PLD2 deficiency alleviates endothelial glycocalyx degradation in LPS-induced ARDS/ALI.

- Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a life-threatening condition marked by severe lung inflammation and increased lung endothelial barrier permeability. Endothelial glycocalyx deterioration is the primary factor of vascular permeability changes in ARDS/ALI. Although previous studies have shown that phospholipase D2 (PLD2) is closely related to the onset and progression of ARDS/ALI, its role and mechanism in the damage of endothelial cell glycocalyx remains unclear. We used LPS-induced ARDS/ALI mice (in vivo) and LPS-stimulated injury models of EA.hy926 endothelial cells (in vitro). We employed C57BL/6 mice, including wild-type and PLD2 knockout (PLD2-/-) mice, to establish the ARDS/ALI model. We applied immunofluorescence and ELISA to examine changes in syndecan-1 (SDC-1), matrix metalloproteinase-9 (MMP9), inflammatory cytokines (TNF-α, IL-6, and IL-1ß) levels and the effect of external factors, such as phosphatidic acid (PA), 1-butanol (a PLD inhibitor), on SDC-1 and MMP9 expression levels. We found that PLD2 deficiency inhibits SDC-1 degradation and MMP9 expression in LPS-induced ARDS/ALI. Externally added PA decreases SDC-1 levels and increases MMP9 in endothelial cells, hence underlining PA's role in SDC-1 degradation. Additionally, PLD2 deficiency decreases the production of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in LPS-induced ARDS/ALI. In summary, these findings suggest that PLD2 deficiency plays a role in inhibiting the inflammatory process and protecting against endothelial glycocalyx injury in LPS-induced ARDS/ALI.

Doxycycline protects against sepsis-induced endothelial glycocalyx shedding.

Endothelial glycocalyx (eGC) covers the inner surface of the vessels and plays a role in vascular homeostasis. Syndecan is considered the "backbone" of this structure. Several studies have shown eGC shedding in sepsis and its involvement in organ dysfunction. Matrix metalloproteinases (MMP) contribute to eGC shedding through their ability for syndecan-1 cleavage. This study aimed to investigate if doxycycline, a potent MMP inhibitor, could protect against eGC shedding in lipopolysaccharide (LPS)-induced sepsis and if it could interrupt the vascular hyperpermeability, neutrophil transmigration, and microvascular impairment. Rats that received pretreatment with doxycycline before LPS displayed ultrastructural preservation of the eGC observed using transmission electronic microscopy of the lung and heart. In addition, these animals exhibited lower serum syndecan-1 levels, a biomarker of eGC injury, and lower perfused boundary region (PBR) in the mesenteric video capillaroscopy, which is inversely related to the eGC thickness compared with rats that only received LPS. Furthermore, this study revealed that doxycycline decreased sepsis-related vascular hyperpermeability in the lung and heart, reduced neutrophil transmigration in the peritoneal lavage and inside the lungs, and improved some microvascular parameters. These findings suggest that doxycycline protects against LPS-induced eGC shedding, and it could reduce vascular hyperpermeability, neutrophils transmigration, and microvascular impairment.

Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

No need to "sugar coat": Removing glycocalyx on apoptotic blebs promotes phagocytosis.

The viscous glycocalyx of mammalian cells, composed of glucosaminoglycans, glycolipids, and glycoproteins, "sugar coat" the outer plasma membrane. In this issue of Developmental Cell, Le et al. (2024) show that the glycocalyx is removed from apoptotic blebs via disassembly of the cortical cytoskeleton, exposing the "eat-me" signals necessary for efferocytosis.

Temporal Alterations in Cerebrovascular Glycocalyx and Cerebral Blood Flow after Exposure to a High-Intensity Blast in Rats.

The glycocalyx is a proteoglycan-glycoprotein structure lining the luminal surface of the vascular endothelium and is susceptible to damage due to blast overpressure (BOP) exposure. The glycocalyx is essential in maintaining the structural and functional integrity of the vasculature and regulation of cerebral blood flow (CBF). Assessment of alterations in the density of the glycocalyx; its components (heparan sulphate proteoglycan (HSPG/syndecan-2), heparan sulphate (HS), and chondroitin sulphate (CS)); CBF; and the effect of hypercapnia on CBF was conducted at 2-3 h, 1, 3, 14, and 28 days after a high-intensity (18.9 PSI/131 kPa peak pressure, 10.95 ms duration, and 70.26 PSI·ms/484.42 kPa·ms impulse) BOP exposure in rats. A significant reduction in the density of the glycocalyx was observed 2-3 h, 1-, and 3 days after the blast exposure. The glycocalyx recovered by 28 days after exposure and was associated with an increase in HS (14 and 28 days) and in HSPG/syndecan-2 and CS (28 days) in the frontal cortex. In separate experiments, we observed significant decreases in CBF and a diminished response to hypercapnia at all time points with some recovery at 3 days. Given the role of the glycocalyx in regulating physiological function of the cerebral vasculature, damage to the glycocalyx after BOP exposure may result in the onset of pathogenesis and progression of cerebrovascular dysfunction leading to neuropathology.

Nafamostat mesylate decreases skin flap necrosis in a mouse model of type 2 diabetes by protecting the endothelial glycocalyx.

The success rate of flap tissue reconstruction has increased in recent years owing to advancements in microsurgical techniques. However, complications, such as necrosis, are still more prevalent in diabetic patients compared to non-diabetic individuals, presenting an ongoing challenge. To address this issue, many previous studies have examined vascular anastomoses dilation and stability, primarily concerning surgical techniques or drugs. In contrast, in the present study, we focused on microvascular damage of the peripheral microvessels in patients with diabetes mellitus and the preventative impact of nafamostat mesylate. Herein, we aimed to investigate the effects of hyperglycemia on glycocalyx (GCX) levels in mice with type 2 diabetes. We examined the endothelial GCX (eGCX) in skin flap tissue of 9-12-week-old type 2 diabetic mice (db/db mice) using a perforator skin flap and explored treatment with nafamostat mesylate. The growth rates were compared after 1 week. Heterotype (db/+) mice were used as the control group. Morphological examination of postoperative tissues was performed at 1, 3, 5, and 7 days post-surgery. In addition, db/db mice were treated with 30 mg/kg/day of nafamostat mesylate daily and were evaluated on postoperative day 7. Seven days after surgery, all db/db mice showed significant partial flap necrosis. Temporal observation of the skin flaps revealed a stasis-like discoloration and necrosis starting from the contralateral side of the remaining perforating branch. The control group did not exhibit flap necrosis, and the flap remained intact. In the quantitative assessment of endothelial glycans using lectins, intensity scoring showed that the eGCX in the db/db group was significantly thinner than that in the db/+ group. These results were consistent with the scanning electron microscopy findings. In contrast, treatment with nafamostat mesylate significantly improved the flap engraftment rate and suppressed eGCX injury. In conclusion, treatment with nafamostat mesylate improves the disrupted eGCX structure of skin flap tissue in db/db mice, potentially ameliorating the impaired capillary-to-venous return in the skin flap tissue.

Nude blebs expose cells to phagocytes.

Displacement of the glycocalyx by membrane blebbing enables macrophages to recognize apoptotic cells.

Autophagy Induced by Low Shear Stress Leads to Endothelial Glycocalyx Disruption.

INTRODUCTION: Previous studies have confirmed that low shear stress (LSS) induces glycocalyx disruption, leading to endothelial dysfunction. However, the role of autophagy in LSS-induced glycocalyx disruption and relevant mechanism are not clear. In this study, we hypothesized that LSS may promote autophagy, disrupting the endothelium glycocalyx. METHODS: Human umbilical vein endothelial cells were subjected to physiological shear stress and LSS treatments, followed by the application of autophagy inducers and inhibitors. Additionally, cells were treated with specific matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) inhibitor. The expression of autophagic markers, glycocalyx, MMP-2, and MMP-9 was measured. RESULTS: LSS impacted the expression of endothelium autophagy markers, increasing the expression of LC3II.LC3I-1 and Beclin-1, and decreasing the levels of p62, accompanied by glycocalyx disturbance. Moreover, LSS upregulated the expression of MMP-2 and MMP-9 and downregulated the levels of syndecan-1 and heparan sulfate (HS). Additionally, expression of MMP-2 and MMP-9 was increased by an autophagy promoter but was decreased by autophagy inhibitor treatment under LSS. Autophagy and MMP-2 and MMP-9 further caused glycocalyx disruption. CONCLUSION: LSS promotes autophagy, leading to glycocalyx disruption. Autophagy increases the expression of MMP-2 and MMP-9, which are correlated with the glycocalyx destruction induced by LSS.

Endothelial CXCR2 deficiency attenuates renal inflammation and glycocalyx shedding through NF-κB signaling in diabetic kidney disease.

BACKGROUND: The incidence of diabetic kidney disease (DKD) continues to rapidly increase, with limited available treatment options. One of the hallmarks of DKD is persistent inflammation, but the underlying molecular mechanisms of early diabetic kidney injury remain poorly understood. C-X-C chemokine receptor 2 (CXCR2), plays an important role in the progression of inflammation-related vascular diseases and may bridge between glomerular endothelium and persistent inflammation in DKD. METHODS: Multiple methods were employed to assess the expression levels of CXCR2 and its ligands, as well as renal inflammatory response and endothelial glycocalyx shedding in patients with DKD. The effects of CXCR2 on glycocalyx shedding, and persistent renal inflammation was examined in a type 2 diabetic mouse model with Cxcr2 knockout specifically in endothelial cells (DKD-Cxcr2 eCKO mice), as well as in glomerular endothelial cells (GECs), cultured in high glucose conditions. RESULTS: CXCR2 was associated with early renal decline in DKD patients, and endothelial-specific knockout of CXCR2 significantly improved renal function in DKD mice, reduced inflammatory cell infiltration, and simultaneously decreased the expression of proinflammatory factors and chemokines in renal tissue. In DKD conditions, glycocalyx shedding was suppressed in endothelial Cxcr2 knockout mice compared to Cxcr2 L/L mice. Modulating CXCR2 expression also affected high glucose-induced inflammation and glycocalyx shedding in GECs. Mechanistically, CXCR2 deficiency inhibited the activation of NF-κB signaling, thereby regulating inflammation, restoring the endothelial glycocalyx, and alleviating DKD. CONCLUSIONS: Taken together, under DKD conditions, activation of CXCR2 exacerbates inflammation through regulation of the NF-κB pathway, leading to endothelial glycocalyx shedding and deteriorating renal function. Endothelial CXCR2 deficiency has a protective role in inflammation and glycocalyx dysfunction, suggesting its potential as a promising therapeutic target for DKD treatment.

Down-regulating lncRNA KCNQ1OT1 relieves type II alveolar epithelial cell apoptosis during one-lung ventilation via modulating miR-129-5p/HMGB1 axis induced pulmonary endothelial glycocalyx.

OBJECTIVE: Endothelial glycocalyx (EG) maintains vascular homeostasis and is destroyed after one-lung ventilation (OLV)-induced lung injury. Long noncoding RNAs (lncRNAs) are critically involved in various lung injuries. This study aimed to investigate the role and regulatory mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in OLV-induced lung injury and LPS-induced type II alveolar epithelial cell (AECII) apoptosis. METHODS: The rat OLV model was established, and the effects of KCNQ1OT1 on OLV-induced ALI in vivo were explored. Bax and Caspase-3 expression in rat lung tissues was measured by immunochemistry (IHC). AECIIs were isolated from rat lungs and treated with LPS or normal saline (control) for in vitro analysis. The expression of KCNQ1OT1, miR-129-5p, and HMGB1 was measured by quantitative real-time PCR (qRT-PCR) or Western blot (WB). Cell proliferation and apoptosis were examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and flow cytometry. The downstream targets of KCNQ1OT1 were predicted by bioinformatics, and the binding relationship between KCNQ1OT1 and miR-129-3p was verified by dual-luciferase reporter assays. The potential target of miR-129-5p was further explored on the Targetscan website and revealed to target HMGB1. Enzyme-linked immunosorbent assay (ELISA) or WB was adopted to determine the levels of IL-1ß, TNF-α, MDA, SOD, heparanase (HPA), matrix metalloproteinase 9 (MMP9), heparan sulfate (HS) and syndecan-1 (SDC-1). RESULTS: KCNQ1OT1 and HMGB1 were up-regulated during OLV-induced lung injury, and their expression was positively correlated. KCNQ1OT1 knockdown reduced OLV-induced pulmonary edema and lung epithelial cell apoptosis, increased vascular permeability, reduced IL-1ß, TNF-α, MDA, and SOD levels and glycocalyx markers by targeting miR-129-5p or upregulating HMGB1. Overexpressing KCNQ1OT1 promoted cell apoptosis, reduced cell proliferation, aggravated inflammation and oxidative stress, and up-regulated HMGB1, HPA and MMP9 in LPS-treated AECIIs, while the HMGB1 silencing showed the opposite effects. MiR-129-5p mimics partially eliminated the KCNQ1OT1-induced effects, while recombinant HMGB1 restored the effects of miR-129-5p overexpression on AECIIs. Additionally, KCNQ1OT1 was demonstrated to promote the activation of the p38 MAPK/Akt/ERK signaling pathways in AECIIs via HMGB1. CONCLUSION: KCNQ1OT1 knockdown alleviated AECII apoptosis and EG damage during OLV by targeting miR-129-5p/HMGB1 to inactivate the p38 MAPK/Akt/ERK signaling. The findings of our study might deepen our understanding of the molecular basis in OLV-induced lung injury and provide clues for the targeted disease management.

