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Bioactive glasses (BGs) have shown great potential for tissue regeneration and their composition flexibility allows the incorporation of different ions with physiological activities and therapeutic properties in the glass network. Among the many ions that could be incorporated, cobalt (Co) is a significant one, as it mimics hypoxia, triggering the formation of new blood vessels by the vascular endothelial growth factor A (VEGFA), due to the stabilizing effect on the hypoxia inducible factor 1 subunit alpha (HIF1A), an activator of angiogenesis-related genes, and is therefore of great interest for tissue engineering applications. However, despite its promising properties, the effects of glasses incorporated with Co on angiogenesis, through human umbilical cord vein endothelial cells (HUVECs) studies, need to be further investigated. Therefore, this work aimed to evaluate the biocompatibility and angiogenic potential of a new sol-gel BG, derived from the SiO 2 -CaO-P 2 O 5 -CoO system. The structural evaluation showed the predominance of an amorphous glass structure, and the homogeneous presence of cobalt in the samples was confirmed. in vitro experiments showed that Co-containing glasses did not affect the viability of HUVECs, stimulated the formation of tubes and the gene expression of HIF1A and VEGFA. in vivo experiments showed that Co-containing glasses stimulated VEGFA and HIF1A expression in blood vessels and cell nuclei, respectively, in the deep dermis layer of the dorsal region of rats, featuring considerable local stimulation of the angiogenesis process due to Co-release. Co-containing glasses showed therapeutic effect, and Co incorporation is a promising strategy for obtaining materials with superior angiogenesis properties for tissue engineering applications., (© 2020 Wiley Periodicals LLC.)

Background: The present study was designed to investigate the effects of Berberis vulgaris (BV) juice consumption on plasma levels of insulin-like growth factor (IGF-1), IGF-binding proteins (IGFBPs), and the expression of PPAR-γ, VEGF and HIF in women with benign breast disease., Methods: This parallel design randomized, double-blind controlled clinical trial was conducted on 85 eligible patients diagnosed with benign breast disease. They were assigned randomly into either BV juice group (n = 44, BV juice: 480 ml/day) or placebo group (n = 41, BV placebo juice: 480 ml/day) for 8 weeks intervention. Participants, caregivers and those who assessed laboratory analyses were blinded to the assignments. Plasma levels of biomarkers were measured at baseline and after 8 weeks by ELISA. Quantitative real-time PCR was used to measure the fold change in the expression of each interested gene., Results: The compliance of participants was 95.2% and 40 available subjects analyzed in each group at last. Relative treatment (RT) effects for BV juice caused 16% fall in IGF-1 concentration and 37% reduction in the ratio of IGF-1/1GFBP1. Absolute treatment effect expressed 111 ng/ml increased mean differences of IGFBP-3 between BV group and placebo. Plasma level of PPAR-γ increased in both groups but it was not significant. Fold changes in the expressions of PPAR-γ, VEGF and HIF showed down-regulation in the intervention group compared to placebos (P < 0.05)., Conclusions: The BV juice intervention over 8 weeks was accompanied by acceptable efficacy and decreased plasma IGF-1, and IGF-1/IGFBP-1 ratio partly could be assigned to enhanced IGFBP-1 level in women with BBD. The intervention caused reductions in the expression levels of PPAR, VEGF, and HIF which are remarkable genomic changes to potentially prevent breast tumorigenesis., Trial Registration: IRCT2012110511335N2. Registered 10 July 2013 (retrospectively registered).

Objectives: This study aimed to investigate the utility of fluorodeoxyglucose (FDG) positron emission tomography for solid pseudopapillary neoplasm (SPN) diagnosis., Methods: The subjects included 53 cases of SPN. We compared the maximal standardized uptake volume (SUVmax) with those of 25 cases of pancreatic duct cancer and 18 cases of pancreatic neuroendocrine neoplasm. In addition, immunopathological testing for SPN with regard to FDG uptake was undertaken., Results: An increase in SUVmax was observed in all tumors with increased tumor diameter. Among tumors of 20 mm or smaller, the SUVmax of SPN was significantly higher than those of pancreatic duct cancer and pancreatic neuroendocrine neoplasm. The results of a pathological study of FDG uptake in SPN revealed increased glucose transporter protein type 1 expression with tumor enlargement. Furthermore, increased hypoxia-inducible factor-1 and vascular endothelial growth factor expression under hypoxic conditions were observed in the areas of necrosis., Conclusions: In cases in which high FDG uptake is observed in small pancreatic tumors, FDG positron emission tomography is potentially useful for SPN differentiation. The factors involved in FDG uptake in SPN include cell density and glucose transporter protein expression, as well as hypoxia-inducible factor and vascular endothelia growth factor expression in the hypoxic environment of necrotic areas.

Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.

BACKGROUND The timing of parturition is an important determinant of labor and delivery care. Early parturition is associated with increased neonatal morbidity and mortality. Most existing studies analyzed a single factor for the initiation of parturition, and the role of multiple factors in initiating parturition has not been comprehensively analyzed. MATERIAL AND METHODS We measured the levels of proinflammatory mediators, hypoxia factor, matrix metalloproteinases, hormones, and oxytocin, as well as fetal umbilical blood flow, before and after labor, and their associations with parturition. We also built a statistical model to predict the timing of parturition based on the measurement data. RESULTS IL-1ß, IL-6, TNF-alpha, MMP-9, and HIF-1alpha concentrations significantly increased from full term to labor. The PRL level significantly decreased from full term to parturition. There was no significant change in MCP-1, E3, and OT concentrations from full term to parturition. IL-1ß, IL-6, TNF-alpha, and MMP-9 concentrations were negatively correlated with the initiation of parturition. There was a small but nonsignificant increase in umbilical venous blood flow before parturition. Multiple factors showed a close correlation with the initiation of parturition, and area under the curve analysis showed that a multiple factor model was superior to single factors in the establishment of a model to predict initiation of parturition; however, these results need further confirmation. CONCLUSIONS Combined proinflammatory biomarkers have better predictive value for term labor than single biomarkers.

