FASEB bioAdvances最新文献

Endothelial colony forming cells (ECFCs) derived from peripheral blood have been shown to retain disease phenotype in several conditions thus possessing great translational potential for regenerative medicine. Hyperglycaemia may alter the phenotype of ECFCs yet the characteristics of ECFCs isolated from people with type 1 diabetes (T1D) have not been described. Here, we establish whether ECFCs can be successfully isolated from donors with T1D and we characterize their functional properties. Human ECFCs were isolated from peripheral blood of up to 9 control and T1D donors. Expression of cell markers and cytokines was analyzed using immunocytochemistry, RT-PCR and ELISA. Ca²⁺ signaling and contraction were studied using Fluo-4-AM in cells treated with serial concentrations of histamine (1 × 10⁻⁷-1 × 10⁻⁴M). T1D ECFCs showed robust endothelial marker expression and displayed normal morphology but had a reduced size compared to those from control donors. In response to inflammatory stimuli, T1D ECFCs exhibited exaggerated pro-atherogenic/pro-inflammatory cytokine (IL-6 and MCP-1) and adhesion molecule gene induction (VCAM-1 and ICAM-1) but suppressed induction of interferon signaling markers (IP-10). Histamine stimulated a concentration-dependent increase in Ca²⁺ influx in ECFCs which was significantly reduced in ECFCs from T1D donors, independent of differences in H1 receptor expression levels. Histamine-induced contraction was significantly enhanced in T1D ECFCs. ECFCs from control and T1D donors exhibit distinct phenotypic differences redolent of the vascular pathologies associated with T1D. This establishes the utility of T1D ECFCs for modeling vascular complications but also highlights the need to understand the potential limitations of autologous ECFCs to treat diabetic complications.

Since Yamanaka's landmark achievement in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) using the four key transcription factors—OCT4, SOX2, KLF4, and c-Myc (OSKM)—iPSC technology has made significant strides. Notable advancements include refining reprogramming factors, delivery systems, somatic cell selection, and optimization of reprogramming conditions, along with developing chemical reprogramming methods. With their unparalleled proliferative capacity and near-pluripotent differentiation potential, iPSCs have become invaluable tools for investigating neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Neuronal models derived from ALS patient-specific iPSCs, particularly iPSC-derived motor neurons (iPSC-MNs), offer a robust platform to recapitulate disease-specific pathology and investigate the molecular mechanisms underpinning ALS, thereby accelerating the discovery of novel therapeutic strategies. This review highlights the evolution of iPSC technology and its transformative applications in ALS modeling, drug discovery, and therapeutic development.

Pilocarpine, a muscarinic receptor agonist, is clinically used to reduce intraocular pressure via ciliary muscle contraction. However, the intracellular signaling pathways mediating pilocarpine-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) synthesis in the ciliary muscle are not fully understood. This study aimed to characterize the muscarinic receptor subtypes and intracellular mechanisms involved in pilocarpine-induced NO and PGE₂ production in porcine ciliary muscle and to explore the potential implications of these pathways in age-related ocular changes, including presbyopia. Ciliary muscle strips from adult porcine eyes were incubated in Krebs-Ringer buffer with pharmacological agents. Nitrate levels were measured using a Griess reaction-based colorimetric assay, and PGE₂ concentrations were quantified via ELISA. Selective receptor antagonists and enzyme inhibitors were used to investigate receptor involvement and signaling cascades. Statistical significance was determined using unpaired t-tests (p < 0.05). Pilocarpine elicited a concentration-dependent increase in nitrate and PGE₂ production, peaking at 1 × 10⁻⁷ M. This effect was inhibited by atropine, pirenzepine (M₁ antagonist), and J104129 (M₃ antagonist), but not by the M₂ antagonist AFDX 116. Inhibitors of nitric oxide synthase (L-NMMA, L-NIO), phospholipase C (U-73122), cyclooxygenase (diclofenac), and calcium signaling (verapamil, TFP) all reduced mediator production, while calcium ionophore A23187 enhanced it. Pilocarpine activates M₁ and M₃ muscarinic receptors in the porcine ciliary muscle, stimulating NO and PGE₂ production via calcium-dependent PLC–NOS–COX signaling. These pathways may influence ciliary muscle function and lens physiology, offering potential therapeutic targets for glaucoma and presbyopia.

