Regulation of genes involved in the metabolic adaptation of murine microglial cells in response to elevated HIF-1α mediated activation.

Microglia cells are activated in response to different stress signals. Several metabolic adaptations underlie microglia activation in the brain. Among these, in conditions like ischemic stroke and, hypoxic stress stimuli activate microglia cells. Hypoxic stress is mediated by HIF-1α. Although HIF-1α has been implicated in the alteration of metabolic pathways, changes in microglia lipid metabolism during M1 activation of microglia induced by elevated HIF-1α levels are yet to be understood. This can also merit interest in the development of novel targets to mitigate chronic inflammation. Our study aims to elucidate the transcriptional regulation of metabolic pathways in microglia cells during HIF-1α mediated activation. To study the adaptations in the metabolic pathways we induced microglia activation, by activating HIF-1α. Here, we show that microglia cells activated in response to elevated HIF-1α require ongoing lipogenesis and fatty acid breakdown. Notably, autophagy is activated during the initial stages of microglia activation. Inhibition of autophagy in activated microglia affects their viability and phagocytic activity. Collectively, our study expands the understanding of the molecular link between autophagy, lipid metabolism, and inflammation during HIF-1α mediated microglial activation that can lead to the development of promising strategies for controlling maladaptive activation states of microglia responsible for neuroinflammation. Together, our findings suggest that the role of HIF-1α in regulating metabolic pathways during hypoxia in microglia is beyond optimization of glucose utilization and distinctly regulates lipid metabolism during pro-inflammatory activation.

Liposome-encapsulated hemoglobin rescues hemorrhagic shock heart through anti-ischemic and anti-arrhythmogenic effects on myocardium in repetitive 65% bleeding rat model.

BACKGROUND: Phase I clinical trial of an artificial oxygen carrier (liposome-encapsulated hemoglobin vesicles [HbVs]) is safely completed, considering the other clinical application. Herein, we aimed to investigate the resuscitation effects of HbVs in cases of lethal hemorrhage, including the mechanisms involved. METHODS: Optical mapping analysis (OMP) and electrophysiological studies (EPS), immunostaining pathological examination for hypoxia-inducible factor-1α (HIF1-alpha) in the heart tissue, and blood troponin I (TnI) level measurements were performed in rats that underwent five rounds of spontaneous arterial bleeding with up to 65% hemorrhage. RESULTS: Ten rats in each group were resuscitated by a transvenous infusion of 5% albumin (ALB), washed erythrocytes (wRBC), HbVs (HbV), 50% HbV diluted by 5% albumin (50% HbV), and 66% HbV diluted by 5% albumin (66% HbV). The rats in the ALB and 50% HbV groups died, whereas those in the other groups survived. OMP showed impaired action potential duration dispersion (APDd) in the left ventricle in the ALB and 50% HbV groups, which was attenuated in the other groups. Lethal arrhythmias were provoked by EPS in the ALB and 50% HbV groups but not in the other groups. HIF1-alpha was positively stained only in the ALB and 50% HbV groups. TnI levels were elevated in the ALB and 50% HbV groups. CONCLUSIONS: Acute lethal hemorrhage causes myocardial ischemia with hypoxia and arrhythmias, which may be induced by impaired APDd and myocardial damage, reflected in the increased levels of HIF1-alpha and TnI. HbVs could be useful for resuscitation and may help save patients with injuries such as gunshot wounds.

A Phase I Trial of Bevacizumab and Temsirolimus in Combination With Valproic Acid in Advanced Solid Tumors.

