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.

Toll-like receptor 4 (TLR4) contributes to the pathogenesis of coronary ischemia/reperfusion (IR). To test whether the new TLR4 antagonist, ApTOLL, may prevent coronary IR damage, we administered 0.078 mg/kg ApTOLL or Placebo in pigs subjected to IR, analyzing the levels of cardiac troponins, matrix metalloproteinases, pro-, and anti-inflammatory cytokines, heart function, and tissue integrity over a period of 7 days after IR. Our results show that ApTOLL reduced cardiac troponin-1 24 h after administration, improving heart function, as detected by a significant recovery of the left ventricle ejection fraction (LVEF) and the shortening fraction (FS) cardiac parameters. The extension of necrotic and fibrotic areas was also reduced, as detected by Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin/Eosine, and Masson Trichrome staining of heart sections, together with a significant reduction in the expression of the extracellular matrix-degrading, matrix metalloproteinase 9. Finally, the expression of the following cytokines, CCL1, CCL2, MIP1-A-B, CCL5, CD40L, C5/C5A, CXCL1, CXCL10, CXCL11, CXCL12, G-CSF, GM-CSF, ICAM-1, INF-g, IL1-a, ILI-b, IL-1Ra, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL13, IL16, IL17-A, IL17- E, IL18, IL21, IL27, IL32, MIF, SERPIN-E1, TNF-a, and TREM-1, were also assayed, detecting a pronounced decrease of pro-inflammatory cytokines after 7 days of treatment with ApTOLL. Altogether, our results show that ApTOLL is a promising new tool for the treatment of acute myocardial infarction (AMI).

Lack of endothelial nitric oxide causes endothelial dysfunction and circulating monocyte infiltration, contributing to systemic atheroma plaque formation in arterial territories. Among the different inflammatory products, macrophage-derived foam cells and smooth muscle cells synthesize matrix metalloproteinases (MMPs), playing a pivotal role in early plaque formation and enlargement. We found increased levels of MMP-9 and MMP-13 in human endarterectomies with advanced atherosclerosis, together with significant amounts of extracellular matrix (ECM) metalloproteinase inducer EMMPRIN. To test whether the absence of NO may aggravate atherosclerosis through EMMPRIN activation, double NOS3/apoE knockout (KO) mice expressed high levels of EMMPRIN in carotid plaques, suggesting that targeting extracellular matrix degradation may represent a new mechanism by which endothelial NO prevents atherosclerosis. Based on our previous experience, by using gadolinium-enriched paramagnetic fluorescence micellar nanoparticles conjugated with AP9 (NAP9), an EMMPRIN-specific binding peptide, magnetic resonance sequences allowed non-invasive visualization of carotid EMMPRIN in NOS3/apoE over apoE control mice, in which atheroma plaques were significantly reduced. Taken together, these results point to EMMPRIN as a new therapeutic target of NO-mediated protection against atherosclerosis, and NAP9 as a non-invasive molecular tool to target atherosclerosis.

(1) Background: Early response after acute myocardial infarction (AMI) prevents extensive cardiac necrosis, in which inflammation resolution, including expression of anti-inflammatory interleukin-10 (IL-10), may play a key role. (2) Methods: We synthesized NIL10, a micelle-based nanoparticle, to target IL-10 receptor in mice and pigs subjected to AMI. (3) Results: Administration of NIL10 induced cardiac protection of wild-type and IL-10 knockout mice and pigs subjected to AMI. Cardiac protection was not induced in IL-10-receptor null mice, as shown by a significant recovery of cardiac function, in which inflammatory foci and fibrosis were strongly reduced, together with the finding that resolving M2-like macrophage populations were increased after day 3 of reperfusion. In addition, anti-inflammatory cytokines, including IL-4, IL-7, IL-10, IL-13, IL-16, and IL-27 were also elevated. Mechanistically, NIL10 induced activation of the IL-10 receptor/STAT-3 signaling pathway, and STAT3-dependent inhibition of nuclear translocation of pro-inflammatory NF-ĸB transcription factor. (4) Conclusions: Taken together, we propose using NIL10 as a novel therapeutic tool against AMI-induced cardiac damage.

Background: Rapid screening and accurate diagnosis of acute myocardial infarction are critical to reduce the progression of myocardial necrosis, in which proteolytic degradation of myocardial extracellular matrix plays a major role. In previous studies, we found that targeting the extracellular matrix metalloprotease inducer (EMMPRIN) by injecting nanoparticles conjugated with the specific EMMPRIN-binding peptide AP9 significantly improved cardiac function in mice subjected to ischemia/reperfusion. Methods: In a porcine model of coronary ischemia/reperfusion, we tested the theragnostic effects of administering 0.1 mg/kg gadolinium-containing nanoparticles conjugated with AP9 (NAP9), a synthetic peptide that targets EMMPRIN or a control nanoparticle (NAPSC). Cardiac magnetic resonance assessment of the infarct progression, ventricular function, and nanoparticle distribution was performed the next 7 days. We also measured the infarcted area of the heart and cardiac remodeling at 7 or 21 days after ischemia/reperfusion. Results: After 21 days of ischemia/reperfusion, NAP9 reduced the extension of cardiac necrosis (14.1±9.7 versus 35.5±1.8) and the levels of collagenolytic activity of MMPs (matrix metalloproteases), along with a significant reduction in collagen deposition (7.5±4.5 versus 41.3±20); including the ratio of type I versus III collagen fibers in the necrotic myocardium. In terms of cardiac function, the response to NAP9 administration resulted in a significant improvement of cardiac performance overtime, as evidenced by the left ventricle ejection fraction (64.0±7.8), when compared with those present in the NAPSC group (47.3±4.7). As shown by magnetic resonance imaging, noninvasive molecular imaging of NAP9 enabled us to find a significant reduction in cardiac necrosis, myocardial edema, hemorrhage, and microvascular obstruction, suggesting that NAP9 may reduce myocardial injury and preserve left ventricular function, at least, by preventing the effect of EMMPRIN on extracellular matrix degradation. Conclusions: Our data point towards NAP9 as a promising theragnostic tool in managing acute myocardial infarction, by inhibiting EMMPRIN-induced extracellular matrix degradation and allowing noninvasive visualization of cardiac necrosis progression over time.

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.