Expression Of Vascular Endothelial Cadherin Research Articles

Pseudomonas aeruginosa N-3-Oxododecanoyl Homoserine Lactone Disrupts Endothelial Integrity by Activating the Angiopoietin-Tie System.

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.

Infantile Hemangiomas Lose Vascular Endothelial Cadherin During Involution: Potential Role in Cell Death?

Background: Infantile hemangiomas (IHs) are benign endothelial cell (EC) tumors that undergo a predictable natural history, with rapid proliferation, stabilization, and involution. However, mechanisms regulating these transitions are not well understood. We have observed loss of vascular endothelial cadherin (VECAD) in involuting/involuted IHs. VECAD plays a critical role in angiogenesis, cell cycle progression, and EC survival. We hypothesize that loss of VECAD is associated with apoptosis occurring during IH involution. Methods: Resected IH samples were clinically categorized as proliferating (n = 4), stable (n = 4), or involuting/involuted (n = 5). Neonatal dermal tissues were used as controls (n = 5). Immunohistochemistry was conducted on sectioned specimens using antibodies against EC markers VECAD and CD31. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Results: CD31 signal intensity in proliferating, stable, and involuting/involuted IH ECs was unchanged relative to each other and to control ECs. VECAD signal significantly and progressively diminished as IHs progressed from proliferation to involution. Involuting/involuted IHs had significantly reduced VECAD expression compared with control ECs (P < 0.0001), proliferating IHs (P < 0.0001), and stable IHs (P < 0.001). As expected, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive ECs was significantly higher in involuting/involuted IHs (P < 0.05) relative to control ECs and proliferating IHs. Conclusions: Loss of VECAD expression in IH endothelium corresponded to IH involution and increased apoptosis. It is unclear whether loss of VECAD is causative of IH involution; further studies are needed to elucidate the role of VECAD function in EC survival.

Role of endothelial hyaluronan in peritoneal membrane transport and disease conditions during peritoneal dialysis

Peritoneal membrane dysfunction in peritoneal dialysis (PD) is primarily attributed to angiogenesis; however, the integrity of vascular endothelial cells can affect peritoneal permeability. Hyaluronan, a component of the endothelial glycocalyx, is reportedly involved in preventing proteinuria in the normal glomerulus. One hypothesis suggests that development of encapsulating peritoneal sclerosis (EPS) is triggered by protein leakage due to vascular endothelial injury. We therefore investigated the effect of hyaluronan in the glycocalyx on peritoneal permeability and disease conditions. After hyaluronidase-mediated degradation of hyaluronan on the endothelial cells of mice, macromolecules, including albumin and β2 microglobulin, leaked into the dialysate. However, peritoneal transport of small solute molecules was not affected. Pathologically, hyaluronan expression was diminished; however, expression of vascular endothelial cadherin and heparan sulfate, a core protein of the glycocalyx, was preserved. Hyaluronan expression on endothelial cells was studied using 254 human peritoneal membrane samples. Hyaluronan expression decreased in patients undergoing long-term PD treatment and EPS patients treated with conventional solutions. Furthermore, the extent of hyaluronan loss correlated with the severity of vasculopathy. Hyaluronan on endothelial cells is involved in the peritoneal transport of macromolecules. Treatment strategies that preserve hyaluronan in the glycocalyx could prevent the leakage of macromolecules and subsequent related complications.

Carnosol alleviates sepsis-induced pulmonary endothelial barrier dysfunction by targeting nuclear factor erythroid2-related factor 2/sirtuin-3 signaling pathway to attenuate oxidative damage.

Excessive reactive oxygen species production during acute lung injury (ALI) will aggravate the inflammatory process and endothelial barrier dysfunction. Carnosol is a natural phenolic diterpene with antioxidant and anti-inflammatory properties, but its role in treating sepsis-induced ALI remains unclear. This study aims to explore the protective effects and underlying mechanisms of carnosol in sepsis-induced ALI. C57BL/6 mouse were preconditioned with carnosol for 1 h, then the model of lipopolysaccharide (LPS)-induced sepsis was established. The degree of pulmonary edema, oxidative stress, and inflammation were detected. Endothelial barrier function was evaluated by apoptosis and cell junctions. In vitro, Mito Tracker Green probe, JC-1 staining, and MitoSOX staining were conducted to investigate the effect of carnosol on mitochondria. Finally, we investigated the role of nuclear factor-erythroid 2-related factor (Nrf2)/sirtuin-3 (SIRT3) in carnosol against ALI. Carnosol alleviated LPS-induced pulmonary oxidative stress and inflammation by inhibiting excess mitochondrial reactive oxygen species production and maintaining mitochondrial homeostasis. Furthermore, carnosol also attenuated LPS-induced endothelial cell barrier damage by reducing vascular endothelial cell apoptosis and restoring occludin, ZO-1, and vascular endothelial-Cadherin expression in vitro and in vivo. In addition, carnosol increased Nrf2 nuclear translocation to promote SIRT3 expression. The protective effects of carnosol on ALI were largely abolished by inhibition of Nrf2/SIRT3. Our study has provided the first evidence that the Nrf2/SIRT3 pathway is a protective target of the endothelial barrier in ALI, and carnosol can serve as a potential therapeutic candidate for ALI by utilizing its ability to target this pathway.

Comparative effects of canagliflozin and sitagliptin in chronically ischemic myocardium

Aim: Recent studies demonstrate that sodium-glucose cotransporter 2 inhibitors (SGLT2i) and dipeptidyl peptidase-4 inhibitors (DPP4i), two classes of antidiabetic drugs, are cardioprotective. However, the mechanisms of these benefits and their comparative efficacy remain unclear. We aimed to compare the effects of these antidiabetic agents on cardiac function, perfusion, and microvascular density using a swine model of chronic myocardial ischemia. Methods: Chronic myocardial ischemia was induced in Yorkshire swine by ameroid constrictor placement to the left circumflex artery. Two weeks later, pigs were administered vehicle (“CON”, 8 pigs), 300 mg SGLT2i canagliflozin, (“CANA”, 8 pigs), or 100 mg DPP4i sitagliptin (“SIT”, 5 pigs) daily. Five weeks later, pigs were euthanized. Cardiac function, perfusion, collateralization, and protein expression were determined by pressure-volume catheter, microsphere analysis, immunofluorescence, and immunoblotting, respectively. Results: Compared with SIT, CANA was associated with improved stroke volume and cardiac output, with a trend towards reduced left ventricular stiffness. Both CANA and SIT trended towards improved perfusion compared to CON, but there were no differences between the two treatment groups. SIT was associated with improved capillary density with a trend towards improved arteriolar density compared to CANA. Both CANA and SIT were associated with increased expression of vascular endothelial cadherin compared to CON, without differences in treatment groups. SIT pigs had decreased 5′ adenosine monophosphate-activated protein kinase activation compared to CON and CANA. There was a trend towards increased endothelial nitric oxide synthase activation in the SIT group compared to CON. There were no differences in activation of extracellular signal-regulated kinase 1/2 across groups. Conclusions: In the setting of chronic myocardial ischemia, canagliflozin is associated with improved cardiac function compared to sitagliptin, with similar effects on perfusion despite differences in microvascular collateralization.

Hydrogels with tunable mechanical plasticity regulate endothelial cell outgrowth in vasculogenesis and angiogenesis

The endothelial cell (EC) outgrowth in both vasculogenesis and angiogenesis starts with remodeling surrounding matrix and proceeds with the crosstalk between cells for the multicellular vasculature formation. The mechanical plasticity of matrix, defined as the ability to permanently deform by external traction, is pivotal in modulating cell behaviors. Nevertheless, the implications of matrix plasticity on cell-to-cell interactions during EC outgrowth, along with the molecular pathways involved, remain elusive. Here we develop a collagen-hyaluronic acid based hydrogel platform with tunable plasticity by using compositing strategy of dynamic and covalent networks. We show that although the increasing plasticity of the hydrogel facilitates the matrix remodeling by ECs, the largest tubular lumens and the longest invading distance unexpectedly appear in hydrogels with medium plasticity instead of the highest ones. We unravel that the high plasticity of the hydrogels promotes stable integrin cluster of ECs and recruitment of focal adhesion kinase with an overenhanced contractility which downregulates the vascular endothelial cadherin expression and destabilizes the adherens junctions between individual ECs. Our results, further validated with mathematical simulations and in vivo angiogenic tests, demonstrate that a balance of matrix plasticity facilitates both cell-matrix binding and cell-to-cell adherens, for promoting vascular assembly and invasion.

Circulating Exosomes from Alzheimer's Disease Suppress Vascular Endothelial-Cadherin Expression and Induce Barrier Dysfunction in Recipient Brain Microvascular Endothelial Cell.

Blood-brain barrier (BBB) breakdown is a crucial aspect of Alzheimer's disease (AD) progression. Dysfunction in BBB is primarily caused by impaired tight junction and adherens junction proteins in brain microvascular endothelial cells (BMECs). The role of adherens junctions in AD-related BBB dysfunction remains unclear. Exosomes from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells. However, it remains unknown if and how these exosomes cause BMEC dysfunction in AD. This study aimed to investigate the impact of AD circulating exosomes on brain endothelial dysfunction. Exosomes were isolated from sera of AD patients and age- and sex-matched cognitively normal controls using size-exclusion chromatography. The study measured the biomechanical nature of BMECs' endothelial barrier, the lateral binding forces between live BMECs. Paracellular expressions of the key adherens junction protein vascular endothelial (VE)-cadherin were visualized in BMEC cultures and a 3D BBB model using human BMECs and pericytes. VE-cadherin signals were also examined in brain tissues from AD patients and normal controls. Circulating exosomes from AD patients reduced VE-cadherin expression levels and impaired barrier function in recipient BMECs. Immunostaining analysis demonstrated that AD exosomes damaged VE-cadherin integrity in a 3D microvascular tubule formation model. The study found that AD exosomes weakened BBB integrity depending on their RNA content. Additionally, diminished microvascular VE-cadherin expression was observed in AD brains compared to controls. These findings highlight the significant role of circulating exosomes from AD patients in damaging adherens junctions of recipient BMECs, dependent on exosomal RNA.

Evaluation of the effects of glucose and oxygen on the vascular endothelial cell migration.

Glucose is essential as the main energy source for living organisms. However, excessive elevation of blood sugar levels can lead to diabetes and serious complications such as arteriosclerosis. Even though blood sugar levels as well as hypoxia associated with hyperglycemia are known to be closely related to diabetes complications, the responses of vascular endothelial cells to glucose and oxygen have not been fully investigated. In this study, using a microfluidic device that can control the oxygen concentration, we observed the behavior of vascular endothelial cell monolayers while simultaneously controlling glucose and oxygen levels. Results showed that the cell migration speed was increased by high-glucose exposure in an oxygen-rich environment, but was decreased in a hypoxic environment regardless of glucose condition. The expression of vascular endothelial-cadherin at the cell periphery, which plays a role in cell-cell adhesion, was increased by hypoxic exposure, but was largely independent of glucose condition. This suggested that cell-cell adhesion is less involved in the increase in migration caused by high glucose. Furthermore, stabilization and nuclear translocation of hypoxia-inducible factor-1α, which is involved in cellular hypoxia sensing, increased 5 h after exposure to high glucose, but decreased 3 days after the exposure. This indicated that intracellular hypoxia was generated by increased oxygen consumption in mitochondria just after the high-glucose exposure, but it was moderated within 3 days.

Reduction in disialyl-T antigen levels in mice deficient for both St6galnac3 and St6galnac4 results in blood filling of lymph nodes

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galβ1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis

Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis. In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined. In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells. E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis. A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vesselproliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

Neutrophil extracellular traps contribute to coagulopathy after traumatic brain injury.