EFFECT OF MIR-21-3P ON INTESTINAL INJURY IN RATS WITH TRAUMATIC HEMORRHAGIC SHOCK RESUSCITATED WITH THE SODIUM BICARBONATE RINGER'S SOLUTION.

ABSTRACT: Background : This study aims to determine the impact and mechanism of miR-21-3p on intestinal injury and intestinal glycocalyx during fluid resuscitation in traumatic hemorrhagic shock (THS), and the different impacts of sodium lactate Ringer's solution (LRS) and sodium bicarbonate Ringer's solution (BRS) for resuscitation on intestinal damage. Methods : A rat model of THS was induced by hemorrhage from the left femur fracture. The pathological changes of intestinal tissues and glycocalyx structure were observed by hematoxylin-eosin staining and transmission electron microscope. MiR-21-3p expression in intestinal tissues was detected by real-time quantitative polymerase chain reaction. The expression of glycocalyx-, cell junction-, and PI3K/Akt/NF-κB signaling pathway-related proteins was analyzed by western blot. Results : MiR-21-3p expression was increased in THS rats, which was suppressed by resuscitation with BRS. BRS or LRS aggravated the intestinal injury and damaged intestinal glycocalyx in THS rats. The expression of SDC-1, HPA, ß-catenin, MMP2, and MMP9 was upregulated, the expression of E-cad was downregulated, and the PI3K/Akt/NF-κB signaling pathway was activated in THS rats, which were further aggravated by BRS or LRS. The adverse effect of LRS was more serious than BRS. MiR-21-3p overexpression deteriorated the injury of intestinal tissues and intestinal glycocalyx; increased the expression of SDC-1, HPA, ß-catenin, MMP2, and MMP9 while decreasing E-cad expression; and activated the PI3K/Akt/NF-κB signaling pathway in BRS-resuscitated THS rats. Conclusion : MiR-21-3p aggravated intestinal tissue injury and intestinal glycocalyx damage through activating PI3K/Akt/NF-κB signaling pathway in rats with THS resuscitated with BRS.

The potential involvement of glycocalyx disruption in abdominal aortic aneurysm pathogenesis.

BACKGROUND: Abdominal aortic aneurysm is a weakening and expansion of the abdominal aorta. Currently, there is no drug treatment to limit abdominal aortic aneurysm growth. The glycocalyx is the outermost layer of the cell surface, mainly composed of glycosaminoglycans and proteoglycans. OBJECTIVE: The aim of this review was to identify a potential relationship between glycocalyx disruption and abdominal aortic aneurysm pathogenesis. METHODS: A narrative review of relevant published research was conducted. RESULTS: Glycocalyx disruption has been reported to enhance vascular permeability, impair immune responses, dysregulate endothelial function, promote extracellular matrix remodeling and modulate mechanotransduction. All these effects are implicated in abdominal aortic aneurysm pathogenesis. Glycocalyx disruption promotes inflammation through exposure of adhesion molecules and release of proinflammatory mediators. Glycocalyx disruption affects how the endothelium responds to shear stress by reducing nitric oxide availabilty and adversely affecting the storage and release of several antioxidants, growth factors, and antithromotic proteins. These changes exacerbate oxidative stress, stimulate vascular smooth muscle cell dysfunction, and promote thrombosis, all effects implicated in abdominal aortic aneurysm pathogenesis. Deficiency of key component of the glycocalyx, such as syndecan-4, were reported to promote aneurysm formation and rupture in the angiotensin-II and calcium chloride induced mouse models of abdominal aortic aneurysm. CONCLUSION: This review provides a summary of past research which suggests that glycocalyx disruption may play a role in abdominal aortic aneurysm pathogenesis. Further research is needed to establish a causal link between glycocalyx disruption and abdominal aortic aneurysm development.

