American journal of physiology. Cell physiology最新文献

Radiation-induced aortic valve deleterious remodeling may occur years after radiotherapy. The transient receptor potential melastatin 4 (TRPM4) cation channel participates in aortic valve radiation-induced remodeling in mice in vivo. Valvular interstitial cells (VICs) are involved in valve leaflet thickening and calcification leading to aortic stenosis. TRPM4 favors their remodeling toward an osteogenic phenotype in vitro. Here, we evaluated whether radiation-induced remodeling involves TRPM4 in human valvular interstitial cells (hVICs). VICs were isolated from aortic valves and maintained in procalcifying media supplemented or not with 9-phenanthrol (a TRPM4 inhibitor) or small hairpin RNA (shRNA)-TRPM4. Cells were irradiated at 0 Gy or 8 Gy. Ten days after irradiation, cell surface, viability, cycle, and proliferation were measured. Senescence was evaluated by β-galactosidase activity measurements. Osteogenic markers [bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), and alkaline phosphatase (ALP)] and TRPM4 mRNA levels were quantified by quantitative polymerase chain reaction (qPCR). VIC surface increased after radiation, whereas cell density decreased. Radiation had no effect on viability but induced an increase in the proportion of cells in G0 cell cycle phase. An increase of cell senescence was observed after irradiation. Finally, irradiation induced an increase of TRPM4, BMP2, Runx2, and ALP mRNA. All these effects were partly prevented by 9-phenanthrol or shRNA-TRPM4. Interestingly, VIC density on aortic valve leaflets from mice submitted to X-ray treatment in vivo was decreased in treated animals compared with untreated ones, and this was not observed in animals with disruption of the Trpm4 gene. TRPM4 participates in radiation-induced hVICs remodeling by promoting cell senescence and osteogenic transition. TRPM4 may, thus, be evaluated as a therapeutic target to diminish valvular effects of radiotherapy.NEW & NOTEWORTHY The paper demonstrates, by pharmacological and molecular approaches, that the TRPM4 cation channel is involved in radiation-induced osteogenic remodeling of human valvular interstitial cells in culture. Osteogenic remodeling and cell senescence are prevented by TRPM4 inhibition. Since interstitial cells participate in aortic valve remodeling leading to aortic stenosis such as observed in some patients treated for cancer with radiotherapy, TRPM4 inhibition might be evaluated as an interesting new target to avoid this deleterious side effect.

Although the content of mitochondrial enzymes in different tissues can vary greatly, understanding the regulation behind these differences has been hampered by a lack of quantitative knowledge in relation to postnatal development. Here we report a quantitative analysis of developing brain, heart, kidneys, and muscle tissue of C57BL/6J mice, focusing on the content of mitochondrial complex I, a key component of the respiratory chain. We found that in all tissues except kidneys, complex I content gradually increases after birth, reaching a plateau level at around 25 days. Complex I content in muscles does not change significantly until postnatal day 7-10, and then also increases. The greatest increment was found in kidneys, where a 16-fold increase in complex I level after birth was observed. We also found that content of complex I in all postnatal tissues, but muscle, is higher in males than in females. These baseline dynamics of this key mitochondrial flavoprotein serve as a reference for evaluating genetic influences on development and provide a standard for assessing mitochondrial complex I function during postnatal growth.NEW & NOTEWORTHY Mitochondrial complex I is a key enzyme of mammalian oxidative phosphorylation. Here, we provide the first quantitative map of mitochondrial complex I maturation in postnatal mouse tissues. Complex I content rises after birth with striking tissue- and sex-specific patterns, including a dramatic 16-fold increase in kidney. These findings establish a baseline for developmental bioenergetics and a reference for evaluating genetic or disease-related mitochondrial dysfunction.

Glycosaminoglycans (GAGs) are major components of the cells glycocalyx and extracellular matrix (ECM), with important roles in both physiological and disease contexts. The biosynthesis of the long and structurally diverse GAG chains is orchestrated by a complex cellular glycosylation machinery and regulated in an organ-, and cell-specific way. Moreover, altered GAG expression levels and structural features have been described in different pathological conditions, including cancer. Noteworthy, GAG chains are endowed with important functional features during cancer progression, such as cancer cell growth, motility, and metastasis formation. Particularly in gastrointestinal (GI) tumors, GAGs have been frequently associated with tumorigenesis and disease progression. This review provides insights on the aberrant GAG expression profiles in GI cancers, highlighting illustrative examples of GAG structural features for each disease model. Relevance is given to the molecular mechanisms underlying altered GAG biosynthesis and post-synthesis editing in GI cancers. Finally, we address the potential of cancer-associated GAG expression signatures for improving GI clinical management.

The A20 binding inhibitor of nuclear factor-kappa B (NF-κB)-1 (ABIN-1) serves as a ubiquitin sensor and autophagy receptor, crucial for modulating inflammation and cell death. Our previous in vitro investigation identified the microtubule-associated protein 1A/1B-light chain 3 (LC3)-interacting region (LIR) motifs 1 and 2 of ABIN-1 as key mitophagy regulators. This study aimed to explore the in vivo biological significance of ABIN1-LIR domains using a novel CRISPR-engineered ABIN1-ΔLIR1/2 mouse model, which lacks both the LIR motifs. Comprehensive morphological, serum, and tissue histochemical analyses revealed increased body fat and liver weights, altered serum and hepatic lipid profiles, and substantial hepatic lipid droplet accumulation, indicative of altered hepatic lipid metabolism, dyslipidemia, and hepatic steatosis in ABIN1-ΔLIR1/2 mice. Transcriptomic, metabolomic, and lipidomic analyses indicated dysregulated hepatic mitochondrial metabolism, favoring lipogenesis. Mechanistically, LIR1/2 deletion inhibited the expression and activity of transcription factor EB (TFEB) and AMP-activated protein kinase β1 (AMPKβ1), resulting in compromised autophagy and lipophagy. ABIN1 interacted with TFEB and colocalization was observed in both the cytoplasmic and nuclear compartments of hepatocytes. Impaired mitophagy was evidenced by the decreased expression of parkin and optineurin, along with increased levels of mitochondrial cytochrome c oxidase subunit II. These findings were corroborated by liver biopsies of patients with metabolic dysfunction-associated steatotic liver disease. Thus, this study underscores the functional role of ABIN1-LIR motifs in modulating the ABIN1-AMPK-TFEB axis, which is critical for mitochondria-associated lipid metabolism and mitophagy, offering insights into the mechanistic pathways contributing to the pathogenesis of steatosis-associated liver diseases with potential therapeutic implications.NEW & NOTEWORTHY Having identified LC3-interacting region (LIR) motifs 1/2 of A20 binding inhibitor of NF-κB-1 (ABIN-1) as mitophagy regulators in vitro, this study generated CRISPR-engineered ABIN1-ΔLIR1/2 mice lacking both LIR motifs to elucidate its in vivo significance. These mice exhibit enhanced hepatic lipid droplet accumulation, dysregulated mitochondrial metabolism, and impaired mitophagy, through modulation of the AMPK-TFEB axis. Liver biopsies from patients with metabolic dysfunction-associated steatotic liver disease corroborate these findings, suggesting therapeutic implications for liver diseases.

Cancer-associated cachexia decreases the quality of life, reduces therapy response, and diminishes survival prospects. Effective cachexia countermeasures remain a significant unmet need. Research into cancer cachexia has made extensive use of models of colon, lung, and pancreatic cancers. However, although cachexia also affects people with metastatic breast cancer, the mechanisms underlying breast cancer-associated cachexia are relatively understudied. Thus, we sought to investigate orthotopic mouse models of metastatic breast cancer for the progression of cachexia, with a focus on muscle wasting given its role in the frailty that is a hallmark of the condition. Female Balb/c mice received an intramammary fat pad injection of 4T1.2 or EMT6.5 cells, and NOD.SCID.γ (NSG) mice received MDA-MB-231-HM (231-HM) cells, to induce primary breast tumors that were subsequently excised. The resultant metastatic burden after approximately 4 wk led to variable loss of muscle mass (tibialis anterior: EMT6.5: -17.1%, 231-HM: -13.5%, and 4T1.2: -9.5%) and fat mass (gonadal fat: EMT6.5: -75.1%, 231-HM: -62.5%, and 4T1.2: -30.2%). Muscle protein synthesis markers were decreased in EMT6.5 tumor-bearing mice. Distinct increases in the abundance of mRNA for E3-ubiquitin ligase and autophagy-related genes were observed between models. Neuromuscular junction perturbations were observed in EMT6.5 and 4T1.2 tumor-bearing mice. Neutrophilia was noted in the muscles of EMT6.5 tumor-bearing mice. The findings show that muscle mass and function are reduced in mouse models of metastatic breast cancer. Further study of these models could provide useful insights with which to better understand the diversity of cachexia progression across different cancer types.NEW & NOTEWORTHY Cancer-associated cachexia is a significant contributor to poor prognosis for many cancer patients. However, mechanisms underlying cachexia associated with breast cancer are relatively understudied. This project examined mouse models of metastatic triple-negative breast cancer and found that different models exhibited varying degrees of cachexia severity, which were associated with distinct effects upon markers of protein synthesis and breakdown. Further study of these models could help to better understand the diversity of cachexia across cancer types.

Extracellular vesicles (EVs), composed of proteins, lipids, and nucleic acids, are key mediators of intercellular communication across various tissues. However, research on EVs isolated directly from bone tissue remains limited. Here, we identified for the first time and characterized two EV subpopulations extracted from bone tissue and enhanced our understanding of their roles in bone physiology. These two EV subpopulations were reproducibly isolated: a large EV population (lEV; 237.7 ± 8.8 nm) and a small EV population (sEV; 109.2 ± 8.3 nm), both exhibiting the expected shape and presence of EV and bone cell markers. Comparative analysis of their cargos revealed unique or enriched proteins and miRNA profiles for each, suggesting shared functional characteristics with bone cells, including osteocytes, osteoblasts, and osteoclasts. Notably, lEVs contained proteins such as FHL2 and pleiotrophin, along with miRNAs including miR-15b-5p, miR-29a-3p, and miR-128-3p, all of which are involved in early osteogenic signaling pathways such as Hippo, TGF-β, and Wnt. Furthermore, sEVs contained ALP, PAPSS2, and miR-125b, both known regulators of matrix mineralization. Both EV subpopulations were internalized by stromal ST2 and preosteoblastic MC3T3-E1 cells. We present evidence that lEVs significantly enhanced ALP activity in ST2 cells, indicating early osteogenic stimulation, whereas sEVs partially promoted matrix mineralization in primary osteoblasts. Our findings provide novel insights into the role of bone-derived EVs as possible complementary mediators of osteogenesis in vivo and highlight the potential of their cargos in advancing bone regeneration strategies.NEW & NOTEWORTHY This study identifies for the first time two extracellular vesicle (EV) subpopulations isolated from young male mice bone tissue, with distinct mean size and molecular signature. Analysis of the protein and miRNA cargos of these EVs indicates that the large and small EVs are involved in different stages of osteogenesis, from commitment to the formation of mineralized tissue. These findings provide new insights into the role of bone-derived EVs in bone physiology.

Autonomic imbalance is a key driver of cardiovascular disease progression, arising from disrupted interactions between sympathetic and parasympathetic signaling. This review explores the molecular mechanisms underpinning autonomic dysfunction, emphasizing the roles of β-adrenergic receptor (βAR) signaling, cyclic AMP (cAMP) compartmentation, and cholinergic regulation. Dysregulated cAMP nanodomain signaling, βAR desensitization, impaired vagal tone, and maladaptive autonomic nerve remodeling collectively promote structural, electrophysiological, and functional deterioration. Advances in high-resolution imaging and molecular mapping have revealed previously unrecognized pathways governing second-messenger compartmentation and neuromodulatory feedback loops. These insights are driving the development of next-generation therapeutics designed to selectively restore autonomic balance. Promising strategies include isoform-specific phosphodiesterase inhibitors, vagus nerve stimulation, and axonal modulation therapy, which target norepinephrine and acetylcholine pathways while preserving physiological responsiveness. Integrating pharmacological, neuromodulatory, and molecular approaches represents an evolving frontier for cardiovascular therapeutics. Future strategies will benefit from precision mapping of autonomic circuits, patient-specific profiling, and optimization of therapeutic timing. By linking fundamental molecular signaling with translational advances, this review highlights opportunities to improve treatment precision and efficacy for autonomic dysfunction in cardiovascular disease.

Zinc is a commonly used antidiarrheal supplement; however, its exact mechanism of action is not well understood. Calcium-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for secretory diarrhea. CaSR is activated by various cations and here we investigated the roles of CaSR in the antidiarrheal effects of the divalent metal zinc (Zn²⁺). In human intestinal T84 cells expressing CaSR, zinc (100 µM) inhibited forskolin-induced secretory short-circuit current (I_sc) by 60% and its effect was comparable with CaSR activator cinacalcet. Zinc effect was via inhibition of apical CFTR Cl^- channel and basolateral K⁺ channels. In cell models, zinc was a CaSR agonist and its antisecretory effects were CaSR-dependent. Similarly, 100 µM zinc inhibited forskolin-induced secretory I_sc by 40% in wild-type mouse intestine with no antisecretory effects in intestinal epithelia-specific CaSR knockout mice (Casr^flox/flox;Vil1-cre). Zinc inhibited I_sc induced by clinically-relevant cAMP agonists (cholera toxin and vasoactive intestinal peptide) by 65% in T84 cells. Interestingly, zinc had no effect on cGMP agonists (heat-stable E. coli enterotoxin and linaclotide)-induced secretory I_sc, suggesting that its antisecretory effects are specific to cAMP. The mechanisms of zinc effect in T84 cells involved intracellular Ca²⁺ release via ryanodine receptors and inhibition of cAMP synthesis. Our findings suggest that CaSR activation is a major mechanism for the antidiarrheal effects of zinc, which specifically reduces cAMP levels. In addition to its use in cholera, zinc can be effective in other cAMP-mediated secretory diarrheas.NEW & NOTEWORTHY Zinc is a commonly used antidiarrheal supplement; however, its exact mechanisms of antisecretory effects remain unknown. In this study, we demonstrated that zinc is an agonist of the extracellular calcium-sensing receptor (CaSR) and its antidiarrheal effects are via reduced cAMP synthesis in intestinal epithelial cells. In addition to elucidating the mechanism of action of this antidiarrheal supplement, our data support the use of zinc as a simple and effective treatment option for all cAMP-mediated diarrheas.

Chlorine (Cl₂) is a highly reactive halogen gas that undergoes rapid hydrolysis in lung epithelial lining fluid (ELF) upon inhalation, forming hypochlorous acid (HOCl) and hydrochloric acid (HCl). These products subsequently, through chemical reactions, modify the structure and the function of membrane proteins. Herein, we investigated the effects of Cl₂ on connexin-hemichannels and the release of ATP in the ELF. Adult C57BL/6 mice were subjected to 400 ppm Cl₂ for 30 min. Subsequent analysis revealed a marked increase in ATP levels within the BAL, with concentrations reaching 43.952 ± 9.553 nM at 2 h and 30.554 ± 7.383 nM at 24 h post exposure, relative to control. In addition, at 24 h post exposure, the lung wet/dry (W/D) ratio significantly increased from 4.48 ± 0.142 to 5.067 ± 0.359, whereas alveolar fluid clearance (AFC) decreased from 0.249 ± 0.019 to 0.145 ± 0.018. Electrophysiological recordings in alveolar type 2 (AT2) cells revealed reduced open probabilities (P_o) of both ENaC (4 pS) and a cation channel (18 pS), declining from 0.323 ± 0.021 and 0.202 ± 0.022 to 0.151 ± 0.042 and 0.091 ± 0.019, respectively. Instillation of 50 µL of 100 µg/mL Gap27-a connexin mimetic peptide selectively inhibiting connexin-hemichannels-administered 30 min post exposure, restored ATP to control, normalized the W/D ratio, improved AFC, and reestablished ENaC function. Moreover, Gap27 normalized airway resistance following methacholine challenge. In human airway smooth muscle cells (hASMCs), 100 μM ATP induced [Formula: see text] elevation and depolarized V_m to -40 mV, with both effects partially reversed by P2X₇R inhibitor, A804598.NEW & NOTEWORTHY Inhaled chlorine gas reacts with lung epithelial lining fluid to form hypochlorous and hydrochloric acids that alter membrane protein structure and function. Under oxidative stress, connexin hemichannels open, releasing ions and metabolites, such as ATP. The released ATP signals danger, cell death, and tissue injury. Early administration of Gap27, a connexin-hemichannel inhibitor, at 30 min post exposure preserves ENaC function and prevents the subsequent development of pulmonary edema. These compelling findings underscore a promising therapeutic strategy.

Proliferative retinopathies are associated with abnormal angiogenesis that can result in visual impairment or vision loss. The tight junction complex regulates blood-retinal barrier integrity; however, its role in proliferative retinopathies is still at an early stage. Here, we used human retinal microvascular endothelial cells (HRMVECs) and a mouse model of oxygen-induced retinopathy (OIR) to investigate the impact of interleukin-33 (IL-33) signaling on tight junction disintegration and pathological angiogenesis. Our experimental findings demonstrate that IL-33 induces zonula occludens-1 (ZO-1) serine/threonine phosphorylation and tight junction disruption in HRMVECs. In addition, mass spectroscopy analysis revealed that treating HRMVECs with IL-33 induces ZO-1 phosphorylation at the Thr861 residue. Furthermore, we observed that NOX1-PKCδ (protein kinase C-δ) signaling modulates IL-33-induced ZO-1 phosphorylation and tight junction integrity in HRMVECs. We also observed that IL-33 depletion significantly reduces OIR-induced NOX1-PKCδ-ZO-1 signaling and vascular leakage in the ischemic retina. We also observed that the NOX1-specific inhibitor, fluoflavine (ML-090), attenuated OIR-induced NADPH oxidase activity and pathological retinal neovascularization in the ischemic retina. Thus, we infer that IL-33-mediated NOX1-PKCδ-ZO-1 signaling regulates ischemia-induced retinal endothelial cell tight junction disruption and retinal neovascularization.NEW & NOTEWORTHY The significance of blood-retinal barrier integrity in proliferative retinopathies, including retinopathy of prematurity and diabetic retinopathy, is still in its nascent phase. Using human retinal microvascular endothelial cells and an oxygen-induced retinopathy animal model, we demonstrate that IL-33-mediated NOX1-PKCδ-ZO-1 signaling regulates tight junction disruption and blood-retinal barrier integrity in ischemic retinal diseases.