Proinflammatory mediators, TNFα, IFNγ, and thrombin, directly induce lymphatic capillary tube regression

In this work, we sought to investigate the direct effects of proinflammatory mediators on lymphatic endothelial cell (LEC) capillaries and whether they might induce regression. Our laboratory has developed novel in-vitro, serum-free, lymphatic tubulogenesis assay models whereby human LEC tube networks readily form in either three-dimensional collagen or fibrin matrices. These systems were initially conceptualized in the hopes of better understanding the influence of proinflammatory mediators on LEC capillaries. In this work, we have screened and identified proinflammatory mediators that cause regression of LEC tube networks, the most potent of which is TNFα (tumor necrosis factor alpha), followed by IFNγ (interferon gamma) and thrombin. When these mediators were combined, even greater and more rapid lymphatic capillary regression occurred. Surprisingly, IL-1β (interleukin-1 beta), one of the most potent and pathologic cytokines known, had no regressive effect on these tube networks. Finally, we identified new pharmacological drug combinations capable of rescuing LEC capillaries from regression in response to the potent combination of TNFα, IFNγ, and thrombin. We speculate that protecting lymphatic capillaries from regression may be an important step toward mitigating a wide variety of acute and chronic disease states, as lymphatics are believed to clear both proinflammatory cells and mediators from inflamed and damaged tissue beds. Overall, these studies identify key proinflammatory mediators, including TNFα, IFNγ, and thrombin, that induce regression of LEC tube networks, as well as identify potential therapeutic agents to diminish LEC capillary regression responses

Obesity Prevalence and Dietary Factors Among Preschool-Aged Head Start Children in Remote Alaska Native Communities: Baseline Data from the ‘‘Got Neqpiaq?’’ Study

Background: American Indian and Alaska Native preschool-aged children experience a high prevalence of obesity, yet are underrepresented in obesity prevention research. This study examined obesity prevalence and dietary risk factors among Alaska Native preschool-aged children in southwest Alaska. Methods: The study used baseline data from ‘‘Got Neqpiaq?’’ a culturally centered multilevel intervention focused on Yup’ik Alaska Native children, aged 3–5 years, enrolled in Head Start in 12 communities in southwest Alaska (n = 155). The primary outcomes were BMI percentile, overweight, and obesity. Dietary factors of interest were measured using biomarkers: traditional food intake (nitrogen stable isotope ratio biomarker), ultraprocessed food intake (carbon stable isotope ratio biomarker), and vegetable and fruit intake (skin carotenoid status biomarker measured by the Veggie Meter). Cardiometabolic markers (glycated hemoglobin [HbA1c] and blood cholesterol) were also measured. Results: Among the Yup’ik preschool-aged children in the study, the median BMI percentile was 91, and the prevalence of overweight or obesity was 70%. The traditional food intake biomarker was negatively associated with BMI, whereas the ultraprocessed foods and vegetable and fruit biomarkers were not associated with BMI. HbA1c and blood cholesterol were within healthy levels. Conclusions: The burden of overweight and obesity is high among Yup’ik preschool-aged children. Traditional food intake is inversely associated with BMI, which underscores the need for culturally grounded interventions that emphasize traditional values and knowledge to support the traditional food systems in Alaska Native communities in southwest Alaska. Registered with ClinicalTrials.gov #NCT03601299.Ye

Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus

SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis

Expression of <i>abl1</i> and <i>abl2</i> in different cell types during zebrafish embryogenesis based on single-cell RNA seq expression atlas.

Data were generated by Daniocell atlas https://daniocell.nichd.nih.gov/ [31]. (TIF)</p

Consensus ABL phosphorylation sites YXXP are required for SHE function.

(A) Consensus ABL phosphorylation sites are conserved between zebrafish, mice and humans. (B) Vascular endothelial expression of a mutant construct fli1:sheFXXP-2A-mCherry, where all four consensus tyrosines have been substituted into phenylalanine, fails to rescue the pericardial edema in she mutants at 4 dpf. The first bar showing she-/- embryos (no Tg) is copied from Fig 3I. ****pfli1:sheFXXP-2A-mCherry embryos and sibling mCherry-negative embryos at 28 hpf. 5 measurements were performed in each embryo, which were then averaged for statistical calculations. 2 replicate experiments were performed, shown in different colors. n corresponds to the number of embryos. Mean ± SD is shown. *p<0.05, Student’s t-test.</p

Hybridization chain reaction (HCR) analysis of <i>cldn5b</i> mRNA expression at 24 hpf.

(A,B) cldn5b (purple) and kdrl:GFP fluorescence in the trunk region of she mutant and wild-type sibling embryos. DA, dorsal aorta; PCV, posterior cardinal vein. cldn5b fluorescence is shown in A’,B’. (C) Quantification of cldn5b fluorescence in the DA. p = 0.17, Student’s t-test. Error bars show SEM. Data show combined results from two independent experiments. (TIF)</p

Blood flow in the tail region of wild-type zebrafish embryos.

Wild-type siblings obtained from an incross of sheci26 heterozygous adults in kdrl:GFP; gata1:dsRed background were imaged at 4 dpf using confocal microscopy. (MP4)</p

A proposed model for SHE and ABL signaling during vascular tubulogenesis.

Activated ABL promotes enlarged vascular lumen through a downstream effector P-CRKL which increases endothelial cell proliferation and increases Cldn5 expression, thus affecting tight junctions and cell adhesion. Activated ABL phosphorylates SHE, which then interacts with ABL to dampen its activity resulting in the lumen of appropriate size.</p