VCAM-1 Targeted Lipopolyplexes as Vehicles for Efficient Delivery of shRNA-Runx2 to Osteoblast-Differentiated Valvular Interstitial Cells; Implications in Calcific Valve Disease Treatment.

Calcific aortic valve disease (CAVD) is a progressive inflammatory disorder characterized by extracellular matrix remodeling and valvular interstitial cells (VIC) osteodifferentiation leading to valve leaflets calcification and impairment movement. Runx2, the master transcription factor involved in VIC osteodifferentiation, modulates the expression of other osteogenic molecules. Previously, we have demonstrated that the osteoblastic phenotypic shift of cultured VIC is impeded by Runx2 silencing using fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 (shRunx2) polyplexes. Since the use of polyplexes for in vivo delivery is limited by their instability in the plasma and the non-specific tissue interactions, we designed and obtained targeted, lipid-enveloped polyplexes (lipopolyplexes) suitable for (1) systemic administration and (2) targeted delivery of shRunx2 to osteoblast-differentiated VIC (oVIC). Vascular cell adhesion molecule (VCAM)-1 expressed on the surface of oVIC was used as a target, and a peptide with high affinity for VCAM-1 was coupled to the surface of lipopolyplexes encapsulating C60-PEI/shRunx2 (V-LPP/shRunx2). We report here that V-LPP/shRunx2 lipopolyplexes are cyto- and hemo-compatible and specifically taken up by oVIC. These lipopolyplexes are functional as they downregulate the Runx2 gene and protein expression, and their uptake leads to a significant decrease in the expression of osteogenic molecules (OSP, BSP, BMP-2). These results identify V-LPP/shRunx2 as a new, appropriately directed vehicle that could be instrumental in developing novel strategies for blocking the progression of CAVD using a targeted nanomedicine approach.

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.

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.

Biomimetic Nanocarriers of Pro-Resolving Lipid Mediators for Resolution of Inflammation in Atherosclerosis.

Atherosclerosis (ATH) is a systemic disease characterized by a chronic inflammatory process and lipid deposition in the arterial walls. The chronic inflammation within ATH lesions results, at least in part, from the failed resolution of inflammation. This process is controlled actively by specialized pro-resolving lipid mediators (SPMs), namely lipoxins, resolvins, protectins, and maresins. Herein, biomimetic nanocarriers are produced comprising a cocktail of SPMs-loaded lipid nanoemulsions (LN) covered with macrophage membranes (Bio-LN/SPMs). Bio-LN/SPMs retain on their surface the macrophage receptors involved in cellular interactions and the "marker of self" CD47, which impede their recognition and uptake by other macrophages. The binding of Bio-LN/SPMs to the surface of endothelial cells (EC) and smooth muscle cells (SMC) is facilitated by the receptors on the macrophage membranes and partly by SPMs receptors. In addition, Bio-LN/SPMs prove functional by reducing monocyte adhesion and transmigration to/through activated EC and by stimulating macrophage phagocytic activity. After intravenous administration, Bio-LN/SPMs accumulate in the aorta of ApoE-deficient mice at the level of atherosclerotic lesions. Also, the safety assessment testing reveals no side effects or immunotoxicity of Bio-LN/SPMs. Thus, the newly developed Bio-LN/SPMs represent a reliable targeted nanomedicine for the resolution of inflammation in atherosclerosis.

Monocytes and smooth muscle cells cross-talk activates STAT3 and induces resistin and reactive oxygen species production [corrected].

