P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs’ inflammation, in a mouse model of acute inflammation

The Specific Molecular Changes Induced by Diabetic Conditions in Valvular Endothelial Cells and upon Their Interactions with Monocytes Contribute to Endothelial Dysfunction

Aortic valve disease (AVD) represents a global public health challenge. Research indicates a higher prevalence of diabetes in AVD patients, accelerating disease advancement. Although the specific mechanisms linking diabetes to valve dysfunction remain unclear, alterations of valvular endothelial cells (VECs) homeostasis due to high glucose (HG) or their crosstalk with monocytes play pivotal roles. The aim of this study was to determine the molecular signatures of VECs in HG and upon their interaction with monocytes in normal (NG) or high glucose conditions and to propose novel mechanisms underlying valvular dysfunction in diabetes. VECs and THP-1 monocytes cultured in NG/HG conditions were used. The RNAseq analysis revealed transcriptomic changes in VECs, in processes related to cytoskeleton regulation, focal adhesions, cellular junctions, and cell adhesion. Key molecules were validated by qPCR, Western blot, and immunofluorescence assays. The alterations in cytoskeleton and intercellular junctions impacted VEC function, leading to changes in VECs adherence to extracellular matrix, endothelial permeability, monocyte adhesion, and transmigration. The findings uncover new molecular mechanisms of VEC dysfunction in HG conditions and upon their interaction with monocytes in NG/HG conditions and may help to understand mechanisms of valvular dysfunction in diabetes and to develop novel therapeutic strategies in AVD

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs’ inflammation, in a mouse model of acute inflammation

Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve

Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD

Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis

Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE−/− mouse model by early type 1 diabetes onset (at 4 and 7 days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1 week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE−/− diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies

Supplementary_figure_1 – Supplemental material for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis

Supplemental material, Supplementary_figure_1 for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis by Monica Madalina Tucureanu, Alexandru Filippi, Nicoleta Alexandru, Cristina Ana Constantinescu, Letitia Ciortan, Razvan Macarie, Mihaela Vadana, Geanina Voicu, Sabina Frunza, Dan Nistor, Agneta Simionescu, Dan Teodor Simionescu, Adriana Georgescu and Ileana Manduteanu in Diabetes & Vascular Disease Researc

Supplementary_Table_1 – Supplemental material for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis

Supplemental material, Supplementary_Table_1 for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis by Monica Madalina Tucureanu, Alexandru Filippi, Nicoleta Alexandru, Cristina Ana Constantinescu, Letitia Ciortan, Razvan Macarie, Mihaela Vadana, Geanina Voicu, Sabina Frunza, Dan Nistor, Agneta Simionescu, Dan Teodor Simionescu, Adriana Georgescu and Ileana Manduteanu in Diabetes & Vascular Disease Researc

Supplementary_figure_1 – Supplemental material for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis

Supplemental material, Supplementary_figure_1 for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis by Monica Madalina Tucureanu, Alexandru Filippi, Nicoleta Alexandru, Cristina Ana Constantinescu, Letitia Ciortan, Razvan Macarie, Mihaela Vadana, Geanina Voicu, Sabina Frunza, Dan Nistor, Agneta Simionescu, Dan Teodor Simionescu, Adriana Georgescu and Ileana Manduteanu in Diabetes & Vascular Disease Researc

Supplementary_Table_1 – Supplemental material for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis

Supplemental material, Supplementary_Table_1 for Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis by Monica Madalina Tucureanu, Alexandru Filippi, Nicoleta Alexandru, Cristina Ana Constantinescu, Letitia Ciortan, Razvan Macarie, Mihaela Vadana, Geanina Voicu, Sabina Frunza, Dan Nistor, Agneta Simionescu, Dan Teodor Simionescu, Adriana Georgescu and Ileana Manduteanu in Diabetes & Vascular Disease Researc