HIF-1α mRNA Expression Research Articles

Role of microphthalmia-associated transcription factor in uveal melanoma and its association with clinicopathological parameters.

153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (UM). In the Asian population, Microphthalmia-Associated Transcription Factor (MITF) regulates the melanogenesis pathway and activates Silver Protein (SILV) for eumelanin synthesis. MITF also stimulates the hypoxia–inducible factor 1-alpha (HIF-1α) gene that regulates hypoxia around the tumor microenvironment. Although their oncogenic potential has been observed in a variety of malignancies, MITF and SILV have not been investigated in UM in the Asian population. Therefore, our study aimed to detect the prognostic significance of MITF, SILV, and HIF1-α gene/protein expression levels in UM patients. Methods: Immunohistochemistry of MITF, SILV & HIF-1 α was performed on 82 UM tissue samples. Western blot of all three markers was carried out on representative cases. The mRNA expression level was done by qRT-PCR in 70 fresh UM cases. Cox proportional hazard model and log-rank test were used to determine the prognostic outcome of these markers. Results: MITF and SILV proteins were expressed in 56% and 70% of cases, respectively. High pigmentation, BAP1 loss, and HIF-1α expression were statistically correlated with MITF and SILV protein expression. Upregulation of both genes was found in more than 50% of cases at the mRNA level. This was also statistically significant with high pigmentation and HIF-1α mRNA expression. High expression of MITF protein showed reduced disease-free survival. Conclusions: Our study highlighted the fact that MITF and SILV could be potential biomarkers in UM development. Hence, it might play a key role in future therapeutic target.

Hypoxia Activates HIF-1α and Affects Gene Expression and Transcriptional Regulation of PHD in Tegillarca granosa

Hypoxia is a common challenge faced by mollusks, and the role of hypoxia-inducible factor-1α (HIF-1α) in regulating related target genes under hypoxia in Tegillarca granosa (Tg) remains unclear. In this study, we identified gene HIF-1α and further explored its function. qRT-PCR was performed to determine the mRNA expression of HIF-1α, prolyl 4-hydroxylases (PHD), hemoglobin (Hb), and myoglobin (Mb) in response to hypoxia. Dual-luciferase reporter analysis was used to assess the transcriptional activity of HIF-1α on the PHD and Hb genes during hypoxia. Results showed that the expression levels of HIF-1α and PHD mRNAs were highest in the gill and lowest in the adductor muscle. Under hypoxic conditions, HIF-1α and PHD mRNAs were both induced, and their expression levels increased significantly, peaking at 8 h and gradually decreasing thereafter. The transcription of Tg-PHD was induced by hypoxia and was Tg-HIF-1α dependent. Notably, the expression of Hb decreased in hemocytes (p < 0.01) after 8 h at 0.5 mg/L, while the expression of MbI and MbII increased (p < 0.01) in the hepatopancreas after 24 h and 120 h, respectively. Moreover, Tg-HIF-1α could transactivate the PHD promoter but not that of Hb. These findings provide valuable insights into the regulatory role of Tg-HIF-1α on Tg-PHD, safeguarding it from degradation by PHD and offering significant contributions to the understanding of HIF-1α’s function. Nonetheless, the expression of Tg-HIF-1α protein was not detected under hypoxic conditions, and additional investigations are required to quantify Tg-HIF-1α protein dynamics and establish the correlation between mRNA expression and protein levels.

Effects of resveratrol on HIF-1α/VEGF pathway and apoptosis in vitrified duck ovary transplantation

Preservation of ovarian tissues is an effective way to ensure genetic diversity of susceptible natural bird populations that are in danger of extinction. We examined whether the addition of the plant phenol resveratrol to vitrification solutions ameliorates the damaging effects of tissue hypoxia and reperfusion injury when the tissues are transplanted. Duck ovary tissues were frozen in the presence of varying concentrations of resveratrol in cryopreservation solutions and then transplanted under the renal capsules of 2-day-old Shelducks. Samples of the transplanted tissues were examined on days 3- and 9- post transplantation for activation of hypoxia-, antioxidant- and apoptosis-related gene expression and apoptosis. Resveratrol significantly increased expression of VEGF, HIF-1α, Nrf2, CAT and Bcl-2 mRNA and decreased BAX and Caspase-3 mRNA and reduced numbers of TUNEL-positive cells after vitrification and heterotopic ovarian transplantation. Resveratrol improved the antioxidant capacity, reduced apoptosis and activated the HIF-1α/VEGF pathway to promote angiogenesis 3- and 9-days following transplantation. These results indicated that the addition of resveratrol to vitrification solutions intended for long-term cryopreservation of ovary tissues improves survival in storage and the grafts following transplantation. This study provides a theoretical basis for the successful transplantation of avian ovarian tissue after vitrification.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.

#6153 SODIUM-GLUCOSE COTRANSPORTER 2 INHIBITORS VERSUS DIPEPTIDYL PEPTIDASE 4 INHIBITORS AS STABILIZERS OF HIF PATHWAY IN DIABETIC NEPHROPATHY RATS

Abstract Background and Aims With the diabetes pandemic, chronic kidney disease is a burden, with diabetic nephropathy (DN) being the primary leading cause of end-stage renal disease. Antidiabetic drugs have shown renoprotective effects via different mechanisms, and among those are SGLT2 inhibitors and DPP-4 inhibitors. Hypoxia-inducible factor (HIF) pathway, a significant contributor to inflammation development in the kidneys, is a major pathway that regulates erythropoietin synthesis. Prolyl hydroxylase domain (PHD) decreases erythropoietin synthesis by inhibiting HIFs. PHD inhibitors are anticipated to have additional effects, such as protection against metabolic diseases, due to HIF's impact on many genes. The diabetic kidney is characterized by enhanced HIF-1α activation but decreased HIF-2α activity. These actions may significantly contribute to glomerular and renal tubular dysfunction, renal inflammation and fibrosis, and decreased erythropoietin synthesis observed in DN. This work aims to test the renoprotective effect of SGLT2 inhibitors and DPP-4 inhibitors by controlling the proinflammatory and anti-inflammatory domains of the HIF pathway. Method Type 1 DM rat model was induced in 24-hour-fasted Sprague Dawley rats by a single intraperitoneal injection of 45 mg/kg streptozotocin (STZ). DM was verified by measuring blood glucose seven days following STZ injection, and only animals with glucose levels of ≥15 mM were included (n = 24), randomly and equally distributed, then received either saline or vildagliptin (3 mg/Kg) or empagliflozin (30 mg/Kg) once daily by oral gavage for three months. An additional group was used as a non-diabetic control. At the end of the experiment, rats were placed in metabolic cages for 24-h urine collection, then sacrificed. Blood samples and kidney tissue were collected. Kidney function was assessed by measuring serum creatinine and urinary albumin: creatinine ratio. HIF1α, HIF2α, and PHD3 mRNA expression in kidney tissues were assessed using quantitative real-time PCR, and to indirectly assess HIF2α protein stability, immunohistochemical staining for PHD3 was also performed. Results Vildagliptin and empagliflozin could significantly ameliorate the diabetic disturbance of renal function, as demonstrated in serum creatinine (F = 23.518, p<0.001) with a non-significant difference between both treated groups, and in the significantly reduced albumin/creatinine ratio (F = 14.453, p<0.001), with a significantly lower ratio for empagliflozin (4.73±0.54) than vildagliptin-treated group (9.09±1.04). Regarding the HIF pathway as a potential mechanism, empagliflozin-treated groups showed a significantly higher HIF2α gene expression in kidney tissue samples (0.98±0.10) than the vildagliptin-treated group (0.58±0.05). For the expression of its regulator, PHD3, there was a significant difference between groups (F = 5.694, p = 0.005). However, the vildagliptin-treated group (0.65±0.06) didn′t differ significantly from the untreated group (0.60±0.03), in contrast to the empagliflozin-treated group (2.01±0.50), which showed a significantly higher expression than the other groups (Figure 1). Conclusion Empagliflozin significantly increased the level of HIF-2α in renal tissue. In addition, it nearly neutralizes the tubular expression of PHD3 compared to non-treated and vildagliptin-treated rats. SGLT2 inhibition is effective for correcting the proinflammatory/anti-inflammatory imbalance in DN.

Effect of Intermittent Hypobaric Hypoxia Exposure on HIF-1α, VEGF, and Angiogenesis in the Healing Process of Post-Tooth Extraction Sockets in Rats.

