. Expression of human wild-type and mutant ILK under the control of the cardiac-specific cmlc2 promotor in zebrafish shows that p.H77Y and p.P70L, a variant previously reported in a dilated cardiomyopathy family, cause cardiac dysfunction and death by about 2-3 weeks of age. Our findings provide genetic and functional evidence that ILK is a cardiomyopathy disease gene and highlight its relevance for diagnosis and genetic counseling of inherited cardiomyopathies.

Biotherapeutic Antibody Subunit LC-MS and Peptide Mapping LC-MS Measurements to Study Possible Biotransformation and Critical Quality Attributes In Vivo.

Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography-mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.