Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions.

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions.

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts.

Several functions of autophagy associated with proliferation, differentiation, and migration of endothelial cells have been reported. Due to lack of models recapitulating angiogenic sprouting, functional heterogeneity of autophagy in endothelial cells along angiogenic sprouts remains elusive. Here, we apply an angiogenesis-on-a-chip to reconstruct 3D sprouts with clear endpoints. We perform single-cell RNA sequencing of sprouting endothelial cells from our chip to reveal high activation of autophagy in two endothelial cell populations- proliferating endothelial cells in sprout basements and stalk-like endothelial cells near sprout endpoints- and further the reciprocal expression pattern of autophagy-related genes between stalk- and tip-like endothelial cells near sprout endpoints, implying an association of autophagy with tip-stalk cell specification. Our results suggest a model describing spatially differential roles of autophagy: quality control of proliferating endothelial cells in sprout basements for sprout elongation and tip-stalk cell specification near sprout endpoints, which may change strategies for developing autophagy-based anti-angiogenic therapeutics.

Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.

Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

BBB-on-a-Chip: Modeling Functional Human Blood-Brain Barrier by Mimicking 3D Brain Angiogenesis Using Microfluidic Chip.

Organ-on-a-chip enables human cell-based 3D tissue culture, which recapitulates the physiological structure and function of the tissue. In terms of the blood-brain barrier (BBB) modeling, the 3D structure of the vessel is essential for studying the cellular interactions among BBB composing cells and investigating the barrier function. Here, we describe a BBB-on-a-chip model with 3D perfusable human vasculature tri-cultured with pericytes and astrocytes. The culture method is based on mimicking angiogenic sprouting since the barrier formation is parallel with angiogenesis during the developmental process. This microfluidic-based 3D tri-culture system enables the comparative study on how surrounding BBB-related cells affect brain angiogenic sprouting. Moreover, the engineered perfusable vasculature is eligible for quantitative analysis on barrier function such as efflux transport system. We expect the BBB-on-a-chip could be used to enhance understanding BBB-related pathologies as well as the drug modulating barrier function of BBB.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

3D brain angiogenesis model to reconstitute functional human blood-brain barrier in vitro.

The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood-brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three-dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri-culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri-culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p-glycoprotein (p-gp), which is highly responsible for restrictive drug transport. By conducting the calcein-AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri-culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Microfluidic-based vascularized microphysiological systems.

Microphysiological systems have emerged in the last decade to provide an alternative to in vivo models in basic science and pharmaceutical research. In the field of vascular biology, in particular, there has been a lack of a suitable in vitro model exhibiting a three-dimensional structure and the physiological function of vasculature integrated with organ-on-a-chip models. The rapid development of organ-on-a-chip technology is well positioned to fulfill unmet needs. Recently, functional integration of vasculature with diverse microphysiological systems has been increasing. This recent trend corresponds to emerging research interest in how the vascular system contributes to various physiological and pathological conditions. This innovative platform has undergone significant development, but adoption of this technology by end-users and researchers in biology is still a work in progress. Therefore, it is critical to focus on simplification and standardization to promote the distribution and acceptance of this technology by the end-users. In this review, we will introduce the latest developments in vascularized microphysiological systems and summarize their outlook in basic research and drug screening applications.

Microfluidics within a well: an injection-molded plastic array 3D culture platform.

Polydimethylsiloxane (PDMS) has been widely used in fabricating microfluidic devices for prototyping and proof-of-concept experiments. Due to several material limitations, PDMS has not been widely adopted for commercial applications that require large-scale production. This paper describes a novel injection-molded plastic array 3D culture (IMPACT) platform that incorporates a microfluidic design to integrate patterned 3D cell cultures within a single 96-well (diameter = 9 mm) plate. Cell containing gels can be sequentially patterned by capillary-guided flow along the corner and narrow gaps designed within the 96-well form factor. Compared to PDMS-based hydrophobic burst valve designs, this work utilizes hydrophilic liquid guides to obtain rapid and reproducible patterned gels for co-cultures. When a liquid droplet (i.e. cell containing fibrin or collagen gel) is placed on a corner, spontaneous patterning is achieved within 1 second. Optimal dimensionless parameters required for successful capillary loading have been determined. To demonstrate the utility of the platform for 3D co-culture, angiogenesis experiments were performed by patterning HUVEC (human umbilical endothelial cells) and LF (lung fibroblasts) embedded in 3D fibrin gels. The angiogenic sprouts (with open lumen tip cells expressing junctional proteins) are comparable to those observed in PDMS based devices. The IMPACT device has the potential to provide a robust high-throughput experimental platform for vascularized microphysiological systems.

Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.

Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal stimulation. Using this platform, we observed that static Epidermal growth factor (EGF) stimulation led to transient ERK activation with a significant cell-to-cell variation, while dynamic stimulation of 3' EGF pulse led to faster adaptation kinetics with no discrepancy. Multiple EGF pulses retriggered ERK activity with respect to frequency of stimulation. We also observed oscillation of ERK activity of each cell at basal state. Introducing of Mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, was not only dropping the average of basal activity for 7.5%, but also diminishing oscillatory behavior. Activity level raised up when inhibitor was removed, followed by transient peak of ERK kinetics. We expect this platform to probe Mitogen-associated protein kinase (MAPK) signaling network for systems biology research at single cellular level.

Microfluidic platform for single cell analysis under dynamic spatial and temporal stimulation.

Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF.

Relationship between Pericytes and Endothelial Cells in Retinal Neovascularization: A Histological and Immunofluorescent Study of Retinal Angiogenesis.

PURPOSE: To evaluate the relationship between pericytes and endothelial cells in retinal neovascularization through histological and immunofluorescent studies. METHODS: C57BL/6J mice were exposed to hyperoxia from postnatal day (P) 7 to P12 and were returned to room air at P12 to induce a model of oxygen-induced retinopathy (OIR). The cross sections of enucleated eyes were processed with hematoxylin and eosin. Immunofluorescent staining of pericytes, endothelial cells, and N-cadherin was performed. Microfluidic devices were fabricated out of polydimethylsiloxane using soft lithography and replica molding. Human retinal microvascular endothelial cells, human brain microvascular endothelial cells, human umbilical vein endothelial cells and human placenta pericyte were mixed and co-cultured. RESULTS: Unlike the three-layered vascular plexus found in retinal angiogenesis of a normal mouse, angiogenesis in the OIR model is identified by the neovascular tuft extending into the vitreous. Neovascular tufts and the three-layered vascular plexus were both covered with pericytes in the OIR model. In this pathologic vascularization, N-cadherin, known to be crucial intercellular adhesion molecule, was also present. Further evaluation using the microfluidic in vitro model, successfully developed a microvascular network of endothelial cells covered with pericytes, mimicking normal retinal angiogenesis within 6 days. CONCLUSIONS: Pericytes covering endothelial cells were observed not only in vasculature of normal retina but also pathologic neovascularization of OIR mouse at P17. Factors involved in the endothelial cell-pericyte interaction can be evaluated as an attractive novel treatment target. These future studies can be performed using microfluidic systems, which can shorten the study time and provide three-dimensional structural evaluation.

Wet-AMD on a Chip: Modeling Outer Blood-Retinal Barrier In Vitro.

Choroidal neovascularization (CNV) in the retinal pigment epithelium (RPE)-choroid complex constituting outer blood retinal barrier (oBRB) is a critical pathological step in various ophthalmic diseases, which results in blindness, such as wet type age-related macula degeneration. Current in vitro experimental models using petri dishes or transwell are unable to study CNV morphogenesis. Here, a unique organotypic eye-on-a-chip model is described that mimics the RPE-choroid complex in vitro. This model consists of an RPE monolayer and adjacent perfusable blood vessel network, which is supporting barrier function of oBRB. The intact barrier function of the RPE-choroid complex is reconstituted while maintaining important structural features. Further, this model can successfully mimic the pathogenesis of CNV especially in terms of morphogenesis, which is penetrating angiogenic sprouts from pre-existing choroidal vessels that result in breakdown of RPE monolayer. The alleviation of the pathological angiogenesis can be modeled with bevacizumab, a clinical drug for CNV treatment. It is believed that this model can be used to aid in the development of advanced in vitro eye drug evaluation in conjunction with animal models.

Biomimetic Model of Tumor Microenvironment on Microfluidic Platform.

The "Tumor microenvironment" (TME) is a complex, interacting system of the tumor and its surrounding environment. The TME has drawn more attention recently in attempts to overcome current drug resistance and the recurrence of cancer by understanding the cancer and its microenvironment systematically, beyond past reductionist approaches. However, a lack of experimental tools to dissect the intricate interactions has hampered in-depth research into the TME. Here, a biomimetic TME model using a microfluidic platform is presented, which enables the interaction between TME constituents to be studied in a comprehensive manner. Paracrine interactions of cocultured tumor cell lines (SK-OV-3, MKN-74, and SW620) with primary fibroblasts show marked morphological changes in the tumor cells, depending on the type of tumor cells, and, importantly, the composition of the extracellular matrix. Furthermore, this model allows direct observation of angiogenesis induced by the tumor-stroma interaction. Finally, reconstituting simultaneous angiogenesis and lymphangiogenesis induced by the tumor-stromal interaction with TME mimicking extrinsic factors is enabled. It is believed that the in vitro biomimetic model and the experimental concepts described will help to shed light on the complex biology of the TME.

Identification of the First Selective Activin Receptor-Like Kinase 1 Inhibitor, a Reversible Version of L-783277.

We synthesized 1 (San78-130), a reversible version of L-783277, as a selective and potent ALK1 inhibitor. Our study showed that 1 possesses great kinase selectivity against a panel of 342 kinases and more potent activity against ALK1 than L-783277. Among the six ALK isotypes (ALK1-6), ALK1 is most significantly inhibited by compound 1. Compound 1 suppresses the BMP9-induced Smad1/5 pathway by mainly inhibiting ALK1 in C2C12 cells. Our molecular dynamics simulations suggest that H-bonding interaction between the C-4' hydroxyl group of 1 and Arg334 of ALK1 substantially contributes to the ALK1 inhibition. To the best of our knowledge, 1 is the first selective ALK1 inhibitor. Furthermore, compound 1 promoted angiogenesis in both endothelial tube formation and microfluidic chip based 3D angiogenesis assays, suggesting that 1 could be a lead compound for therapeutic angiogenesis agents. Our study may provide an insight into designing selective and potent inhibitors against ALK1.

Interstitial flow regulates the angiogenic response and phenotype of endothelial cells in a 3D culture model.

A crucial yet ill-defined phenomenon involved in the remodeling of vascular networks, including angiogenic sprouting, is flow-mediated endothelial dynamics and phenotype changes. Despite interstitial flow (IF) being ubiquitously present in living tissues surrounding blood capillaries, it is rarely investigated and poorly understood how endothelial cells respond to this flow during morphogenesis. Here we develop a microfluidic 3D in vitro model to investigate the role of IF during vasculogenic formation and angiogenic remodeling of microvascular networks. In the presented model, human blood endothelial cells co-cultured with stromal fibroblasts spontaneously organize into an interconnected microvascular network and then further expand to adjacent avascular regions in a manner of neovessel sprouting. We found that in the presence of IF, vasculogenic organization of the microvascular network was significantly facilitated regardless of the flow direction, whereas angiogenic sprouting was promoted only when the directions of flow and sprouting were opposite while angiogenic activity was suppressed into the direction of flow. We also observed that the vasculatures switch between active angiogenic remodeling and quiescent/non-sprouting state in the contexts provided by IF. This regulatory effect can be utilized to examine the role of anti-angiogenic compounds, clearly distinguishing the differential influences of the compounds depending on their mechanisms of action. Collectively, these results suggest that IF may serve as a critical regulator in tissue vascularization and pathological angiogenesis.

Three-dimensional biomimetic model to reconstitute sprouting lymphangiogenesis in vitro.

Formation of new lymphatic vessels, termed lymphangiogenesis, is central for diverse biological processes during development, inflammation and tumor metastasis. However, reliable in vitro model is still under demand for detailed elucidation of how sprouting lymphangiogenesis is initiated and coordinated. Here, we describe a microfluidic platform optimized for close reconstitution of lymphangiogenesis, achieved by on-chip integration of salient constituents of lymphatic microenvironment found in vivo. With flexible and precise control over the factors that include biochemical cues, interstitial flow (IF), and endothelial-stromal interactions, we found that orchestrated efforts of multiple environmental factors are necessary for robust lymphatic sprouting in 3D extracellular matrix. Especially, we demonstrate that IF serves as a central regulatory cue which defines lymphangiogenic responses and phenotypes of lymphatic endothelial cells. When synergized with pro-lymphangiogenic factors, IF significantly augmented initiation and outgrowth of lymphatic sprouts toward upstream of the flow while suppressing downstream-directed sprouting. In an appropriate synergism, lymphatic sprouts exhibited structural, molecular signatures and cellular phenotypes that closely approximate sprouting lymphatic neovessels in vivo, and precisely reflected the modulatory effects of pro- and anti-lymphangiogenic stimuli. Our study not only reveals critical but unappreciated role of mechanical cue that regulates lymphangiogenic sprouting, but also provides a novel biomimetic model that may leverage further biological studies as well as phenotypic drug screening.