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.

Anti-Inflammatory Neutrophils Reprogram Macrophages toward a Pro-Healing Phenotype with Increased Efferocytosis Capacity.

Following myocardial infarction (MI), blood neutrophils quickly and extensively infiltrate the heart, where they are temporally polarized into pro-inflammatory (N1) and anti-inflammatory (N2) subpopulations. Neutrophil transmigration is rapidly followed by the accrual of macrophages (MACs), which are believed to undergo local phenotypic transformations from pro-inflammatory to pro-healing MACs that mediate inflammation resolution. We hypothesized that N2 neutrophils can reprogram MACs toward a healing phenotype with increased efferocytosis capacity. To examine this, human neutrophils isolated from healthy subjects were polarized in N1 and N2 neutrophils, and their secretome was added to human MACs derived from THP monocytes. The impact of neutrophil factors on macrophages was investigated using qPCR, ELISA, Western blot, immunofluorescence, or an efferocytosis assay. The results show that the MACs exposed to N2 neutrophil secretome exhibited (i) increased expression of the anti-inflammatory molecules CD206, TGF-ß, and IL-10 and the nuclear factors associated with reparatory macrophages (PPARγ, Nur77, and KLF4); (ii) enhanced expression of efferocytosis receptors (MerTK, CD36, CX3CR1, and integrins αv/ß5) and of the bridge molecules Mfage8 and Gas6; and (iii) enhanced efferocytosis. In conclusion, factors released by N2 neutrophils induce a pro-healing phenotype of MACs by upregulating anti-inflammatory molecules and efferocytosis receptors and ensuing the efferocytosis capacity. The data suggest that molecular therapy to foster N2 polarization, which boosts macrophages' pro-healing phenotype, could be a promising strategy to speed up inflammation resolution and tissue repair.

Ficolin-2 amplifies inflammation in macrophage-smooth muscle cell cross-talk and increases monocyte transmigration by mechanisms involving IL-1ß and IL-6.

Ficolin-2, recently identified in atherosclerotic plaques, has been correlated with future acute cardiovascular events, but its role remains unknown. We hypothesize that it could influence plaque vulnerability by interfering in the cross-talk between macrophages (MØ) and smooth muscle cells (SMC). To examine its role and mechanism of action, we exposed an in-vitro co-culture system of SMC and MØ to ficolin-2 (10 µg/mL) and then performed cytokine array, protease array, ELISA, qPCR, Western Blot, and monocyte transmigration assay. Carotid plaque samples from atherosclerotic patients with high plasma levels of ficolin-2 were analyzed by immunofluorescence. We show that ficolin-2: (i) promotes a pro-inflammatory phenotype in SMC following interaction with MØ by elevating the gene expression of MCP-1, upregulating gene and protein expression of IL-6 and TLR4, and by activating ERK/MAPK and NF-KB signaling pathways; (ii) increased IL-1ß, IL-6, and MIP-1ß in MØ beyond the level induced by cellular interaction with SMC; (iii) elevated the secretion of IL-1ß, IL-6, and CCL4 in the conditioned medium; (iv) enhanced monocyte transmigration and (v) in atherosclerotic plaques from patients with high plasma levels of ficolin-2, we observed co-localization of ficolin-2 with SMC marker αSMA and the cytokines IL-1ß and IL-6. These findings shed light on previously unknown mechanisms underlying ficolin-2-dependent pathological inflammation in atherosclerotic plaques.

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.

Lipopolysaccharide-induced inflammation in monocytes/macrophages is blocked by liposomal delivery of Gi-protein inhibitor.

BACKGROUND: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of Gi protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin. PURPOSE: The aim of the present work was to evaluate the potential of a Gi-protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages. MATERIALS AND METHODS: Guanosine 5'-O-(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1ß, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated. RESULTS: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1ß, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS. CONCLUSION: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages.

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-1ß, 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.

Amendment of the cytokine profile in macrophages subsequent to their interaction with smooth muscle cells: Differential modulation by fractalkine and resistin.

In atherosclerotic plaques, macrophages (MAC) and smooth muscle cells (SMC) frequently reside in close proximity and resistin (Rs) and fractalkine (Fk) are present at increased levels, resistin being associated with CD68 macrophages and fractalkine predominantly associated with intimal SMC; however, their role in this location is not clear, yet. The objective of this study was to determine whether the cross-talk between MAC-SMC induces changes in MAC cytokine phenotype and if Fk and Rs have a role in the process. To this purpose, macrophages (THP-1 monocytes differentiated with phorbol myristate acetate) were interacted with SMC cultured on the membrane inserts in the presence or absence of Rs or Fk. After 24h, MAC were removed from the co-culture and the gene and protein expression of 57 cytokines was assessed by QPCR and Proteome Profiler™ Array. Fk secreted in the culture medium following MAC-SMC interaction was determined (ELISA assay) and the role of Fk in MAC cytokine gene expression was assessed by silencing the Fk receptor in both cell types. The results showed that subsequent to the interaction with SMC, MAC exhibit: (1) a general increased expression of chemokines (the highest fold increase: VCC-1 and GRO-α) and of some interleukins, such as interleukins IL-5 (∼8-fold) and IL-6; (2) an increased Fk expression that in turn induces expression of: CXCL17, CCL19, CCL2, CXCL10, CXCL12, CXCL4, CXCL7, CCL4, CCL18, CXCL16, CXCL1 and IL-27; (3) in the presence of Rs, a predominant increased expression of interleukins (the highest fold increase: IL-6, IL-27, IL-23 and IL-5) and an augmented expression of some chemokines such as MIP-1ß, GRO-α and CCL1. In addition, the secretome collected from the SMC-MAC co-culture increased human monocytes chemotaxis. DAVID analysis of the data revealed that the switch of MAC to a pro-inflammatory phenotype, prime the cells to intervene in the immune response, chemotaxis and inflammatory response. In conclusion, MAC cytokines expression is considerable augmented upon their interaction with SMC and Fk and Rs have distinct immunomodulatory roles: Fk predominantly increases the pro-angiogenic and inflammatory chemokines expression and Rs mostly the pro-inflammatory interleukins with consequences on monocyte chemotaxis. The novel data could help to develop targeted nanotherapies to reduce leukocyte chemotaxis and the ensuing inflammatory process associated with atherosclerosis.

Conjugation of curcumin-loaded lipid nanoemulsions with cell-penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells.

OBJECTIVES: To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell-penetrating peptide. METHODS: Curcumin-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells, and in vivo biodistribution studies were performed using C57BL6 mice. KEY FINDINGS: When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 h. CONCLUSIONS: Cell-penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in HEC and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.