Micro-RNA (miRNA)-based regulation of hypoxia, angiogenesis and tumour growth provides promising targets for effective therapy in malignant glioblastoma multiforme (GBM). Accumulating evidence suggests a potential role of melatonin in miRNA expression in cancer cells. Despite these findings, the melatonin-miRNA interaction in GBM and the effect of this interaction on GBM tumour development and invasion are not clearly understood. The aim of the present study was to evaluate the effects of melatonin on human GBM tumour spheroid tumorigenesis and invasion in vitro, and to analyse the interaction between 36 angio-miRNAs and the HIF1/VEGF/MMP9 axis, which is known to be associated with the antitumour effect of melatonin. We found that melatonin is able to selectively induce cell death in single-layer U87-MG cells (a GBM cell line) in a dose- and time-dependent manner, as characterized by MTT assay. The use of tumour spheroids and a Matrigel invasion assay revealed that melatonin impairs tumorigenesis, and it significantly reduced both the tumour spheroid area and invasion rate, especially at the 0.5 mM and 1 mM concentrations. This inhibition was accompanied by strong reductions in hypoxia-inducible factor 1-α (HIF1-α) and vascular endothelial growth factor (VEGF) gene expression and protein levels in GBM tumour spheroids. In addition, melatonin significantly reduced the relative gene expression and protein levels of matrix metalloproteinase-9 (MMP-9). This study revealed that six differentially expressed angio-miRs (miR-15b, miR-18a-5p, miR-23a-3p, miR-92a-3p, miR-130a-5p, miR-200b-3p) may play important roles in GBM tumorigenesis and invasion, and all respond to melatonin therapy. Our results suggest that melatonin inhibits tumorigenesis and invasion of human GBM tumour spheroids, possibly by suppressing HIF1-α/VEGF/MMP9 signalling via regulation of angio-miRNAs.

Keywords: Angiogenesis; Glioblastoma multiforme; Hypoxia; Melatonin; microRNA.

There has been increasing interest in the role of hypoxia in the microenvironment of organs, because of the discovery of hypoxia-inducible factor-1 (HIF1), which acts as a transcription factor for many genes activated specifically under hypoxic conditions. The ovary changes day by day during the estrous cycle as it goes through phases of follicular growth, ovulation, and formation and regression of the corpus luteum (CL). These phenomena are regulated by hypothalamic and pituitary hormones, sex steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis via transcription of a potent angiogenic factor, vascular endothelial growth factor (VEGF), that is regulated by HIF1. A CL forms with a rapid increase of angiogenesis that is mainly induced by HIF1-VEGF signaling. Hypoxia also contributes to luteolysis by down-regulating progesterone synthesis and by up-regulating apoptosis of luteal cells. This review focuses on recent studies on the roles of hypoxia- and HIF1-regulated genes in the regulation of bovine CL function.

In the field of bone defect repair, 3D printed scaffolds have the characteristics of personalized customization and accurate internal structure. However, how to construct a well-structured vascular network quickly and effectively inside the scaffold is essential for bone repair after transplantation. Herein, inspired by the unique biological structure of "lotus seedpod", hydrogel microspheres encapsulating deferoxamine (DFO) liposomes were prepared through microfluidic technology as "lotus seeds", and skillfully combined with a three-dimensional (3D) printed bioceramic scaffold with biomimetic "lotus" biological structure which can internally grow blood vessels. In this composite scaffold system, DFO was effectively released by 36% in the first 6 h, which was conducive to promote the growth of blood vessels inside the scaffold quickly. In the following 7 days, the release rate of DFO reached 69%, which was fundamental in the formation of blood vessels inside the scaffold as well as osteogenic differentiation of bone mesenchymal stem cells (BMSCs). It was confirmed that the composite scaffold could significantly promote the human umbilical vein endothelial cells (HUVECs) to form the vascular morphology within 6 h in vitro. In vivo, the composite scaffold increased the expression of vascularization and osteogenic related proteins Hif1-α, CD31, OPN, and OCN in the rat femoral defect model, significantly cutting down the time of bone repair. To sum up, this "lotus seedpod" inspired porous bioceramic 3D printed scaffold with internal vascularization functionality has broad application prospects in the future.

Keywords: 3D scaffolds; Drug release; Neovascularization; Osteogenesis; Regeneration.

Intronic microRNAs (miRNAs) are considered to be transcribed using their host gene promoter. However, about one third of intronic miRNAs are predicted to have independent promoter elements.

Human breast cancer cells were cultured under normoxia or hypoxia, and expression levels of intronic miR-106b-25 cluster miRNAs and their host gene minichromosome maintenance complex component 7 (MCM7) transcripts were analyzed by semi-quantitative polymerase chain reaction. The putative promoter element of miR-106b-25 cluster was analyzed by chromatin immunoprecipitation and luciferase assays.

Exposure to hypoxia reduced the expression of MCM7 mRNA and a primary transcript of miR-106b-25 cluster, but did not affect that of mature miRNAs. The putative promoter element of miR-106b-25 cluster was not bound by hypoxia-inducible factor 1-alpha (HIF1-α), and was not activated under hypoxia.

Maintenance of miR-106b-25 cluster miRNA levels under hypoxia was not caused by the activation of an independent promoter element.

Hepatocellular carcinoma (HCC) is the most common visceral neoplasms with its heterogeneity and high rate of recurrence. HCC is characterized to be delayed diagnosis and the development of resistant disease. However, the molecular mechanism for HCC pathogenesis and progression remains largely unknown. Here, we demonstrated that ubiquitin-specific protease14 (USP14) is highly expressed in HCC samples, and the higher expression of USP14 is positively correlated with poor prognosis. Interestingly, USP14 is involved in the maintenance of HIF1-α stability to activate HIF1-α-induced transactivation via its deubiquitinase activity. USP14 depletion or its specific inhibitor IU1 treatment decreased cell proliferation, invasion, migration, and Vascular Mimicry (VM) formation even under hypoxia conditions in HCC cell lines. Moreover, we provided the evidence to show that knockdown of USP14 or USP14 inhibitor (IU1) treatment inhibited tumor growth in tumor-bearing nude mice. Our findings suggest that USP14 maintains HIF1-α stability through its deubiquitination activity, providing a potential biomarker for the early diagnosis and therapy of HCC.

<strong cl<em>a</em>ss="sub-title"> B<em>a</em>ckground: </strong> We previously showed th<em>a</em>t selected single-nucleotide-polymorphisms (SNPs) of genes involved in <em>a</em>ngiogenesis influence the <em>a</em>ggressiveness of thymic epitheli<em>a</em>l tumors (TETs). This study <em>a</em>n<em>a</em>lyzes their role in TETs <em>a</em>nd in thymic benign lesions, in order to investig<em>a</em>te potenti<em>a</em>l correl<em>a</em>tion with risk <em>a</em>nd outcome.

<strong cl<em>a</em>ss="sub-title"> Methods: </strong> Genomic DNA w<em>a</em>s extr<em>a</em>cted from p<em>a</em>r<em>a</em>ffin-embedded tissue of 92 p<em>a</em>tients, undergoing surgery <em>a</em>t our Institution. We investig<em>a</em>ted by Re<em>a</em>l-Time PCR the SNPs of the following genes: pl<em>a</em>telet-derived growth f<em>a</em>ctor receptor<i>-</i>α (<i>PDGFR</i>α), hypoxi<em>a</em>-inducible f<em>a</em>ctor-1α (<i>HIF-1</i>α), v<em>a</em>scul<em>a</em>r endotheli<em>a</em>l growth f<em>a</em>ctor <i>A</i> (<i>VEGF-A</i>), v<em>a</em>scul<em>a</em>r endotheli<em>a</em>l growth f<em>a</em>ctor receptor-2 <em>a</em>nd 3 (<i>VEGF-2, VEGFR-3</i>), excision rep<em>a</em>ir cross-complement<em>a</em>tion group-1 (<i>ERCC1</i>).