Adiponectin Reduces Glomerular Endothelial Glycocalyx Disruption and Restores Glomerular Barrier Function in a Mouse Model of Type 2 Diabetes.

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.

GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17.

Acute lung injury (ALI) has received considerable attention in intensive care owing to its high mortality rate. It has been demonstrated that the selective alpha7 nicotinic acetylcholine receptor agonist Gainesville Tokushima scientists (GTS)-21 is promising for treating ALI caused by lipopolysaccharides (LPS). However, the precise underlying mechanism remains unknown. This study aimed to investigate the potential efficacy of GTS-21 in the treatment of ALI. We developed mouse models of ALI and alveolar epithelial type II cells (AT2s) injury following treatment with LPS and different polarized macrophage supernatants, respectively. Pathological changes, pulmonary edema, and lung compliance were assessed. Inflammatory cells count, protein content, and pro-inflammatory cytokine levels were analysed in the bronchoalveolar lavage fluid. The expression of angiotensin-converting enzyme (ACE), ACE2, syndecan-1 (SDC-1), heparan sulphate (HS), heparanase (HPA), exostosin (EXT)-1, and NF-κB were tested in lung tissues and cells. GTS-21-induced changes in macrophage polarization were verified in vivo and in vitro. Polarized macrophage supernatants with or without recombination a disintegrin and metalloproteinase-17 (ADAM-17) and small interfering (si)RNA ADAM-17 were used to verify the role of ADAM-17 in AT2 injury. By reducing pathological alterations, lung permeability, inflammatory response, ACE/ACE2 ratio, and glycocalyx shedding, as well as by downregulating the HPA and NF-κB pathways and upregulating EXT1 expression in vivo, GTS-21 significantly diminished LPS-induced ALI compared to that of the LPS group. GTS-21 significantly attenuated macrophage M1 polarization and augmented M2 polarization in vitro and in vivo. The destructive effects of M1 polarization supernatant can be inhibited by GTS-21 and siRNA ADAM-17. GTS-21 exerted a protective effect against LPS-induced ALI, which was reversed by recombinant ADAM-17. Collectively, GTS-21 alleviates LPS-induced ALI by attenuating AT2s ACE/ACE2 ratio and glycocalyx shedding through the inhibition of macrophage M1 polarization derived ADAM-17.

Improving impact of heparan sulfate on the endothelial glycocalyx abnormalities in atherosclerosis as revealed by glycan-protein interactome.

Endothelial dysfunction induced by oxidative stress is an early predictor of atherosclerosis, which can cause various cardiovascular diseases. The glycocalyx layer on the endothelial cell surface acts as a barrier to maintain endothelial biological function, and it can be impaired by oxidative stress. However, the mechanism of glycocalyx damage during the development of atherosclerosis remains largely unclear. Herein, we established a novel strategy to address these issues from the glycomic perspective that has long been neglected. Using countercharged fluorescence protein staining and quantitative mass spectrometry, we found that heparan sulfate, a major component of the glycocalyx, was structurally altered by oxidative stress. Comparative proteomics and protein microarray analysis revealed several new heparan sulfate-binding proteins, among which alpha-2-Heremans-Schmid glycoprotein (AHSG) was identified as a critical protein. The molecular mechanism of AHSG with heparin was characterized through several methods. A heparan analog could relieve atherosclerosis by protecting heparan sulfate from degradation during oxidative stress and by reducing the accumulation of AHSG at lesion sites. In the present study, the molecular mechanism of anti-atherosclerotic effect of heparin through interaction with AHSG was revealed. These findings provide new insights into understanding of glycocalyx damage in atherosclerosis and lead to the development of corresponding therapeutics.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes.

BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.