ABSTRACT
We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1ß, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.
Subject(s)
COVID-19 Drug Treatment , Leukocytes/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Pyrimidines/pharmacology , Respiratory Distress Syndrome/drug therapy , Transendothelial and Transepithelial Migration/drug effects , Animals , COVID-19/pathology , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Movement/drug effects , Cytokine Release Syndrome/drug therapy , Cytokines/metabolism , Disease Models, Animal , Female , Humans , Leukocytes/immunology , Lipopolysaccharides/adverse effects , Mice , Mice, Inbred BALB C , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Pyrimidines/chemistry , Respiratory Distress Syndrome/chemically induced , SARS-CoV-2ABSTRACT
Atherosclerosis is a non-resolving inflammatory condition that underlies major cardiovascular diseases.Recent clinical trial using an anti-inflammatory drug has showna reduction of cardiovascular mortality, but increased the susceptibility to infections. For this reason, tissue target anti-inflammatory therapies can represent a better option to regress atherosclerotic plaques. Docosahexaenoic acid (DHA) is a natural omega 3 fatty acidcomponentof algae oil and acts asaprecursor of several anti-inflammatory compounds, such the specialized proresolving lipid mediators(SPMs). During the atherosclerosis process, the inflammatory condition of the endothelium leads to the higher expression of adhesion molecules, such as Endothelial Cell Adhesion Molecule Plate 1 (PECAM-1 or CD31), as part of the innate immune response. Thus, the objective of this study was to develop lipid-core nanocapsules with DHA constituting the nucleus and anti-PECAM-1 on their surface and drive this structure to the inflamed endothelium. Nanocapsules were prepared by interfacial deposition of pre-formed polymer method. Zinc-II was added to bind anti-PECAM-1 to the nanocapsule surface by forming an organometallic complex. Swelling experiment showed that the algae oil act as non-solvent for the polymer (weight constant weight for 60 days, p > 0.428) indicating an adequate material to produce kinetically stable lipid-core nanocapsules (LNC). Five formulations were synthesized: Lipid-core nanocapsules containing DHA (LNC-DHA) or containing Medium-chain triglycerides (LNC-MCT), multi-wall nanocapsules containing DHA (MLNC-DHA) or containing MCT (MLNC-MCT) and the surface-functionalized (anti-PECAM-1) metal-complex multi-wall nanocapsules containing DHA (MCMN-DHA-a1). All formulations showed homogeneous macroscopic aspects without aggregation. The mean size of the nanocapsules measured by laser diffraction did not show difference among the samples (p = 0.241). Multi-wall nanocapsules (MLNC) showed a slight increase in the mean diameter and polydispersity index (PDI) measured by DLS, lower pH and an inversion in the zeta-potential (ξP) compared to LNCs. Conjugation test for anti-PECAM-1 showed 94.80% of efficiency. The mean diameter of the formulation had slightly increased from 160 nm (LCN-DHA) and 162 nm (MLNC-DHA) to 164 nm (MCMN-DHA-a1) indicating that the surface functionalization did not induce aggregation of the nanocapsules. Biological assays showed that the MCMN-DHA-a1 were uptaken by the HUVEC cells and did not decrease their viability. The surface-functionalized (anti- PECAM-1) metal-complex multi-wall nanocapsules containing DHA (MCMN-DHA-a1) can be considered adequate for pharmaceutical approaches.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Atherosclerosis/drug therapy , Docosahexaenoic Acids/administration & dosage , Nanocapsules/chemistry , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Drug Combinations , Drug Compounding/methods , Human Umbilical Vein Endothelial Cells , Humans , Lipids/chemistry , Organometallic Compounds/chemistry , Particle Size , Zinc/chemistryABSTRACT
Low shear stress serves an important role in the initiation and progression of atherosclerotic lesions, with an impact on progression, but its detailed mechanisms are .not yet fully known. The present study aimed to investigate endothelial cell (EC) apoptosis, as well as monocyte adhesion induced by low shear stress and the potential underlying mechanisms. The expression of platelet endothelial cell adhesion molecule1 (PECAM1) was demonstrated to be enhanced in human umbilical vascular ECs with a trend that was associated with time when stimulated by low shear stress compared with unstimulated cells. EC apoptosis was increased under low shear stress compared with unstimulated cells, and knockdown of PECAM1 inhibited this process. Furthermore, downregulation of PECAM1 reduced monocyte adhesion induced by low shear stress compared with that in the negative control cells. Mechanistically, PECAM1 small interfering RNA transfection increased Akt and forkhead box O1 phosphorylation under low shear stress conditions compared with that in the negative control cells. Collectively, the findings of the present study revealed that low shear stress induced EC apoptosis and monocyte adhesion by upregulating PECAM1 expression, which suggested that PECAM1 may be a potential therapeutic target for atherosclerosis.