<strong cl<em>a</em>ss="sub-title"> Results: </strong> Fifty-seven TETs <em>a</em>nd 35 thymic benign lesions were included into the study. Frequency of SNPs w<em>a</em>s <em>a</em>s follows: rs2057482 C, rs11158358 C <em>a</em>nd rs11549465 C polymorphisms of <i><em>HIF1</em>-<em>a</em></i>: thymom<em>a</em>s < gener<em>a</em>l popul<em>a</em>tion (P=0.008, P=0.007, <em>a</em>nd P=0.044 respectively). <i><em>HIF1</em>-<em>a</em></i> <em>a</em>lleles: gener<em>a</em>l popul<em>a</em>tion > study groups, rs1951795C SNP (P=0.026 for benign lesions <em>a</em>nd P=0.0007 for thymom<em>a</em>s), rs10873142T SNP (P=0.008 <em>a</em>nd P=0.001 respectively), rs12434438 A SNP (P=0.034 <em>a</em>nd P=0.0007) <em>a</em>nd rs2301113A SNP (P=0.027 <em>a</em>nd P=0.010). rs699947C polymorphism of VEGF-A: benign lesions > gener<em>a</em>l popul<em>a</em>tion (P=0.012).

<strong cl<em>a</em>ss="sub-title"> Conclusions: </strong> This is the first study investig<em>a</em>ting the <em>a</em>ngiogenetic polymorphisms in thymic benign lesions <em>a</em>nd TETs. SNPs <em>a</em>n<em>a</em>lysis m<em>a</em>y represent <em>a</em> further <em>a</em>sset in identific<em>a</em>tion of p<em>a</em>tients who could benefit from <em>a</em>nti-<em>a</em>ngiogenetic ther<em>a</em>py.

<strong cl<em>a</em>ss="sub-title"> Keywords: </strong> Thymic epitheli<em>a</em>l tumor (TETs); outcome; thymic cyst; thymic hyperpl<em>a</em>si<em>a</em>; thymic prognostic f<em>a</em>ctor; thymolipom<em>a</em>.

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Müller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-α (HIF1-α), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation.

Keywords: epiretinal membrane; human retina; internal limiting membrane; macular hole; proliferative diabetic retinopathy; vitreoretinal surgery.

OBJECTIVE

To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia.

METHODS

Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei.

CONCLUSIONS

HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.

To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1-α (HIF1-α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1-α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl₂ ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl₂ for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl₂ -treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl₂ treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.

Keywords: cellular reprogramming; hypoxia inducible factor; metabolism; porcine; somatic cell nuclear transfer.

Background: Distraction osteogenesis has been broadly used to treat various structural bone deformities and defects. However, prolonged healing time remains a major problem. Various approaches including the use of low-intensity pulsed ultrasound, parathyroid hormone, and bone morphogenetic proteins (BMPs) have been studied to shorten the treatment period with limited success. Our previous studies of rats have reported that the transcutaneous application of CO2 accelerates fracture repair and bone-defect healing in rats by promoting angiogenesis, blood flow, and endochondral ossification. This therapy may also accelerate bone generation during distraction osteogenesis, but, to our knowledge, no study investigating CO2 therapy on distraction osteogenesis has been reported.

Questions/purposes: We aimed to investigate the effect of transcutaneous CO2 during distraction osteogenesis in rabbits, which are the most suitable animal as a distraction osteogenesis model for a lengthener in terms of limb size. We asked: Does transcutaneous CO2 during distraction osteogenesis alter (1) radiographic bone density in the distraction gap during healing; (2) callus parameters, including callus bone mineral content, volumetric bone mineral density, and bone volume fraction; (3) the newly formed bone area, cartilage area, and angiogenesis, as well as the expression of interleukin-6 (IL-6), BMP-2, BMP-7, hypoxia-inducible factor (HIF) -1α, and vascular endothelial growth factor (VEGF); and (4) three-point bend biomechanical strength, stiffness, and energy?

Methods: Forty 24-week-old female New Zealand white rabbits were used according to a research protocol approved by our institutional ethical committee. A distraction osteogenesis rabbit tibia model was created as previously described. Briefly, an external lengthener was applied to the right tibia, and a transverse osteotomy was performed at the mid-shaft. The osteotomy stumps were connected by adjusting the fixator to make no gap. After a 7-day latency phase, distraction was continued at 1 mm per day for 10 days. Beginning the day after the osteotomy, a 20-minute transcutaneous application of CO2 on the operated leg using a CO2 absorption-enhancing hydrogel was performed five times per week in the CO2 group (n = 20). Sham treatment with air was administered in the control group (n = 20). Animals were euthanized immediately after the distraction period (n = 10), 2 weeks (n = 10), and 4 weeks (n = 20) after completion of distraction. We performed bone density quantification on the plain radiographs to evaluate consolidation in the distraction gap with image analyzing software. Callus parameters were measured with micro-CT to assess callus microstructure. The newly formed bone area and cartilage area were measured histologically with safranin O/fast green staining to assess the progress of ossification. We also performed immunohistochemical staining of endothelial cells with fluorescein-labeled isolectin B4 and examined capillary density to evaluate angiogenesis. Gene expressions in newly generated callus were analyzed by real-time polymerase chain reaction. Biomechanical strength, stiffness, and energy were determined from a three-point bend test to assess the mechanical strength of the callus.

Results: Radiographs showed higher pixel values in the distracted area in the CO2 group than the control group at Week 4 of the consolidation phase (0.98 ± 0.11 [95% confidence interval 0.89 to 1.06] versus 1.19 ± 0.23 [95% CI 1.05 to 1.34]; p = 0.013). Micro-CT demonstrated that bone volume fraction in the CO2 group was higher than that in the control group at Week 4 (5.56 ± 3.21 % [95% CI 4.32 to 6.12 %] versus 11.90 ± 3.33 % [95% CI 9.63 to 14.25 %]; p = 0.035). There were no differences in any other parameters (that is, callus bone mineral content at Weeks 2 and 4; volumetric bone mineral density at Weeks 2 and 4; bone volume fraction at Week 2). At Week 2, rabbits in the CO2 group had a larger cartilage area compared with those in the control group (2.09 ± 1.34 mm [95% CI 1.26 to 2.92 mm] versus 5.10 ± 3.91 mm [95% CI 2.68 to 7.52 mm]; p = 0.011). More newly formed bone was observed in the CO2 group than the control group at Week 4 (68.31 ± 16.32 mm [95% CI 58.19 to 78.44 mm] versus 96.26 ± 19.37 mm [95% CI 84.25 to 108.26 mm]; p < 0.001). There were no differences in any other parameters (cartilage area at Weeks 0 and 4; newly formed bone area at Weeks 0 and 2). Immunohistochemical isolectin B4 staining showed greater capillary densities in rabbits in the CO2 group than the control group in the distraction area at Week 0 and surrounding tissue at Weeks 0 and 2 (distraction area at Week 0, 286.54 ± 61.55 /mm [95% CI 232.58 to 340.49] versus 410.24 ± 55.29 /mm [95% CI 361.78 to 458.71]; p < 0.001; surrounding tissue at Week 0 395.09 ± 68.16/mm [95% CI 335.34 to 454.83] versus 589.75 ± 174.42/mm [95% CI 436.86 to 742.64]; p = 0.003; at Week 2 271.22 ± 169.42 /mm [95% CI 122.71 to 419.73] versus 508.46 ± 49.06/mm [95% CI 465.45 to 551.47]; p < 0.001 respectively). There was no difference in the distraction area at Week 2. The expressions of BMP -2 at Week 2, HIF1-α at Week 2 and VEGF at Week 0 and 2 were greater in the CO2 group than in the control group (BMP -2 at Week 2 3.84 ± 0.83 fold [95% CI 3.11 to 4.58] versus 7.32 ± 1.63 fold [95% CI 5.88 to 8.75]; p < 0.001; HIF1-α at Week 2, 10.49 ± 2.93 fold [95% CI 7.91 to 13.06] versus 20.74 ± 11.01 fold [95% CI 11.09 to 30.40]; p < 0.001; VEGF at Week 0 4.80 ± 1.56 fold [95% CI 3.43 to 6.18] versus 11.36 ± 4.82 fold [95% CI 7.13 to 15.59]; p < 0.001; at Week 2 31.52 ± 8.26 fold [95% CI 24.27 to 38.76] versus 51.05 ± 15.52 fold [95% CI 37.44 to 64.66]; p = 0.034, respectively). There were no differences in any other parameters (BMP-2 at Week 0 and 4; BMP -7 at Weeks 0, 2 and 4; HIF-1α at Weeks 0 and 4; IL-6 at Weeks 0, 2 and 4; VEGF at Week 4). In the biomechanical assessment, ultimate stress and failure energy were greater in the CO2 group than in the control group at Week 4 (ultimate stress 259.96 ± 74.33 N [95% CI 167.66 to 352.25] versus 422.45 ± 99.32 N [95% CI 299.13 to 545.77]; p < 0.001, failure energy 311.32 ± 99.01 Nmm [95% CI 188.37 to 434.25] versus 954.97 ± 484.39 Nmm [95% CI 353.51 to 1556.42]; p = 0.003, respectively). There was no difference in stiffness (216.77 ± 143.39 N/mm [95% CI 38.73 to 394.81] versus 223.68 ± 122.17 N/mm [95% CI 71.99 to 375.37]; p = 0.92).

Conclusion: Transcutaneous application of CO2 accelerated bone generation in a distraction osteogenesis model of rabbit tibias. As demonstrated in previous studies, CO2 treatment might affect bone regeneration in distraction osteogenesis by promoting angiogenesis, blood flow, and endochondral ossification.

Clinical relevance: The use of the transcutaneous application of CO2 may open new possibilities for shortening healing time in patients with distraction osteogenesis. However, a deeper insight into the mechanism of CO2 in the local tissue is required before it can be used in future clinical practice.

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tβ-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tβ-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tβ-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tβ-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens.

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.

Long noncoding RNAs (lncRNAs) have been recently recognized as key players of gene expression in cerebral pathogenesis. Thus, their potential use in stroke diagnosis, prognosis, and therapy is actively pursued. Due to the complexity of the disease, identifying stroke-specific lncRNAs remains a challenge. This study investigated the expression of lncRNAs HIF1A-AS2 and LINK-A, and their target gene hypoxia-inducible factor-1 (HIF-1) in Egyptian stroke patients. It also aimed to determine the molecular mechanism implicated in the disease. A total of 75 stroke patients were divided into three clinical subgroups, besides 25 healthy controls of age-matched and sex-matched. Remarkable upregulation of lncRNA HIF1A-AS2 and HIF1-α along with a downregulation of lncRNA LINK-A was noticed in all stroke groups relative to controls. Serum levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated-Akt (p-Akt), vascular endothelial growth factor (VEGF), and angiopoietin-1 (ANG1) as well as their receptors, malondialdehyde (MDA), and total antioxidant capacity (TAC) were significantly increased, whereas brain-derived neurotrophic factor (BDNF) levels were significantly decreased particularly in hemorrhagic stroke versus ischemic groups. Eventually, these findings support the role of lncRNAs HIF1A-AS2 and LINK-A as well as HIF1-α in activation of angiogenesis, neovascularization, and better prognosis of stroke, especially the hemorrhagic type.

Keywords: Cerebral stroke; HIF1-α; Non-invasive stroke biomarkers; lncRNA HIF1A-AS2; lncRNA LINK-A.

Promoting angiogenesis is a key strategy for stimulating the repair of damaged tissues, including bone. Among other proangiogenic factors, ions have recently been considered a potent element that can be incorporated into biomaterials and then released at therapeutic doses. Silicate-based biomaterials have been reported to induce neovascularization through vascular endothelial growth factor signaling pathway, potentiating acceleration of bone regeneration. Here, we designed a silicate-shelled hydrogel fiber scaffold with a hard/soft layered structure to investigate the possibility of silicate coating on biopolymer for enhancing biological properties. An alginate hydrogel was injected to form a fiber scaffold with shape-tunability that was then coated with a thin silicate layer with various sol-gel compositions. The silicate/alginate scaffold could release calcium and silicate ions, and in particular, silicate ion release was highly sustainable for over one week at therapeutically relevant levels. The ionic release was highly effective in stimulating the mRNA expression of angiogenic markers (VEGF, KDR, eNOS, bFGF, and HIF1-α) in endothelial cells (HUVECs). Moreover, the in vitro tubular networking of cells was significantly enhanced (1.5 times). In vivo implantation in subcutaneous tissue revealed more pronounced blood vessel formation around the silicate-shelled scaffolds than around silicate-free scaffolds. The presence of a silicate shell was also shown to accelerate acellular mineral (hydroxyapatite) formation. The cellular osteogenesis potential of the silicate/alginate scaffold was further proven by the enhanced expression of osteogenic genes (Col1a1, ALP and OCN). When implanted in a rat calvarium defect, the silicate-shelled scaffold demonstrated significantly improved bone formation (2-3 times higher in bone volume and density) with a concurrent sign of proangiogenesis. This work highlights that the surface-layering of silicate composition is an effective approach for improving the bone regeneration capacity of polymeric hydrogel scaffolds by stimulating ion-induced angiogenesis and providing bone bioactivity to the surface.

A stem cell niche is a microenvironment where stem cells reside in a quiescent state, until activated. In a previous rat model, we combined 5-bromo-2-deoxy-uridine labeling with activation of endogenous stem cells by physical exercise and revealed a distinct region, in the atrioventricular junction (AVj), with features of a stem cell niche. In this study, we aim to investigate whether a similar niche exists in the human heart. Paired biopsies from AVj and left ventricle (LV) were collected both from explanted hearts of organ donors, not used for transplantation (N = 7) and from severely failing hearts from patients undergoing heart transplantation (N = 7). Using antibodies, we investigated the expression of stem cell, hypoxia, proliferation and migration biomarkers. In the collagen-dense region of the AVj in donor hearts, progenitor markers, MDR1, SSEA4, ISL1, WT1, and hypoxia marker, HIF1-α, were clearly detected. The expression gradually decreased with distance from the valve. At the myocardium border in the AVj costaining of the proliferation marker Ki67 with cardiomyocyte nuclei marker PCM1 and cardiac Troponin-T (cTnT) indicated proliferation of small cardiomyocytes. In the same site we also detected ISL1⁺/WT1⁺/cTnT cells. In addition, heterogeneity in cardiomyocyte sizes was noted. Altogether, these findings indicate different developmental stages of cardiomyocytes below the region dense in stem cell marker expression. In patients suffering from heart failure the AVj region showed signs of impairment generally displaying much weaker or no expression of progenitor markers. We describe an anatomic structure in the human hearts, with features of a progenitor niche that coincided with the same region previously identified in rats with densely packed cells expressing progenitor and hypoxia markers. The data provided in this study indicate that the adult heart contains progenitor cells and that AVj might be a specific niche region from which the progenitors migrate at the time of regeneration.

İntroduction: Familial Mediterranean Fever (FMF) is an autoinflammatory disease. Prolidase is a specific imidodipeptidase that plays a role in collagen degradation. Prolidase plays an important role in inflammation and wound healing. Hypoxia-inducible factor-1α (HIF-1) is an important protein in the regulation of immunological response, hemostasis, vascularization. The aim of the study was to compare serum prolidase and HIF-1α levels in patients with FMF in attack-free period and healthy control group. Methods: Between August 2017 and December 2017, sixty patients who diagnosed FMF according to the criteria of the Tel-hashomer who admitting to Sivas Cumhuriyet University Medical Faculty Internal Medicine Rheumatology Department and sixty healthy volunteers were enrolled in the study. Results: Median serum prolidase level were 72.1 (25.1-114.9) ng/ml in FMF group and 30.7 (21.3-86.2) ng/mL in healthy control (HC) group (p=0.018). ROC analysis showed that the sensitivity was 65% and the specificity was 68.3% at serum prolidase levels 54.03 ng/mL (p<0.05). The median serum levels of HIF-1α in the FMF group was 482.0(292.0-3967.0) pg/mL and 632.0(362.0-927.0) pg/mL in the HC group (p>0.05). There was no significant correlation between laboratory findings, sex, age, and prolidase (p>0.05). \Conclusion: Serum prolidase enzyme levels in FMF patients with attack-free period were significantly higher than in the HC group. However, the role of prolidase and HIF1-α in the FMF disease needs to be clarified with more extensive and comprehensive studies.

OBJECTIVE

The purpose of this study was to investigate the pathology and mechanism of immobilization-induced skin fibrosis in a rat joint contracture model.

METHODS

Rats were randomly divided into control and immobilization groups. In the immobilization groups, knee joints of the rats were immobilized for 1, 2, and 4 weeks. After each immobilization, skin was dissected. To assess fibrosis in the skin, the thickness and area of adipocytes and connective tissue fibers were measured. Myofibroblasts were analyzed by immunohistochemistry by using anti-α-SMA as a marker. Gene expression levels of type I and III collagen, TGF-ß1, and HIF-1α were measured by using RT-PCR.

RESULTS

One week after immobilization, there was a marked increase in the area of connective tissue fibers in the immobilization group. Type I and type III collagen were significantly increased with prolonged immobilization. Higher numbers of α-SMA-positive cells were noted in the immobilized group at 2 and 4 weeks after immobilization. The expression level of TGF-β1 mRNA in the immobilization group increased after one week of immobilization. In contrast, the expression level of HIF1-α mRNA increased after 2 weeks of immobilization, and a greater increase was seen at 4 weeks after immobilization.

CONCLUSIONS

These results suggest that immobilization induces skin fibrosis with accumulation of types I and III collagen. These fibrotic changes may be evoked by upregulation of TGF-β1 after one week of immobilization. Additionally, upregulation of HIF-1α may relate to skin fibrosis by accelerating the differentiation of fibroblasts to myofibroblasts starting at 2 weeks after immobilization.

Individuals carrying a pathogenic germline variant in the breast cancer predisposition gene BRCA1 (gBRCA1+) are prone to developing breast cancer. Apart from its well-known role in DNA repair, BRCA1 has been shown to powerfully impact cellular metabolism. While, in general, metabolic reprogramming was named a hallmark of cancer, disrupted metabolism has also been suggested to drive cancer cell evolution and malignant transformation by critically altering microenvironmental tissue integrity. Systemic metabolic effects induced by germline variants in cancer predisposition genes have been demonstrated before. Whether or not systemic metabolic alterations exist in gBRCA1+ individuals independent of cancer incidence has not been investigated yet. We therefore profiled the plasma metabolome of 72 gBRCA1+ women and 72 age-matched female controls, none of whom (carriers and non-carriers) had a prior cancer diagnosis and all of whom were cancer-free during the follow-up period. We detected one single metabolite, pyruvate, and two metabolite ratios involving pyruvate, lactate, and a metabolite of yet unknown structure, significantly altered between the two cohorts. A machine learning signature of metabolite ratios was able to correctly distinguish between gBRCA1+ and controls in ~82%. The results of this study point to innate systemic metabolic differences in gBRCA1+ women independent of cancer incidence and raise the question as to whether or not constitutional alterations in energy metabolism may be involved in the etiology of BRCA1-associated breast cancer.

Keywords: BRCA1 germline mutation; HIF1 alpha; NAD+ balance; aerobic glycolysis; breast cancer; energy metabolism; lactate; plasma metabolome.

Phosphoinositide metabolism is crucial intracellular signaling system that regulates a plethora of biological functions including mitogenesis, cell proliferation and division. Phospholipase C gamma 1 (PLCγ1) which belongs to phosphoinositide-specific phospholipase C (PLC) family, is activated by many extracellular stimuli including hormones, neurotransmitters, growth factors and modulates several cellular and physiological functions necessary for tumorigenesis such as cell survival, migration, invasion and angiogenesis by generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) via hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2). Cancer remains as a leading cause of global mortality and aberrant expression and regulation of PLCγ1 is linked to a plethora of deadly human cancers including carcinomas of the breast, lung, pancreas, stomach, prostate and ovary. Although PLCγ1 cross-talks with many onco-drivers and signaling circuits including PI3K, AKT, HIF1-α and RAF/MEK/ERK cascade, its precise role in carcinogenesis is not completely understood. This review comprehensively discussed the status quo of this ubiquitously expressed phospholipase as a tumor driver and highlighted its significance as a novel therapeutic target in cancer. Furthermore, we have highlighted the significance of somatic driver mutations in PLCG1 gene and molecular roles of PLCγ1 in several major human cancers, a knowledgebase that can be utilized to develop novel, isoform-specific small molecule inhibitors of PLCγ1.

Keywords: Cancer therapeutics; Growth factor receptor; Molecular oncology; Phosphoinositide signaling pathways; Phospholipase Cγ1; Receptor tyrosine kinases.

Hypoxia is a nonphysiological oxygen tension which is common in most malignant tumors. Hypoxia stimulates complicated cell signaling networks in cancer cells, e.g., the HIF, PI3K, MAPK, and NFκB pathways. Then, cells release a number of cytokines such as VEGFA to promote the growth of peripheral blood vessels and lead to metastasis. In the current work, understanding of the internal hypoxic environment in solid tumor tissue was attempted by developing a folding paper system. A paper-based solid tumor was constructed by folding a filter paper cultured with cancer cells. The cellular response in each layer could be analyzed by disassembling the folded paper after the culture course. The result showed that an internal hypoxic environment was successfully reproduced in the paper-based solid tumor. The cells in the inner layer expressed high levels of HIF1-α and VEGFA. Hence, proliferation and migration of endothelial cells were shown to be induced by the cells located in the internal hypoxic environment. Moreover, the paper-based solid tumor was transplanted into nude mice for the study of hypoxic response and angiogenesis. The crosstalk between internal and external parts of solid tumor tissue could be analyzed by sectioning each layer of the paper-based solid tumor. This approach provides a favorable analytical method for the discovery of the interaction between cancer cells, hypoxia, and peripheral angiogenesis.

Keywords: angiogenesis; cancer cells; cancer metastasis; hypoxia; paper-based microfluidics; solid tumor.

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.

Keywords: Acute kidney injury; Apoptosis; Autophagy; Hypoxia/reoxygenation; PTEN.

Hypoxic areas are typically resistant to treatment. However, the fluorine-18-fluoroazomycin-arabinoside (FAZA) and fluorine 18 misonidazole (FMISO) tracers have never been compared in non small cell lung cancer (NSCLC). This study compares the capability of 18F-FAZA PET/CT with that of 18F-FMISO PET/CT for detecting hypoxic tumour regions in early and locally advanced NSCLC patients. We prospectively evaluated patients who underwent preoperative PET scans before surgery for localised NSCLC (i.e., fluorodeoxyglucose (FDG)-PET, FMISO-PET, and FAZA-PET). The PET data of the three tracers were compared with each other and then compared to immunohistochemical analysis (GLUT-1, CAIX, LDH-5, and HIF1-Alpha) after tumour resection. Overall, 19 patients with a mean age of 68.2 ± 8 years were included. There were 18 lesions with significant uptake (i.e., SUVmax >1.4) for the F-MISO and 17 for FAZA. The mean SUVmax was 3 (±1.4) with a mean volume of 25.8 cc (±25.8) for FMISO and 2.2 (±0.7) with a mean volume of 13.06 cc (±13.76) for FAZA. The SUVmax of F-MISO was greater than that of FAZA (p = 0.0003). The SUVmax of F-MISO shows a good correlation with that of FAZA at 0.86 (0.66-0.94). Immunohistochemical results are not correlated to hypoxia PET regardless of the staining. The two tracers show a good correlation with hypoxia, with FMISO being superior to FAZA. FMISO, therefore, remains the reference tracer for defining hypoxic volumes.

Keywords: FAZA; FMISO; PET; hypoxia; lung cancer.