The aim of this study was to investigate the effect of intermittent hypobaric hypoxia (IHH) exposure on the expression of hypoxia-induced factor-1α (HIF-1α) messenger RNA (mRNA), vascular endothelial growth factor-a (VEGF-a) mRNA, and angiogenesis after tooth extraction in rats. On 45 male Sprague-Dawley rats were performed the removal of the maxillary left first molar, and then they were randomly divided into 9 groups, namely: 4 groups that were exposed to IHH for 30 minutes every day in the Hypobaric Chamber at an altitude of 18,000 feet, with 1 time hypobaric hypoxia (HH), 3 times HH, 5 times HH, and 7 times HH; 4 normoxia groups that were terminated on days 1, 3, 5, and 7 after tooth extraction; and the 1 control group. Real-time polymerase chain reaction measured the molecular changes in the socket tissue after tooth extraction in rats to evaluate the expression of HIF-1α mRNA and VEGF mRNA. Histological changes with hematoxylin and eosin staining were noted to evaluate the amount of angiogenesis in the socket after tooth extraction. Molecular and histological parameters were calculated at the end of each experiment on days 0, 1, 3, 5, and 7 after tooth extraction, which exhibited the improvement phase of the wound-healing process. Increases in the expression of HIF-1α mRNA, VEGF mRNA, and angiogenesis were found in the IHH group compared with the normoxia group and the control group. The expression of HIF-1α mRNA increased significantly (p < 0.05) in the group after one time HH exposure on day 1, then decreased in the IHH group (three times HH exposure, five times HH exposure, and seven times HH exposure) approaching the control group. The expression of VEGF mRNA and angiogenesis began to increase after one time HH exposure on day 1, and increased again after three times HH exposure on day 3, then increased even more after five times HH exposure on day 5, and increased very significantly (**p < 0.05) after seven times HH exposure on day 7. It showed that repeated or intermittent exposure to HH conditions induced a protective response that made cells adapt under hypoxia conditions. IHH exposure accelerates the socket healing of post-tooth extraction, which is proven by changes in HIF-1α mRNA expression and increase in VEGF mRNA expression as stimuli for angiogenesis in post-tooth extraction sockets under hypobaric hypoxic condition, which also stimulates the formation of new blood vessels, thereby increasing blood supply and accelerating wound healing.

Immunometabolic changes of β-glucan-trained immunity induction and inhibition on neonatal calf immune innate cells

The growing antibiotic resistance and low-efficient vaccines make searching for alternatives a need to fight infectious diseases in newborn calves. Thus, trained immunity could be used as a tool to optimize immune response against a wide range of pathogens. Although β-glucans have shown to induce trained immunity, it has not been demonstrated in bovines yet. Uncontrolled trained immunity activation can generate chronic inflammation in mice and humans, and inhibiting it might reduce excessive immune activation. The aim of this study is to demonstrate that in vitro β-glucan training induces metabolic changes in calf monocytes, characterized by an increase in lactate production and glucose consumption upon restimulation with lipopolysaccharide. These metabolic shifts can be abolished by co-incubation with MCC950, a trained immunity inhibitor. Moreover, the dose-response relationship of β-glucan on the viability of calf monocytes was demonstrated. In newborn calves, in vivo β-glucan oral administration also induced a trained phenotype in innate immune cells, leading to immunometabolic changes, upon ex vivo challenge with E.coli. β-glucan-induced trained immunity improved phagocytosis, nitric oxide production, myeloperoxidase activity, and TNF-α gene expression through up-regulation genes of the TLR2/NF-κB pathway. Furthermore, β-glucan oral doses enhanced consumption and production of glycolysis metabolites (glucose and lactate, respectively), as well as up-regulated expression of mTOR and HIF1-α mRNA. Therefore, the results suggest that β-glucan immune training may confer calf protection from a secondary bacterial challenge, and trained phenotype induced by β-glucan can be inhibited.

Protective effect of dihydromyricetin against lipopolysaccharide-induced HK2 cells by upregulating HIF-1α

ABSTRACT Sepsis complicated by acute kidney injury (AKI) carries an extremely high mortality rate. The present study aimed to investigate the protective effect and underlying mechanism of dihydromyricetin (DHM) on human renal tubular epithelial cells (HK2) during AKI. To simulate an in vitro model of AKI, HK2 cells were treated with lipopolysaccharide (LPS) and divided into four groups: Control, LPS, LPS+DHM, and LPS+DHM+si-HIF-1α. The cellular viability of HK2 cells was determined by the CCK-8 assay after treatment with LPS and DHM (60 μmol/L). The expression of Bcl-2, Bax, Cleaved Caspase-3, and HIF-1α was measured by Western blotting. The expression of Bcl-2, Bax, and HIF-1α mRNA was assessed by PCR. The apoptosis rate of each group was determined by flow cytometry, while different kits were used to measure the levels of MDA, SOD, and LDH in each group of HK2 cells. DHM was found to increase the expression of HIF-1α in HK2 cells after treatment with LPS. the expression of HIF-1α mRNA and protein, Cleaved Caspase-3, Bax protein, MDA and LDH levels, and apoptosis rate were significantly decreased, while Bcl-2 protein, cell viability, and SOD activity were markedly increased in the LPS+DHM group compared with the LPS and LPS+DHM+si-HIF-1α groups. Thus, DHM can reduce apoptosis and oxidative stress damage in HK2 cells by increasing HIF-1α expression after LPS treatment. DHM may be a treatment for AKI, but in vitro studies must be validated in animal models and clinical trials before drawing conclusions. Caution must be exercised in interpreting in vitro results.

Association between HIF-1α, BNIP3, and autophagy in the chorionic villi of missed abortion.

To investigate the expression of autophagy mediated by the hypoxia-inducible factor 1α (HIF-1α)/BNIP3 signaling pathway in villus tissues of missed abortion and HTR-8/SVneo cells and to elucidate the association of HIF-1α and BNIP3 in autophagy of missed abortion. Villus tissues from 30 healthy women with induced abortion and 35 patients with missed abortion were collected, and HTR-8/SVneo cells were cultured under hypoxia and transfected with HIF-1α-siRNA. Real-time polymerase chain reaction was utilized to measure the mRNA levels of HIF-1α and BNIP3; Western blotting was performed to determine the protein levels of HIF-1α, BNIP3, LC3 II/I, and Beclin 1 in villus tissues and HTR-8/SVneo cells. Cellular invasion activity was detected by transwell matrigel assay. The level of autophagy was confirmed by transmission electron microscopy of autophagosome formation. The mRNA levels of HIF-1α and BNIP3 were significantly lower in the missed abortion villi than in the induced abortion samples. The protein levels of HIF-1α, BNIP3, Beclin 1, and LC3II/I were significantly decreased in villus tissues from missed abortion, and autophagosomes were significantly decreased in villus tissues from missed abortion. Under hypoxia, the mRNA expression of HIF-1α and BNIP3 was inhibited after silencing HIF-1α by RNAi, while the protein expression of HIF-1α, BNIP3, Beclin1, and LC3II/I was significantly downregulated. The number of invading cells was significantly decreased, and autophagosomes were significantly decreased after silencing HIF-1α by RNAi in HTR-8/SVneo cells. Autophagy mediated by the HIF-1α/BNIP3 signaling pathway in villous trophoblast cells may be associated with the progression and development of missed abortion.

Hypoxia-inducible lncRNA MIR210HG promotes HIF1α expression by inhibiting miR-93-5p in renal tubular cells.

Chronic hypoxia in the renal tubular interstitium has been reported to contribute to the progression of chronic kidney disease. Recently, long-noncoding RNAs have been shown to be involved in various pathological conditions, including hypoxia, one of which is the MIR210 host gene (MIR210HG). To elucidate the function of MIR210HG in renal hypoxia, we exposed primary cultured renal proximal tubular epithelial cells to hypoxia and examined the temporal profile of MIR210HG expression and the role of MIR210HG interaction with hypoxia-inducible factor1α (HIF1α, encoded by HIF1A). MIR210HG expression was induced by hypoxia. HIF1A silencing and cobalt chloride exposure showed that MIR210HG expression in hypoxia is HIF1α-dependent. MIR210HG silencing significantly reduced both the mRNA and protein levels of HIF1α, pointing to positive feedback regulation. To further investigate the details of this regulation, we turned to the in-silico miRNA targets of MIR210HG. We found that miR-93-5p levels increased when MIR210HG was knocked down. We then showed that miR-93-5p reduced the expression of HIF1A mRNA and MIR210HG. Furthermore, a dual luciferase assay confirmed that miR-93-5p binds to MIR210HG and HIF1A 3' UTR, inhibiting their expression. In conclusion, the long-noncoding RNA MIR210HG is induced shortly after hypoxia, and it promotes HIF1α expression by competing for miR-93-5p and inhibiting it. MIR210HG plays a crucial role in the biological response to hypoxia in renal tubular epithelial cells.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Curcumin alleviates nuclear factor-κB/NOD-like receptor protein 3 mediated renal injury caused by acute respiratory distress syndrome through reducing mitochondrial oxidative stress

To evaluate the effect of curcumin on renal mitochondrial oxidative stress, nuclear factor-κB/NOD-like receptor protein 3 (NF-κB/NLRP3) inflammatory body signaling pathway and tissue cell injury in rats with acute respiratory distress syndrome (ARDS). A total of 24 specific pathogen free (SPF)-grade healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ARDS model group, and low-dose and high-dose curcumin groups, with 6 rats in each group. The ARDS rat model was reproduced by intratracheal administration of lipopolysaccharide (LPS) at 4 mg/kg via aerosol inhalation. The control group was given 2 mL/kg of normal saline. The low-dose and high-dose curcumin groups were administered 100 mg/kg or 200 mg/kg curcumin by gavage 24 hours after model reproduction, once a day. The control group and ARDS model group were given an equivalent amount of normal saline. After 7 days, blood samples were collected from the inferior vena cava, and the levels of neutrophil gelatinase-associated lipocalin (NGAL) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The rats were sacrificed, and kidney tissues were collected. Reactive oxygen species (ROS) levels were determined by ELISA, superoxide dismutase (SOD) activity was detected using the xanthine oxidase method, and malondialdehyde (MDA) levels were determined by colorimetric method. The protein expressions of hypoxia-inducible factor-1α (HIF-1α), caspase-3, NF-κB p65, and Toll-like receptor 4 (TLR4) were detected by Western blotting. The mRNA expressions of HIF-1α, NLRP3, and interleukin-1β (IL-1β) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Renal cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). The morphological changes in renal tubular epithelial cells and mitochondria were observed under a transmission electron microscope. Compared with the control group, the ARDS model group exhibited kidney oxidative stress and inflammatory response, significantly elevated serum levels of kidney injury biomarker NGAL, activated NF-κB/NLRP3 inflammasome signaling pathway, increased kidney tissue cell apoptosis rate, and renal tubular epithelial cell damage and mitochondrial integrity destruction under transmission electron microscopy, indicating successful induction of kidney injury. Following curcumin intervention, the injury to renal tubular epithelial cells and mitochondria in the rats was significantly mitigated, along with a noticeable reduction in oxidative stress, inhibition of the NF-κB/NLRP3 inflammasome signaling pathway, and a significant decrease in kidney tissue cell apoptosis rate, demonstrating a certain dose-dependency. Compared with the ARDS model group, the high-dose curcumin group exhibited significantly reduced serum NGAL levels and kidney tissue MDA and ROS levels [NGAL (μg/L): 13.8±1.7 vs. 29.6±2.7, MDA (nmol/g): 115±18 vs. 300±47, ROS (kU/L): 75±19 vs. 260±15, all P < 0.05], significantly down-regulated protein expressions of HIF-1α, caspase-3, NF-κB p65, and TLR4 in the kidney tissue [HIF-1α protein (HIF-1α/β-actin): 0.515±0.064 vs. 0.888±0.055, caspase-3 protein (caspase-3/β-actin): 0.549±0.105 vs. 0.958±0.054, NF-κB p65 protein (NF-κB p65/β-actin): 0.428±0.166 vs. 0.900±0.059, TLR4 protein (TLR4/β-actin): 0.683±0.048 vs. 1.093±0.097, all P < 0.05], and significantly down-regulated mRNA expressions of HIF-1α, NLRP3, and IL-1β [HIF-1α mRNA (2-ΔΔCt): 2.90±0.39 vs. 9.49±1.87, NLRP3 mRNA (2-ΔΔCt): 2.07±0.21 vs. 6.13±1.32, IL-1β mRNA (2-ΔΔCt): 1.43±0.24 vs. 3.95±0.51, all P < 0.05], and significantly decreased kidney tissue cell apoptosis rate [(4.36±0.92)% vs. (27.75±8.31)%, P < 0.05], and significantly increased SOD activity (kU/g: 648±34 vs. 430±47, P < 0.05). Curcumin can alleviate kidney injury in ARDS rats, and its mechanism may be related to the increasing in SOD activity, reduction of oxidative stress, and inhibition of the activation of the NF-κB/NLRP3 inflammasome signaling pathway.

Changes in superoxide dismutase 3 (SOD3) expression in periodontal tissue during orthodontic tooth movement of rat molars and the effect of SOD3 on in vitro hypoxia-exposed rat periodontal ligament cells.

Hypoxia during orthodontic tooth movement (OTM) induces reactive oxygen species (ROS) production in periodontal tissues. Superoxide dismutase 3 (SOD3) is an anti-inflammatory enzyme that protects cells from ROS. This study investigated the expression and function of SOD3 during rat OTM and in hypoxia-exposed rat periodontal ligament (PDL) cells. OTM of right maxillary first molars were performed in 8-week-old male Sprague-Dawley rats using closed-coil spring for 1 and 14 days (n = 6 per group). SOD3 and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression was evaluated by immunohistochemistry. The effects of SOD3 on cell viability and proliferation, ROS production, and mRNA expression of Hif1-α, receptor activator of nuclear factor kappa-Β ligand (Rankl), and osteoprotegerin (Opg) in PDL cells and osteoclast differentiation were investigated under normal and hypoxic conditions. SOD3 expression in PDL tissues significantly decreased on the compression side on day 1 and on both sides on day 14 of OTM. HIF-1α levels significantly increased on the compression side on day 14. Cell viability, cell proliferation, and Opg mRNA expression decreased, whereas ROS production and Hif1-α and Rankl mRNA expression increased in the PDL cells upon SOD3 silencing. Hypoxia reduced Sod3 and Opg mRNA expression and increased ROS, Rankl mRNA expression, and osteoclast formation; SOD3 treatment attenuated these effects. SOD3 plays a role in periodontal tissue remodelling during OTM and in hypoxia-exposed PDL cells through ROS, HIF-1α, and RANKL/OPG pathways. Moreover, SOD3 treatment could attenuate the negative effects of hypoxia on the PDL cells.

Sympathetic dysregulation induced by postnatal intermittent hypoxia.

Exposure to postnatal chronic intermittent hypoxia (pCIH), as experienced in sleep-disordered breathing, is a risk factor for developing cardiorespiratory diseases in adulthood. pCIH causes respiratory instability and motor dysfunction that persist until adult life. In this study, we investigated the impact of pCIH on the sympathetic control of arterial pressure in rats. Neonate male Holtzman rats (P0-1) were exposed to pCIH (6% O2 for 30 seconds, every 10 minutes, 8 h/day) during their first 10-15 days of life, while control animals were maintained under normoxia. In early adult life (P25-40), freely behaving pCIH animals (n = 13) showed higher baseline arterial pressure levels linked to augmented sympathetic-mediated variability than control animals (n = 12, p < 0.05). Using decerebrated in situ preparations, we found that juvenile pCIH rats exhibited a twofold increase in thoracic sympathetic nerve activity (n = 14) and elevated firing frequency of ventromedullary presympathetic neurons (n = 7) compared to control rats (n = 6-7, p < 0.05). This pCIH-induced sympathetic dysregulation was associated with increased HIF-1α (hypoxia-inducible factor 1 alpha) mRNA expression in catecholaminergic presympathetic neurons (n = 5, p < 0.05). At older age (P90-99), pCIH rats displayed higher arterial pressure levels and larger depressor responses to ganglionic blockade (n = 6-8, p < 0.05), confirming the sympathetic overactivity state. pCIH facilitates the vasoconstrictor sympathetic drive by mechanisms associated with enhanced firing activity and HIF-1α expression in ventromedullary presympathetic neurons. This excessive sympathetic activity persists until adulthood resulting in high blood pressure levels and variability, which contribute to developing cardiovascular diseases.

LncRNA FRMD6-AS1 promotes hepatocellular carcinoma cell migration and stemness by regulating SENP1/HIF-1α axis.

Long non-cording RNAs (lncRNAs) drive the malignant progression of hepatocellular carcinoma (HCC), a cancer with high mortality rates but the function of FERM Domain Containing 6 antisense RNA 1 (FRMD6-AS1) in HCC has not been fully addressed. Hypoxia-inducible factors (HIFs) are transcription factors relevant to HCC under hypoxia and are regulated by SUMO-specific protease 1 (SENP1) through its deSUMOylation of HIF-1α. The current study investigated the role of FRMD6-AS1 in the regulation of SENP1-mediated deSUMOylation of HIF-1α. HUH7 and MHCC97H cells were treated with CoCl2 to mimic hypoxia in vitro and lentiviral vector-mediated FRMD6-AS1 overexpressing HCC cells were established. Wound-healing, Transwell, sphere formation assay, Western blotting analysis and animal experiments were performed. Expression of FRMD6-AS1, SENP1 mRNA and HIF-1α mRNA was assessed by RT-qPCR and of HIF-1α and SENP1 protein by Western blot. DeSUMOylation of HIF-1α was detected by immunoprecipitation. RNA immunoprecipitation with SENP1 antibody or IgG was performed to assess endogenous interactions between SENP1 and FRMD6-AS1. FRMD6-AS1 was upregulated in HCC tissues and cells and its upregulation indicated poor prognosis for HCC patients. FRMD6-AS1 promoted HCC cells migration and stemness in vitro and also promoted tumor growth in an in vivo mouse xenograft model. Mechanistic studies showed that FRMD6-AS1 regulated the level of HIF-1α protein but not the mRNA and this effect was achieved by binding to SENP1 protein and enhancing its protease activity. Rescue experiments demonstrated the oncogenic role of the FRMD6-AS1/SENP1/ HIF-1α axis in HCC cells. High FRMD6-AS1 expression was associated with poor prognosis of HCC patients. FRMD6-AS1 may have an oncogenic role in HCC via regulation of the SENP1/HIF-1α axis and may be a prognostic biomarker for HCC. Blockade of FRMD6-AS1 may offer a novel therapeutic approach to restrict HCC progression.

Coenzyme Q0 defeats NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects by inhibiting HIF-1α expression in human triple-negative breast cancer cells.

Coenzyme Q0 (CoQ0) is a derivative quinone from Antrodia camphorata (AC) that exerts anticancer activities. This study examined the anticancer attributes of CoQ0 (0-4µM) on inhibited anti-EMT/metastasis and NLRP3 inflammasome, and altered Warburg effects via HIF-1α inhibition in triple-negative breast cancer (MDA-MB-231 and 468) cells. MTT assay, cell migration/invasion assays, Western blotting, immunofluorescence, metabolic reprogramming, and LC-ESI-MS were carried out to assess the therapy potential of CoQ0. CoQ0 inhibited HIF-1α expression and suppressed the NLRP3 inflammasome and ASC/caspase-1 expression, followed by downregulation of IL-1β and IL-18 expression in MDA-MB-231 and 468 cells. CoQ0 ameliorated cancer stem-like markers by decreasing CD44 and increasing CD24 expression. Notably, CoQ0 modulated EMT by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin. CoQ0 inhibited glucose uptake and lactate accumulation. CoQ0 also inhibited HIF-1α downstream genes involved in glycolysis, such as HK-2, LDH-A, PDK-1, and PKM-2 enzymes. CoQ0 decreased extracellular acidification rate (ECAR), glycolysis, glycolytic capacity, and glycolytic reserve in MDA-MB-231 and 468 cells under normoxic and hypoxic (CoCl2) conditions. CoQ0 inhibited the glycolytic intermediates lactate, FBP, and 2/3-PG, and PEP levels. CoQ0 increased oxygen consumption rate (OCR), basal respiration, ATP production, maximal respiration, and spare capacity under normoxic and hypoxic (CoCl2) conditions. CoQ0 increased TCA cycle metabolites, such as citrate, isocitrate, and succinate. CoQ0 inhibited aerobic glycolysis and enhanced mitochondrial oxidative phosphorylation in TNBC cells. Under hypoxic conditions, CoQ0 also mitigated HIF-1α, GLUT1, glycolytic-related (HK-2, LDH-A, and PFK-1), and metastasis-related (E-cadherin, N-cadherin, and MMP-9) protein or mRNA expression in MDA-MB-231 and/or 468 cells. Under LPS/ATP stimulation, CoQ0 inhibited NLRP3 inflammasome/procaspase-1/IL-18 activation and NFκB/iNOS expression. CoQ0 also hindered LPS/ATP-stimulated tumor migration and downregulated LPS/ATP-stimulated N-cadherin and MMP-2/-9 expression. The present study revealed that suppression of HIF-1α expression caused by CoQ0 may contribute to inhibition of NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects of triple-negative breast cancers.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization

To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) . Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope. The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood. The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

Expression and Clinical Significance of HIF-1α in Follicular Fluid and Granulosa Cells in Infertile PCOS Patients.

The present study aimed to determine the clinical predictive significance of HIF-1α in follicular development and assisted reproductive technology (ART). We collected follicular fluid (FF) and granulosa cells (GCs) from PCOS (polycystic ovary syndrome) patients (experimental group) and other patients who were infertile due to tubal factors or male factors (control group) with IVF/ICSI-ET. The localization and expression of HIF-1α in GCs were determined by immunofluorescence staining. HIF-1α protein and mRNA expression were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. To clarify the regulation of HIF-1α by TGF-β1, we added the HIF-1α-specific blocker YC-1 to GCs. The serum AMH, LH, LH/FSH, testosterone, BMI and the number of oocytes retrieved in the PCOS group were significantly higher, while the cleavage rate was significantly lower, than those in the control group. HIF-1α protein was expressed in the cytoplasm of GCs. The expression of HIF-1α protein in the FF of the PCOS group was significantly lower than that in the control group. However, the expression of HIF-1α protein in GCs between the two groups was not significantly different. HIF-1α protein was highly expressed in large FF (follicular diameter ≥ 14mm). Compared with the control group, the expression of HIF-1α mRNA in GCs of the PCOS group was significantly lower. The results showed a significant positive correlation between HIF-1α and TGF-β1 expression. We found that both HIF-1α and TGF-β1 were involved in the development of PCOS follicular development. The mutual regulation of HIF-1α and TGF-β1 may be one of the important mechanisms of the occurrence and development of PCOS.

Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey.

Many whey proteins, peptides and protein-derived amino acids have been suggested to improve gut health through their anti-oxidant, anti-microbial, barrier-protective and immune-modulating effects. Interestingly, although the degree of hydrolysis influences peptide composition and, thereby, biological function, this important aspect is often overlooked. In the current study, we aimed to investigate the effects of whey protein fractions with different degrees of enzymatic hydrolysis on the intestinal epithelium in health and disease with a novel 2D human intestinal organoid (HIO) monolayer model. In addition, we aimed to assess the anti-microbial activity and immune effects of the whey protein fractions. Human intestinal organoids were cultured from adult small intestines, and a model enabling apical administration of nutritional components during hypoxia-induced intestinal inflammation and normoxia (control) in crypt-like and villus-like HIO was established. Subsequently, the potential beneficial effects of whey protein isolate (WPI) and two whey protein hydrolysates with a 27.7% degree of hydrolysis (DH28) and a 50.9% degree of hydrolysis (DH51) were assessed. In addition, possible immune modulatory effects on human peripheral immune cells and anti-microbial activity on four microbial strains of the whey protein fractions were investigated. Exposure to DH28 prevented paracellular barrier loss of crypt-like HIO following hypoxia-induced intestinal inflammation with a concomitant decrease in hypoxia inducible factor 1 alpha (HIF1α) mRNA expression. WPI increased Treg numbers and Treg expression of cluster of differentiation 25 (CD25) and CD69 and reduced CD4+ T cell proliferation, whereas no anti-microbial effects were observed. The observed biological effects were differentially mediated by diverse whey protein fractions, indicating that (degree of) hydrolysis influences their biological effects. Moreover, these new insights may provide opportunities to improve immune tolerance and promote intestinal health.

Reversal of maternal obesity attenuates hypoxia and improves placental development in the preeclamptic-like BPH/5 mouse model.

Women with obesity have higher risk of adverse pregnancy outcomes, including preeclampsia (PE). Late-gestational hypertension, aberrant fetoplacental development, and fetal growth restriction (FGR), hallmarks of PE, are observed spontaneously in BPH/5 mice. Similar to obese preeclamptic women, BPH/5 mice have higher visceral white adipose tissue (WAT) and circulating leptin. We hypothesized that attenuation of maternal obesity and serum leptin in pregnant BPH/5 mice will improve fetoplacental development by decreasing hypoxia markers and leptin expression at the maternal-fetal interface. To test this hypothesis, BPH/5 mice were fed ad libitum (lib) and pair-fed (PF) to C57 ad lib controls beginning at embryonic day (e) 0.5. Hypoxia-related genes, hypoxia inducible factor (Hif) 1α, stem cell factor (Scf), heme oxygenase-1 (Ho-1), leptin (Lep), and leptin receptor (LepR) were assessed in e7.5 implantation sites. BPH/5 ad lib had 1.5 to 2-fold increase in Hif1α, Scf, and Ho-1 mRNA and a greater than 3-fold increase in leptin mRNA vs. C57 that was attenuated with PF. Exogenous leptin promoted Hif1α and Ho-1 mRNA expression in e7.5 decidua in vitro. While hypoxic conditions in vitro did not change decidual leptin mRNA. Furthermore, BPH/5 PF mice demonstrated improved fetal and placental outcomes later in gestation, with greater placental vascular area by e18.5 and attenuation of FGR. In conclusion, pair-feeding BPH/5 mice beginning at conception may improve placental vasculature formation via decreased leptin and hypoxia-associated markers in this model. Future investigations are needed to better determine the effect of hypoxia and leptin on pregnancy outcomes in obese pregnant women.