Promoters and Antagonists of Phagocytosis: A Plastic and Tunable Response.

Recent observations indicate that, rather than being an all-or-none response, phagocytosis is finely tuned by a host of developmental and environmental factors. The expression of key phagocytic determinants is regulated via transcriptional and epigenetic means that confer memory on the process. Membrane traffic, the cytoskeleton, and inside-out signaling control the activation of phagocytic receptors and their ability to access their targets. An exquisite extra layer of complexity is introduced by the coexistence of distinct "eat-me" and "don't-eat-me" signals on targets and of corresponding "eat" and "don't-eat" receptors on the phagocyte surface. Moreover, assorted physical barriers constitute "don't-come-close-to-me" hurdles that obstruct the engagement of ligands by receptors. The expression, mobility, and accessibility of all these determinants can be modulated, conferring extreme plasticity on phagocytosis and providing attractive targets for therapeutic intervention in cancer, atherosclerosis, and dementia.

Glycocalyx Curving the Membrane: Forces Emerging from the Cell Exterior.

Morphological transitions are typically attributed to the actions of proteins and lipids. Largely overlooked in membrane shape regulation is the glycocalyx, a pericellular membrane coat that resides on all cells in the human body. Comprised of complex sugar polymers known as glycans as well as glycosylated lipids and proteins, the glycocalyx is ideally positioned to impart forces on the plasma membrane. Large, unstructured polysaccharides and glycoproteins in the glycocalyx can generate crowding pressures strong enough to induce membrane curvature. Stress may also originate from glycan chains that convey curvature preference on asymmetrically distributed lipids, which are exploited by binding factors and infectious agents to induce morphological changes. Through such forces, the glycocalyx can have profound effects on the biogenesis of functional cell surface structures as well as the secretion of extracellular vesicles. In this review, we discuss recent evidence and examples of these mechanisms in normal health and disease.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.

Siglec-15/sialic acid axis as a central glyco-immune checkpoint in breast cancer bone metastasis.

Immunotherapy is a promising approach for treating metastatic breast cancer (MBC), offering new possibilities for therapy. While checkpoint inhibitors have shown great progress in the treatment of metastatic breast cancer, their effectiveness in patients with bone metastases has been disappointing. This lack of efficacy seems to be specific to the bone environment, which exhibits immunosuppressive features. In this study, we elucidate the multiple roles of the sialic acid-binding Ig-like lectin (Siglec)-15/sialic acid glyco-immune checkpoint axis in the bone metastatic niche and explore potential therapeutic strategies targeting this glyco-immune checkpoint. Our research reveals that elevated levels of Siglec-15 in the bone metastatic niche can promote tumor-induced osteoclastogenesis as well as suppress antigen-specific T cell responses. Next, we demonstrate that antibody blockade of the Siglec-15/sialic acid glyco-immune checkpoint axis can act as a potential treatment for breast cancer bone metastasis. By targeting this pathway, we not only aim to treat bone metastasis but also inhibit the spread of metastatic cancer cells from bone lesions to other organs.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Steric pressure between glycosylated transmembrane proteins inhibits internalization by endocytosis.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

Engineered molecular sensors for quantifying cell surface crowding.

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

Chondroitin sulfate enhances the barrier function of basement membrane assembled by heparan sulfate.

Glycosaminoglycans are ubiquitously expressed polysaccharides that are attached to proteoglycans. Here, we showed that ablation of the heparan sulfate (HS) polymerase Ext1 in retinal progenitor cells did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which could be rescued by exogenous vascular endothelial growth factor (VEGF), consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing-specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the basement membrane. Lastly, we showed that further deletion of C4st1, a chondroitin sulfate (CS) sulfation enzyme, did not affect the assembly of the ILM but, when combined with Ext1 deletion, it aggravated the retinal permeability by disrupting the retinal glycocalyx. These results demonstrate an important role of CS and HS in establishing the barrier function of the extracellular matrix.

Mechanisms of triple-negative breast cancer extravasation: Impact of the physical environment and endothelial glycocalyx.

Cancer metastasis is the leading cause of death for those afflicted with cancer. In cancer metastasis, the cancer cells break off from the primary tumor, penetrate nearby blood vessels, and attach and extravasate out of the vessels to form secondary tumors at distant organs. This makes extravasation a critical step of the metastatic cascade. Herein, with a focus on triple-negative breast cancer, the role that the prospective secondary tumor microenvironment's mechanical properties play in circulating tumor cells' extravasation is reviewed. Specifically, the effects of the physically regulated vascular endothelial glycocalyx barrier element, vascular flow factors, and subendothelial extracellular matrix mechanical properties on cancer cell extravasation are examined. The ultimate goal of this review is to clarify the physical mechanisms that drive triple-negative breast cancer extravasation, as these mechanisms may be potential new targets for anti-metastasis therapy.

Acute brain injury increases pulmonary capillary permeability via sympathetic activation-mediated high fluid shear stress and destruction of the endothelial glycocalyx layer.

Neurogenic pulmonary edema secondary to acute brain injury (ABI) is a common and fatal disease condition. However, the pathophysiology of brain-lung interactions is incompletely understood. This study aims to investigate whether sympathetic activation-mediated high fluid shear stress after ABI would damage pulmonary endothelial glycocalyx thus leading to increased pulmonary capillary permeability. The tricuspid annular plane systolic excursion (TAPSE) was detected in a rat model of controlled cortical impact (CCI) and CCI + transection of the cervical sympathetic trunk (TCST). Changes in pulmonary capillary permeability were assessed by analyzing the Evans blue, measuring the dry/wet weight ratio of the lungs and altering protein levels in the bronchoalveolar lavage fluid (BALF). The parallel-plate flow chamber system was used to simulate the fluid shear stress in vitro. Western blotting and immunofluorescence staining were used to determine the expression levels of hyaluronan-binding protein (CEMIP), syndecan-1 and tight junction proteins (TJPs, including claudin-5 and occludin). TCST could restrain cardiac overdrive and sympathetic activation in a rat model of CCI. Compared to the CCI group, the CCI + TCST group showed a reduction of CEMPI (which degrades hyaluronic acid), along with an increase of syndecan-1 and TJPs. CCI + TCST group presented decreasing pulmonary capillary permeability. In vitro, high shear stress (HSS) increased the expression of CEMIP and reduced syndecan-1 and TJPs, which was coordinated with the results in vivo. Our findings show that sympathetic activation-mediated high fluid shear stress after ABI would damage pulmonary endothelial glycocalyx thus leading to increased pulmonary capillary permeability.

Impaired instructive and protective barrier functions of the endothelial cell glycocalyx pericellular matrix is impacted in COVID-19 disease.

The aim of this study was to review the roles of endothelial cells in normal tissue function and to show how COVID-19 disease impacts on endothelial cell properties that lead to much of its associated symptomatology. This places the endothelial cell as a prominent cell type to target therapeutically in the treatment of this disorder. Advances in glycosaminoglycan analytical techniques and functional glycomics have improved glycosaminoglycan mimetics development, providing agents that can more appropriately target various aspects of the behaviour of the endothelial cell in-situ and have also provided polymers with potential to prevent viral infection. Thus, promising approaches are being developed to combat COVID-19 disease and the plethora of symptoms this disease produces. Glycosaminoglycan mimetics that improve endothelial glycocalyx boundary functions have promising properties in the prevention of viral infection, improve endothelial cell function and have disease-modifying potential. Endothelial cell integrity, forming tight junctions in cerebral cell populations in the blood-brain barrier, prevents the exposure of the central nervous system to circulating toxins and harmful chemicals, which may contribute to the troublesome brain fogging phenomena reported in cognitive processing in long COVID disease.

Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures.