BACKGROUND: Preclinical models suggest synergy between anti-angiogenesis therapy, mammalian target of rapamycin (mTOR), and histone deacetylase inhibitors to promote anticancer activity. METHODS: This phase I study enrolled 47 patients between April 2012 and 2018 and determined safety, maximum tolerated dose (MTD), and dose-limiting toxicities (DLTs) when combining bevacizumab, temsirolimus, and valproic acid in patients with advanced cancer. RESULTS: Median age of enrolled patients was 56 years. Patients were heavily pretreated with a median of 4 lines of prior therapy. Forty-five patients (95.7%) experienced one or more treatment-related adverse events (TRAEs). Grade 3 TRAEs were lymphopenia (14.9%), thrombocytopenia (8.5%), and mucositis (6.4%). Grade 4 TRAEs included lymphopenia (2.1%) and CNS cerebrovascular ischemia (2.1%). Six patients developed DLTs across 10 dose levels with grade 3 infection, rash, mucositis, bowel perforation, elevated lipase, and grade 4 cerebrovascular ischemia. The MTD was dose level 9 (bevacizumab 5 mg/kg days 1 and 15 intravenously (IV) plus temsirolimus 25 mg days 1, 8, 15, and 22 IV and valproic acid 5 mg/kg on days 1-7 and 15-21 per orally (PO)). Objective response rate (ORR) was 7.9% with confirmed partial response (PRs) in 3 patients (one each in parotid gland, ovarian, and vaginal cancers). Stable disease (SD) ≥+6 months was seen in 5 patients (13.1%). Clinical benefit state (CBR: PR + SD ≥+6 months) was 21%. CONCLUSION: Combination therapy with bevacizumab, temsirolimus, and valproic acid was feasible, but there were numerous toxicities, which will require careful management for future clinical development (ClinicalTrials.gov Identifier: NCT01552434).

Analyses of Elaeocarpus sphaericus Extract for Antioxidant, Antiproliferative and Gene Repression Activities against HIF-1α and VEGF.

The study presents antioxidant, phytochemical, anti-proliferative, and gene repression activities against Hypoxia-inducible factor (HIF-1) alpha and Vascular endothelial growth factor (VEGF) of Elaeocarpus sphaericus extract. Elaeocarpus sphaericus dried and crushed plant leaves were extracted using water and methanol by ASE (Accelerated Solvent Extraction) method. Total phenolic content (TPC) and total flavonoid content (TFC) were used to measure the extracts' phytochemical activity (TFC). Antioxidant potential of the extracts was measured through DPPH, ABTS, FRAP, and TRP. Methanolic extract of the leaves of E. sphaericus has shown a higher amount of TPC (94.666±4.040 mg/gm GAE) and TFC value (172.33±3.21 mg/gm RE). The antioxidant properties of extracts in the yeast model (Drug Rescue assay) showed promising results. Ascorbic acid, gallic acid, hesperidin, and quercetin were found in the aqueous and methanolic extracts of E. sphaericus at varying amounts, according to a densiometric chromatogram generated by HPTLC analysis. Methanolic extract of E. sphaericus (10 mg/ml) has shown good antimicrobial potential against all bacterial strains used in the study except E. coli. The anticancer activity of the extract in HeLa cell lines ranged from 77.94±1.03 % to 66.85±1.95 %, while it ranged from 52.83±2.57 % to 5.44 % in Vero cell lines at varying concentration (1000 µg/ml-31.2 µg/ml). A promising effect of extract was observed on the expression activity of HIF-1 and VEGF gene through RT-PCR assay.

miR-210 Expression Is Strongly Hypoxia-Induced in Anaplastic Thyroid Cancer Cell Lines and Is Associated with Extracellular Vesicles and Argonaute-2.

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.

Adipose-Derived Stem Cells Improve Angiogenesis and Lymphangiogenesis in a Hypoxic Dermal Regeneration Model In Vitro.

