HIF-1α mRNA Levels Research Articles

Sanguinarine Reverses Pulmonary Vascular Remolding of Hypoxia-Induced PH via Survivin/HIF1α-Attenuating Kv Channels.

Background: Similarities in the biology of pulmonary hypertension and cancer suggest that anticancer therapies, such as sanguinarine, may also be effective in treating pulmonary hypertension. This, along with underlying biochemical pathways, is investigated in this study. Methods: Rats were subjected to 4-week hypoxia (or control) with or without sanguinarine treatment. In addition, pulmonary artery smooth muscle cells (PASMCs) were examined after 24–48 h hypoxia (with normoxic controls) and with or without sanguinirine. Pulmonary artery pressures and plasma survivin levels were measured in vivo. Ex vivo tissues were examined histologically with appropriate staining. mRNA and protein levels of survivin, HIF-1α, TGFb1, BMPR2, Smad3, P53, and Kv 1.2, 1.5, 2.1 were determined by real-time PCR and Western blot in PASMCs and distal PAs tissue. PASMC proliferation and changes of TGFb1 and pSmad3 induced by sanguinarine were studied using MTT and Western blot. Electrophysiology for Kv functions was measured by patch-clamp experiments. Results: Four-week hypoxia resulted in an increase in serum survivin and HIF-1α, pulmonary artery pressures, and pulmonary vascular remodeling with hypertrophy. These changes were all decreased by treatment with sanguinarine. Hypoxia induced a rise of proliferation in PASMCs which was prevented by sanguinarine treatment. Hypoxic PASMCs had elevated TGFb1, pSmad3, BMPR2, and HIF1α. These increases were all ameliorated by sanguinarine treatment. Hypoxia treatment resulted in reduced expression and function of Kv 1.2, 1.5, 2.1 channels, and these changes were also modulated by sanguinarine. Conclusion: Sanguinarine is effective in modulating hypoxic pulmonary vascular hypertrophy via the survivin pathway and Kv channels.

UDCA Inhibits Hypoxic Hepatocellular Carcinoma Cell-Induced Angiogenesis Through Suppressing HIF-1α/VEGF/IL-8 Intercellular Signaling.

Background: A hypoxic microenvironment may induce angiogenesis and promote the development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate whether ursodeoxycholic acid (UDCA) may inhibit hypoxic HCC cell–induced angiogenesis and the possible mechanisms. Methods: Tube formation and matrigel plug angiogenesis assays were used to evaluate angiogenesis in vitro and in vivo, respectively. Real-time PCR, enzyme-linked immunosorbent assay, and Western blot were used to evaluate the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and IL-8, respectively. Dual-luciferase reporter assay was applied to assess the reporter gene expression of hypoxia-response element (HRE). Results: UDCA antagonized hypoxic Huh 7 cell-induced tube formation of EA.hy 926 cells. In HCC cells, UDCA inhibited hypoxia-induced upregulation of VEGF and IL-8 both in mRNA and protein levels. UDCA also inhibited IL-8–induced angiogenesis in vitro and in vivo through suppressing IL-8–induced phosphorylation of ERK. The levels of HIF-1α mRNA and protein and HRE-driven luciferase activity in HCC cells were upregulated by hypoxia and were all inhibited by UDCA. The proteasome inhibitor MG132 antagonized the effect of UDCA on HIF-1α degradation. In hypoxic condition, the phosphorylation of ERK and AKT was obviously increased in HCC cells, which was suppressed by UDCA. Transfection of the HIF-1α overexpression plasmid reversed the effects of UDCA on hypoxic HCC cell–induced angiogenesis, HRE activity, and expressions of IL-8 and VEGF. Conclusions: Our results demonstrated that UDCA could inhibit hypoxic HCC cell–induced angiogenesis through suppressing HIF-1α/VEGF/IL-8–mediated intercellular signaling between HCC cells and endothelial cells.

Capsaicin inhibits HIF-1α accumulation through suppression of mitochondrial respiration in lung cancer cells

Hypoxia inducible factor (HIF)-1α is an important transcription factor regulating cancer metabolism in hypoxic environment. Capsaicin is known to inhibit hypoxia-induced HIF activity in lung cancer. Hence, in this study we tried to elucidate its inhibitory mechanism of action. In lung cancer cells, including H1299, H23, A549, and H2009 cells, capsaicin inhibited cell growth and HIF activation. Under hypoxic conditions, capsaicin reduced the accumulation of HIF-1α protein and the expression of its target genes, including pyruvate dehydrogenase kinase 1 (PDK1) and glucose transporter 1 (GLUT1), with no effect on overall HIF-1α mRNA levels in the H1299 cells. In addition, capsaicin increased intracellular oxygen levels by suppressing mitochondrial respiration, resulting in a reduction of HIF-1α accumulation. Furthermore, mitochondrial ATP production was reduced by capsaicin through the inhibition of mitochondrial respiration in the H1299, H23, A549, and H2009 cells. These results indicate that capsaicin potentially exhibits anticancer therapeutic effects in lung cancer under hypoxic conditions.

Pyruvate Kinase M2 Regulates Hif-1alpha Activity in Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Hypoxia-inducible factor 1 (HIF-1) is a nuclear protein with transcriptional activity. HIF-1 can be activated by immune cells exposed to hypoxia and regulate glycolysis. In this regulation, pyruvate kinase M2 (PKM2), a key enzyme in the cell glycolytic pathway, is an important regulatory site. Our previous studies suggest that PKM expression is increased in myeloid dendritic cells (mDCs) in SAA patients. Therefore, we investigated the expression level of HIF-1α in mDCs and its interaction with PKM2 in SAA patients. 1.The HIF-1α expression of mRNA and protein on mDCs in SAA untreated patients was significantly higher than that in SAA remission patients and normal controls.2.In the SAA patients, the HIF-1α expression on mDCs was positively correlated with the numbers of mDCs (p < 0.01), CD80+mDC/mDC (r = 0.689, p < 0.01), and CD86+mDC/mDC in PB (p < 0.05).3.In SAA patients, the HIF-1α expression on mDCs was negatively correlated with the CD4+T/CD8+T ratio in peripheral blood (PB) (p < 0.05), the percentage of granulocytoid and erythroid cells in bone marrow (p < 0.05), the WBC count in PB (p < 0.05), ANC in PB (p < 0.05), and the percentage of Rets in the PB (p < 0.05); was positively correlated with the percentage of lymphoid cells in bone marrow(p < 0.05); and was not statistically correlated with the megakaryocyte number in bone marrow, absolute PBL count, HGB in PB, absolute Ret count in PB, or PLT count in PB.4.In the correlation analysis we observed that there was a positively correlation between HIF-1α and PKM2 expression on mDCs in SAA patients (p < 0.001). To evaluate whether there is a mutual adjustment relationship between HIF-1α and PKM2, we successfully reduced PKM2 gene expression in this cell population via siRNA transfection. This process resulted in significantly lower levels of PKM2 protein expression relative to cells transfected with siControl which were evaluated by western blotting. We observed that the relative expression levels of HIF-1α mRNA in mDCs transfected with PKM2 siRNA was lower than siControl group（P< 0.01）.Conclusions 1.In this study, we found that untreated patients with SAA had higher HIF-1α expression on mDCs compared with recovering SAA patients and normal controls. The expression of HIF-1α was correlated with the number and function in mDCs and the severity of pancytopenia of SAA.2.The results indicated that the mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2.3.The mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The above experimental results confirmed that HIF-1α plays an important role in the abnormal immune response in patients with severe aplastic anemia, mainly by regulating the activity of PKM2 and thereby affecting the energy metabolism in immune cells. DisclosuresNo relevant conflicts of interest to declare.

Dexmetomidine promotes the activity of breast cancer cells through miR-199a/HIF-1α axis.

BackgroundBreast cancer, as one of the most common malignant tumors in women, is still a great threat to women all over the world. Dexmetomidine (DMED) is a highly selective α2-adrenergic receptor agonist, which has attracted much attention in recent years. This study aimed to clarify the potential mechanism of DMED in regulating the activity of breast cancer cells.MethodsBreast cancer cell lines MCF-7 and MDA-MB-231 were treated with DMED. The levels of miR-199a and HIF-1α mRNA were detected using quantitative real-time polymerase chain reaction (QRT-PCR); the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and transwell assays were applied to monitor the activity of breast cancer cells; the apoptosis of breast cancer cells was detected using the caspase-3 activity assay and flow cytometry; binding of miR-199a and HIF-1α was assessed using double luciferase reporter gene assay, and western blot was employed to monitor the level of HIF-1α in cells.ResultsThe cytotoxicity and apoptosis of MCF-7 and MDA-MB-231 cells was inhibited by DMED. It also downregulated the expression of miR-199a in breast cancer cells and enhanced the downregulation of miR-199a to promote the activity of breast cancer cells and inhibit apoptosis. Also, miR-199a targeted HIF-1α. Further functional experiments confirmed that DMED promoted the progression of breast cancer through the miR-199a/HIF-1α axis.ConclusionsDMED promotes the activity of breast cancer cells through miR-199a/HIF-1αaxis. This can provide some reference for DMED in the clinical treatment of breast cancer.

Digitoxin promotes apoptosis and inhibits proliferation and migration by reducing HIF-1α and STAT3 in KRAS mutant human colon cancer cells

Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor. New treatment options are needed to improve survival in patients with KRAS mutated colorectal cancer. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers. However, the anticancer mechanisms of digitoxin in KRAS mutant human colon cancer cells remain elusive. Our result demonstrated that digitoxin but not cetuximab markedly decreased the expression of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 protein in KRAS mutant colon cancer cells. Further analysis revealed that digitoxin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. The phosphorylation levels of ribosomal protein S6 kinase (p70S6K) and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by digitoxin. Digitoxin inhibited the expression and activation of STAT3 through upregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN), SHP1 and protein inhibitors of activated STAT3 (PIAS3) and direct binding to STAT3. Meanwhile, digitoxin inhibited HIF-1α in STAT3-independent manner in KRAS mutant colon cancer cells. Moreover, digitoxin promoted apoptosis and inhibited proliferation and migration, which was potentially mediated by suppression of HIF-1α and STAT3. We also found that digitoxin administration inhibited tumor growth in a mouse xenograft model. Taken together, our findings highlight the therapeutic potential of digitoxin for the treatment of cetuximab-resistant human colon cancer.

Overexpression of HIF1α in Hunner Lesions of Interstitial Cystitis: Pathophysiological Implications.

The aim of our study was to elucidate biological changes in Hunner lesions, which underlie the pathophysiology of Hunner-type interstitial cystitis, by characterizing their whole transcriptome and immunopathological profiles. Paired bladder mucosal biopsies, 1 sample each from the Hunner lesion and nonlesion area, were obtained from 25 patients with Hunner-type interstitial cystitis. The samples were subjected to whole-transcriptome profiling; immunohistochemical quantification of CD3, CD4, CD8, CD20, CD138, mast cell tryptase, cytokeratin and HIF1α; and quantitative polymerase chain reaction for IFN-α, IFN-β, IFN-γ, TNF, TGF-β1, HIF1α, IL-2, IL-4, IL-6, IL-10 and IL-12A. The results were compared between the lesion and nonlesion areas. RNA sequencing identified 109 differentially expressed genes and 30 significantly enriched biological pathways in Hunner lesions. Up-regulated pathways (24) included HIF1α signaling pathway, PI3K-Akt signaling pathway, RAS signaling pathway and MAPK signaling pathway. By contrast, down-regulated pathways (6) included basal cell carcinoma and protein digestion and absorption. The mRNA levels of HIF1α, IFN-γ and IL-2, and the HIF1α protein level were significantly higher in lesion areas. Otherwise, there were no significant differences between the lesion and nonlesion samples in terms of mRNA levels of inflammatory cytokines or histological features such as lymphoplasmacytic and mast cell infiltration, epithelial denudation and CD4/CD8 T-lymphocyte ratio. Our findings demonstrate significant overexpression of HIF1α and up-regulation of its related biological pathways in Hunner lesions. The results indicate that ischemia, in conjunction with inflammation, plays a pathophysiological role in this subtype of interstitial cystitis/bladder pain syndrome, particularly in Hunner lesions.

Protein arginine N-methyltransferase 1 gene polymorphism is associated with proliferative diabetic retinopathy in a Japanese population.

To investigate the effects of single-nucleotide polymorphisms (SNPs) around the protein arginine N-methyltransferase 1 (PRMT1) gene on the incidence and severity of diabetic retinopathy (DR). A total of 310 Japanese patients with type 2 diabetes mellitus (T2DM) were investigated. Genotyping of ten tagged SNPs were performed by quantitative real-time polymerase chain reaction (qRT-PCR). The association between each SNP genotype and diabetic microangiopathy was assessed using univariate analysis in a dominant model of the minor alleles followed by multivariate logistic regression analysis with the propensity score matching (PSM) method. The effect of disease-related SNP on PRMT1 and hypoxia-inducible factor-1α (HIF-1α) mRNA levels in vivo was evaluated by qRT-PCR. In the univariate analysis, the minor A allele at rs374569 and the minor C allele at rs3745468 were associated with DR severity (P = 0.047 and P = 0.003, respectively), but not diabetic nephropathy and peripheral polyneuropathy severity. Multivariate analysis showed that the rs3745468 variant caused an increased incidence of proliferative DR (PDR) (odds ratio 9.37, 95% confidence interval 1.12-78.0, P = 0.039). In the PSM cohort, the patients carrying the rs3745468 variant had lower PRMT1 mRNA levels compared to those without the variant (P = 0.037), and there was an inverse correlation between PRMT1 and HIF-1α mRNA levels (r = -0.233, P = 0.035). The rs3745468 variant in the PRMT1 gene was associated with an increased incidence of PDR in Japanese patients with T2DM and might be involved in the HIF-1-dependent hypoxic pathway through altered PRMT1 levels.

Nobiletin, a hexamethoxyflavonoid from citrus pomace, attenuates G1 cell cycle arrest and apoptosis in hypoxia-induced human trophoblast cells of JEG-3 and BeWo via regulating the p53 signaling pathway.

BackgroundHypoxia is associated with abnormal cell apoptosis in trophoblast cells, which causes fetal growth restriction and related placental pathologies. Few effective methods for the prevention and treatment of placenta-related diseases exist. Natural products and functional foods have always been a rich source of potential anti-apoptotic drugs. Nobiletin (NOB), a hexamethoxyflavonoid derived from the citrus pomace, shows an anti-apoptotic activity, which is a non-toxic constituent of dietary phytochemicals approved by the Food and Drug Administration. However, their effects on hypoxia-induced human trophoblast cells have not been fully studied.ObjectiveThe aim of this study was to investigate the protective effects of NOB on hypoxia-induced apoptosis of human trophoblast JEG-3 and BeWo cells, and their underlying mechanisms.DesignFirst, the protective effect of NOB on hypoxia-induced apoptosis of JEG-3 and BeWo cells was studied. Cell viability and membrane integrity were determined by CCK-8 assay and lactate dehydrogenase activity, respectively. Real Time Quantitative PCR (RT-qPCR) and Western blot analysis were used to detect the mRNA and protein levels of HIF1α. Propidium iodide (PI)-labeled flow cytometry was used to detect cell cycle distribution. Cell apoptosis was detected by flow cytometry with Annexin V-FITC and PI double staining, and the expression of apoptosis marker protein cl-PARP was detected by Western blot analysis. Then, the molecular mechanism of NOB against apoptosis was investigated. Computer molecular docking and dynamics were used to simulate the interaction between NOB and p53 protein, and this interaction was verified in vitro by Ultraviolet and visible spectrum (UV-visible spectroscopy), fluorescence spectroscopy and circular dichroism. Furthermore, the changes in the expression of p53 signaling pathway genes and proteins were detected by RT-qPCR and Western blot analysis, respectively.ResultsHypoxia treatment resulted in a decreased cell viability and cell membrane integrity in JEG-3 and BeWo cell lines, and an increased expression of HIF1α, cell cycle arrest in the G1 phase, and massive cell apoptosis, which were alleviated after NOB treatment. Molecular docking and dynamics simulations found that NOB spontaneously bonded to human p53 protein, leading to the change of protein conformation. The intermolecular interaction between NOB and human p53 protein was further confirmed by UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism. After the treatment of 100 μM NOB, a down-regulation of mRNA and protein levels of p53 and p21 and an up-regulation of BCL2/BAX mRNA and protein ratio were observed in JEG-3 cells; however, there was also a down-regulation of mRNA and protein levels observed for p53 and p21 in BeWo cells after the treatment of NOB. The BCL2/BAX ratio of BeWo cells did not change after the treatment of 100 μM NOB.ConclusionNOB attenuated hypoxia-induced apoptosis in JEG-3 and BeWo cell lines and might be a potential functional ingredient to prevent pregnancy-related diseases caused by hypoxia-induced apoptosis. These findings would also suggest the exploration and utilization of citrus resources, and the development of citrus industry.

Phenothiazine Inhibits Neuroinflammation and Inflammasome Activation Independent of Hypothermia After Ischemic Stroke.

A depressive or hibernation-like effect of chlorpromazine and promethazine (C + P) on brain activity was reported to induce neuroprotection, with or without induced-hypothermia. However, the underlying mechanisms remain unclear. The current study evaluated the pharmacological function of C + P on the inhibition of neuroinflammatory response and inflammasome activation after ischemia/reperfusion. A total of 72 adult male Sprague-Dawley rats were subjected to 2h middle cerebral artery occlusion (MCAO) followed by 6 or 24h reperfusion. At the onset of reperfusion, rats received C + P (8mg/kg) with temperature control. Brain cell death was detected by measuring CD68 and myeloperoxidase (MPO) levels. Inflammasome activation was measured by mRNA levels of NLRP3, IL-1β, and TXNIP, and protein quantities of NLRP3, IL-1β, TXNIP, cleaved-Caspase-1, and IL-18. Activation of JAK2/STAT3 pathway was detected by the phosphorylation of STAT3 (p-STAT3) and JAK2 (p-JAK2), and the co-localization of p-STAT3 and NLRP3. Activation of the p38 pathway was assessed with the protein levels of p-p38/p38. The mRNA and protein levels of HIF-1α, FoxO1, and p-FoxO1, and the co-localization of p-STAT3 with HIF-1α or FoxO1 were quantitated. As expected, C + P significantly reduced cell death and attenuated the neuroinflammatory response as determined by reduced CD68 and MPO. C + P decreased ischemia-induced inflammasome activation, shown by reduced mRNA and protein expressions of NLRP3, IL-1β, TXNIP, cleaved-Caspase-1, and IL-18. Phosphorylation of JAK2/STAT3 and p38 pathways and the co-localization of p-STAT3 with NLRP3 were also inhibited by C + P. Furthermore, mRNA levels of HIF-1α and FoxO1 were decreased in the C + P group. While C + P inhibited HIF-1α protein expression, it increased FoxO1 phosphorylation, which promoted the exclusion of FoxO1 from the nucleus and inhibited FoxO1 activity. At the same time, C + P reduced the co-localization of p-STAT3 with HIF-1α or FoxO1. In conclusion, C + P treatment conferred neuroprotection in stroke by suppressing neuroinflammation and NLRP3 inflammasome activation. The present study suggests that JAK2/STAT3/p38/HIF-1α/FoxO1 are vital regulators and potential targets for efficacious therapy following ischemic stroke.

Acteoside isolated from Colebrookea oppositifolia attenuates I/R brain injury in Wistar rats via modulation of HIF-1α, NF-κB, and VEGF pathways.

The objective of this study was to assess the anti-stroke activity of acteoside isolated from methanolic root extract of C. oppositifolia METHODS: Ischemia-reperfusion(I/R) brain injury was induced in Wistar rats to assess the anti-stroke activity of acteoside. Rats were pretreated with acteoside (10, 25 & 50mg/kg, p.o.) before the induction of I/R injury. Parameters such as neurological, motor-cognitive functionswere evaluated along with morphological (brain volume, infarct size), biochemical (SOD, Catalase, GSH, lipid peroxidation, TNF-α, IL-6, IL-10, ICAM-1, HIF-1α, VEGF, and NF-κB), histopathological, and gene expression studies (HIF-1α, VEGF) were performed to study the protective effect of acteoside against I/R induced brain injury. I/R injury caused significant deterioration of neurological (p < 0.01), motor (p < 0.01) and cognitive (p < 0.01) functions, associated with increase in the brain volume (p < 0.01), and infarct size (p < 0.01); increase in the levels of MDA, TNF-α, IL-6, ICAM-1, HIF-1α, VEGF, and NF-κBalong with significant decrease in SOD, catalase, GSH, and IL-10 (p < 0.01 for all parameters) compared to Sham control group. Histology of brain tissue of disease control group exhibited significant vascular changes, neutrophil infiltration, cerebral oedema, and necrosis of the neuronal cells. Further, the gene-expression studies showed significant increase in the HIF-1α (p < 0.01) and VEGF (p < 0.01) mRNA levels in the I/R control compared to Sham control. Interestingly, the acteoside (10, 25 & 50mg/kg) has prevented the neurological, motor and cognitive dysfunctions, along with inhibiting the morphological, biochemical, histological and gene expression changes induced by I/R-injury (p < 0.05 for 10mg; p < 0.01 for 25 & 50mg/kg of acteoside for all the parameters). These findings suggest that acteoside possess potent anti-stroke activity through modulation of HIF-1α, NF-κB, and VEGF pathway along with its potent antioxidant activity.

Can hypoxia-inducible factor-1α overexpression discriminate human colorectal cancers with different microsatellite instability?

Clinicopathological features of high-frequency microsatellite instability (MSI-H) colorectal cancers (CRCs) are different from low-frequency MSI (MSI-L) and microsatellite stable (MSS) CRCs. The clinical features of MSI-L cases are unknown, and although the tumors usually show instability for dinucleotide markers, evaluation based on dinucleotides alone could lead to the misclassification of MSI-L or MSS as MSI-H. In this research, we investigated the usefulness of hypoxia-inducible factor-1α (HIF-1α) expression to discriminate MSI-L from MSS and MSI-H in human CRC. Tumor tissue from 94 CRC patients was used to determine the expression level of HIF-1α mRNA and HIF-1α protein using quantitative real-time PCR and immunohistochemistry analyses, respectively. The results indicated that HIF-1α mRNA and HIF-1α protein levels were upregulated in CRC patients compared with controls (P < 0.0001). Average HIF-1α expression in tissues with advanced stages and grades was also higher than that in earlier stages and grades. Expression of HIF-1α mRNA varied between CRC patients with different types of microsatellite instability (MSS, MSI-L and MSI-H). Taken together, our findings provide preliminary evidence that HIF-1α expression level in CRC tumors correlates with different MSI categories. HIF-1α expression may therefore represent a novel marker to separate the MSI-L group from the MSS and MSI-H groups.

Chronic Kidney Disease Induced by Cisplatin, Folic Acid and Renal Ischemia Reperfusion Induces Anemia and Promotes GATA-2 Activation in Mice.

Anemia is a common feature of chronic kidney disease (CKD). It is a process related to erythropoietin deficiency, shortened erythrocyte survival, uremic erythropoiesis inhibitors, and disordered iron homeostasis. Animal models of CKD-induced anemia are missing and would be desirable in order to study anemia mechanisms and facilitate the development of novel therapeutic tools. We induced three different models of CKD in mice and evaluated the development of anemia characteristics. Mice were subjected to unilateral ischemia-reperfusion or received repeated low doses of cisplatin or folic acid to induce nephropathy. Renal function, kidney injury and fibrotic markers were measured to confirm CKD. Moreover, serum hemoglobin, ferritin and erythropoietin were analyzed. Renal mRNA levels of HIF-2α, erythropoietin, hepcidin, GATA-2, and GATA-2 target genes were also determined. All three CKD models presented increased levels of creatinine, urea, and proteinuria. Renal up-regulation of NGAL, KIM-1, and TNF-α mRNA levels was observed. Moreover, the three CKD models developed fibrosis and presented increased fibrotic markers and α-SMA protein levels. CKD induced decreased hemoglobin and ferritin levels and increased erythropoietin levels in the serum. Renal tissue showed decreased erythropoietin and HIF-2α mRNA levels, while an increase in the iron metabolism regulator hepcidin was observed. GATA-2 transcription factor (erythropoietin repressor) mRNA levels were increased in all CKD models, as well as its target genes. We established three models of CKD-induced anemia, regardless of the mechanism and severity of kidney injury.

Abstract LB256: Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) invasion, metastasis and decreased cancer-specific survival. Kindlins are integrin adaptors and mechanosensory focal adhesion (FA) proteins that activate and cluster integrins in response to structural ECM alterations in the tumor microenvironment. Hypoxia is a well-known inducer of tumor migration and invasion, results in ECM remodeling, and is physiologically relevant for PCa. The study's objective was to determine if integrin-kindlin-2 adhesion complexes were responsive to hypoxia and the dynamic distribution of integrin-kindlin-2 complexes using immunofluorescence microscopy (IFM). The methods included a combination of biochemical analysis of kindlin-2 adhesion complexes, qRT-PCR, immunoblotting, and immunocytochemistry. DU145 cells were analyzed in HEPES-containing media with 10% FBS under hypoxic conditions (1% O2) continuously exposed for 4, 8, 12, and 16 hours. The results showed that hypoxia increased the mRNA expression of CAIX, a well-known HIF-1α target gene, within 4 hours, resulting in 7-fold maximum expression. Similarly, VEGF-A mRNA was responsive to hypoxia as expected. In contrast, HIF-1α, α6 integrin, and kindlin-2 mRNA levels remained unchanged as compared to normoxia. Strikingly, we found that kindlin-2-containing adhesion complexes increased under hypoxia conditions as compared to normoxia and detected by immunoprecipitation, the complexes contained α6β1 integrin. It is important to note that the constitutive levels of either α6 integrin or kindlin-2 were not altered by hypoxia. Using IFM under normoxic conditions, we confirmed the location of kindlin-2 within phosphorylated (Y31) paxillin (pPXN)-containing FAs and within extended plasma membrane domains exclusive of pPXN. However, under hypoxic conditions, an increased reorganization of pPXN-containing kindlin-2 complexes occurred within 4 hours, was continuously changing up to 16 hours, with increasing fibrillar forms of FAs. In addition, kindlin-2 was observed as increased in plasma membrane protrusions devoid pPXN. The dynamic nature of the hypoxia-driven FAs was observed by a time course analysis and indicated the apparent assembly and disassembly of the structures during 16-hours of PCa cells exposed to hypoxia. We conclude that kindlin-2-integrin complexes are responsive to hypoxia and contain the unexpected α6β1 integrin in the membrane exclusive of FAs. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB256.

Biogenic silver nanoparticles synthesized from Piper longum fruit extract inhibit HIF-1α/VEGF mediated angiogenesis in prostate cancer cells

In the present study, biogenic silver nanoparticles (PL-AgNPs) were synthesized by using Piper longum (PL) dried fruit extract and investigated the effect of PL-AgNPs on angiogenesis and signal transduction pathway of hypoxia-inducible factor alpha (HIF-1α)/vascular endothelial growth factor (VEGF) on human prostate cancer cells. The prepared PL-AgNPs were characterized by ultraviolet-visible (UV-vis) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and dynamic light scattering (DLS) analysis. Potential cytotoxic and anti-angiogenic effects of PL-AgNPs were evaluated on PC-3 and DU-145 prostate cancer cells. Cytotoxicity was evaluated by MTT assay. The anti-angiogenic effect was investigated via a cell migration assay. Protein and mRNA levels of key angiogenesis related molecules such as VEGF and HIF-1α were evaluated via ELISA and qRT-PCR assays. UV-vis spectroscopy showed an absorbance peak at 450 nm confirming the PL-AgNPs synthesis. Various characterization techniques revealed that the average size of synthesized PL-AgNPs was below 100 nm. The cytotoxic effect was elevated in a concentration-dependent manner (p˂0.05). The biosynthesized PL-AgNPs inhibited cell migration and reduced the levels of both protein and mRNA levels of VEGF and HIF-1α in prostate cancer cells (p˂0.05). Results revealed that the PL extract with AgNO3 nanoparticles may be a potential candidate for developing novel anticancer and antiangiogenic compounds for prostate cancer.

Melittin inhibits the expression of key genes involved in tumor microenvironment formation by suppressing HIF-1α signaling in breast cancer cells.

HIF-1α has critical roles in the formation of tumor microenvironment by regulating genes involved in angiogenesis and anaerobic respiration. TME fuels tumors' growth and metastasis and presents therapy with several challenges. Therefore, we aimed to investigate if Melittin disrupts HIF-1α signaling pathway in breast adenocarcinoma cell line MDA-MB-231. Breast adenocarcinoma cell line MDA-MB-231 was cultured in the presence of different doses of Melittin, and MTT assay was carried out to measure Melittin's cytotoxic effects. Cells were exposed to 5% O2 to mimic hypoxic conditions and Melittin. Western blot was used to measure HIF-1α protein levels. Gene expression analysis was performed using real-time PCR to measure relative mRNA abundance of genes involved in tumor microenvironment formation. Our results revealed that Melittin effectively inhibits HIF-1α at transcriptional and translational/post-translational level. HIF-1α protein and mRNA level were significantly decreased in Melittin-treated groups. It is found that inhibition of HIF-1α by Melittin is through downregulation of NFκB gene expression. Furthermore, gene expression analysis showed a downregulation in VEGFA and LDHA expression due to inhibition of HIF-1α protein by Melittin. In addition, cell toxicity assay showed that Melittin inhibits the growth of MDA-MB-231 cell line through activation of extrinsic and intrinsic apoptotic pathways by upregulating TNFA and BAX expression. Melittin suppresses the expression of genes responsible for formation of TME physiological hallmarks by suppressing HIF-1α signaling pathway. Our results suggest that Melittin can modulate tumor microenvironment by inhibition of VEGFA and LDHA.

Multitarget neuroprotection by quercetin: Changes in gene expression in two perinatal asphyxia models

Hypoxic-ischemic encephalopathy (HIE) causes mortality and long-term neurologic morbidities in newborns, affecting pathways related to energy failure, excitotoxicity and oxidative stress that often lead to cell death. The whole process of HIE injury is coupled to changes in the expression of a great array of proteins. A nanoliposomal preparation of the flavonoid quercetin has been shown to exert neuroprotective effects in perinatal asphyxia models.This study aimed to identify neonatal HIE markers and explore the effect of quercetin administration in two perinatal asphyxia models: newborn rats and piglets. In the rat model, nanoliposomal quercetin administration reduced mortality after asphyxia. In the piglet model, quercetin partially overrode the reduction of HIF-1α mRNA levels in the cortex induced by asphyxia. Quercetin administration also reduced increased level of HO-1 mRNA in asphyctic piglets. These results suggest that quercetin neuroprotection might be involved in the regulation of HIF-1α, HO-1 and their targets.A proteomic approach revealed that the glycolytic pathway is strongly regulated by quercetin in both species. We also identified a set of proteins differentially expressed that could be further considered as markers. In piglets, this set includes Acidic Leucine-rich nuclear phosphoprotein 32 (ANP32A), associated with nervous system differentiation, proteins related with death pathways and alpha-enolase which can be converted to neuron-specific enolase, a glycolytic enzyme that may promote neuroprotection. In newborn rats, other promising proteins associated with neurogenesis and neuroprotection emerged, such as dihydropyrimidinase-related proteins, catalytic and regulatory subunits of phosphatases and heterogeneous nuclear ribonucleoprotein K (hnRNPK).Our results show that a nanoliposomal preparation of quercetin, with protective effect in two HIE mammal models, modulates the expression of proteins involved in energy metabolism and other putative neuroprotective signals in the cortex. Identification of these signals could reveal potential molecular pathways involved in disease onset and the novel quercetin neuroprotective strategy.

Hypoxia Inducible Factor (HIF)‐1α is a Transcriptional Mediator for Thiamine Insufficiency Induced Neurotoxicity

Age-dependent decline in vitamin B1 (thiamine) levels has a devastating impact on the brain parenchyma. The neurotoxicity promoted by chronic thiamine insufficiency (TI) is a well-established comorbidity of Alzheimer's Disease (AD), the most prevalent age-related metabolic neurodegenerative disease. Inadequate levels of blood thiamine and derivates highly correlate with senile dementia prognosis as predictive peripheral AD biomarker. Impaired activity of thiamine-dependent enzymes due to TI, dramatically decreased cerebral oxidative metabolism promoting oxidative stress, mitochondrial damage, inflammation, and eventually neuronal loss. Furthermore, age-related decline in TI potentiates the AD neuropathology triggering the amyloid plaque formation and hyperphosphorylation of tau. We previously showed that TI stabilizes Hypoxia Inducible Factor -1α (HIF-1α), the main transcription factor involved in hypoxic stress, leading to HIF-1α induced pro-apoptotic and pro-inflammatory response. Among the transcriptionally activated HIF-1α target genes, TI triggered the expression of the pro-apoptotic protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (BNIP-3). Induction of BNIP-3 leads to its mitochondria migration where eventually triggers cell death via loss of membrane potential and increase in reactive oxygen species. To date, despite the well-established correlation between TI and neuropathogenesis, the molecular mechanism underlying the TI mediated neurotoxicity is still unknown. Therefore, we hypothesize that HIF-1α may be a critical upstream mediator between thiamine insufficiency and neurotoxicity. In this study, hippocampal murine cells (HT22) treated in TI conditions exhibited a significant increase in protein and mRNA levels of HIF-1α. Increase in BNIP-3 and other HIF-1α target genes (LDHA, VEGF, BACE-1, MCP-1) implicated in pro-inflammatory and pro-apoptotic response was also detected. Treatment with 50 μM and 100 μM of the thiamine antagonist Pyrithiamine (PT) up to 5 days significantly decrease HT22 viability to 30% and 20%, respectively. HIF-1α silencing via shRNA attenuated the neurotoxic response improving viability up to 60% with the same PT doses. In order to better characterize the HIF-1α response, BNIP-3 was also silenced via shRNA. In congruency with HIF-1α shRNA result, we found a 2-fold decrease in PT-induced toxicity in BNIP-3 shRNA transduced cells. Localization of BNIP-3 in mitochondria and loss of mitochondrial potential after PT treatment further highlighted the involvement of the HIF-1α - BNIP-3 response in TI induced neuronal death. Overall, these findings suggest a central role for HIF-1α as upstream regulator in TI mediated neurotoxicity.

CELF1 promotes vascular endothelial growth factor degradation resulting in impaired microvasculature in heart failure.

Vascular rarefaction due to impaired angiogenesis is associated with contractile dysfunction and the transition from compensation to decompensation and heart failure. The regulatory mechanism controlling vascular rarefaction during the transition remains elusive. Increased expression of a nuclear RNA-binding protein CUGBP Elav-like family member 1 (CELF1) in the adult heart is associated with the transition from compensated hypertrophy to decompensated heart failure. Elevated CELF1 level resulted in degradation of the major cardiac gap junction protein, connexin 43, in dilated cardiomyopathy (DCM), the most common cause of heart failure. In the present study, we investigated the role of increased CELF1 expression in causing vascular rarefaction in DCM. CELF1 overexpression (CELF1-OE) in cardiomyocytes resulted in reduced capillary density. CELF1-OE mice administered hypoxyprobe showed immunoreactivity and increased mRNA levels of HIF1α, Glut-1, and Pdk-1, which suggested the association of a reduced capillary density-induced hypoxic condition with CELF1 overexpression. Vegfa mRNA level was downregulated in mouse hearts exhibiting DCM, including CELF1-OE and infarcted hearts. Vegfa mRNA level was also downregulated to a similar extent in cardiomyocytes isolated from infarcted hearts by Langendorff preparation, which suggested cardiomyocyte-derived Vegfa expression mediated by CELF1. Cardiomyocyte-specific depletion of CELF1 preserved the capillary density and Vegfa mRNA level in infarcted mouse hearts. Also, CELF1 bound to Vegfa mRNA and regulated Vegfa mRNA stability via the 3' untranslated region. These results suggest that elevated CELF1 level has dual effects on impairing the functions of cardiomyocytes and microvasculature in DCM.

Excisanin A suppresses proliferation by inhibiting hypoxiainducible factor-1α expression in human hepatocellular carcinoma cells

Purpose: To investigate the effect of excisanin A on human hepatocellular carcinoma cells as well as to elucidate its mechanism of action.&#x0D; Methods: Molecular docking was used to determine the binding characteristics of excisanin A to HIF-1α protein. The transcriptional activation and viability of excisanin A were assessed using Luciferase reporter and MTT assay. The HIF-1α protein in the nucleus was assayed using western blot and immunofluorescence. HIF-1α and VEGF mRNA levels were evaluated using reverse-transcription polymerase chain reaction (RT-PCR). Cell proliferation was determined by flow cytometry, as well as by EdU and clonogenic assays. In vivo tumor growth was assessed in a murine xenograft model of SKHep1 cells.&#x0D; Results: Excisanin A inhibited HIF-1α transcriptional activation, as well as HIF-1α protein synthesis (p &lt; 0.001). Excisanin A also reduced VEGF protein and mRNA expressions (p &lt; 0.001). In addition, the compound inhibited the proliferation of hepatocellular carcinoma cells. and tumor growth in the xenograft tumor model.&#x0D; Conclusion: Excisanin A is a potent HIF-1α inhibitor, supporting its potential development for human hepatoma therapy.&#x0D; Keywords: Excisanin A, HIF-1α, Protein synthesis, Hepatoma therapy