The authors describe a significantly improved colorimetric nanoprobe for the determination of transcription factors (TFs). It is making use of click-mediated growth of gold nanoparticles (AuNPs) to amplify the signal-to-noise ratio. Hypoxia-inducible factor-1 (HIF-1) is an important TF that acts as a mediator of cell response to hypoxia. So, the detection of HIF-1 was chosen as the model analyte. Specifically, target HIF-1 is designed to bind to the hypoxia response element within DNA duplex. The click chemistry between the DNA duplex and alkynyl-functionalized AuNPs (AF-AuNPs) is then inhibited because of significant steric hindrance. As a result, the AF-AuNPs grow into larger-sized highly-aggregated irregular nanostructures, which in turn enable colorimetric determination. The ratio of absorbances at 620 and 560 nm increases in the 0.5 to 10 nM HIF-1 concentration range, and the detection limit is 0.27 nM. This is better by a factor of 100 than that of aggregation-based colorimetric assays. The nanoprobe is selective and can be used in complex samples. Conceivably, it may also be extended to the determination of other TFs by simply changing the used DNA duplex. Graphical abstract Schematic of a nanoprobe for detecting hypoxia-inducible factor-1 (HIF-1). Three concepts are involved: the binding of HIF-1 and hypoxia response element, the Cu + -catalyzed click chemistry between P1/P2 duplex and alkynyl-functionalized AuNPs (AF-AuNPs), and the AuNPs growth with hydroxylamine and HAuCl 4 .

Objective: Grape seed proanthocyanidine extract (GSPE) is a strong antioxidant derived from the grape seeds (Vitis vinifera, Terral J.F.) and has a polyphenolic structure with a wide range of biological activity. The aim of the present study was to evaluate the effects of GSPE on alveolar bone loss and histopathological changes in rats with diabetes mellitus and ligature-induced periodontitis., Material and Methods: Forty rats were divided into 6 study groups. Control (C, 6 rats) group, periodontitis (P, 6 rats) group, diabetes (D, 6 rats) group, diabetes and periodontitis (D+P, 6 rats) group, diabetes, periodontitis and 100 mg/kg/day GSPE (GSPE-100, 8 rats), and diabetes, periodontitis and 200 mg/kg/day GSPE (GSPE-200, 8 rats) group. Diabetes mellitus was induced by intraperitoneal injection of a single dose of streptozotocin (60 mg/kg). Periodontitis was induced via ligation method. Silk ligatures were placed at the mandibular right first molars. GSPE was administered by oral gavage. After 30 days, all rats were killed. Alveolar bone loss was measured morphometrically via a stereomicroscope. For histopathological analyses, Alizarin red staining, and matrix metalloproteinase (MMP)-8, vascular endothelial growth factor and hypoxia inducible factor (HIF)-1α immunohistochemistry were performed. Tartrate-resistant acid phosphatase-positive osteoclast cells and relative total inflammatory cells were also determined., Results: The highest alveolar bone loss was observed in the D+P group (P < .05). GSP-200 group decreased alveolar bone loss (P < .05). The D+P group had the highest osteoclast counts, but the difference was not significant compared to the P, GSPE-100 and GSPE-200 groups (P > .05). The inflammation in the D+P group was also higher than the other groups (P < .05). The osteoblast numbers increased in the GSPE-100 and GSPE-200 groups compared to the P and D+P groups (P < .05). MMP-8 and HIF-1α levels were highest in the D+P group and GSPE significantly decreased these levels (P < .05)., Conclusion: Within the limits of this animal study, it can be suggested that GSPE administration may decrease periodontal inflammation and alveolar bone loss via decreasing MMP-8 and HIF-1α levels and increase osteoblastic activity in diabetic rats with experimental periodontitis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The complex cross-talk of intricate intercellular signaling networks between the tumor and stromal cells promotes cancer progression. Hypoxia is one of the most common conditions encountered within the tumor microenvironment that drives tumorigenesis. Most responses to hypoxia are elicited by a family of transcription factors called hypoxia-inducible factors (HIFs), which induce expression of a diverse set of genes that assist cells to adapt to hypoxic environments. Among the three HIF protein family members, the role of HIF-1 is well established in cancer progression. HIF-1 functions as a signaling hub to coordinate the activities of many transcription factors and signaling molecules that impact tumorigenesis. This mini review discusses the complex role of HIF-1 and its context-dependent partners under various cancer-promoting events including inflammation and generation of cancer stem cells, which are implicated in tumor metastasis and relapse. In addition, the review highlights the importance of therapeutic targeting of HIF-1 for cancer prevention., (© 2015 UICC.)

