FASEB bioAdvances最新文献

Cardiolipin (CL), a mitochondria-specific non-bilayer phospholipid, plays an essential role in the assembly and structural dynamics of the respiratory chain, affecting the membrane morphology and functional activity of inner mitochondria membrane (IMM)-embedded proteins. CL forms CL-rich domains on the IMM where negative curvature is required to increase the stability of cristae. However, CL constantly transitions between lamellar bilayer and non-bilayer phases, such as inverted CL hexagonal phases and inverted CL micelles. Non-bilayer phases of CL promote mitochondrial fission and fragmentation, transition of CL to the outer mitochondrial membrane (OMM), and mitophagy. In addition, non-bilayer phases of CL can increase proton leakage, which leads to mitochondrial depolarization and decreased mitochondrial ATP synthesis. Thus, therapeutic applications for minimizing non-bilayer CL phases should be able to optimize mitochondrial stability during various stresses. We have developed a novel, high-density aromatic peptide (HDAP2) that targets CL and enhances the stability of CL within the lipid core of bilayers in CL-POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. We also demonstrated that HDAP2 interacts with inverted CL micelles, forming HDAP2-CL micelles. This suggests that HDAP2 interacts with the non-bilayer phase of CL, thereby stabilizing CL in the bilayer configuration. Scanning electron microscopy confirmed that HDAP2 assembles into spherical micelles approximately 1–3 μm in diameter. We have also demonstrated that this novel, water-soluble peptide is cell-permeable and targets mitochondria without causing cell toxicity. Furthermore, we used a well-known mitochondrial toxicity model of serum starvation to demonstrate that HDAP2 significantly promoted cell survival in a dose-dependent manner in mitochondria-dependent Madin-Darby bovine kidney (MDBK) cells. Importantly, HDAP2 preserved mitochondrial membrane potential and mitigated oxidative stress during serum deprivation. These protective effects suggest that, through its unique mechanism of action, HDAP2 can enhance cellular homeostasis, which would offer broad therapeutic potential for the prevention, recovery, and reversal of many acute and chronic disease conditions, including neurodegeneration, ischemia–reperfusion injury, and inflammation.

Receptor-interacting protein kinase 1 (RIP1) plays a regulatory role in inflammation and cell survival, but its involvement in colorectal cancer (CRC), particularly in relation to lymphatic changes within the tumor microenvironment, remains poorly defined. The expressions of RIP1 and VEGF-C were examined in CRC tissues and adjacent normal samples by qRT-PCR and Western blot. Their correlation was analyzed, and functional assays were conducted using RIP1-silenced and reexpressed HT29 and SW480 cells. Cell proliferation, migration, and colony formation were evaluated, alongside NF-κB reporter activity. Conditioned media from LPS-stimulated CRC cells were applied to assess tube formation by lymphatic endothelial cells. A xenograft model was applied to verify tumor growth and vascular changes in vivo. RIP1 was found to be elevated in CRC tissues and positively associated with VEGF-C expression. Knockdown of RIP1 reduced cell growth, migration, VEGF-C level, and NF-κB activity. These changes were partially reversed by restoring RIP1 overexpression. Conditioned media from RIP1-deficient cells impaired tubule formation in lymphatic endothelial cells. In mice, RIP1 silencing suppressed tumor growth and reduced microvessel density and Ki67-positive cells. RIP1 promotes CRC progression and is associated with elevated VEGF-C expression and NF-κB pathway activation. It may contribute to CRC growth and lymphatic remodeling, suggesting RIP1 as a potential target for CRC intervention.

Myocardial infarction remains one of the leading causes of mortality. Reperfusion of the infarcted myocardium restores blood flow and reduces primary ischemic injury. However, despite its protective function, reperfusion is also associated with several deleterious outcomes that can result in ischemia–reperfusion (I/R) injury to cardiac tissue. Although negative outcomes such as reactive oxygen species generation are strongly associated with I/R injury, cardiac energy metabolism is also greatly disrupted. Furthermore, previous studies have shown that the restoration of normal fuel oxidation in the myocardium regulates the extent of contractile recovery. A better understanding of the pathophysiological mechanisms underlying I/R injury may allow us to develop new treatments that limit the negative aspects of the process. In this study, we examined the role played by GCN5L1, a protein implicated in the regulation of energy metabolism, in I/R injury. We demonstrate that cardiac-specific loss of GCN5L1 promotes the inhibitory phosphorylation of pyruvate dehydrogenase in vitro and in vivo, a process likely to inhibit glucose oxidation, and that this corresponds to increased myocardial damage following ischemia–reperfusion (I/R) injury.

Vascular endothelial growth factor (VEGF) is important in both developmental and pathologic angiogenesis in retinopathy of prematurity (ROP). Using a rat model representative of ROP, we found that regulation of VEGF signaling through VEGF receptor 2 (VEGFR2) in retinal microvascular endothelial cells (RMVECs) extended developmental angiogenesis but reduced pathologic angiogenesis, that is, intravitreal neovascularization (IVNV). We identified an adaptor protein, MEMO1, in IVNV in the rat model and tested the hypothesis that MEMO1 in RMVECs was important in IVNV by regulating signaling through VEGFR2. Instead, we found MEMO1 knockdown enhanced phosphorylation of VEGF-induced VEGFR2 and STAT3 and increased wound closure in vitro using cultured human RMVECs. Furthermore, MEMO1 overexpression suppressed VEGF-induced VEGFR2 and STAT3 phosphorylation and dampened VEGF-induced RMVEC wound closure. In contrast, in the absence of VEGF, MEMO1 overexpression promoted RMVEC proliferation in the wound closure assay and AKT phosphorylation, supporting a role for MEMO1 in VEGF-independent angiogenic processes. In vivo, retinal endothelial cell-specific knockdown of MEMO1 in the rat ROP model significantly increased IVNV but did not affect developmental angiogenesis. Our findings support a novel regulatory role for MEMO1 where MEMO1 limits VEGF-driven IVNV and promotes VEGF-independent angiogenic signaling. These results suggest MEMO1 may serve as a protective modulator of pathological angiogenesis in ROP and represent a potential therapeutic target to limit IVNV while preserving physiologic angiogenesis.

Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor (GPCR) that mediates chemotaxis and bactericidal activities in phagocytes. The monoclonal antibody 5F1 is generated against full-length FPR1 and used widely for detection of FPR1 expression. This study aimed to characterize 5F1 for its functions. We found that 5F1 is highly selective for human FPR1 over the homologous FPR2. Epitope mapping led to the identification of extracellular loop 2 (ECL2) as a major epitope, and the synthetic peptide of ECL2 interfered with 5F1 binding to FPR1. Using a NanoLuc Bioluminescence Resonance Energy Transfer approach, we found that 5F1 binding induced FPR1 conformational changes. Although less potent than fMLF, 5F1 binding induced FPR1 internalization, Gi protein dissociation, and β-arrestins membrane translocation. Alanine substitution of F110 and R205 markedly reduced 5F1 binding without affecting FPR1 cell surface expression, suggesting that 5F1 is sensitive to conformational changes in FPR1 as these residues are not present in ECL2. Altogether, mAb 5F1 can alter FPR1 conformation and modulate transmembrane signaling, features that may be explored for potential use beyond the detection of FPR1 expression.

Oxidative stress is characterized by an imbalance between the production and elimination of free radicals, where the rate of free radical generation surpasses the rate of their removal. This imbalance can lead to tissue and organ damage, contributing to the pathogenesis of various diseases. The nervous system, due to its high oxygen consumption, is particularly susceptible to oxidative stress. Numerous neurotoxins can induce neurotoxicity through oxidative stress, thereby contributing to the onset of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Furthermore, neurotoxicity can exacerbate oxidative stress by disrupting mitochondrial metabolism and impairing the activity of antioxidant enzymes, thereby intensifying neurotoxic effects. This review examines the mechanisms underlying the interaction between oxidative stress and neurotoxicity and explores strategies to mitigate neurotoxicity by reducing oxidative stress, with the aim of informing future clinical approaches for the treatment of neurodegenerative diseases.

Previous studies reported the pro-osteogenic ability of L-Tryptophan (L-Trp) and Calcium-Sensing RCeceptor (CaSR) respectively. Recent researchers found L-Trp could activate CaSR. Therefore, this study investigated the osteogenic mechanisms of L-Trp through CaSR activation. Using in vivo and in vitro models, we evaluated L-Trp's effects on bone formation and osteoblast activity. Levo-Trp solution was injected into the temporomandibular joint of 3-week-old mice, and the mandibular development was observed by Micro-CT at 6 weeks of age. The pre-osteoblast cell line MC3T3-E1 cells were stimulated by L-Trp in vitro, and their proliferation, migration, and osteogenic ability were detected by CCK8 assay, alizarin red staining, etc. Transcriptome sequencing was used to investigate the underlying mechanism of L-Trp stimulation and validated by qPCR and Western blot analyses. Local injection of 0.5% L-Trp in juvenile mice significantly increased mandibular bone mineral density. In vitro, L-Trp enhanced MC3T3-E1 pre-osteoblast proliferation, migration, and differentiation, with upregulated osteogenic markers (Runx2, Sp7, Alp) and mineralization. CaSR antagonism (NPS-2143) abolished these effects, confirming CaSR's pivotal role. Transcriptome sequencing revealed L-Trp activation of the focal adhesion pathway, characterized by increased Ptk2, Rhoa, Itga11, and Clec11a expression. These findings established L-Trp as a CaSR-dependent osteogenic enhancer, mediated via the focal adhesion pathway.

Hemophilia A is an X-linked monogenic disease resulting in insufficient pro-coagulant factor VIII (FVIII) levels. Hemophiliac infants are at risk for life-threatening hemorrhage, especially during birth. No perinatal treatment for Hemophilia A is currently available. It has been previously shown that the transamniotic route is a viable option to deliver exogenous mRNA to the fetus. We sought to determine whether FVIII mRNA so delivered could be translated by the fetus, leading to the presence of FVIII in the fetal circulation. Time-dated pregnant Sprague Dawley dams underwent volume-matched intra-amniotic injections in all their fetuses (n = 166) of either a human FVIII (hFVIII) mRNA encapsulated by lipopolyplex (mRNA; n = 115) or of the same lipopolyplex without mRNA (control; n = 51) on gestational day 17 (E17; term = E21–22). Fetal liver and serum samples were procured daily until term and screened for hFVIII protein by ELISA. There was no maternal mortality. Overall survival was 90% (149/166). Controlled by the mRNA-free injections, fetal serum levels of hFVIII were statistically significantly higher overall in the mRNA group (p = 0.002), peaking at E20 (24.4 ± 2.4 ng/mL in the mRNA group vs. 10.5 ± 1.9 ng/mL for control; p < 0.001). In the fetal liver, there was variability in statistically significant differences between the groups, with the shorter time point showing significance (p = 0.003). Encapsulated exogenous mRNA encoding for factor VIII can be incorporated and translated by the fetus following simple intra-amniotic injection in a rat model. Transamniotic mRNA delivery could become a novel strategy for the perinatal management of Hemophilia A.

Mutations in the ABCA4 gene in Stargardt disease (STGD1) cause enhanced accumulation of cytotoxic lipofuscin, manifesting in RPE atrophy and photoreceptor dysfunction. One component of lipofuscin is the all-trans-retinal derivative, pyridinium bisretinoid A2E. Since ocular A2E biosynthesis relies on all-trans-retinal, which is obtained from circulating all-trans-retinol bound to retinol binding protein 4 (RBP4-ROL), we hypothesized that modulating vitamin A receptors, such as RBPR2, which regulate serum RBP4-ROL homeostasis, should in principle attenuate A2E production. In silico analysis revealed multiple retinoic acid response element (RARE) binding sites on the murine Rbpr2 gene promoter, which was confirmed in vitro by EMSA and ChIP assays. In vitro luciferase assays showed that Rbpr2 promoter activity was induced by exogenous β-carotene (BC) metabolites. Dietary BC supplementation of Abca4^−/− mice, a mouse model for STGD1, increased hepatic all-trans-retinoic acid and 9-cis-retinoic acid production, which induced Rbpr2 mRNA expression. This mechanism decreased serum RBP4 protein levels, fundus lipofuscin autofluorescence, and ocular A2E accumulation, altogether improving photoreceptor and RPE function. Conversely, such a rescue was not observed in either Abca4^−/− mice fed a diet devoid of BC or in double knockout Rbpr2^−/−; Abca4^−/− mice. Thus, there was a significant inverse correlation between dietary BC supplementation and Rbpr2 gene presence in Abca4^−/− mice, to that of lipofuscin accumulation in Abca4^−/− mice on diets devoid of BC or in Rbpr2^−/−; Abca4^−/− mice. Our results provide impetus to include dietary obtained BC for STGD1 patients with ABCA4 gene mutations and identify a novel role for the vitamin A receptor RBPR2 in this process.

Interest in the endogenous role of methane has grown rapidly over the past decade, driven both by its relevance for disease detection (including intestinal methanogen overgrowth) as well as discoveries that raise the possibility of endogenous sources of methane and suggestive evidence of methane effects relevant to physiology. This review explores both established and emerging origins of breath methane, its physiological relevance, and the evolving landscape of detection methods. We aim to summarize current understanding and provide a platform to outline key directions for future research. Evidence supports the existence of non-microbial, endogenous methane production pathways and potential biological effects beyond the gut. However, the concentrations generated via these pathways and the levels required to elicit physiological responses remain under investigation. Recent technological advances have enabled more accessible and longitudinal breath methane monitoring, opening new avenues for research and clinical application.