Pancreatic ß-Cells Communicate With Vagal Sensory Neurons.

BACKGROUND AND AIMS: Destroying visceral sensory nerves impacts pancreatic islet function, glucose metabolism, and diabetes onset, but how islet endocrine cells interact with sensory neurons has not been studied. METHODS: We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of ß-cells with vagal sensory neurons, we recorded Ca2+ responses in individual nodose neurons in vivo while selectively stimulating ß-cells with chemogenetic and pharmacologic approaches. RESULTS: We found that pancreatic islets are innervated by vagal sensory axons expressing Phox2b, substance P, calcitonin-gene related peptide, and the serotonin receptor 5-HT3R. Centrally, vagal neurons projecting to the pancreas terminate in the commissural nucleus of the solitary tract. Nodose neurons responded in vivo to chemogenetic stimulation of ß-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of ß-cells were blocked by a 5-HT3R antagonist and were enhanced by increasing serotonin levels in ß-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin. CONCLUSIONS: Our study establishes that pancreatic ß-cells communicate with vagal sensory neurons, likely using serotonin signaling as a transduction mechanism. Serotonin is coreleased with insulin and may therefore convey information about the secretory state of ß-cells via vagal afferent nerves.

Live imaging of multicolor-labeled cells in Drosophila.

We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution.

Upregulation of Caveolae-Associated Proteins in Lesional Samples of Hidradenitis Suppurativa: A Case Series Study.

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin condition. HS disease management has proven difficult owing to an insufficient understanding of the immunological processes that drive its pathogenesis. We have demonstrated that misregulation of caveolae perturbs inflammatory responses, inhibits cutaneous wound healing, and contributes to immune privilege collapse in other hair follicle-related diseases. However, nothing is known about its role or the role of structural components of caveolae (caveolin [Cav1] 1, Cav2, and Cavin-1) in the pathophysiology of HS. We aimed to identify whether Cav1, Cav2, and Cavin-1 may serve as immunohistochemical markers of HS. Lesional and perilesional HS skin samples from patients (n = 7, mean age = 35.7 years, range = 20-57 years) with active HS and normal skin from control participants (n = 4, mean age = 36.7 years, range = 23-49 years) were used to assess Cav1, Cav2, and Cavin-1 expression and localization by immunofluorescence staining. HS samples demonstrated increased levels of Cav1 compared with normal skin, whereas Cav1, Cav2, and Cavin-1 were all elevated in hair follicles of lesional versus perilesional HS samples, suggesting a potentially novel therapeutic target and highlighting caveolae as potential biomarkers of HS.

Pericyte dysfunction and impaired vasomotion are hallmarks of islets during the pathogenesis of type 1 diabetes.

Pancreatic islets are endocrine organs that depend on their microvasculature to function. Along with endothelial cells, pericytes comprise the islet microvascular network. These mural cells are crucial for microvascular stability and function, but it is not known if/how they are affected during the development of type 1 diabetes (T1D). Here, we investigate islet pericyte density, phenotype, and function using living pancreas slices from donors without diabetes, donors with a single T1D-associated autoantibody (GADA+), and recent onset T1D cases. Our data show that islet pericyte and capillary responses to vasoactive stimuli are impaired early on in T1D. Microvascular dysfunction is associated with a switch in the phenotype of islet pericytes toward myofibroblasts. Using publicly available RNA sequencing (RNA-seq) data, we further found that transcriptional alterations related to endothelin-1 signaling and vascular and extracellular matrix (ECM) remodeling are hallmarks of single autoantibody (Aab)+ donor pancreata. Our data show that microvascular dysfunction is present at early stages of islet autoimmunity.

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control.

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors.

A Practical Approach to Quantitative Processing and Analysis of Small Biological Structures by Fluorescent Imaging.

Standards in quantitative fluorescent imaging are vaguely recognized and receive insufficient discussion. A common best practice is to acquire images at Nyquist rate, where highest signal frequency is assumed to be the highest obtainable resolution of the imaging system. However, this particular standard is set to insure that all obtainable information is being collected. The objective of the current study was to demonstrate that for quantification purposes, these correctly set acquisition rates can be redundant; instead, linear size of the objects of interest can be used to calculate sufficient information density in the image. We describe optimized image acquisition parameters and unbiased methods for processing and quantification of medium-size cellular structures. Sections of rabbit aortas were immunohistochemically stained to identify and quantify sympathetic varicosities, >2 µm in diameter. Images were processed to reduce background noise and segment objects using free, open-access software. Calculations of the optimal sampling rate for the experiment were based on the size of the objects of interest. The effect of differing sampling rates and processing techniques on object quantification was demonstrated. Oversampling led to a substantial increase in file size, whereas undersampling hindered reliable quantification. Quantification of raw and incorrectly processed images generated false structures, misrepresenting the underlying data. The current study emphasizes the importance of defining image-acquisition parameters based on the structure(s) of interest. The proposed postacquisition processing steps effectively removed background and noise, allowed for reliable quantification, and eliminated user bias. This customizable, reliable method for background subtraction and structure quantification provides a reproducible tool for researchers across biologic disciplines.

BMP-7 Induces Adult Human Pancreatic Exocrine-to-Endocrine Conversion.

The exocrine pancreas can give rise to endocrine insulin-producing cells upon ectopic expression of key transcription factors. However, the need for genetic manipulation remains a translational hurdle for diabetes therapy. Here we report the conversion of adult human nonendocrine pancreatic tissue into endocrine cell types by exposure to bone morphogenetic protein 7. The use of this U.S. Food and Drug Administration-approved agent, without any genetic manipulation, results in the neogenesis of clusters that exhibit high insulin content and glucose responsiveness both in vitro and in vivo. In vitro lineage tracing confirmed that BMP-7-induced insulin-expressing cells arise mainly from extrainsular PDX-1(+), carbonic anhydrase II(-) (mature ductal), elastase 3a (acinar)(-) , and insulin(-) subpopulations. The nongenetic conversion of human pancreatic exocrine cells to endocrine cells is novel and represents a safer and simpler alternative to genetic reprogramming.

Imaging dynamic molecular signaling by the Cdc42 GTPase within the developing CNS.

Protein interactions underlie the complexity of neuronal function. Potential interactions between specific proteins in the brain are predicted from assays based on genetic interaction and/or biochemistry. Genetic interaction reveals endogenous, but not necessarily direct, interactions between the proteins. Biochemistry-based assays, on the other hand, demonstrate direct interactions between proteins, but often outside their native environment or without a subcellular context. We aimed to achieve the best of both approaches by visualizing protein interaction directly within the brain of a live animal. Here, we show a proof-of-principle experiment in which the Cdc42 GTPase associates with its alleged partner WASp within neurons during the time and space that coincide with the newly developing CNS.

Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed.

KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.