Coagulopathy contributes to the majority of deaths and disabilities associated with traumatic brain injury (TBI). Whether neutrophil extracellular traps (NETs) contribute to an abnormal coagulation state in the acute process of TBI remains unknown. Our objectives were to demonstrate the definitive role of NETs in coagulopathy in TBI. We detected NET markers in 128 TBI patients and 34 healthy subjects. Neutrophil-platelet (PLT) aggregates were detected in blood samples from TBI patients and healthy subjects using flow cytometry and staining with CD41 and CD66b. Endothelial cells (ECs) were incubated with isolate NETs and detected the expression of vascular endothelial (VE)-cadherin, syncanden-1, thrombomodulin, von Willebrand factor (VWF), phosphatidylserine (PS) and and tissue factor (TF). In addition, we established a TBI mouse model to detect the potential role of NETs in TBI associated coagulopathy. NETs generation was mediated by High Mobility Group Box 1(HMGB1) from activated platelets and contribute to procoagulant activity in TBI. Furthermore, coculture experiments indicated that NETs damaged the endothelial barrier and caused these cells to assume a procoagulant phenotype. Moreover, the administration of DNase I before or after brain trauma markedly reduced coagulopathy and improved the survival and clinical outcome of mice with TBI.

Activating transcription factor 4 protects mice against sepsis-induced intestinal injury by regulating gut-resident macrophages differentiation.

Gut-resident macrophages (gMacs) supplemented by monocytes-to-gMacs differentiation play a critical role in maintaining intestinal homeostasis. Activating transcription factor 4 (ATF4) is involved in immune cell differentiation. We therefore set out to investigate the role of ATF4-regulated monocytes-to-gMacs differentiation in sepsis-induced intestinal injury. Sepsis was induced in C57BL/6 wild type (WT) mice and Atf4- knockdown ( Atf4+/ - ) mice by cecal ligation and puncture or administration of lipopolysaccharide (LPS). Colon, peripheral blood mononuclear cells, sera, lung, liver, and mesenteric lymph nodes were collected for flow cytometry, hematoxylin and eosin staining, immunohistochemistry, quantitative reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. CD64, CD11b, Ly6C, major histocompatibility complex-II (MHC-II), CX3CR1, Ly6G, and SSC were identified as optimal primary markers for detecting the process of monocytes-to-gMacs differentiation in the colon of WT mice. Monocytes-to-gMacs differentiation was impaired in the colon during sepsis and was associated with decreased expression of ATF4 in P1 (Ly6C hi monocytes), the precursor cells of gMacs. Atf4 knockdown exacerbated the impairment of monocytes-to-gMacs differentiation in response to LPS, resulting in a significant reduction of gMacs in the colon. Furthermore, compared with WT mice, Atf4+/- mice exhibited higher pathology scores, increased expression of inflammatory factor genes ( TNF-α, IL-1β ), suppressed expression of CD31 and vascular endothelial-cadherin in the colon, and increased translocation of intestinal bacteria to lymph nodes and lungs following exposure to LPS. However, the aggravation of sepsis-induced intestinal injury resulting from Atf4 knockdown was not caused by the enhanced inflammatory effect of Ly6C hi monocytes and gMacs. ATF4, as a novel regulator of monocytes-to-gMacs differentiation, plays a critical role in protecting mice against sepsis-induced intestinal injury, suggesting that ATF4 might be a potential therapeutic target for sepsis treatment.

Construction of functional magnetic scaffold with temperature control switch for long-distance vascular injury

Fusion of endothelial monolayer for rapid reendothelialization is the key to vascular transplantation, which is crucial for remodeling long-distance (≥10 mm) vascular injury (LDVJ) and reducing the long-term repair period. Although silk fibroin (SF) has been widely prepared as a scaffold to repair vascular injuries, it is still challenging to remodel LDVJs. The design and manufacture of functional scaffolds with a rapid reendothelialization enhancement effect are helpful to solve this problem. Here, we developed a well-designed functional magnetic scaffold (MDP) with a temperature-controlled switch, intelligently regulating the release of magnetic nanoparticles (MNPs) to induce macrophages migration and M2 polarization. The results show that the radial support force of MDP is appropriately five times higher than that of pure silk fibroin scaffold (SFC), and the degradation of MDP is delayed, which is beneficial to maintaining the integrity of the scaffold. MDP induces the migration of macrophages, upregulates the expression of repair cytokines such as TGF-β, IL-10 and VEGF, and promotes the proliferation of mouse aortic vascular smooth muscle (MOVAS) cells. More importantly, the enhanced migration of macrophages at the phase transition temperature is more conducive to the proliferation and accumulation of vascular repair-related cells, which is conducive to rapid reendothelialization. In addition, MDP also significantly regulates the polarization of macrophages to M2 type, which is conducive to the secretion of repair cytokines to further promote vascular recovery. The evaluation of endothelial cells' adhesion and function also proved that MDP could increase the expression of vascular endothelial cadherin (VE-Cad) and collagen IV (COL IV) and has the potential for rapid reendothelialization. The replacement model in vivo animal experiments further proved that MDP exhibited a better repair effect than SFC, providing a new option for the repair of LDVJ in the future.