Vimentin intermediate filaments form part of the cytoskeleton of mesenchymal cells, but under pathological conditions often associated with inflammation, vimentin filaments depolymerize as the result of phosphorylation or citrullination, and vimentin oligomers are secreted or released into the extracellular environment. In the extracellular space, vimentin can bind surfaces of cells and the extracellular matrix, and the interaction between extracellular vimentin and cells can trigger changes in cellular functions, such as activation of fibroblasts to a fibrotic phenotype. The mechanism by which extracellular vimentin binds external cell membranes and whether vimentin alone can act as an adhesive anchor for cells is largely uncharacterized. Here, we show that various cell types (normal and vimentin null fibroblasts, mesenchymal stem cells, and A549 lung carcinoma cells) attach to and spread on polyacrylamide hydrogel substrates covalently linked to vimentin. Using traction force microscopy and spheroid expansion assays, we characterize how different cell types respond to extracellular vimentin. Cell attachment to and spreading on vimentin-coated surfaces is inhibited by hyaluronic acid degrading enzymes, hyaluronic acid synthase inhibitors, soluble heparin or N-acetyl glucosamine, all of which are treatments that have little or no effect on the same cell types binding to collagen-coated hydrogels. These studies highlight the effectiveness of substrate-bound vimentin as a ligand for cells and suggest that carbohydrate structures, including the glycocalyx and glycosylated cell surface proteins that contain N-acetyl glucosamine, form a novel class of adhesion receptors for extracellular vimentin that can either directly support cell adhesion to a substrate or fine-tune the glycocalyx adhesive properties.

Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Insights into the Molecular Mechanism of Endothelial Glycocalyx Dysfunction during Heart Surgery.

The endothelial glycocalyx (EGC) is a layer of proteoglycans (associated with glycosaminoglycans) and glycoproteins, which adsorbs plasma proteins on the luminal surface of endothelial cells. Its main function is to participate in separating the circulating blood from the inner layers of the vessels and the surrounding tissues. Physiologically, the EGC stimulates mechanotransduction, the endothelial charge, thrombocyte adhesion, leukocyte tissue recruitment, and molecule extravasation. Hence, severe impairment of the EGC has been implicated in various pathological conditions, including sepsis, diabetes, chronic kidney disease, inflammatory disorders, hypernatremia, hypervolemia, atherosclerosis, and ischemia/reperfusion injury. Moreover, alterations in EGC have been associated with altered responses to therapeutic interventions in conditions such as cardiovascular diseases. Investigation into the function of the glycocalyx has expanded knowledge about vascular disorders and indicated the need to consider new approaches in the treatment of severe endothelial dysfunction. This review aims to present the current understanding of the molecular mechanisms underlying cardiovascular diseases and to elucidate the impact of heart surgery on EGC dysfunction.

Interleukin-6 drives endothelial glycocalyx damage in COVID-19 and bacterial sepsis.

Damage of the endothelial glycocalyx (eGC) plays a central role in the development of vascular hyperpermeability and organ damage during systemic inflammation. However, the specific signalling pathways for eGC damage remain poorly defined. Aim of this study was to combine sublingual video-microscopy, plasma proteomics and live cell imaging to uncover further pathways of eGC damage in patients with coronavirus disease 2019 (COVID-19) or bacterial sepsis. This secondary analysis of the prospective multicenter MICROCODE study included 22 patients with COVID-19 and 43 patients with bacterial sepsis admitted to intermediate or intensive care units and 10 healthy controls. Interleukin-6 (IL-6) was strongly associated with damaged eGC and correlated both with eGC dimensions (rs=0.36, p = 0.0015) and circulating eGC biomarkers. In vitro, IL-6 reduced eGC height and coverage, which was inhibited by blocking IL-6 signalling with the anti-IL-6 receptor antibody tocilizumab or the Janus kinase inhibitor tofacitinib. Exposure of endothelial cells to 5% serum from COVID-19 or sepsis patients resulted in a significant decrease in eGC height, which was attenuated by co-incubation with tocilizumab. In an external COVID-19 cohort of 219 patients from Massachusetts General Hospital, a previously identified proteomic eGC signature correlated with IL-6 (rs=-0.58, p < 0.0001) and predicted the combined endpoint of 28-day mortality and/or intubation (ROC-AUC: 0.86 [95% CI: 0.81-0.91], p < 0.001). The data suggest that IL-6 may significantly drive eGC damage in COVID-19 and bacterial sepsis. Our findings provide valuable insights into pathomechanisms of vascular dysfunction during systemic inflammation and highlight the need for further in vivo studies.

