Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a ...property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.
Although reactive oxidants have long been stigmatized as unwanted metabolic byproducts, the expression of oxidases specifically functioning to produce these same molecules in a regulated fashion is ...surprisingly pervasive throughout metazoan and plant evolution. Although the involvement of oxidants in many signaling pathways is well documented, the cellular strategies for conferring pathway specificity to such reactive molecules have remained more recondite. Recent studies now suggest that cells may spatially restrict oxidant production to allow microdomain-specific signaling.
Shimozawa, Y, Kurihara, T, Kusagawa, Y, Hori, M, Numasawa, S, Sugiyama, T, Tanaka, T, Suga, T, Terada, RS, Isaka, T, and Terada, M. Point prevalence of the biomechanical dimension of dysfunctional ...breathing patterns among competitive athletes. J Strength Cond Res 37(2): 270-276, 2023-There is growing evidence of associations between altered biomechanical breathing patterns and numerous musculoskeletal and psychological conditions. The prevalence of dysfunctional and diaphragmatic breathing patterns is unknown among athletic populations. The purpose of this study was to examine the prevalence of dysfunctional and diaphragmatic breathing patterns among athletic populations with a clinical measure to assess the biomechanical dimension of breathing patterns. Using a cross-sectional design, 1,933 athletes across multiple sports and ages were screened from 2017 to 2020. Breathing patterns were assessed using the Hi-Lo test in the standing position. Scores of the Hi-Lo test were determined based on the presence or absence of abdominal excursion, anterior-posterior chest expansion, superior rib cage migration, and shoulder elevation. The Hi-Lo test scores were used to categorize observational breathing mechanics as dysfunctional and diaphragmatic breathing patterns. The prevalence of athletes with dysfunctional breathing patterns was 90.6% (1,751 of 1,933). Athletes with diaphragmatic breathing patterns accounted for 9.4% of all athletes in our sample (182 of 1,933). There were no differences in the proportion of breathing patterns between male and female athletes ( p = 0.424). Breathing patterns observations were associated with sport-setting categories ( p = 0.002). The highest percentages of dysfunctional breathers were in middle school student athletes (93.7%), followed by elementary school student athletes (91.2%), high school student athletes (90.6%), professional/semiprofessional athletes (87.5%), and collegiate athletes (84.8%). The current study observed that dysfunctional breathing patterns (90.6%) in the biomechanical dimension were more prevalent than diaphragmatic breathing pattern (9.4%) among competitive athletes. These results suggest that clinicians may need to consider screening breathing patterns and implementing intervention programs aimed to improve the efficiency of biomechanical dimensions of breathing patterns in athletic populations. This study may help raise awareness of impacts of dysfunctional breathing patterns on athletes' health and performance.
Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second ...messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.
The endoplasmic reticulum (ER) produces the vast majority of all proteins secreted into the extracellular space, including hormones and cytokines, as well as cell surface receptors and other proteins ...which interact with the environment. Accordingly, this organelle controls essentially all vital links to a cell's external milieu, responding to systemic metabolic, inflammatory, endocrine, and mechanical stimuli. The central role the ER plays in meeting protein synthetic and quality control requirements in the face of such demands is matched by an extensive and versatile ER stress response signaling network. ROS mediate several critical aspects of this response. Nox4, an ER resident capable of producing ROS, acts as a proximal signaling intermediate to transduce ER stress-related conditions to the unfolded protein response, a homeostatic corrective mechanism. However, chronic ER stress caused by unrelenting internal or external demands produces a secondary rise in ROS, generally resulting in cell death. Sorting out the involvement of ROS at different levels of the ER stress response in specific cell types is key to understanding the molecular basis for chronic diseases such as atherosclerosis, hypertension, and diabetes. Here, we provide an overview of ER stress signaling with an emphasis on the role of ROS.
Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute ...that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.
In response to endoplasmic reticulum (ER) stress, endothelial cells initiate corrective pathways such as the unfolded protein response. Recent studies suggest that reactive oxygen species produced on ...the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway.
In endothelial cells, we found that ER stress induced by tunicamycin activates the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the unfolded protein response. In contrast, treatment with the growth factor VEGF (vascular endothelial growth factor) results in Ras activation and reactive oxygen species production confined instead to the plasma membrane and not to the ER, demonstrating local coupling of reactive oxygen species and Ras signals. We further identify the calcium-responsive, ER-resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the sarcoendoplasmic reticulum calcium ATPase and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the unfolded protein response.
ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF.
•The respiratory rate was continuously monitored following cesarean delivery.•Mild bradypnea and desaturation were common after 3 mg of epidural morphine.•The incidence of bradypnea was almost ...constant until ambulation.
Epidural morphine is widely used for postoperative analgesia after cesarean delivery. However, respiratory depression can occur after neuraxial administration of morphine. Previous reports describing respiratory depression in obstetric patients have relied on intermittent visual counting of the respiratory rate. In this study, we estimated the incidence of respiratory depression in patients who had received epidural morphine after cesarean delivery, using a continuous respiratory rate monitoring system with a finger sensor.
One hundred patients scheduled to undergo elective cesarean delivery and receive intraoperative neuraxial morphine between April and December 2016 were recruited for this single-center, prospective observational study. Postoperatively, all patients received epidural morphine 3 mg and were equipped with the Nellcor respiratory rate monitoring system. Respiratory depression was defined as both bradypnea (respiratory rate ≤10 breaths/min) and oxygen desaturation (mild ≤95%; moderate ≤90%; severe ≤85%) for longer than one minute. The number of patients with respiratory depression between administration of morphine and first ambulation was recorded hourly.
Complete monitoring was obtained for 89 of 100 women. The median duration of monitoring was 19.0 hours. Forty-six patients (52%) developed mild respiratory depression at least once before ambulation, but only one (1%) developed moderate respiratory depression. None required supplemental oxygen or naloxone.
Approximately half the women experienced mild respiratory depression, but only one developed moderate respiratory depression. Continuous respiratory rate monitoring until ambulation may assist in early identification of respiratory depression after neuraxial administration of morphine.
Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of ...Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.
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