The Elevated Inflammatory Status of Neutrophils Is Related to In-Hospital Complications in Patients with Acute Coronary Syndrome and Has Important Prognosis Value for Diabetic Patients.

Despite neutrophil involvement in inflammation and tissue repair, little is understood about their inflammatory status in acute coronary syndrome (ACS) patients with poor outcomes. Hence, we investigated the potential correlation between neutrophil inflammatory markers and the prognosis of ACS patients with/without diabetes and explored whether neutrophils demonstrate a unique inflammatory phenotype in patients experiencing an adverse in-hospital outcome. The study enrolled 229 ACS patients with or without diabetes. Poor evolution was defined as either death, left ventricular ejection fraction (LVEF) <40%, Killip Class 3/4, ventricular arrhythmias, or mechanical complications. Univariate and multivariate analyses were employed to identify clinical and paraclinical factors associated with in-hospital outcomes. Neutrophils isolated from fresh blood were investigated using qPCR, Western blot, enzymatic assay, and immunofluorescence. Poor evolution post-myocardial infarction (MI) was associated with increased number, activity, and inflammatory status of neutrophils, as indicated by significant increase of Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), fibrinogen, interleukin-1ß (IL-1ß), and, interleukin-6 (IL-6). Among the patients with complicated evolution, neutrophil activity had an important prognosis value for diabetics. Neutrophils from patients with unfavorable evolution revealed a pro-inflammatory phenotype with increased expression of CCL3, IL-1ß, interleukin-18 (IL-18), S100A9, intracellular cell adhesion molecule-1 (ICAM-1), matrix metalloprotease (MMP-9), of molecules essential in reactive oxygen species (ROS) production p22phox and Nox2, and increased capacity to form neutrophil extracellular traps. Inflammation is associated with adverse short-term prognosis in acute ACS, and inflammatory biomarkers exhibit greater specificity in predicting short-term outcomes in diabetics. Moreover, neutrophils from patients with unfavorable evolution exhibit distinct inflammatory patterns, suggesting that alterations in the innate immune response in this subgroup may exert detrimental effects on disease progression.

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

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.

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.

Peroxisome Proliferator-Activated Receptor α in Lipoprotein Metabolism and Atherosclerotic Cardiovascular Disease.

Peroxisome proliferator-activated receptors (PPARs) are a group of ligand-binding transcription factors with pivotal action in regulating pleiotropic signaling pathways of energetic metabolism, immune responses and cell proliferation and differentiation. A significant body of evidence indicates that the PPARα receptor is an important modulator of plasma lipid and lipoprotein metabolism, with pluripotent effects influencing the lipid and apolipoprotein cargo of both atherogenic and antiatherogenic lipoproteins and their functionality. Clinical evidence supports an important role of PPARα agonists (fibric acid derivatives) in the treatment of hypertriglyceridemia and/or low high-density lipoprotein (HDL) cholesterol levels, although the effects of clinical trials are contradictory and point to a reduction in the risk of nonfatal and fatal myocardial infarction events. In this manuscript, we provide an up-to-date critical review of the existing relevant literature.

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.

Pharmacological Inhibition of Lysine-Specific Demethylase 1A Reduces Atherosclerotic Lesion Formation in Apolipoprotein E-Deficient Mice by a Mechanism Involving Decreased Oxidative Stress and Inflammation; Potential Implications in Human Atherosclerosis.

Dysregulated epigenetic mechanisms promote transcriptomic and phenotypic alterations in cardiovascular diseases. The role of histone methylation-related pathways in atherosclerosis is largely unknown. We hypothesize that lysine-specific demethylase 1A (LSD1/KDM1A) regulates key molecular effectors and pathways linked to atherosclerotic plaque formation. Human non-atherosclerotic and atherosclerotic tissue specimens, ApoE-/- mice, and in vitro polarized macrophages (Mac) were examined. Male ApoE-/- mice fed a normal/atherogenic diet were randomized to receive GSK2879552, a highly specific LSD1 inhibitor, or its vehicle, for 4 weeks. The mRNA and protein expression levels of LSD1/KDM1A were significantly elevated in atherosclerotic human carotid arteries, atherosclerotic aortas of ApoE-/- mice, and M1-Mac. Treatment of ApoE-/- mice with GSK2879552 significantly reduced the extent of atherosclerotic lesions and the aortic expression of NADPH oxidase subunits (Nox1/2/4, p22phox) and 4-hydroxynonenal-protein adducts. Concomitantly, the markers of immune cell infiltration and vascular inflammation were significantly decreased. LSD1 blockade down-regulated the expression of genes associated with Mac pro-inflammatory phenotype. Nox subunit transcript levels were significantly elevated in HEK293 reporter cells overexpressing LSD1. In experimental atherosclerosis, LSD1 mediates the up-regulation of molecular effectors connected to oxidative stress and inflammation. Together, these data indicate that LSD1-pharmacological interventions are novel targets for supportive therapeutic strategies in atherosclerosis.

Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy.

Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells' lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.

Synthetic lipoproteins based on apolipoprotein E coupled to fullerenol have anti-atherosclerotic properties.