Subject(s)
Apoptosis , Cell Adhesion/physiology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Shear Strength , Cell Line , Coculture Techniques , Endothelial Cells/cytology , Endothelial Cells/metabolism , Forkhead Box Protein O1/metabolism , Humans , Monocytes/cytology , Monocytes/metabolism , Phosphorylation , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Nanoemulsion-based synthesis has been introduced to enhance the bioavailability of natural compounds at target sites for their various biomedical applications. In this study, we synthesized carvacrol nanoemulsion (CN) an oil-in-water (O/W) as a nano-emulsion vehicle system by using ultrasonication emulsification for anti-angiogenesis therapy formulated by combining MCT, lecithin, and polysorbate 80 at the O/W interface called carvacrol encapsulated nanoemulsion (CEN). The diameter of CEN determined by TEM analysis was 105.32â¯nm. The hydrodynamic droplet size was 101.0â¯nm with a -39.38-mV zeta potential. The stability of the synthesized CEN was approved till 100 days without any change in diameter size distribution and encapsulation efficiency. We evaluated the role of CEN on angiogenesis in lung adenocarcinoma A549 cells both in vitro and in vivo and observed that it reduced the growth and MMP levels of A549 cells in a dose-dependent manner. Exposure to CEN decreased the activation of MAPK p38 as well as ERK. Moreover, we found that CEN reduced the expression of VEGF and CD31 in A549 cells both in vitro and in vivo. Our in-silico study also indicated the binding of carvacrol to COX-2 and VEGF at the active and allosteric sites of CD31 with low binding energy. Overall, CEN induced anti-angiogenic effects in A549 cells in vitro, in silico, and in vivo, thereby establishing its potential as targeted drug delivery vehicle against angiogenesis.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents/pharmacology , Cymenes/pharmacology , Lung Neoplasms/drug therapy , Nanoparticles/chemistry , Neovascularization, Pathologic/drug therapy , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/pathology , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cyclooxygenase 2/metabolism , Cymenes/chemistry , Down-Regulation/drug effects , Drug Carriers/chemistry , Drug Screening Assays, Antitumor , Emulsions/chemistry , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Particle Size , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Surface Properties , Tumor Cells, Cultured , Vascular Endothelial Growth Factors/antagonists & inhibitors , Vascular Endothelial Growth Factors/metabolismABSTRACT
OBJECTIVES: To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin ß1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). METHODS: Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion. RESULTS: Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively). CONCLUSIONS: Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.
Subject(s)
Allyl Compounds/pharmacology , Disulfides/pharmacology , Integrin beta1/physiology , Ischemic Postconditioning , Myocardial Reperfusion Injury/prevention & control , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Animals , Disease Models, Animal , Integrin beta1/analysis , Integrin beta1/genetics , Male , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Platelet Endothelial Cell Adhesion Molecule-1/genetics , RNA, Messenger/analysis , SwineABSTRACT
Peripheral nerve injury has been suggested to up-regulate mRNA for the vascular endothelial growth factor (VEGF) which enhances nerve regeneration. VEGF is known to regulate angiogenesis by binding with a specific receptor, the vascular endothelial growth factor receptor (VEGFR). However, little is known about the involvement of VEGF-VEGFR signaling in the nerve regeneration at early stages though previous studies contained a lengthy observation. The present study examined that relationship between angiogenesis and peripheral nerve regeneration at the early stage after nerve transection by focusing on the chronological changes in the expression patterns of VEGF-VEGFR signaling. This study used our previously reported experimental model for nerve regeneration following the transection of the inferior alveolar nerve (IAN) in mice. In a double staining of PGP9.5 and CD31, respective markers for the nerve fibers and endothelial cells, CD31 immunoreactions first appeared in the injury site on postoperative (PO) day 2 when the transected nerve fibers had not been re-connected. The most intense immunoreaction for CD31 was found around the regenerating nerve fibers extending from the proximal stump on PO day 3, but it gradually lessened to disappear by PO day 7. The expression patterns of VEGFR1 and VEGFR2 showed similar chronological changes through the observation periods, with most intense immunoreaction found on PO day 3. Western blotting of total protein extracted from the injury site demonstrated the clear bands for VEGF-A and VEGF-B on PO day 2, indicating a time lag for the expression of ligands and receptors. A local administration of antibody to VEGF-A inhibited the elongation of the nerve fibers from the proximal stump. Furthermore, this administration of VEGF-A antibody inhibited the expression of CD31 in the gap between proximal and distal stumps. These results indicated that a nerve injury initiates productions in VEGF-A and VEFG-B, followed with the expression of VEGFR1 and VEGFR2 at early stages after the nerve injury. Taken these findings together, it is reasonable to postulate that immediate response of VEGF-VEGFR signaling to nerve injury plays a crucial role in local angiogenesis, resulting in a trigger for the regeneration of the nerve fibers in mouse IAN.
Subject(s)
Endothelial Growth Factors/metabolism , Nerve Regeneration , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Animals , Endothelial Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Nerve Fibers , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Ubiquitin Thiolesterase/metabolism , Vascular Endothelial Growth Factor B/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Background: Pneumococci are the major cause of bacterial meningitis globally. To cause meningitis pneumococci interact with the 2 endothelial receptors, polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), to penetrate the blood-brain barrier (BBB) and invade the brain. Methods: C57BL/6 mice were infected intravenously with bioluminescent pneumococci, and treated with ceftriaxone (1 hour postinfection) and anti-pIgR and PECAM-1 antibodies (1 or 5 hours postinfection), then monitored for 5 and 10 days. Bacterial brain invasion was analyzed using IVIS imaging and bacterial counts. Results: Ceftriaxone, given early after pneumococcal challenge, cleared pneumococci from the blood but not from the brain. After combining ceftriaxone with receptor blockade, using anti-pIgR and PECAM-1 antibodies, we found 100% survival after 5 and 10 days of infection, in contrast to 60% for ceftriaxone alone. Combined antibiotic and antibody treatment resulted in no or few viable bacteria in the brain and no microglia activation. Antibodies remained bound to the receptors during the study period. Receptor blockade did not interfere with antibiotic permeability through the BBB. Conclusions: We suggest that adjunct treatment with pIgR and PECAM-1 antibodies to antibiotics may prevent pneumococcal meningitis development and associated brain damages. However, further evaluations are required.
Subject(s)
Meningitis, Pneumococcal/drug therapy , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Receptors, Polymeric Immunoglobulin/antagonists & inhibitors , Animals , Anti-Bacterial Agents/administration & dosage , Antibodies/administration & dosage , Bacterial Load , Ceftriaxone/administration & dosage , Disease Models, Animal , Drug Therapy, Combination , Intravital Microscopy , Mice, Inbred C57BL , Survival Analysis , Treatment OutcomeABSTRACT
Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.