High-fat diets (HFDs) are a well-established cause of hepatic steatosis, a condition associated with altered hepatic fatty acid metabolism and reduced choline availability. Folic acid (FA) deficiency can also promote steatosis, in part by impairing choline metabolism. Although FA supplementation has been found to reduce liver fat in mice with hepatic steatosis, it is unclear if this effect is due to increased hepatic choline levels, changes in fatty acid profiles, or a combination of both. In this study, four-week-old male C57BL/6J mice were fed 45 kcal% HFDs with total FA content adjusted to onefold, fivefold, or tenfold AIN-93G recommended level (2 mg/kg diet) for 15 weeks ad libitum. Hepatic triacylglycerol (TAG), choline concentrations, expression of key genes in choline metabolism, and TAG-bound fatty acid profiles were analyzed. Mice receiving tenfold FA had lower liver weight and hepatic TAG levels compared to the onefold control group (p < 0.05). Both fivefold and tenfold FA supplementation increased hepatic choline concentrations and upregulated mRNA expression of choline-metabolizing genes (p < 0.05), suggesting enhanced choline utilization. Additionally, tenfold FA supplementation altered the hepatic TAG fatty acid profile, reducing levels of palmitoleic acid and oleic acid (p < 0.05), fatty acids typically associated with de novo lipogenesis. A strong inverse correlation was observed between hepatic choline and TAG levels (p < 0.001, adjusted R² = 0.56), supporting a potential role for choline availability in mediating FA's protective effects. Folic acid supplementation protects against hepatic steatosis by enhancing choline availability, modulating lipid metabolism, and reducing liver fat accumulation.

As part of a systemic inflammatory response, acute phase proteins (APPs) are released into the blood to support the body's immune response. Once in the bloodstream, the APPs can also interact with immune cells, such as neutrophil granulocytes (PMNs). However, this interaction is not yet fully understood. This study aims to investigate the effects of specific APPs on various functions of neutrophil granulocytes in vitro. PMNs were isolated from peripheral blood of healthy volunteers and subsequently exposed to varying concentrations of CRP, fibrinogen, or ferritin. As activating agents, TNF-α (TNFα)/N-formylmethionine-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), or ionomycin were used. Triggered oxidative burst and the expression of surface antigens CD11b, CD62L, and CD66b were measured by flow cytometry. Live cell imaging (LCI) determined the influence of ferritin on migration behavior, time-resolved MPO release, and NET formation. CRP had a certain, non-significant activating effect on PMN oxidative burst and surface epitope expression. Ferritin led to a moderate increase in the oxidative burst, especially after activation with TNF-α/fMLP, PMA, or ionomycin. Ferritin reduced PMN migration without TNFα and enhanced PMN migration in the presence of TNFα. Without TNFα, ferritin prolonged NETosis and had a certain dose-specific effect on MPO release. Fibrinogen mainly influenced the expression of CD11b, CD62L, and CD66b. The observed effects of acute phase proteins on PMNs showed plausible, concentration-dependent, and differential effects for the tested APPs, but only of moderate magnitude. Future experiments should focus on intracellular signaling pathways and on the determination of PMN gene expression profiles. Given the broad context in which APPs are elevated, their interaction with PMNs is of considerable scientific interest for a multitude of clinical conditions.

Abdominal adiposity is associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are non-lysosomal calcium-dependent cysteine proteases that are highly expressed in human and experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we previously demonstrated that calpain-2 (a major ubiquitous isoform) deficiency mitigated angiotensin II (AngII)-induced AAA formation in hypercholesterolemic mice. In addition, we also demonstrated that calpain inhibition strongly suppressed adipose tissue inflammation in obese mice. Here, we evaluated the contribution of adipocyte-specific calpain-2 on obesity-accelerated AAA in mice. Calpain-2 protein is highly expressed in the periaortic adipose tissue (PAAT) of AngII-induced AAAs in obese mice. To determine the relative contribution of calpain-2 in obesity-accelerated AAA development, calpain-2 floxed mice were bred to mice with a tamoxifen-inducible form of Cre under control of either the ubiquitous promoter, chicken β-actin, or adipocyte-specific promoter, Adipoq. Ubiquitous or adipocyte-specific depletion of calpain-2 in mice significantly suppressed Ang II–induced AAA formation in obese mice. In addition, calpain-2 depletion reduced the incidence of AngII-induced AAAs in mice. Furthermore, calpain-2 deficiency prevented AngII-induced aortic medial elastin fragmentation, adventitial collagen disruption, and periaortic leukocytic accumulation. These results suggest that adipocyte-derived calpain-2 plays a critical role in AngII-induced AAA development in diet-induced obese mice.

Calumenin (CALU), a multifunctional E-F-hand protein of the CREC family involved in various cellular processes, is ubiquitously expressed in almost all human tissues, although its expression level is tissue-specific. CALU plays an effective role in calcium (Ca) homeostasis throughout various Ca2⁺-related processes, and its correlations with various ER-related functions are evident. Due to its propensity to bind Ca, participation in calcium ion (Ca²⁺)-dependent activities, major cellular processes, such as the production and maintenance of extracellular matrix, have been attributed to this multifaceted protein. In this regard, CALU's association with various normal calcification processes, as well as pathologic Ca deposits, is apparent. Additionally, its key role as a crossroads among various normal cellular processes and many inflammatory diseases and cancers is also evident. The important correlation of CALU with cancer-related proteins through its regulatory role in γ-carboxylation of known cancer-related vitamin K-dependent proteins has also been noted. The opposing and pleiotropic functions of the CALU isoforms might play balancing roles in establishing a state of equilibrium in the cell. Given the contradictory functions of CALU isoforms during cancer, the need for a balance between these isoforms, as well as the existence of mechanisms to regulate their ratio in normal cells, is speculated. The relationship between CALU and immune response, tumor-infiltrating immune cells, and cancer patients' responsiveness to various cancer therapies is also described. In this regard, the involvement of the CALU isoform in response to cancer treatment and various immune pathways is discussed. This comprehensive review addresses the outstanding features and the latest findings on CALU's molecular aspects and diverse functions in various physiological processes and pathological conditions.

Apical periodontitis (AP) is an inflammatory/infectious disease that leads to alveolar bone resorption. Cyanidin-3-glucoside (C3G) is an anthocyanin known for its anti-inflammatory, antioxidant, and bone metabolism-regulating properties, suggesting benefits during AP development. This study aimed to evaluate the influence of C3G supplementation on bone resorption and inflammation during the progression of AP in Wistar rats. Sixteen male albino Wistar rats were divided into two groups: a control group (C) that received potable water, and a treatment group (C3G) that received a C3G solution. Supplements were administered daily via oral gavage for 45 days. AP was induced in both groups on Day 15, and it was allowed to develop for 30 days. After the experimental period, the animals were euthanized, and the upper and lower jaws were collected to assess lesion volume, bone microstructure, the intensity of the inflammatory infiltrate, and the expression of IL-1β, TNF-α, IL-10, the RANKL/OPG ratio, and TRAP immunolabeling at the AP site. Statistical analysis was performed with a 5% significance level. Compared to controls, C3G-treated rats exhibited reduced lesion volume, increased bone volume fraction and trabecular thickness, lower RANKL/OPG ratio, and fewer TRAP-positive cells/mm (p < 0.05). No significant differences were observed in inflammatory infiltrate or cytokine expression (IL-1β, TNF-α, and IL-10) at the lesion site (p > 0.05). C3G supplementation modulated bone resorption by reducing lesion volume and osteoclast activity, while enhancing bone microstructural parameters, without significantly affecting local inflammatory markers.

Xiaoxianxiong decoction (XXXD) is a widely used clinical prescription effective in the treatment of pneumonia. The study investigates the efficacy and mechanism of XXXD in pneumonia. In the study, an in vivo pneumonia model was induced by lipopolysaccharide (LPS) in mice, and an in vitro pneumonia model was induced by LPS in A549 cells. The study assessed the potential therapeutic effect of XXXD by detecting inflammatory cells in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, lung wet/dry weight ratio, and histology. ELISA kits were used to detect the release of inflammatory factors. Apoptosis was identified by TUNEL labeling and flow cytometry, and c-caspase3/caspase3 expression was analyzed by Western Blot. SIRT1 expression was detected by immunofluorescence labeling, while Western blotting measured the expression levels of SIRT1 and its downstream proteins. Finally, the SIRT1 inhibitor EX527 was used in LPS-induced mice, and a rescue assay was performed in LPS-induced A549 cells by interfering with SIRT1 to investigate the regulatory mechanism of XXXD. XXXD greatly reduced the effect of LPS on apoptosis, significantly decreased the expression of p66shc and p-NF-κB/NF-κB downstream of SIRT1, and also significantly increased the expression of SIRT1. Interestingly, the anti-inflammatory effect of XXXD on the pneumonia model disappeared after interfering with SIRT1. XXXD protects against LPS-induced pneumonia by inhibiting the inflammatory response through activating SIRT1, offering a fresh approach to the creation of focused pneumonia treatment plans.