During the early phase of atherosclerosis, monocytes attach to and migrate through the vessel wall where they activate and communicate with smooth muscle cells (SMC) affecting plaque progression by largely unknown mechanisms. Activation of STAT3 transcription factor is suggested to be critically involved in dedifferentiation, migration, and proliferation of SMC in the neointima formation after vascular injury. Monocytes-SMC cross-talk induces an inflammatory phenotype of the resident SMC, but the involvement of STAT3 in phenotype switching is not known. Resistin is a cytokine found in human atheroma associated to monocytes/macrophages with role in inflammation associated with cardiovascular disease. The aim of this study was to follow the effect of activated monocytes-SMC cross-talk on STAT3 activation and subsequent resistin and reactive oxygen species (ROS) production. Our results showed that the interaction of activated monocytes with SMC determines: (i) phosphorylation of STAT3 and reduction of SOCS3 expression in both cell types; (ii) intracellular ROS production dependent on NADPH oxidase (by increased Nox1 expression) and STAT3 activation in SMC; (iii) up-regulation of resistin expression in monocytes dependent on STAT3 activation. Furthermore, exposure of SMC to resistin induces ROS by increasing NADPH oxidase activity and the p22phox and Nox1 expression. In conclusion, the cross-talk between SMC and monocytes activates STAT3 transcription factor and lead to resistin up-regulation in monocytes and ROS production in SMC. Moreover, resistin increases the ROS levels in SMC. These data indicate that monocyte-SMC communication may represent an important factor for progression of the atherosclerotic lesion.

Inflammatory effects of resistin on human smooth muscle cells: up-regulation of fractalkine and its receptor, CX3CR1 expression by TLR4 and Gi-protein pathways.

In the atherosclerotic plaque, smooth muscle cells (SMC) acquire an inflammatory phenotype. Resistin and fractalkine (CX3CL1) are found in human atheroma and not in normal arteries. CX3CL1 and CX3CR1 are predominately associated with SMC. We have questioned whether resistin has a role in the expression of CX3CL1 and CX3CR1 in SMC thus contributing to the pro-inflammatory status of these cells. Cultured human aortic SMC were stimulated with 100 ng/ml resistin for 4, 6, 12, and 24 h, and then CX3CL1 and CX3CR1 expression was assessed by quantitative reverse transcription with the polymerase chain reaction and Western blot. We found that resistin up-regulated CX3CL1 and CX3CR1 in SMC and induced the phosphorylation of p38MAPK and STAT3. Inhibitors of p38MAPK, JAK-STAT, NF-kB, and AP-1 significantly reduced CX3CL1 and CX3CR1 expression. Knockdown of STAT1 and STAT3 with decoy oligodeoxinucleotides and the silencing of p65 and cjun with short interfering RNA decreased CX3CL1 and CX3CR1 expression. Anti-TLR4 antibody and pertussis toxin also reduced CX3CL1 and CX3CR1 protein expression. xCELLigence experiments revealed that resistin probably uses Gi-proteins for its effect on SMC. The CX3CL1 induced by resistin exhibited a chemotactic effect on monocyte transmigration. Thus, (1) resistin contributes to the pro-inflammatory state of SMC by the up-regulation of CX3CL1 and CX3CR1 expression via a mechanism involving NF-kB, AP-1, and STAT1/3 transcription factors, (2) resistin employs TLR4 and Gi-protein signaling for its effect on SMC, (3) CX3CL1 induced by resistin is functional in monocyte chemotaxis. The data reveal new mechanisms by which resistin promotes the inflammatory phenotype of SMC.

Advances in Natural and Bio-Inspired Nanoparticles for the Treatment of Cardiovascular Diseases.

Cardiovascular diseases (CVD) is a general term for disorders affecting the heart or blood vessels and represent a major cause of disability and death worldwide [...].

Multilayer gold nanoparticles as non-viral vectors for targeting MCF-7 cancer cells.

Cargocomplexes play a vital role in non-viral delivery methods due to their capacity to target certain cells (or cells through the cell-division cycle) and inject their (macro)molecular "cargo" into them. The development of gene carriers that can efficiently transport and deliver genetic material into human-targeted cells with minimal toxicity is an important challenge in the field. The present study reports the straightforward preparation and testing of a modular non-viral gene carrier based on AuNPs. The design, synthesis, and in vitro evaluation of multilayer gold nanoparticles (AuNPs) as non-viral gene carriers with high transfection efficiency, reduced cytotoxicity for targeted therapeutic delivery of nucleic acids to MCF-7 cancer cells are presented. The developed non-viral vector is based on supramolecular "host-guest" inclusion complexes of ß-cyclodextrin, positioned on the AuNPs surface over a layer of polyethyleneimine, and adamantyl moiety from polyethylene glycol conjugated decapeptide (WXEAAYQRFL). First, the ß-CD functionalized PEI was utilized as the template for the synthesis of AuNPs of controlled sizes. The reaction produced small AuNPs with a cationic layer which is known for efficient condensation of genetic material and ß-CD suitable for the decoration of the carrier with targeting moieties using "host-guest" inclusion complexation. Subsequently, adamantine-polyethylene glycol conjugated decapeptide was attached to the AuNPs. The in vitro results have validated the ability of the proposed systems to selectively target tumor cells with high efficacy and low toxicity due to the unique affinity of the aptamer-functionalized nanoparticles toward breast cancer cells. The findings of this work demonstrated that the proposed modular system may represent a very promising platform for the AuNP-based non-viral vectors mainly due to the versatility of the system, which allows for the facile exchange of several types of ligands for improving the targeting properties and transfection efficiency, or for providing better protection from the endocytotic systems.

Tri-Component Hydrogel as Template for Nanocrystalline Hydroxyapatite Deposition Using Alternate Soaking Method for Bone Tissue Engineering Applications.

Composite hydrogels containing apatite-like particles can act as scaffolds for osteoblast proliferation, with applications in bone tissue engineering. In this respect, porous biocompatible hydrogels were obtained from chitosan, oxidized pullulan, and PVA in different ratios. The stability of the hydrogels was ensured both by covalent bonds between aldehyde groups of oxidized pullulan and free amino groups of chitosan, and by physical bonds formed during freeze-thaw cycles and lyophilization. The deposition of calcium phosphates was performed by alternate soaking of the porous hydrogels into solutions with calcium and phosphate ions, assuring a basic pH required for hydroxyapatite formation. The mineralized hydrogels were characterized using FTIR spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis, showing that inorganic particles containing between 80 and 92% hydroxyapatite were deposited in a high amount on the pore walls of the polymeric matrix. The composition of the organic matrix influenced the crystallization of calcium phosphates and the mechanical properties of the composite hydrogels. In vitro biological tests showed that mineralized hydrogels support the proliferation of MG-63 osteoblast-like cells to a greater extent compared to pristine hydrogels.

Nanocarriers of shRNA-Runx2 directed to collagen IV as a nanotherapeutic system to target calcific aortic valve disease.

Runx2 is a key transcription factor involved in valvular interstitial cells (VIC) osteodifferentiation, a process actively entwined with the calcific aortic valve disease (CAVD). We hypothesize that a strategy intended to silence Runx2 could be a valuable novel therapeutic option for CAVD. To this intent, we aimed at (i) developing targeted nanoparticles for efficient delivery of short hairpin (sh)RNA sequences specific for Runx2 to the aortic valve employing a relevant mouse model for CAVD and (ii) investigate their therapeutic potential in osteoblast-differentiated VIC (oVIC) cultivated into a 3D scaffold. Since collagen IV was used as a target, a peptide that binds specifically to collagen IV (Cp) was conjugated to the surface of lipopolyplexes encapsulating shRNA-Runx2 (Cp-LPP/shRunx2). The results showed that Cp-LPP/shRunx2 were (i) cytocompatible; (ii) efficiently taken up by 3D-cultured oVIC; (iii) diminished the osteodifferentiation of human VIC (cultured in a 3D hydrogel-derived from native aortic root) by reducing osteogenic molecules expression, alkaline phosphatase activity, and calcium concentration; and (iv) were recruited in aortic valve leaflets in a murine model of atherosclerosis. Taken together, these data recommend Cp-LPP/shRunx2 as a novel targeted nanotherapy to block the progression of CAVD, with a good perspective to be introduced in practical use.

Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules.

OBJECTIVE: In atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages, mediate the heterotypic anchorage and chemotaxis of these cells. We questioned whether, during the close interaction of monocytes with SMC, the CX3CL1/CX3CR1 pair modulates the expression of pro-atherogenic molecules in these cells. METHODS AND RESULTS: SMC were co-cultured with monocytes or LPS-activated monocytes (18h) and then the cells were separated and individually investigated for the gene and protein expression of TNFα, IL-1ß, IL-6, CX3CR1 and metalloproteinases (MMP-2, MMP-9). We found that SMC-monocyte interaction induced, in each cell type, an increased mRNA and protein expression of TNFα, IL-1ß, IL-6, CX3CR1, MMP-2 and MMP-9. Blocking the binding of fractalkine to CX3CR1 (by pre-incubation of monocytes with anti-CX3CR1 or by CX3CR1 siRNA transfection) before cell co-culture decreased the production of TNFα, CX3CR1 and MMP-9. Monocyte-SMC interaction induced the phosphorylation of p38MAPK and activation of AP-1 transcription factor. Silencing the p65 (NF-kB subunit) inhibited the IL-1ß and IL-6 and silencing c-jun inhibited the TNFα, CX3CR1 and MMP-9 induced by SMC-monocyte interaction. CONCLUSIONS: The cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1ß, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis.

A novel pro-inflammatory mechanism of action of resistin in human endothelial cells: up-regulation of SOCS3 expression through STAT3 activation.

Resistin is a significant local and systemic regulatory cytokine involved in inflammation. Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction induced by several cytokines in a cytokine and cell specific manner. Resistin up-regulates SOCS3 expression in mice adipocytes but it is not known whether this is a common occurrence in other cells. We questioned whether resistin-induces SOCS3 in human endothelial cells and if signal transducer and activator of transcription (STAT) proteins are involved in the process. The Real-Time PCR and Western blot analysis showed that in resistin-activated HEC the gene and protein expression of SOCS3 were significantly increased. Furthermore, resistin induced activation of STAT3 as characterized by increased tyrosine phosphorylation. Resistin-induced SOCS3 expression was blocked by specific inhibitors of STAT3 signaling and by the transfection of siRNA specific for STAT3. Silencing of SOCS3 gene expression by transfection with SOCS3 siRNA reduced the expression of resistin induced-P-selectin and fractalkine in HEC. Together, our results demonstrate that in HEC (1) resistin up-regulates SOCS3 expression and activates STAT3 transcription factor; (2) the increase in SOCS3 mRNA and protein expression as well as STAT3 activation have a long-lasting effect (up to 18h); (3) inhibition of SOCS3 function prevents resistin-induced expression of cell adhesion molecules P-selectin and fractalkine and thus activation of endothelial cells. The data uncover a new resistin-mediated mechanism in human endothelial cells and designate SOCS3 as a novel therapeutic target to modulate resistin-dependent inflammation in vessel wall diseases.

Bio-Responsive Carriers for Controlled Delivery of Doxorubicin to Cancer Cells.

The cellular internalization of drug carriers occurs via different endocytic pathways that ultimately involve the endosomes and the lysosomes, organelles where the pH value drops to 6.0 and 5.0, respectively. We aimed to design and characterize pH/temperature-responsive carriers for the effective delivery of the anti-tumoral drug doxorubicin. To this purpose, poly(N-isopropylacrylamide-co-vinylimidazole) was synthesized as an attractive pH/temperature-sensitive copolymer. Microspheres made of this copolymer, loaded with doxorubicin (MS-DXR), disintegrate in monodisperse nanospheres (NS-DXR) under conditions similar to that found in the bloodstream (pH = 7.4, temperature of 36 °C) releasing a small amount of payload. However, in environments that simulate the endosomal and lysosomal conditions, nanospheres solubilize, releasing the entire amount of drug. We followed the NS-DXR internalization using two cancer cell lines, hepatic carcinoma HepG2 cells and lung adenocarcinoma A549 cells. The data showed that NS-DXR are internalized to a greater extent by HepG2 cells than A549 cells, and this correlated with increased cytotoxicity induced by NS-DXR in HepG2 cells compared with A549 cells. Moreover, NS-DXR particles do not cause hemolysis and erythrocytes aggregation. Administered in vivo, NS-DXR localized in the liver and kidneys of mice, and the loading of DXR into NS resulted in the reduced renal clearance of DXR. In conclusion, the newly developed poly(N-isopropylacrylamide-co-vinyl imidazole) particles are biocompatible and may be introduced as carriers for doxorubicin to hepatic tumors.

Antitumor Properties of a New Macrocyclic Tetranuclear Oxidovanadium(V) Complex with 3-Methoxysalicylidenvaline Ligand.

A wide variety of metal-based compounds have been obtained and studied for their antitumor activity since the intensely used cytostatic drugs (e.g., cisplatin) failed to accomplish their expected pharmacological properties. Thus, we aimed to develop a new vanadium-based drug and assess its antitumor properties using the human hepatocarcinoma (HepG2) cell line. The compound was synthesized from vanadyl sulfate, DL-valine, and o-vanillin and was spectrally and structurally characterized (UV-Vis, IR, CD, and single-crystal/powder-XRD). Compound stability in biological media, cell uptake, and the interaction with albumin were assessed. The mechanisms of its antitumor activity were determined compared to cisplatin by performing cytotoxicity, oxidative and mitochondrial status, DNA fragmentation, ß-Tubulin synthesis investigation, and cell cycle studies. Herein, we developed a macrocyclic tetranuclear oxidovanadium(V) compound, [(VVO)(L)(CH3O)]4, having coordinated four Schiff base (H2L) ligands, 3-methoxysalicylidenvaline. We showed that [(VVO)(L)(CH3O)]4: (i) has pH-dependent stability in biological media, (ii) binds to albumin in a dose-dependent manner, (iii) is taken up by cells in a time-dependent way, (iv) has a higher capacity to induce cell death compared to cisplatin (IC50 = 6 µM vs. 10 µM), by altering the oxidative and mitochondrial status in HepG2 cells. Unlike cisplatin, which blocks the cell cycle in the S-phase, the new vanadium-based compound arrests it in S and G2/M-phase, whereas no differences in the induction of DNA fragmentation and reduction of ß-Tubulin synthesis between the two were determined. Thus, the [(VVO)(L)(CH3O)]4 antitumor mechanism involved corroboration between the generation of oxidative species, mitochondrial dysfunction, degradation of DNA, cell cycle arrest in the S and G2/M-phase, and ß-Tubulin synthesis reduction. Our studies demonstrate the potent antitumor activity of [(VVO)(L)(CH3O)]4 and propose it as an attractive candidate for anticancer therapy.

Folic acid-decorated PEGylated magnetite nanoparticles as efficient drug carriers to tumor cells overexpressing folic acid receptor.

The improved drug delivery systems (DDS) are needed for the targeted delivery of their therapeutic cargo (biologically active protein/peptide molecules, nucleic acids, vaccines, etc.) to diseased cells. Thus, we aimed to develop magnetite nanoparticles (Fe3O4), stabilized with polyethylene glycol (PEG) and decorated (surface-functionalized) with folic acid (FA) (Fe3O4@PEG@FA) to ensure targeted internalization in cells expressing the folic acid receptors (FR). The Fe3O4@PEG@FA nanoparticles were synthesized by co-precipitation in a one-pot methodology. Curcumin (Curc), a polyphenol with anti-tumoral activity, was loaded on the nanoparticles, and FA-targeted (Fe3O4@PEG@FA@Curc) and non-targeted (Fe3O4@PEG@Curc) systems were obtained. The internalization of Fe3O4@PEG@FA@Curc and Fe3O4@PEG@Curc nanoparticles was determined in two tumor cell lines, the FR-positive MCF-7 human breast carcinoma cell line and A549 human lung adenocarcinoma cell line, expressing a low level of FR. The results showed that MCF-7 cells internalize FA-functionalized nanoparticles to a greater extent than non-targeted ones and also than A549 cells. The competitive studies performed in the presence of FA in excess suggested that internalization is an FR-dependent process. The increased internalization of Fe3O4@PEG@FA@Curc nanoparticles in MCF-7 cells is correlated with increased cytotoxicity in this cell line compared to A549 cells. In conclusion, the FA-functionalized magnetic systems can ensure a better internalization of the nanoparticles and can be used to deliver various therapeutic agents, both in cancer treatment and also in the treatment of other inflammation-associated diseases such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, Crohn's disease or atherosclerosis.

Chemical Attachment of 5-Nitrosalicylaldimine Motif to Silatrane Resulting in an Organic-Inorganic Structure with High Medicinal Significance.

Two chemical motifs of interest for medicinal chemistry, silatrane as 1-(3-aminopropyl) silatrane (SIL M), and nitro group attached in position 5 to salicylaldehyde, are coupled in a new structure, 1-(3-{[(2-hydroxy-5-nitrophenyl)methylidene]amino}propyl)silatrane (SIL-BS), through an azomethine moiety, also known as a versatile pharmacophore. The high purity isolated compound was structurally characterized by an elemental, spectral, and single crystal X-ray diffraction analysis. Given the structural premises for being a biologically active compound, different specific techniques and protocols have been used to evaluate their in vitro hydrolytic stability in simulated physiological conditions, the cytotoxicity on two cancer cell lines (HepG2 and MCF7), and protein binding ability-with a major role in drug ADME (Absorption, Distribution, Metabolism and Excretion), in parallel with those of the SIL M. While the latter had a good biocompatibility, the nitro-silatrane derivative, SIL-BS, exhibited a higher cytotoxic activity on HepG2 and MCF7 cell lines, performance assigned, among others, to the known capacity of the nitro group to promote a specific cytotoxicity by a "activation by reduction" mechanism. Both compounds exhibited increased bio- and muco-adhesiveness, which can favor an optimized therapeutic effect by increased drug permeation and residence time in tumor location. Additional benefits of these compounds have been demonstrated by their antimicrobial activity on several fungi and bacteria species. Molecular docking computations on Human Serum Albumin (HSA) and MPRO COVID-19 protease demonstrated their potential in the development of new drugs for combined therapy.

High glucose induces enhanced expression of resistin in human U937 monocyte-like cell line by MAPK- and NF-kB-dependent mechanisms; the modulating effect of insulin.

Resistin has emerged as a significant local and systemic regulatory cytokine involved in inflammation. In diabetic patients, the serum resistin level is increased, monocytes/macrophages being an important source of resistin production. We therefore hypothesize that high glucose concentrations (HG) regulate resistin expression in human monocytes. Our aim has been to uncover the potential signalling pathways involved in this process. We have also questioned whether insulin has an effect on the regulation of resistin expression induced by HG. Human monocytes (U937 cell line) were exposed to 25 mM glucose for 24 h and then resistin gene expression and protein levels were determined by reverse transcription with the polymerase chain reaction and Western blot assays. We found that (1) the gene expression and protein level of resistin were up-regulated by HG; (2) the inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580), extracellular signal-regulated kinases 1/2 (ERK1/2; PD98059) and c-Jun N-terminal kinase (SP600125) and of the transcription factor nuclear factor kappa-B (PDTC) inhibited HG-induced resistin protein production and (3) insulin reduced HG-induced resistin expression via a mechanism independent of phosphatidylinositol 3-kinase (PI3K) or p38 and ERK1/2. Therefore, HG significantly increases resistin gene expression and protein production in the U937 cell line by mechanisms involving MAPKs and the transcription factor NF-kB, whereas insulin reduces its expression. This study adds new data concerning the molecular mechanisms involved in the pro-inflammatory effects of HG on human monocytes.

Curcumin and a Morus alba extract reduce pro-inflammatory effects of resistin in human endothelial cells.

Resistin is a cytokine which plays an important role in cardiovascular disease by influencing systemic inflammation and endothelial activation. In human endothelial cells (HEC) it increases the expression of P-selectin and fractalkine, and enhances monocyte adhesion by antioxidant mechanisms. This study investigated whether the natural antioxidants curcumin (CC) and an extract of Morus alba leaves (MA) have protective effects in resistin-activated HEC. HEC were exposed to 100 ng/mL resistin for 6 and 18 h in the absence or presence of MA or CC and the expression of fractalkine and P-selectin was determined by RT-PCR and western blot. Intracellular accumulation of reactive oxygen species (ROS) was monitored by fluorimetry and NADPH oxidase activity by a lucigenin-enhanced chemiluminescence assay. In addition, adhesion assays using the monocytic U937 cells were performed. The results showed that treatment of HEC exposed to resistin with MA and CC: (1) inhibited significantly P-selectin and fractalkine expression, (2) inhibited the increase in the intracellular ROS level, (3) reduced NADPH activation and (4) reduced monocytes adhesion to HEC. The results indicate that MA and curcumin target resistin-induced human endothelial activation partly via antioxidant mechanisms and suggest that they may represent therapeutic agents in vascular disease mediated by resistin.

