NOS3 prevents MMP-9, and MMP-13 induced extracellular matrix proteolytic degradation through specific microRNA-targeted expression of extracellular matrix metalloproteinase inducer in hypertension-related atherosclerosis.

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.

New environmental factors related to diabetes risk in humans: Emerging bisphenols used in synthesis of plastics.

BACKGROUND: Diabetes mellitus (DM) is one of the largest global health emergencies of the 21st century. In recent years, its connection with environmental pollutants, such as bisphenol A (BPA), has been demonstrated; consequently, new structurally similar molecules are used to replace BPA in the plastics industry (BPS, BPF and BPAF). AIM: To carry out a systematic review to allow coherent evaluation of the state of the art. Subsequently, a meta-analysis was performed to unify the existing quantitative data. METHODS: Firstly, a systematic review was carried out, using the terms "(bisphenol) AND (Diabetes OR Hyperglycemia)", to maximize the number of results. Subsequently, three authors analyzed the set of articles. Finally, a meta-analysis was performed for each BP, using RevMan software. In addition, funnel plots were developed to study publication bias. RESULTS: The systematic analysis of the literature revealed 13 recent articles (2017-2023) related to the study paradigm. The qualitative analysis showed interesting data linking diabetes to the three most widely used substitute BPs in the industry: BPS, BPF and BPAF. Finally, the meta-analysis determined a positive relationship with BPS, BPF and BPAF, which was only statistically significant with BPS. CONCLUSION: There is a need to apply the precautionary principle, regulating the use of new BPs. Therefore, replacing BPA with BPS, BPF or BPAF is unlikely to protect the population from potential health risks, such as DM.

NIL10: A New IL10-Receptor Binding Nanoparticle That Induces Cardiac Protection in Mice and Pigs Subjected to Acute Myocardial Infarction through STAT3/NF-κB Activation.

(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.

Theranostic Contribution of Extracellular Matrix Metalloprotease Inducer-Paramagnetic Nanoparticles Against Acute Myocardial Infarction in a Pig Model of Coronary Ischemia-Reperfusion.

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.

Ivabradine Induces Cardiac Protection against Myocardial Infarction by Preventing Cyclophilin-A Secretion in Pigs under Coronary Ischemia/Reperfusion.

In response to cardiac ischemia/reperfusion, proteolysis mediated by extracellular matrix metalloproteinase inducer (EMMPRIN) and its secreted ligand cyclophilin-A (CyPA) significantly contributes to cardiac injury and necrosis. Here, we aimed to investigate if, in addition to the effect on the funny current (I(f)), Ivabradine may also play a role against cardiac necrosis by reducing EMMPRIN/CyPA-mediated cardiac inflammation. In a porcine model of cardiac ischemia/reperfusion (IR), we found that administration of 0.3 mg/kg Ivabradine significantly improved cardiac function and reduced cardiac necrosis by day 7 after IR, detecting a significant increase in cardiac CyPA in the necrotic compared to the risk areas, which was inversely correlated with the levels of circulating CyPA detected in plasma samples from the same subjects. In testing whether Ivabradine may regulate the levels of CyPA, no changes in tissue CyPA were found in healthy pigs treated with 0.3 mg/kg Ivabradine, but interestingly, when analyzing the complex EMMPRIN/CyPA, rather high glycosylated EMMPRIN, which is required for EMMPRIN-mediated matrix metalloproteinase (MMP) activation and increased CyPA bonding to low-glycosylated forms of EMMPRIN were detected by day 7 after IR in pigs treated with Ivabradine. To study the mechanism by which Ivabradine may prevent secretion of CyPA, we first found that Ivabradine was time-dependent in inhibiting co-localization of CyPA with the granule exocytosis marker vesicle-associated membrane protein 1 (VAMP1). However, Ivabradine had no effect on mRNA expression nor in the proteasome and lysosome degradation of CyPA. In conclusion, our results point toward CyPA, its ligand EMMPRIN, and the complex CyPA/EMMPRIN as important targets of Ivabradine in cardiac protection against IR.

