Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1

The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.     

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

The fission yeast FHIT homolog affects checkpoint control of proliferation and is regulated by mitochondrial electron transport

Genetic analysis has strongly implicated human FHIT (Fragile Histidine Triad) as a tumor suppressor gene, being mutated in a large proportion of early‐stage cancers. The functions of the FHIT protein have, however, remained elusive. Here, we investigated aph1⁺, the fission yeast homolog of FHIT, for functions related to checkpoint control and oxidative metabolism. In sublethal concentrations of DNA damaging agents, aph1Δ mutants grew with a substantially shorter lag phase. In aph1Δ mutants carrying a hypomorphic allele of cds1 (the fission yeast homolog of Chk2), in addition, increased chromosome fragmentation and missegregation were found. We also found that under hypoxia or impaired electron transport function, the Aph1 protein level was strongly depressed. Previously, FHIT has been linked to regulation of the human 9‐1‐1 checkpoint complex constituted by Hus1, Rad1, and Rad9. In Schizosaccharomyces pombe, the levels of all three 9‐1‐1 proteins are all downregulated by hypoxia in similarity with Aph1. Moreover, deletion of the aph1⁺ gene reduced the Rad1 protein level, indicating a direct relationship between these two proteins. We conclude that the fission yeast FHIT homolog has a role in modulating DNA damage checkpoint function, possibly through an effect on the 9‐1‐1 complex, and that this effect may be critical under conditions of limiting oxidative metabolism and reoxygenation.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.     

Effects of YC-1 targeting hypoxia-inducible factor 1 alpha in oesophageal squamous carcinoma cell line Eca109 cells

HIF-1α (hypoxia-inducible factor 1 alpha) is believed to promote oesophageal squamous tumour growth. Thus, an HIF-1α inhibitor is viewed as a therapeutic target in treating oesophageal cancer. Recently, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] has been widely used as a potential HIF-1α inhibitor and is being developed as a novel anticancer drug. However, little is known about the effects of YC-1 in human oesophageal cancer. In the present study, we aimed to investigate these effects in an esophageal squamous cancer cell line; i.e. Eca109 cells. We found that YC-1 abolished the hypoxia-induced up-regulation of HIF-1α. YC-1 arrested cell growth and inhibited cell migration activities in Eca109 cells. These results suggest that YC-1 may be a chemotherapy candidate against oesophageal squamous cancers.     

β-N-oxalyl-L-α,β-diaminopropionic acid regulates mitogen-activated protein kinase signaling by down-regulation of phosphatidylethanolamine-binding protein 1

β-N-Oxalyl-L-α,β-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis

Endometriosis is a common gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells. Hypoxia has been implicated as a crucial microenvironmental factor that contributes to endometriosis. It has been reported that long non‐coding RNA MALAT1 (lncRNA‐MALAT1) highly expressed in endometriosis and up‐regulated by hypoxia. Hypoxia may also induce autophagy, which might act as cell protective mechanism. However, the relationship between lncRNA‐MALAT1 and autophagy under hypoxia conditions in endometriosis remains unknown. In the present study, we found that both lncRNA‐MALAT1 and autophagy level were up‐regulated in ectopic endometrium from patients with endometriosis, and its expression level correlates positively with that of hypoxia‐inducible factor‐1α (HIF‐1α). In cultured human endometrial stromal cells, both lncRNA‐MALAT1 and autophagy were induced by hypoxia in a time‐dependent manner and lncRNA‐MALAT1 up‐regulation was dependent on HIF‐1α signalling. Our analyses also show that knockdown of lncRNA‐MALAT1 suppressed hypoxia induced autophagy. Furthermore, inhibiting autophagy with specific inhibitor 3‐Methyladenine (3‐MA) and Beclin1 siRNA enhanced apoptosis of human endometrial stromal cells under hypoxia condition. Collectively, our findings identify that lncRNA‐MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis.