Myocardial infarction (MI) triggers complex pathological processes, including inflammation, hypoxia, and fibrotic remodeling. MicroRNAs (miRNAs) have emerged as promising biomarkers for cardiovascular injury; however, their expression dynamics along processes remain underexplored. We used an in vivo rat model of permanent coronary occlusion to study the molecular alterations associated with MI and its resolution in a temporal mode, including five experimental groups with five animals in each: sham, PO 24 h, PO 72 h, PO 7 d, PO 1 month. Histological analysis, serum biomarkers, and miRNA/gene expression profiles were analyzed in a time-dependent manner post-occlusion. Subsequent analysis revealed early depletion of selected circulating miRNAs (PO 24 h). Transient upregulation in cardiac tissue miRNAs, inflammatory and fibrotic gene expression (Fibronectin, Collagen, Vimentin, E-Cadherin) were observed at PO 72 h. These molecular alterations correlated with histological evidence of myocardial injury and repair. Taken together, our findings delineate the molecular timeline of MI progression and resolution and identify candidate miRNAs as sensitive and time-dependent indicators of myocardial stress, including miR-107, miR-122-5p and miR-221-3p. This integrative approach supports the use of miRNA signatures for noninvasive monitoring of cardiac injury and resolution and unveils potential therapeutic targets to reduce pathological remodeling.

BACKGROUND:Abnormal levels of VWF (von Willebrand Factor) are a risk factor for venous thromboembolism (VTE) and bleeding. Genome-wide association studies for VWF have identified novel candidate genes that may regulate VWF levels in humans, including RAB5C (RAS [rat sarcoma]-associated protein RAB5C). We hypothesized that RAB5C regulates VWF release from endothelial cells. METHODS:We studied the effect of RAB5C on vesicle trafficking in human endothelial cells. We performed CRISPR (clustered regularly interspaced short palindromic repeats) interference targeting 2 genetic variants linked to altered VWF levels and evaluated RAB5C expression by reverse transcription-quantitative polymerase chain reaction. We silenced RAB5C or overexpressed RAB5C wild-type, constitutive active or dominant negative; and then, we measured VWF exocytosis from human umbilical vein endothelial cells to the media by ELISA. We performed proximity labeling and mass spectrometry to identify intracellular signaling pathways mediating the effects of RAB5C on VWF exocytosis. RESULTS:We found that 2 genetic variants (rs9915255 and rs9912088 identified by genome-wide association studies for VWF levels) regulate RAB5C expression in stem cell-derived endothelial cells. We next silenced or overexpressed RAB5C in endothelial cells to assess its effect on VWF release. RAB5C silencing decreased VWF release after histamine stimulation, whereas overexpression of RAB5C or constitutively active RAB5C increased endothelial VWF release. To explore the intracellular signaling pathway mediating the effects of RAB5C on VWF exocytosis, we performed proximity labeling and mass spectrometry. We identified 147 proteins proximal to RAB5C, many of which are involved in vesicle trafficking. From this screen, we identified SNAP29 (synaptosome-associated protein 29), a SNARE (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor)-associated protein that plays a crucial role in vesicle fusion, as a key RAB5C interactor regulating VWF exocytosis. CONCLUSIONS:Taken together, our data demonstrate that RAB5C regulates VWF release in part through SNAP29 control of vesicle trafficking in endothelial cells. These findings validate genetic epidemiology data linking RAB5C to VWF levels in humans and provide new insights into the molecular mechanisms regulating VWF exocytosis.

BACKGROUND:Human amniotic membrane mesenchymal stem cells (hAMSCs) hold strong cardioprotective potential, yet their mechanisms of action remain largely elusive. METHODS AND RESULTS:C57BL/6 mice were subjected to cardiac ischemia/reperfusion (I/R) and received intravenous (IV) 2 × 105 hAMSCs at 2, 7 or 14 days post-reperfusion. Cardiac function and MIAT/miR-150/HOXA4 signalling were assessed. Mice treated 2 days post-I/R markedly improved LVEF and reduced myocardial necrosis and fibrosis by Day 21. Minimal hAMSC engraftment, evidenced by SSEA-4 immunostaining, suggests paracrine rather than direct cellular effects. Guided by GWAS implicating miRNAs in myocardial infarction, we identified miR-150 as a key effector, finding that hAMSC upregulated cardiac miR-150 and suppressed HOXA4, a profibrotic target in ischemic myocardium. In parallel, hAMSC treatment reduced cardiac MIAT, a lncRNA that sequesters miR-150, uncovering a mechanism of cardioprotection via MIAT downregulation post-reperfusion. Notably, CRISPR-Cas9 miR-150-silenced hAMSC exhibited severely impaired cardioprotective effects compared to wild-type cells, confirming the functional role of miR-150. miR-150 was identified as a key extracellular vesicle (EV) cargo released by hAMSC under hypoxic conditions, both in vitro and in hAMSC-injected I/R mice. Strickingly, administration of miR-150-enriched EVs to mice recapitulated the therapeutic benefits of hAMSC, underscoring miR-150-5p as central mediator of hAMSC-iduced cardioprotection. CONCLUSIONS:hAMSCs promote cardioprotection following I/R via the MIAT/miR-150-5p/HOXA4 axis, in which miR-150-5p plays a central role. These findings provide loss-of-function evidence about the therapeutic potential of hAMSC-derived EVs as a novel cell-free exosome-based strategy for the treatment of acute myocardial infarction.

BACKGROUND:Genome-wide association studies have identified multiple novel loci that contribute to coronary artery disease pathogenesis, but the mechanisms of these associations remain largely unknown. METHODS:In this study, we used a multitrait colocalization approach to prioritize novel endothelial-specific loci for atherosclerosis. We combined computational methods with in vitro assays and mouse models to study one of those new loci targeting the gene REST (RE1-silencing transcription factor). RESULTS:A multitrait colocalization approach across expression quantitative trait loci in atherosclerosis-relevant cell types, followed by in vitro CRISPR interference, revealed that a conserved regulatory element in a chromosome 4 genetic locus increases the risk of coronary artery disease and decreases the expression of REST, a transcriptional repressor, in endothelial cells. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared with littermate controls. RNA-seq in human aortic endothelial cells after REST silencing, followed by assessment of protein expression, revealed that REST silencing triggers endothelial-to-mesenchymal transition. Consistently, REST silencing increased endothelial permeability and migration in vitro. Single-nucleus RNA sequencing in endothelial lineage traced atherosclerotic mice with Rest knock-out revealed evidence of endothelial TGFb signaling activation and of transition smooth muscle-like cells in atherosclerotic aortas on genetic knockout of Rest. Cleavage under targets and tagmentation (CUT&Tag) sequencing did not identify any known TGFb (transforming growth factor-beta) effector genes as direct REST transcriptional targets. Instead, joint analysis of CUT&Tag with RNA-seq highlighted L1CAM (L1 cell adhesion molecule), a known endothelial-to-mesenchymal transition activator, and its interactors as the most significant gene set directly affected by REST in the endothelium. Simultaneous silencing of L1CAM and REST in human aortic endothelial cells inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing and diminished the upregulation of several TGFb effectors overexpressed on REST silencing. CONCLUSIONS:In summary, our data reveal the novel role of REST as a repressor in endothelial cells that functions to constitutively inhibit endothelial-to-mesenchymal transition and protect against atherosclerosis.

Introduction: Human amniotic mesenchymal stem cells (hAMSC) possess substantial therapeutic potential in hepatic, oncological, and degenerative disorders; however, their role in cardiovascular diseases in the context of acute myocardial infarction still remains unexplored. Methods and results: We investigated the contribution of hAMSC in a murine model of myocardial ischemia/reperfusion (I/R). C57BL/6 mice were subjected to 40 minutes transient ligation of the left anterior descending coronary artery, followed by intravenous administration of 2x10^5 hAMSC at 2, 7, or 14 days post-I/R. Cardiac function and molecular signaling involving the MIAT/miR-150/Hoxa4 axis were evaluated. Mice receiving hAMSC two days after I/R exhibited significant cardioprotection, evidenced by an improved left ventricular ejection fraction (LVEF) at 21 days post-injection (64.5 ± 2.7 vs. 50.0 ± 3.6), and a marked reduction in myocardial necrosis and fibrosis. Immunohistochemical analysis of cardiac tissue using a human-specific anti-hAMSC antibody, SSEA-4, revealed minimal cellular engraftment, limiting the assessment of direct cell-mediated effects. Interestingly, hAMSC-treated hearts showed elevated levels of miR-150 and concomitant downregulation of Hoxa4, consistent with its cardioprotective role. This effect is likely mediated by suppression of MIAT effect, a lncRNA known to upregulate Hoxa4 by sponging miR-150. To assess the functional role of miR-150, we employed CRISPR-Cas9 to generate miR-150-deficient hAMSC. Notably, these modified cells lost their cardioprotective effects when administered to I/R-injured mice (56.45 ± 4.94 vs 44 ± 5.29). Further analysis revealed that miR-150 is present in extracellular vesicles (EVs) secreted by hAMSC in response to I/R stimuli. Administration of purified EVs reproduced the cardioprotective effects observed with intact hAMSC (57.33 ± 2.51 vs 49.66 ± 3.50), underscoring a paracrine mechanism mediated by the miR-150 cargo. Following the above, engineered lipid nanoparticles containing miR150 also exerted the same levels of protection (55 ± 2 vs 44.58 ± 3.73). Conclusions: hAMSC promote cardiac repair following acute myocardial infarction through mechanisms involving the MIAT/miR-150/Hoxa4 axis. These findings highlight the therapeutic relevance of hAMSC-derived EVs and suggest that miR-150-based interventions, such as exosome therapy or nanoparticle delivery, may serve as effective alternatives to conventional cell-based treatments.

Introduction: Tricuspid regurgitation (TR) contributes to severe cardiovascular complications by causing atrial and ventricular dilation, retrograde blood flow, and abnormal cardiac remodeling, leading to electrical disturbances and disease progression. The lack of reliable preclinical models limits our understanding of TR pathophysiology and hinders early biomarkers for timely intervention of disease. Research Questions: Our study aims to establish a new reproducible, minimally-invasive, and valve-harmless porcine model to mimic TR, enabling the investigation of disease progression and the identification of novel molecular signatures underlying this condition. Methods: TR was induced by catheter-based placement of an inferior vena cava filter to prevent tricuspid leaflet coaptation between right chambers. Hemodynamic, echocardiographic and electrophysiological assessments, jet flow, heart rate, and chamber dimensions, were weekly assessed over 30- and 60-days post implantation. Results: Following device implantation, TR led to significant increases in heart rate, right chamber dilation, and arrhythmogenic events. Early manifestations included sinus tachycardia and multifocal atrial tachycardia, progressing to brief episodes of paroxysmal atrial fibrillation. Histological analysis also revealed cellular hypertrophy and fibrosis in both the right atrium (RA) and sinoatrial node (SAN), accompanied by upregulation of the TGF-β/Smad2/3, and -4 signaling axis, along with increased MMP2, and MMP9, suggesting its involvement in disease progression. To investigate in further detail, progressive downregulation and spatial redistribution of the pacemaker HCN4 channel were observed over time (Figure 1), accompanied by increased phosphorylation of Gap junction Connexins -43, and -45, along with elevated CaMKII and PKA levels, while SERCA2A and PLN levels remained unchanged. These findings addresses for the first time, how TR signaling disrupts cardiac conduction velocity and pacemaker function through dual pathways: (1) impairing SAN electrophysiology via HCN4 dysregulation, and (2) creating electrical uncoupling through connexin remodeling mediated by CaMKII/PKA-dependent phosphorylation. Conclusions: We successfully established a percutaneous porcine model of TR that recapitulates human disease pathophysiology. This experimental breakthrough bridges the gap between mechanistic and phenotyping manifestations, accelerating therapeutic discovery for TR-related cardiac complications.

Endothelial senescence (ES) contributes to aging-related disorders and triggers a senescence-associated secretory-pattern (SASP), releasing Extracellular Vesicles (EVs), potentially impacting atherosclerosis. We used EVs from young (8 weeks) and aged (24 months) ApoE-knockout mice to detect ES in human aortic (HAEC) and coronary (CAEC) endothelial cells. Age-related atherosclerosis was confirmed by increased atheroma plaque formation in aged compared to young ApoE-knockout mice fed a high-fat diet, and the contribution of EVs from aged ApoE-knockout mice on ES was evidenced by a replicative senescence assay in cultured HAEC and CAEC, starting with the promotion of ES. A proteomic analysis depicted the recently PCSK9-associated CAP1 protein as a cargo component in EVs from aged animals and highly expressed in mouse and human endarterectomy plaques. Gene silencing of CAP1 inhibited HAEC and CAEC ES while overexpressing CAP1 in these cells restored the senescent-phenotype. The in vivo contribution of CAP1 was assessed by injecting CAP1-containing EVs isolated from aged ApoE-knockout mice into wild-type (WT) mice fed either a regular or high-fat diet. Compared to the EVs from young mice, the CAP1-containing EVs led to a pronounced ES along with the formation of intraluminal atheroma plaques. Similarly, young ApoE-knockout mice developed thickened and calcified atheroma plaques, along with increased ß-Gal-positive aortic staining when injected with EVs isolated from aged ApoE-knockout mice, like the atheroma plaques observed in aged ApoE-knockout animals. In conclusion, early molecular targets of ES may contribute to better management of atherosclerosis, in which here we unveiled CAP1 as a new molecular target.

Vascular aging is associated with the development of cardiovascular complications, in which endothelial cell senescence (ES) may play a critical role. Nitric oxide (NO) prevents human ES through inhibition of oxidative stress, and inflammatory signaling by mechanisms yet to be elucidated. Endothelial cells undergo an irreversible growth arrest and alter their functional state after a finite number of divisions, a phenomenon called replicative senescence. We assessed the contribution of NO during replicative senescence of human aortic (HAEC) and coronary (CAEC) endothelial cells, in which accumulation of the senescence marker SA-β-Gal was quantified by β-galactosidase staining on cultured cells. We found a negative correlation in passaged cell cultures from P0 to P12, between a reduction in NO production with increased ES and the formation of reactive oxygen (ROS) and nitrogen (ONOO−) species, indicative of oxidative and nitrosative stress. The effect of ES was evidenced by reduced expression of endothelial Nitric Oxide Synthase (eNOS), Interleukin Linked Kinase (ILK), and Heat shock protein 90 (Hsp90), alongside a significant increase in the BH2/BH4 ratio, inducing the uncoupling of eNOS, favoring the production of superoxide and peroxynitrite species, and fostering an inflammatory environment, as confirmed by the levels of Cyclophilin A (CypA) and its receptor Extracellular Matrix Metalloprotease Inducer (EMMPRIN). NO prevents ES by preventing the uncoupling of eNOS, in which oxidation of BH4, which plays a key role in eNOS producing NO, may play a critical role in launching the release of free radical species, triggering an aging-related inflammatory response.

Introduction: Abnormal levels of the coagulation factor VIII (FVIII) are a risk factor for venous thromboembolism (VTE). Genome-wide association studies have identified novel candidate gene associations that may regulate FVIII levels in humans, including CD36. Hypothesis: We hypothesized that CD36 regulates FVIII release from endothelial cells. Methods and Results: We identified a subset of human liver endothelial cells (HLEC) expressing FVIII and purified them using a differentially expressed cell surface protein CD32. CD32+ HLEC expressed FVIII, transported FVIII in intracellular vesicles, and secreted FVIII. We stimulated CD36 signaling or silenced CD36 to test the effect of CD36 on FVIII release. Oxidized LDL (oxLDL), a CD36 agonist, increased endothelial release of FVIII. Conversely, silencing CD36 decreased FVIII release. We searched for an intracellular signaling pathway that mediates CD36 stimulation of FVIII secretion and found that oxLDL increased p38 activity and a p38 inhibitor decreased FVIII release. Conclusions: Taken together, our data show that CD36 stimulates FVIII release through a p38 dependent pathway in specialized liver endothelial cells. These results validate genetic epidemiology data linking CD36 to FVIII levels in humans and provide new insights into the regulation of FVIII levels.

Background: Endothelial nitric oxide synthase (NOS3) elicits atheroprotection by preventing extracellular matrix (ECM) proteolytic degradation through inhibition of extracellular matrix metalloproteinase inducer (EMMPRIN) and collagenase MMP-13 by still unknown mechanisms. Methods: C57BL/6 mice lacking ApoE, NOS3, and/or MMP13 were fed with a high-fat diet for 6 weeks. Entire aortas were extracted and frozen to analyze protein and nucleic acid expression. Atherosclerotic plaques were detected by ultrasound imaging, Oil Red O (ORO) staining, and Western Blot. RNA-seq and RT-qPCR were performed to evaluate EMMPRIN, MMP-9, and EMMPRIN-targeting miRNAs. Mouse aortic endothelial cells (MAEC) were incubated to assess the role of active MMP-13 over MMP-9. One-way ANOVA or Kruskal-Wallis tests were performed to determine statistical differences. Results: Lack of NOS3 in ApoE null mice fed with a high-fat diet increased severe plaque accumulation, vessel wall widening, and high mortality, along with EMMPRIN-induced expression by upregulation of miRNAs 46a-5p and 486-5p. However, knocking out MMP-13 in ApoE/NOS3-deficient mice was sufficient to prevent mortality (66.6 vs. 26.6%), plaque progression (23.1 vs. 8.8%), and MMP-9 expression, as confirmed in murine aortic endothelial cell (MAEC) cultures, in which MMP-9 was upregulated by incubation with active recombinant MMP-13, suggesting MMP-9 as a new target of MMP-13 in atherosclerosis. Conclusion: We describe a novel mechanism by which the absence of NOS3 may worsen atherosclerosis through EMMPRIN-induced ECM proteolytic degradation by targeting the expression of miRNAs 146a-5p and 485-5p. Focusing on NOS3 regulation of ECM degradation could be a promising approach in the management of atherosclerosis.

Introduction: Genome-wide association studies (GWAS) have identified multiple novel loci that contribute to CAD pathogenesis, but the mechanisms of these associations remain largely unknown. Research Question: This study aims to functionally characterize the association between the chromosomal GWAS locus for 4q12 and CAD. Methods: We combined computational methods with in vitro assays and mouse models to study REST. Results: A meta-analysis of expression quantitative trait locus (eQTL) data and multi-trait colocalization across atherosclerosis-relevant cell types revealed that the CAD-associated 4q12 locus regulates the expression of REST, a transcriptional repressor, in human aortic endothelial cells (HAEC). This analysis identified two enhancer variants as candidate causal variants for the GWAS association. CRISPR interference confirmed that the rs6853156 variant regulates REST expression in inducible human endothelial cells. Pathway analysis in HAEC after REST silencing indicated that epithelial-to-mesenchymal transition was the most upregulated pathway. Endothelial-to-mesenchymal transition (endMT) was validated in HAEC after REST silencing, as evidenced by rt-qPCR, western blot, and immunofluorescence. Consistently, REST silencing increased endothelial permeability and migration in vitro. CUT&Tag sequencing, jointly analyzed with RNA-sequencing, highlighted L1CAM and its interactors as the most significant gene-set directly affected by REST in the endothelium. L1CAM, a known endMT activator, was expressed in HAEC only upon REST silencing and direct binding of REST to its promoter was confirmed. Simultaneous silencing of L1CAM and REST in HAEC inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared to littermate controls. Conclusion: In summary, our data reveal the novel role of REST in atherosclerosis as a repressor that functions to constitutively inhibit endMT and protect against atherosclerosis.

Introduction: Adverse cardiac remodeling following acute myocardial infarction (AMI) depends on how promptly the phagocytic-nuclear system responds. Among strategies to limit the inflammatory response, we have developed a nanoparticle (NIL10) which binds to IL-10 receptor (IL-10R) in-vitro and promotes macrophage polarization towards a resolutive phenotype in animal models of myocardial ischemia-reperfusion (I/R). However, its therapeutic use still depends on understanding its ability to regulate monocyte recruitment. Hypothesis: Targeting the IL-10R in circulating monocytes may represent a new therapeutic approach to improve cardiac function in patients undergoing AMI. Objective: To explore the molecular mechanisms leading to the inhibition of monocyte recruitment by NIL10 in pigs undergoing cardiac I/R. Methods: By using a porcine model of myocardial I/R, we arranged the following groups: (1) control group (2) intravenous administration (IV) 1 mg/kg NIL10, (3) IV of 4x10 5 isolated monocytes from group 2, (4) IV of 4x10 5 isolated monocytes from group 1 and then incubated with NIL10 (A). Results: Groups 2-4 exhibited better cardiac function compared to group 1 (B), which resulted in a decrease in myocardial fibrosis (C), along with a reduction of circulating CCR2+ monocytes (D) and a decrease in classical-monocyte infiltration, as shown by a reduction of CCR2+ macrophages in the necrotic area of the heart (E). As a result, CCL2 plasma expression, a functional ligand of CCR2, was decreased by day 7 post-I/R (F) in contrast to the necrotic tissue expression profile in which we detected large amounts of CCL2 (G) and STAT3 activation, indicative of IL10R activity (H). Conclusion: In addition to macrophage polarization, NIL10 induces cardiac protection by limiting classical monocyte infiltration after myocardial I/R, which opens a new window for using, in combination with endogenous monocyte therapy, to treat AMI.

Introduction: Despite the emergence of new lipid-lowering therapies, coronary artery disease (CAD) remains the leading cause of death worldwide. Genome-wide association studies (GWAS) for CAD have discovered multiple novel loci that contribute to CAD pathogenesis. However, limitations in the interpretability of those GWAS associations have hindered the clinical translation of those findings. Hypothesis: In this study, we sought to evaluate the mechanisms of the association between chromosomal locus 4q12 and CAD, replicated in multiple independent GWAS for CAD. Methods: We utilized a combination of computational methods and in vitro assays to achieve our objectives. Results: Meta-analysis of public expression quantitative trait locus (eQTL) data and multi-trait colocalization across atherosclerosis-relevant cell types revealed that the CAD 4q12 locus regulates the expression of REST, a transcriptional repressor, in human aortic endothelial cells (HAEC) and prioritized 2 enhancer variants as candidate causal variants for the GWAS association. CRISPR interference confirmed that the rs6853156 variant regulates REST expression in inducible human endothelial cells. Pathway enrichment analysis in HAEC after REST silencing showed that epithelial to mesenchymal transition was the most upregulated pathway suggesting a potential mechanism via which silencing REST contributes to atherosclerosis. Endothelial to mesenchymal transition (endMT) was confirmed in HAEC after REST silencing at the RNA and protein level by rt-qPCR, western blot, and immunofluorescence. Concordantly, REST silencing increased endothelial permeability and matrix metalloproteinase activity in endothelial cell supernatants in vitro. L1CAM, a known endMT activator was only expressed in HAEC upon REST silencing. Chromatin immunoprecipitation sequencing supports direct binding of REST to the L1CAM promoter. Simultaneous silencing of L1CAM and REST in HAEC inhibited the observed upregulation of mesenchymal genes induced by REST silencing. Conclusions: In summary, our data reveal a novel role of REST as a repressor that functions as a constitutive inhibitor of endMT in human arterial endothelial cells.