Background and Objectives: Impaired wound healing represents an unsolved medical issue with a high impact on patients' quality of life and global health care. Even though hypoxia is a significant limiting factor for wound healing, it reveals stimulating effects in gene and protein expression at cellular levels. In particular, hypoxically treated human adipose tissue-derived stem cells (ASCs) have previously been used to stimulate tissue regeneration. Therefore, we hypothesized that they could promote lymphangiogenesis or angiogenesis. Materials and Methods: Dermal regeneration matrices were seeded with human umbilical vein endothelial cells (HUVECs) or human dermal lymphatic endothelial cells (LECs) that were merged with ASCs. Cultures were maintained for 24 h and 7 days under normoxic or hypoxic conditions. Finally, gene and protein expression were measured regarding subtypes of VEGF, corresponding receptors, and intracellular signaling pathways, especially hypoxia-inducible factor-mediated pathways using multiplex-RT-qPCR and ELISA assays. Results: All cell types reacted to hypoxia with an alteration of gene expression. In particular, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), vascular endothelial growth factor receptor 1 (VEGFR1/FLT1), vascular endothelial growth factor receptor 2 (VEGFR2/KDR), vascular endothelial growth factor receptor 3 (VEGFR3/FLT4), and prospero homeobox 1 (PROX1) were overexpressed significantly depending on upregulation of hypoxia-inducible factor 1 alpha (HIF-1a). Moreover, co-cultures with ASCs showed a more intense change in gene and protein expression profiles and gained enhanced angiogenic and lymphangiogenic potential. In particular, long-term hypoxia led to continuous stimulation of HUVECs by ASCs. Conclusions: Our findings demonstrated the benefit of hypoxic conditioned ASCs in dermal regeneration concerning angiogenesis and lymphangiogenesis. Even a short hypoxic treatment of 24 h led to the stimulation of LECs and HUVECs in an ASC-co-culture. Long-term hypoxia showed a continuous influence on gene expressions. Therefore, this work emphasizes the supporting effects of hypoxia-conditioned-ASC-loaded collagen scaffolds on wound healing in dermal regeneration.

[Immunohistochemical analysis of a hypoxia-associated signature in melanomas with positive and negative sentinel lymph nodes : Hypoxia-associated signature of primary cutaneous melanomas]. / Immunhistochemische Analyse einer Hypoxie-assoziierten Signatur in Melanomen mit positivem und negativem Schildwächterlymphknoten : Hypoxie-assoziierte Signatur primär kutaner Melanome.

Metabolic reprogramming mediated by hypoxia-inducible factors and its downstream targets plays a crucial role in many human malignancies. Excessive proliferation of tumor cells under hypoxic conditions leads to metabolic reprogramming and altered gene expression enabling tumors to adapt to their hypoxic environment. Here we analyzed the metabolic signatures of primary cutaneous melanomas with positive and negative sentinel node status in order to evaluate potential differences in their metabolic signature. We found a positive correlation of the expression of glucose transporter 1 (GLUT-1) with tumor thickness and ulceration in all melanomas with subgroup analyses as well as in the subgroup with a negative sentinel node. Furthermore, the expression of vascular endothelial growth factor (VEGF) was positively correlated with the presence of ulceration in melanomas with positive sentinel node.

Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†.

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

Sustained Hypoxia Suppresses Joint Destruction in a Rat Model of Rheumatoid Arthritis via Negative Feedback of Hypoxia Inducible Factor-1α.

Hypoxia inducible factor (HIF)-1α has been implicated in the pathogenesis of rheumatoid arthritis (RA). HIF-1α, which is expressed in hypoxia, is reversely suppressed in sustained hypoxia. Here, we investigated the inhibitory effect of hypoxia on arthritis by controlling HIF-1α. Rheumatoid fibroblast-like synoviocyte MH7A cells were cultured in a hypoxic incubator for up to 72 h to evaluate the expression of HIF-1. Furthermore, collagen-induced arthritis (CIA) model rats were maintained under 12% hypoxia in a hypoxic chamber for 28 days to evaluate the effect on arthritis. In MH7A cells, HIF-1α protein level increased at 3 h, peaked at 6 h, and subsequently decreased in a time-dependent manner. The transcription of pro-inflammatory cytokines increased at 1 h; however, they decreased after 3 h (p < 0.05). Deferoxamine-mediated activation of HIF-1α abolished the inhibitory effect of sustained hypoxia on pro-inflammatory cytokines. In the rat CIA model, the onset of joint swelling was delayed and arthritis was suppressed in the hypoxia group compared with the normoxia group (p < 0.05). Histologically, joint destruction was suppressed primarily in the cartilage. Thus, sustained hypoxia may represent a new safe, and potent therapeutic approach for high-risk patients with RA by suppressing HIF-1α expression.

Yersiniabactin Siderophore of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Is Involved in Autophagy Activation in Host Cells.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) have been implicated in the etiology of Crohn's disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of Yersinia spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection. METHODS: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression. RESULTS: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells. CONCLUSION: Our study highlights a novel role for yersiniabactin siderophore in AIEC-host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.

COVID-19-driven endothelial damage: complement, HIF-1, and ABL2 are potential pathways of damage and targets for cure.

COVID-19 pandemia is a major health emergency causing hundreds of deaths worldwide. The high reported morbidity has been related to hypoxia and inflammation leading to endothelial dysfunction and aberrant coagulation in small and large vessels. This review addresses some of the pathways leading to endothelial derangement, such as complement, HIF-1α, and ABL tyrosine kinases. This review also highlights potential targets for prevention and therapy of COVID-19-related organ damage and discusses the role of marketed drugs, such as eculizumab and imatinib, as suitable candidates for clinical trials.

RBM38 induces SIRT1 expression during hypoxia in non-small cell lung cancer cells by suppressing MIR34A expression.

OBJECTIVE: The study is to research how miR-34-SIRT1 is regulated during hypoxia in lung cancer cells. RESULTS: Analysis of publicly available datasets from patients with NSCLC did not reveal significant genomic alterations in RBM38, SIRT1, HIF1A, MIR34A, MIR34B, and MIR34C, but expectedly revealed alterations in TP53. Overall survival in NSCLC patients with or without alterations in these genes was not significantly different. When expanded to include all lung cancer patients, overall survival was significantly lower in patients with genomic alterations in these genes. CONCLUSIONS: Cumulatively, our results reveal a novel mechanism of RBM38-mediated regulation of the HIF1A/miR-34a/SIRT1/p53 axis under hypoxia in NSCLC cells.

Hypoxia Compromises Anti-Cancer Immune Responses.

Hypoxia, one of the hallmarks of cancer, is caused by an insufficient oxygen supply, mostly due to a chaotic, deficient tumor microcirculation. Apart from a hypoxia-mediated resistance to standard therapies, modulated gene and protein expression, genetic instability and malignant progression, hypoxia also plays a pivotal role in anti-cancer immune responses by (a) reducing survival, cytolytic and migratory activity of effector cells such as CD4+ cells, CD8+ cytotoxic T cells, natural killer-like T cells and natural killer cells, (b) reducing the production and release of effector cytokines, (c) supporting immunosuppressive cells such as regulatory T cells, myeloid-derived suppressor cells and M2 macrophages, (d) increasing the production and release of immunosuppressive cytokines, and (e) inducing the expression of immune checkpoint inhibitors. In this minireview, immunosuppressive effects of hypoxia- and HIF-1a-driven traits in cancers are described.

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis.

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

Could HIF-1α be a novel biomarker for the clinical course and treatment of pulmonary embolism?

