Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells

Background

Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells.

Methods and Findings

Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells.

Conclusions

Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.     

Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment

Background

Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.

Methodology/Principal Findings

We analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.

Conclusions/Significance

Hypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

Increased C-C Chemokine Receptor 2 Gene Expression in Monocytes of Severe Obstructive Sleep Apnea Patients and under Intermittent Hypoxia

Background

Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes.

Methods

Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes.

Results

Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia.

Conclusions

This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

Characterization of the Activation of Protein Tyrosine Phosphatase,Receptor-Type,Z Polypeptide 1 (PTPRZ1) by Hypoxia Inducible Factor-2 Alpha

Background

Hypoxia inducible factors (HIFs) are the principal means by which cells upregulate genes in response to hypoxia and certain other stresses. There are two major HIFs, HIF-1 and HIF-2. We previously found that certain genes are preferentially activated by HIF-2. One was protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). PTPRZ1 is overexpressed in a number of tumors and has been implicated in glioblastoma pathogenesis.

Methodology/Principal Findings

To understand the preferential activation of PTPRZ1 by HIF-2, we studied the PTPRZ1 promoter in HEK293T cells and Hep3B cells. Through deletion and mutational analysis, we identified the principal hypoxia response element. This element bound to both HIF-1 and HIF-2. We further identified a role for ELK1, an E26 transformation-specific (Ets) factor that can bind to HIF-2α but not HIF-1α, in the HIF-2 responsiveness. Knock-down experiments using siRNA to ELK1 decreased HIF-2 activation by over 50%. Also, a deletion mutation of one of the two Ets binding motifs located near the principal hypoxia response element similarly decreased activation of the PTPRZ1 promoter by HIF-2. Finally, chromatin immunoprecipitation assays showed binding of HIF and ELK1 to the PTPRZ1 promoter region.

Conclusions/Significance

These results identify HIF-binding and Ets-binding motifs on the PTPRZ1 promoter and provide evidence that preferential activation of PTPRZ1 by HIF-2 results at least in part from cooperative binding of HIF-2 and ELK1 to nearby sites on the PTPRZ1 promoter region. These results may have implications in tumor pathogenesis and in understanding neurobiology, and may help inform the development of novel tumor therapy.     

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

Background

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.

Results

Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.

Conclusions

Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.     

PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells

Background

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.

Methodology/Principal Findings

In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.

Conclusions/Significance

Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.     

Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

Background

Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.

Methodology/Principal Findings

Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.

Conclusion and Implications

This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.     

Radiosensitization of normoxic and hypoxic h1339 lung tumor cells by heat shock protein 90 inhibition is independent of hypoxia inducible factor-1α

Background

Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy.

Methodology/Principal Findings

Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity.

Conclusions/Significance

In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

Melittin Suppresses HIF-1α/VEGF Expression through Inhibition of ERK and mTOR/p70S6K Pathway in Human Cervical Carcinoma Cells

Objective

Melittin (MEL), a major component of bee venom, has been associated with various diseases including arthritis, rheumatism and various cancers. In this study, the anti-angiogenic effects of MEL in CaSki cells that were responsive to the epidermal growth factor (EGF) were examined.

Methodology/Principal Findings

MEL decreased the EGF-induced hypoxia-inducible factor-1α (HIF-1α) protein and significantly regulated angiogenesis and tumor progression. We found that inhibition of the HIF-1α protein level is due to the shortened half-life by MEL. Mechanistically, MEL specifically inhibited the EGF-induced HIF-1α expression by suppressing the phosphorylation of ERK, mTOR and p70S6K. It also blocked the EGF-induced DNA binding activity of HIF-1α and the secretion of the vascular endothelial growth factor (VEGF). Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MEL reduced the binding of HIF-1α to the VEGF promoter HRE region. The anti-angiogenesis effects of MEL were confirmed through a matrigel plus assay.

Conclusions

MEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF-1α. These results suggest that MEL may inhibit human cervical cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression.     

Prolonged Mechanical Ventilation Alters the Expression Pattern of Angio-neogenetic Factors in a Pre-Clinical Rat Model

Objective

Mechanical ventilation (MV) is a life saving intervention for patients with respiratory failure. Even after 6 hours of MV, diaphragm atrophy and dysfunction (collectively referred to as ventilator-induced diaphragmatic dysfunction, VIDD) occurs in concert with a blunted blood flow and oxygen delivery. The regulation of hypoxia sensitive factors (i.e. hypoxia inducible factor 1α, 2α (HIF-1α,–2α), vascular endothelial growth factor (VEGF)) and angio-neogenetic factors (angiopoietin 1–3, Ang) might contribute to reactive and compensatory alterations in diaphragm muscle.

Methods

Male Wistar rats (n = 8) were ventilated for 24 hours or directly sacrificed (n = 8), diaphragm and mixed gastrocnemius muscle tissue was removed. Quantitative real time PCR and western blot analyses were performed to detect changes in angio-neogenetic factors and inflammatory markers. Tissues were stained using Isolectin (IB 4) to determine capillarity and calculate the capillary/fiber ratio.

Results

MV resulted in up-regulation of Ang 2 and HIF-1α mRNA in both diaphragm and gastrocnemius, while VEGF mRNA was down-regulated in both tissues. HIF-2α mRNA was reduced in both tissues, while GLUT 4 mRNA was increased in gastrocnemius and reduced in diaphragm samples. Protein levels of VEGF, HIF-1α, -2α and 4 did not change significantly. Additionally, inflammatory cytokine mRNA (Interleukin (IL)-6, IL-1β and TNF α) were elevated in diaphragm tissue.

Conclusion

The results demonstrate that 24 hrs of MV and the associated limb disuse induce an up-regulation of angio-neogenetic factors that are connected to HIF-1α. Changes in HIF-1α expression may be due to several interactions occurring during MV.     

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

Background

Stem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.

Methods

The MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.

Results

rAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.

Conclusions

Vacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.     

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

Background

The endothelin B receptor (ET_BR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.

Principal Findings

Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET_BR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET_BR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET_BR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ET_BR expression is associated with low PHD2 levels. In melanoma xenografts, ET_BR blockade by ET_BR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.

Conclusions

In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET_BR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.     

Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background

Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling.

Methods and Results

Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO₂ = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression.

Conclusion

The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression.