Activation of PPAR-α attenuates myocardial ischemia/reperfusion injury by inhibiting ferroptosis and mitochondrial injury via upregulating 14-3-3η.

This study aimed to explore the effects of peroxisome proliferator-activated receptor α (PPAR-α), a known inhibitor of ferroptosis, in Myocardial ischemia/reperfusion injury (MIRI) and its related mechanisms. In vivo and in vitro MIRI models were established. Our results showed that activation of PPAR-α decreased the size of the myocardial infarct, maintained cardiac function, and decreased the serum contents of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and Fe2+ in ischemia/reperfusion (I/R)-treated mice. Additionally, the results of H&E staining, DHE staining, TUNEL staining, and transmission electron microscopy demonstrated that activation of PPAR-α inhibited MIRI-induced heart tissue and mitochondrial damage. It was also found that activation of PPAR-α attenuated MIRI-induced ferroptosis as shown by a reduction in malondialdehyde, total iron, and reactive oxygen species (ROS). In vitro experiments showed that intracellular contents of malondialdehyde, total iron, LDH, reactive oxygen species (ROS), lipid ROS, oxidized glutathione disulphide (GSSG), and Fe2+ were reduced by the activation of PPAR-α in H9c2 cells treated with anoxia/reoxygenation (A/R), while the cell viability and GSH were increased after PPAR-α activation. Additionally, changes in protein levels of the ferroptosis marker further confirmed the beneficial effects of PPAR-α activation on MIRI-induced ferroptosis. Moreover, the results of immunofluorescence and dual-luciferase reporter assay revealed that PPAR-α achieved its activity via binding to the 14-3-3η promoter, promoting its expression level. Moreover, the cardioprotective effects of PPAR-α could be canceled by pAd/14-3-3η-shRNA or Compound C11 (14-3-3η inhibitor). In conclusion, our results indicated that ferroptosis plays a key role in aggravating MIRI, and PPAR-α/14-3-3η pathway-mediated ferroptosis and mitochondrial injury might be an effective therapeutic target against MIRI.

Role of dysregulated ferroptosis­related genes in cardiomyocyte ischemia­reperfusion injury: Experimental verification and bioinformatics analysis.

Acute myocardial infarction is a life-threatening condition with high mortality and complication rates. Although myocardial reperfusion can preserve ischemic myocardial tissue, it frequently exacerbates tissue injury, a phenomenon known as ischemia-reperfusion injury (IRI). However, the underlying pathological mechanisms of IRI remain to be completely understood. Ferroptosis is a novel type of regulated cell death that is associated with various pathological conditions, including angiocardiopathy. The purpose of this article was to elucidate the possible mechanistic role of ferroptosis in IRI through bioinformatics analysis and experimental validation. Healthy and IRI heart samples were screened for differentially expressed ferroptosis-related genes and functional enrichment analysis was performed to determine the potential crosstalk and pathways involved. A protein-protein interaction network was established for IRI, and 10 hub genes that regulate ferroptosis, including HIF1A, EGFR, HMOX1, and ATF3 were identified. In vitro, an anoxia/reoxygenation (A/R) injury model was established using H9c2 cardiomyoblasts to validate the bioinformatics analysis results, and extensive ferroptosis was detected. A total of 4 key hub genes and 3 key miRNAs were also validated. It was found that IRI was related to the aberrant infiltration of immune cells and the small-molecule drugs that may protect against IRI by preventing ferroptosis were identified. These results provide novel insights into the role of ferroptosis in IRI, which can help identify novel therapeutic targets.

The biofunctionalization of titanium nanotube with chitosan/genipin heparin hydrogel and the controlled release of IL-4 for anti-coagulation and anti-thrombus through accelerating endothelialization.