Integrating molecular and cellular components of endothelial shear stress mechanotransduction.

The lining of blood vessels is constantly exposed to mechanical forces exerted by blood flow against the endothelium. Endothelial cells detect these tangential forces (i.e., shear stress), initiating a host of intracellular signaling cascades that regulate vascular physiology. Thus, vascular health is tethered to the endothelial cells' capacity to transduce shear stress. Indeed, the mechanotransduction of shear stress underlies a variety of cardiovascular benefits, including some of those associated with increased physical activity. However, endothelial mechanotransduction is impaired in aging and disease states such as obesity and type 2 diabetes, precipitating the development of vascular disease. Understanding endothelial mechanotransduction of shear stress, as well as the molecular and cellular mechanisms by which this process becomes defective, is critical for the identification and development of novel therapeutic targets against cardiovascular disease. In this review, we detail the primary mechanosensitive structures that have been implicated in detecting shear stress, including junctional proteins such as PECAM-1, the extracellular glycocalyx and its components, and ion channels such as Piezo1. We delineate which molecules are truly mechanosenstive and which may simply be indispensable for the downstream transmission of force. Furthermore, we discuss how these mechanosensors interact with other cellular structures, such as the cytoskeleton and membrane lipid rafts, which are implicated in translating shear forces to biochemical signals. Based on findings to date, we also seek to integrate these cellular and molecular mechanisms with a view of deciphering endothelial mechanotransduction of shear stress, a tenet of vascular physiology.

Liposomal nanocarriers of preassembled glycocalyx restore normal venular permeability and shear stress sensitivity in sepsis: assessed quantitatively with a novel microchamber system.

The endothelial glycocalyx (EG), covering the luminal side of endothelial cells, regulates vascular permeability and senses wall shear stress. In sepsis, EG undergoes degradation leading to increased permeability and edema formation. We hypothesized that restoring EG integrity using liposomal nanocarriers of preassembled glycocalyx (LNPG) will restore normal venular permeability in lipopolysaccharide (LPS)-induced sepsis model of mice. To test this hypothesis, we designed a unique perfusion microchamber in which the permeability of isolated venules could be assessed by measuring the concentration of Evans blue dye (EBD) in microliter samples of extravascular solution (ES). Histamine-induced time- and dose-dependent increases in EBD in the ES could be measured, confirming the sensitivity of the microchamber system. Notably, the histamine-induced increase in permeability was significantly attenuated by histamine receptor (H1) antagonist, triprolidine hydrochloride. Subsequently, mice were treated with LPS or LPS + LNPG. When compared with control mice, venules from LPS-treated mice showed a significant increased permeability, which was significantly reduced by LNPG administration. Moreover, in the presence of wall shear stress, intraluminal administration of LNPG significantly reduced the permeability in isolated venules from LPS-treated mice. We have found no sex differences. In conclusion, our newly developed microchamber system allows us to quantitatively measure the permeability of isolated venules. LPS-induced sepsis increases permeability of mesenteric venules that is attenuated by in vivo LNPG administration, which also reestablished endothelial responses to shear stress. Thus, LNPG presents a promising therapeutic potential for restoring EG function and thereby mitigating vasogenic edema due to increased permeability in sepsis.NEW & NOTEWORTHY In sepsis, the degradation of the endothelial glycocalyx leads to increased venular permeability. In this study, we developed a potentially new therapeutic approach by in vivo administration of liposomal nanocarriers of preassembled glycocalyx to mice, which restored venular sensitivity to wall shear stress and permeability in lipopolysaccharide-induced sepsis, likely by restoring the integrity of the endothelial glycocalyx. Using a new microchamber system, the permeability of Evans blue dye could be quantitatively determined.