Calcific aortic valve disease (CAVD) is a common malady with few treatment options other than valve replacement by surgery or transcatheter aortic valve implantation (TAVI). Endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells and osteogenic differentiation of valvular interstitial cells are crucial processes of CAVD. Smad3 and Runx2 are key transcription factors (TFs) that drive these processes by regulating gene expression and cellular functions. We hypothesize that downregulation of these TFs with nanoparticle-mediated RNA interference could mitigate aortic valve stenosis and calcification. We engineered dual-targeted lipid-polymer hybrid nanocarriers (lipopolyplexes, LPP) to deliver short-hairpin RNA (shRNA) for gene silencing in pathologically remodeled aortic valve. The nanocarriers simultaneously target vascular cell adhesion molecule-1 (VCAM-1) and collagen IV, enhancing specificity toward inflamed and fibrotic valvular tissue. Encapsulated shRNA constructs were designed to silence either Smad3 or Runx2 (yielding formulations V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2). Therapeutic efficacy was evaluated in a mouse model of atherosclerosis aggravated by diabetes, mimicking the pathological environment of CAVD. The dual-targeted lipopolyplexes effectively facilitated gene delivery to the aortic valve, ensuring efficient transfection. Treatment with V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2 resulted in marked silencing of Smad3 and Runx2, accompanied by significant suppression of osteogenic markers, including osteopontin, alkaline phosphatase, and osteocalcin, as well as reduced α-smooth muscle actin (αSMA) expression in valve tissue. Our data further identify Runx2 as a novel upstream modulator of Smad3 expression, unveiling a previously unrecognized Runx2-Smad3 regulatory axis with important implications for valvular pathology and targeted therapy. Beyond localized effects, systemic administration of these lipopolyplexes resulted in reduced plasma concentrations of alkaline phosphatase, cholesterol, and triglycerides, while maintaining hepatic and renal function, suggesting additional benefits for systemic metabolic homeostasis. These findings highlight the pivotal role of Smad3 and Runx2 downregulation in mitigating aortic valve calcification, unveiling both molecules as compelling therapeutic targets in CAVD.

BackgroundDiabetic foot syndrome (DFS) is characterized by chronic inflammation, thrombotic imbalance, and impaired wound healing, yet systemic molecular alterations underlying this complication remain incompletely defined. In this study, we combined clinical plasma proteomics with experimental pharmacological modulation to characterize a circulating damage-associated molecular pattern (DAMP)-related signature linked to systemic inflammatory signaling in DFS.MethodsPlasma samples from patients with type 2 diabetes with and without DFS, including individuals with varying degrees of limb ischemia, were analyzed using liquid chromatography-tandem mass spectrometry. Differentially abundant proteins were evaluated in relation to inflammatory, hematological, and metabolic parameters. Pharmacological responsiveness was assessed in a murine diabetic ischemic wound model treated with a selective Toll-like receptor 4 (TLR4) inhibitor.ResultsProteomic analysis identified coordinated differences in the abundance of multiple acute-phase and stress-associated proteins, including serum amyloid A1, serum amyloid A2, serum amyloid P component, S100A8, defensin alpha 1B, fibrinogen chains, heat shock protein family A member 5, thymosin beta 4, fibronectin 1, and tenascins. These proteins exhibited differences between DFS patients and diabetic controls and were explored in relation to systemic inflammatory variables. Several DAMPs demonstrated reproducible patterns across the studied groups, suggesting the presence of a coordinated circulating molecular pattern rather than isolated changes in individual proteins. In diabetic ischemic mice, TLR4 inhibition was associated with altered abundance of several circulating proteins, including reductions in selected amyloid-associated proteins. These observations suggest an association between modulation of innate immune signaling pathways and circulating protein profiles.ConclusionOverall, these findings support a systemic alteration in circulating DAMP abundance in DFS and provide exploratory clinical and experimental evidence to guide future investigations into DAMP-mediated inflammatory pathways in diabetic ischemic complications. Proteomics data are available via ProteomeXchange with identifier PXD073507.

MiR-210 is widely recognized as the quintessential hypoxia-responsive miRNA and is thought to fine-tune various facets of cellular homeostasis. We hereby present an integrative appraisal of the phenotypic and molecular repercussions of disrupting the corresponding locus in human and mouse cells using multiple genetic strategies. In brief, MIR210 deletion led to decreased cellular fitness and suboptimal responses to several stress types. Transcriptomic comparisons via different profiling platforms, performed independently by members of this collaboration, revealed consistent deregulation of neighboring genes, in locus-disrupted cells. Interestingly, the anticipated enrichment of miR-210 targets failed to materialize in unbiased analyses. Our results point to the biological significance of unrecognized regulatory elements that overlap miRNA genes and should serve as a note of caution for studies based on the genetic disruption of such loci.

Despite the recognized importance of neutrophils in cardiac repair following myocardial infarction (MI), their interaction with mesenchymal stromal cells (MSCs), particularly regarding polarization phenotypes and functional impacts, remains unclear. Here, we investigated these interactions across controlled in vitro systems and an in vivo MI model. Human HL-60 cells were differentiated into neutrophil-like cells (dHL-60) and polarized toward N1/N2 states to test the MSC paracrine effects using indirect transwell coculture. Readouts included gene expression, cytokine profiling and functional assays. To increase translational relevance, indirect ex vivo cocultures of human MSCs with primary neutrophils isolated from MI mice or from MI patients were analysed for gene expression and cytokine profiles in conditioned media. In vivo, syngeneic mouse MSCs were transplanted subcutaneously immediately after MI, and early cardiac function was evaluated by echocardiography. Cardiac neutrophils where quantified by flow cytometry, and Ly6G+ neutrophils from infarcted hearts and peripheral blood were purified by MACS for bulk RNA-seq with targeted RT-qPCR validation. In vitro, MSCs suppressed pro-inflammatory mediators in N1-like neutrophils and enhanced reparative factors in N2-like cells. In vivo, remote MSC transplantation improved early cardiac performance, and reduced neutrophil accumulation in the infarct. Paradoxically, cardiac neutrophils showed transcriptomic enrichment of inflammatory pathways, whereas blood neutrophils showed reduced interferon-related programs. Our findings indicate that MSCs can modulate neutrophil responses, underscoring the nuanced effects of MSC-based approaches in ischemic heart disease. These results suggest that anti-inflammatory effects observed under controlled conditions may not fully translate in vivo, highlighting the importance of context when evaluating MSC-based therapies.

BACKGROUND Calcific aortic valve disease (CAVD) is a common malady with few treatment options other than valve replacement by surgery or transcatheter aortic valve implantation (TAVI). Endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells and osteogenic differentiation of valvular interstitial cells are crucial processes of CAVD. Smad3 and Runx2 are key transcription factors (TFs) that drive these processes by regulating gene expression and cellular functions. We hypothesize that downregulation of these TFs with nanoparticle-mediated RNA interference could mitigate aortic valve stenosis and calcification. METHODS We engineered dual-targeted lipid-polymer hybrid nanocarriers (lipopolyplexes, LPP) to deliver short-hairpin RNA (shRNA) for gene silencing in pathologically remodeled aortic valve. The nanocarriers simultaneously target vascular cell adhesion molecule-1 (VCAM-1) and collagen IV, enhancing specificity toward inflamed and fibrotic valvular tissue. Encapsulated shRNA constructs were designed to silence either Smad3 or Runx2 (yielding formulations V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2). Therapeutic efficacy was evaluated in a mouse model of atherosclerosis aggravated by diabetes, mimicking the pathological environment of CAVD. RESULTS The dual-targeted lipopolyplexes effectively facilitated gene delivery to the aortic valve, ensuring efficient transfection. Treatment with V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2 resulted in marked silencing of Smad3 and Runx2, accompanied by significant suppression of osteogenic markers, including osteopontin, alkaline phosphatase, and osteocalcin, as well as reduced αSMA expression in valve tissue. Our data further identify Runx2 as a novel upstream modulator of Smad3 expression, unveiling a previously unrecognized Runx2-Smad3 regulatory axis with important implications for valvular pathology and targeted therapy. Beyond localized effects, systemic administration of these lipopolyplexes led to reduced plasma concentrations of alkaline phosphatase, cholesterol, and triglycerides, while maintaining hepatic and renal function, suggesting additional benefits on systemic metabolic homeostasis. CONCLUSIONS These findings highlight the pivotal role of Smad3 and Runx2 downregulation in mitigating aortic valve calcification, unveiling both molecules as compelling therapeutic targets in CAVD. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Inflammatory pathways, particularly those involving vascular cell adhesion molecule-1 (VCAM-1), play a central role in post-ischemic myocardial remodeling. However, its relationship with left ventricle (LV) performance or volumetric changes has not been systematically examined. This study aimed to investigate the association between circulating VCAM-1 levels, LV volumes, and LV ejection fraction (LVEF), quantified using three-dimensional echocardiography (3-DE), in patients with acute coronary syndrome (ACS). All patients underwent comprehensive clinical evaluation and 3-DE assessment within the first 24 hours of hospital admission. Concurrently, a full panel of locally available laboratory tests was performed, including serum sampling for VCAM-1 analysis. A follow-up evaluation, comprising repeated biological and echocardiographic measurements, was conducted two months after the index event. A total of 90 patients with ACS (mean age 54 ± 9 years; 75 males) were included in the analysis. Among these, 30 patients (33.3%) had a ≥10% increase in left ventricular end-diastolic volume (LVEDV) at follow-up, indicative of adverse left ventricular remodeling. Baseline VCAM-1 levels were significantly correlated with subsequent changes in LVEDV and LVEF from admission to follow-up (r = -0.42, P < 0.05, and r = -0.43, P < 0.05, respectively). Furthermore, the dynamic changes in VCAM-1 between assessments also showed significant correlations with changes in LVEDV and LVEF (r = 0.41, P < 0.05; r = -0.46, P < 0.05). This study suggests that VCAM-1, an inflammatory biomarker, may be a prognostic indicator of LV remodeling and dysfunction in patients with acute coronary syndromes. The findings support further exploration of VCAM-1 for risk stratification and therapy.

IntroductionMacrophages play fundamental roles in immune regulation and tissue homeostasis, serving as one of the primary cell types that orchestrate tissue repair after injury. MiR-210 is a hypoxia-inducible, small non-coding RNA involved in regulating metabolic adaptation and inflammatory responses during normal repair processes. However, its role in macrophage polarization is not fully understood. Here, we report the impact of miR-210 deletion on macrophage polarization towards a pro-reparatory phenotype.MethodsBone marrow-derived macrophages were obtained from miR-210 knockout (KO) and wild-type (WT) mice and polarized toward the pro-reparative M2 phenotype. The transcriptomic profile of these cells, as well as their phagocytic capacity, cell energy phenotype, and cytokine production were assessed to determine the impact of miR-210 on the macrophage polarization process into a M2-like phenotype.ResultsCompared with their WT counterparts, miR-210 KO M0 macrophages presented a reduced glycolytic activity and a diminished metabolic flexibility. However, miR-210 KO cells exhibited increased phagocytosis in both M0 and M2 states, potentially as an adaptive response to their metabolic limitations. Transcriptomic analysis revealed distinct clustering between the M0 and M2 states, along with several notable differences in the transcriptional patterns between the two genotypes. Analysis of differentially expressed genes indicated an increased pro-inflammatory state in resting miR-210 KO macrophages compared to WT control cells. These data were further confirmed by the higher levels of IL-6, TNF-α, and IL-1b secreted by miR-210 KO M0 macrophages compared to WT cells. Analysis of the biological processes activated during the polarization process towards the M2 phenotype revealed an incomplete polarization of miR-210 KO cells, which may be attributed, at least in part, to reduced activation of mitotic regulators, leading to slower cell cycle progression and diminished proliferation.DiscussionOur data offers new insights into the role of miR-210 in promoting a macrophage shift toward the anti-inflammatory, pro-reparative M2 phenotype. The fine-tuned involvement of miR-210 in immune responses may have potential implications for chronic inflammation, immune dysfunction, and tissue repair.

Recent studies have shown that chronic inflammation in atherosclerotic (ATH) lesions is due to an inability to resolve the inflammatory response. We evaluated the therapeutic potential of specialized pro-resolving mediators (SPMs) incorporated into biomimetic lipid nanoemulsions covered with macrophage membranes (Bio-LN/SPMs) to enhance their stability, targeting, and bioactivity in resolving atherosclerotic plaque inflammation. We utilized both in vitro and in vivo experimental models to test this hypothesis. In vitro, we found that Bio-LN/SPMs significantly reduced the inflammatory markers VCAM-1, MCP-1 in TNF-α-activated endothelial and smooth muscle cells, and iNOS, and NLRP3 in LPS-activated macrophages. In contrast, free SPMs exhibited a more modest effect. In vivo, the i.v. administration of Bio-LN/SPMs in ApoE-deficient mice with progressive atherosclerotic lesions developed after administration for 4 and 8 weeks of a high-fat diet, reduced plasma triglycerides, improved renal function, and decreased plasma proteins associated with complement activation and inflammation (i.e. C4d, C5b-9, IL-6, and MCP-1) to a greater extent than other treatment groups. Bio-LN/SPMs also affected circulated monocyte subpopulations by increasing the percentage of anti-inflammatory Ly6Clow monocytes and reducing that of pro-inflammatory Ly6Chigh monocytes. Additionally, they promoted the transition of macrophages in atherosclerotic plaques to a reparative M2 phenotype. They decreased the production of TNF-α, IL-1β, and IL-6 cytokines, along with lipid deposits in the aorta of ApoE-deficient mice. These findings demonstrate the improved therapeutic efficacy of Bio-LN/SPMs compared to unincorporated SPMs and standard nanoemulsions (LN/SPMs), emphasizing their potential as a novel approach for treating atherosclerosis and other inflammatory diseases.

Oxidative stress and alterations in redox signalling have been implicated in the pathophysiology of myocardial infarction (MI). NADPH oxidase (Nox) is an important source of reactive oxygen species (ROS) in the infarcted myocardium. Alarmin S100A8/A9 amplifies acute myocardial inflammation in MI and has been shown to be a promising therapeutic target to improve cardiac function post-MI. We aimed to elucidate the underlying mechanisms linking S100A8/A9, oxidative stress and the inflammatory response in MI. MI was induced by permanent left coronary artery ligation in C57BL/6J mice, followed by treatment with the S100A8/A9 inhibitor ABR-238901 (30 mg/kg) or PBS for 3 days. The in-vivo experiments were complemented with mechanistic studies on cultured macrophages (Mac), important cellular effectors in MI. Compared to sham-operated animals, we detected significant increases in the Nox1, Nox2, Nox4 catalytic subunits at mRNA and protein levels, and NADPH-dependent ROS production in the left ventricle of MI mice. S100A8/A9 blockade prevented the up-regulation of Nox1/2/4 expression, reduced ROS formation, suppressed NF-kB activation and prevented NLRP3 inflammasome priming and activation, leading to reduced levels of active IL-1β. In-vitro, S100A8/A9 induced gene expression of Nox catalytic subtypes and NLRP3 in Mac in a TLR4-dependent and dose-dependent manner. These effects were counteracted by pharmacological inhibition of S100A8/9, TLR4, Nox1/4 and Nox2. In conclusion, we show that Nox upregulation and ROS formation triggered by S100A8/A9 contributes to NLRP3 inflammasome priming and increased IL-1β production in the infarcted myocardium. These mechanisms can be therapeutically targeted to prevent inflammatory and oxidant myocardial damage in acute MI.

Dysregulation of histone methylation-based epigenetic mechanisms leads to either transient or long-lasting transcriptomic alterations in vascular and immune cells with important consequences on atherosclerotic plaque development and stability. We hypothesized that the epigenetic enzyme SET7 lysine methyltransferase contributes to the up-regulation of NADPH oxidase (Nox) and NLRP3 inflammasome expression in atherosclerosis. To test this hypothesis, we examined human non-atherosclerotic and atherosclerotic tissue samples, apolipoprotein E-deficient (ApoE-/-) mice, and human macrophages (Mac) employing real-time PCR, Western blot, immunofluorescence microscopy, and histological techniques. Male ApoE-/- mice with established atherosclerosis were randomized to receive concomitant with the high-fat diet, 5 mg/kg (R)-PFI-2, a selective SET7 pharmacological inhibitor, or its vehicle, every other day for 4 weeks. The results revealed that SET7 mRNA and protein, and H3K4me1 levels were significantly elevated in human carotid atherosclerotic lesions, aorta of atherosclerotic mice, and in cultured pro-inflammatory Mac. In the atherosclerotic mice, pharmacological blockade of SET7 catalytic activity with the specific inhibitor, significantly reduced atherosclerotic plaque development, decreased the aortic up-regulation of mRNA and protein levels of Nox catalytic subunits, mitigated the formation of NT-/4HNE-protein adducts, attenuated NLRP3 gene and protein expression, and reduced pro-caspase-1 and pro-IL18 cleavage. In polarized pro-inflammatory human M1-Mac, SET7-oriented pharmacological intervention reduced the transcriptional up-regulation of Nox catalytic subunits, NLRP3, caspase-1, IL1β, and IL18, and the secretion IL1β and TNFα. Transient overexpression of SET7 in human endothelial cells enhanced mRNA levels of Nox1, Nox2, Nox4, Nox5, and p22phox. The novel results show that SET7 regulates important mechanisms leading to enhanced formation of reactive oxygen species and pro-inflammatory cytokines release in atherosclerosis. The data recommend SET7 as a promising target for pharmacological interventions and as supportive therapeutic strategy in atherosclerotic cardiovascular diseases.

Chronic wounds represent a major therapeutic challenge, with limited effective treatment options currently available. Both cellular and acellular approaches are being explored to address this issue, with mesenchymal stromal cells (MSCs) emerging as a promising option. While these cells have been extensively studied, alternative stromal cell sources, such as fibroblasts (Fbs), may also possess comparable therapeutic potential. Thus, this review focuses on stromal cell-derived secretomes (conditioned medium) as a source of acellular therapy for chronic wounds and presents the available wound-healing models (in vitro, ex vivo, and in vivo) suitable for evaluating their therapeutic efficacy, prior to clinical application. By conducting an analysis of the existing studies, we present the impact of the cell culture conditions on the enhancement in the bioactivity of the MSC/Fb-derived conditioned medium, a research area that continues to evolve.

BACKGROUND:Left ventricular (LV) remodeling plays a pivotal role in the pathophysiology of heart failure (HF) following acute coronary syndrome (ACS). Left ventricular ejection fraction (LVEF), left ventricular (LV) volumes and, more recently, speckle tracking echocardiography (STE) are used to describe LV performance. Myocardial work (MW) is a new noninvasive imaging method that integrates loading conditions and can be used to detect early myocardial dysfunction before LVEF decreases. AIM:This study aims to characterize the relationship between MW, high-sensitivity troponin (hs-cTn I) and C-reactive protein (CRP) as an inflammation biomarker. Their use as predictors for LV dysfunction in the acute phase of ACS is of particular interest but is still under debate. METHOD:Complete clinical examination and two-dimensional echocardiography (2-DE), with speckle-tracking and myocardial work measurements, were performed in the first 24 hours after admission. Locally available biomarkers were assessed in the same timeframe, with special interest in hs-cTn I and CRP, as a marker for inflammation. A follow-up visit, including the same clinical, biological, and echocardiographic measurements, was performed six to eight weeks after the index event. RESULTS:We evaluated 56 patients (53 ± 10 years, 45 men) with ACS. Baseline hs-cTn I significantly correlated with baseline global longitudinal strain (GLS) (r=0.43, p=0.001) and baseline MW parameters (GWI: r=-0.44, p=0.001; GCW: r=-0.40, p=0.002). A correlation between hs-cTn I and LVEF was not statistically relevant. C-reactive protein, which was used to assess systemic inflammation, also failed to correlate with LVEF. However, CRP significantly correlated with relevant MW parameters (GWE: r=-0.53, p<0.001 and GWW: r=0.48, p<0.001). C-reactive protein levels above 28 mg/L correlated with a decrease in MW performance assessed by GWE, suggesting a possible tendency to adverse remodeling. CONCLUSIONS:C-reactive protein level in the first 24 hours after ACS and its correlation with MW parameters may be a potential indicator of future LV dysfunction and heart failure.

INTRODUCTION:The infarcted heart is energetically compromised exhibiting a deficient production of adenosine triphosphate (ATP) and the ensuing impaired contractile function. Short-term blockade of the protein S100A9 improves cardiac performance in mice after myocardial infarction (MI). The implications upon ATP production during this process are not known. OBJECTIVES:This study evaluates whether S100A9 blockade effects ATP synthesis and cardiac contractility in C57BL/6 mice at seven days post-MI. METHODS:Three experimental groups were used: (i) mice with MI, induced by permanent left coronary ligation, (ii) mice with MI, short-term treated with the S100A9 blocker ABR-238901, and (iii) sham (control) mice. After removing the left ventricle, mass spectrometry, pathway enrichment analysis, Western blot, RT-PCR and pharmacological network analysis were performed. RESULTS:A number of 600 differentially abundant proteins (DAPs) was significantly altered by the S100A9 blocker in MI-treated mice compared with MI mice. Some of these proteins were associated with oxidative phosphorylation, citrate cycle (TCA), mitochondrial fatty acid beta-oxidation, glycolysis and cardiac muscle contraction pathways. In the ischemic ventricle, ABR-238901 treatment increased (1.8- to 38-fold) the abundance of proteins NDUFAB1, UQCRC1, HADHA, ACAA2, ALDOA, PKM1, DLD, DLAT, PDHX, ACO2, IDH3A, FH1, CKM, CKMT2, TNNC1, crucial for early cellular metabolic changes, ATP distribution and contractility. The cardiac level of ATP increased (1.8-fold, p < 0.05) in MI mice treated with ABR-238901 compared to MI mice. The network pharmacology analysis uncovered potential pharmacologic targets of ABR-238901 that may interact with DAPs related to ATP production and contractility. CONCLUSION:Short-term S100A9 blockade effectively regulates the proteins implicated in ATP production and cardiac contractility post-MI, providing a framework for future cardiac energy metabolism studies.

MicroRNAs, involved in a large variety of pathological conditions, tend to be potential specific biomarkers in cardiovascular diseases. Moreover, these short, non-coding RNAs, regulate post-transcriptional gene expression and protein synthesis, making them ideal for therapeutic targets. Down-regulation and upregulation of specific microRNAs are currently studied as a novel approach to the diagnosis and treatment of cardiovascular diseases, such as chronic and acute coronary syndromes, atherosclerosis, heart failure, and arrhythmia. MicroRNAs are interesting and attractive targets for cardiovascular-associated therapeutics because of their stability, tissue-specific expression pattern, and secretion of body fluids. Extended research on their isolation, detection, and function will provide the standardization needed for using microRNAs as biomarkers and potential therapeutic targets. This review will summarize recent data on the implication of microRNAs in cardiovascular diseases, their potential role as biomarkers for diagnosis, and also the challenges of using microRNAs as future therapeutic targets.

Recent studies reported that circulating microRNAs (miRNAs) can target different metalloproteases (MMPs) involved in matrix remodeling and plaque vulnerability. Consequently, they might have a role in the diagnosis and prognosis of coronary artery disease. To quantify circulating miRNAs (miRNA126, miRNA146, and miRNA21) suggested to have possible cardiovascular implications, as well as levels of MMP-1 and MMP-9, and to determine their association with left ventricular (LV) function and with arterial function, in patients with either ST-segment elevation acute myocardial infarction (STEMI) or stable ischemic heart disease (SIHD). A total of 90 patients with coronary artery disease (61% men, 58 ± 12 years), including 60 patients with STEMI and 30 patients with SIHD, were assessed within 24 h of admission, by measuring serum microRNAs, and serum MMP-1 and MMP-9. LV function was assessed by measuring ejection fraction (EF) by 2D and 3D echocardiography, and global longitudinal strain (GLS) by speckle tracking. Arterial function was assessed by echo tracking, CAVI, and peripheral Doppler. Circulating levels of miRNA146, miRNA21, and MMP1 were significantly increased in patients with STEMI vs. SIHD (p = 0.0001, p = 0.0001, p = 0.04, respectively). MiRNA126 negatively correlated with LVEF (r = −0.33, p = 0.01) and LV deformation parameters (r = −0.31, p = 0.03) in patients with STEMI and negatively correlated with ABI parameters (r = −0.39, p = 0.03, r = −0.40, p = 0.03, respectively) in patients with SIHD. MiRNA146 did not have any significant correlations, while higher values of miRNA21 were associated with lower values of GLS in STEMI patients and with higher values of GLS in SIHD patients. Both MMP1 and MMP9 correlated negatively with LVEF (r = −0.27, p = 0.04, r = −0.40, p = 0.001, respectively) and GLS in patients with STEMI, and positively with arterial stiffness in patients with SIHD (r = 0.40 and r = 0.32, respectively; both p < 0.05). MiRNA126, miRNA21, and both MMP1 and MMP9 are associated with LV and arterial function parameters in patients with acute coronary syndrome. Meanwhile, they inversely correlate with arterial function in patients with chronic atherosclerotic disease. However, further studies are needed to establish whether these novel biomarkers have diagnosis and prognosis significance.

Endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VEC) is a key process in the development and progression of calcific aortic valve disease (CAVD). High expression of the Smad3 transcription factor is crucial in the transition process. We hypothesize that silencing Smad3 could hinder EndMT and provide a novel treatment for CAVD. We aimed at developing nanoparticles encapsulating short-hairpin (sh)RNA sequences specific for Smad3 targeted to the aortic valve. We synthesized VCAM-1-targeted lipopolyplexes encapsulating shRNA-Smad3 plasmid (V-LPP/shSmad3) and investigated their potential to reduce the EndMT of human VEC. VEC incubation in a medium containing high glucose concentrations and osteogenic factors (HGOM) triggers EndMT and increased expression of Smad3. Exposed to lipopolyplexes, VEC took up efficiently the V-LPP/shSmad3. The latter reduced the EndMT process in VEC exposed to HGOM by downregulating the expression of αSMA and S100A4 mesenchymal markers and increasing the expression of the CD31 endothelial marker. In vivo, V-LPP/shSmad3 accumulated in the aortic root and aorta of a murine model of atherosclerosis complicated with diabetes, without affecting the liver and kidney function. The results suggest that targeting activated VEC with lipopolyplexes to silence Smad3 could be an effective, novel treatment for CAVD mediated by the EndMT process.

Acute-phase inhibition of the pro-inflammatory alarmin S100A8/A9 improves cardiac function post-myocardial infarction (MI), but the mechanisms underlying the long-term benefits of this short-term treatment remain to be elucidated. Here, we assessed the effects of S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 on myocardial neovascularization in mice with induced MI. The treatment significantly reduced S100A9 and increased neovascularization in the myocardium, assessed by CD31 staining. Proteomic analysis by mass-spectrometry showed strong myocardial upregulation of the pro-angiogenic proteins filamin A ( 10-fold) and reticulon 4 ( 5-fold), and downregulation of the anti-angiogenic proteins Ras homolog gene family member A (RhoA, 4.7-fold), neutrophilic granule protein (Ngp, 4.0-fold), and cathelicidin antimicrobial peptide (Camp, 4.4-fold) versus controls. In-vitro, ABR-238901 protected against apoptosis induced by recombinant human S100A8/A9 in human umbilical vein endothelial cells (HUVECs). In conclusion, S100A8/A9 blockade promotes post-MI myocardial neovascularization by favorably modulating pro-angiogenic proteins in the myocardium and by inhibiting endothelial cell apoptosis.

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-β, 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/β5) 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.

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.

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-1β (IL-1β), 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-1β, 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.