Target Gene Vascular Endothelial Growth Factor Research Articles

CRISPR-dcas9 Optogenetic Nanosystem for the Blue Light-Mediated Treatment of Neovascular Lesions.

Vascular endothelial growth factor (VEGF) is the key regulator in neovascular lesions. The anti-VEGF injection is a major way to relieve retinal neovascularization and treat these diseases. However, current anti-VEGF therapeutics show significant drawbacks. The reason is the inability to effectively control its therapeutic effect. Therefore, how to controllably inhibit the VEGF target is a key point for preventing angiogenesis. Here, a CRISPR-dCas9 optogenetic nanosystem was designed for the precise regulation of pathologic neovascularization. This system is composed of a light-controlled regulatory component and transcription inhibition component. They work together to controllably and effectively inhibit the target gene's VEGF. The opto-CRISPR nanosystem achieved precise regulation according to individual differences, whereby the expression and interaction of gene was activated by light. The following representative model laser-induced choroid neovascularization and oxygen-induced retinopathy were taken as examples to verify the effect of this nanosystem. The results showed that the opto-CRISPR nanosystem was more efficacious in the light control group (NV area effectively reduced by 41.54%) than in the dark control group without light treatment. This strategy for the CRISPR-optogenetic gene nanosystem led to the development of approaches for treating severe eye diseases. Besides, any target gene of interest can be designed by merely replacing the guide RNA sequences in this system, which provided a method for light-controlled gene transcriptional repression.

HIF-1α and VEGF Are Involved in Deferoxamine-Ameliorated Traumatic Brain Injury

BackgroundDeferoxamine (DFX) has been reported to have neuroprotective effect. This study aimed to investigate the neuroprotective effect of DFX and its effect on hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in rats after traumatic brain injury (TBI). Materials and methodsRats were randomly divided into sham operation, TBI + DFX, and TBI + vehicle groups. The rats in the TBI + DFX group were intraperitoneally injected with DFX 2 and 6 h after injury, thereafter once every 12 h. The rats in the TBI + vehicle group were intraperitoneally injected with saline at the same time points. At 6, 12, 24, and 48 h after TBI, 6 rats in each group were euthanized, and the brains were harvested. The expression of HIF-1α and VEGF in the pericontusional area was detected using real-time polymerase chain reaction and Western blot analysis. TBI-induced apoptosis was investigated using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Three days after TBI, the density of microvessels was examined via immunohistochemical staining. ResultsDFX treatment upregulated the expression of HIF-1α and VEGF after TBI. DFX treatment reduced apoptosis and improved the neurobehavioral score after TBI. The density of microvessels was higher in the TBI + DFX group than that in the TBI + vehicle group 3 d after TBI. ConclusionsDFX can stimulate angiogenesis, inhibit apoptosis, and play a protective role after TBI. The protective effect of DFX may, at least in part, be through upregulating the expression of HIF-1α and its downstream target gene VEGF.

Opposite effects of demethylating treatment on VEGFA and HIF1A expression in MDA-MB-231 breast cancer cell line in hypoxic microenvironment

Hypoxic microenvironment in breast tumors has been associated with tumor aggressiveness, therapy resistance, and worse prognosis. HIF-1α (hypoxia inducible factor-1α) plays important roles in hypoxia by regulating a set of genes, such as VEGFA (vascular endothelial growth factor), which is related to angiogenesis. Studies have shown that DNA methylation acts on the gene control of HIF1A in hypoxia. However, little is known about the HIF1A transcriptional regulation in aggressive breast cancer MDA-MB-231 cell line. Therefore, the present study analyzed the effect of a demethylating agent on the transcription levels of HIF1A, and its target gene VEGFA, in the MDA-MB-231 cell line, in hypoxia. Herein, we demonstrated that 5′-aza-2′-deoxycitidine treatment in hypoxia reverted and raised HIF1A mRNA levels, but decreased VEGFA mRNA levels. Additionally, in both normoxia and hypoxia, HIF-1α protein levels increased in response to the demethylating treatment. The results of the current study indicated that HIF1A could be regulated by DNA methylation in normoxia and hypoxia. Furthermore, our results indicated a distinct regulation of VEGFA transcripts, independent of HIF-1α, in 5′-aza-dC-treated MDA-MB-231 cells in hypoxia.

Effects of EGCG on proliferation and apoptosis of gastric cancer SGC7901 cells via down-regulation of HIF-1α and VEGF under a hypoxic state.

To investigate the effects of epigallocatechin-3-gallate (EGCG) on proliferation and apoptosis of human gastric cancer SGC7901 cells under a hypoxic state. Human gastric cancer SGC7901 cells were sub-cultured, and the cobalt chloride (CoCl2) hypoxia model was established. The blank control group (normoxia group), hypoxia control group (hypoxia group) and hypoxia + different concentrations of EGCG subgroups (20, 40, 60, 80, 100 μg/mL EGCG) were set up. Cell viability was detected via methyl thiazolyl tetrazolium (MTT) assay, apoptosis was detected via flow cytometry, and expressions of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Relatively low concentrations of EGCG (20-80 μg/mL) presented no significant inhibiting effect on SGC7901 cell growth within a short time (24 h) (p>0.05). The increasing concentration of EGCG inhibited cell proliferation under a hypoxia state (p<0.05). EGCG induced apoptosis in a dose-dependent manner under hypoxia (p<0.05). EGCG could significantly impede expressions of HIF-1α and VEGF proteins (p<0.05), and down-regulate the level of VEGF mRNA (p<0.05), but it showed no significant effect on the HIF-1α mRNA expression (p>0.05). EGCG inhibited cell proliferation under hypoxia via the downregulation of HIF-1α and its downstream target gene VEGF levels, providing a theoretical basis for the early diagnosis and treatment of gastric cancer in clinic.

Syntenin promotes VEGF-induced VEGFR2 endocytosis and angiogenesis by increasing ephrin-B2 function in endothelial cells.

Syntenin, a tandem PDZ-domain-containing scaffold protein, is involved in the regulation of diverse biological functions, including protein trafficking, exosome biogenesis, and cancer metastasis. Here, we present the first study to explore the significance of syntenin in endothelial cells. Syntenin knockdown in human umbilical vein endothelial cells (HUVECs) impaired vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, vascular permeability, and nitric oxide (NO) production. Syntenin knockdown also suppressed expression of the VEGFR2 target genes VEGF, MMP2, and Nurr77 as well as VEGF-induced angiogenesis in vitro and in vivo. And it decreased cell-surface levels of ephrin-B2. Biochemical analyses revealed that syntenin exists in complex with VEGFR2 and ephrin-B2. Syntenin knockdown abolished the association between VEGFR2 and ephrin-B2, suggesting syntenin functions as a scaffold protein facilitating their association in HUVECs. Consistent with these observations, knocking down syntenin or ephrin-B2 abolished VEGF-induced endocytosis and VEGFR2 phosphorylation and activation of its downstream signaling molecules. Treatment with MG132, a proteasome inhibitor, rescued the downregulation of ephrin-B2 and VEGFR2 signaling induced by syntenin knockdown. These findings demonstrate that syntenin promotes VEGF signaling and, through its PDZ-dependent interaction with ephrin-B2, enhances VEGF-mediated VEGFR2 endocytosis and subsequent downstream signaling and angiogenesis in endothelial cells.

Expression of hypoxia-inducible factor 1α, vascular endothelial growth factor and protein kinase B in lichen planus lesions

Objective To explore relationships of expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and protein kinase B (P-Akt) with angiopoiesis and cell apoptosis. Methods Biopsy specimens were collected from skin lesions of 32 patients with lichen planus and normal skin of 20 patients with lipomyoma, and subjected to paraffin embedding. Immunohistochemical staining was performed to measure expression of HIF-1α, VEGF and P-Akt, and TUNEL technique was used to detect apoptosis of keratinocytes in these paraffin-embedded tissue sections. Microvessel density (MVD) was assessed by counting CD34-labeled vascular endothelial cells. Results HIF-1α, VEGF and P-Akt were moderately or strongly expressed in lichen planus lesions, but absent or weakly expressed in normal skin of controls, and the expression of HIF-1α, VEGF and P-Akt was significantly higher in the lichen planus group than in the control group (all P < 0.01). HIF-1α was mainly expressed in nuclei of keratinocytes, while VEGF and P-Akt were expressed in the cytoplasm of keratinocytes. In addition, the lichen planus group showed significantly increased MVD (21.27 ± 6.54 vs. 10.26 ± 1.10 microvessels/high-power (200 × ) field, t = 5.607, P < 0.01) and apoptosis rate of keratinocytes (72.81% ± 9.234% vs. 28.16% ± 3.464%, t = 8.431, P < 0.01) compared with the control group. Pearson correlation analysis showed that there were positive correlations between HIF-1α and VEGF expression, between VEGF and P-Akt expression, and between P-Akt and HIF-1α expression in the lichen planus group (r = 0.625, 0.453, 0.455, respectively, all P < 0.01), and expression of HIF-1α, VEGF and P-Akt was all positively correlated with MVD (r = 0.721, 0.646, 0.671, respectively, all P < 0.01). Conclusion HIF-1α and its downstream target genes VEGF and P-Akt may play a certain role in the occurrence of lichen planus. Key words: Lichen planus; Hypoxia-inducible factor 1, alpha subunit; Vascular endothelial growth factors; Protein kinases; Apoptosis

Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-κB activation in PDL cells and periodontal diseases.

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1β, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1β, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1β, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

Hedgehog inhibition reduces angiogenesis by downregulation of tumoral VEGF-A expression in hepatocellular carcinoma.

Dysregulation and activation of Hedgehog (Hh) signalling may contribute to tumorigenesis, angiogenesis, and metastatic seeding in several solid tumours. We investigated the impact of Hh inhibition on tumour growth and angiogenesis using in-vitro and in-vivo models of hepatocellular carcinoma (HCC). The effect of the Hh pathway inhibitor GDC-0449 on tumour growth was investigated using an orthotopic rat model. Effects on angiogenesis were determined by immunohistochemical staining of von Willebrand factor antigen and by assessing the mRNA expression of several angiogenic factors. Invitro, HCC cell lines were treated with GDC-0449 and evaluated for viability and expression of vascular endothelial growth factor (VEGF). Endothelial cells were evaluated for viability, migration, and tube formation. In the orthotopic HCC model, GDC-0449 significantly decreased tumoral VEGF expression which was accompanied by a significant reduction of microvessel density and tumour growth. In HCC cells, GDC-0449 had no effect on cell growth but significantly reduced target gene regulation and VEGF expression while having no direct effect on endothelial cell viability, migration, and tube formation. Hh inhibition with GDC-0449 downregulates tumoral VEGF production invitro and reduces tumoral VEGF expression, angiogenesis, and tumour growth in an orthotopic HCC model.

Detection of protein secretion and expression and osteogenic activity of adipose tissue-derived stem cells after in vitro transfection of vascular endothelial growth factor

To examine vascular endothelial growth factor (VEGF) protein secretion and expression and explore the osteogenic activity of adipose tissue-derived stem cells (ADSCs) after transfection of human VEGF. The ADSCs were isolated from human adipose tissue after the digestion of collagenase.After identification by flow cytometry, the cells were cultured and passaged in nutritive medium. Gene sequence encoding human VEGF mature peptide was obtained by Trizol reagent method from human vascular tissue. Target gene VEGF was connected with bicistronic expression vector containing green fluorescent protein to form pSELECT-GFP zeo-VEGF for transfecting 2nd, 3rd, 4th, 5th generation ADSCs mediated by liposome. The transfection results were verified under fluorescence microscope. VEGF protein secretion by transfected cells was detected by enzyme-linked immunosorbent assay (ELISA). Second-generation transfected ADSCs were cultured under osteogenic conditions.The supernatant levels of alkaline phosphatase (ALP) and osteocalcin (OC) were detected. Liposome-mediated VEGF target gene fragment could transfect ADSCs successfully. ELISA quantitative detection showed that VEGF mRNA expression levels in supernatant of the transfected group was significantly higher than the control group. And there were significant differences. After osteogenic culturing, the detections of ELISA, real-time PCR and Western blot showed that the secretion of ALP and OC of VEGF transfected group was significantly higher than that of empty vector transfected and blank cell groups.And there were significant differences (P < 0.01). After transfected by liposome-mediated VEGF target gene fragment, human ADSCs can express biologically active VEGF mRNA in vitro continuously and effectively. Directional differentiation capacity of transfected ADSCs is significantly enhanced.

C-myc but not Hif-1α-dependent downregulation of VEGF influences the proliferation and differentiation of HL-60 cells induced by ATRA

Vascular endothelial growth factor (VEGF) plays an important role in solid tumor growth, progression and metastasis as well as in the proliferation and differentiation of hematological malignancies. However, the molecular mechanism that modulates VEGF expression and secretion in leukemia cells has not yet to be elucidated. The purpose of the present study was to investigate the role of the signal pathway in modulating the expression of VEGF in HL-60 cells. Specific siRNAs targeting VEGF were transfected into HL-60 cells and the VEGF expression was measured with reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. The cell proliferation of HL-60 cells was detected by the cell counting kit-8 (CCK-8) assay and the differentiation of HL-60 cells induced by all-trans-retinoic acid (ATRA) was detected by the RT-PCR assay and flow cytometry assay for CD11b. The upstream transcription factors that were related to VEGF expression such as P53, SP-1, c-jun, VHL, cox-2, c-myc and stat3 were detected by RT-PCR assay. In addition, the chromatin immunoprecipitation (ChIP) assay was used to reveal the role of c-myc by binding the target gene VEGF. The results demonstrated the hypoxia-inducible factor1α-related signaling pathway, not the same as in solid tumors, might not play a key role in modulating VEGF expression. c-myc contributes to the modulation of VEGF expression by targeting the promoter of VEGF, which was indicated by the ChIP assay. In conclusion, our data demonstrate that VEGF plays an important role in the differentiation and proliferation of HL-60 cells; c-myc-dependent downregulation of VEGF induced by ATRA contributes to the differentiation of HL-60 cells.

Transient hypoxia‐inducible factor activation in rat renal ablation and reduced fibrosis with L‐mimosine

Hypoxia-inducible factor (HIF) activity during the course of chronic kidney disease (CKD) development is poorly defined, and the effect of HIF activation on CKD is still controversial. The purpose of the present study was to characterize HIF expression during the course of CKD development, and to investigate the effect of HIF activation on CKD by using prolyl hydroxylase (PHD) inhibitor L-mimosine. Rats with remnant kidneys (RK) were killed at week 1, 2, 4, 6, 8, 12 after subtotal nephrectomy. An additional group of RK rats was treated with L-mimosine to study the effect of HIF-α activation. Tubulointerstitial hypoxia in the remnant kidney began at week 1 and continued, albeit attenuated, until week 12, the last time point examined. The nuclear expression of HIF-1α and HIF-2α, as well as typical HIF target genes VEGF (vascular endothelial growth factor), HO-1 (heme oxygenase-1), GLUT-1 (glucose transporter-1) and EPO (erythropoietin), were all upregulated in the early stage of RK when renal function was stable, and returned to the basal level later, accompanied by impaired renal function and interstitial fibrosis. L-mimosine administered from week 5 to week 12 led to accumulation of HIF-1α and HIF-2α proteins, increased expression of VEGF, HO-1 and GLUT-1, and improved renal function. Furthermore, fibrosis markers α-smooth muscle actin (α-SMA) and Collagen III, as well as peritubular capillary rarefaction index, were all significantly decreased after L-mimosine treatment. There was a transient HIF-α activation in the remnant kidney of rats at the early stage following subtotal nephrectomy. L-mimosine administered in later stages re-activated HIF-α and reduced tubulointerstitial fibrosis.