BACKGROUND: Apolipoprotein E (apoE) is an anti-atherosclerotic protein associated with almost all plasma lipoproteins. Fullerenol (Full-OH) contains the fullerene hydrophobic cage and several hydroxyl groups that could be derivatized to covalently bind various molecules. Herein, we aimed to produce fullerenol-based nanoparticles carrying apoE3 (Full-apoE) and test their anti-atherosclerotic effects. METHODS: Full-apoE nanoparticles were obtained from Full-OH activated to reactive cyanide ester fullerenol derivative that was further reacted with apoE protein. To test their effect, the nanoparticles were administered to apoE-deficient mice for 24 h or 3 weeks. ApoE part of the nanoparticles was determined by Western Blot and quantified by ELISA. Atherosclerotic plaque size was evaluated after Oil Red O staining and the gene expression was determined by Real-Time PCR. RESULTS: Full-apoE nanoparticles were detected mainly in the liver, and to a lesser extent in the kidney, lung, and brain. In the plasma of the Full-apoE-treated mice, apoE was found associated with very-low-density lipoproteins and high-density lipoproteins. Treatment for 3 weeks with Full-apoE nanoparticles decreased plasma cholesterol levels, increased the expression of apolipoprotein A-I, ABCA1 transporter, scavenger receptor-B1, and sortilin, and reduced the evolution of the atheromatous plaques in the atherosclerotic mice. CONCLUSIONS: In experimental atherosclerosis, the administration of Full-apoE nanoparticles limits the evolution of the atheromatous plaques by decreasing the plasma cholesterol level and increasing the expression of major proteins involved in lipid metabolism. Thus, they represent a novel promising strategy for atherosclerosis therapy.

Short-Term Blockade of Pro-Inflammatory Alarmin S100A9 Favorably Modulates Left Ventricle Proteome and Related Signaling Pathways Involved in Post-Myocardial Infarction Recovery.

Prognosis after myocardial infarction (MI) varies greatly depending on the extent of damaged area and the management of biological processes during recovery. Reportedly, the inhibition of the pro-inflammatory S100A9 reduces myocardial damage after MI. We hypothesize that a S100A9 blockade induces changes of major signaling pathways implicated in post-MI healing. Mass spectrometry-based proteomics and gene analyses of infarcted mice left ventricle were performed. The S100A9 blocker (ABR-23890) was given for 3 days after coronary ligation. At 3 and 7 days post-MI, ventricle samples were analyzed versus control and Sham-operated mice. Blockade of S100A9 modulated the expressed proteins involved in five biological processes: leukocyte cell-cell adhesion, regulation of the muscle cell apoptotic process, regulation of the intrinsic apoptotic signaling pathway, sarcomere organization and cardiac muscle hypertrophy. The blocker induced regulation of 36 proteins interacting with or targeted by the cellular tumor antigen p53, prevented myocardial compensatory hypertrophy, and reduced cardiac markers of post-ischemic stress. The blockade effect was prominent at day 7 post-MI when the quantitative features of the ventricle proteome were closer to controls. Blockade of S100A9 restores key biological processes altered post-MI. These processes could be valuable new pharmacological targets for the treatment of ischemic heart. Mass spectrometry data are available via ProteomeXchange with identifier PXD033683.

Parathyroid Hormone Induces Human Valvular Endothelial Cells Dysfunction That Impacts the Osteogenic Phenotype of Valvular Interstitial Cells.

Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-ß1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification.

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.

P-selectin targeted RAGE-shRNA lipoplexes alleviate atherosclerosis-associated inflammation.

The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1ß, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.

Activated Histone Acetyltransferase p300/CBP-Related Signalling Pathways Mediate Up-Regulation of NADPH Oxidase, Inflammation, and Fibrosis in Diabetic Kidney.

Accumulating evidence implicates the histone acetylation-based epigenetic mechanisms in the pathoetiology of diabetes-associated micro-/macrovascular complications. Diabetic kidney disease (DKD) is a progressive chronic inflammatory microvascular disorder ultimately leading to glomerulosclerosis and kidney failure. We hypothesized that histone acetyltransferase p300/CBP may be involved in mediating diabetes-accelerated renal damage. In this study, we aimed at investigating the potential role of p300/CBP in the up-regulation of renal NADPH oxidase (Nox), reactive oxygen species (ROS) production, inflammation, and fibrosis in diabetic mice. Diabetic C57BL/6J mice were randomized to receive 10 mg/kg C646, a selective p300/CBP inhibitor, or its vehicle for 4 weeks. We found that in the kidney of C646-treated diabetic mice, the level of H3K27ac, an epigenetic mark of active gene expression, was significantly reduced. Pharmacological inhibition of p300/CBP significantly down-regulated the diabetes-induced enhanced expression of Nox subtypes, pro-inflammatory, and pro-fibrotic molecules in the kidney of mice, and the glomerular ROS overproduction. Our study provides evidence that the activation of p300/CBP enhances ROS production, potentially generated by up-regulated Nox, inflammation, and the production of extracellular matrix proteins in the diabetic kidney. The data suggest that p300/CBP-pharmacological inhibitors may be attractive tools to modulate diabetes-associated pathological processes to efficiently reduce the burden of DKD.

Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo.

Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton's jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17ß-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.

Aortic valve disease in diabetes: Molecular mechanisms and novel therapies.

Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non-diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic-hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes-induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.

Transcriptional Profiling and Functional Analysis of N1/N2 Neutrophils Reveal an Immunomodulatory Effect of S100A9-Blockade on the Pro-Inflammatory N1 Subpopulation.

Neutrophils have been classically viewed as a homogenous population. Recently, neutrophils were phenotypically classified into pro-inflammatory N1 and anti-inflammatory N2 sub-populations, but the functional differences between the two subtypes are not completely understood. We aimed to investigate the phenotypic and functional differences between N1 and N2 neutrophils, and to identify the potential contribution of the S100A9 alarmin in neutrophil polarization. We describe distinct transcriptomic profiles and functional differences between N1 and N2 neutrophils. Compared to N2, the N1 neutrophils exhibited: i) higher levels of ROS and oxidative burst, ii) increased activity of MPO and MMP-9, and iii) enhanced chemotactic response. N1 neutrophils were also characterized by elevated expression of NADPH oxidase subunits, as well as activation of the signaling molecules ERK and the p65 subunit of NF-kB. Moreover, we found that the S100A9 alarmin promotes the chemotactic and enzymatic activity of N1 neutrophils. S100A9 inhibition with a specific small-molecule blocker, reduced CCL2, CCL3 and CCL5 chemokine expression and decreased MPO and MMP-9 activity, by interfering with the NF-kB signaling pathway. Together, these findings reveal that N1 neutrophils are pro-inflammatory effectors of the innate immune response. Pharmacological blockade of S100A9 dampens the function of the pro-inflammatory N1 phenotype, promoting the alarmin as a novel target for therapeutic intervention in inflammatory diseases.

A novel platform for drug testing: Biomimetic three-dimensional hyaluronic acid-based scaffold seeded with human hepatocarcinoma cells.

Hepatic cancer is one of the most widespread maladies worldwide that requires urgent therapies and thus reliable means for testing anti-cancer drugs. The switch from two-dimensional (2D) to three-dimensional (3D) cell cultures produced an improvement in the in vitro outcomes for testing anti-cancer drugs. We aimed to develop a novel hyaluronic acid (HA)-based 3D cell model of human hepatocellular carcinoma (HepG2 cells) for drug testing and to assess comparatively in 3D vs. 2D, the cytotoxicity and the apoptotic response to the anti-tumor agent, cisplatin. The 3D model was developed by seeding HepG2 cells in a HA/poly(methylvinylether-alt-maleic acid) (HA3P50)-based scaffold. Compared to 2D, the cells grown in the HA3P50 scaffold proliferate into larger-cellular aggregates that exhibit liver-like functions by controlling the release of hepatocyte-specific biomarkers (albumin, urea, bile acids, transaminases) and the synthesis of cytochrome-P450 (CYP)7A1 enzyme. Also, growing the cells in the scaffold sensitize the hepatocytes to the anti-tumor effect of cisplatin, by a mechanism involving the activation of ERK/p38α-MAPK and dysregulation of NF-kB/STAT3/Bcl-2 pathways. In conclusion, the newly developed HA-based 3D model is suitable for chemotherapeutic drug testing on hepatocellular carcinoma. Moreover, the system can be adapted and employed as experimental platform functioning as a proper tissue/tumor surrogate.

Human Mesenchymal Stromal Cell-Derived Exosomes Promote In Vitro Wound Healing by Modulating the Biological Properties of Skin Keratinocytes and Fibroblasts and Stimulating Angiogenesis.

Bone marrow-derived mesenchymal stromal cells (MSCs) are major players in regenerative therapies for wound healing via their paracrine activity, mediated partially by exosomes. Our purpose was to test if MSC-derived exosomes could accelerate wound healing by enhancing the biological properties of the main cell types involved in the key phases of this process. Thus, the effects of exosomes on (i) macrophage activation, (ii) angiogenesis, (iii) keratinocytes and dermal fibroblasts proliferation and migration, and (iv) the capacity of myofibroblasts to regulate the turnover of the extracellular matrix were evaluated. The results showed that, although exosomes did not exhibit anti-inflammatory properties, they stimulated angiogenesis. Exposure of keratinocytes and dermal (myo)fibroblasts to exosomes enhanced their proliferation and migratory capacity. Additionally, exosomes prevented the upregulation of gene expression for type I and III collagen, α-smooth muscle actin, and MMP2 and 14, and they increased MMP13 expression during the fibroblast-myofibroblast transition. The regenerative properties of exosomes were validated using a wound healing skin organotypic model, which exhibited full re-epithelialization upon exosomes exposure. In summary, these data indicate that exosomes enhance the biological properties of keratinocytes, fibroblasts, and endothelial cells, thus providing a reliable therapeutic tool for skin regeneration.

Molecular Research in Cardiovascular Disease.

Cardiovascular diseases have attracted our full attention not only because they are the main cause of mortality and morbidity in many countries but also because the therapy for and cure of these maladies are among the major challenges of the medicine in the 21st century [...].