In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many ...Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35's primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development.
Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely studied, area of ...endothelial cell regulation is vesicular trafficking, also termed sorting. After moving through the Golgi apparatus, proteins are shuttled to organelles, plugged into membranes, recycled, or degraded depending on the internal and extrinsic cues. A snapshot of these protein-sorting systems can be viewed as a trafficking signature that is not only unique to endothelial tissue, but critically important for blood vessel form and function. In this review, we will cover how vesicular trafficking impacts various aspects of angiogenesis, such as sprouting, lumen formation, vessel stabilization, and secretion, emphasizing the role of Rab GTPase family members and their various effectors.
Objectives
Vesicular trafficking dictates protein localization, functional activity, and half‐life, providing a critically important regulatory step in tissue development; however, there is little ...information detailing endothelial‐specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3‐dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events.
Methods
Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed‐cell microscopy.
Results
Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture.
Conclusions
Our results establish that the fibrin bead sprouting assay is well‐suited for imaging of trafficking events during angiogenic growth.
Endocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy ...and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin-mediated and clathrin-independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis.
Primary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently-tagged constructs for live-cell imaging.
We found that Arf6 localized to both filamentous actin and sites of endocytosis in two-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence.
Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation.
Objective
Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is ...regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta‐like ligand 4 (Dll4) endocytosis during Notch activation.
Approach and Results
Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane‐bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE‐cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling.
Conclusions
Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.
Palatants may be added to equine feed and medication either during or after manufacturing to enhance product acceptance. Prior studies have examined a variety of palatants but results have been ...limited and inconsistent. Therefore, the objective of this research was to evaluate topically applied oil–based palatants on feeding preferences in horses. Stock-type horses (n = 10) were used in this paired preference test across a two-phase study. Phase one compared six palatants (banana, anise, peppermint, apple, spearmint, and orange) to a control (corn oil), whereas phase two compared preference among palatants (anise, apple, and peppermint). Feeding stocks were utilized and horses were allowed 15 seconds for olfaction followed by 3 minutes for consumption. Variables recorded included first diet sniffed and consumed, first action, aversive behaviors, excessive salivation, and consumption. Each trial was also video-recorded and number of chews were counted. Data were analyzed using chi-square and t tests in SAS version 9.4 with P < .05 established as significant. Findings from phase one reveal excessive salivation was observed less frequently (P < .05) for most palatants with the exception of peppermint and orange. Orange negatively impacted palatability indicated by less consumption when compared with the control (P = .02), although there was no impact on chews per gram. No difference between control and treatment diets for first sniff or first consumed was observed when analyzed individually in either phase one or two, although there was a moderate positive correlation (ф = 0.39, P = .04) between olfaction and consumption during the peppermint and anise comparison. Consumption as the first action was consistent across all trials (P < .05). Anise was preferred over apple and peppermint as indicated by higher total consumption (P < .05) in phase two. These data indicate that oil-based palatants can affect feed preferences in horses with increased palatability from anise and decreased palatability from orange flavors.
•Topically applied oil–based palatants influence the preference for concentrate diet.•Orange flavor decreased preference for concentrate.•Anise flavor increased preference compared with apple or peppermint flavors.
ObjectiveThere are no globally agreed on strategies on early detection and first response management of postpartum haemorrhage (PPH) during and after caesarean birth. Our study aimed to develop an ...international expert’s consensus on evidence-based approaches for early detection and obstetric first response management of PPH intraoperatively and postoperatively in caesarean birth.DesignSystematic review and three-stage modified Delphi expert consensus.SettingInternational.PopulationPanel of 22 global experts in PPH with diverse backgrounds, and gender, professional and geographic balance.Outcome measuresAgreement or disagreement on strategies for early detection and first response management of PPH at caesarean birth.ResultsExperts agreed that the same PPH definition should apply to both vaginal and caesarean birth. For the intraoperative phase, the experts agreed that early detection should be accomplished via quantitative blood loss measurement, complemented by monitoring the woman’s haemodynamic status; and that first response should be triggered once the woman loses at least 500 mL of blood with continued bleeding or when she exhibits clinical signs of haemodynamic instability, whichever occurs first. For the first response, experts agreed on immediate administration of uterotonics and tranexamic acid, examination to determine aetiology and rapid initiation of cause-specific responses. In the postoperative phase, the experts agreed that caesarean birth-related PPH should be detected primarily via frequently monitoring the woman’s haemodynamic status and clinical signs and symptoms of internal bleeding, supplemented by cumulative blood loss assessment performed quantitatively or by visual estimation. Postoperative first response was determined to require an individualised approach.ConclusionThese agreed on proposed approaches could help improve the detection of PPH in the intraoperative and postoperative phases of caesarean birth and the first response management of intraoperative PPH. Determining how best to implement these strategies is a critical next step.
To determine the rate of long-term cochlear implant (CI) use in children.
Consecutive case series.
Tertiary referral center.
Approximately 474 patients younger than 18 years who received a first CI ...from 1999 to 2011.
Cochlear implantation.
Regular CI use, defined as using the CI for 8 hours or greater per day.
We successfully contacted and obtained follow-up data on 402 patients (85%) via email, telephone, and postal survey. The rate of regular CI use was 93.2% (95% CI, 90.0-95.4) at 5 years postimplantation and 87.7% (95% CI, 82.9-91.3) at 10 years postimplantation. The mean number of hours of use per day was 12.0 hours (SD, 4.1 h). Cox proportional hazard regression analysis demonstrated a linear association between the age at implantation and the risk of discontinuing regular CI use. Rates of CI discontinuation increased by 18.2% per year of age at implantation (95% CI, 7.2%-30.4%). Reported reasons for CI use less than 8 hours per day include poor hearing benefit (53.2%), social pressure (21.3%), and recurrent displacement of the transmitter coil (17.0%).
High rates of regular CI use are sustained after childhood implantation, and younger age at implantation is associated with a higher rate of continued device usage.
PDF
