Synergistic effect of HIF-1α gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia

Ischemia induces the production of angiogenic cytokines and the homing of bone-marrow-derived angiogenic cells (BMDACs), but these adaptive responses become impaired with aging because of reduced expression of hypoxia-inducible factor (HIF)-1α. In this study, we analyzed the effect of augmenting HIF-1α levels in ischemic limb by intramuscular injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, and intravenous administration of BMDACs that were cultured in the presence of the prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to induce HIF-1 expression. The combined therapy increased perfusion, motor function, and limb salvage in old mice subjected to femoral artery ligation. Homing of BMDACs to the ischemic limb was dramatically enhanced by intramuscular AdCA5 administration. DMOG treatment of BMDACs increased cell surface expression of β₂ integrins, which mediated increased adherence of BMDACs to endothelial cells. The effect of DMOG was abolished by coadministration of the HIF-1 inhibitor digoxin or by preincubation with a β₂ integrin-blocking antibody. Transduction of BMDACs with lentivirus LvCA5 induced effects similar to DMOG treatment. Thus, HIF-1α gene therapy increases homing of BMDACs to ischemic muscle, whereas HIF-1 induction in BMDACs enhances their adhesion to vascular endothelium, leading to synergistic effects of combined therapy on tissue perfusion.     

HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells

AIM: To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC).METHODS: Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction.RESULTS: Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo.CONCLUSION: HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2.     

The effect of hypoxia-inducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes

Aim

To study the role of hypoxia-inducible factor 1-alpha (HIF-1α) on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes.

Methods

Primary neonatal rat ventricular myocytes were exposed to hypoxia for 24 hours. HIF-1α activity was suppressed by treating the cells with 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1). The degree of cell apoptosis was assessed by Hoechst 33258 DNA staining. The levels of HIF-1α and the pro-apoptotic proteins Bnip3, Bax and Bad were measured with western blotting.

Results

On exposure to hypoxia, there was an increase in the expression levels of HIF-1α, and the pro-apoptotic protein Bnip3 was upregulated. Suppression of HIF-1α activity by YC-1 treatment was followed by blockade of hypoxia-induced apoptosis and Bnip3 expression; however, the changes in the levels of Bax and Bad expression were unclear.

Conclusion

Acute hypoxia enhanced primary neonatal rat ventricular myocyte apoptosis through the activation of HIF-1α and a mechanism that perhaps involved Bnip3. Targeting HIF-1α may be a new strategy for reducing the degree of hypoxia-induced apoptosis in ventricular myocytes.     

Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells

AIM: To investigate the influence of phosphatidylinositol-3-kinase protein kinase B(PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. METHODS: Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, m RNA and activity levels of hypoxia inducible factor-1 alpha(HIF-1α), glucose transporter 1, hexokinase-Ⅱ, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting si RNA to assess impact of the high expression of HIF-1α on glycolysis.RESULTS: HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymesand the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions.CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.     

Hypoxia induces cardiac fibroblast proliferation and phenotypic switch: a role for caveolae and caveolin-1/PTEN mediated pathway

Background

Cardiac fibrosis following myocardial infarction (MI) results in heart failure. Caveolin-1, the main structural protein of caveolae, regulates signal transduction pathways controlling cell proliferation and apoptosis. Meanwhile, low phosphatase and tensin homolog (PTEN) activity enhances the PI3K/Akt signal pathway to induce cell proliferation. But whether caveolin-1 and PTEN activation regulates cardiac fibroblast proliferation and contributes to cardiac fibrosis from ischemic injury is incompletely understood. This study investigates whether hypoxia inducing cardiac fibroblast proliferation and phenotypic switch is caveolin-dependent.

Methods

We used in vitro and in vivo models of ischemic injury, immunohistochemical staining, and cell proliferation assays to address this hypothesis.

Results

We found that MI induced collagen deposition and cardiac dysfunction. After MI, mice displayed reduced caveolin-1 and PTEN expression and increased α-smooth muscle actin (α-SMA) expression in the infarct zone. Qualitative and quantitative analyses indicated that caveolin-1 expression was lowest at 7 days after MI, accompanied by increased collagen deposition and attenuated cardiac function. We cultured cardiac fibroblasts of mice were in hypoxia or normoxia conditions for 12, 24 and 48 hours. At all the time points, caveolin-1 and PTEN expression were gradually reduced, whereas, α-SMA was gradually increased. We also observed that cell viability was increased at 12 and 24 h after hypoxia then lightly decreased at 48 h. Additionally, disruption of caveolae with methyl-β-cyclodextrin (MβCD) enhanced p-Akt and α-SMA expression and fibroblast proliferation and phenotypic switch.

Conclusions

These findings suggest a key role for caveolae, perhaps through the caveolin-1/PTEN signaling pathway, in cardiac fibroblast proliferation and phenotypic switch under hypoxia.     

Altering β-cell number through stable alteration of miR-21 and miR-34a expression

Aim: An insufficient functional β-cell mass is a prerequisite to develop diabetes. Thus, means to protect or restore β-cell mass are important goals in diabetes research. Inflammation and proinflammatory cytokines play important roles in β-cell dysfunction and death, and recent data show that 2 miRNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed miR-21 and knocked down miR-34a, and investigated essential cellular processes.   Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a increased net β-cell number in the absence of cytokines, and reduced apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was decreased upon miR-34a knockdown. Conclusion: As overexpression of miR-21 increased proliferation, but also apoptosis and NO synthesis, the potential of miR-21 as a therapeutic agent to increase β-cell survival is doubtful. Knockdown of miR-34a slightly decreased proliferation, but as apoptosis and NO synthesis were highly reduced, miR-34a may be further investigated as a therapeutic target to reduce β-cell death and dysfunction.     

Biological and Prognostic Significance of the Morphological Types and Vascular Patterns in Colorectal Liver Metastases (CRLM): Looking Beyond the Tumor Margin

Patients with encapsulated colorectal liver metastases (CRLM) have a better prognosis than those without a capsule. The reason for the encapsulation is unknown. Hypoxia inducible factor-1α (HIF-1α) increases tumor angiogenesis and tumor tissue expression is associated with reduced survival. Our aim was to determine whether the good prognosis of encapsulated CRLM is associated with reduced HIF-1α expression by the cancer.The study selected only patients who had not undergone neoadjuvant chemotherapy prior to a potentially curative hepatectomy for CRLM. From 30 selected patients, serial sections were cut from a single randomly selected metastasis. Morphology was assessed following H&E staining. Tumor hypoxia, vascular endothelial growth factor (VEGF), proliferation, and microvascular density (MVD) were assessed by immunostaining for HIF-1α and carbonic anhydrase-9 (CA-9), VEGF, Ki67, and cluster of differentiation-31, respectively. MVD was calculated in the vascular hot spots. Pathology was reported without clinical outcome information. Actual long-term survival was recorded.Thirteen (43%) of the cancers were encapsulated CRLM containing glands which were large, complex, and cribriform. Thirteen (43%) were infiltrative CRLM and their glands were small, closely packed, and rounded with vessels in the interglandular fibrous tissue with no capsule; 3 (10%) had a mixed picture. Encapsulated CRLM had a higher expression of HIF-1α (58% vs 8%, P = 0.03), CA-9 (42% vs 0%, P = 0.04), and VEGF (92% vs 25%, P = 0.02). MVD was lower in the encapsulated CRLM group (37 mm² vs 143 mm², P < 0.001). The median follow-up was 115 months. The encapsulated CRLM group had a better overall and 5-year survival (relative hazard: 0.58, P = 0.057 and hazard ratio: 0.52, P = 0.044).There are 2 main morphological appearances of CRLM which have very different long-term survival following liver resection surgery. The morphology is associated with differences in expression of HIF-1α, CA-9, VEGF, and angiogenesis.     

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α–, but not HIF-1α–, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.     

miR-206在大鼠肺动脉高压模型中的表达和意义

目的 探讨miR-206在大鼠肺动脉高压（pulmonary hypertension，PH）模型中的表达和意义。 方法 构建肺动脉高压大鼠模型，按缺氧暴露时间分别标记为(1、7、14、21和28 d)组，并将常压常氧（FIO₂为0.21）作为对照组（n = 5），提取大鼠右下肺叶组织及外周血，实时定量PCR检测miR-206表达；分离大鼠原代肺动脉平滑肌细胞（pulmonary arterial smooth muscle cell，PASMC），以miR-206 mimics转染细胞并进行缺氧暴露，检测细胞增殖能力。生物信息学分析发现并选择缺氧诱导因子（hypoxia-inducible factor，HIF）-1α作为miR-206的候选靶基因，以双荧光素酶报告实验验证miR-206是否可直接调控HIF-1α，共转染miR-206mimics与HIF-1α过表达质粒，检测PASMC细胞增殖能力变化。 结果 与对照组相比，大鼠肺组织中miR-206的表达在缺氧暴露后即可出现miR-206表达降低（P < 0.05），大鼠血清miR-206亦明显降低（P < 0.05）；缺氧暴露后的大鼠肺组织分离培养的PASMC miR-206表达与对照组相比也明显降低（P < 0.05），miR-206低表达时PASMC增殖能力明显增加（P < 0.05）；miR-206 mimics转染可拮抗因缺氧暴露引起的PASMC增殖能力增加（P < 0.05）。生物信息学发现HIF-1α的3’非编码区（3’ untranslated region，3'UTR）具有miR-206的结合位点，用miR-206和HIF-1α（野生型3'UTR）共转染的PASMC中荧光素酶报告基因活性较对照组显著下调（P < 0.05），而miR-206 mimics和HIF-1α（突变型3'UTR）共转染组则较对照组无明显统计学差异。共转染miR-206 mimics后，HIF-1α上调引起的细胞增殖被逆转（P < 0.05）。 结论 缺氧诱导的miR-206下调通过靶向PASMC中的HIF-1α途径来促进PH，miR-206可能是缺氧诱导PH早期的触发因素。     

Hypoxia-inducible factor-1αPro-582-Ser polymorphism prevents iron deprivation in healthy blood donors

Background

Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α^Pro-582-Ser variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α^Pro-582-Ser polymorphism might influence the response to repeated blood withdrawals.

Materials and methods

Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α^Pro-582-Ser in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene.

Results

We found that male carriers of the HIF-1α^Pro-582-Ser polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α^Pro-582-Ser polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity.

Discussion

These findings show for the first time that the HIF-1α^Pro-582-Ser polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α^Pro-582-Ser polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.