Ivabradine induces cardiac protection by preventing cardiogenic shock-induced extracellular matrix degradation.

INTRODUCTION AND OBJECTIVES: Ivabradine reduces heart rate by blocking the I(f) current and preserves blood pressure and stroke volume through unknown mechanisms. Caveolin-3 protects the heart by forming protein complexes with several proteins, including extracellular matrix (ECM)-metalloproteinase-inducer (EMMPRIN) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HN4), a target of ivabradine. We hypothesized that ivabradine might also exert cardioprotective effects through inhibition of ECM degradation. METHODS: In a porcine model of cardiogenic shock, we studied the effects of ivabradine on heart integrity, the levels of MMP-9 and EMMPRIN, and the stability of caveolin-3/HCN4 protein complexes with EMMPRIN. RESULTS: Administration of 0.3 mg/kg ivabradine significantly reduced cardiogenic shock-induced ventricular necrosis and expression of MMP-9 without affecting EMMPRIN mRNA, protein, or protein glycosylation (required for MMP activation). However, ivabradine increased the levels of the caveolin-3/LG-EMMPRIN (low-glycosylated EMMPRIN) and caveolin-3/HCN4 protein complexes and decreased that of a new complex between HCN4 and high-glycosylated EMMPRIN formed in response to cardiogenic shock. We next tested whether caveolin-3 can bind to HCN4 and EMMPRIN and found that the HCN4/EMMPRIN complex was preserved when we silenced caveolin-3 expression, indicating a direct interaction between these 2 proteins. Similarly, EMMPRIN-silenced cells showed a significant reduction in the binding of caveolin-3/HCN4, which regulates the I(f) current, suggesting that, rather than a direct interaction, both proteins bind to EMMPRIN. CONCLUSIONS: In addition to inhibition of the I(f) current, ivabradine may induce cardiac protection by inhibiting ECM degradation through preservation of the caveolin-3/LG-EMMPRIN complex and control heart rate by stabilizing the caveolin-3/HCN4 complex.

Ivabradine-Stimulated Microvesicle Release Induces Cardiac Protection against Acute Myocardial Infarction.

Ivabradine can reduce heart rate through inhibition of the current I(f) by still unexplored mechanisms. In a porcine model of ischemia reperfusion (IR), we found that treatment with 0.3 mg/kg Ivabradine increased plasma release of microvesicles (MVs) over Placebo, as detected by flow cytometry of plasma isolated from pigs 7 days after IR, in which a tenfold increase of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) containing (both high and low-glycosylated) MVs, was detected in response to Ivabradine. The source of MVs was investigated, finding a 37% decrease of CD31+ endothelial cell derived MVs, while CD41+ platelet MVs remained unchanged. By contrast, Ivabradine induced the release of HCN4+ (mostly cardiac) MVs. While no differences respect to EMMPRIN as a cargo component were found in endothelial and platelet derived MVs, Ivabradine induced a significant release of EMMPRIN+/HCN4+ MVs by day 7 after IR. To test the role of EMMPRIN+ cardiac MVs (EMCMV), H9c2 cell monolayers were incubated for 24 h with 107 EMCMVs, reducing apoptosis, and increasing 2 times cell proliferation and 1.5 times cell migration. The in vivo contribution of Ivabradine-induced plasma MVs was also tested, in which 108 MVs isolated from the plasma of pigs treated with Ivabradine or Placebo 7 days after IR, were injected in pigs under IR, finding a significant cardiac protection by increasing left ventricle ejection fraction and a significant reduction of the necrotic area. In conclusion ivabradine induces cardiac protection by increasing at least the release of EMMPRIN containing cardiac microvesicles.

Targeting TLR4 with ApTOLL Improves Heart Function in Response to Coronary Ischemia Reperfusion in Pigs Undergoing Acute Myocardial Infarction.

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).

Non-Invasive Detection of Extracellular Matrix Metalloproteinase Inducer EMMPRIN, a New Therapeutic Target against Atherosclerosis, Inhibited by Endothelial Nitric Oxide.

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.