Gentianella acuta improves TAC-induced cardiac remodelling by regulating the Notch and PI3K/Akt/FOXO1/3 pathways

Cardiac remodelling mainly manifests as excessive myocardial hypertrophy and fibrosis, which are associated with heart failure. Gentianella acuta (G. acuta) is reportedly effective in cardiac protection; however, the mechanism by which it protects against cardiac remodelling is not fully understood. Here, we discuss the effects and mechanisms of G. acuta in transverse aortic constriction (TAC)-induced cardiac remodelling in rats. Cardiac function was analysed using echocardiography and electrocardiography. Haematoxylin and eosin, Masson′s trichrome, and wheat germ agglutinin staining were used to observe pathophysiological changes. Additionally, real-time quantitative reverse transcription polymerase chain reaction and western blotting were used to measure protein levels and mRNA levels of genes related to myocardial hypertrophy and fibrosis. Immunofluorescence double staining was used to investigate the co-expression of endothelial and interstitial markers. Western blotting was used to estimate the expression and phosphorylation levels of the regulatory proteins involved in autophagy and endothelial-mesenchymal transition (EndMT). The results showed that G. acuta alleviated cardiac dysfunction and remodelling. The elevated levels of myocardial hypertrophy and fibrosis markers, induced by TAC, decreased significantly after G. acuta intervention. G. acuta decreased the expression of LC3 II and Beclin1, and increased p62 expression. G. acuta upregulated the expression of CD31 and vascular endothelial-cadherin, and prevented the expression of α-smooth muscle actin and vimentin. Furthermore, G. acuta inhibited the PI3K/Akt/FOXO1/3a pathway and activated the Notch signalling. These findings demonstrated that G. acuta has cardioprotective effects, such as alleviating myocardial fibrosis, inhibiting hypertrophy, reducing autophagy, and blocking EndMT by regulating the PI3K/Akt/FOXO1/3a and Notch signalling pathways.

Serum expression of Vascular Endothelial-Cadherin, CD44, Human High mobility group B1, Kallikrein 6 proteins in different stages of laryngeal intraepithelial lesions and early glottis cancer.

BackgroundThe study was designed to evaluate the potential validity and utility of selected molecular markers in serum samples from patients with specific stages of laryngeal intraepithelial lesions that could serve as diagnostic tools in differentiation of benign and dysplastic lesions from invasive pathologies.MethodsProspective study included 80 consecutive patients with vocal fold lesions treated at the single otorhinolaryngology centre. All participants had surgical resection of the lesion. Blood samples were collected from each patient before the surgery. Final diagnosis was confirmed on histopathological examination and included 39 (48.75%) non-dysplastic lesions, eight (10%) low-grade dysplasia, six (7.5%) high-grade dysplasia and 27 (33.75%) invasive cancers. The ELISA procedures were performed according to the manufacturer’s instruction. Individual serum concentration of selected proteins was reported in ng/ml: Vascular Endothelial-Cadherin Complex (VE-cad), CD44, Human High mobility group protein B1(HMGB1), Kallikrein 6.ResultsThe highest mean levels of HMGB1, KLK6 and VE-cad were detected in sera of patients with low-grade dysplasia (81.14, 24.33, 14.17 respectively). Soluble CD44 was the most elevated in patients with non-dysplastic lesions (2.49). The HMGB1, KLK6 and VE-cad serum levels were increasing from non-dysplastic to low-grade dysplasia and followed by the decrease for high-grade dysplasia and invasive cancer, however the differences were not significant (p-values 0.897, 0.354, 0.1 respectively). Patients’ serum had the highest CD44 concentration in non-dysplastic and low-grade dysplasia with the following decrease through high-grade dysplasia and invasive cancer. GERD symptomatic patients had higher levels of KLK6 and CD44 than other patients (p-value 0.06 and 0.084 respectively). There were no significant differences of biomarkers levels related to patients’ gender (p-value from 0.243 to 1) or smoking status (p-value from 0.22 to 0.706).ConclusionsVE-cad, HMGB1, CD44 and KLK6 did not prove to be reliable biomarkers implicating malignant potential within vocal fold hypertrophic intraepithelial lesions.

