EAhy926 Endothelial Cells Research Articles

Methotrexate-Loaded Chitosan Oligosaccharide-ES2 for Targeted Cancer Therapy.

Cancer presents a significant health threat, necessitating the development of more precise, efficient, and less damaging treatment approaches. To address this challenge, we employed the 1-ethyl-(3-dimethyl aminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) catalytic system and utilized quaternized chitosan oligosaccharide (HTCOSC) as a drug carrier to construct a nanoparticle delivery system termed HTCOSC-cRGD-ES2-MTX (CREM). This system specifically targets integrin αvβ3 on tumor cell surfaces and enables simultaneous loading of the antiangiogenic agent ES2 (IVRRADRAAVP) and the chemotherapy drug methotrexate (MTX). Due to its amphiphilic properties, CREM self-assembles into nanoparticles in aqueous solution, exhibiting an average diameter of 179.47 nm. Comparative studies demonstrated that CREM, in contrast to free ES2 and MTX-free nanoparticles (CRE), significantly suppressed the proliferation of EAhy926 endothelial cells and B16 melanoma cells in vitro, resulting in inhibition rates of 71.18 and 82.25%, respectively. Furthermore, CREM exhibited a hemolysis rate below 2%, indicating excellent in vitro antiangiogenic and antitumor activity as well as favorable blood compatibility. Additionally, both CRE and CREM demonstrated favorable tumor targeting capabilities through the specific binding action of cyclic RGD (cRGD) to integrin αvβ3. Further in vivo investigations revealed that CREM induced apoptosis in tumor cells via the mitochondrial apoptotic pathway and reduced the expression of angiogenic factors such as vascular endothelial growth factor (VEGF), thereby inhibiting tumor angiogenesis. This potent antitumor effect was evident through a tumor suppression rate of 80.19%. Importantly, histopathological staining (HE staining) demonstrated the absence of significant toxic side effects of CREM on various organs compared to MTX. In conclusion, the CREM nano drug delivery system synergistically enhances the therapeutic efficacy of antiangiogenic drugs and chemotherapeutic agents, thus offering a novel targeted approach for cancer treatment.

#2660 WWP2-induced pericytes to myofibroblast transition contributes to the progression of fibrosis in CKD in diabetes

Abstract Background and Aims CKD in diabetes can be characterized by different clinical and histological phenotypes, associated to different molecular pathways specifically involved in the progression of kidney disease. CKD in diabetes includes different phenotypes, commonly classified according to Mazzucco et al (PMID:11920336) as diabetic Nephropathy (DN), non-diabetic renal disease (NDRD) and mixed forms (DN+NDRD). Among these, DN represents the primary cause of end stage renal disease (ESRD) and our group demonstrated that renal damage in DN is characterized by a specific molecular feature, represented by the activation of lysine63 (K63)-ubiquitination (Ub) pathway. In particular, we demonstrated that accumulation of K63-Ub proteins is involved in the progression of renal fibrosis (PMID: 27881486). WWP2 is an E3 ubiquitin-protein ligase that modulates myofibroblast pro-fibrotic activation in cardiac fibrosis (PMID: 31399586), however its specific role in kidney fibrosis in diabetes and its subsequent involvement in K63-Ub pathway has not been described so far. Thus aim of this study was to evaluate the involvement of WWP2 in the progression of kidney fibrosis in diabetes and its involvement in K63-Ub pathway. Method WWP2, K-63Ub, alpha-sma and PDGFbeta-receptor expression were evaluated in: i) 22 kidney biopsies from patients with a biopsy proven diagnosis of DN (n = 11), NDRD (n = 6) and CKD without diabetes (n = 5), by immunohistochemistry and immunofluorescence; ii) streptozotocin (STZ)-treated DBA/2J mice, a model of human DN, in the presence or absence of a specific inhibitor of K63Ub (NSC697923) by immunohistochemistry (n = 3 for each group); iii) in HK2 tubular cells, EAHY926 endothelial cells and in human-derived pericytes under hyperglycemic conditions by western blotting and qPCR. Results Immunohistochemistry showed that WWP2 was expressed both in CKD and in DN patients’ biopsies when compared to patients with NDRD (p < 0.05 and p < 0.01 respectively), both at tubular, glomerular and in the vascular compartment. Immunofluorescence confirmed that WWP2 increased expression in DN patients compared to NDRD, was associated to accumulation of K63Ub proteins in the tubular compartment as well as in the vascular compartment in particular in pericytes, cells that envelop the surface of vessels and have been described as the major source of scar-forming myofibroblasts in CKD (PMID: 19008372). The involvement of WWP2 in the K63-Ub pathway was confirmed in vivo in DBA2J diabetic mice in which the specific inhibition by NSC697923 of the K63Ub pathway, significantly reduced WWP2 expression in kidney tissues from diabetic mice (p < 0.05). These results were confirmed in vitro where specific inhibition of K63Ub pathway by NSC697923 significantly reduced hyperglycaemia-induced WWP2 expression in HK2 cells by western blotting and qPCR (p < 0.01). Hyperglycaemic conditions did not influence WWP2 expression in EAHY926 endothelial cells (p = n.s.). Conversely, using qPCR, in pericytes we observed an hyperglycemia-induced expression of both WWP2 levels (RR>2) and alpha-sma levels (RR>3) at different time points. Moreover, western blotting showed that hyperglycemia also induced a decrease of PDGF-beta-receptor marker of pericytes, thus suggesting their transition to myofibroblasts. Conclusion These findings demonstrate the influence of WWP2 on the k63-Ub pathway thus driving fibrogenesis in DN patients, in particular through pericyte to myofibroblast transition. WWP2 could represent a potential novel target for therapeutic intervention in the treatment of chronic kidney disease in diabetes.

Heterobivalent Peptide Agonists Comprising Covalently Linked Protease Activated Receptor 1 and Protease Activated Receptor 3 Peptides Mimic the Cytoprotective Cell Signaling Activities of Activated Protein C

Introduction: Activated protein C (APC) exhibits both anticoagulant and cytoprotective cell signaling actions, including anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Extensive in vitro and in vivo data for APC's cell signaling benefits identified requirements for two G-protein coupled receptors (GPCR), protease-activated receptor (PAR) PAR1 and PAR3 which are cleaved by APC at the noncanonical sites (R46 and R41, respectively). Agonism of PARs following proteolysis of the N-terminal tail is driven by the newly revealed N-terminus and related synthetic peptides, e.g., for APC, PAR1 peptides (P1) beginning with N47 or PAR3 peptides (P3) beginning with G42. We found that the non-canonical P1-47-66 and P3-42-65 peptides mimic APC in that each is anti-inflammatory by reducing NLRP3-driven caspase-1 activity in human THP-1 cells (Healy LD et al, J Thromb Haemost 2021;19:269-280). Remarkably, these two peptides act synergistically to attenuate activity of caspase-1 at low concentrations where either peptide alone has little activity. Thus, we undertook studies of P1 peptides that were covalently linked to P3 peptides for ability to mimic APC's anti-inflammatory and endothelial barrier stabilization actions. Methods: Monocytes (THP-1 cells) were differentiated into macrophages using PMA, then pretreated with APC or peptides prior to stimulation with lipopolysaccharide and adenosine triphosphate to induce caspase-1 whose activity was measured using a luminescent caspase-1 substrate. Endothelial barrier stability was monitored for transendothelial electrical resistance (TEER) using an electric cell-substrate sensing system (iCelligence apparatus) when cultured human EAhy926 or murine aortic endothelial cells (EC) were exposed to thrombin treatment (0.25 nM) plus or minus treatment with P1 and/or P3 peptides or G10-related peptides. Synthetic P1 and P3 sequence-derived peptides of various compositions were made, and bivalent P1:P3 peptides were made using oligo-Gly-peptide linkers containing from 4 to 14 Gly residues. Results: Using THP-1 cell anti-inflammatory assays for various peptides, studies showed that P1-47-55 was as potent as P1-47-66 and that the N-terminal N47 was essential. In anti-inflammatory activity assays, some P3 peptides shorter than P3-42-65 (e.g., P3-44-54, P3-51-65, and P3-51-59) were as active as P3-42-65. Remarkably, this is the first example for any PAR-agonist peptide that does not require a native-like sequence with an intact free alpha-amino N-terminus to agonize the PAR response. For bivalent P1:P3 studies, we covalently linked P3-47-55 to P3-51-65 using oligo-Gly linkers with 4-14 Gly residues. In anti-inflammatory activity assays, the maximal activity was seen for a 10-Gly linker. The bivalent peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65, designated as the G10 peptide, was much more potent than any P1 peptide or P3 peptide in anti-inflammatory THP-1 cell assays in dose-response studies. In TEER studies of thrombin-challenged EAhy926 EC where APC markedly attenuates thrombin-induced endothelial barrier dysfunction, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was much more potent than the P1-47-55 peptide or any combination of P1-47-55 with a P3 peptide. The P3 peptides alone were very weak. In TEER studies of thrombin-challenged murine aortic EC where murine APC markedly attenuates thrombin-induced endothelial barrier dysfunction, the G10 peptide comprising murine PAR sequences mimicked murine APC's protective actions. Conclusions: Covalent linkage of a PAR1-derived peptide agonist to a PAR3-derived peptide agonist using a 10-Gly-residue linker provided the first heterobivalent PAR peptide GPCR agonist. This heterobivalent G10 peptide potently mimicked APC's cytoprotective activities of anti-inflammation and endothelial barrier stabilization. Molecular mechanisms for G10 likely involve both orthosteric and allosteric effects for PAR1 and PAR3 which can form heterodimers. Given APC's beneficial cell signaling effects in many preclinical injury models and given the increasing usage of peptide-based drugs, studies of the G10 peptide for beneficial APC-mimicking effects may help to identify important directions for future translational research.

SAT651 Human Adipose-derived Extracellular Vesicles (AdEVs) Increase Proinflammatory Markers In Renal And Endothelial Cells: An In Vitro Preliminary Study

Abstract Disclosure: C.A. Carvajal: None. M.P. Hernandez: None. P. Carrion: None. A. Tapia: None. J.A. Lopez: None. A. Vecchiola: None. C.E. Fardella: None. A. Sandoval-Borquez: None. During obesity, white adipose tissue (WAT), undergoes hypertrophic and hyperplastic changes by differentiation of preadipocytes into adipocytes, which increase the expression of PPARγ, FASN, FABP4, and adiponectin genes. These adipokines play a key role in obesity progression and in cell communication. WAT also causes a chronic inflammatory state that modifies the gene expression and secretome, including releasing of adipose-derived extracellular vesicles (AdEVs) that carry a specific cargo thought to modify different signaling pathways in target cells. Aim: To evaluate the effect of AdEVs in renal and endothelial cells and its inflammatory phenotype. Methods: Human SW872 preadipocytes and differentiated adipocytes were cultured and characterized by optical microscopy and Oil-Red-O stain. EVs from both preadipocytes and adipocytes were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, electron microscopy and w-blot. AdEVs (1x10^3) were added to renal HCD and endothelial EaHy926 cells for 24 hours. Expression of adipogenic differentiation genes in preadipocytes, adipocytes and their EVs, as well as the expression of inflammatory markers (IL-6 and IL-1B) were perfomed by RT-qPCR. Results: SW872 differentiated cells showed a classical adipocyte morphology and important accumulation of lipid-droplets. Isolated AdEVs have a donut shape morphology and size (50 - 150 nm) in accordance to the MISEV2018 guidelines, including also the EVs markers CD9 and Tsg101. A higher EVs concentration released from preadipocytes compared to adipocytes was observed (5.21x10^10 vs 0.44x10^10 particles/mL, p ≤0.05). The analysis of relative gene expression (2^-ΔΔCT), showed that adipocytes increase FAPB4 and adiponectin and decrease in PPARγ and FASN gene expression, with respect to preadipocytes (p ≤0.05). EVs from preadipocytes and adipocytes present similar relative expression of adiponectin, PPARγ and FASN than their parental cells. Finally, both renal or endothelial cells treated with AdEVs express higher levels of IL-6 and IL-1B that untreated cells (p≤ 0.05). Conclusion: EVs released from SW872 adipocytes and preadipocytes have similar gene expression as their parental cells. Treatment of both renal and endothelial cells with AdEVs showed an increase in inflammatory markers, as IL-6 and IL-1B. These preliminary results reveals novel insights about the impact of AdEVs in renal collecting duct and endothelial cells, which support novel roles of AdEVs -and its cargo- in human pathophysiology associated to obesity. Acknowledgements: This study was supported by grants ANID-FONDECYT 1212006, 3200646; CONICYT-FONDEQUIP EQM150023, SOCHED & CETREN UC. Presentation: Saturday, June 17, 2023

Anti-malaria drug artesunate prevents development of amyloid-β pathology in mice by upregulating PICALM at the blood-brain barrier

BackgroundPICALM is one of the most significant susceptibility factors for Alzheimer’s disease (AD). In humans and mice, PICALM is highly expressed in brain endothelium. PICALM endothelial levels are reduced in AD brains. PICALM controls several steps in Aβ transcytosis across the blood-brain barrier (BBB). Its loss from brain endothelium in mice diminishes Aβ clearance at the BBB, which worsens Aβ pathology, but is reversible by endothelial PICALM re-expression. Thus, increasing PICALM at the BBB holds potential to slow down development of Aβ pathology.MethodsTo identify a drug that could increase PICALM expression, we screened a library of 2007 FDA-approved drugs in HEK293t cells expressing luciferase driven by a human PICALM promoter, followed by a secondary mRNA screen in human Eahy926 endothelial cell line. In vivo studies with the lead hit were carried out in Picalm-deficient (Picalm+/−) mice, Picalm+/−; 5XFAD mice and Picalmlox/lox; Cdh5-Cre; 5XFAD mice with endothelial-specific Picalm knockout. We studied PICALM expression at the BBB, Aβ pathology and clearance from brain to blood, cerebral blood flow (CBF) responses, BBB integrity and behavior.ResultsOur screen identified anti-malaria drug artesunate as the lead hit. Artesunate elevated PICALM mRNA and protein levels in Eahy926 endothelial cells and in vivo in brain capillaries of Picalm+/− mice by 2–3-fold. Artesunate treatment (32 mg/kg/day for 2 months) of 3-month old Picalm+/−; 5XFAD mice compared to vehicle increased brain capillary PICALM levels by 2-fold, and reduced Aβ42 and Aβ40 levels and Aβ and thioflavin S-load in the cortex and hippocampus, and vascular Aβ load by 34–51%. Artesunate also increased circulating Aβ42 and Aβ40 levels by 2-fold confirming accelerated Aβ clearance from brain to blood. Consistent with reduced Aβ pathology, treatment of Picalm+/−; 5XFAD mice with artesunate improved CBF responses, BBB integrity and behavior on novel object location and recognition, burrowing and nesting. Endothelial-specific knockout of PICALM abolished all beneficial effects of artesunate in 5XFAD mice indicating that endothelial PICALM is required for its therapeutic effects.ConclusionsArtesunate increases PICALM levels and Aβ clearance at the BBB which prevents development of Aβ pathology and functional deficits in mice and holds potential for translation to human AD.

Nimbolide, a Neem Limonoid, Inhibits Angiogenesis in Breast Cancer by Abrogating Aldose Reductase Mediated IGF-1/PI3K/Akt Signalling.

The insulin/IGF-1R/PI3K/Akt signalling cascade is increasingly being linked to breast cancer development, with aldose reductase (AR) playing a key role in mediating the crosstalk between this pathway and angiogenesis. The current study was designed to investigate whether nimbolide, a neem limonoid, targets the oncogenic signaling network to prevent angiogenesis in breast cancer. Breast cancer cells (MCF-7, MDA-MB-231), EAhy926 endothelial cells, MDA-MB-231 xenografted nude mice, and tumour tissues from breast cancer patients were used for the study. The expression of AR and key players in IGF-1/PI3K/Akt signaling and angiogenesis was evaluated by qRT-PCR, immunoblotting, and immunohistochemistry. Molecular docking and simulation, overexpression, and knockdown experiments were performed to determine whether nimbolide targets AR and IGF-1R. Nimbolide inhibited AR with consequent blockade of the IGF-1/PI3K/Akt and /HIF-1alpha/VEGF signalling circuit by influencing the phosphorylation and intracellular localisation of key signaling molecules. The downregulation of DNMT-1, HDAC-6, miR-21, HOTAIR, and H19 with the upregulation of miR-148a/miR-152 indicated that nimbolide regulates AR and IGF-1/PI3K/Akt signaling via epigenetic modifications. Coadministration of nimbolide with metformin and the chemotherapeutic drugs tamoxifen/cisplatin displayed higher efficacy than single agents in inhibiting IGF-1/PI3K/Akt/AR signaling. Grade-wise increases in IGF-1R and AR expression in breast cancer tissues underscore their value as biomarkers of progression. This study provides evidence for the anticancer effects of nimbolide in cellular and mouse models of breast cancer besides providing leads for new drug combinations. It has also opened up avenues for investigating potential molecules such as AR for therapeutic targeting of cancer.

Tocilizumab (TCZ) Decreases Angiogenesis in Rheumatoid Arthritis Through Its Regulatory Effect on miR-146a-5p and EMMPRIN/CD147.

