Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

��-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that β-actin is associated with eNOS oxygenase domain and that association of eNOS with β-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that β-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326–333 of human eNOS, one of the putative ABSs, specifically bound to β-actin and prevented eNOS association with β-actin in vitro. Peptide 326 also prevented β-actin-induced decrease in superoxide formation and increase in NO and l-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-β-actin binding and to prevent β-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-β-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-β-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that β-actin binding to eNOS through residues 326–333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by β-actin plays an important role in endothelial function.     

Hypoxia-induced expression of complement receptor type 1(CR1, CD35) in human vascular endothelial cells

Reoxygenation of hypoxic human umbilical vein endothelial cells(HUVECs) increases protein expression of the complement regulators CD46and CD55. As the receptor for C3b is known to be present on injuredbovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expressionincreased 3.7 ± 0.6-fold as measured by ELISA on HUVECsfollowing hypoxia (48 h, 1%O₂). Colocalization of CD35 andvon Willebrand factor by confocal microscopy confirmed that CD35 waspredominantly intracellular. Lipopolysaccharide or tumor necrosisfactor- also significantly increased HUVEC CR1 proteinexpression. Western blot analysis of neutrophil or hypoxicHUVEC lysates revealed a 221-kDa CR1 band under nonreducingconditions. RT-PCR of hypoxic HUVEC mRNA revealed a singleband that, after sequencing, was identified as CD35. In situhybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA.Hypoxic HUVECs bound immune complexes and acted as a cofactorfor factor I-mediated cleavage of C3b. Thus hypoxia induces functionalHUVEC CR1 expression.     

Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells

Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl(2)) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl(2). Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl(2). Hypoxia also represses HO-1 mRNA expression in human coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types.     

Prevention of CCAAT/Enhancer-binding Protein �� DNA Binding by Hypoxia during Adipogenesis

Upon exposure to adipogenesis-inducing hormones, confluent 3T3-L1 preadipocytes express C/EBPβ (CCAAT/enhancer binding protein β). Early induced C/EBPβ is inactive but, after a lag period, acquires its DNA-binding capability by sequential phosphorylation. During this period, preadipocytes pass the G₁/S checkpoint synchronously. Thr¹⁸⁸ of C/EBPβ is phosphorylated initially to prime the factor for subsequent phosphorylation at Ser¹⁸⁴ or Thr¹⁷⁹ by GSK3β, which translocates into the nuclei during the G₁/S transition. Many events take place during the G₁/S transition, including reduction in p27^Kip1 protein levels, retinoblastoma (Rb) phosphorylation, GSK3β nuclear translocation, and C/EBPβ binding to target promoters. During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is stabilized, thus maintaining expression of p27^Kip1, which inhibits Rb phosphorylation. Even under normoxic conditions, constitutive expression of p27^Kip1 blocks the nuclear translocation of GSK3β and DNA binding capability of C/EBPβ. Hypoxia also blocks nuclear translocation of GSK3β and DNA binding capability of C/EBPβ in HIF-1α knockdown 3T3-L1 cells that fail to induce p27^Kip1. Nonetheless, under hypoxia, these cells can block Rb phosphorylation and the G₁/S transition. Altogether, these findings suggest that hypoxia prevents the nuclear translocation of GSK3β and the DNA binding capability of C/EBPβ by blocking the G₁/S transition through HIF-1α-dependent induction of p27^Kip1 and an HIF-1α/p27-independent mechanism.     

The Tyrosine Phosphatase SHP-1 Regulates Hypoxia Inducible Factor-1α (HIF-1α) Protein Levels in Endothelial Cells under Hypoxia

Introduction

The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.

Results

SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H₂-DCF and DHE fluorescence.

Conclusions

SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.     

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.     

Opposite effects of prostaglandin-J2 on VEGF in normoxia and hypoxia: role of HIF-1

The vascular endothelial growth factor (VEGF) is produced in response to hypoxia or inflammatory cytokines. In normoxia VEGF synthesis is upregulated by 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) via induction of heme oxygenase-1 (HO-1). Here we compared the influence of 15d-PGJ(2) on VEGF expression in human microvascular endothelial cells in normoxia (approximately 20% O(2)) and hypoxia ( approximately 2% O(2)). Regardless of the oxygen concentration, 15d-PGJ(2) inhibited activity of hypoxia inducible factor-1 (HIF-1), the major hypoxic regulator of VEGF. However, in normoxic conditions 15d-PGJ(2) (1-10microM) activated the VEGF promoter and increased synthesis of the VEGF protein. Concomitantly, it strongly induced expression of HO-1. In contrast, in hypoxia, 15d-PGJ(2) decreased VEGF promoter activity and reduced VEGF release by 50%. Inhibition of HO-1 activity additionally attenuated VEGF synthesis in hypoxia. We conclude that induction of HO-1 by 15d-PGJ(2) results in augmentation of VEGF synthesis in normoxia. In hypoxia, however, the stimulatory effect of HO-1 is outweighed by 15d-PGJ(2)-mediated inhibition of the HIF-1 pathway.     

Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes

Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of Mafa, Pdx1, Slc2a2, Ndufa5, Kcnj11, Ins1, Wfs1, Foxa2, and Neurod1, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1–independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.