The valve replacement is the main treatment of heart valve disease. However, thrombus formation following valve replacement has always been a major clinical drawback. Accelerating the endothelialization of cardiac valve prosthesis is the main approach to reduce thrombus. In the current study, a titanium nanotube was biofunctionalized with a chitosan/genipin heparin hydrogel and the controlled release of interleukin-4 (IL-4), and its regulation of macrophages was investigated to see if it could influence endothelial cells to eventually accelerate endothelialization. TNT60 (titanium dioxide nanotubes, 60 V) with nanoarray was obtained by anodic oxidation of 60 V, and IL-4 was loaded into the nanotube by vacuum drying. The hydrogel (chitosan : genipin = 4 : 1) was applied to the surface of the nanotubes following drying, and the heparin drops were placed on the hydrogel surface with chitosan as the polycation and heparin as the polyanion. A TNT/IL-4/G (G = gel, chitosan/genipin heparin) delivery system was prepared. Our results demonstrated that the biofunctionalization of titanium nanotube with chitosan/genipin heparin hydrogel and the controlled release of IL-4 had a significant regulatory effect on macrophage M2 polarization, reducing the inflammatory factor release and higher secretion of VEGF (vascular endothelial growth factor), which can accelerate the endothelialization of the implant.

Inorganic phosphate-osteogenic induction medium promotes osteogenic differentiation of valvular interstitial cells via the BMP-2/Smad1/5/9 and RhoA/ROCK-1 signaling pathways.

Calcific aortic valve disease (CAVD) currently lacks a highly effective in vitro model. The presence of high concentrations of serum inorganic phosphate in patients with end-stage renal disease leads to calcification of vascular and aortic valves. Therefore, we applied inorganic phosphate to induce the osteogenic differentiation of valvular interstitial cells (VICs) and mimic its in vivo pathophysiological effects. Calcification and inflammatory response assays determined that inorganic phosphate-osteogenic induction medium (IP-OIM) was more efficient than classic osteogenic induction medium (OIM) containing organic glycerophosphate. Levels of BMP-2, RhoA, and ROCK-1 were significantly increased in IP-OIM cells. Knockdown efficiency of BMP-2- and RhoA-siRNA in VICs was evaluated, and expression of RhoA and its downstream target ROCK-1 was decreased after BMP-2-siRNA transfection. Moreover, ROCK-1 was significantly downregulated after RhoA knockdown, whereas expression of BMP-2 was unchanged. Interference of BMP-2 had a stronger anti-calcification effect than RhoA, further identifying BMP-2 as an upstream regulator of RhoA/ROCK-1. Stimulation of VICs by IP-OIM led to increased Smad1/5/9 phosphorylation, which peaked at 60 min, while pre-treatment of VICs with the Smad1/5/9 inhibitor Compound C attenuated VICs calcification. These results suggest that IP-OIM induced VICs osteogenic differentiation via Smad1/5/9 signaling. Knockdown of BMP-2 or RhoA also decreased Smad1/5/9 phosphorylation also decreased. We conclude that the RhoA/ROCK-1 axis participates in VICs osteogenic differentiation as a "bypass mediator" of the BMP-2/Smad1/5/9 signaling pathway.

Biofunctionalization of decellularized porcine aortic valve with OPG-loaded PCL nanoparticles for anti-calcification.

Decellularized valve stents are widely used in tissue-engineered heart valves because they maintain the morphological structure of natural valves, have good histocompatibility and low immunogenicity. However, the surface of the cell valve loses the original endothelial cell coverage, exposing collagen and causing calcification and decay of the valve in advance. In this study, poly ε-caprolactone (PCL) nanoparticles loaded with osteoprotegerin (OPG) were bridged to a decellularized valve using a nanoparticle drug delivery system and tissue engineering technology to construct a new anti-calcification composite valve with sustained release function. The PCL nanoparticles loaded with OPG were prepared via an emulsion solvent evaporation method, which had a particle size of 133 nm and zeta potential of -27.8 mV. Transmission electron microscopy demonstrated that the prepared nanoparticles were round in shape, regular in size, and uniformly distributed, with an encapsulation efficiency of 75%, slow release in vitro, no burst release, no cytotoxicity to BMSCs, and contained OPG nanoparticles in vitro. There was a delay in the differentiation of BMSCs into osteoblasts. The decellularized valve modified by nanoparticles remained intact and its collagen fibers were continuous. After 8 weeks of subcutaneous implantation in rats, the morphological structure of the valve was almost complete, and the composite valve showed anti-calcification ability to a certain extent.