The FASEB Journal最新文献

This study aims to develop a novel therapeutic strategy for osteoporosis (OP) by enhancing the osteogenic differentiation potential of mesenchymal stem cells (MSCs). A three-channel biomimetic bone organ-on-a-chip model was constructed. Through bioinformatics analysis, TPD52L1, a key upregulated gene involved in the osteogenic differentiation of MSCs, was identified. Molecular experiments were conducted to verify the effects of its overexpression on the Wnt/β-catenin pathway. Critically, systematic knockdown experiments were performed to validate its necessity in mechanotransduction. Subsequently, functional experiments were performed to evaluate its role in the osteogenic and adipogenic differentiation of MSCs in a GIOP model. Safety assessments for TPD52L1-overexpressing MSCs covered subcellular localization, proliferation, anchorage-independent growth, oncogene expression, and short-term in vivo tumorigenicity. TPD52L1 overexpression activated the Wnt/β-catenin pathway and promoted osteogenic differentiation of MSCs. Its knockdown blocked mechanical stimulation-induced pathway activation and osteogenic marker upregulation, confirming its necessity in mechano-osteogenic conversion. TPD52L1 upregulated osteoblast markers osteocalcin (OCN) and alkaline phosphatase (ALP), downregulated the osteoclast marker tartrate-resistant acid phosphatase (TRAP), and reversed the osteoporosis phenotype. When combined with cyclic mechanical force stimulation in the organ-on-a-chip system, a synergistic effect was observed, enhancing bone repair. TPD52L1 was localized in the cytoplasm, and its endogenous expression was upregulated by mechanical stimulation, indicating mechanosensitivity. TPD52L1 overexpression did not affect MSCs' proliferation, anchorage-independent growth, or oncogene expression. Subcutaneous transplantation experiments confirmed that it did not induce tumor formation or significant pathological changes, demonstrating favorable biosafety. TPD52L1 serves as a key target for promoting the osteogenic differentiation of MSCs.

Kinesin KIF16B, as a molecular motor protein within cells, primarily utilizes energy derived from ATP hydrolysis to transport intracellular cargo along microtubules, thereby participating in material transport, organelle dynamics, and cytoskeletal organization. However, the mechanism by which KIF16B regulates the maturation process of cytoplasm in mouse oocytes remains unclear. This study was to investigate the potential role of KIF16B in modulating organelle dynamics in mouse oocytes. Our findings suggest that depletion of KIF16B impairs oocyte developmental competence following parthenogenesis, implying potential abnormalities in oocyte maturation. We observed that oocytes with diminished KIF16B exhibited disrupted mitochondrial distribution and function, and further analysis revealed that this may be due to KIF16B involvement in p-Drp1 and Fis1-mediated mitochondrial fission. Besides, impaired mitochondrial function also resulted in oxidative stress. Additionally, abnormal distribution of the ER and ER stress were observed in oocytes lacking KIF16B. This was accompanied by elevated expression of ER stress-related genes CHOP and ATF4. Concurrently, KIF16B knockdown affected the distribution and function of the Golgi apparatus, leading to abnormalities in Golgi-based vesicular transport processes. In summary, our data suggest that the kinesin KIF16B modulates organelle dynamics during oocyte maturation.

Marked trabecular bone loss of the distal femoral and proximal tibial regions is one of the most common and devastating complications of spinal cord injury (SCI) with highest incidence of fracture and no efficacious treatment to date. Osteocytes exhibit increased transforming growth factor-β (TGF-β) signaling 5 weeks after SCI. Whether reducing TGF-β signaling will restore trabecular bone structure after development of bone loss with SCI has not been tested. To address this, male mice underwent laminectomy (sham) or thoracic (T10) contusion SCI resulting in complete hindlimb paralysis. Five weeks later, sham and SCI mice received either control (IgG) or anti-TGF-β neutralizing (ID11) antibody (10 mg/kg/day, twice weekly) for 5 weeks before sacrifice. Micro-computed tomography (micro-CT) analysis of the distal femoral region showed that, compared to sham-IgG animals, treatment with ID11 significantly (p < 0.05) restored trabecular fractional bone volume (107% SCI-ID11 vs. 65% SCI-IgG), thickness (109% SCI-ID11 vs. 88% SCI-IgG), connectivity (99% SCI-ID11 vs. 78% SCI-IgG), and structure model index (101% SCI-ID11 vs. 118% SCI-IgG). In contrast, analysis of femoral mid-shaft regions showed that both SCI-1D11 and SCI-IgG mice exhibited reduced cortical thickness (91% vs. 84%). Histomorphometric analysis revealed no differences in indices of osteoblast or osteoclast numbers or surfaces between SCI-IgG, SCI-1D11, and sham-IgG groups. Similarly, analysis of femoral bone marrow supernatants demonstrated no significant difference in levels of procollagen type 1 intact N-terminal propeptide (P1NP) or tartrate-resistant acid phosphatase 5b (TRAcP-5b). Thus, TGF-β signaling may be a promising therapeutic target to regain trabecular bone architecture and prevent fractures after SCI.

Pre-implantation embryonic loss constitutes a major barrier to reproductive efficiency in livestock, yet the extrinsic determinants of embryonic survival remain poorly defined. Intra-organoid fluid (IOF) faithfully recapitulates native tissue secretions across multiple organ systems. We hypothesized that bovine endometrial epithelial organoids (BEEO) would produce IOF that mirrored in vivo uterine luminal fluid composition and extend embryo culture duration in vitro. We pursued three objectives: (a) establish and morphologically characterize BEEO, (b) define BEEO transcriptomic and secretory responses to 17β-estradiol (E2), medroxyprogesterone acetate (MPA), and interferon-tau (IFNτ), and (c) determine whether BEEO-derived IOF can support in vitro embryonic development beyond Day 8 (hatched blastocyst stage) under conventional culture conditions. BEEO were established from primary endometrial tissue (n = 4) and maintained a stable epithelial phenotype through multiple passages. Transcriptomic profiling revealed robust responses to stimulation, with E2, MPA, and IFNτ inducing distinct gene expression programs consistent with in vivo effects. IOF metabolomic analysis confirmed hormone-dependent regulation of IOF secretory output, with E2 + MPA (diestrus mimic) enhancing the production of metabolites implicated in conceptus development. Remarkably, IOF from diestrus mimic-stimulated BEEO, despite being diluted approximately seven-fold in PBS, maintained embryo survival rates comparable to optimized commercial medium and exceeded PBS-only controls. These findings position BEEO as a physiologically relevant model for dissecting maternal-embryo interactions in vitro and identifying targets to improve fertility in cattle and other livestock.

Although immunotherapy has shown promise in improving outcomes for bladder cancer (BCa) patients, treatment responses remain highly variable. A comparative examination of the tumor microenvironment (TME) between responders and non-responders may reveal key resistance mechanisms and identify potential therapeutic targets. We integrated spatial transcriptomics, single-cell RNA sequencing, and multiplexed immunofluorescence to characterize spatial structures within the TME that influence response to anti-PD-1 therapy in BCa patients. In non-responders, we observed an accumulation of stem-like malignant epithelial cells with high MYBL2 expression near the tumor boundary. Furthermore, we identified a spatial triad structure—composed of SPP1⁺ tumor-associated macrophages (TAMs), POSTN⁺ cancer-associated fibroblasts (CAFs), and endothelial cells—located at the tumor periphery. This structure was associated with T-cell exclusion and reduced efficacy of immune checkpoint blockade. In a preclinical model, inhibiting SPP1 enhanced the response to anti-PD-1 therapy, resulting in reduced CAF infiltration and increased recruitment of cytotoxic T cells. Our study reveals a triad cellular structure mediated by SPP1⁺ TAMs, POSTN⁺ CAFs, and endothelial cells that contribute to immunotherapy resistance in BCa. Targeting this structure, particularly through SPP1 blockade, represents a promising strategy to augment the efficacy of immune checkpoint inhibitors.

Cholesterol lowering through diet, lifestyle, and pharmacologic therapy remains central for limiting atherosclerosis and prevention of major adverse cardiovascular events. Yet, 33%–50% of individuals on lipid-lowering therapy continue to exhibit elevated inflammation. Middle age (MA) represents a critical window for disease acceleration, underscoring a need to better understand nonresolving inflammation in this time frame. Here, we rendered young (2 months) and MA mice (10 months) hypercholesterolemic with an AAV8-PCSK9 virus and western diet (WD). Following 20 weeks on WD, mice were switched to a chow diet for 6 weeks to induce lipid lowering. This design models dietary cholesterol reduction to dissect lipid-driven versus inflammation-driven pathways in atherosclerosis. At baseline atherosclerosis, we found that MA mice had increased plaque necrosis as well as increased circulating and bone marrow PMN compared to young mice. After chow switch, unlike in young mice, MA mice had increased plaque necrosis and reduced remodeling, as well as increased circulating white blood cells and bone marrow hematopoietic stem cell progenitors (HSPCs). In MA chow-switched mice, circulating neutrophils correlated with necrosis whereas young mice exhibited no correlation. Furthermore, MA atherosclerotic mice had bone marrow HSPCs and neutrophils that exhibited a more activated phenotype relative to young after chow switch. In addition, we observed elevated neutrophil–endothelial contacts in the hippocampal vasculature of MA mice after chow switch. While dietary intervention and lowered plasma cholesterol restrained atheroprogression in young, it was inadequate in MA mice, failing to reduce systemic inflammation and indicating the need for complementary therapies during this time frame.

Hexokinase 2 (HK2) is a pivotal enzyme in glycolytic metabolism and plays a crucial role in the activation of hepatic stellate cells (HSCs). Despite its importance, specific inhibitors targeting HK2 are still lacking in clinical practice. In this study, we utilized a human protein microarray to identify a significant interaction between limonin (Lim) and HK2. Subsequent analyses confirmed that Lim effectively inhibits HK2 activity and the modification of histone lactylation. Moreover, Lim's systemic effects include the downregulation of gene expression associated with HSC activation, which is closely linked to its modulation of HK2 and histone lactylation. Additionally, Lim exhibits anti-fibrotic properties, influencing the expression of genes involved in HSC activation and histone lactylation in vivo. These findings provide novel mechanistic insights into how Lim derivatives may mitigate hepatic fibrosis through the suppression of HK2 and targeted regulation of histone lactylation in activated HSCs.

The cardiac burden associated with doxorubicin (DOX) significantly limits its application in cancer treatment. Therefore, it is essential to identify effective strategies to protect the heart from cardiotoxic damage caused by chemotherapy. As sex is among the risk factors associated with DOX-induced cardiotoxicity, whether the cardiac beneficial effects observed from male mice can be applied to female mice remains unknown. We established a two-week DOX-induced cardiotoxicity model, in which the cumulative DOX dose administered to mice was comparable to that used in previous research. This model effectively induces cardiotoxicity and fibrosis while allowing for a sufficiently long monitoring period to evaluate the chemotherapeutic effects of DOX on tumors, without imposing an excessive physiological burden on the mice from prolonged tumor growth. Utilizing this tumor-bearing murine model, we employed TC-1 cancer cells, which express HPV16-E6 and HPV16-E7 proteins, to investigate the cardioprotective effects of circ-ZNF609 inhibition in DOX-treated tumor-bearing female mice. Our findings indicate that cardiac inhibition of circ-ZNF609 protects against DOX-induced cardiotoxicity without compromising the anti-tumor efficacy of DOX in females. These results suggest that targeting circ-ZNF609 in the heart may represent a promising and viable therapeutic strategy for preventing DOX-induced cardiotoxicity.