Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

NIH workshop report on the trans-agency blood-brain interface workshop 2016: exploring key challenges and opportunities associated with the blood, brain and their interface.

A trans-agency workshop on the blood-brain interface (BBI), sponsored by the National Heart, Lung and Blood Institute, the National Cancer Institute and the Combat Casualty Care Research Program at the Department of Defense, was conducted in Bethesda MD on June 7-8, 2016. The workshop was structured into four sessions: (1) blood sciences; (2) exosome therapeutics; (3) next generation in vitro blood-brain barrier (BBB) models; and (4) BBB delivery and targeting. The first day of the workshop focused on the physiology of the blood and neuro-vascular unit, blood or biofluid-based molecular markers, extracellular vesicles associated with brain injury, and how these entities can be employed to better evaluate injury states and/or deliver therapeutics. The second day of the workshop focused on technical advances in in vitro models, BBB manipulations and nanoparticle-based drug carrier designs, with the goal of improving drug delivery to the central nervous system. The presentations and discussions underscored the role of the BBI in brain injury, as well as the role of the BBB as both a limiting factor and a potential conduit for drug delivery to the brain. At the conclusion of the meeting, the participants discussed challenges and opportunities confronting BBI translational researchers. In particular, the participants recommended using BBI translational research to stimulate advances in diagnostics, as well as targeted delivery approaches for detection and therapy of both brain injury and disease.

NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies.

NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the pineal gland and retina. At 2 months of age, NeuroD1 cKO retinas have a dramatic reduction in rod- and cone-driven electroretinograms and contain shortened and disorganized outer segments; by 4 months, NeuroD1 cKO retinas are devoid of photoreceptors. In contrast, the NeuroD1 cKO pineal gland appears histologically normal. Microarray analysis of 2-month-old NeuroD1 cKO retina and pineal gland identified a subset of genes that were affected 2-100-fold; in addition, a small group of genes exhibit altered differential night/day expression. Included in the down-regulated genes are Aipl1, which is necessary to prevent retinal degeneration, and Ankrd33, whose protein product is selectively expressed in the outer segments. These findings suggest that NeuroD1 may act through Aipl1 and other genes to maintain photoreceptor homeostasis.

Using the Tg(nrd:egfp)/albino zebrafish line to characterize in vivo expression of neurod.

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.

Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors.

Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes beta-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable beta-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulin-like growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IGFBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation.