CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-ß (TGF-ß) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-ß signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-ß-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-ß-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-ß-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Extracellular matrix components and elasticity regulate mouse vaginal epithelial differentiation induced by mesenchymal cells†.

Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM's components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.

Transcriptomic Analysis of Breast Cancer Stem Cells and Development of a pALDH1A1:mNeptune Reporter System for Live Tracking.

Many solid cancers are hierarchically organized with a small number of cancer stem cells (CSCs) able to regrow a tumor, while their progeny lacks this feature. Breast CSC is known to contribute to therapy resistance. The study of those cells is usually based on their cell-surface markers like CD44high /CD24low/neg or their aldehyde dehydrogenase (ALDH) activity. However, these markers cannot be used to track the dynamics of CSC. Here, a transcriptomic analysis is performed to identify segregating gene expression in CSCs and non-CSCs, sorted by Aldefluor assay. It is observed that among ALDH-associated genes, only ALDH1A1 isoform is increased in CSCs. A CSC reporter system is then developed by using a far red-fluorescent protein (mNeptune) under the control of ALDH1A1 promoter. mNeptune-positive cells exhibit higher sphere-forming capacity, tumor formation, and increased resistance to anticancer therapies. These results indicate that the reporter identifies cells with stemness characteristics. Moreover, live tracking of cells in a microfluidic system reveals a higher extravasation potential of CSCs. Live tracking of non-CSCs under irradiation treatment show, for the first time, live reprogramming of non-CSCs into CSCs. Therefore, the reporter will allow for cell tracking to better understand the implication of CSCs in breast cancer development and recurrence.

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.

Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.

Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state.

Placement of a tag sequence is usually limited to either terminal end of the target protein, reducing the potential of epitope tags for various labeling applications. The PA tag is a dodecapeptide (GVAMPGAEDDVV) that is recognized by a high-affinity antibody NZ-1. We determined the crystal structure of the PA-tag-NZ-1 complex and found that NZ-1 recognizes a central segment of the PA tag peptide in a tight ß-turn configuration, suggesting that it is compatible with the insertion into a loop. This possibility was tested and confirmed using multiple integrin subunits and semaphorin. More specifically, the PA tag can be inserted at multiple locations within the integrin αIIb subunit (encoded by ITGA2B) of the fibrinogen receptor αIIbß3 integrin (of which the ß3 subunit is encoded by ITGB3) without affecting the structural and functional integrity, while maintaining its high affinity for NZ-1. The large choice of the sites for 'epitope grafting' enabled the placement of the PA tag at a location whose accessibility is modulated during the biological action of the receptor. Thus, we succeeded in converting a general anti-tag antibody into a special anti-integrin antibody that can be classified as a ligand-induced binding site antibody.

Facile Synthesis of Dimeric Thioether-Macrocyclic Peptides with Antibody-like Affinity against Plexin-B1.

Macrocyclic peptides have gained increasing attention due to their ease of discovery through various in vitro display platforms as well as their potential in possessing favorable properties of both small molecule and antibody drug classes. It is well-known that the avidity achieved through the bivalent binding mode of antibodies gives rise to their slow dissociation rates and thus high potency as drug molecules. Here, we report the synthesis of dimeric thioether-macrocyclic peptides through a branched synthesis approach allowing for synthesis of dimeric peptides in a comparable number of steps as monomers and tunability of linker lengths from 30 to 200 Å. Applying this method to synthesize dimers of a model PlexinB1-binding macrocyclic peptide showed close to 300-fold increases in their apparent binding affinity, bringing the KD down from 8 nM to 30 pM as well as affording improved biological activities when compared to their monomeric counterparts. These enhancements demonstrate that this is a simple synthetic strategy to harness the benefits of bivalence that antibodies naturally possess.

Prospective study of removing solar lentigines in Asians using a novel dual-wavelength and dual-pulse width picosecond laser.

OBJECTIVES: Quality-switched (QS) lasers are known to be an effective treatment for removing solar lentigines, however, high incidence of post-inflammatory hyperpigmentation (PIH) is a concern in darker skin types. The objective of this study was to evaluate the efficacy and safety of a dual-wavelength and dual-pulse width picosecond Nd:YAG laser for removing solar lentigines in Asians. METHODS: This was a prospective, IRB-approved study. Twenty cases with solar lentigines on the face were enrolled for treatment and evaluated at 1- and 3-month after the final treatment. Results were assessed by blinded evaluators using a 5-grade percentage improvement scale and Melanin index (MI) measured by a reflectance spectrophotometer. A patient self-assessment questionnaire was also administered using a 5-grade improvement scale. Additional treatment was performed if the improvement was less than 75% or the lentigo partially remained after 4 weeks. Histological evaluation was performed to compare the differences between the current picosecond laser and a QS Nd:YAG laser 532-nm using light and electron microscopy. RESULTS: Forty-three lesions in 20 females, skin type III or IV, age 53.7 ± 9.75 were treated and evaluated. The laser setting was: 532-nm, 750 picoseconds, average fluence of 0.35 ± 0.06 J/cm [2] using a spot size of 3 or 4 mm. Forty lesions (93.02%) achieved over 75% clearance with a single treatment and the other three lesions (6.98%) needed two treatments. PIH occurred only in 4.65% of lesions. The average score of the blinded evaluators' assessment was 4.77 and 4.58 on a 5-grade percentage improvement scale. The patients' self-assessment rating was 4.76 and 4.67 on a 5-grade scale at 1- and 3-month follow-up, respectively. The improvement rate of relative MI (MI in the lesion minus that of the normal area) was 77.60 ± 36.27% and 76.93 ± 20.95% at 1-and 3-month follow-up. Histology showed vacuolar formation by both lasers in the epidermis that were different sizes between lasers. Electron microscopy showed destruction of melanosomes with surrounding tissue damage with the QS laser and without particular damage with the picosecond laser. CONCLUSIONS: To the best of our knowledge, this is the first study using a picosecond Nd:YAG laser 532-nm for removing solar lentigines in darker skin types that includes histological evaluation. Although there are many options to treat solar lentigines, our results suggest that picosecond laser with preferable endpoint determination can be a safer and more effective treatment over conventional treatments in Asian patients. Lasers Surg. Med. 50:851-858, 2018. © 2018 Wiley Periodicals, Inc.

Structural basis for semaphorin signalling through the plexin receptor.

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.

Mesotrypsin and caspase-14 participate in prosaposin processing: potential relevance to epidermal permeability barrier formation.

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A-D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A-D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.

Metre-long cell-laden microfibres exhibit tissue morphologies and functions.

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Protocol for fabricating and characterizing microvessel-on-a-chip for human umbilical vein endothelial cells.

Organ-on-a-chip technologies enable the fabrication of endothelial tissues, so-called microvessels (MVs), which emulate the endothelial barrier function in healthy or disease conditions. In this protocol, we describe the fabrication of perfusable open-chamber style MVs embedded in collagen gels. We then report a simple technology to characterize the MV barrier properties in static or under pressure based on fluorescence confocal imaging. Finally, we provide quantification techniques that enable us to infer the structure of MV paracellular pores. For complete details on the use and execution of this protocol, please refer to Cacheux et al.1.

Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring.

Fluorescent microbeads hold great promise for in vivo continuous glucose monitoring with wireless transdermal transmission and long-lasting activity. The full potential of fluorescent microbeads has yet to be realized due to insufficient intensity for transdermal transmission and material toxicity. This paper illustrates the highly-sensitive, biostable, long-lasting, and injectable fluorescent microbeads for in vivo continuous glucose monitoring. We synthesized a fluorescent monomer composed of glucose-recognition sites, a fluorogenic site, spacers, and polymerization sites. The spacers are designed to be long and hydrophilic for increasing opportunities to bind glucose molecules; consequently, the fluorescent monomers enable high-intensive responsiveness to glucose. We then fabricated injectable-sized fluorescent polyacrylamide hydrogel beads with high uniformity and high throughput. We found that our fluorescent beads provide sufficient intensity to transdermally monitor glucose concentrations in vivo. The fluorescence intensity successfully traced the blood glucose concentration fluctuation, indicating our method has potential uses in highly-sensitive and minimally invasive continuous blood glucose monitoring.

Phenotypic and transcriptional characterization of oligodendrocyte precursor cells in a 3D culture.

Remyelination of the central nervous system (CNS) is a regenerative response that depends on the development of oligodendrocyte precursor cells (OPCs), which are generated from neural stem cells in developmental stages and exist as tissue stem cells in the adult CNS. Three-dimensional (3D) culture systems that recapitulate the complexity of the in vivo microenvironment are important for understanding the behavior of OPCs in remyelination and for exploring effective therapeutic approaches. In general, functional analysis of OPCs has mainly used two-dimensional (2D) culture systems; however, the differences between the properties of OPCs cultured in 2D and 3D have not been fully elucidated despite cellular functions being affected by the scaffold. In this study, we analyzed the phenotypic and transcriptomic differences in OPCs from 2D and collagen gel-based 3D cultures. In the 3D culture, the OPCs exhibited less than half ratio of proliferation and almost half ratio of differentiation to mature oligodendrocytes, compared to the 2D culture in the same culturing period. RNA-seq data showed robust changes in the expression level of genes associated with oligodendrocyte differentiation, and there were more up-regulated genes than down-regulated genes in 3D cultures compared to 2D cultures. In addition, the OPCs cultured in collagen gel scaffolds at lower collagen fiber densities showed higher proliferation activity compared with those cultured in collagen gel with higher collagen fiber densities. Our findings have identified the effect of culture dimension as well as the complexity of the scaffold on OPC responses at the cellular and molecular levels.

Solute transport in the brain tissue: what are the key biophysical parameters tying in vivo and in vitro studies together?

The mechanisms of solute transport in brain tissues are still under debate. The medical relevance of this topic has put the blood-brain barrier and the mechanisms of solute transport through the brain parenchyma in the spotlight, notably in the context of brain clearance. In the last decade, the classical view of pure diffusive flow across the brain parenchyma was tested against the recent proposal of an active, convectional fluid flow model known as the glymphatic model. Experimental studies of brain transport on living humans and animals have temporal and spatial limitations to validate any of these models. Therefore, detailed microscopic observations, mostly ex vivo tissue and simplified in vitro brain models with the support from computational models, are necessary to understand transport mechanisms in brain tissues. However, standardization is lacking between these experimental approaches, which tends to limit the generality of conclusions. In this review, we provide an overview of the output and limitations of modern brain solute transport studies to search for key parameters comparable across experimental setups. We emphasize that in vitro models relying on physiological material and reproducing the biophysical setting of the brain, as well as computational/mathematical models constitute powerful solutions to understand the solute transport phenomena inside of the brain tissue. Finally, we suggest the blood-brain barrier permeability and the apparent diffusion coefficient through the brain parenchyma to be robust biophysical parameters for the extraction of cross-model conclusion.

Asymmetry of tensile versus compressive elasticity and permeability contributes to the regulation of exchanges in collagen gels.

The Starling principle describes exchanges between blood and tissues based on the balance of hydrostatic and osmotic flows. However, the permeation properties of the main constituent of tissues, namely, collagen, in response to the stress exerted by blood pressure remain poorly characterized. Here, we develop an instrument to determine the elasticity and permeability of collagen gels under tensile and compressive stress based on measuring the temporal change in pressure in an air cavity sealed at the outlet of a collagen slab. Data analysis with an analytical model reveals a drop in the permeability and enhanced strain stiffening of native collagen gels under compression versus tension, both effects being essentially lost after chemical cross-linking. Furthermore, we report the control of the permeability of native collagen gels using sinusoidal fluid injection, an effect explained by the asymmetric response in tension and compression. We lastly suggest that blood-associated pulsations could contribute to exchanges within tissues.

Endothelial tissue remodeling induced by intraluminal pressure enhances paracellular solute transport.

The endothelial layers of the microvasculature regulate the transport of solutes to the surrounding tissues. It remains unclear how this barrier function is affected by blood flow-induced intraluminal pressure. Using a 3D microvessel model, we compare the transport of macromolecules through endothelial tissues at mechanical rest or with intraluminal pressure, and correlate these data with electron microscopy of endothelial junctions. On application of an intraluminal pressure of 100 Pa, we demonstrate that the flow through the tissue increases by 2.35 times. This increase is associated with a 25% expansion of microvessel diameter, which leads to tissue remodeling and thinning of the paracellular junctions. We recapitulate these data with the deformable monopore model, in which the increase in paracellular transport is explained by the augmentation of the diffusion rate across thinned junctions under mechanical stress. We therefore suggest that the deformation of microvasculatures contributes to regulate their barrier function.