Background: Although the function of hypoxia-inducible factor 1 (HIF1) in many kinds of solid tumor has been revealed, the significance of HIF1 in osteosarcoma is still controversial and not well understood., Methods: Immunohistochemistry was used to detect HIF1 expression. The correlation between HIF1 and clinicopathology factors was analyzed by use of chi-squared tests. The prognostic value of HIF1 was evaluated by univariate and multivariate analysis. Moreover, the function of HIF1 in osteosarcoma cells was further investigated in in-vitro experiments by regulating HIF1 and vascular endothelial growth factor-A (VEGF-A) expression., Results: Expression of HIF1 was high for 56.82 % of the samples in our investigation. HIF1 expression was significantly associated with positive metastasis (P = 0.037). By use of the Kaplan-Meier method, high expression of HIF1 was proved to be related to poorer overall survival (P = 0.007). By use of a Cox-regression model, HIF1 was identified as an independent prognostic biomarker (P = 0.019). We also proved that HIF1 can promote osteosarcoma invasion in hypoxia by inducing VEGF-A expression., Conclusions: HIF1 was identified as an independent prognostic biomarker in osteosarcoma. It can promote osteosarcoma cell invasion by inducing VEGF-A expression, indicating that HIF1 is a potential drug target in osteosarcoma.

Background: Angiogenesis, which is initiated by certain tumor micro-environmental conditions and diverse protein factors, plays a pivotal role during tumor development and metastasis. Therefore, many efforts have been made to develop effective anti-angiogenic agents as anticancer therapeutics. In the current study, we investigated the anti-angiogenic potential of an ethanol extract of Annona atemoya seeds (EEAA) in vitro and in vivo., Methods: The anti-angiogenic potential of EEAA was evaluated using various in vitro/in vivo models, including cell proliferation, migration, and tube formation by human umbilical vascular endothelial cells (HUVECs); a Matrigel plug assay; and tumor-induced angiogenesis. The expression of hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) was investigated using reverse transcription-polymerase chain reaction, immunoassays, and western blotting., Results: EEAA was able to significantly inhibit the angiogenic properties of HUVECs in vitro as well as angiogenic factor-induced blood vessel formation in vivo. EEAA down-regulated the expression of VEGF and HIF-1alpha/2alpha at the mRNA and protein levels, respectively, in cancer cells under hypoxic conditions., Conclusions: EEAA shows a strong anti-angiogenic potential in both in vitro and in vivo systems, and we suggest that EEAA may be a valuable herbal source for anticancer drug development.

Background: The skin color of human hypertrophic scar changes dynamically during scar progression., Objective: To investigate whether hypoxia is dynamic during scar progression., Methods: Thirty-five patients with early, proliferative, regressive, and mature scars were involved in this study. Tissue oxygen tension was measured before scar surgery. After surgery, the scar stage was further defined using hematoxylin and eosin staining, and microvessel density and hypoxia inducible factor-1 (HIF-1) expression were detected using immunohistochemistry to determine a correlation with oxygen level., Results: Mild hypoxia is present in early scars, moderate hypoxia in proliferative scars, and severe hypoxia in regressive scars. Oxygen levels then return to normal in mature scars, which was consistent with the dynamic change in microvessel density. Meanwhile, HIF-1 expression also changed dynamically along with alteration in oxygen levels., Conclusion: Hypoxia is dynamic in scar tissue and is possibly correlated with scar formation and regression., (© 2014 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc.)

Purpose: We aimed to develop a radiolabeled peptide probe for the imaging of hypoxia-inducible factor-1 (HIF-1)-active tumors., Procedures: We synthesized the peptide probes that contain or lack an essential sequence of the oxygen-dependent degradation of HIF-1α in proteasomes ((123/125)I-DKOP30 or (125)I-mDKOP, respectively). The degradation of probes was evaluated in vitro using cell lysates containing proteasomes. In vivo biodistribution study, planar imaging, autoradiography, and comparison between probe accumulation and HIF-1 transcriptional activity were also performed., Results: The (125)I-DKOP30 underwent degradation in a proteasome-dependent manner, while (125)I-mDKOP was not degraded. Biodistribution analysis showed (125)I-DKOP30 accumulation in tumors. The tumors were clearly visualized by in vivo imaging, and intratumoral distribution of (125)I-DKOP30 coincided with the HIF-1α-positive hypoxic regions. Tumoral accumulation of (125)I-DKOP30 was significantly correlated with HIF-1-dependent luciferase bioluminescence, while that of (125)I-mDKOP was not., Conclusion: (123)I-DKOP30 is a useful peptide probe for the imaging of HIF-1-active tumors.

Purpose: Oral pregnancy tumors (OPTs) arise on the inflamed gingiva of women after the first trimester of pregnancy. The expression of angiogenic markers and female hormone receptors was assessed., Materials and Methods: Immunohistochemistry was used to analyze the expression of estrogen and progesterone receptors and the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and its receptor, fibroblast growth factor (FGF), and hypoxia inducible factors 1α and 3α (HIF1α and HIF3α). Experimental groups included 9 OPTs, 10 oral pyogenic granulomas from nonpregnant women of the same age, and 9 oral pyogenic granulomas from postmenopausal women., Results: VEGF expression in stromal histiocytes and endothelial cells of small vessels was positively correlated in the OPT group (P < .05 by χ(2) test). VEGF receptor also was overexpressed in stromal histiocytes and endothelial cells of OPTs compared with oral pyogenic granulomas from nonpregnant and postmenopausal women (P < .005 by χ(2) test). No correlation was detected among estrogen and progesterone receptors, FGF and HIF1α and HIF3α (ER and PgR respectively) in the 3 experimental groups., Conclusions: VEGF-associated angiogenesis is most likely involved in the pathogenesis of the lesion. These results imply that local inhibition of VEGF activity could be an adjuvant therapeutic approach for OPTs to control hemorrhage, which can be massive at the surgical excision of such lesions during pregnancy., (Copyright © 2013 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Introduction: Human dental pulp cells (HDPCs) are recalcitrant to hypoxic stress. We investigated whether hypoxia-induced autophagy of HDPCs offered these cells a survival advantage and the underlying mechanism of this resistance., Methods: The viability and apoptosis of HDPCs were examined after exposure to hypoxia by Vi-CELL cell viability analyzer and flow cytometry. Autophagy was assessed by using immunofluorescence, acridine orange staining, real-time polymerase chain reaction, and Western blotting. Either 3-methyladenine or expression vectors encoding dominant negative ATG5 were used to inhibit autophagy. Rapamycin was used as an autophagic inducer. To explore the mechanisms of autophagy, adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and hypoxia-inducible transcription factor-1 were suppressed by chemical inhibitors Compound C and YC-1, respectively., Results: The exposure of HDPCs to hypoxia had no effect on viability and resulted in increasing acidic vesicular organelle-positive cells, autophagosome formation, and up-regulation of autophagy genes. Inhibition of autophagy with 3- methyladenine or expression vectors encoding dominant negative ATG5 abrogated the protective effects of HDPCs. The phosphorylation of AMPK was up-regulated, whereas the phosphorylation of mTOR was down-regulated in hypoxia-treated HDPCs, which were both attenuated by Compound C. Furthermore, treatment with Compound C rather than YC-1 reduced the autophagy., Conclusions: Our results suggested that autophagy of HDPCs might be cytoprotective against hypoxic stress via the AMPK/mTOR signaling pathway., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Diaromatic-substituted ortho- and meta-carboranes were synthesized as mimics of manassantin A. Among the carboranes synthesized, compounds 1 and 2 showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity, with IC(50) values of 3.2 and 2.2 μM, respectively. Compounds 1 and 2 similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of HIF-1α mRNA. The hypoxia-induced accumulation and translocation of HIF-1α into nuclei were not observed in HeLa cells treated with compounds 1 and 2 by immunofluorescence analysis, revealing that the inhibition of hypoxia-induced HIF-1 transcriptional activity is induced by compounds 1 and 2 through a degradation pathway of the HIF-1α protein under hypoxic conditions., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Spermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5'-RCGTG-3') identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1., Methods: The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis., Results: The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter., Conclusions: The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.

Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression.

Background: Hypoxia-inducible factor (HIF)-1 is a key transcription factor responding to hypoxia. It is composed of an oxygen-sensitive α subunit (HIF-1α) and a constitutively expressed β subunit. Increasing evidence indicates an essential role for HIF-1α in infection and immunity. Because inflamed periodontium is thought to be hypoxic, we hypothesize that HIF-1α is expressed and related to its upstream regulator tumor necrosis factor (TNF)-α and downstream effecter vascular endothelial growth factor (VEGF)., Methods: Human gingival biopsies were collected from advanced periodontitis sites and clinically healthy sites, and immunohistochemically examined for HIF-1α and VEGF peptides. The messenger ribonucleic acid (mRNA) and protein levels of HIF-1α, VEGF, and TNF-α in the biopsies were then assessed by reverse transcription polymerase chain reaction and Western blotting., Results: HIF-1α-positive immunoreactivity was detected in the nuclei of epithelial and endothelial cells. In periodontal pockets, there was a marked increase in the proportion of fibroblast-like cells and leukocyte-like cells expressing HIF-1α. Protein levels of HIF-1α, VEGF, and TNF-α were significantly higher in periodontal pockets than in control gingival samples. The mRNA expression of VEGF and TNF-α was also increased in periodontal pockets., Conclusion: HIF-1α is expressed in healthy and diseased periodontium and may be related to TNF-α and VEGF function during periodontitis.

The purpose of this paper is to explore neovascularization in the biomaterials implanted in vivo. Capillaries formation of dermis defect filled with porous silk fibroin films (PSFFs) implant were investigated in rat at different times. Vascular endothelial growth factor (VEGF), CD34 and hypoxia inducible factor-1 (HIF) were observed in inflammatory cells and fibroblast in PSFFs by day 1, but expression in HIF and VEGF decreased by day 7, otherwise VEGF expressed in vascular endothelial cell increased by day 15 and 25, respectively. VEGF, angiopoietin 1 (Ang-1) and integrins αv and integrins β3 in PSFFs were observed by Reverse transcription-polymerase chain reaction (RT-PCR). Polyvinyl alcohol (PVA) sponge was control. PSFFs have better neovascularization than that of PVA expression of VEGF, HIF and angiogenesis in immunohistochemical and mRNA level.

Aims: The present study investigated whether transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) in renal capillary network improves renal interstitial fibrosis in unilateral ureteral obstruction (UUO) model in mice., Main Methods: Ex vivo generated, characterized, and cultivated mice BM-EPCs were identified by their vasculogenic properties in vitro. BM-EPCs were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) before transplantation. The animal models of UUO were used. Histological changes in renal tubular interstitium were observed with HE and Masson staining. The protein levels of vascular endothelial growth factor(VEGF), hypoxia inducible factor-1alpha (HIF-1alpha) and connective tissue growth factor (CTGF) were analyzed by western blotting and immunohistochemistry. Transforming growth factor-beta1 (TGF-beta1) was detected by immunohistochemistry. Peritubular capillary (PTC) density was determined by CD31 immunostaining., Key Findings: Transplanted BM-EPCs were successfully incorporated into the capillary network in the obstructed kidney in vivo. UUO induced a significant decrease in VEGF levels and PTC density in the kidney tissue, which was accompanied by a significant increase in HIF-1alpha, CTGF and TGF-beta1. Transplantation of BM-EPCs increased PTC density, VEGF expression and alleviated the development of renal interstitial fibrosis in UUO mice. No significant pathological changes were found in control mice., Significance: The reduction of PTC density and up-regulation of HIF-1alpha are the important mechanisms of interstitial fibrosis in UUO mice. BM-EPCs transplantation may increase the number of capillary density and alleviate the development of renal fibrosis in obstructive nephropathy in mice.

Objectives: To uncover novel biomarkers that will help predict which patients are at risk and may guide future treatment decisions. A significant proportion of prostate cancer patients treated with radical prostatectomy will experience a recurrence of the disease. Given the substantial role of hypoxia in prostate cancer development and treatment, this investigation focuses on the Hypoxia Inducible Factor (HIF-1alpha) pathway., Methods: A tissue microarray was constructed of prostate cancer tissue collected from 71 patients undergoing radical prostatectomy. The expression of proteins involved in the HIF-1alpha pathway was investigated by an immunohistochemical approach and correlated to clinical features including the time to biochemical recurrence., Results: Expression of GLUT1 correlated significantly (P <.05) with a shorter time to biochemical recurrence after radical prostatectomy and was independent from the Gleason grade and stage of cancer. Furthermore, our studies revealed for the first time that accumulation of prolyl-4-hydroxylases 1 especially in the nucleus, is a significant indicator for a worse prognosis (P <.001)., Conclusions: This study confirms the upregulation of proteins involved in the HIF-1alpha hypoxia pathway in prostate cancer cells, indicative of a hypoxic tumor state. Importantly, we report the identification of 2 novel markers, GLUT1 and prolyl-4-hydroxylases 1, with prognostic significance for patients undergoing radical prostatectomy., (Copyright 2010 Elsevier Inc. All rights reserved.)

Unlabelled: Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumor progression and in the development of resistance to radiotherapy. We designed a novel fusion protein (PTD-ODD-SAV [POS]) consisting of a protein transduction domain (PTD), streptavidin (SAV), and a portion of the oxygen-dependent degradation domain (ODD) of HIF-1alpha that confers the same oxygen-dependent regulation as HIF-1alpha on POS. (3-(123/125)I-iodobenzoyl)norbiotinamide ((123/125)I-IBB) was conjugated to the SAV moiety of POS to synthesize (123/125)I-IBB-labeled POS ((123/125)I-IPOS). The purpose of this study was to evaluate the feasibility of (123)I-IPOS as an imaging probe for HIF-1-active tumor hypoxia., Methods: After a 24-h incubation of (125)I-IPOS with various tumor cell lines under either normoxic (20% O(2)) or hypoxic (0.1% O(2)) conditions, the intracellular radioactivity was investigated. Then, the biodistribution of (123/125)I-IPOS was examined with tumor-implanted mice, and an in vivo imaging study was performed. The tumoral accumulation of (125)I-IPOS was compared with HIF-1 activity using the mice carrying tumors with the HIF-1-dependent luciferase reporter gene. Furthermore, the intratumoral localization of (125)I-IPOS was examined by the autoradiographic study, and then the same slide was subjected to immunostaining for pimonidazole, which is the hypoxic marker., Results: The ratios of radioactivity in hypoxic cells to that in normoxic cells were more than 2. These results indicate incorporation of (125)I-IPOS into these cells and degradation of (125)I-IPOS by normoxic tumor cells. In the biodistribution study, (125)I-IPOS accumulated in the tumor (1.4 +/- 0.3 percentage injected dose per gram) 24 h after administration. At that time, (125)I-IPOS showed high tumor-to-blood and tumor-to-muscle ratios (5.1 +/- 0.3 and 14.0 +/- 3.9, respectively). The tumors were clearly visualized by in vivo imaging 24 h after (123)I-IPOS injection (tumor-to-muscle ratio was 9.6). The tumoral accumulation of (125)I-IPOS correlated with HIF-1 activity (R = 0.71, P < 0.05), and its intratumoral distribution coincided with the hypoxic regions., Conclusion: (123)I-IPOS is a potential probe for the imaging of HIF-1 activity in tumors. Given the role of HIF-1 in tumor biology, its detection may be considered an indicator of aggressive cancer phenotypes.

Objective: We have previously demonstrated that the transcription factor hypoxia-inducible factor 1 (HIF-1) promotes the onset of autophagy in chondrocytes. The overall goal of this study was to test the hypothesis that another HIF family transcription factor, HIF-2, modulates the induction of autophagy by chondrocytes., Methods: Expression of HIF-1, HIF-2, and light chain 3 (LC3) in human and murine articular cartilage was visualized by immunohistochemistry. Suppression of HIF-2 was achieved using small interfering RNA technology. Assessments of autophagic flux and lysosomal activity, as well as ultrastructural analysis, were performed in chondrocytes in cell culture., Results: HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2alpha was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1 and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2alpha-knockout mouse growth plate showed an elevated autophagic response throughout the plate., Conclusion: Based on these observations, we conclude that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Our data suggest that this protein acts as a brake on the autophagy-accelerator function of HIF-1.

The number of compounds and doping methods in sports is in a state of constant flux. In addition to 'traditional' doping agents, such as anabolic androgenic steroids or erythropoietin, new therapeutics and emerging drugs have considerable potential for misuse in elite sport. Such compounds are commonly based on new chemical structures, and the mechanisms underlying their modes of action represent new therapeutic approaches arising from recent advances in medical research; therefore, sports drug testing procedures need to be continuously modified and complementary methods developed, preferably based on mass spectrometry, to enable comprehensive doping controls. This tutorial not only discusses emerging drugs that can be categorized as anabolic agents (selective androgen receptor modulators, SARMs), gene doping [hypoxia-inducible factor stabilizers, peroxisome-proliferator-activated receptor (PPAR)delta-agonists] and erythropoietin-mimetics (Hematide) but also compounds with potentially performance-enhancing properties that are not classified in the current list of the World Anti-Doping Agency. Compounds such as ryanodine-calstabin-complex modulators (benzothiazepines) are included, their mass spectrometric properties discussed, and current approaches in sports drug testing outlined., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Optical computed tomography (optical-CT) and optical-emission computed tomography (optical-ECT) are new techniques for imaging the 3D structure and function (including gene expression) of whole unsectioned tissue samples. This work presents a method of improving the quantitative accuracy of optical-ECT by correcting for the 'self'-attenuation of photons emitted within the sample. The correction is analogous to a method commonly applied in single-photon-emission computed tomography reconstruction. The performance of the correction method was investigated by application to a transparent cylindrical gelatin phantom, containing a known distribution of attenuation (a central ink-doped gelatine core) and a known distribution of fluorescing fibres. Attenuation corrected and uncorrected optical-ECT images were reconstructed on the phantom to enable an evaluation of the effectiveness of the correction. Significant attenuation artefacts were observed in the uncorrected images where the central fibre appeared approximately 24% less intense due to greater attenuation from the surrounding ink-doped gelatin. This artefact was almost completely removed in the attenuation-corrected image, where the central fibre was within approximately 4% of the others. The successful phantom test enabled application of attenuation correction to optical-ECT images of an unsectioned human breast xenograft tumour grown subcutaneously on the hind leg of a nude mouse. This tumour cell line had been genetically labelled (pre-implantation) with fluorescent reporter genes such that all viable tumour cells expressed constitutive red fluorescent protein and hypoxia-inducible factor 1 transcription-produced green fluorescent protein. In addition to the fluorescent reporter labelling of gene expression, the tumour microvasculature was labelled by a light-absorbing vasculature contrast agent delivered in vivo by tail-vein injection. Optical-CT transmission images yielded high-resolution 3D images of the absorbing contrast agent, and revealed highly inhomogeneous vasculature perfusion within the tumour. Optical-ECT emission images yielded high-resolution 3D images of the fluorescent protein distribution in the tumour. Attenuation-uncorrected optical-ECT images showed clear loss of signal in regions of high attenuation, including regions of high perfusion, where attenuation is increased by increased vascular ink stain. Application of attenuation correction showed significant changes in an apparent expression of fluorescent proteins, confirming the importance of the attenuation correction. In conclusion, this work presents the first development and application of an attenuation correction for optical-ECT imaging. The results suggest that successful attenuation correction for optical-ECT is feasible and is essential for quantitatively accurate optical-ECT imaging.

The aim of this study is to investigate the plasticity of human epithelial ovarian cancer cell SKOV3ip and formation of vasculogenic mimicry (VM) in vivo. SKOV3ip was transfected with lentiviral vector carrying green fluorescence protein (GFP). Female nude mice were implanted intraperitoneally with GFP-labled SKOV3ip. When the transplanted tumor reached a volume of approximately 1 cm(3), paraffin-embedded, formaldehyde-fixed tissue was prepared and stained with hematoxylin and eosin (H & E). Tumor tissues were also studied by electron microscopy and fluorescence microscopy. The results of H & E staining, electron microscopy, and fluorescence microscopy indicated SKOV3ip formed patterned networks with erythrocytes in them, in the absence of vascular epithelial cells, which was a sign that SKOV3ip engaged in VM in vivo. Expression of vascular epithelium marker CD31 was investigated by immunohistochemical staining, immunofluorescence assay, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometric analysis (FACS). Factor VIII and vascular endothelial growth factor (VEGF) were also analyzed by FACS. Weak and focal CD31 immunohistochemical staining was found along the channels of tumor cells. Immunofluorescence assay and RT-PCR demonstrated that CD31 was expressed in primary-cultured SKOV3ip. CD31 and Factor VIII, but not VEGF were detected in primary-cultured SKOV3ip by FACS. The present study has shown that human ovarian cancer cell line SKOV3ip may be able to express some specific markers of vascular epithelial cells and has plasticity to form VM in vivo. In the following study, we indicated that hypoxia-inducible factor (HIF)-1alpha inhibitor, rapamycin, could possibly prevent VM and phenotype transformation of SKOV3ip, reflected by down-regulating expression of CD31 and Factor VIII. HIF-1alpha protein expression correlated with CD31 and Factor VIII protein expression in SKOV3ip. These results indicated that VM might be associated with HIF-1alpha.

Oral squamous cell carcinoma (SCC) can be vascularized through a process called "tumor cell-lined vessels". Currently, the tumor microenvironment, which is recognized as hypoxic and orchestrated largely by inflammatory cells and defective blood vessels, is considered an important participant in the neoplastic process. We sought to determine their clinicopathologic significance and prognostic implication in oral SCC. Vascular structure was investigated by multistaining with pan-cytokeratin, CD34, and alpha-smooth actin/type IV collagen. Immunohistochemical staining of the hypoxia inducible factor-1 alpha (HIF-1 alpha) and CD68 was used to reflect hypoxia and tumor-associated macrophages (TAM). Our results showed that in a high percentage of vessels in cancer tissue. There is absence of pericyte coverage and loss of basement membrane lining. Significant association between the integrity of vascular structure and lymph node involvement and presence of tumor cell-lined vessel was found. HIF-1 alpha overexpression was frequently observed in cancer cells (78/112) and correlated with tumor progress index. In cancer tissues, the TAM ranged from 28 to 296 cells/mm2 with a mean of 144.6+/-64.3 cells/mm2. There was a significant correlation between TAM and lymph node involvement (P=0.004) and tumor size (P=0.004). Also, a close association was found between TAM count and integrity of vascular structure. In addition, survival analysis revealed that tumor cell-lined vessels (P=0.001), HIF-1 alpha expression (P=0.004), and TAM (P=0.001) correlated significantly with poor survival. We conclude that in the cancer microenvironment, HIF-1 alpha expression and the TAM are induced and contributed to malignant behavior of tumor cells. Furthermore, the presence of tumor cell-lined vessel, HIF-1 alpha overexpression, and high TAM could be the potential markers of prognosis for patients with oral SCC.

Hypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes.

Object: To observe the inhibitory effect of Hypoxia inducible factor-1 antisense oligonuclecotide on growth of human hepatocellular carcinoma cells and gene expression of HIF-1, in order to seek a new gene therapy for hepatocellular carcinoma., Methods: Six Hypoxia inducible factor-1 antisense oligonuclecotides with various concentrations (0.2 micromol/l, 0.4 micromol/l, and 0.8 micromol/l) were transformed into HepG2 cells by lipofectamine reagent. 72 h after transfection, MTS assay was used to detect cellular proliferation. In addition, Hypoxia inducible factor-1 antisense oligonuclecotide2 with various concentrations (0.2, 0.4, 0.8, and 1.0 micromol/l) were transformed into HepG2 cells. About 48 h after transfection, reverse transcription-polymerase chain reaction assay and Western Blot assay were employed to detect the expression of Hypoxia inducible factor-1 gene and the synthesis of Hypoxia inducible factor-1 protein respectively., Results: HepG2 cell growth was inhibited by 6 Hypoxia inducible factor-1 antisense oligonuclecotides at various concentrations. Among them, Hypoxia inducible factor-1 antisense oligonuclecotide2 showed the most effective inhibition ability (P < 0.01), the inhibitory rate was 89.66% at the concentration of 1.0 micromol/l. About 48 h after transfection, Hypoxia inducible factor-1 mRNA expression was downregulated and Hypoxia inducible factor-1 protein synthesis was decreased by antisense oligonuclecotide2., Conclusions: The hepatocellular carcinoma cell proliferation was inhibited by Hypoxia inducible factor-1 antisense oligonuclecotide. Moreover, the gene expression and protein synthesis of Hypoxia inducible factor-1 were reduced by Hypoxia inducible factor-1 antisense oligonuclecotide. The findings suggested that antisense technique targeting Hypoxia inducible factor-1 might be an effective gene therapy of human hepatocellular carcinoma.

Uncontrolled expression of vascular endothelial growth factor (VEGF) in vivo may cause unexpected side effects, such as brain hemangioma or tumor growth. Because hypoxia-inducible factor-1 (HIF-1) is upregulated during cerebral ischemia and regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we therefore used a novel HRE, originating in the 3'-end of the erythropoietin (Epo) gene, to control gene expression in the ischemic brain. A concatemer of nine copies (H9) of the consensus sequence of HRE was used to mediate hypoxia induction. Three groups of adult CD-1 mice received AAVH9-VEGF, AAVH9-lacZ or saline injection, and then underwent 45 min of transient middle cerebral artery occlusion (tMCAO). Results show that HIF-1 was persistently expressed in the ischemic brain. VEGF was overexpressed in the ischemic perifocal region in AAVH9-VEGF-transduced mice. Double-labeled immunostaining showed that VEGF expressed in neurons and astrocytes but not endothelial cells, suggesting that adeno-associated virus (AAV) vectors transduced neurons and astrocytes predominantly. The total number of microvessels/enlarged microvessels was greatly increased in the AAVH9-VEGF-transduced mice (180+/-29/27+/-4) compared to the AAVH9-lacZ (118+/-19/14+/-3) or saline-treated (119+/-20/14+/-2) mice after tMCAO (P<0.05). Cell proliferation examination demonstrated that these microvessels were newly formed. Regional cerebral blood flow recovery in the AAVH9-VEGF-transduced mice was also better than in AAVH9-lacZ or saline-treated mice (P<0.05). Our data indicated that HRE is a novel trigger for the control of VEGF expression in the ischemic brain. VEGF overexpression through AAVH9-VEGF gene transfer showed stable focal angiogenic effects in post-ischemic repair process, providing an opportunity to rebuild injured brain tissue.

Background: Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease., Methods: ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques., Results: HIF-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level., Conclusions: HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1 alpha. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.

Leukemoid reaction is defined as a reactive leukocytosis in response to infection, inflammation, or therapeutic agents such as growth factors and malignancy. We report a case of leukemoid reaction resulting from granulocyte colony-stimulating factor producing urothelial carcinoma of the renal pelvis as evidenced on immunohistochemical analysis.

Tumor hypoxia is a feature common to almost all solid tumors due to malformed vasculature and inadequate perfusion. Tumor cells have evolved mechanisms that allow them to respond and adapt to a hypoxic microenvironment. The hypoxia-inducible transcription factor (HIF) family is comprised of oxygen-sensitive alpha (alpha) subunits that respond rapidly to decreased oxygen levels and oxygen-insensitive beta (beta) subunits. HIF binds to specific recognition sequences in the genome and increases the transcription of genes involved in a variety of metabolic and enzymatic pathways that are necessary for cells to respond to an oxygen-poor environment. The critical role of this family of transcriptional regulators in maintaining oxygen homeostasis is supported by multiple regulatory mechanisms that allow the cell to control the levels of HIF as well as its transcriptional activity. This review will focus on how the transcriptional activity of HIF is studied and how it can be exploited for cancer therapy.

Background: Chronic low oxygen in the tubulointerstitial area is a crucial cause of renal degradation and tubulointerstitial damage. Previous reports have suggested that the maintenance of renal blood flow plays a role in the suppression of progressive renal damage. Neovascularization is important for the maintenance of blood flow. We studied the production of angiogenic factors by culturing renal proximal tubular epithelial cells (PTEC) under hypoxic conditions., Methods: Cultured PTEC were exposed to normal and low-oxygen conditions. The levels of angiogenin (ANG) and vascular endothelial growth factor (VEGF) in the cell supernatants were measured by enzyme-linked immunosorbent assay. The messenger RNAs (mRNAs) of ANG and VEGF in the PTEC were examined by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). The presence of ANG, VEGF and hypoxia-inducible factor-1 (HIF-1) was studied by immunofluorescence techniques. The effect of cobalt chloride (CoCl(2)), which is an HIF-1 inducer, on the production of ANG and VEGF was also examined in order to elucidate the contribution of the HIF-1 pathway to the production of these cytokines., Results: ANG and VEGF were demonstrated to exist in the cell supernatants, and ANG and VEGF mRNAs were detected in the PTEC. Hypoxic conditions stimulated the secretion of ANG (2.5-fold vs normoxia, P<0.001) and VEGF (3.2-fold vs normoxia, P<0.001) by PTEC. Hypoxic conditions increased the mRNA expression of ANG for 6 h (1.38-fold vs normoxia, P<0.05) and VEGF for 24 h (2.04-fold vs normoxia, P<0.01). Hypoxic conditions also enhanced ANG, VEGF and HIF-1 protein expression in PTEC. The CoCl(2) increased the secretion of ANG (5.2-fold vs control, P<0.0001) and VEGF (2.3-fold vs control, P<0.0001) by PTEC., Conclusion: Under hypoxic conditions, the ANG and VEGF secreted by PTEC may modulate angiogenesis and vascular remodeling in the renal interstitium via an increase in the production of HIF-1.

Pituitary autotransplantation eliminates direct vascular contact between the hypothalamus and the adenohypophysis, and enables us to study the role of the hypothalamus in regulating adenohypophysial endocrine activity. The aim of this study was to investigate vascularization of the pituitary autografts. Three-month-old male Wistar rats were hypophysectomized, and their adenohypophyses were autotransplanted under the renal capsule. The animals were killed 3 weeks after autotransplantation. The grafts were removed and studied by using histology, immunohistochemistry and transmission electron microscopy. In the central portion of the grafts, organizing necrosis was apparent. The peripheral portion of the graft contained all adenohypophysial cell types, with a predominance of lactotrophs. Vascular endothelial growth factor and hypoxia-inducible factor were expressed in the graft mainly in the perinecrotic areas. Several capillaries inside the grafts were lined by continuous unfenestrated epithelium, while others were lined by fenestrated endothelium, suggesting that neovascularization is the result of two processes: ingrowths of capillaries from the renal capsule to the graft, and neoformation of capillaries from pre-existing adenohypophysial vessels. In conclusion, hypoxia seems to be an important factor in the vascularization of pituitary autografts. Mediated via hypoxia-inducible factor, hypoxia stimulates vascular endothelial growth factor secretion, which plays a crucial role in angiogenesis.

The human hypoxia inducible factor 1 (HIF-1) system is activated under various pathological conditions, yet less is known about its physiological regulation in healthy human tissue. We have studied the effect of exercise on the activation of HIF-1 in human skeletal muscle. Employing a model where oxygen consumption increases and oxygen tension can be manipulated, nine healthy male subjects performed 45 min of one-legged knee-extension exercise. Biopsies were taken before, directly after, and 30, 120, and 360 min after exercise. Exercise led to elevated HIF-1alpha protein levels and a more prevalent nuclear staining of HIF-1alpha. Interestingly, a concurrent decrease in von Hippel-Lindau tumor suppressor protein (VHL) levels was detected in some subjects. Moreover, exercise induced an increase in the DNA binding activity of HIF-1alpha. Characterization of gene expression by real-time PCR demonstrated that the HIF-1 target genes VEGF and EPO were activated. VEGF mRNA was further increased when blood flow to the exercising leg was restricted. In conclusion, these data clearly demonstrate that physical activity induces the HIF-1-mediated signaling pathway in human skeletal muscle, providing the first evidence that human HIF-1alpha can be activated during physiologically relevant conditions.