American journal of physiology. Cell physiology最新文献

Aging is an intricate and gradual process characterized by tissue and cellular dysfunction. Adipose-derived mesenchymal stem cells (ADMSCs) experience a functional decline as part of systemic aging. However, the alterations in ADMSCs across various anatomical sites throughout an individual's lifespan remain unclear. To shed light on these changes, we collected white adipose tissue and brown adipose tissue samples from the epididymis, perirenal, inguinal, and scapular regions of young, adult, and aged rats and subsequently isolated ADMSCs for RNA sequencing. As aging progressed, we observed a reduction in the number of ADMSCs at all anatomical sites. Marker genes of ADMSCs from different sites were identified. Aging triggered notable activation of inflammatory and immune responses while diminishing the ADMSC differentiation capacity and ability to maintain a normal tissue morphology. Furthermore, miR-195-5p and miR-497-3p, which promoted cell senescence and apoptosis while inhibiting proliferation and differentiation, were positively correlated with aging. These findings increase our understanding of ADMSC senescence and underscore the unique physiological changes and functions of ADMSCs across different anatomical sites during aging.NEW & NOTEWORTHY Dynamic changes in mRNAs and miRNAs of ADMSCs during aging are shown. As aging progressed, we observed a reduction in the number of ADMSCs at all anatomical sites. Aging leads to the activation of inflammatory and cellular dysfunction. miR-195-5p and miR-497-3p are positively correlated with aging, which promoted cell senescence and apoptosis while inhibiting proliferation and differentiation. ADMSCs associated with different anatomical sites have site-specific markers.

Inhalation exposure to airborne fine particulate matter (aerodynamic diameter: <2.5 µm, PM_2.5) is known to cause metabolic dysfunction-associated steatohepatitis (MASH) and the associated metabolic syndrome. Hepatic lipid accumulation and inflammation are the key characteristics of MASH. However, the mechanism by which PM_2.5 exposure induces lipid accumulation and inflammation in the liver remains to be further elucidated. In this study, we revealed that inhalation exposure to PM_2.5 induces nitrosative stress in mouse livers by suppressing hepatic S-nitrosoglutathione reductase activities, which leads to S-nitrosylation modification of the primary unfolded protein response (UPR) transducer inositol-requiring 1 α (IRE1α), an endoplasmic reticulum-resident protein kinase and endoribonuclease (RNase). S-nitrosylation suppresses the RNase activity of IRE1α and subsequently decreases IRE1α-mediated splicing of the mRNA encoding X-box binding protein 1 (XBP1) and IRE1α-dependent degradation of select microRNAs (miRNAs), including miR-200 family members, miR-34, miR-223, miR-155, and miR-146, in the livers of the mice exposed to PM_2.5. Elevation of IRE1α-target miRNAs, due to impaired IRE1α RNase activity by PM_2.5-triggered S-nitrosylation, leads to decreased expression of the major regulators of fatty acid oxidation, lipolysis, and anti-inflammatory response, including XBP1, sirtuin 1, peroxisome proliferator-activated receptor α, and peroxisome proliferator-activated receptor γ, in the liver, which account at least partially for hepatic lipid accumulation and inflammation in mice exposed to airborne PM_2.5. In summary, our study revealed a novel pathway by which PM_2.5 causes cytotoxicity and promotes MASH-like phenotypes through inducing hepatic nitrosative stress and S-nitrosylation of the primary UPR transducer and subsequent elevation of select miRNAs involved in metabolism and inflammation in the liver.NEW & NOTEWORTHY Exposure to fine airborne particulate matter PM_2.5 causes metabolic dysfunction-associated steatohepatitis characterized by hepatic steatosis, inflammation, and fibrosis. Here, we discovered that inhalation exposure to environmental PM_2.5 induces nitrosative stress in livers by suppressing hepatic S-nitrosoglutathione reductase activities, which leads to S-nitrosylation of the unfolded protein response transducer IRE1α. S-nitrosylation decreases IRE1α-dependent degradation of miRNAs in the livers of mice exposed to PM_2.5, leading to downregulation of major regulators of energy metabolism and anti-inflammatory response.

A complication of type 1 diabetes mellitus (T1DM) is diabetic myopathy that includes reduced regenerative capacity of skeletal muscle. Sphingolipids are a diverse family of lipids with roles in skeletal muscle regeneration. Some studies have found changes in sphingolipid species levels in T1DM, however, the effect of T1DM on a sphingolipid panel in regenerating skeletal muscle has not been examined. Wild-type (WT) and diabetic Ins2^Akita+/- (Akita) mice received cardiotoxin-induced muscle injury in their left quadriceps, gastrocnemius-plantaris-soleus, and tibialis anterior muscles with the contralateral muscles serving as uninjured controls. Muscles were collected at 1, 3, 5, or 7 days postinjury. In regenerating muscle from Akita mice, lipid staining with BODIPY 493/503 revealed increased intramyocellular and total lipids and perilipin-1-positive cell numbers as compared with WT. Liquid chromatography-mass spectrometry of quadriceps was used to identify sphingolipid levels in skeletal muscle. The C22:0 and C24:0 ceramides were significantly elevated in uninjured Akita, whereas ceramide C24:1 was decreased in injured Akita compared with WT. Ceramide-1-phosphate was increased in Akita compared with WT regardless of injury, whereas sphingosine-1-phosphate (S1P) was elevated with injury in WT but this response was muted in Akita mice. Western blotting of key enzymes involved in sphingolipid metabolism revealed S1P lyase, the enzyme that degrades S1P irreversibly, was significantly elevated in the injured muscle in Akita mice during regeneration, in accordance with lower S1P levels. This mouse model of T1DM demonstrates sphingolipidomic changes that may contribute to delayed muscle regeneration.NEW & NOTEWORTHY Muscle lipids become elevated, and the sphingolipid profile is altered by T1DM in skeletal muscle regeneration. A loss of S1P is accompanied by greater expression of sphingosine-1-phosphate lyase (SPL) in response to injury in Akita mice, suggesting a role for sphingolipids in the attenuated repair of skeletal muscle in T1DM rodent models. Although ceramide-1-phosphate (C1P) is increased with T1DM, there was no increase in ceramide kinase (CerK) suggesting an alternative route of ceramide phosphorylation in skeletal muscle.

As cardiomyocyte loss causes heart failure, inhibition of cardiomyocyte death may be a therapeutic strategy against heart failure. In this study, we have identified defender against cell death 1 (Dad1) as a candidate regulator of cardiomyocyte death, using complementary DNA microarray and siRNA knockdown screening. Dad1 is a subunit of oligosaccharyltransferase (OST) complex that is responsible for protein N-glycosylation; however, its function in cardiomyocytes remains unknown. Importantly, the knockdown of Dad1 using siRNA reduced the viability of neonatal rat cardiomyocytes (NRCMs), accompanied by cleaved caspase3 expression, independent of endoplasmic reticulum stress. Dad1 knockdown impaired cell spreading and reduced myofibrillogenesis in NRCMs, suggesting that Dad1 knockdown induced anoikis, apoptosis by disrupting cell-matrix interactions. Consistently, knockdown of Dad1 impaired N-glycosylation of integrins α5 and β1, accompanied by inactivation of focal adhesion kinase. When cell adhesion was enhanced using adhesamine, fibronectin, or collagen type IV, cardiomyocyte death induced by Dad1 knockdown was reduced. Dad1 knockdown decreased the expression of staurosporine and temperature-sensitive 3 A (Stt3A), a catalytic subunit of OST complex. Interestingly, Stt3A knockdown using Stt3A siRNA reduced the expression of Dad1, indicating that both Dad1 and Stt3A were required for OST stabilization. In conclusion, Dad1 plays an important role in maintaining the expression of mature N-glycosylated integrins and their downstream signaling molecules to suppress cardiomyocyte anoikis.NEW & NOTEWORTHY This study found for the first time that the knockdown of Dad1 induced cardiomyocyte death, accompanied by impairment of myofibrillogenesis and cell spreading. Dad1 regulates the N-glycosylation of integrins in cooperation with Stt3A and preserves cell adhesion activity, promoting cardiomyocyte survival. This is the first demonstration that Dad1 contributes to the maintenance of cardiac homeostasis through the posttranslational modification of integrins, providing a novel insight into the biological significance of OST complex in cardiomyocytes.

The present study investigated the role of endothelial brain-derived neurotrophic factor (BDNF) in cognition. Male adult mice with a selective knockout of BDNF in endothelial cells (BDNF^ECKO) and their wild-type (WT) littermates were subjected to tests for detection of anxiety- and depression-like behaviors and impaired recognition memory. Neuronal activity and synaptogenesis were assessed from hippocampal levels of c-fos and synaptophysin, respectively, and cerebral capillary density from forebrain levels of CD31. BDNF/TrkB (tropomyosin-related kinase type B) receptor signaling was investigated through hippocampal levels of BDNF and activated TrkB receptors coupled with their immunolabeling by neurons and endothelial cells from both cerebrovascular fractions enriched in capillaries and hippocampal arterioles. Endothelial nitric oxide (NO) production was assessed from the expression of endothelial NO synthase phosphorylated at serine 1177. BDNF^ECKO mice exhibited anxio-depressive phenotype, impaired memory, and reduced synaptogenesis. Neither neuronal activity, neuronal BDNF/TrkB signaling, nor capillary density differed between BDNF^ECKO and WT mice. However, endothelial-activated TrkB receptors as well as endothelial NO production and hippocampal BDNF levels were lower in BDNF^ECKO than those in WT mice. We conclude that endothelial BDNF is involved in cognition through mechanisms independent of neuronal BDNF/TrkB signaling and that endothelial NO might be a driver of the procognitive effect of endothelial BDNF.NEW & NOTEWORTHY The study provides the proof of concept that endothelial brain-derived neurotrophic factor (BDNF) plays a crucial role in postnatal synaptogenesis and development of behavior/memory. It also shows that neuronal tropomyosin-related kinase type B (TrkB) receptors are not a target of endothelium-derived BDNF.

Osteosarcoma (OS) is a highly malignant tumor, and chemotherapy resistance is a major challenge in the treatment of this disease. This study aims to explore the role of the CLTC-VMP1 gene fusion in the mechanism of chemotherapy resistance in OS and investigate its molecular mechanisms in mediating energy metabolism reprogramming by regulating autophagy and apoptosis balance. Using single-cell transcriptome analysis, the heterogeneity of OS cells and their correlation with resistance to platinum drugs were revealed. Cisplatin-resistant cell lines were established in human OS cell lines for subsequent experiments. Based on transcriptomic analysis, the importance of VMP1 in chemotherapy resistance was confirmed. Lentiviral vectors overexpressing or interfering with VMP1 were used, and it was observed that inhibiting VMP1 could reverse cisplatin resistance, promote cell apoptosis, and inhibit autophagy, and mitochondrial respiration and glycolysis. Furthermore, the presence of CLTC-VMP1 gene fusion was validated, and its ability to regulate autophagy and apoptosis balance, promote mitochondrial respiration, and glycolysis was demonstrated. Mouse model experiments further confirmed the promoting effect of CLTC-VMP1 on tumor growth and chemotherapy resistance. In summary, the CLTC-VMP1 gene fusion mediates energy metabolism reprogramming by regulating autophagy and apoptosis balance, which promotes chemotherapy resistance in OS.NEW & NOTEWORTHY This study identifies the CLTC-VMP1 gene fusion as a key driver of chemotherapy resistance in osteosarcoma by regulating autophagy and reprogramming cellular energy metabolism. Through single-cell transcriptomics, the research reveals the heterogeneity of tumor cells and the role of VMP1 in promoting resistance to cisplatin. The findings suggest that targeting the CLTC-VMP1 fusion gene may offer new therapeutic strategies to overcome chemotherapy resistance in osteosarcoma.

Cancer cachexia affects up to 80% of patients with cancer and results in reduced quality of life and survival. We previously demonstrated that the transcriptional repressor Forkhead box P1 (FoxP1) is upregulated in the skeletal muscle of cachectic mice and people with cancer, and when overexpressed in skeletal muscle, it is sufficient to induce pathological features characteristic of cachexia. However, the role of myofiber-derived FoxP1 in both normal muscle physiology and cancer-induced muscle wasting remains largely unexplored. To address this gap, we generated a conditional mouse line with myofiber-specific ablation of FoxP1 (FoxP1^SkmKO) and found that in cancer-free mice, deletion of FoxP1 in skeletal myofibers resulted in increased myofiber size in both males and females, with a significant increase in muscle mass in males. In response to murine KPC pancreatic tumor burden, we found that myofiber-derived FoxP1 mediates cancer-induced muscle wasting and diaphragm muscle weakness in male but not female mice. In summary, our findings identify myofiber-specific FoxP1 as a negative regulator of skeletal muscle with sex-specific differences in the context of cancer.NEW & NOTEWORTHY Here we identify myofiber-derived FoxP1 as a negative regulator of skeletal muscle with sex-specific effects in cancer. Under cancer-free conditions, FoxP1 knockout increased myofiber size in male and female mice. However, in response to pancreatic cancer, FoxP1 myofiber-specific deletion attenuated muscle wasting and weakness in males but not females. This highlights the need to consider sexual dimorphism in cancer-induced muscle pathologies and provides evidence suggesting that targeting FoxP1 could help mitigate these effects in males.

Human skeletal muscle displays an epigenetic memory of resistance exercise induced-hypertrophy. It is unknown, however, whether high-intensity interval training (HIIT) also evokes an epigenetic muscle memory. This study used repeated training intervention interspersed with a detraining period to assess epigenetic memory of HIIT. Twenty healthy subjects (25 ± 5 yr) completed two HIIT interventions (training and retraining) lasting 2 mo, separated by 3 mo of detraining. Measurements at baseline, after training, detraining, and retraining included maximal oxygen consumption (V̇o_2max). Vastus lateralis biopsies were taken for genome-wide DNA methylation and targeted gene expression analyses. V̇o_2max improved during training and retraining (P < 0.001) without differences between interventions (P > 0.58). Thousands of differentially methylated positions (DMPs) predominantly demonstrated a hypomethylated state after training, retained even after 3-mo of exercise cessation and into retraining. Five genes, ADAM19, INPP5a, MTHFD1L, CAPN2, and SLC16A3, possessed differentially methylated regions (DMRs) with retained hypomethylated memory profiles and increased gene expression. The retained hypomethylation during detraining was associated with an enhancement in expression of the same genes even after 3 mo of detraining. SLC16A3, INPP5a, and CAPN2 are involved in lactate transport and calcium signaling. Despite similar physiological adaptations between training and retraining, memory profiles were found at epigenetic and gene expression level, characterized by retained hypomethylation and increased gene expression after training into long-term detraining and retraining. These genes were associated with calcium signaling and lactate transport. Although significant memory was not observed in physiological parameters, our novel findings indicate that human skeletal muscle possesses an epigenetic memory of HIIT.NEW & NOTEWORTHY Cells possess a "memory" such that adaptations can be more quickly regained when a previously encountered challenge is reintroduced. Exercise provides an excellent experimental model to explore the concept of cellular memory to physiologically relevant stressors in humans. This study highlights molecular mechanisms that contribute to muscle memory in response to high-intensity interval training in humans, showing retention of DNA methylation and gene expression profiles from earlier training into detraining and retraining.

Oxidative stress from placental ischemia/reperfusion and hypoxia/reoxygenation (H/R) in preeclampsia is accompanied by Na⁺-K⁺ pump inhibition and S-glutathionylation of its β1 subunit (GSS-β1), a modification that inhibits the pump. β3-adrenergic receptor (β3-AR) agonists can reverse GSS-β1. We examined the effects of the agonist CL316,243 on GSS-β1 and sources of H/R-induced oxidative stress in immortalized first-trimester human trophoblast (HTR-8/SVneo) and freshly isolated placental explants from normal-term pregnancies. H/R increased GSS-β1 and, reflecting compromised α1/β1 subunit interaction, reduced α1/β1 pump subunit coimmunoprecipitation. H/R increased p47^phox/p22^phox NADPH oxidase subunit coimmunoprecipitation, reflecting membrane translocation of cytosolic p47^phox that is needed to activate NADPH oxidase. Fluorescence of O₂^•--sensitive dihydroethidium increased in parallel. H/R increased S-glutathionylation of endothelial nitric oxide synthase (GSS-eNOS) that uncouples nitric oxide synthesis toward the synthesis of O₂^•- and reduced trophoblast migration. Oxidative stress induced by tumor necrosis factor α increased soluble fms-like tyrosine kinase receptor 1 (sFlt-1) trophoblast release, a marker of preeclampsia, and reduced trophoblast integration into endothelial cellular networks. CL316,243 eliminated H/R-induced GSS-β1 and decreases of α1/β1 subunit coimmunoprecipitation, eliminated NADPH oxidase activation and increases in GSS-eNOS, restored trophoblast migration, eliminated increased sFlt-1 release, and restored trophoblast integration in endothelial cell networks. H/R-induced GSS-β1, α1/β1 subunit coimmunoprecipitation, and NADPH oxidase activation of placental explants reflected effects of H/R for trophoblasts and CL316,243 eliminated these changes. We conclude a β3-AR agonist counters key pathophysiological features of preeclampsia in vitro. β3 agonists already in human use for another purpose are potential candidates for repurposing to treat preeclampsia.NEW & NOTEWORTHY H/R-induced oxidative stress and deficient NO-dependent placentation are features of preeclampsia, yet nonspecific antioxidants and NO donors are ineffective. Here, activation of the microdomain-confined signaling pathway with an agonist for the eNOS-coupled β3-AR eliminates inhibitory glutathionylation of the Na⁺-K⁺ pump's β1 subunit, uncoupling of eNOS, and activation of NADPH oxidase that are sources of H/R-induced oxidative stress. The agonist also eliminates H/R-induced inhibition of trophoblast migration and their integration into an endothelial network.

Emerging studies have reported the vital role of histone modification in the dysfunction of pulmonary vascular endothelial cells, which acts as the key reason to drive the hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension (PH). This study aims to investigate the role of a histone 3 lysine 9 (H3K9) methyltransferase, SET domain bifurcated 1 (SETDB1), in hypoxia-induced functional and phenotypical changes of pulmonary vascular endothelial cells. Primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as cell model. Specific knockdown and overexpression strategies were used to systematically determine the molecular regulation and function of SETDB1 in PMVECs. SETDB1 is highly expressed and significantly upregulated in the pulmonary vascular endothelium of lung tissue isolated from SU5416/hypoxia-induced PH (SuHx-PH) rats and also in pulmonary arterial endothelial cells (PAECs) from patients with idiopathic pulmonary arterial hypertension (IPAH), comparing with their respective controls. In primarily cultured rat PMVECs, treatment of hypoxia or CoCl₂ induces significant upregulation of HIF2α, SETDB1, and H3K9me3. Specific knockdown and overexpression strategies indicate that the hypoxia- or CoCl₂-induced upregulation of SETDB1 is mediated through a HIF2α-dependent mechanism. Knockdown of SETDB1 significantly inhibits the hypoxia- or CoCl₂-induced apoptosis, senescence, and endothelial to mesenchymal transition (EndoMT) in rat PMVECs. Moreover, treatment of the specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of SuHx-PH in rat. Our results suggest that the HIF2α-dependent upregulation of SETDB1 facilitates hypoxia-induced functional and phenotypical changes of PMVECs, potentially contributing to the hypoxia-induced pulmonary vascular remodeling and PH.NEW & NOTEWORTHY Abnormal histone modification plays vital role in pulmonary hypertension (PH). This study reports the regulation and role of a histone 3 lysine 9 (H3K9) methyltransferase, SETDB1, in primarily cultured rat pulmonary microvascular endothelial cells (PMVECs). Hypoxia induces significant upregulation of SETDB1 at both mRNA and protein levels, in a HIF2α-dependent manner. The hypoxic upregulation of SETDB1 leads to significant apoptosis, senescence, and endothelial-to-mesenchymal transition in PMVECs. Treatment of a specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of PH rat model induced by SU5416/hypoxia.