Development of Dextran-Coated Magnetic Nanoparticles Loaded with Protocatechuic Acid for Vascular Inflammation Therapy.

Vascular inflammation plays a crucial role in the progression of various pathologies, including atherosclerosis (AS), and thus it has become an attractive therapeutic target. The protocatechuic acid (PCA), one of the main metabolites of complex polyphenols, is endowed with anti-inflammatory activity, but its formulation into nanocarriers may increase its bioavailability. In this study, we developed and characterized dextran shell‒iron oxide core nanoparticles loaded with PCA (MNP-Dex/PCA) and assessed their cytotoxicity and anti-inflammatory potential on cells acting as key players in the onset and progression of AS, namely, endothelial cells (EC) and monocytes/macrophages. The results showed that MNP-Dex/PCA exert an anti-inflammatory activity at non-cytotoxic and therapeutically relevant concentrations of PCA (350 µM) as supported by the reduced levels of inflammatory molecules such as MCP-1, IL-1ß, TNF-α, IL-6, and CCR2 in activated EC and M1-type macrophages and functional monocyte adhesion assay. The anti-inflammatory effect of MNP-Dex/PCA was associated with the reduction in the levels of ERK1/2 and p38-α mitogen-activated protein kinases (MAPKs) and NF-kB transcription factor. Our data support the further development of dextran shell-magnetic core nanoparticles as theranostic nanoparticles for guidance, imaging, and therapy of vascular inflammation using PCA or other anti-inflammatory compounds.

Detection of Vascular Reactive Oxygen Species in Experimental Atherosclerosis by High-Resolution Near-Infrared Fluorescence Imaging Using VCAM-1-Targeted Liposomes Entrapping a Fluorogenic Redox-Sensitive Probe.

Excessive production of reactive oxygen species (ROS) and the ensuing oxidative stress are instrumental in all phases of atherosclerosis. Despite the major achievements in understanding the regulatory pathways and molecular sources of ROS in the vasculature, the specific detection and quantification of ROS in experimental models of disease remain a challenge. We aimed to develop a reliable and straightforward imaging procedure to interrogate the ROS overproduction in the vasculature and in various organs/tissues in atherosclerosis. To this purpose, the cell-impermeant ROS Brite™ 700 (RB700) probe that produces bright near-infrared fluorescence upon ROS oxidation was encapsulated into VCAM-1-targeted, sterically stabilized liposomes (VLp). Cultured human endothelial cells (EC) and macrophages (Mac) were used for in vitro experiments. C57BL6/J and ApoE-/- mice were randomized to receive normal or high-fat, cholesterol-rich diet for 10 or 32 weeks. The mice received a retroorbital injection with fluorescent tagged VLp incorporating RB700 (VLp-RB700). After two hours, the specific signals of the oxidized RB700 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DSPE), inserted into liposome bilayers, were measured ex vivo in the mouse aorta and various organs by high-resolution fluorescent imaging. VLp-RB700 was efficiently taken up by cultured human EC and Mac, as confirmed by fluorescence microscopy and spectrofluorimetry. After systemic administration in atherosclerotic ApoE-/- mice, VLp-RB700 were efficiently concentrated at the sites of aortic lesions, as indicated by the augmented NBD fluorescence. Significant increases in oxidized RB700 signal were detected in the aorta and in the liver and kidney of atherosclerotic ApoE-/- mice. RB700 encapsulation into sterically stabilized VCAM-1-sensitive Lp could be a novel strategy for the qualitative and quantitative detection of ROS in the vasculature and various organs and tissues in animal models of disease. The accurate and precise detection of ROS in experimental models of disease could ease the translation of the results to human pathologies.