Background/aim: Pulmonary embolism (PE) is associated with high morbidity and mortality rates if not diagnosed and treated rapidly. The aim of our study was to investigate the relationship between levels of hypoxia-induced factor-1 alpha (HIF-1α) and clinical course and prognosis in patients with intermediate low-risk, intermediate high-risk, and high-risk PE. Materials and methods: The study included 240 subjects in 4 groups: a healthy control group (n = 60, mean age = 60 ± 15.2, female/male = 30/30 ), intermediate low-risk PE group (n = 60, mean age = 60 ± 12,5, female/male = 27/33), intermediate high-risk PE group (n = 60, mean age = 61,4 ± 14,8, female/male = 36/24), and high-risk PE group (n = 60, mean age = 62,3 ± 15, female/male = 33/27). Plasma HIF-1α levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kit. Results: Comparison of HIF-1α levels revealed a statistically significant difference between the groups in proportion to clinical scoring (P = 0.001 for all). Comparison of initial HIF-1α and troponin levels in intermediate high-risk PE patients given thrombolytic therapy and those treated with enoxaparin sodium showed that HIF-1α levels were significantly higher in the group that received thrombolytic therapy (P = 0.001), while there was no difference in troponin levels (P = 0.146). Conclusion: HIF-1α can be used in the PE clinical risk stratification and monitoring of PE and may also serve as a valuable early indicator in intermediate high-risk PE, for which early reperfusion therapy is important.

The Milk Protein Alpha-Casein Suppresses Triple Negative Breast Cancer Stem Cell Activity Via STAT and HIF-1alpha Signalling Pathways in Breast Cancer Cells and Fibroblasts.

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

Functional Proteomics of Breast Cancer Metabolism Identifies GLUL as Responder during Hypoxic Adaptation.

Hypoxia as well as metabolism are central hallmarks of cancer, and hypoxia-inducible factors (HIFs) and metabolic effectors are crucial elements in oxygen-compromised tumor environments. Knowledge of changes in the expression of metabolic proteins in response to HIF function could provide mechanistic insights into adaptation to hypoxic stress, tumorigenesis, and disease progression. We analyzed time-resolved alterations in metabolism-associated protein levels in response to different oxygen potentials across breast cancer cell lines. Effects on the cellular metabolism of both HIF-dependent and -independent processes were analyzed by reverse-phase protein array profiling and a custom statistical model. We revealed a strong induction of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) as well as reduced glutamate-ammonia ligase (GLUL) protein levels across all cell lines tested as consistent changes upon hypoxia induction. Low GLUL protein levels were correlated with aggressive molecular subtypes in breast cancer patient data sets and also with hypoxic tumor regions in a xenograft mouse tumor model. Moreover, low GLUL expression was associated with poor survival in breast cancer patients and with high HIF-1α-expressing patient subgroups. Our data reveal time-resolved changes in the regulation of metabolic proteins under oxygen-deprived conditions and elucidate GLUL as a strong responder to HIFs and the hypoxic environment.

Angiopoietin-like 4 production upon treatment with hypoxia and L-mimosine in periodontal fibroblasts.

BACKGROUND AND OBJECTIVE: A key factor in the modulation of angiogenesis as well as in bone resorption is angiopoietin-like 4. However, the role of angiopoietin-like 4 in periodontal tissue is unknown. Here, we hypothesized that hypoxia and the hypoxia mimetic agent L-mimosine can induce the production of angiopoietin-like 4 in periodontal fibroblasts. METHODS: Human periodontal ligament fibroblasts (PDLF) were cultured in monolayer and spheroid cultures. The cultures were incubated in the presence of hypoxia or L-mimosine. Angiopoietin-like 4 mRNA and protein levels were measured by qPCR and ELISA, respectively. Also, the impact of Lipopolysaccharides of E. coli and P. gingivalis, interleukin (IL)-1ß and tumor necrosis factor (TNF)α was evaluated. Furthermore, we tested dependency on hypoxia-inducible factor (HIF)-1 activity by Western blotting for HIF-1 and inhibitor studies with echinomycin. Potential autocrine effects were assessed by exposure of PDLF to recombinant angiopoietin-like 4 in full length, C-terminal and N-terminal fragments. The impact on viability, DNA synthesis, alkaline phosphatase, and matrix mineralization was evaluated. RESULTS: Both hypoxia and L-mimosine elevated angiopoietin-like 4 mRNA and protein levels in monolayer cultures of PDLF. HIF-1 was elevated after both hypoxia and L-mimosine treatment. LPS, IL-1ß, and TNFα did not modulate angiopoietin-like 4 levels significantly. Addition of echinomycin in the cultures inhibited the production of angiopoietin-like 4. In spheroid cultures of PDLF, the increase did not reach the level of significance at mRNA and protein levels. Angiopoietin-like 4 in full length, C-terminal, and N-terminal fragments did not modulate viability, DNA synthesis, alkaline phosphatase, and matrix mineralization. CONCLUSION: Overall, we found that hypoxia and the hypoxia mimetic agent L-mimosine can stimulate angiopoietin-like 4 production in monolayer cultures of PDLF. This increase depends on HIF-1 activity. Future studies will reveal how the modulation of angiopoietin-like 4 in the periodontium contributes to periodontal disease and regeneration.

Expression of the oxygen-sensitive transcription factor subunit HIF-1α in patients suffering from secondary Raynaud syndrome.

Anti-ischemic therapy remains a challenge due to the complexity of hypoxia response pathways. Hypoxia-inducible factor (HIF)-1 is a heterodimer transcription factor consisting of 2 subunits, HIF-1α and HIF-1ß. Hypoxia-dependent activation of HIF-1α regulates cellular O2 homeostasis. Raynaud syndrome (RS), as a comorbidity of the autoimmune disease systemic sclerosis (SS), is characterized by vasospasms that limit blood flow to the limbs, resulting in hypoxia. A single-center randomized study was conducted to compare prostaglandin E1 (PgE1) therapy with a treatment combining PgE1 and an endothelin-1 blocker, bosentan. A total of 30 patients suffering from SS with RS were enrolled. We examined the regulation of HIF-1α, its target heme oxygenase-1 (HMOX-1), and the serum levels of the HIF-1α protein in a subset of patients as well as in ten healthy individuals. The expression of HIF-1α and HMOX-1 in monocytes was measured using absolute plasmid-based quantitative real-time PCR, whereas serum HIF-1α levels were measured with ELISA. Samples were taken at the time of randomization and after 24 weeks. We found that HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels were significantly higher in the SS/RS patients compared to the healthy control group. Single-drug therapy significantly increased HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels in the SS/RS patients compared to those at the time of randomization, whereas combining PgE1 with an endothelin-1 blocker prevented the further increases in HIF-1α and HMOX-1 expression. We propose HIF-1α and HMOX-1 as novel markers for anti-ischemic therapy in RS.

The Relevance of Vascular Endothelial Growth Factor, Hypoxia Inducible Factor-1 Alpha, and Clusterin in Carotid Plaque Instability.

BACKGROUND: Stroke is one of the leading causes of morbidity and mortality. Thromboembolism, as a major cause of carotid artery-related stroke, can be caused by plaque rupture which is associated with neoangiogenesis within the carotid plaque. AIM: We sought to investigate a possible correlation between angiogenesis-related factors and preoperative neurological manifestations in patients with internal carotid artery stenosis, for a better understanding of thromboembolism in internal carotid artery stenosis-related stroke. METHODS: This study included 54 patients (asymptomatic, n = 20 and symptomatic, n = 34) undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. In the retrieved carotid plaques, angiogenesis-related factors (vascular endothelial growth factor [VEGF], hypoxia inducible factor-1 alpha [HIF-1α], and Clusterin) were measured by immunohistochemistry and quantified by real-time polymerase chain reaction. RESULTS: We demonstrated the expression of VEGF, HIF-1α, and Clusterin by endothelial cells and smooth muscle cells in the carotid plaques. Noteworthy, mRNA VEGF levels were .7-fold higher in symptomatic patients (P = .017) compared to asymptomatic patients. In contrast, mRNA Clusterin levels were 1.8-fold lower (P = .021). Levels of mRNA HIF-1α were 1.5-fold higher in asymptomatic patients, but no statistical significance was reached between the 2 groups. CONCLUSIONS: Our results show an association between VEGF and Clusterin and neurological symptoms of patients with high-grade carotid artery stenosis.