The novel peptide F29 facilitates the DNA-binding ability of hypoxia-inducible factor-1α

Hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta) is a heterodimeric transcriptional activator that mediates gene expression in response to hypoxia. HIF-1alpha has been noted as an effective therapeutic target for ischemic diseases such as myocardiac infarction, stroke and cancer. By using a yeast two-hybrid system and a random peptide library, we found a 16-mer peptide named F29 that directly interacts with the bHLH-PAS domain of HIF-1alpha. We found that F29 facilitates the interaction of the HIF-1alpha/beta heterodimer with its target DNA sequence, hypoxia-responsive element (HRE). The transient transfection of an F29-expressing plasmid increases the expression of both an HRE-driven luciferase gene and the endogenous HIF-1 target gene, vascular endothelial growth factor (VEGF). Taken together, we conclude that F29 increases the DNA-binding ability of HIF-1alpha, leading to increased expression of its target gene VEGF. Our results suggest that F29 can be a lead compound that directly targets HIF-1alpha and increases its activity.

Hypoxia-Inducible Factor-1 in Multiple Myeloma Progression.

Abstract 1812Poster Board I-838Multiple Myeloma (MM) is a malignancy of immunoglobulin (Ig)-synthesizing plasma cells, that home to and expand in the bone marrow. Similarly to other tumours its development is correlated to the formation of regions of hypoxia, which may be a prognostic indicator and determinant of malignant progression. It is known how in solid tumours the degree of intra-tumoral hypoxia is positively correlated with the expression of the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 is composed of HIF-1á and HIF-1β subunits and its production has been identified as a key element in allowing cells to adapt and survive in a hostile hypoxic environment via a variety of pathways. In hypoxia conditions, the HIF-1á subunit becomes stable and regulates the expression of target genes. When activated HIF-1á also targets those genes which are required for angiogenesis, the development of new blood vessels from an existing vascular network. Angiogenesis represents a constant hallmark of MM progression. In response to hypoxia plasma cells and stromal cells (endothelial cells [ECs], macrophages, mast cells) within the tumour express Vascular Endothelial Growth Factor (VEGF), a mitogen and survival factor specific for endothelial cells. VEGF is the major regulator of tumor-associated angiogenesis. HIF-1á directly activates transcription of the VEGF gene and this leads to autocrine signal transduction that is critical for angiogenesis.In this study we demonstrate the role of HIF-1á in MM angiogenesis. The constitutive stabilization of HIF-1á contributes to increase angiogenesis in MM. Our data show that HIF-1á is stabilized in the nucleus of MM endothelial cells (MMECs) but not in ECs of Monoclonal Gammopathies of Undetermined Significance (MGECs) and in Human Umbilical Vein ECs (HUVECs) used as controls. Western Blot and Enzyme-Linked Immunosorbent Assay (ELISA) analyses show the overexpression of HIF-1á and the proteic products of its target genes VEGF and VEGF Receptor (VEGFR)-1, in patients with relapsed disease and in MM progression but not in patients with nonactive MM (avascular phase). Moreover, immunofluorescent staining confirm the nuclear stabilization of HIF-1á in MMECs.At mRNA level all ECs express same quantity of HIF-1á mRNA, as confirmed by RT-PCR and Real-time RT-PCR, indicating that in MMECs the post-trascriptional control is affected. Finally, we show that the inhibition of HIF-1á by siRNA suppresses vessel formation in vitro and promote ECs apoptosis. Our findings indicate that HIF-1á plays an important role in MM progression and that it is correlated to the angiogenic switch from nonactive MM to active MM. Furthermore these data suggest that HIF-1á may represent a target for the MM antiangiogenic treatment. DisclosuresNo relevant conflicts of interest to declare.

Expression of HIF-1α, VEGF and EPO in peripheral blood from patients with two cardiac abnormalities associated with hypoxia

Objectives HIF-1α (hypoxia-inducible factor-1 alpha) mediates the responses of mammalian cells to hypoxia/ischemia by inducing the expression of adaptive gene products (e.g., vascular endothelial growth factor (VEGF) and erythropoietin (EPO)). Persistent pulmonary hypertension of the newborn (PPHN) and cyanotic congenital heart disease (CCHD) are common neonatal diseases considered as paradigms of hypoxemia. Since the expression HIF-1α, VEGF and EPO in newborns diagnosed with these diseases has yet to be studied, we set out to define the expression of these genes in peripheral blood from newborn infants diagnosed with PPHN and CCHD. Design and methods The mRNA transcripts encoding HIF-1α, VEGF and EPO were measured by RT-PCR in healthy newborn infants and infants diagnosed with PPHN and CCHD. Results An important increase in HIF-1α expression was observed in both pathological conditions, accompanied by significant increases in VEGF and EPO expression when compared to healthy infants. Conclusions HIF-1α mRNA expression increases in newborn infants with PPHN or CCHD, as does the expression of its target genes VEGF and EPO.

Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy

Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1α (hypoxia inducible factor-1α) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1α and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1α was assessed by quantitative RT-PCR and protein levels of HIF-1α and VEGF were assessed by western blot. HIF-1α DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48 h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1α and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1α and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1α activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1α and VEGF induced by partial hepatectomy alone.

Dexamethasone impairs hypoxia-inducible factor-1 function

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of α- and β-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1α levels in the cytosol of HepG2 cells, while nuclear HIF-1α levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

The involvement of phosphoinositid 3-kinase/Akt pathway in the activation of hypoxia-inducible factor-1α in the developing rat brain after hypoxia–ischemia

Hypoxia inducible transcription factor (HIF)-1α plays an important role in maintaining oxygen homeostasis. However, the pathways involved in the regulation of HIF-1α are not clear. Since phosphoinositid 3-kinase/Akt (PI3K/Akt) pathway has been shown to be a common pathway involved in cell signaling, we therefore hypothesized that PI3K/Akt pathway is involved in the regulation of HIF-1α in developing rat brain after hypoxia–ischemia (HI). To test this hypothesis, we subjected postnatal day 10 rats to HI by ligating common carotid artery followed by hypoxia. Rat brains were collected to detect the expression of HIF-1α and its target gene, vascular endothelial growth factor (VEGF), as well as PI3K/Akt using immunohistochemistry and Western blot analysis. We found that the expression of HIF-1α and VEGF was significantly upregulated and peaked at 8 h after HI compared with sham controls. However, the expression of p-Akt peaked at 4 h, earlier than that seen in HIF-1α expression. Furthermore, we found that HIF-1α and VEGF protein were significantly inhibited after blocking the PI3K/Akt pathway using a specific inhibitor, wortmannin. Our findings suggest that the PI3K/Akt pathway is involved in the regulation of HIF-1α and its target gene VEGF in the developing rat brain after HI.

Hif-1α Independent Down-Regulation of VEGF Expression in HL60 Cells after In Vitro Exposure to ATRA.

Background: Although the biologic effects of ATRA so far are well characterized, the molecular mechanisms regulating these processes are largely unknown. The expression of VEGF has been widely demonstrated in various human malignant tumors including haematological malignancies. Some results demonstrated that high expression of VEGF can promote the proliferation and colony formation and inhibit the apoptosis of leukemic cells, which promotes the progression of hematopoietic tumors not only by stimulating vascular endothelial growth. But the molecular mechanism of modulation of VEGF secretion and expression by ATRA in leukemia cells, for example HL60 cells, has not been elucidated clearly. The study was aimed to investigate the anti-leukemic mechanism of ATRA in respect of vascular endothelial growth factor (VEGF) production and which pathway contributed to the molecular mechanism of VEGF expression modulation.Methods: HL60 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, glutamine, and antibiotics in a humidified 5% CO2 in air atmosphere at 37°C. The MTT assay was performed to detect the proliferation of HL60 cells. Analysis of cell cycle phase distribution was analyzed using a FACScan (Becton Dickinson, Mountain View, CA) and the MacCycle software (CELLQUEST). The differentiation of HL60 cells after exposure to ATRA was assessed by NBT reduction assay and percent of CD11b positive cells. Total MMP2/MMP-9 enzyme-linked immunosorbent assay of secretion. Semi-quantitative RT-PCR and ELISA assay were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by ATRA respectively. ChIP assay was taken to detect the binding of c-Myc on target gene VEGF.Results: The results showed that the proliferation of HL60 cells was obviously inhibited with the time and density dependent. The differentiation was indicated by the up-regulation of NBT positive and CD11b positive cells. The expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells, which could be down-regulated by exposure to ATRA for 48 and 72 hours. Level of Metalloproteinase MMP9 but not MMP2 in the culture supernatant of HL60 cells was correlated with VEGF secretion, which was demonstrated by the special inhibition of MMP inhibitor batimastat (BB-94) or ATRA treatment. It indicated that MMP9 played a role in the release of biologically active VEGF. ATRA exerts its anti-leukemia effects by reduction of the expression of VEGF mRNA and VEGF protein secretion. The VEGF mRNA or c-Myc mRNA expression was down-regulated, otherwise, HIF-1α mRNA was up-regulated in HL-60 cells after exposure to ATRA. The antisense c-Myc could block the VEGF mRNA expression, but there is not obvious variation of c-Myc binding on promoter of target gene VEGF. The results indicated that the down-regulation of VEGF by ATRA was not Hif-1α dependent, otherwise it was related to the indirect pathway of c-Myc.Conclusions: c-Myc but not HIF-1α indirect pathway contributes to the down-regulation of VEGF expression in HL60 cells induced by ATRA.

The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects

The response to hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) which leads to the induction of a variety of adaptive gene products including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). This study was designed to test the hypothesis that HIF-1 and its target genes would be upregulated in the ventricular myocardium of infants with cyanotic congenital cardiac defects. 14 infants with cyanotic (n = 7) or acyanotic cardiac defects (n = 7) were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were studied for protein expression and DNA-binding activity. Protein levels of HIF-1alpha were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease and inversely correlated with the degree of hypoxemia. This response was accompanied by significantly enhanced HIF-1 DNA binding activity. Furthermore, protein levels of VEGF and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic infants. To test the potential involvement of upstream regulatory pathways, activation of MAP kinases was determined. Intramyocardial levels of phosphorylated p38 MAP kinase, but not of ERK1/2 were significantly higher in infants with cyanotic compared to those with acyanotic congenital heart disease and inversely correlated to hypoxemia. These findings show that chronic hypoxemia is associated with the induction and stabilization of the transcription factor HIF-1 as well as its target genes VEGF and eNOS in the myocardium of infants with cyanotic cardiac defects. Thus, stabilization of HIF-1 and induction of the adaptive hypoxia response could particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia.

The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects

The response to hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) which leads to the induction of a variety of adaptive gene products including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). This study was designed to test the hypothesis that HIF-1 and its target genes would be upregulated in the ventricular myocardium of infants with cyanotic congenital cardiac defects. 14 infants with cyanotic (n = 7) or acyanotic cardiac defects (n = 7) were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were studied for protein expression and DNA-binding activity. Protein levels of HIF-1α were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease and inversely correlated with the degree of hypoxemia. This response was accompanied by significantly enhanced HIF-1 DNA binding activity. Furthermore, protein levels of VEGF and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic infants. To test the potential involvement of upstream regulatory pathways, activation of MAP kinases was determined. Intramyocardial levels of phosphorylated p38 MAP kinase, but not of ERK1/2 were significantly higher in infants with cyanotic compared to those with acyanotic congenital heart disease and inversely correlated to hypoxemia. These findings show that chronic hypoxemia is associated with the induction and stabilization of the transcription factor HIF-1 as well as its target genes VEGF and eNOS in the myocardium of infants with cyanotic cardiac defects. Thus, stabilization of HIF-1 and induction of the adaptive hypoxia response could particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia.