Hypoxia Treatment Research Articles

Cellular Redox Status of Dermal fibroblasts as a Model to Explore Hypoxia Responses Across Diverse Mammals

Specializations in energy production pathways can define phenotype in mammals, enabling resilience to environmental stressors and survival in extreme habitats. Most mammals rely on oxygen-based mitochondrial respiration to sustain high rates of ATP production necessary for homeothermy; oxidative phosphorylation is fueled by donation of reducing equivalents (NADH, FADH2) to the electron transport chain. As such, relative redox levels within the cell (NAD+/NADH ratio) indicate cell health and metabolic status. In hypoxic conditions, cells can experience reductive stress, evidenced by a buildup of NADH and altered NAD+/NADH ratio. We are using primary dermal fibroblasts isolated from skin biopsies in n=9 species to compare metabolic changes via the ratio between NAD+ and NADH when exposed to 0.5% O2 for 6-h and 24-h. Species were selected to encompass diverse phylogeny as well as to include species that are adapted to low oxygen environments (high altitude gelada baboons, diving humpback whales and Weddell seals). Control species without adaptation to hypoxic environments included human, dromedary camel, little brown bat, honey badger, and rhinoceros. We expect fibroblasts from species adapted to living with variable oxygen availability to have more stability in NAD+/ NADH ratios when exposed to hypoxia in cell culture compared to those from species that are not adapted to low oxygen environments. We quantified NAD+ and NADH following exposure to hypoxia as well as control normoxic samples. Preliminary data with n=3 biological replicates per species show that fibroblasts from the control animals display significant differences in NAD+/NADH ratio between normoxic and hypoxic treatments (Kruskal-Wallis test, p=0.01), however, there are no significant differences in the ratios presented for tolerant animals (Kruskal-Wallis test, p=0.518). This finding supports our hypothesis that animals adapted to low oxygen reduce variability in the NAD+/NADH ratios. Understanding the metabolic patterns when cells are exposed to reduced oxygen levels can provide a framework for estimating a species resilience to environmental variation. Funded by NSF #2022046, CSUN-OUR Summer Undergraduate Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

APOE and sex impacts functional recovery after cervical spinal cord injury

Genetic factors are key to understanding recovery following spinal cord injury (SCI). Prior work conducted in our laboratory demonstrated that APOE genotype impacts respiratory plasticity after cervical SCI. Specifically, female mice expressing the APOE4 allele had impairments in their ability to generate and maintain robust diaphragmatic activity following cervical SCI and intermittent hypoxia treatment relative to APOE3 counterparts. Subsequently we showed that different APOE alleles leads to altered neural respiratory control even without a spinal cord injury and that the effect is sex dependent. To extend these findings, we are now investigating the impact of APOE genotype and sex on locomotor and respiratory motor recovery in a humanized APOE knock-in mouse model homozygous for either the APOE3 and E4 alleles. Further, we examined how genotype and sex effected plasticity and serotonin levels below the lesion. We hypothesized that animals with the APOE4 allele will demonstrate impaired recovery following SCI across all measures. We performed a left C2 hemisection injury on male and female mice of both APOE3 and APOE4 genotype (n = 8/group). We recorded whole body plethysmography and CatWalk gait analysis weekly beginning at pre-injury through three weeks post injury. Consistent with our previous findings male APOE3 mice responded better than APOE4 counterparts during respiratory challenge even following SCI. Specifically APOE3 male mice showed greater improvements in respiratory rate during both normoxia and hypoxia challenge at early timepoints following injury. Interestingly during catwalk gait analysis APOE4 male mice recovered function faster than APOE3 counterparts in the weeks following SCI. This work is critical because it lays the foundation for understanding how diverse genotypes in the clinical SCI population may indicate the need to personalize treatment for individuals following injury. AG065220 AG060056 Nielsen Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ethanol pre-exposure attenuates the hypoxic ventilatory response and may attenuate expression of long term facilitation following intermittent hypoxia treatment in adult rats

Obstructive sleep apnea (OSA) is a highly prevalent breathing disorder in which airway collapse or obstruction during sleep leads to periodic bouts of inadequate (hypopneic) or absent (apneic) ventilation despite neurorespiratory effort. Repetitive apneic and hypopneic exposures during sleep can induce intermittent hypoxemia, sympathetic activation, and sleep fragmentation which can lead to a host of maladaptive behavioral and physiological outcomes. Intermittent hypoxia, which consists of alternating exposure to hypoxia and normal oxygen levels (normoxia), can induce a long-lasting increase in breathing motor output called long term facilitation (LTF) and is currently being investigated as a therapeutic treatment. Interestingly, experts in the field of LTF have noted how IH models some key aspects of OSA. It has been well established through clinical studies that ethanol consumption prior to sleep exacerbates existing OSA, increasing the apnea/hypopnea index and worsening arterial blood oxygen desaturations during the night. However, it is unknown whether ethanol affects expression of LTF following IH treatment. If LTF might serve as a means of increasing ventilatory output to compensate for periods of apnea and hypopnea during OSA, ethanol might not only worsen the severity of OSA, but impede individuals’ compensatory response. Thus, for this study we hypothesized that ethanol treatment would attenuate LTF expression and the magnitude of the hypoxic ventilatory response during IH treatment. Using adult female Sprague-Dawley rats, we administered either low-dose (0.8 g/kg) or high-dose (3 g/kg) ethanol through intraperitoneal injection to model a 2-drink nightcap or binge drinking-type ethanol exposure, respectively. Immediately afterwards, we measured subjects’ ventilatory output during baseline, then during a 5 by 3-minute moderate IH protocol, and for one hour following the end of IH using whole-body plethysmography. Finally, we took venous blood samples to assay serum ethanol concentration at the end of the approximately 2.5 hour plethysmography measurement. Results from the high-dose ethanol group indicate that ethanol pre-exposure might attenuate respiratory rate and minute volume LTF in comparison to saline-treated control. Furthermore, high-dose ethanol attenuated the hypoxic ventilatory response with respect to respiratory rate and minute volume. Low dosage of ethanol attenuated subjects’ HVR of respiratory rate and minute volume, but conflictingly trends towards increasing LTF of tidal volume while diminishing LTF of respiratory rate when compared to saline control. Overall, our findings indicate that high levels of ethanol exposure dramatically diminish the acute ventilatory response to hypoxic exposure and may impede long-term increases in ventilatory output following IH treatment. On the other hand, lower doses of ethanol have a subtler effect on the ventilatory response to hypoxia. Future directions will include analysis of additional parameters of ventilatory function to gain a more detailed understanding of how ethanol effects respiratory plasticity in the context of intermittent hypoxic exposure. NIH 5T32 AA027488 to ALSUniversity of Kentucky Endowment to WJA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MicroRNA-135b-5p Is a Pathologic Biomarker in the Endothelial Cells of Arteriovenous Malformations.

Arteriovenous malformations (AVMs) are congenital vascular anomalies with a poor prognosis. AVMs are considered intractable diseases, as there is no established approach for early diagnosis and treatment. Therefore, this study aimed to provide new evidence by analyzing microRNAs (miRNAs) associated with AVM. We present fundamental evidence for the early diagnosis and treatment of AVM by analyzing miRNAs in the endothelial cells of AVMs. This study performed sequencing and validation of miRNAs in endothelial cells from normal and AVM tissues. Five upregulated and two downregulated miRNAs were subsequently analyzed under hypoxia and vascular endothelial growth factor (VEGF) treatment by one-way analysis of variance (ANOVA). Under hypoxic conditions, miR-135b-5p was significantly upregulated in the AVM compared to that under normal conditions, corresponding to increased endothelial activity (p-value = 0.0238). VEGF treatment showed no significant increase in miR-135b-5p under normal conditions, however, a surge in AVM was observed. Under both hypoxia and VEGF treatment, comparison indicated a downregulation of miR-135b-5p in AVM. Therefore, miR-135b-5p was assumed to affect the pathophysiological process of AVM and might play a vital role as a potential biomarker of AVMs for application related to diagnosis and treatment.

Regulation of Erythroid Differentiation via the HIF1α-NFIL3-PIM1 Signaling Axis Under Hypoxia.

Aims: Erythropoiesis is controlled by several factors, including oxygen level under different circumstances. However, the role of hypoxia in erythroid differentiation and the underlying mechanisms are poorly understood. We studied the effect and mechanism of hypoxia on erythroid differentiation of K562 cells and observed the effect of hypoxia on early erythropoiesis of zebrafish. Results: Compared with normal oxygen culture, both hemin-induced erythroid differentiation of K562 cells and the early erythropoiesis of zebrafish were inhibited under hypoxic treatment conditions. Hypoxia-inducible factor 1 alpha (HIF1α) plays a major role in the response to hypoxia. Here, we obtained a stable HIF1α knockout K562 cell line using the CRISPR-Cas9 technology and further demonstrated that HIF1α knockout promoted hemin-induced erythroid differentiation of K562 cells under hypoxia. We demonstrated an HIF1-mediated induction of the nuclear factor interleukin-3 (NFIL3) regulated in K562 cells under hypoxia. Interestingly, a gradual decrease in NFIL3 expression was detected during erythroid differentiation of erythropoietin-induced CD34+ hematopoietic stem/progenitor cells (HSPCs) and hemin-induced K562 cells. Notably, erythroid differentiation was inhibited by enforced expression of NFIL3 under normoxia and was promoted by the knockdown of NFIL3 under hypoxia in hemin-treated K562 cells. In addition, a target of NFIL3, pim-1 proto-oncogene, serine/threonine kinase (PIM1), was obtained by RNA microarray after NFIL3 knockdown. PIM1 can rescue the inhibitory effect of NFIL3 on hemin-induced erythroid differentiation of K562 cells. Innovation and Conclusion: Our findings demonstrate that the HIF1α-NFIL3-PIM1 signaling axis plays an important role in erythroid differentiation under hypoxia. These results will provide useful clues for preventing the damage of acute hypoxia to erythropoiesis.

TRPC4 aggravates hypoxic pulmonary hypertension by promoting pulmonary endothelial cell apoptosis

Pulmonary hypertension (PH) is a devastating disease that lacks effective treatment options and is characterized by severe pulmonary vascular remodeling. Pulmonary arterial endothelial cell (PAEC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension. Canonical transient receptor potential (TRPC) channels, a family of Ca2+-permeable channels, play an important role in various diseases. However, the effect and mechanism of TRPCs on PH development have not been fully elucidated. Among the TRPC family members, TRPC4 expression was markedly upregulated in PAECs from hypoxia combined with SU5416 (HySu)-induced PH mice and monocrotaline (MCT)-treated PH rats, as well as in hypoxia-exposed PAECs, suggesting that TRPC4 in PAECs may participate in the occurrence and development of PH. In this study, we aimed to investigate whether TRPC4 in PAECs has an aggravating effect on PH and elucidate the molecular mechanisms. We observed that hypoxia treatment promoted PAEC apoptosis through a caspase-12/endoplasmic reticulum stress (ERS)-dependent pathway. Knockdown of TRPC4 attenuated hypoxia-induced apoptosis and caspase-3/caspase-12 activity in PAECs. Accordingly, adeno-associated virus (AAV) serotype 6-mediated pulmonary endothelial TRPC4 silencing (AAV6-Tie-shRNA-TRPC4) or TRPC4 antagonist suppressed PH progression as evidenced by reduced right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, PAEC apoptosis and reactive oxygen species (ROS) production. Mechanistically, unbiased RNA sequencing (RNA-seq) suggested that TRPC4 deficiency suppressed the expression of the proapoptotic protein sushi domain containing 2 (Susd2) in hypoxia-exposed mouse PAECs. Moreover, TRPC4 activated hypoxia-induced PAEC apoptosis by promoting Susd2 expression. Therefore, inhibiting TRPC4 ameliorated PAEC apoptosis and hypoxic PH in animals by repressing Susd2 signaling, which may serve as a therapeutic target for the management of PH.

The Evaluation of the N-cadherin Promoter's ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines.

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-β and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and β-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of β-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-β in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

Hypoxia treatment and resistance training alters microRNA profiling in rats skeletal muscle

MicroRNAs (miRNAs) may play a crucial regulatory role in the process of muscle atrophy induced by high-altitude hypoxia and its amelioration through resistance training. However, research in this aspect is still lacking. Therefore, this study aimed to employ miRNA microarray analysis to investigate the expression profile of miRNAs in skeletal muscle from an animal model of hypoxia-induced muscle atrophy and resistance training aimed at mitigating muscle atrophy. The study utilized a simulated hypoxic environment (oxygen concentration at 11.2%) to induce muscle atrophy and established a rat model of resistance training using ladder climbing, with a total intervention period of 4 weeks. The miRNA expression profile revealed 9 differentially expressed miRNAs influenced by hypoxia (e.g., miR-341, miR-32-5p, miR-465-5p) and 14 differentially expressed miRNAs influenced by resistance training under hypoxic conditions (e.g., miR-338-5p, miR-203a-3p, miR-92b-3p) (∣log2(FC)∣ ≥ 1.5, p < 0.05). The differentially expressed miRNAs were found to target genes involved in muscle protein synthesis and degradation (such as Utrn, mdm2, eIF4E), biological processes (such as negative regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-dependent), and signaling pathways (such as Wnt signaling pathway, MAPK signaling pathway, ubiquitin-mediated proteolysis, mTOR signaling pathway). This study provides a foundation for understanding and further exploring the molecular mechanisms underlying hypoxia-induced rats muscle atrophy and the mitigation of atrophy through resistance training.

Unveiling the hidden effects of hypoxia: Pituitary damage and hormonal imbalance in fat greenling (Hexagrammos otakii)

BackgroundIn fisheries, hypoxia stress is one of the most common environmental stresses that often lead to the death of large numbers of fish and cause significant economic losses. The pituitary, an important endocrine gland, lies below the hypothalamus region of the brain. It plays a crucial part in controlling vital physiological functions in fish, such as growth, reproduction, and responses to stress. However, the detailed mechanisms of how hypoxia affects these physiological processes via the pituitary remain largely unknown. MethodsFat greenlings (Hexagrammous otakii) were exposed to different dissolved oxygen (DO = 7. 6 mg/L and DO = 2 mg/L) for 24 h. miRNA-mRNA association analysis of H. otakii pituitary after hypoxia stress. Detecting apoptosis in H. otakii pituitary using Tunel and qPCR. Subsequent detection of hormones in H. otakii liver, gonads and serum by ELISA. ResultsIn this study, hypoxia causes immune system disorders and inflammatory responses through the combined analysis of miRNAs and mRNAs. Subsequent verification indicated a significant accumulation of reactive oxygen species (ROS) subsequent to hypoxia treatment. The overproduction of ROS cause oxidative stress and apoptosis in the pituitary, ultimately causing pituitary damage and reduced growth hormone and luteinising hormone release. ConclusionsAccording to the association study of miRNA-mRNA, apoptosis problems caused by hypoxia stress result in H. otakii pituitary damage. In the meantime, this work clarifies the possible impact of hypoxia-stress on the pituitary cells, as well as on the gonadal development and growth of H. otakii.

WTAP-MEDIATED M6A MODIFICATION OF KLF6 AGGRAVATES HYPOXIA/REOXYGENATION-INDUCED HUMAN CARDIOMYOCYTE INJURY.

Background: Myocardial infarction (MI) is a severe condition that typically results from the ischemia and necrosis of heart muscle. Kruppel-like factor 6 (KLF6) can aggravate myocardial ischemia/reperfusion injury. This work aims to reveal its role and mechanism in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. Methods: Human cardiomyocyte (AC16) was exposed to hypoxic treatment to mimic MI-like cell injury. mRNA expression levels of KLF6 and WT1-associated protein (WTAP) were detected by quantitative real-time polymerase chain reaction. Protein expression was detected by western blotting assay. Cell viability was assessed by CCK-8 assay. Cell apoptosis and cell cycle were investigated by flow cytometry. Enzyme-linked immunosorbent assays were conducted to detect IL-1β, TNF-α and IL-6 levels. Fe 2+ colorimetric assay kit was used to detect Fe 2+ level. MDA Content Assay Kit was used to detect MDA level. Cellular ROS Assay kit was applied to assess ROS level. The association of KLF6 and WTAP was identified by RNA immunoprecipitation assay and dual-luciferase reporter assay. Results: KLF6 and WTAP expression at mRNA and protein levels were significantly upregulated in serum samples of MI patients and H/R-induced AC16 cells when compared with control groups. KLF6 silencing attenuated H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis. Additionally, WTAP stabilized KLF6 mRNA by regulating its m6A modification. Furthermore, WTAP knockdown rescued H/R-induced AC16 cell apoptosis, inflammatory response, oxidative stress, and ferroptosis by decreasing KLF6 expression. Conclusion: WTAP-mediated m6A modification of KLF6 aggravated hypoxia/reoxygenation-induced apoptosis, inflammatory response, oxidative stress, and ferroptosis of human cardiomyocytes, providing a therapeutic strategy for MI.

WITHDRAWN: Maresin-1 promotes mitochondrial function and autophagy after myocardial I/R injury by modulating the SIRT1/NRF2 signaling

Maresin-1 (MaR1) is a derived metabolite of Docosahexaenoic acid (DHA) and has been proved to play a protective role in ischemic reperfusion (I/R) damage. Nevertheless, the role and potential mechanism of MaR1 in myocardial ischemic reperfusion injury (MIRI) remain obscure. In this study, hypoxia-reoxygenation (H/R) cell model was established by hypoxia treatment. CCK-8, western blot and flow cytometry were used to estimate cell proliferation and apoptosis. LDH, CK-MB and inflammation factors were detected by corresponding kits. Mitochondrial function impairment was estimated through ROS, ATP, mPTP, OCR and JC-1 staining and DCFH-DA staining. Monodansylcadaverine (MDC) staining and western blot were carried out to identify the autophagy. In addition, the SIRT1/NRF2 signaling was estimated with western blot. The data showed that MaR1 pretreatment elevated cell viability, reduced the contents of LDH, CK-MB, and inflammation factors after H/R treatment. In addition, MaR1 mitigated the functional damage of mitochondria, ameliorated H/R-induced abnormal level of autophagy and hindered the cell apoptosis rate. MaR1 also declined the contents of pro-apoptotic proteins, and inhibited oxidative stress in H/R-challenged H9c2 cells. Meanwhile, the contents of Nuclear Nrf2, Nrf2 and SIRT1 were enhanced following MaR1 treatment. SIRT1 inhibitor Selisistat (EX-527) reversed the protective influences of MaR1 on H/R-induced H9c2 cells. To sum up, MaR1 may ameliorate H/R-stimulated H9c2 cell injury via the modulation of SIRT1/NRF2 signaling.

Effects of hypothermia–hypoxia pulping, high hydrostatic pressure processing, and repeat freeze–thaw cycles on the quality of raw kiwifruit puree

AbstractThis study evaluated the effects of the whole process of non‐thermal processing (hypothermia–hypoxia pulping and high hydrostatic pressure (HHP) pasteurization), different freezing methods, and repeat freeze–thaw cycles on physicochemical and nutritional quality of kiwifruit puree. Results showed that hypothermia–hypoxia treatment can retain the vitamin C, total phenol content, and antioxidant capacity of the puree better. Different pasteurization treatments can effectively kill microorganisms and improve the total phenol content and antioxidant capacity of the raw puree. Compared with thermal treatment, HHP can better retain the color, viscosity, and aroma components of the raw puree. The freezing methods have little impact on the quality of the raw puree, but peroxidase enzyme activity after quick freezing was significantly higher than that of slow freezing. Different freeze–thaw treatments had adverse effects on the quality of kiwifruit, and the damage of 25°C thawing conditions was more serious than that of 4°C thawing conditions. This research would provide new ideas for the production and storage of high‐quality kiwifruit puree and potential technical support for kiwifruit puree commercial production.Practical ApplicationsThe results of this study showed that non‐thermal processing can maintain the quality of kiwifruit puree better than traditional thermal processing, which provides a new idea for the production of high‐quality kiwifruit puree. In addition, this study suggests that temperature fluctuations should be minimized during storage to better maintain kiwifruit quality. This will provide potential technical support for the commercialization of kiwifruit puree.

Use of CPAP Ventilation in Non-ICU Wards May Influence Outcomes in Patients with Severe Respiratory COVID-19.

Background and Objectives: The COVID-19 disease has significantly burdened the healthcare system, including all units of severe patient treatment. Non-intensive care units were established to rationalize the capacity within the Intensive Care Unit (ICU) and to create a unit where patients with Acute Respiratory Distress Syndrome (ARDS) could be treated with non-invasive Continuous Positive Air Pressure (CPAP) outside the ICU. This unicentric retrospective study aimed to assess the efficacy of NIV Treatment in Patients of the fourth pandemic wave and how its application affects the frequency and mortality of ICU-treated patients at University Hospital Rijeka compared to earlier waves of the COVID-19 pandemic. Furthermore, the study showcases the effect of the Patient/Nurse ratio (P/N ratio) on overall mortality in the ICU. Materials and Methods: The study was conducted on two groups of patients with respiratory insufficiency in the second and third pandemic waves, treated in the COVID Respiratory Centre (CRC) (153 patients). We also reviewed a cohort of patients from the fourth pandemic wave who were initially hospitalized in a COVID-6 non-intensive unit from 1 October 2021 to 1 November 2022 (102 patients), and some of them escalated to CRC. Results: The introduction of the CPAP non-invasive ventilation method as a means of hypoxic respiratory failure treatment in non-intensive care units has decreased the strain, overall number of admissions, and CRC patient mortality. The overall fourth wave mortality was 29.4%, compared to the 58.2% overall mortality of the second and third waves. Conclusions: As a result, this has decreased CRC patient admissions and, by itself, overall mortality.

Hypoxic environment promotes angiogenesis and bone bridge formation by activating Notch/RBPJ signaling pathway in HUVECs

After epiphyseal fracture, the epiphyseal plate is prone to ischemia and hypoxia, leading to the formation of bone bridge and deformity. However, the exact mechanism controlling the bone bridge formation remains unclear. Notch/RBPJ signaling axis has been indicated to regulate angiogenesis and osteogenic differentiation. Our study aims to investigate the mechanism of bone bridge formation after epiphyseal plate injury, and to provide a theoretical basis for new therapeutic approaches to prevent the bone bridge formation. The expression of DLL4 and RBPJ was significantly up-regulated in HUVECs after ischemia and hypoxia treatment. Notch/RBPJ pathway positively regulated the osteogenic differentiation of BMSCs. HUVECs can induce osteogenic differentiation of BMSCs under ischemia and hypoxia. Notch/RBPJ pathway is involved in the regulation of the trans-epiphyseal bridge formation. Notch/RBPJ in HUVECs is associated with osteogenic differentiation of BMSCs and may participate in the regulation of the bone bridge formation across the epiphyseal plate.

Abstract 2769: Long non coding RNA H19/cell adhesion molecules circuitry as novel target and diagnostic tool for metastatic prostate cancer

Abstract In the complex tumor biology of prostate cancer (PCa), we previously contributed by demonstrating that estrogen receptor and hypoxia signaling promoted transcription of progression-associated genes. Notably, combined treatment, estrogens plus hypoxia, caused a significant down regulation of the long noncoding RNA H19, one of the most sensitive to these pro-tumoral stimuli. Mechanistically, H19 acts as transcriptional repressor: H19 over-expression decreases E-cadherin (CDH1) level causing epithelial-to-mesenchymal transition whereas H19 reduction promotes the expression of E-cadherin and β4 integrin (ITGB4) switching the tumor dissemination program toward an alternative mechanism, specifically the cohesive metastatic phenotype. We focused on this H19/cell adhesion molecules circuitry by investigating its role as: 1.Novel prognostic biomarker at the time of biopsies. eNOS, HIF2a and β4 integrin expression was evaluated by immunohistochemistry, H19 and CDH1 transcripts by Droplet Digital PCR on FFPE-biopsies in a retrospective cohort of PCa patients [n = 30; Gleason score GS≤ 7(3+4) n= 17, GS ≥7(4+3) n= 13; biochemical, local, or metastatic recurrence defining disease progression (n=8/30; time of recurrence: range 2-6 years)]. Importantly, multivariate analysis of combined biomarkers, named “Bioscore”, defined by the presence of at least 3 among HIF-2α, eNOS, CDH1, β4 integrin ≥cut off or H19≤cut off revealed its prognostic value for progression and Progression-Free Survival determined using the ROC analysis (p=0.003) and Kaplan Meier curve (p= 0.005), respectively. 2.Master regulator of metastatic program in vivo and ex vivo PCa experimental models. Upon H19 silencing, mimicking combined estrogens plus hypoxia treatment, luciferase-positive PC-3 and 22Rv1 cells led to increased E-cadherin and β4 integrin expression, enhanced tumor growth and metastasis formation at bone, lung, and liver in PCa mouse models. Further, treatment with H3K27 demethylase inhibitor, GSK-J4, rescued cell adhesion molecules expression by modulating H3K27me3 level at gene promoters, reduced metastasis number and size, and induced cell death in PCa-derived Organotypic Slice Cultures. Additionally, treatment with JQ1 and dBET6 BET protein family inhibitors) markedly reduced both E-cadherin and β4 integrin levels, cell proliferation and in vivo tumor growth. Intriguingly, H19 silencing increased BRD3 and BRD4 levels, implicating H19 in BRD transcription. Overall, this research enhances our understanding of the molecular mechanisms driving aggressive prostate cancer phenotypes, providing a foundation for new diagnostic and therapeutic strategies in prostate cancer management. Citation Format: Valeria Pecci, Aurora Aiello, Sara De Martino, Cristian Ripoli, Dante Rotili, Francesco Pierconti, Francesco Pinto, Claudio Grassi, Carlo Gaetano, Alfredo Pontecorvi, Lidia Strigari, Antonella Farsetti, Simona Nanni. Long non coding RNA H19/cell adhesion molecules circuitry as novel target and diagnostic tool for metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2769.

Modeling hypoxia-induced radiation resistance and the impact of radiation sources

Hypoxia contributes significantly to resistance in radiotherapy. Our research rigorously examines the influence of microvascular morphology on radiotherapy outcome, specifically focusing on how microvasculature shapes hypoxia within the microenvironment and affects resistance to a standard treatment regimen (30×2GyRBE). Our computational modeling extends to the effects of different radiation sources. For photons and protons, our analysis establishes a clear correlation between hypoxic volume distribution and treatment effectiveness, with vascular density and regularity playing a crucial role in treatment success. On the contrary, carbon ions exhibit distinct effectiveness, even in areas of intense hypoxia and poor vascularization. This finding points to the potential of carbon-based hadron therapy in overcoming hypoxia-induced resistance to RT. Considering that the spatial scale analyzed in this study is closely aligned with that of imaging data voxels, we also address the implications of these findings in a clinical context envisioning the possibility of detecting subvoxel hypoxia.

Active Anchoring Stimuli-Responsive Nano-Craft to Relieve Pulmonary Vasoconstriction by Targeting Smooth Muscle Cell for Hypoxic Pulmonary Hypertension Treatment.

Recently, nanotechnology-based drug delivery platforms in treating pulmonary arterial hypertension (PAH) have gradually emerged. However, large mechanical stress and shear stress in blood vessels greatly affect the retention of nanopreparative materials at lesion sites, severely limiting nanotechnology-based drug delivery. Herein, a stimuli-responsive nanocraft is rationally designed by actively anchoring E-selectin overexpressed on pulmonary arterial endothelial cells (PAECs), under hypoxic conditions, allowing effective accumulation and retention of the drug at the lesion site. Briefly, a nitrobenzene group is incorporated into the framework of a nanocarrier, and then it is simultaneously linked with chitosan. Additionally, the surface of the nanocarrier with sialic acid (SA) and encapsulated the clinically used drug ambrisentan (Am), which enables the anchoring of E-selectin and subsequent drug delivery is modifed. This system facilitates intercellular transport to pulmonary artery smooth muscle cells (PASMCs) when targeting PAECs and specifically responds to a reductive hypoxic microenvironment with elevated nitroreductase in PASMCs. Moreover, compared with free Am, nanoencapsulation and SA-PEG2000-NH2 prolong the blood circulation time, achieving better therapeutic outcomes in preventing vascular remodeling and reversing systolic dysfunction. The originality and contribution of this work reveal the promising value of this pulmonary arterial anchoring stimuli-responsive nanocraft as a novel therapeutic strategy for satisfactory PAH treatment.

Hypoxia expedites the progression of papillary thyroid carcinoma by promoting the CPT1A-mediated fatty acid oxidative pathway.

Hypoxia has been reported to promote the proliferation and migration of thyroid cancer, while the special mechanism was still unclear. HIF-1α/carnitine palmitoyl-transferase 1A (CPT1A) was found to be associated with papillary thyroid carcinoma (PTC) but the biological role of CPT1A in PTC was not explored. The effects of hypoxia and carnitine palmitoyl-transferase 1A (CPT1A) expression on PTC cells were determined by cell counting kit-8 assay, detection of oxidative stress, inflammation response and mitochondrial membrane motential (MMP). Oil Red O staining and the detection of free fatty acids were performed to assess the status of lipid metabolism. Flow cytometric analysis was performed to assess cell apoptosis. Quantitative polymerase chain reaction (qPCR) and western blot analysis were applied to investigate the expressions of CPT1A and HIF-1α and the molecules involved cell function. The expressions of CPT1A and HIF-1α were significantly increased in PTC cells with or without hypoxia treatment. CPT1A overexpression or silencing promoted or inhibited cell viability, and hypoxia further repressed cell viability. In addition, CPT1A overexpression alleviates hypoxia-induced increased oxidative stress, inflammation response and elevated MMP. CPT1A overexpression enhanced palmitic acid-induced decreased cell growth, enhanced the metabolic capacity of free fatty acid and suppressed cell apoptosis. Animal experiments showed that CPT1A overexpression promoted PTC tumor growth, reduced lipid deposition, oxidative stress and inflammation, as well as enhancing cell function indicators. However, CPT1A silencing showed the opposite effects both in vitro and in vivo. Hypoxia induces the high expression of HIF-1α/CPT1A, thereby reprogramming the lipid metabolism of PTC cells for adapting the hypoxia environment, meanwhile inhibiting the cell damage and apoptosis caused by oxidative stress.

Potential Impact of Hypoxic Astrocytes on the Aggravation of Depressive Symptoms in Parkinson's Disease.

Mounting evidence suggests a significant correlation between depressive disorders and neurodegenerative conditions, encompassing Alzheimer's disease and Parkinson's disease (PD). Depression represents a substantial non-motor manifestation frequently identified in individuals with PD, posing a significant threat to patients' overall well-being and necessitating the implementation of effective management strategies. Despite its high prevalence, impacting over 40% of PD patients, the precise cellular and molecular mechanisms underlying depression and its relationship to dopaminergic system degeneration remain largely ambiguous. In this study, we presented our findings demonstrating distinct characteristics of cortical astrocytes in PD patients compared to reactivated glial cells in the substantia nigra. We identified a subset of differentially expressed genes associated with depressive disorders from PD-associated cortical astrocytes. Furthermore, we uncovered the potential involvement of the hypoxia signaling in driving cortical astrocytic dysfunctions. Through a comprehensive investigation utilizing transcriptome and chromatin accessibility analyses on cultured human astrocytes, we revealed that hypoxic treatment could induce similar expression changes observed in cortex from PD patients. Additionally, we provided evidence that activation of the HIF-1 signaling pathway suppressed the expression of key components of mitochondrial ribosomes and electron transport chain proteins COX2 and CYTB, resulting in abnormal mitochondrial membrane potential. Our results underscore the potential impact of glial metabolic abnormalities on the development of depressive disorders associated with Parkinson's disease.

Transient sleep apnea results in long-lasting increase in β-amyloid generation and tau hyperphosphorylation

Sleep apnea is regarded as an important risk factor in the pathogenesis of Alzheimer disease (AD). Chronic intermittent hypoxia treatment (IHT) given during the sleep period of the circadian cycle in experimental animals is a well-established sleep apnea model. Here we report that transient IHT for 4 days on AD model mice causes Aβ overproduction 2 months after IHT presumably via upregulation of synaptic BACE1, side-by-side with tau hyperphosphorylation. These results suggest that even transient IHT may be sufficient to cause long-lasting changes in the molecules measured as AD biomarkers in the brain.