BackgroundAngiogenesis is a major contributor to the development of inflammation during Rheumatoid arthritis (RA), as the vascularization of the pannus provides nutrients and oxygen for the infiltrating immune cells and proliferating synoviocytes. Tocilizumab (TCZ) is an anti-IL-6 receptor antibody that is used in the treatment of RA patients, and has been shown to exert anti-inflammatory effects. However, its effects on angiogenesis are not fully elucidated, and the molecular mechanisms regulating this effect are unknown.MethodsWe evaluated the concentrations of several pro- and anti-angiogenic factors and the expression levels of several microRNA molecules that are associated with RA and angiogenesis in serum samples obtained from 40 RA patients, before and 4 months after the initiation of TCZ treatment. Additionally, we used an in vitro co-culture system of fibroblasts (the HT1080 cell line) and monocytes (the U937 cell line) to explore the mechanisms of TCZ action.ResultsSerum samples from RA patients treated with TCZ exhibited reduced circulating levels of EMMPRIN/CD147, enhanced expression of circulating miR-146a-5p and miR-150-5p, and reduced the angiogenic potential as was manifested by the lower number of tube-like structures that were formed by EaHy926 endothelial cell line. In vitro, the accumulation in the supernatants of the pro-angiogenic factors EMMPRIN, VEGF and MMP-9 was increased by co-culturing the HT1080 fibroblasts and the U937 monocytes, while the accumulation of the anti-angiogenic factor thrombospondin-1 (Tsp-1) and the expression levels of miR-146a-5p were reduced. Transfection of HT1080 cells with the miR-146a-5p mimic, decreased the accumulation of EMMPRIN, VEGF and MMP-9. When we neutralized EMMPRIN with a blocking antibody, the supernatants derived from these co-cultures displayed reduced migration, proliferation and tube formation in the functional assays.ConclusionsOur findings implicate miR-146a-5p in the regulation of EMMPRIN and propose that TCZ affects angiogenesis through its effects on EMMPRIN and miR-146a-5p.

Natural Compound Resveratrol Attenuates TNF-Alpha-Induced Vascular Dysfunction in Mice and Human Endothelial Cells: The Involvement of the NF-κB Signaling Pathway.

Resveratrol, a natural compound in grapes and red wine, has drawn attention due to potential cardiovascular-related health benefits. However, its effect on vascular inflammation at physiologically achievable concentrations is largely unknown. In this study, resveratrol in concentrations as low as 1 μm suppressed TNF-α-induced monocyte adhesion to human EA.hy926 endothelial cells (ECs), a key event in the initiation and development of atherosclerosis. Low concentrations of resveratrol (0.25–2 μm) also significantly attenuated TNF-α-stimulated mRNA expressions of MCP-1/CCL2 and ICAM-1, which are vital mediators of EC-monocyte adhesion molecules and cytokines for cardiovascular plaque formation. Additionally, resveratrol diminished TNF-α-induced IκB-α degradation and subsequent nuclear translocation of NF-κB p65 in ECs. In the animal study, resveratrol supplementation in diet significantly diminished TNF-α-induced increases in circulating levels of adhesion molecules and cytokines, monocyte adhesion to mouse aortic ECs, F4/80-positive macrophages and VCAM-1 expression in mice aortas and restored the disruption in aortic elastin fiber caused by TNF-α treatment. The animal study also confirmed that resveratrol blocks the activation of NF-κB In Vivo. In conclusion, resveratrol at physiologically achievable concentrations displayed protective effects against TNF-α-induced vascular endothelial inflammation in vitro and In Vivo. The ability of resveratrol in reducing inflammation may be associated with its role as a down-regulator of the NF-κB pathway.

Synergistic Protective Effect of Curcumin and Resveratrol against Oxidative Stress in Endothelial EAhy926 Cells.

Curcumin (C) and resveratrol (R) are two well-known nutraceuticals with strong antioxidant activity that can protect cells from oxidative stress. This study aims to investigate the synergy of CR combinations in protecting human endothelial EAhy926 cells against H2O2-induced oxidative stress and its related mechanisms. C and R as individual compounds as well as CR combinations at different ratios were screened for their protective effects against H2O2 (2.5 mM) induced cell death assessed by cell viability assays. The synergistic interaction was analysed using the combination index model. The effects of optimal CR combinations on caspase-3 activity, ROS level, SOD activity, NAD cellular production, expression of Nrf2 and HO-1, and Nrf2 translocation were determined. CR combinations produced a synergistic protection against that of H2O2-induced changes in cell viability, caspase-3 activity, and ROS production. The strongest effect was observed for CR with the ratio of 8 : 2. Further experiments showed that CR 8 : 2 exhibited significantly greater effects in increasing Nrf2 translocation and expressions of Nrf2 and HO-1 proteins, as well as SOD activity and total cellular NAD production, than that of C or R alone. The findings demonstrate that combination of C and R produced a strong synergy in activity against H2O2-induced oxidative stress in EAhy926 cells. The mechanism of this synergy involves the activation of Nrf2-HO-1 signaling pathway and promotion of antioxidant enzymes. Further studies on CR synergy may help develop a new combination therapy for endothelial dysfunction and other conditions related to oxidative stress.

Curcumin Suppresses TGF-β1-Induced Myofibroblast Differentiation and Attenuates Angiogenic Activity of Orbital Fibroblasts.

Orbital fibrosis, a hallmark of tissue remodeling in Graves’ ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor β1 (TGF-β1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-β1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-β1 for 24 h. The effects of curcumin on TGF-β1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-β1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-β1. Curcumin, at the concentration of 5 μg/mL, suppressed 5 ng/mL TGF-β1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-β1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.

Spent Hen Muscle Protein-Derived RAS Regulating Peptides Show Antioxidant Activity in Vascular Cells.

Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)—upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR-dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.

Conjugating homogenized liver-extracellular matrix into decellularized hepatic scaffold for liver tissue engineering.

The generation of a transplantable liver scaffold is crucial for the treatment of end-stage liver failure. Unfortunately, decellularized liver scaffolds suffer from lack of bioactive molecules and functionality. In this study, we conjugated homogenized liver-extracellular matrix (ECM) into a decellularized liver in a rat model to improve its structural and functional properties. The homogenized ECM was prepared, characterized, and subsequently perfused into ethyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) activated liver scaffolds. Various techniques were performed to confirm the improvements that were accomplished through the conjugation process; these included micro/ultra-structural analyses, biochemical analysis of ECM components, DNA quantification, swelling ratio, structural stability, calcification properties, platelet activation study, static and dynamic seeding with EAhy926 endothelial cells and HepG2 hepatocarcinoma cells, subcutaneous implantation and intrahepatic transplantation. The results showed that the conjugated scaffolds have superior micro- and ultrastructural and biochemical characteristics. In addition, DNA contents, swelling ratios, calcification properties, platelet reactions, and host inflammatory reactions were not altered with the conjugation process. The conjugated scaffolds revealed better cellular spreading and popularity compared to the non-conjugated scaffolds. Intrahepatic transplantation showed that the conjugated scaffold had higher popularity of hepatic regenerative cells with better angiogenesis. The conjugation of the decellularized liver scaffold with homogenized liver-ECM is a promising tool to improve the quality of the generated scaffold for further transplantation.

Characterization of cytotoxic effects of aristolochic acids on the vascular endothelium

Aristolochic acid nephropathy (AAN) is characterized by interstitial fibrosis, proximal tubular atrophy, and hypoxia. A correlation between a reduced peritubular capillary density and the severity of fibrosis has been demonstrated. As calcium, redox and energetic homeostasis are crucial in maintaining endothelial cell function and survival, we aimed to investigate AA-induced disturbances involved in endothelial cell injury. Our results showed a cytotoxic effect of AA on EAhy926 endothelial cells. Exposure of aortic rings to AA impaired vascular relaxation to Acetylcholine (ACh). Increased levels of intracellular reactive oxygen species (ROS) were observed in cells exposed to AA. Pre-treatment with antioxidant N-acetyl cysteine inhibited AA-induced cell death. Superoxide dismutase resulted in restoring ACh-induced relaxation. An increase in intracellular calcium level ([Ca2+]i) was observed on endothelial cells. Calcium chelators BAPTA-AM or APB, a specific inhibitor of IP3R, improved cell viability. Moreover, AA exposure led to reduced AMP-activated protein kinase (AMPK) expression. AICAR, an activator of AMPK, improved the viability of AA-intoxicated cells and inhibited the rise of cytosolic [Ca2+]i levels.This study provides evidence that AA exposure increases ROS generation, disrupts calcium homeostasis and decreases AMPK activity. It also suggests that significant damage observed in endothelial cells may enhance microcirculation defects, worsening hypoxia and tubulointerstitial lesions.

