bims-midhyp Biomed News
on Mitochondrial dysfunction and hypoxia
Issue of 2023‒06‒11
24 papers selected by
Alia Ablieh, Universität Heidelberg



  1. Biophys Rep. 2021 Jun 30. 7(3): 239-249
      Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.
    Keywords:  Astrocyte; Hypoxia; Hypoxia-inducible factor 1; Nitric oxide; Reactive oxygen species
    DOI:  https://doi.org/10.52601/bpr.2021.200016
  2. Proc Natl Acad Sci U S A. 2023 Jun 13. 120(24): e2216310120
      Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating F1Fo-ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal.
    Keywords:  F1Fo-ATP synthase; IGF/insulin signaling; mitochondrial membrane potential; reactive oxygen species; serum- and glucocorticoid-regulated kinase 1
    DOI:  https://doi.org/10.1073/pnas.2216310120
  3. Nature. 2023 Jun 07.
      The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.
    DOI:  https://doi.org/10.1038/s41586-023-06142-0
  4. Plant J. 2023 Jun 09.
      Arabidopsis mitochondria-targeted heat shock protein 70 (mtHSC70-1) plays important roles in the establishment of cytochrome c oxidase-dependent respiration and redox homeostasis during vegetative growth of plants. Here, we report that knocking out the mtHSC70-1 gene led to a decrease of plant fertility; the fertility defect of the mutant was completely rescued by introducing the mtHSC70-1 gene. mtHSC70-1 mutants also showed defects in female gametophyte (FG) development, including delayed mitosis, abnormal nuclear position and ectopic gene expression in the embryo sacs. In addition, we found that an Arabidopsis mitochondrial J-protein gene (DjA30) mutant, j30+/- , had defects in FG development and fertility similar to those of mtHSC70-1 mutant. mtHSC70-1 and DjA30 had similar expression patterns in FGs and interacted in vivo, suggesting that these two proteins might cooperate during female gametogenesis. Further, respiratory chain complex IV activity in mtHSC70-1 and DjA30 mutant embryo sacs was markedly downregulated; this led to the accumulation of mitochondrial reactive oxygen species (ROS). Scavenging excess ROS by introducing Mn-superoxide dismutase 1 or catalase 1 gene into the mtHSC70-1 mutant rescued FG development and fertility. Altogether, our results suggest that mtHSC70-1 and DjA30 are essential for the maintenance of ROS homostasis in the embryo sacs, and provide direct evidence for the roles of ROS homostasis in embryo sac maturation and nuclear patterning, which might determine the fate of gametic and accessory cells.
    Keywords:  Arabidopsis thaliana; DjA30; cell fate; female gametophyte; fertility; mtHSC70-1; reactive oxygen species
    DOI:  https://doi.org/10.1111/tpj.16347
  5. Comput Struct Biotechnol J. 2023 ;21 3103-3108
      Heat shock proteins (HSPs) are part of the cell's molecular chaperone system responsible for the proper folding (or refolding) of proteins. They are expressed in cells of a wide variety of organisms, from bacteria and fungi to humans. While some HSPs require metal ions for proper functioning, others are expressed as a response of the organism to either essential or toxic metal ions. Their presence can influence the occurrence of cellular processes, even those as significant as programmed cell death. The development of research methods and structural modeling has enabled increasingly accurate recognition of new HSP functions, including their role in maintaining metal ion homeostasis. Current investigations on the expression of HSPs in response to heavy metal ions include not only the direct effect of these ions on the cell but also analysis of reactive oxygen species (ROS) and the increased production of HSPs with increasing ROS concentration. This minireview contains information about the direct and indirect interactions of heat shock proteins with metal ions, both those of biological importance and heavy metals.
    Keywords:  Calcium homeostasis; Heat shock proteins; Metal ions; ROS sensors
    DOI:  https://doi.org/10.1016/j.csbj.2023.05.024
  6. Biochem J. 2023 Jun 15. 480(11): 773-789
      Glucose-regulated insulin secretion becomes defective in all forms of diabetes. The signaling mechanisms through which the sugar acts on the ensemble of beta cells within the islet remain a vigorous area of research after more than 60 years. Here, we focus firstly on the role that the privileged oxidative metabolism of glucose plays in glucose detection, discussing the importance of 'disallowing' in the beta cell the expression of genes including Lactate dehydrogenase (Ldha) and the lactate transporter Mct1/Slc16a1 to restrict other metabolic fates for glucose. We next explore the regulation of mitochondrial metabolism by Ca2+ and its possible role in sustaining glucose signaling towards insulin secretion. Finally, we discuss in depth the importance of mitochondrial structure and dynamics in the beta cell, and their potential for therapeutic targeting by incretin hormones or direct regulators of mitochondrial fusion. This review, and the 2023 Sir Philip Randle Lecture which GAR will give at the Islet Study Group meeting in Vancouver, Canada in June 2023, honor the foundational, and sometimes under-appreciated, contributions made by Professor Randle and his colleagues towards our understanding of the regulation of insulin secretion.
    Keywords:  diabetes; glucose homeostasis; hormone secretion; insulin; mitochondria; pancreatic beta cell
    DOI:  https://doi.org/10.1042/BCJ20230167
  7. Redox Rep. 2023 Dec;28(1): 2218684
      OBJECTIVE: To investigate the effects of glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide on endothelial dysfunction in LDL receptor-deficient (LDLR-KO) mice and ox-LDL-challenged human umbilical vein endothelial cells (HUVECs) and its possible mechanism.METHODS: LDLR-KO mice were randomly treated with normal saline, liraglutide, or liraglutide plus a GLP-1R antagonist exendin-9 for four weeks. In parallel, HUVECs were cultured with ox-LDL alone or combined with liraglutide, in the presence or absence of lectin-like ox-LDL receptor-1(LOX-1) overexpression or GLP-1R knockdown. Endothelial-dependent relaxation and LOX-1 protein expression of thoracic aorta, circulating levels of oxidative and inflammatory markers in mice, and cell survival, reactive oxygen species production, and expression of adhesion molecules and signal regulators in ox-LDL cultured endothelial cells were measured.
    RESULTS: liraglutide effectively enhanced acetylcholine-induced vasodilation, reduced LOX-1 expression in aortas, and decreased circulatory oxidative and inflammatory levels in LDLR-KO mice, which were abolished by cotreatment with exendin-9. HUVECs exposed to ox-LDL exhibited reduced cell viability, increased reactive oxygen species production and apoptosis, and elevated protein expression of ICAM-1, VCAM-1, LOX-1, NOX4, and NF-κB, which were markedly ameliorated by liraglutide treatment. The protective effects of liraglutide against ox-LDL-induced cell injury were abrogated in HUVECs overexpressing LOX-1 or silencing GLP-1R.
    CONCLUSIONS: Liraglutide improved oxidized LDL-induced endothelial dysfunction via GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation.
    Keywords:  Liraglutide; endothelial dysfunction; inflammation; lectin-like ox-LDL receptor-1; oxidative stress; oxidized low-density lipoprotein
    DOI:  https://doi.org/10.1080/13510002.2023.2218684
  8. Am J Physiol Cell Physiol. 2023 Jun 05.
      Mitochondrial function is widely recognized as a major determinant of health, emphasizing the importance of understanding the mechanisms promoting mitochondrial quality in various tissues. Recently, the mitochondrial unfolded protein response (UPRmt) has come into focus as a modulator of mitochondrial homeostasis, particularly in stress conditions. In muscle, the necessity for ATF4 and its role in regulating mitochondrial quality control (MQC) has yet to be determined. We overexpressed (OE) and knocked down ATF4 in C2C12 myoblasts, differentiated them to myotubes for 5 days, and subjected them to acute (ACA) or chronic (CCA) contractile activity. ATF4 mediated myotube formation through the regulated expression of myogenic factors, mainly Myc and MyoD, and supressed mitochondrial biogenesis basally through PGC-1a. However, our data also show that ATF4 expression levels are directly related to mitochondrial fusion and dynamics, UPRmt activation, as well as lysosomal biogenesis and autophagy. Thus, ATF4 promoted enhanced mitochondrial networking, protein handling, and capacity for clearance of dysfunctional organelles under stress conditions, despite lower levels of mitophagy flux with OE. Indeed, we found that ATF4 promoted the formation of a smaller pool of high functioning mitochondria that are more responsive to contractile activity, have higher oxygen consumption rates and lower reactive oxygen species levels. These data provide evidence that ATF4 is both necessary and sufficient for mitochondrial quality control and adaptation during both differentiation and contractile activity, thus advancing the current understanding of ATF4 beyond its canonical functions, to include the regulation of mitochondrial morphology, lysosomal biogenesis and mitophagy in muscle cells.
    Keywords:  ATF4; mitochondrial quality control; mitochondrial unfolded protein response; mitophagy and lysosomal biogenesis; skeletal muscle C2C12
    DOI:  https://doi.org/10.1152/ajpcell.00080.2023
  9. Development. 2023 Jun 01. pii: dev201408. [Epub ahead of print]150(11):
      The central nervous system contains a myriad of different cell types produced from multipotent neural progenitors. Neural progenitors acquire distinct cell identities depending on their spatial position, but they are also influenced by temporal cues to give rise to different cell populations over time. For instance, the progenitors of the cerebral neocortex generate different populations of excitatory projection neurons following a well-known sequence. The Notch signaling pathway plays crucial roles during this process, but the molecular mechanisms by which Notch impacts progenitor fate decisions have not been fully resolved. Here, we show that Notch signaling is essential for neocortical and hippocampal morphogenesis, and for the development of the corpus callosum and choroid plexus. Our data also indicate that, in the neocortex, Notch controls projection neuron fate determination through the regulation of two microRNA clusters that include let-7, miR-99a/100 and miR-125b. Our findings collectively suggest that balanced Notch signaling is crucial for telencephalic development and that the interplay between Notch and miRNAs is essential for the control of neocortical progenitor behaviors and neuron cell fate decisions.
    Keywords:  Cell fate; Cortical development; Mouse; Neurogenesis; Notch; miRNA
    DOI:  https://doi.org/10.1242/dev.201408
  10. Int Endod J. 2023 Jun 09.
      AIM: Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells, and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vascular ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model.METHODOLOGY: HUVECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-β1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM), and HUVEC+DPSC cocultures (E+D-CM) were analyzed by enzyme-linked immunosorbent assay. TGF-β RI kinase inhibitor VI, SB431542, was used to block TGF-β1/ALK5 signaling in DPSCs.
    RESULTS: The expression of SMC-specific markers, α-SMA, SM22α, and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-β1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-β1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-β1/ALK5 signaling inhibited the expression of α-SMA, SM22α, and Calponin in DPSCs.
    CONCLUSIONS: TGF-β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-β1/ALK5 signaling pathway played a vital role in this process.
    Keywords:  TGF-β1; dental pulp stem cells; differentiation; smooth muscle cells
    DOI:  https://doi.org/10.1111/iej.13943
  11. Sci Rep. 2023 06 03. 13(1): 9044
      Proper lipid metabolism is crucial to maintain alveolar epithelial cell (AEC) function, and excessive AEC death plays a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The mRNA expression of fatty acid synthase (FASN), a key enzyme in the production of palmitate and other fatty acids, is downregulated in the lungs of IPF patients. However, the precise role of FASN in IPF and its mechanism of action remain unclear. In this study, we showed that FASN expression is significantly reduced in the lungs of IPF patients and bleomycin (BLM)-treated mice. Overexpression of FASN significantly inhibited BLM-induced AEC death, which was significantly potentiated by FASN knockdown. Moreover, FASN overexpression reduced BLM-induced loss of mitochondrial membrane potential and the production of mitochondrial reactive oxygen species (ROS). Oleic acid, a fatty acid component increased by FASN overexpression, inhibited BLM-induced cell death in primary murine AECs and rescue BLM induced mouse lung injury/fibrosis. FASN transgenic mice exposed to BLM exhibited attenuated lung inflammation and collagen deposition compared to controls. Our findings suggest that defects in FASN production may be associated with the pathogenesis of IPF, especially mitochondrial dysfunction, and augmentation of FASN in the lung may have therapeutic potential in preventing lung fibrosis.
    DOI:  https://doi.org/10.1038/s41598-023-36009-3
  12. FEBS Lett. 2023 Jun 07.
      Fluctuations in nutrient and biomass availability, often as a result of disease, impart metabolic challenges that must be overcome in order to sustain cell survival and promote proliferation. Cells adapt to these environmental changes and stresses by adjusting their metabolic networks through a series of regulatory mechanisms. Our understanding of these rewiring events has largely been focused on those genetic transformations that alter protein expression and the biochemical mechanisms that change protein behavior, such as post-translational modifications and metabolite-based allosteric modulators. Mounting evidence suggests that a class of proteome surveillance proteins called molecular chaperones also can influence metabolic processes. Here, we summarize several ways the Hsp90 and Hsp70 chaperone families act on human metabolic enzymes and their supramolecular assemblies to change enzymatic activities and metabolite flux. We further highlight how these chaperones can assist in the translocation and degradation of metabolic enzymes. Collectively, these studies provide a new view for how metabolic processes are regulated to meet cellular demand and inspire new avenues for therapeutic intervention.
    Keywords:  cellular metabolism; metabolons; molecular chaperones; protein degradation; protein folding; supramolecular complexes
    DOI:  https://doi.org/10.1002/1873-3468.14682
  13. Mol Metab. 2023 Jun 06. pii: S2212-8778(23)00082-0. [Epub ahead of print] 101748
      OBJECTIVE: Cancer cells convert more glucose into lactate than healthy cells, what results in their growth advantage. Pyruvate kinase (PK) is a key rate limiting enzyme in this process, what makes it a promising potential therapeutic target. However, currently it is still unclear what consequences the inhibition of PK has on cellular processes. Here, we systematically investigate the consequences of PK depletion for gene expression, histone modifications and metabolism.METHODS: Epigenetic, transcriptional and metabolic targets were analysed in different cellular and animal models with stable knockdown or knockout of PK.
    RESULTS: Depleting PK activity reduces the glycolytic flux and causes accumulation of glucose-6-phosphate (G6P). Such metabolic perturbation results in stimulation of the activity of a heterodimeric pair of transcription factors MondoA and MLX but not in a major reprogramming of the global H3K9ac and H3K4me3 histone modification landscape. The MondoA:MLX heterodimer upregulates expression of thioredoxin-interacting protein (TXNIP) - a tumour suppressor with multifaceted anticancer activity. This effect of TXNIP upregulation extends beyond immortalised cancer cell lines and is applicable to multiple cellular and animal models.
    CONCLUSIONS: Our work shows that actions of often pro-tumorigenic PK and anti-tumorigenic TXNIP are tightly linked via a glycolytic intermediate. We suggest that PK depletion stimulates the activity of MondoA:MLX transcription factor heterodimers and subsequently, increases cellular TXNIP levels. TXNIP-mediated inhibition of thioredoxin (TXN) can reduce the ability of cells to scavenge reactive oxygen species (ROS) leading to the oxidative damage of cellular structures including DNA. These findings highlight an important regulatory axis affecting tumour suppression mechanisms and provide an attractive opportunity for combination cancer therapies targeting glycolytic activity and ROS-generating pathways.
    Keywords:  ROS; arrestins; cancer; glycolysis; metabolic flux; pyruvate kinase; thioredoxin-interacting protein
    DOI:  https://doi.org/10.1016/j.molmet.2023.101748
  14. Med Oncol. 2023 Jun 09. 40(7): 199
      Colorectal cancer (CRC) is a prevalent gastrointestinal neoplasm that ranks fourth in terms of cancer-related deaths worldwide. In the process of CRC progression, multiple ubiquitin-conjugating enzymes (E2s) are involved; UBE2Q1 is one of those newly identified E2s that is markedly expressed in human colorectal tumors. Since p53 is a well-known tumor suppressor and defined as a key factor to be targeted by the ubiquitin-proteasome system, we hypothesized that UBE2Q1 might contribute to CRC progression through the modulation of p53. Using the lipofection method, the cultured SW480 and LS180 cells were transfected with the UBE2Q1 ORF-containing pCMV6-AN-GFP vector. Then, quantitative RT-PCR was used to assay the mRNA expression levels of p53's target genes, i.e., Mdm2, Bcl2, and Cyclin E. Moreover, Western blot analysis was performed to confirm the cellular overexpression of UBE2Q1 and assess the protein levels of p53, pre- and post-transfection. The expression of p53's target genes were cell line-dependent except for Mdm2 that was consistent with the findings of p53. The results of Western blotting demonstrated that the protein levels of p53 were greatly lower in UBE2Q1-transfected SW480 cells compared to the control SW480 cells. However, the reduced levels of p53 protein were not remarkable in the transfected LS180 cells compared to the control cells. The suppression of p53 is believed to be the result of UBE2Q1-dependent ubiquitination and its subsequent proteasomal degradation. Furthermore, the ubiquitination of p53 can act as a signal for degradation-independent functions, such as nuclear export and suppressing the p53's transcriptional activities. In this context, the decreased Mdm2 levels can moderate the proteasome-independent mono-ubiquitination of p53. The ubiquitinated p53 modulates the transcriptional levels of target genes. Therefore, the up-modulation of UBE2Q1 may influence the transcriptional activities depending on p53, and thereby contributes to CRC progression through regulating the p53.
    Keywords:  Bcl2; Colorectal neoplasms; Cyclin E; Mdm2; Tumor suppressor protein p53; UBE2Q1
    DOI:  https://doi.org/10.1007/s12032-023-02039-0
  15. Cells Dev. 2023 Jun 01. pii: S2667-2901(23)00036-0. [Epub ahead of print]175 203860
      Peroxiredoxins (Prdxs) are thiol-dependent enzymes that scavenge peroxides. Previously, we found that Prdxs were hyperoxidized in a Parkinson's disease model induced by paraquat (PQ), which led to their inactivation, perpetuating reactive oxygen species (ROS) formation. Herein, we evaluated the redox state of the typical 2-Cys-Prx subgroup. We found that PQ induces ROS compartmentalization in different organelles, reflected by the 2-Cys-Prdx hyperoxidation pattern detected by redox eastern blotting. 2-Cys Prdxs are most vulnerable to hyperoxidation, while atypical 2-Cys Peroxiredoxin 5 (Prdx5) is resistant and is expressed in multiple organelles, such as mitochondria, peroxisomes, and cytoplasm. Therefore, we overexpressed human Prdx5 in the dopaminergic SHSY-5Y cell line using the adenoviral vector Ad-hPrdx5. Prdx5 overexpression was confirmed by western blotting and immunofluorescence (IF) and effectively decreased PQ-mediated mitochondrial and cytoplasmic ROS assessed with a mitochondrial superoxide indicator and DHE through IF or flow cytometry. Decreased ROS mediated by Prdx5 in the main subcellular compartments led to overall cell protection against PQ-induced cell death, which was demonstrated by flow cytometry using Annexin V labeling and 7-AAD. Therefore, Prdx5 is an attractive therapeutic target for PD, as its overexpression protects dopaminergic cells from ROS and death, which warrants further experimental animal studies for its subsequent application in clinical trials.
    Keywords:  Antioxidant enzymes; Paraquat; Parkinson's disease; Peroxiredoxins; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.cdev.2023.203860
  16. Invest Ophthalmol Vis Sci. 2023 06 01. 64(7): 13
      Purpose: Diabetic retinopathy (DR) is a significant cause of blindness. Most research around DR focus on late-stage developments rather than early changes such as early endothelial dysfunction. Endothelial-to-mesenchymal transition (EndMT), an epigenetically regulated process whereby endothelial cells lose endothelial characteristics and adopt mesenchymal-like phenotypes, contributes to early endothelial changes in DR. The epigenetic regulator microRNA 9 (miR-9) is suppressed in the eyes during DR. MiR-9 plays a role in various diseases and regulates EndMT-related processes in other organs. We investigated the role miR-9 plays in glucose-induced EndMT in DR.Methods: We examined the effects of glucose on miR-9 and EndMT using human retinal endothelial cells (HRECs). We then used HRECs and an endothelial-specific miR-9 transgenic mouse line to investigate the effect of miR-9 on glucose-induced EndMT. Finally, we used HRECs to probe the mechanisms through which miR-9 may regulate EndMT.
    Results: We found that miR-9 inhibition was both necessary and sufficient for glucose-induced EndMT. Overexpression of miR-9 prevented glucose-induced EndMT, whereas suppressing miR-9 caused glucose-like EndMT changes. We also found that preventing EndMT with miR-9 overexpression improved retinal vascular leakage in DR. Finally, we showed that miR-9 regulates EndMT at an early stage by regulating EndMT-inducing signals such as proinflammatory and TGF-β pathways.
    Conclusions: We have shown that miR-9 is an important regulator of EndMT in DR, potentially making it a good target for RNA-based therapy in early DR.
    DOI:  https://doi.org/10.1167/iovs.64.7.13
  17. Discov Med. 2023 Jun;35(176): 361-371
      BACKGROUND: Colorectal cancer is a common digestive tract malignancy. This study aimed to expound the functional role of fatty-acid-binding protein 4 (FABP4) and the potential underlying mechanisms in the development of colorectal cancer.METHODS: Several techniques were utilized to investigate the role of FABP4 in colorectal cancer. FABP4 mRNA expression was quantified using Real time-quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), sphere formation assays and flow cytometry evaluated cell growth, stemness, and apoptosis in SW480 and HT29 cells. Glycolysis was assessed via extracellular acidification rate (ECAR) , lactate production, glucose uptake, adenosine triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio, and Glut1 and Elevated lactate dehydrogenase A (LDHA) protein expression. Reactive oxygen species (ROS) levels were analyzed by flow cytometry. Western blot measured the protein expression of FABP4, Proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Glut1, LDHA, stemness makers (Sox2, Oct4, and ALDHA1), and extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) pathway proteins. In vivo experiments, BALB/c nude mice (n = 12) were inoculated with 200 μL HT29 cells (5 × 106 cells) transfected with sh-FABP4 or short hairpin (sh)-negative control (NC), forming two groups with 6 mice each. The in vivo mice tumor model allowed for evaluating FABP4's impact on tumor growth.
    RESULTS: FABP4 was significantly upregulated in colorectal cancer tissues and cells (p < 0.05). FABP4 knockdown markedly inhibited cell proliferation, stemness, and glycolysis, while promoting apoptosis in these cells (p < 0.05). Additionally, FABP4 depletion led to a significant increase in ROS level (p < 0.05). However, N-acetyl-L-cysteine (NAC) (p < 0.05), a ROS scavenger, mitigates these effects. Furthermore, the effects of FABP4 depletion on cell growth, stemness, glycolysis, and apoptosis in colorectal cancer cells were also retarded by NAC (p < 0.05). Notably, FABP4 knockdown also suppressed the ERK/mTOR pathway, suggesting its regulation via ROS (p < 0.05). In vivo study results showed, FABP4 depletion significantly curbed tumor growth in colorectal cancer (p < 0.05).
    CONCLUSIONS: These results suggest that FABP4 depletion inhibits colorectal cancer progression by modulating cell growth, stemness, glycolysis and apoptosis. This regulation occurs through the ROS/ERK/mTOR pathway.
    Keywords:  ERK/mTOR pathway; FABP4; NAC; colorectal cancer
    DOI:  https://doi.org/10.24976/Discov.Med.202335176.37
  18. Fish Physiol Biochem. 2023 Jun 09.
      Hypoxia is the most significant factor that threatens the health and even survival of freshwater and marine fish. Priority should be given to the investigation of hypoxia adaptation mechanisms and their subsequent modulation. Acute and chronic studies were designed for the current study. Acute hypoxia comprised of normoxia dissolved oxygen (DO) 7.0 ± 0.5 mg/mL (N0), low-oxygen 5.0 ± 0.5 mg/mL(L0), and hypoxia 1.0 ± 0.1 mg/mL (H0) and 300 mg/L Vc for hypoxia regulation (N300, L300, H300). Chronic hypoxia comprised of normoxia (DO 7.0 ± 0.5 mg/mL) with 50 mg/kg Vc in the diet (N50) and low oxygen (5.0 ± 0.5 mg/mL) with 50, 250, 500 mg/kg Vc in the diet (L50, L250, L500) to assess the effect of Vc in hypoxia. The growth, behavior, hematological parameters, metabolism, antioxidants, and related inflammatory factors of channel catfish were investigated, and it was found that channel catfish have a variety of adaptive mechanisms in response to acute and chronic hypoxia. Under acute 5 mg/mL DO, the body color lightened (P < 0.05) and reverted to normal with 300 mg/mL Vc. PLT was significantly elevated after 300 mg/L Vc (P < 0.05), indicating that Vc can effectively restore hemostasis following oxygen-induced tissue damage. Under acute hypoxia, the significantly increased of cortisol, blood glucose, the gene of pyruvate kinase (pk), and phosphofructokinase (pfk), together with the decreased expression of fructose1,6-bisphosphatase (fbp) and the reduction in myoglycogen, suggested that Vc might enhance the glycolytic ability of the channel catfish. And the enzyme activities of superoxide dismutase (SOD) and catalase (CAT) and the gene expression of sod rose significantly, showing that Vc might improve the antioxidant capacity of the channel catfish. The significant up-regulation of tumor necrosis factor-alpha (tnf-α), interleukin-1β (il-1β), and cd68 under acute hypoxia implies that hypoxia may generate inflammation in channel catfish, whereas the addition of Vc and down-regulation of these genes suggests that Vc suppresses inflammation under acute hypoxia. We found that the final weight, WGR, FCR, and FI of channel catfish were significantly reduced under chronic hypoxia, and that feeding 250 mg/kg of Vc in the diet was effective in alleviating the growth retardation caused by hypoxia. The significant increase in cortisol, blood glucose, myoglycogen, and the expression of tnf-α, il-1β, and cd68 (P < 0.05) and the significant decrease in lactate (P < 0.05) under chronic hypoxia indicated that the channel catfish had gradually adapted to the survival threat posed by hypoxia and no longer relied on carbohydrates as their primary source of energy. While the addition of Vc did not appear to increase the energy supply of the fish under hypoxia in terms of glucose metabolism, but the significantly decreased expression of tnf-α, il-1β, and cd68 (P < 0.05) also were found, indicating that chronic hypoxia, similar acute hypoxia, may increase inflammation in the channel catfish. This study indicates that under acute stress, channel catfish withstand stress by raising energy supply through glycolysis, and acute hypoxic stress significantly promotes inflammation in channel catfish, but Vc assists the channel catfish resist stress by raising glycolysis, antioxidant capacity, and decreasing the production of inflammatory markers. Under chronic hypoxia, the channel catfish no longer utilize carbohydrates as their primary energy source, and Vc may still effectively reduce inflammation in the channel catfish under hypoxia.
    Keywords:  Antioxidant; Hypoxia; Ictalurus punctatus; Metabolic response; Vitamin C
    DOI:  https://doi.org/10.1007/s10695-023-01205-5
  19. bioRxiv. 2023 May 24. pii: 2023.05.22.541793. [Epub ahead of print]
      Energy landscapes can provide intuitive depictions of population heterogeneity and dynamics. However, it is unclear whether individual cell behavior, hypothesized to be determined by initial position and noise, is faithfully recapitulated. Using the p21-/Cdk2-dependent quiescence-proliferation decision in breast cancer dormancy as a testbed, we examined single-cell dynamics on the landscape when perturbed by hypoxia, a dormancy-inducing stress. Combining trajectory-based energy landscape generation with single-cell time-lapse microscopy, we found that initial position on a p21/Cdk2 landscape did not fully explain the observed cell-fate heterogeneity under hypoxia. Instead, cells with higher cell state velocities prior to hypoxia, influenced by epigenetic parameters, tended to remain proliferative under hypoxia. Thus, the fate decision on this landscape is significantly influenced by "inertia", a velocity-dependent ability to resist directional changes despite reshaping of the underlying landscape, superseding positional effects. Such inertial effects may markedly influence cell-fate trajectories in tumors and other dynamically changing microenvironments.
    DOI:  https://doi.org/10.1101/2023.05.22.541793
  20. Pflugers Arch. 2023 Jun 05.
      People with sedentary lifestyles engage in minimal or no physical activity. A sedentary lifestyle promotes dysregulation of cellular redox balance, diminishes mitochondrial function, and increases NADPH oxidase activity. These changes collectively increase cellular oxidative stress, which alters endothelial function by oxidizing LDL-C, reducing NO production, and causing eNOS uncoupling. Reduced levels of nitric oxide (NO) leads to vasoconstriction, vascular remodeling, and vascular inflammation. Exercise modulates reactive oxygen species (ROS) to modify NRF2-KEAP signaling, leading to the activation of NRF2 to alleviate oxidative stress. While regular moderate exercise activates NRF2 through ROS production, high-intensity intermittent exercise stimulates NRF2 activation to a greater degree by reducing KEAP levels, which can be more beneficial for sedentary individuals. We review the damaging effects of a sedentary lifestyle on the vascular system and the health benefits of regular and intermittent exercise.
    Keywords:  Cardiovascular disease; Exercise; MOTS-c; NRF-2; Oxidative stress; Sedentary lifestyles
    DOI:  https://doi.org/10.1007/s00424-023-02828-6
  21. Biotechnol Adv. 2023 Jun 06. pii: S0734-9750(23)00091-5. [Epub ahead of print] 108184
      Glycosylation is how proteins and lipids are modified with complex carbohydrates known as glycans. The post-translational modification of proteins with glycans is not a template-driven process in the same way as genetic transcription or protein translation. Glycosylation is instead dynamically regulated by metabolic flux. This metabolic flux is determined by the concentrations and activities of the glycotransferase enzymes, which synthesise glycans, the metabolites that act as their precursors and transporter proteins. This review provides an overview of the metabolic pathways underlying glycan synthesis. Pathological dysregulation of glycosylation, particularly increased glycosylation occurring during inflammation, is also elucidated. The resulting inflammatory hyperglycosylation acts as a glycosignature of disease, and we report on the changes in the metabolic pathways which feed into glycan synthesis, revealing alterations to key enzymes. Finally, we examine studies in developing metabolic inhibitors targeting these critical enzymes. These results provide the tools for researchers investigating the role of glycan metabolism in inflammation and have helped to identify promising glycotherapeutic approaches to inflammation.
    Keywords:  Glycan synthesis; Glycosylation; Inflammation; Metabolic flux
    DOI:  https://doi.org/10.1016/j.biotechadv.2023.108184
  22. J Physiol. 2023 Jun 09.
      Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species (ROS). Since dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subject to single-limb casting (3 or 7 days) and consumed drinking water with or without 1 mM sodium nitrate. Compared to the contralateral control limb, 3 days of immobilization lowered myofibrillar fractional synthesis rates (FSR, p<0.0001), resulting in muscle atrophy. While FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared to intermyofibrillar (IMF) mitochondria, immobilization for 3 days decreased FSR in both SS (p = 0.009) and IMF (p = 0.031) mitochondria. Additionally, 3 days of immobilization reduced maximal mitochondrial respiration and protein content, and increased maximal mitochondrial ROS emission without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS, IMF). While nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics following both 3 and 7 days of immobilization. However, in contrast to 3 days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR following 7 days. Therefore, while nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Since dietary nitrate can improve mitochondrial bioenergetics, we examined if nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3 day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits, and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, while dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse. Abstract figure legend In female mice consuming standard drinking water (H2 O), 3 and 7 days of single-limb immobilization decreased mitochondrial (mito) protein fractional synthesis rate (FSR), myofibrillar (myofib) protein FSR, and mitochondrial respiration, and increased mitochondrial reactive oxygen species (ROS). In contrast, sodium nitrate (NO3 ) prevented the immobilization-induced alterations in mitochondrial bioenergetics (respiration, ROS) at both timepoints (3- and 7-day). In addition, mitochondrial protein FSR was transiently (3 day) preserved in the immobilized limb of nitrate-consuming mice. Combined, while dietary nitrate was not sufficient to prevent muscle atrophy, nitrate preserved mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term muscle disuse in mice. This article is protected by copyright. All rights reserved.
    Keywords:  IMF mitochondria; SS mitochondria; immobilization; mitochondrial ROS; mitochondrial respiration; nitrate; protein synthesis
    DOI:  https://doi.org/10.1113/JP284701
  23. Biol Open. 2023 Jun 15. pii: bio059881. [Epub ahead of print]12(6):
      Genetic studies place Tbx5 at the apex of the sinoatrial node (SAN) transcriptional program. To understand its role in SAN differentiation, clonal embryonic stem (ES) cell lines were made that conditionally overexpress Tbx5, Tbx3, Tbx18, Shox2, Islet-1, and MAP3k7/TAK1. Cardiac cells differentiated using embryoid bodies (EBs). EBs overexpressing Tbx5, Islet1, and TAK1 beat faster than cardiac cells differentiated from control ES cell lines, suggesting possible roles in SAN differentiation. Tbx5 overexpressing EBs showed increased expression of TAK1, but cardiomyocytes did not differentiate as SAN cells. EBs showed no change in the expression of the SAN transcription factors Shox2 and Islet1 and decreased expression of the SAN channel protein HCN4. EBs constitutively overexpressing TAK1 direct cardiac differentiation to the SAN fate but have reduced phosphorylation of its targets, p38 and Jnk. This opens the possibility that blocking the phosphorylation of TAK1 targets may have the same impact as forced overexpression. To test this, we treated EBs with 5z-7-Oxozeanol (OXO), an inhibitor of TAK1 phosphorylation. Like TAK1 overexpressing cardiac cells, cardiomyocytes differentiated in the presence of OXO beat faster and showed increased expression of SAN genes (Shox2, HCN4, and Islet1). This suggests that activation of the SAN transcriptional network can be accomplished by blocking the phosphorylation of TAK1.
    Keywords:  Embryoid body; SAN; Sinoatrial node; TAK1; Tbx5
    DOI:  https://doi.org/10.1242/bio.059881
  24. Pediatr Surg Int. 2023 Jun 06. 39(1): 214
      BACKGROUND: Actin Alpha 2 (ACTA2) is expressed in intestinal smooth muscle cells (iSMCs) and is associated with contractility. Hirschsprung disease (HSCR), one of the most common digested tract malformations, shows peristaltic dysfunction and spasm smooth muscles. The arrangement of the circular and longitudinal smooth muscle (SM) of the aganglionic segments is disorganized. Does ACTA2, as a marker of iSMCs, exhibit abnormal expression in aganglionic segments? Does the ACTA2 expression level affect the contraction function of iSMCs? What are the spatiotemporal expression trends of ACTA2 during different developmental stages of the colon?METHODS: Immunohistochemical staining was used to detect the expression of ACTA2 in iSMCs of children with HSCR and Ednrb-/- mice, and the small interfering RNAs (siRNAs) knockdown technique was employed to investigate how Acta2 affected the systolic function of iSMCs. Additionally, Ednrb-/- mice were used to explore the changes in the expression level of iSMCs ACTA2 at different developmental stages.
    RESULTS: The expression of ACTA2 is higher in circular SM in the aganglionic segments of HSCR patients and Ednrb-/- mice than in normal control children and mice. Down regulation of Acta2 weakens the contraction ability of intestinal smooth muscle cells. Abnormally elevated expression of ACTA2 of circular smooth muscle occurs since embryonic day 15.5 (E15.5d) in aganglionic segments of Ednrb-/- mice.
    CONCLUSIONS: Abnormally elevated expression of ACTA2 in the circular SM leads to hyperactive contraction, which may cause the spasm of aganglionic segments in HSCR.
    Keywords:  ACTA2; Circular and longitudinal muscles; ENCCs; Embryonic development; Hirschsprung disease
    DOI:  https://doi.org/10.1007/s00383-023-05479-x