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

BackgroundThe brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types.MethodsMouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function.ResultsHerein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function.ConclusionsAgo2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Loss of Endothelial Cell Matrix Metalloproteinase 14 Reduces Melanoma Growth and Metastasis by Increasing Tumor Vessel Stability

Matrix metalloproteinase (MMP) 14 belongs to a large family of zinc-dependent endopeptidases and plays a critical role in skin physiological and pathological processes. Complete loss of the protease resulted in severe developmental defects leading to early death. However, because of the premature death of the mice, the functional significance for endothelial cell (EC) expression of MMP14 in skin physiology and pathology invivo after birth is yet unknown. Using a mouse model with constitutive EC-specific deletion of Mmp14 (Mmp14EC‒/‒), we showed that mice developed and bred normal, but melanoma growth and metastasis were reduced. Although vascularity was unaltered, vessel permeability was decreased. Deletion of MMP14 in ECs led to increased vessel coverage by pericytes and vascular endothelial-cadherin expression in mice invivo and invitro but not in human ECs. Endothelial nitric oxide synthase expression and nitric oxide production were significantly reduced in Mmp14EC‒/‒ ECs and MMP14-silenced human umbilical vein ECs. A direct correlation between endothelial nitric oxide synthase and MMP14 expression was detected in intratumoral vessels of human malignant melanomas. Altogether, we show that endothelial MMP14 controls tumor vessel function during melanoma growth. These data suggest that EC-derived MMP14 direct targeting alone or with vascular stabilizing agents may be therapeutically crucial in inhibiting melanoma growth and metastasis.

EMT-cancer cells-derived exosomal miR-27b-3p promotes circulating tumour cells-mediated metastasis by modulating vascular permeability in colorectal cancer.

BackgroundMetastasis is the main cause of death in colorectal cancer (CRC). Circulating tumour cells (CTCs) are regarded as the precursor cells of metastasis. The CTCs, which underwent epithelial‐mesenchymal transition (EMT), are associated with metastasis and responsible for poor prognosis. EMT cancer cells modulate endothelial permeability in the invasive front and facilitate cancer cell intravasation, resulting in CTCs‐mediated distant metastasis. Exosomes derived from cancer cells are key mediators of cancer‐host intercommunication. However, the mechanism by which EMT‐tumour cells‐derived exosomes modulate vascular permeability and promote CTCs generation has remained unclear.MethodsExosomes isolation and purification were conducted by ultra‐centrifugation. Exosomal miRNA was identified by sequencing followed by quantitative PCR. In vitro co‐culture assay experiments were conducted to evaluate the effect of exosomal miR‐27b‐3p on the permeability of blood vessel endothelium. Dual‐luciferase reporter assay, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) were performed to investigate the underlying mechanism by which miR‐27b‐3p is packaged into exosomes. A mouse model was established to determine the role of exosomal miR‐27b‐3p in blood vessel permeability modulation in vivo.ResultsWe found that EMT‐CRC cells attenuate the blood vessel barrier by transferring miR‐27b‐3p to human umbilical vein endothelial cells (HUVECs) in exosomes. Mechanically, miR‐27b‐3p atteuated the expression of vascular endothelial cadherin (VE‐Cad) and p120 at the post‐transcriptional level by binding to 3′‐untranslated region of VE‐Cad and p120 directly. The packaging of miR‐27b‐3p into exosomes was induced by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which activated by STAT3. Clinically, miR‐27b‐3p up‐regulated in CRC tissues. Plasma exosomal miR‐27b‐3p was positively correlated with malignant progression and CTC count in CRC patients. Our study reveals a novel mechanism by which EMT‐CRC cells promote metastasis, increasing blood vessel permeability and facilitating the generation of CTCs.ConclusionExosomal miR‐27b‐3p secreted by EMT‐CRC cells increases blood vessel permeability and facilitates the generation of CTCs. Exosomal miR‐27b‐3p may become a promising biomarker for CRC metastasis.

A reciprocal feedback of miR-548ac/YB-1/Snail induces EndMT of HUVECs during acidity microenvironment

BackgroundResearches indicated the process of Endothelial-Mesenchymal-Transition (EndMT) of vascular endothelial cells (ECs) was critically involved in the progression of tumor. ECs demonstrated functional and phenotypic heterogeneity when located under different microenvironments. The extracellular pH of tumor tissues was acidic compared to that of normal tissues. However, there was still unclear whether the acidic microenvironment affected the EndMT of vascular ECs.MethodsHuman Umbilical Vein Endothelial Cell (HUVECs) was cultured under the normal or acidic medium to evaluate the alteration of morphology, migration, permeability, and EndMT markers. Microarray assay was adopted to analyze the differential expression of miRNAs in the acidity-treated HUVECs. Gain- and loss- of function experiments were performed to evaluate the functional role of miRNA-548ac on acidity-induced EndMT of HUVECs. Luciferase reporter and Chromatin-immunoprecipitation assays were conducted to assess the downstream pathway of miRNA-548ac in acidity-induced EndMT of HUVECs.ResultsOur results showed that HUVECs demonstrated mesenchymal transition under acidic conditions with the increase of migration, permeability, and expression of α-SMA and Vimentin, but the expression of vascular endothelial cadherin (VE-cadherin) and CD31 were reduced. In addition, the acidity-treated HUVECs remarkably facilitated the transmigration of pancreatic cancer cells. The expression of miRNA-548ac was significantly decreased in the acidity-treated HUVECs. Moreover, overexpression of miR-548ac inhibited the EndMT of HUVECs and consequently impeded the transmigration of pancreatic cancer cells. The miR-548ac inhibited the expression of YB-1 by binding to the 3’UTR of its mRNA, and YB-1 promoted the translation of Snail which was a critical regulator of EndMT. What’s more, Snail transcriptionally inhibited the expression of miR-548ac through binding to the promoter of its host gene.ConclusionsOur data implicated that the acidic microenvironment promoted the EndMT of HUVECs by the miR-548ac/YB-1/Snail axis, which could contribute to the metastasis of pancreatic cancer.

Integrating engineered macro vessels with self-assembled capillaries in 3D implantable tissue for promoting vascular integration in-vivo

A functional multi-scale vascular network can promote 3D engineered tissue growth and improve transplantation outcome. In this work, by using a combination of living cells, biological hydrogel, and biodegradable synthetic polymer we fabricated a biocompatible, multi-scale vascular network (MSVT) within thick, implantable engineered tissues. Using a templating technique, macro-vessels were patterned in a 3D biodegradable polymeric scaffold seeded with endothelial and support cells within a collagen gel. The lumen of the macro-vessel was lined with endothelial cells, which further sprouted and anastomosed with the surrounding self-assembled capillaries. Anastomoses between the two-scaled vascular systems displayed tightly bonded cell junctions, as indicated by vascular endothelial cadherin expression. Moreover, MSVT functionality and patency were demonstrated by dextran passage through the interconnected multi-scale vasculature. Additionally, physiological flow conditions were applied with home-designed flow bioreactors, to achieve a MSVT with a natural endothelium structure. Finally, implantation of a multi-scale-vascularized graft in a mouse model resulted in extensive host vessel penetration into the graft and a significant increase in blood perfusion via the engineered vessels compared to control micro-scale-vascularized graft. Designing and fabricating such multi-scale vascular architectures within 3D engineered tissues may benefit both in vitro models and therapeutic translation research.