Heat-treated human platelet pellet lysate modulates microglia activation, favors wound healing and promotes neuronal differentiation in vitro

The neurorestorative efficacy of human platelet lysates in neurodegenerative disorders is still under investigation. Platelets prepared from standard and pathogen reduced platelet concentrates were pelletized, washed, concentrated, and subjected to freeze-thawing. The lysate was heated to 56°C for 30 min and characterized. Toxicity was evaluated using SH-SY5Y neuroblastoma, BV-2 microglial, and EA-hy926 endothelial cells. Inflammatory activity was tested by examining tumor necrosis factor (TNF) and cyclooxygenase (COX)-2 expressions by BV-2 microglia with or without stimulation by lipopolysaccharides (LPS). The capacity to stimulate wound healing was evaluated by a scratch assay, and the capacity to differentiate SH-SY5Y into neurons was also examined. Platelet lysates contained a range of neurotrophins. They were not toxic to SH-SY5Y, EA-hy926, or BV-2 cells, did not induce the expression of TNF or COX-2 inflammatory markers by BV-2 microglia, and decreased inflammation after LPS stimulation. They stimulated the wound closure in the scratch assay and induced SH-SY5Y differentiation as revealed by the increased length of neurites as well as β3-tubulin and neurofilament staining. These data confirm the therapeutic potential of platelet lysates in the treatment of disorders of the central nervous system and support further evaluation as novel neurorestorative biotherapy in preclinical models.

MLCK and ROCK mutualism in endothelial barrier dysfunction

Myosin activation contributes to the contractile forces that induce disturbances in the vascular endothelial integrity and promote protein-rich edema of the underlying tissues. Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) have been reported to phosphorylate myosin regulatory light chains (RLC) for myosin activation. However, the relative contribution and roles of these kinases are debatable and not understood in very detail. In this study, using a combinational inhibitory analysis of MLCK, ROCK, and their antagonist, myosin light chain phosphatase (MLCP), we show that the MLCK-dependent RLC mono-(Ser19)phosphorylation (P-RLC) is sufficient to induce the FITC-dextran hyperpermeability in EA.hy926 endothelial cells (EC) in response to thrombin. However, MLCK relies on the ROCK assistance that attenuates MLCP activity. On the other hand, MLCK supplies P-RLC myosin as an intermediate substrate to ROCK thus adding to a faster accumulation of di-(Thr18/Ser19)phosphorylated RLC (PP-RLC) by the latter kinase. ROCK also produces P-RLC but is solely responsible for the thrombin-induced PP-RLC generation in EA.hy926 EC and other cell types. Still, as a direct myosin activator, ROCK contributes less to endothelial hyperpermeability than MLCK. Our findings are consistent with a concerted complementary mutual interplay between ROCK and MLCK to activate endothelial myosin and elicit the thrombin-mediated EC barrier dysfunction.

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells.

Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour growth. The aim of this research was to determine the potency and efficacy of adenosine A1, A2A and A2B receptor agonists on the rate of wound healing and cell proliferation in human EA.hy926 endothelial cells. Real-time PCR data showed that only adenosine A1, A2A and A2B receptor mRNA were expressed in this cell line. All three adenosine receptor agonists, CPA, CGS21680 and NECA, significantly increased the rate of wound healing in human EAhy926 endothelial cells with the following order of potency CGS21680>CPA>NECA and efficacy CPA>NECA>CGS21680. The selective adenosine A1, A2A and A2B receptor antagonists, DPCPX, ZM241385 and MRS1754 (all at 10nM), reversed the effects of their respective agonists. EAhy926 endothelial cell proliferation was also significantly increased with the adenosine A1 and A2B receptor agonists, CPA and NECA. Western blot analysis demonstrated that adenosine A2A and A1 receptor protein levels were highly expressed compared with the adenosine A2B receptors in the EAhy926 endothelial cell lines. While all three adenosine A1, A2A and A2B receptor subtypes contribute to cell proliferation and wound healing in human EAhy926 endothelial cells, treatments selectively targeting receptor subtypes may further enhance wound healing.

Enhanced effects of curcumin encapsulated in polycaprolactone-grafted oligocarrageenan nanomicelles, a novel nanoparticle drug delivery system

One of the most effective strategies to enhance the bioavailability and the therapeutic effect of hydrophobic drugs is the use of nanocarriers. We have used κ-carrageenan extracted from Kappaphycus alvarezii to produce oligocarrageenan via an enzymatic degradation process. Polycaprolactone (PCL) chains were grafted onto the oligocarrageenans using a protection/deprotection technique yielding polycaprolactone-grafted oligocarrageenan. The resulting amphiphilic copolymers formed spherical nanomicelles with a mean size of 187 ± 21 nm. Hydrophobic drugs such as curcumin were efficiently encapsulated in the micelles and released within 24–72 h in solution. The micelles were non-cytotoxic and facilitated the uptake of curcumin by endothelial EA-hy926 cells. They also increased the anti-inflammatory effect of curcumin in TNF-alpha-induced inflammation experiments. Finally, in vivo experiments supported a lack of toxicity in zebrafish and thus the potential use of polycaprolactone-grafted oligocarrageenan to improve the delivery of hydrophobic compounds to different organs, including liver, lung and brain as shown in mice.

Overexpression of Axl reverses endothelial cells dysfunction in high glucose and hypoxia.

The receptor tyrosine kinase Axl is involved in diabetic vascular disease. This study aims to investigate the effect of high glucose on endothelial cells injury and Axl expression in hypoxia condition in vitro, and we present details of the mechanism associated with overexpression of Axl rescue the high glucose injury. Our results showed that high glucose impaired both human umbilical vein endothelial cells (HUVECs) and EAhy926 cells angiogenesis in hypoxia condition. In addition, high glucose inhibits Axl and hypoxia-inducible factor 1-α (HIF-1α) protein expression in hypoxia condition. Axl overexpression significantly reversed endothelial cells dysfunction in high glucose/hypoxia. Furthermore, Axl overexpression in EAhy926 cells increases HIF-1α protein synthesis through PI3K/Akt/mTOR/p70 S6K signal pathway but not Mek/Erk in high glucose/hypoxia condition. This study demonstrates that high glucose can alter Axl signaling and HIF-1α in hypoxia condition. Overexpression of Axl may rescue endothelial cells dysfunction and HIF-1α expression through its downstream signals in high glucose/hypoxia.

Higher Anti-angiogenesis Activity, Better Cellular Uptake and Longer Half-life of a Novel Glyco-modified Endostatin by Polysulfated Heparin.

Endostatin (ES) is a promising anti-angiogenesis protein and has been approved for the treatment of non-small cell lung cancer, but short half-life, poor stability and nonspecific delivery caused great pain to patients and produced unsatisfactory treatment effectiveness. In this work, in order to overcome these disadvantages, ES was covalently modified by polysulfated heparin (PSH) with the expectancy of longer half-life, higher anti-angiogenesis activity and better cellular uptake. To characterize the cellular uptake, flow cytometry and confocal laser scanning microscopy were used to study the intracellular localization of fluorescein isothiocyanate-labeled ES and PSH-ES in EAhy926 endothelial cells. Zebrafish model was used to study the anti-angiogenesis activities of ES and its derivatives in vivo. The 125I-radiolabeled ES and PSH-ES were administered to healthy BALC/c mice for the pharmacokinetics study. Compared with ES, better cellular uptake effects were detected in PSH-ES group. Both ES and PSH-ES showed inhibition on the intersegmental vessels formation, while PSH-ES displayed a higher one. The half-life of PSH-ES was lengthened and area under the curve (AUC) was increased. At the same time, ES and PSH-ES were both widely and rapidly distributed in the lungs, livers, kidneys and hearts with little difference. The results indicated that PSH displayed good properties as a novel glyco-modifier for protein and peptide. The results also showed that PSH-ES displayed better cellular uptake, higher antiangiogenesis activity and prolonged half-life, which would lead to better anti-tumour effects.

Suspension state promotes extravasation of breast tumor cells by increasing integrin β1 expression

Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells. However, the effect of suspension state on their extravasation and the mechanisms involved are still unclear. To explore the influence of suspension state on extravasation (including adhesion, spreading and transendothelial migration) of breast tumor cells and its relevant molecular mechanism, MDA-MB-231 cells were cultured on poly (2-hydroxyethyl methacrylate) coated 6-well plates to minic the suspension state. Suspension state promoted adhesion, spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells (ECs) monolayer under both the static condition and 0.5 dyne/cm2 flow shear stress (FSS). The number of cells adhered to ECs monolayer reached 2.15 (static condition, 3 d) and 1.67 (FSS, 3 d) times, and the number of migration reached 10.60 times, respectively, as compared to that in adhesion state. Moreover, MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration, as compared with MDA-MB-231 cells. But it made no difference in cell spreading. Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells, but had no significant influence on their spreading.