Low-Dose Near-Infrared Light-Activated Mitochondria-Targeting Photosensitizers for PDT Cancer Therapy.

Phthalocyanines (Pcs) are promising candidates for photodynamic therapy (PDT) due to their absorption in the phototherapeutic window. However, the highly aromatic Pc core leads to undesired aggregation and decreased reactive oxygen species (ROS) production. Therefore, short PEG chain functionalized A3B type asymmetric Pc photosensitizers (PSs) were designed in order to decrease aggregation and increase the aqueous solubility. Here we report the synthesis, characterization, optical properties, cellular localization, and cytotoxicity of three novel Pc-based agents (LC31, MLC31, and DMLC31Pt). The stepwise functionalization of the peripheral moieties has a strong effect on the distribution coefficient (logP), cellular uptake, and localization, as well as photocytotoxicity. Additional experiments have revealed that the presence of the malonic ester moiety in the reported agent series is indispensable in order to induce photocytotoxicity. The best-performing agent, MLC31, showed mitochondrial targeting and an impressive phototoxic index (p.i.) of 748 in the cisplatin-resistant A2780/CP70 cell line, after a low-dose irradiation of 6.95 J/cm2. This is the result of a high photocytotoxicity (IC50 = 157 nM) upon irradiation with near-infrared (NIR) light, and virtually no toxicity in the dark (IC50 = 117 µM). Photocytotoxicity was subsequently determined under hypoxic conditions. Additionally, a preliminarily pathway investigation of the mitochondrial membrane potential (MMP) disruption and induction of apoptosis by MLC31 was carried out. Our results underline how agent design involving both hydrophilic and lipophilic peripheral groups may serve as an effective way to improve the PDT efficiency of highly aromatic PSs for NIR light-mediated cancer therapy.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Vitamin C is dispensable for oxygen sensing in vivo.

Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors. Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here, we report that vitamin C-deprived Gulo(-/-) knockout mice show normal HIF-dependent gene expression. The systemic response of Gulo(-/-) animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to that of animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell-culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all 3 PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity, and endogenous target gene expression in cells exposed to CoCl(2). A Cys201Ser mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface-accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.

Fate-mapping of erythropoietin-producing cells in mouse models of hypoxaemia and renal tissue remodelling reveals repeated recruitment and persistent functionality.

AIM: Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate. Therefore, we tested the hypothesis that REP cell migration, loss and/or differentiation may cause Epo inhibition. METHODS: Using a reporter mouse that allows permanent labelling of active REP cells at any given time point, we analysed the spatiotemporal fate of REP cells following their initial hypoxic recruitment in models of hypoxaemia and renal tissue remodelling. RESULTS: In long-term tracing experiments, tagged REP reporter cells neither died, proliferated, migrated nor transdifferentiated into myofibroblasts. Approximately 60% of tagged cells re-expressed Epo upon a second hypoxic stimulus. In an unilateral model of tissue remodelling, tagged cells proliferated and ceased to produce Epo before a detectable increase in myofibroblast markers. Treatment with a hypoxia-inducible factor (HIF) stabilizing agent (FG-4592/roxadustat) re-induced Epo expression in the previously active REP cells of the damaged kidney to a similar extent as in the contralateral healthy kidney. CONCLUSIONS: Rather than cell death or differentiation, these results suggest cell-intrinsic transient inhibition of Epo transcription: following long-term dormancy, REP cells can repeatedly be recruited by tissue hypoxia, and during myofibrotic tissue remodelling, dormant REP cells are efficiently rescued by a pharmaceutic HIF stabilizer, demonstrating persistent REP cell functionality even during phases of Epo suppression.

Patient-Reported Outcomes in Middle Ear and Active Transcutaneous Bone Conduction Hearing Implants.

OBJECTIVE: This study used questionnaires to examine the patient-reported satisfaction with 2 hearing implant devices to determine the level of overall satisfaction with the devices, which, if any, factors predicted good or poor perceived outcomes, or whether there were any specific aspects of the devices where dissatisfaction was apparent. METHODS: A post-treatment questionnaire survey of 39 adult patients who had received a Vibrant Soundbridge (VSB) or Bonebridge (BB) hearing implant, with at least 3 months of follow-up, was conducted using the Glasgow Benefit Inventory (GBI) and Hearing Device Satisfaction Scale (HDSS). Satisfaction scores were compared to pre- and post-operative audiologic outcomes. The correlation between GBI and HDSS scores was also examined. RESULTS: A total of 28 of the 39 patients (72%) responded: 13 with a BB and 15 with a VSB at a mean of 13 months after implantation. The overall mean total GBI score was 30, with no significant differences across the groups. The responders generally reported that they were "satisfied" across most domains of the HDSS. In the study, 25 of the 28 responders were largely satisfied with their devices but 3 respondents were not. Two were known non-users, while one used the device but did not gain the benefit expected. It is instructive to note that all of these dissatisfied recipients were close to the manufacturer recommended limits for implantation of their respective devices at the time of surgery. Certain themes were identified within the patients' responses, indicating common aspects where satisfaction was poorer. CONCLUSION: This series of 28 implant recipients demonstrates high levels of satisfaction with implantable hearing devices across 2 different validated questionnaires. Implant teams could exercise caution and manage patient expectations if the patients are close to the recommended limits of a particular device.

15-Year review of laryngeal and oral dysplasias and progression to invasive carcinoma.

The objective of this study is to identify a management or follow-up strategy for patients with laryngeal and oral dysplasia. A chart review of all patients with laryngeal and oral dysplasia over a 15-year period was performed. All patients were followed for a minimum period of 5 years from initial diagnosis of oropharyngeal or laryngeal dysplasia. If invasive carcinoma was demonstrated on subsequent biopsies, the exact time of this was recorded and Kaplan-Meier survival curves were plotted. In the laryngeal cohort, 45 patients were identified, 15 (33%) developed carcinoma, 7 of 30 patients (23%) with mild or moderate dysplasia, compared with 8 of 15 (53%) with severe dysplasia or CIS (P = 0.01). Thirteen of the carcinomas (87%) developed within 36 months of original biopsy. In the oral cohort, 32 patients were identified, 17 (53%) developed carcinoma, 1 of 9 patients (11%) with mild dysplasia, compared with 8 of 12 (67%) with severe dysplasia and 8 of 10 (80%) with CIS (P < 0.001). Fifteen of the 17 patients (88%) developed carcinoma within 36 months of original biopsy. In conclusion, although numbers are small, our results show that mild and moderate laryngeal dysplasia behaves differently to severe dysplasia and CIS. Mild oral dysplasia also behaves differently to severe dysplasia or CIS. In general, progress to malignancy happens within a 3-year period. Severe dysplasia or CIS should be managed aggressively.

Device profile of the Bonebridge bone conduction implant system in hearing loss: an overview of its safety and efficacy.

INTRODUCTION: The Bonebridge is an active transcutaneous semi-implantable bone conduction hearing device suitable for several types of hearing loss. It has unique benefits over some more established technologies. It consists of an internal active implant and an external sound processor. It was first launched in 2012, with a newer model released in late 2019. AREAS COVERED: The structure and features of the device are described. Indications, audiological criteria, and contraindications to implantation are discussed. The planning and procedure of implantation surgery are also described. Research outlining the outcomes of implant use and risk of adverse events is highlighted. EXPERT OPINION: The evidence included in this article demonstrates the successful audiological outcomes and patient satisfaction with Bonebridge implantation. The rate of adverse events following surgery is low and compares well with other devices which may be considered for Bonebridge candidates. The device should be considered as an option for suitable candidates and in many cases may be the better option available, given the low incidence of skin complications and the absence of a skin penetrating abutment. Future advances are likely to affect sound processor technology, connectivity, and possibly further reduction in implant size and gain.

Quality of Life Outcomes following Treatment of Hypopharyngeal Cancer.

Quality of life (QoL) is an important consideration in the management of individuals with head and neck cancer. The poor prognosis and significant impact of treatment modalities on function of the salivary glands, larynx and pharynx combine to make hypopharyngeal carcinoma a particularly challenging condition to treat. The impact of diagnosis and treatment on health related QoL is substantial. There is increased understanding that organ preservation does not necessarily correlate with function preservation as was previously expected. The impact on QoL, of chemoradiotherapy (CRT) or surgery, must be taken into account when managing individuals and deciding on treatment. Several QoL tools have been developed to understand the subjective consequences of functional impairment. The number and quality of studies specifically for hypopharyngeal carcinoma are low. The effects on QoL differ for surgery and CRT, as one would expect, but there are no demonstrable significant differences in most domains. Those treated with CRT show higher levels of dry mouth and sticky saliva, while those patients who have undergone surgery report greater levels of sensory disturbance. Significant differences were not noted in speech outcomes or global (general) health scores. The psychological morbidity and lack of good coping strategies are thought to play an important role in the high suicide rates of these patients (12-fold higher than the average population in the USA). Large, long-term, longitudinal studies of patients surviving treatment, answering both general and disease-specific questionnaires are required to direct clinicians towards the least morbid treatment strategies. The ability to cope and the availability of emotional support probably have a greater impact on subjective QoL than the functional outcomes of treatment.

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1.

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.

Glucose-stimulated insulin production in mice deficient for the PAS kinase PASKIN.

The Per-ARNT-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast and regulates glycogen synthase in mammals. A recent report suggested that PASKIN mRNA, protein, and kinase activity are increased in pancreatic islet beta-cells under hyperglycemic conditions and that PASKIN is necessary for insulin gene expression. We previously generated Paskin knockout mice by targeted replacement of the kinase domain with the beta-geo fusion gene encoding beta-galactosidase reporter activity. Here we show that no 5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside (X-gal) staining was observed in islet beta-cells derived from Paskin knockout mice, irrespective of the ambient glucose concentration, whereas adenoviral expression of the lacZ gene in beta-cells showed strong X-gal staining. No induction of PASKIN mRNA could be detected in insulinoma cell lines or in islet beta-cells. Increasing glucose concentrations resulted in PASKIN-independent induction of insulin mRNA levels and insulin release. PASKIN mRNA levels were high in testes but undetectable in pancreas and in islet beta-cells. Finally, blood glucose levels and glucose tolerance after intraperitoneal glucose injection were indistinguishable between Paskin wild-type and knockout mice. These results suggest that Paskin gene expression is not induced by glucose in pancreatic beta-cells and that glucose-stimulated insulin production is independent of PASKIN.

Self-Evaluated Quality of Life and Functional Outcomes After Microsurgery, Stereotactic Radiation or Observation-Only for Vestibular Schwannoma of the Adult Patient: A Systematic Review.

OBJECTIVE: To evaluate the impact of different management options on health-related quality of life (HRQoL) in vestibular schwannoma patients. DATA SOURCES: A systematic search of the Cochrane Database, Database of Abstracts of Reviews of Effectiveness, and the Ovid Medline & EMBASE was performed. English and German language studies published between 1980 and 2015 were considered. STUDY SELECTION: This is a systematic review of HRQoL of patients managed for vestibular schwannoma. Studies in which HRQoL after one management option were evaluated or compared with other managements or with control populations using validated or reliable questionnaires, were included. DATA EXTRACTION: The included studies were independently evaluated by two reviewers. The quality of studies was assessed and graded as per Oxford Centre of Evidence Based Medicine System. RESULTS: Ten prospective and 29 retrospective studies were identified: microsurgery initially exerted a negative effect on HRQoL but this tended to improve with follow up. Radiotherapy had a less negative effect but with minimal change over follow up. A significant limitation was that studies did not present results stratified by tumor size. Many patients will need active treatment despite the potential for negative effects on their QoL. The concept of a minimal clinically important difference has been introduced into this field and was compared with five studies. CONCLUSION: A number of prospective studies are available but none yet with a disease-specific questionnaire. Heterogeneity and the methodological weaknesses of the included studies constitute the principle limitation of this review. The introduction of the minimal clinically important difference should improve the relevance of studies and allow a sensitive comparison of treatments.

Regulated function of the prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins.

Cellular oxygen is sensed by prolyl-4-hydroxylase domain (PHD) proteins that hydroxylate hypoxia-inducible factor (HIF) alpha subunits. Under normoxic conditions, hydroxylated HIFalpha is bound by the von Hippel-Lindau (pVHL) tumor suppressor, leading to ubiquitinylation and proteasomal degradation. Under hypoxic conditions, hydroxylation becomes reduced, leading to HIFalpha stabilization. The authors recently showed that changes in PHD abundance and activity can regulate HIFalpha stability under normoxic as well as under hypoxic conditions. Thus, the PHD oxygen sensors themselves represent effectors of cellular signalling pathways as well as potential drug targets. Here, a cell-free in vitro microtiter plate-based peptide hydroxylation assay was used to investigate the influence of ferrous iron, Krebs cycle intermediates, transition metals, and vitamin C and other antioxidants on the activity of purified PHD1 to 3. PHD activity depends not only on oxygen availability but is also regulated by iron, vitamin C, and Krebs cycle intermediates, suggesting a physiological relevance of their cellular concentrations. Copper but not iron, cobalt, or nickel salts catalyzed vitamin C oxidation. While vitamin C is essential for PHD activity in vitro, N-acetyl-L-cysteine had no effect, and gallic acid or n-propyl gallate efficiently inhibited the activity of all three PHDs, demonstrating different functions of these antioxidants.

Determination and modulation of prolyl-4-hydroxylase domain oxygen sensor activity.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation.

Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 -/- mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.

Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKß phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.