Continuous Lumen Research Articles

Integrating collecting systems in kidney organoids through fusion of distal nephron to ureteric bud.

The kidney maintains homeostasis through an array of parallel nephrons, which all originate in development as isolated epithelial structures that later fuse through their distal poles to a system of collecting ducts (CD). This connection is required to generate functional nephrons by providing a pathway for excretion of metabolic waste and byproducts. Currently, methods for differentiating human pluripotent stem cells into kidney organoids generate nephrons that lack CDs and instead terminate as blind-ended tubules. Here we describe a developmentally inspired system that addresses this deficiency through assembly of induced nephrogenic mesenchyme with ureteric bud (UB) tissues, the embryonic building blocks of the kidney's collecting system. The UB progenitors grow and develop into a network of CDs within the organoid, and importantly, they functionally integrate with the nephrons through recapitulating fusion between the distal tubule and CD to create a continuous epithelial lumen. We further showed that proximal-distal nephron specification, fusion frequency, and maturation of the CD can be augmented through temporal manipulation of developmental signaling pathways. This work provides a platform for interrogating the principles and mechanisms underlying nephron-UB fusion and a framework for engineering unobstructed nephrons with patterned collecting systems, an important step toward the de novo generation of functional kidney tissue.

Oscillatory contractile forces refine endothelial cell-cell interactions for continuous lumen formation governed by Heg1/Ccm1.

The formation and organization of complex blood vessel networks rely on various biophysical forces, yet the mechanisms governing endothelial cell-cell interactions under different mechanical inputs are not well understood. Using the dorsal longitudinal anastomotic vessel (DLAV) in zebrafish as a model, we studied the roles of multiple biophysical inputs and cerebral cavernous malformation (CCM)-related genes in angiogenesis. Our research identifies heg1 and krit1 (ccm1) as crucial for the formation of endothelial cell-cell interfaces during anastomosis. In mutants of these genes, cell-cell interfaces are entangled with fragmented apical domains. A Heg1 live reporter demonstrated that Heg1 is dynamically involved in the oscillatory constrictions along cell-cell junctions, whilst a Myosin live reporter indicated that heg1 and krit1 mutants lack actomyosin contractility along these junctions. In wild-type embryos, the oscillatory contractile forces at junctions refine endothelial cell-cell interactions by straightening junctions and eliminating excessive cell-cell interfaces. Conversely, in the absence of junctional contractility, the cell-cell interfaces become entangled and prone to collapse in both mutants, preventing the formation of a continuous luminal space. By restoring junctional contractility via optogenetic activation of RhoA, contorted junctions are straightened and disentangled. Additionally, haemodynamic forces complement actomyosin contractile forces in resolving entangled cell-cell interfaces in both wild-type and mutant embryos. Overall, our study reveals that oscillatory contractile forces governed by Heg1 and Krit1 are essential for maintaining proper endothelial cell-cell interfaces and thus for the formation of a continuous luminal space, which is essential to generate a functional vasculature.

Rab11 is essential to pancreas morphogenesis, lumen formation and endocrine mass

The molecular links between tissue-level morphogenesis and the differentiation of cell lineages in the pancreas remain elusive despite a decade of studies. We previously showed that in pancreas both processes depend on proper lumenogenesis. The Rab GTPase Rab11 is essential for epithelial lumen formation in vitro, however few studies have addressed its functions in vivo and none have tested its requirement in pancreas. Here, we show that Rab11 is critical for proper pancreas development. Co-deletion of the Rab11 isoforms Rab11A and Rab11B in the developing pancreatic epithelium (Rab11pancDKO) results in ∼50% neonatal lethality and surviving adult Rab11pancDKO mice exhibit defective endocrine function. Loss of both Rab11A and Rab11B in the embryonic pancreas results in morphogenetic defects of the epithelium, including defective lumen formation and lumen interconnection. In contrast to wildtype cells, Rab11pancDKO cells initiate the formation of multiple ectopic lumens, resulting in a failure to coordinate a single apical membrane initiation site (AMIS) between groups of cells. This results in an inability to form ducts with continuous lumens. Here, we show that these defects are due to failures in vesicle trafficking, as apical and junctional components remain trapped within Rab11pancDKO cells. Together, these observations suggest that Rab11 directly regulates epithelial lumen formation and morphogenesis. Our report links intracellular trafficking to organ morphogenesis in vivo and presents a novel framework for decoding pancreatic development.

Meshed neuronal mitochondrial networks empowered by AI-powered classifiers and immersive VR reconstruction.

Mitochondrial networks are defined as a continuous matrix lumen, but the morphological feature of neuronal mitochondrial networks is not clear due to the lack of suitable analysis techniques. The aim of the present study is to develop a framework to capture and analyze the neuronal mitochondrial networks by using 4-step process composed of 2D and 3D observation, primary and secondary virtual reality (VR) analysis, with the help of artificial intelligence (AI)-powered Aivia segmentation an classifiers. In order to fulfill this purpose, we first generated the PCs-Mito-GFP mice, in which green fluorescence protein (GFP) could be expressed on the outer mitochondrial membrane specifically on the cerebellar Purkinje cells (PCs), thus all mitochondria in the giant neuronal soma, complex dendritic arborization trees and long projection axons of Purkinje cells could be easily detected under a laser scanning confocal microscope. The 4-step process resolved the complicated neuronal mitochondrial networks into discrete neuronal mitochondrial meshes. Second, we measured the two parameters of the neuronal mitochondrial meshes, and the results showed that the surface area (μm2) of mitochondrial meshes was the biggest in dendritic trees (45.30 ± 53.21), the smallest in granular-like axons (3.99 ± 1.82), and moderate in soma (27.81 ± 22.22) and silk-like axons (17.50 ± 15.19). These values showed statistically different among different subcellular locations. The volume (μm3) of mitochondrial meshes was the biggest in dendritic trees (9.97 ± 12.34), the smallest in granular-like axons (0.43 ± 0.25), and moderate in soma (6.26 ± 6.46) and silk-like axons (3.52 ± 4.29). These values showed significantly different among different subcellular locations. Finally, we found both the surface area and the volume of mitochondrial meshes in dendritic trees and soma within the Purkinje cells in PCs-Mito-GFP mice after receiving the training with the simulating long-term pilot flight concentrating increased significantly. The precise reconstruction of neuronal mitochondrial networks is extremely laborious, the present 4-step workflow powered by artificial intelligence and virtual reality reconstruction could successfully address these challenges.

1D continuous ZIF-8 tubes incorporated PDMS mixed matrix membrane for superior ethyl acetate pervaporation separation

• 1D ZIF-8 tubes were incorporated in PDMS for membrane preparation. • Continuous permselective molecular pathways constructed by ZIF-8 tubes. • Superior separation performances by tubes than conventional nanoparticles. • Longer ZIF-8 tubes contributed more for enhancing ethyl acetate pervaporation. Mixed matrix membranes (MMMs) filled with dispersed nanoparticles may not favor all requirements for high separation performances due to the discontinuity in molecular permeation pathways. Here, we reported polydimethylsiloxane (PDMS) based MMMs by incorporating one-dimensional (1D) ZIF-8 tubes for the pervaporation (PV) recovery of ethyl acetate (EtOAc) from its diluted aqueous solution. The 1D ZIF-8 is a tubular structure formed by the ZIF-8 crystal particles inlaying each other, some windows between the ZIF-8 crystal particles are seamlessly connected, so that the cavities of the ZIF-8 are connected through the windows. The ZIF-8 tubes not merely possess microporous and molecular sieving properties, but also are inherently continuous in the integral morphology. The morphological continuity renders docking micropores along the tube body, which can act as efficient molecular permselective pathways for EtOAc molecules. Both the short and long-sized ZIF-8 tubes (STZIF-8 and LTZIF-8) played greater roles in facilitating PV performances than that of dispersed nanoparticles. Importantly, the most remarkable effect of the LTZIF-8 on separation performance was obtained since their micron-sized continuous structure, connected cavities and lumen, is more conductive to the separation process. The MMMs with 8% loading of the LTZIF-8 exhibited superior separation factor of 591.9 and EtOAc flux of 0.82 kg·m −2 ·h −1 for separating 1 wt% feed solution at 30 °C, which are 3.11 and 3.45 times of that of PDMS membrane, respectively. This study may give inspiration for developing high-performance MMMs by incorporating such morphologically continuous, microstructurally porous and molecular sieving fillers as permselective pathways for guest molecules.

Non-Standard Management of Target Vessels With the Inner Branch Arch Endograft: A Single-Center Retrospective Study

Purpose: The purpose of this study was to evaluate early and mid-term results of non-standard management of the supraaortic target vessels with the use of the inner branch arch endograft in a single high-volume center. Material and methods: A single-center retrospective study including all patients undergoing implantation of an inner branch arch endograft from December 2012 to March 2021, who presented a non-standard management of the supraaortic target vessels (any bypass other than a left carotid-subclavian or landing in a dissected target vessel). Technical success, mortality, reinterventions, endoleak (EL), and aortic remodeling at follow-up were analyzed. Results: Twenty-four patients were included. In 17 (71%) cases, the non-standard management was related to innominate artery (IA) compromise (12 with IA dissection, 2 with short IA, 2 with short proximal aortic landing zone that required occlusion of IA, 1 with occluded IA after open arch repair). Two (8%) cases were related to an aberrant right subclavian artery (RSA), 1 patient (4%) due to the concomitant presence of a left vertebral artery (LVA) arising from the arch and an occluded left subclavian artery (LSA), and another patient presented with an occluded LSA distal to a dominant vertebral artery. Three (13%) cases were exclusively related to management in patients with genetic aortic syndromes. Twenty (83%) patients had a previous type A aortic dissection. Ten (42%) patients presented a thoracic or thoracoabdominal aortic aneurysm and 8 (33%) patients an arch aneurysm, 6 of them associated to false lumen (FL) perfusion. There were 2 (8%) perioperative minor strokes, and 1 patient with perioperative mortality. Seven patients presented an early type I endoleak, all resolved at follow-up. Seven patients required reinterventions during follow-up (7 reinterventions related to continuous false lumen perfusion, 3 related to Type Ia endoleak, 2 related to surgical bypass). All patients who presented with FL perfusion had complete FL thrombosis at follow-up. No patient presented aneurysm growth at follow-up. Conclusions: The use of the inner branch arch endograft with a non-standard management of the supraaortic target vessels is a possible option. Despite a high reintervention rate, regression or stability of the aneurysmal diameter was achieved in all the patients with follow-up.

Apical PAR complex proteins protect against programmed epithelial assaults to create a continuous and functional intestinal lumen.

Sustained polarity and adhesion of epithelial cells is essential for the protection of our organs and bodies, and this epithelial integrity emerges during organ development amidst numerous programmed morphogenetic assaults. Using the developing Caenorhabditis elegans intestine as an in vivo model, we investigated how epithelia maintain their integrity through cell division and elongation to build a functional tube. Live imaging revealed that apical PAR complex proteins PAR-6/Par6 and PKC-3/aPkc remained apical during mitosis while apical microtubules and microtubule-organizing center (MTOC) proteins were transiently removed. Intestine-specific depletion of PAR-6, PKC-3, and the aPkc regulator CDC-42/Cdc42 caused persistent gaps in the apical MTOC as well as in other apical and junctional proteins after cell division and in non-dividing cells that elongated. Upon hatching, gaps coincided with luminal constrictions that blocked food, and larvae arrested and died. Thus, the apical PAR complex maintains apical and junctional continuity to construct a functional intestinal tube.

Scalable fabrication of renal spheroids and nephron-like tubules by bioprinting and controlled self-assembly of epithelial cells

Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120–131 µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22 µm). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm−2 driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.

Bio-assembling Macro-Scale, Lumenized Airway Tubes of Defined Shape via Multi-Organoid Patterning and Fusion.

Epithelial, stem‐cell derived organoids are ideal building blocks for tissue engineering, however, scalable and shape‐controlled bio‐assembly of epithelial organoids into larger and anatomical structures is yet to be achieved. Here, a robust organoid engineering approach, Multi‐Organoid Patterning and Fusion (MOrPF), is presented to assemble individual airway organoids of different sizes into upscaled, scaffold‐free airway tubes with predefined shapes. Multi‐Organoid Aggregates (MOAs) undergo accelerated fusion in a matrix‐depleted, free‐floating environment, possess a continuous lumen, and maintain prescribed shapes without an exogenous scaffold interface. MOAs in the floating culture exhibit a well‐defined three‐stage process of inter‐organoid surface integration, luminal material clearance, and lumina connection. The observed shape stability of patterned MOAs is confirmed by theoretical modelling based on organoid morphology and the physical forces involved in organoid fusion. Immunofluorescent characterization shows that fused MOA tubes possess an unstratified epithelium consisting mainly of tracheal basal stem cells. By generating large, shape‐controllable organ tubes, MOrPF enables upscaled organoid engineering towards integrated organoid devices and structurally complex organ tubes.

Digestive tract regeneration in the posteriorly eviscerating sea cucumber Holothuria parva (Holothuroidea, Echinodermata)

In the present study, the regeneration process following posterior evisceration along with changes in antioxidant enzymes during regeneration in Holothuria parva was investigated for the first time. In the present study, evisceration was induced after molecular and morphological identification of the species. Most of the digestive tract and the left respiratory tree were removed from the body through the anus. The tissue regeneration of the digestive tract and changes in the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (LPO)] were examined during 75 days. According to the results, evisceration in this species was from the anus and the regeneration process could be divided into four steps based on the histomorphological alterations. The digestive tract grew in the form of a straight cord from the anterior end to the cloaca. During the regeneration process, several cavities were created inside the cord, which gradually joined to form a continuous lumen. For the first time on the 24th day after evisceration, food was found in the digestive tract. Fifty days after evisceration, the tissue structure of different parts derived from regenerated digest tract was found to be similar to that in the intact digestive tract. According to the results, 5 days after evisceration the activity of CAT and SOD in the regenerated digestive tract was significantly higher than in the control. The CAT and SOD levels gradually decreased in the regenerated digestive tract, although by the end of the experiment it was significantly higher than in the control group. The LPO level in eviscerated animals showed a decreasing trend during the experiment and its value decreased to the control level at the end of the experiment.

Fusion of airways during avian lung development constitutes a novel mechanism for the formation of continuous lumena in multicellular epithelia.

During development of the avian lung, the initially terminally branched epithelial tree later forms a continuous network of airways. This occurs via a large-scale epithelial fusion event, wherein airways that originate proximally collide with those that originate distally to form one continuous lumen. Here, we found that prior to fusion, the epithelium of the embryonic chicken lung undergoes a shape change to permit the initiation and extension of new branches which contain the cells that initiate contact. These changes in epithelial shape coincide with the differentiation of smooth muscle cells that wrap the airways. From these nascent epithelial branches, individual cells form cytoskeletal protrusions that extend toward and form a bridge with their target airway. Additional cells then join the fusion site, forming a bilayered epithelium. During this process, the basement membrane around the prefusion epithelium degrades and then reforms after fusion. The epithelial bilayer then undergoes apoptosis, clearing the path between the two lumens. The process of airway epithelial fusion in the developing chicken lung constitutes a novel mechanism for the generation of complex multicellular tubes and suggests a conserved role for smooth muscle in the shaping of airway epithelia.

MO068A KIDNEY-ON-THE-CHIP APPROACH USING PRIMARY HUMAN TUBULAR CELLS IN A 3D CO-CULTURE SYSTEM

Abstract Background and Aims Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, although their value is limited by poor translatability and predictive value for the in vivo or even human situation. The implementation of more sophisticated in vitro assays as routine cell culture systems is often limited by complex protocols and long lasting procedures. We aimed to establish and validate a relatively easy-to-use but yet (patho-) physiologically relevant cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Method We utilized the 3-lane OrganoPlate® system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker (Mimetas), that facilitates continuous gravity-triggered bi-directional perfusion of C1. Within 48h the cells form a leak-tight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of the C2-matrix and – upon continuous perfusion – formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells were isolated and seeded into C3 (see figure). Results Establishing the whole tubule-on-the-chip as described above takes on average three days. We investigated its operational life span by monitoring the barrier integrity of the tubular structure in C1 using a fluorescence-labeled dextran (150 kDa). Over a course of 5 days the tubular integrity did not decline, suggesting that the co-culture system remains stable and functional for at least 5 days. In accordance with other studies, the primary human tubular cells constituting the 3D tubule-on-the-chip expressed higher levels of functionally relevant proteins, e..g the tight-junction protein ZO-1 and the sodium-potassium-pump Na-K-ATPase, compared to standard 2D settings without perfusion. This emphasizes, that even primary cells show a physiologically reduced phenotype in standard 2D settings, which possibly impedes the identification and representative quantification of physiologically and hence also patho-physiologically relevant mechanisms in vitro. To study the interaction of cells in the tubule-on-the-chip, we investigated the recruitment of immune cells from C3 (vessel) across C2 (interstitium) to C1 (renal tubule), which - in vivo - represents a detrimental mechanism of action in intrarenal forms of AKI. Under baseline conditions the immune cells inserted into C3 did not leave their compartment. Upon damaging the tubular cells in C1 with extracellular histones, neutrophils and monocytes left C3 (extravasation), migrated through C2 and could be found in close contact with epithelial cells of C1. This serves as a proof of principle, that the tubule-on-the-chip is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Measuring lactate dehydrogenase release for a number of known nephrotoxic agents revealed, that tubular cells forming a 3D-structure while kept under perfusion show significantly different responses to the same dose compared to standard 2D conditions, suggesting that dose-response studies using target cells out of their tissues context can be misleading when extrapolating results from in vitro to in vivo. Conclusion The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited to allow for in depth mechanistic studies in vitro.

Dashing Through the Maze: Active Mixing in the Endoplasmic Reticulum

The endoplasmic reticulum (ER) serves as the striking example of morphological complexity in cellular organelles. It comprises a series of stacked membranous sheets in the perinuclear region and an expansive network of tubules spanning the cell periphery, interconnected by a single continuous lumen. Its many functions include protein sorting and quality control, lipid distribution, and intracellular calcium modulation. Each of these functions relies on the ability to efficiently deliver molecular components to localized regions of the cell as well as redistributing these components throughout the ER lumen and membrane. While transport within the ER is generally assumed to be diffusive in nature, recent evidenceindicates that proteins in the the peripheral network also exhibit rapid processive movements indicative of an active transport mechanism. We use physical modeling to examine both the plausible underlying causes of this processive motion and the potential effect on ER function. Specifically, we focus on calcium ion distribution through the ER as an exemplar system for transport within an active network. We show that diffusion alone places a hard physical limit on the magnitude of cytoplasmic calcium sparks that can be generated by localized release from an ER tubule, and that processive motion of calcium buffer proteins can greatly enhance these sparks. Furthermore, we demonstrate that refilling the ER lumen after global calcium release would result in a substantial latency period between sequential signaling events unless the mixing of luminal contents in the periphery is enhanced, as expected from sporadic active transport. Our results highlight the importance of calcium buffer protein mobility for the efficiency of ion distribution through the ER lumen, as well as potentially crucial roles for actively driven processive motion within ER tubules.

One or Two Ca2+ Stores in the Neuronal Endoplasmic Reticulum?

It is generally accepted that the endoplasmic reticulum (ER) has one continuous lumen and operates as one unified Ca2+ store, but new data from Chen-Engerer etal. in hippocampal neurons now challenge this idea and indicate that receptors for inositol trisphosphate (IP3) and ryanodine may be located in two functionally distinct Ca2+ stores. Are both these stores in the ER?

Continuous Assessment of Carotid Diameter using an Accelerometer Patch Probe for Ambulatory Arterial Stiffness Monitoring.

We present a system with an accelerometer patch probe design for non-invasive evaluation of carotid arterial stiffness. The proposed system could continuously measure the acceleration signal derived due to the propagation of blood pulse wave through the left carotid artery, double integrating and scaling it to estimate the accelerometer-derived carotid wall displacement. This functional principle was proved by comparing the accelerometer-derived carotid wall displacement with the carotid distension signal from the reference system ARTSENS® (ARTerial Stiffness Evaluation for Noninvasive Screening device) for all the recruited human subjects. Assuming the relationship to be linear, a one-time subject-specific calibration was performed with the simultaneously acquired reference distension signal and the accelerometer-derived carotid displacement signals on its anachrotic limbs data points (at systolic phases) for each subject. This calibration equation was tested with latterly acquired accelerometer signals and results in the measurement of accelerometer-derived carotid distension and lumen-diameter values. The ability of the accelerometer system to measure real-time carotid distension and lumen diameter in a repeatable beat-by-beat manner for arterial stiffness index evaluation was validated in-vivo. The accuracy of the obtained results was studied with our clinically validated reference system. The experimental validation study results exhibit the feasibility of using the developed accelerometer system for continuous carotid distension and lumen diameter measurements, whereby the estimation of carotid arterial stiffness.

Development of in vitro enteroids derived from bovine small intestinal crypts

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host–pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or “mini gut”, cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host–pathogen interactions in the bovine small intestine can be studied.

Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/β-arr1 (β-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

The Lipocalin LPR-1 Cooperates with LIN-3/EGF Signaling To Maintain Narrow Tube Integrity in Caenorhabditis elegans.

Lipocalins are secreted cup-shaped glycoproteins that bind sterols, fatty acids, and other lipophilic molecules. Lipocalins have been implicated in a wide array of processes related to lipophilic cargo transport, sequestration, and signaling, and several are used as biomarkers for human disease, but the functions of most lipocalins remain poorly understood. Here we show that the Caenorhabditis elegans lipocalin LPR-1 is required to maintain apical membrane integrity and a continuous lumen in two narrow unicellular tubes, the excretory duct and pore, during a period of rapid lumen elongation. LPR-1 fusion protein is expressed by the duct and pore and accumulates both intracellularly and in apical extracellular compartments, but it can also function cell nonautonomously when provided from outside of the excretory system. lpr-1 mutant defects can be rescued by increased signaling through the epidermal growth factor (EGF)-Ras-extracellular signal regulated kinase (ERK) pathway, which promotes the more elongated duct vs. less elongated pore tube fate. Spatial and temporal rescue experiments indicate that Ras signaling acts within the duct and pore tubes during or prior to cell fate determination to bypass the requirement for LPR-1 lpr-1 mutations did not disrupt LIN-3/EGF-dependent duct-fate specification, prevent functioning of any specific LIN-3/EGF isoform, or alter LET-23/EGFR localization, and reduced signaling did not phenocopy or enhance lpr-1 mutant defects. These data suggest that LPR-1 protects lumen integrity through a LIN-3/EGF-independent mechanism, but that increased signaling upregulates some target(s) that can compensate for lpr-1 absence.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

Role of CD34 family members in lumen formation in the developing kidney

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.