Fluoxetine protects against monocrotaline-induced pulmonary arterial remodeling by inhibition of hypoxia-inducible factor-1α and vascular endothelial growth factor

The selective serotonin re-uptake inhibitor fluoxetine has been shown to protect against monocrotaline (MCT)-induced pulmonary hypertension in rats. To investigate the possible role of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in mediating this protective effect, MCT-treated rats were administered fluoxetine by gavage, at doses of 2?mg/kg body mass or 10?mg/kg once daily for 3 weeks. Changes in pulmonary hemodynamic parameters, pulmonary artery morphologies, and expressions of HIF-1α and VEGF were assessed. Fluoxetine at the 10?mg/kg dose, but not at the 2?mg/kg dose, attenuated the effects of MCT on pulmonary artery pressure, right ventricle index, and medial wall thickness. In addition, 10?mg/kg fluoxetine mitigated the MCT-induced up-regulation of HIF-1α and VEGF protein and reactive oxygen species (ROS) in the lungs. This dosage also decreased pERK1/2 levels and inhibited proliferation of pulmonary arterial smooth muscle cells in MCT-treated rats. In conclusion, fluoxetine can protect against MCT-induced pulmonary arterial remodeling, which linked to reduced ROS generation and decreased HIF-1α and VEGF protein levels via the ERK1/2 phosphorylation pathway.     

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.     

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1α and vascular endothelial growth factor in pressure-overloaded rat heart

Summary The use of -blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1 (HIF-1) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1 in cardiac hypertrophy due to pressure overload and after treatment with -blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1 expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor- (NGF-) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1 and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1, VEGF and BNP increased and mRNA of NGF- decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1, VEGF, BNP, and NGF- to the baseline values. Increased immunohistochemical labeling of HIF-1, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF- mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1,VEGF, BNP, and NGF- in the hypertrophic myocardium.     

High temperature requirement factor A1 (HTRA1) gene regulates angiogenesis through transforming growth factor-β family member growth differentiation factor 6

Genome-wide association study (GWAS) has identified genetic variants in the promoter region of the high temperature requirement factor A1 (HTRA1) gene associated with age-related macular degeneration (AMD). As a secreted serine protease, HTRA1 has been reported to interact with members of the transforming growth factor-β (TGF-β) family and regulate their signaling pathways. Growth differentiation factor 6 (GDF6), a member of the TGF-β family, is involved in ectoderm patterning and eye development. Mutations in GDF6 have been associated with abnormal eye development that may result in microphthalmia and anophthalmia. In this report, we identified a single nucleotide polymorphism (SNP) rs6982567 A/G near the GDF6 gene that is significantly associated with AMD (p value = 3.54 × 10(-8)). We demonstrated that the GDF6 AMD risk allele (rs6982567 A) is associated with decreased expression of the GDF6 and increased expression of HTRA1. Similarly, the HTRA1 AMD risk allele (rs10490924 T) is associated with decreased GDF6 and increased HTRA1 expression. We observed decreased vascular development in the retina and significant up-regulation of GDF6 gene in the RPE layer, retinal and brain tissues in HTRA1 knock-out (htra1(-/-)) mice as compared with the wild-type counterparts. Furthermore, we showed enhanced SMAD signaling in htra1(-/-) mice. Our data suggests a critical role of HTRA1 in the regulation of angiogenesis via TGF-β signaling and identified GDF6 as a novel disease gene for AMD.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

Overexpressed homeobox B9 regulates oncogenic activities by transforming growth factor-β1 in gliomas

Glioma is the leading cause of deaths related to tumors in the central nervous system. The mechanisms of gliomagenesis remain elusive to date. Homeobox B9 (HOXB9) has a crucial function in the regulation of gene expression and cell survival, but its functions in glioma formation and development have yet to be elucidated. This study showed that HOXB9 expression in glioma tissues was significantly higher than that in nontumor tissues. Higher HOXB9 expression was also significantly associated with advanced clinical stage in glioma patients. HOXB9 overexpression stimulated the proliferation, migration, and sphere formation of glioma cells, whereas HOXB9 knockdown elicited an opposite effect. HOXB9 overexpression also increased the tumorigenicity of glioma cells in vivo. Moreover, the activation of transforming growth factor-β1 contributed to HOXB9-induced oncogenic activities. HOXB9 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in glioma for diagnosis or prognosis.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α in asthenozoospermia

Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.     

Autocrine modulation of glucose transporter SGLT2 by IL-6 and TNF-α in LLC-PK1 cells

We determined in cultured kidney epithelial cells (LLC-PK(1)) the effects of high glucose, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) on mRNA and protein expression of the renal glucose transporters SGLT1 and SGLT2. Cultured monolayers were incubated with similar concentrations of IL-6 and TNF-α to those produced by LLC-PK(1) in the presence of 20 mM glucose. Confluent monolayers with either 5 (controls, C) or 20 mM glucose (high glucose, HG) were incubated in the presence of 5 mM glucose, 20 mM glucose, 10 pg/ml IL-6, or TNF-α alone or in combination. Separate groups with IL-6 and TNF-α were incubated with antibodies to their respective receptors. HG induced an increased SGLT1 mRNA at 48 h (p<0.05 vs. C) and protein expression in 120 h (p<0.05 vs. C). HG also induced an increased SGLT2 mRNA at 72 and 96 h (P<0.05 vs. C) and SGLT2 protein expression at 120 h (p<0.05 vs. C). In C, 10 pg/ml IL-6 or TNF-α did not modify SGLT1 mRNA (n.s vs. in the absence of cytokines). In contrast, cytokines induced an increased expression of SGLT1 protein at 120 h (p<0.05 vs. in the absence of cytokines), and SGLT2 mRNA and protein were increased at 96 and 120 h, respectively (p<0.05 vs. in absence of cytokines). No changes were observed when cells were incubated with cytokines and HG (n.s vs. C). In conclusion, this study showed that SGLT2 increased in the presence of IL-6 and TNF-α, indicating an autocrine modulation of the expression of this transporter by cytokines.     

Inflammation stimulates hypoxia-inducible factor-1α regulatory activity in 3T3-L1 adipocytes with conditioned medium from lipopolysaccharide-activated RAW 264.7 macrophages

Obesity is a multifactorial, chronic, inflammatory disease that involves different processes, such as adipose tissue hypoxia. The aim of the current study was to characterize the effects of conditioned medium (CM) from lipopolysaccharide (LPS)-activated macrophages on the regulation of hypoxia-inducible factor 1α (HIF-1α)-related genes in murine adipocytes. For the in vitro analyses, 3T3-L1 murine adipocytes (9 days postdifferentiation) were incubated either in CM (25% medium of RAW 264.7 murine macrophages with 24 hr 500 ng/ml LPS), LPS at 500 ng/ml, or hypoxia (Hx; 1% O₂, 94% N₂, 5% CO₂) for 24 hr. For the in vivo experiments, mice were fed a high-fat diet. Both epididymal white adipose tissue (eWAT) and adipocytes in CM showed upregulation of Glut1, Mcp1, Il10, Tnf, and Il1b. The secretion of IL-6, TNF-α, and MCP-1 was also increased in CM-treated adipocytes. Moreover, increased levels of HIF-1α subunit and nuclear factor kappa B p65 were found after CM treatment, linking Hx, and inflammation. HIF-1α directly bound vascular endothelial growth factor A (Vegfa) and uncoupling protein 2 (Ucp2) genes, up- and downregulating its expression, respectively. Furthermore, the oxygen consumption rate was 30% lower in CM. The siRNA knockdown of mammalian target of rapamycin (Mtor) reversed the induction of HIF-1α found in CM. The macrophage infiltration simulated through CM seems to be a similar environment to an abnormally enlarged eWAT. We have evidenced that HIF-1α plays a regulatory role in the expression of Vegfa and Ucp2 in CM. Finally, the inhibition of the mTOR pathway prevented the HIF-1α activation induced by CM. The involvement of HIF-1α under proinflammatory conditions provides insight into the origins of Hx in obesity.