A Comparative Study Of The Wild-type And Various Mutants Of HIF-1α On Angiogenesis In Vitro And The Role Of CBP/p300 And HDAC In HIF-1α-induced Angiogenic Genes Transcription

BackgroundThe concept of reconstituting the microvasculature as part of strategy for treating ischemic tissue (ie, therapeutic angiogenesis) evolved soon after pioneering work by Folkman and colleagues documented the existence of angiogenic growth factors. Recognition that therapies designed to repair the microcirculation may enhance cardiovascular function induced paradigm shift in treatment strategies for acute and chronic ischemia. The new microvascular strategies were first investigated through series of preclinical studies that evaluated recombinant growth factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF) in models of myocardial and lower-extremity ischemia; however, maintaining adequate levels of the recombinant protein in the target zone was technically challenging and considered prohibitively expensive. Accordingly, subsequent experiments evaluated the potential use of gene therapy for therapeutic angiogenesis. The first in-human studies of gene therapy for treatment of peripheral and coronary artery disease (PAD and CAD, respectively) were reported in the late 1990s. Since these initial reports, much has been learned about the mechanism of new blood vessel formation, and the safety of angiogenic gene therapy has been supported by substantial evidence.Fifteen years have elapsed since the earliest reports of angiogenic gene therapy in humans. The preclinical evidence demonstrating the bioactivity of transplanted genes and several early clinical trials indicating that gene therapy is safe, feasible, and potentially efficacious. Despite great expectations, randomized controlled clinical trials on therapeutic angiogenesis with the vascular endothelial growth factor (VEGF) and related molecules for the treatment of ischemic heart and peripheral arterial occlusive disease have not consistently produced conclusive evidence of benefit, implicating that it is difficult to complete the "angiogenic program" relying on a single growth factor, so alternative strategies to stimulate revascularization of ischemic tissues are required.Angiogenesis is an intricate multistep and temporally ordered process that involves a great number of genes, modifiers and pathways. Hypoxia-inducible factor-1αis a key nuclear factor, can directly or indirectly regulate more than 100 angiogenic genes to take part in neovascularization. In recent years, it has become clear that HIF-1 is a "master switch" and may play a central role in angiogenesis. HIF is a heterodimer that consists of one of the regulatable HIF-a subunit and the constitutively expressed HIF-1βsubunit. Upon activation by the hypoxic signal, HIF-αtranslocates to the nucleus, dimerizes with HIF-P, and induces the expression of its transcriptional targets. HIF-1αcontains a unique O-dependent degradation domain (ODDD) that mediates oxygen-dependent stability, and two transactivation domains:the N-terminal activation domain (NAD) and C-terminal activation domain (CAD) involving in regulating transcriptional activity. The stability and transcriptional activities of HIF-1αare precisely regulated by the intracellular oxygen concentration, however, HIF-1αis very unstable, low transcriptional activity and unable to induce functional mature neovascularization in normoxia. Under normoxic conditions, HIF-1αhas an exceptionally short half-life and steady-state levels are very low. The destruction of HIF-1αis regulated by a prolyl hydroxylase (termed PHD, prolyl hydroxylase domain) at two specific prolyl residues (Pro402 and Pro564 in human HIF-1α), which can interact independently with VHL E3; these interactions contribute to the extremely rapid proteolysis of HIF-1α. The transcriptional activity of HIF-1αis regulated by an asparaginyl hydroxylase (termed FIH-1, factor inhibiting HIF-1) and blocks the interaction of C-TAD with transcriptional co-activator CBP/p300 via hydroxylation of the asparagine residue (Asn803 in human HIF-1α) in normoxia. Under hypoxic conditions, PHD and FIH-1 are inactive; the lack of hydroxylation results in stable HIF-1α, which can bind with HIF-1βand recruit CBP/p300 at the C-TAD to enhance target angiogenic gene expression. Hence, according to the oxygen-dependent regulation mechanisms of HIF-la stability and transcriptional activity, target PHD or/and FIH-1 by mutation of either or both of Pro402 and Pro564 in HIF-1α, especially accompanying mutation of Asn803 in HIF-1α, could be a potential strategy for stabilizing HIF-1αand increasing HIF-1αtranscriptional activity for angiogenesis in normoxia.How many downstream target angiogenic genes HIF-la as a nuclear transcription factor can regulate depends on the interaction of HIF-la with its corresponding co-activators. The TAD function is critical dependent on transcriptional co-activator CREB-binding protein/adenovirus E1A-binding protein p300 (CBP/p300) to up-regulate HIF-la-induced gene transcription, as well as histone deacetylase (HDAC) is also essential for HIF-la-induced angiogenic gene transcription. Although HDAC is usually associated with transcriptional repression, it may also function as co-activator. It had been reported that mutant mice bearing deletions in the CHI domains (DeltaCHl) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CHI domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CHI domain was only required for an average of approximately 35-50% of global HIF-1-responsive gene expression, whereas another HIF transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) accounts for approximately 70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription. Nevertheless, is there any quantitative relationships between CBP/p300 or/and HDAC with HIF-la-induced angiogenic genes? How to play a role in these transcriptional processes? These problems need be further studied.ObjectWe sought to investigate the stability and transcriptional activity of the wild-type and various mutants of HIF-la, the influence of mRNA stability on itself induced angiogenic genes and the angiogenic potential in vitro under normoxic conditions, and hope to filter out the optimal HIF-la mutant for angiogenesis in vivo. We also wish to explore the rough quantitative relationships of CBP/p300 and HDAC with HIF-la-induced angiogenic genes. Methods1) Human lung micro vascular endothelial cells (HMVEC-Ls) were purchased from Clonetics and cultured using the optimized growth medium developed by Clonetics according to the manufacturer’s instructions. The cells were then viewed with a transmission electron microscope. Fluorescent staining analysis the specific antigen of HMVEC-Ls with the von Willebrand FactorⅧ(vWF) and platelet endothelial cell adhesion molecule (PECAM or CD31) antibodies.2) Recombinant adenovirus Ad-HIF-1α-Trip were produced by homologous recombination in HEK293 cells, amplified also in HEK293 cells on a large scale, and purified by ultracentrifugation in CsCl step gradient solutions. The viral particles were then viewed with a transmission electron microscope, the virus titres of the purified Ad-HIF-1α-Trip were determined by End-point Dilution Assay, and the transfection efficiency was assessed by X-gal staining. PCR assay was then used to identify the sequence integrity of the triple mutation of HIF-1αgene mediated by recombinant adenovirus.3) Expression of HIF-1αand HIF-1α-induced angiogenic genes (1) mRNA Level:HMVEC-Ls (8.0×103 cells/well in 96-well plate) maintained in EBM supplemented with 2% FBS (without SingleQuots) were treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip or Ad-Null (MOI=200) for 72 h. Cells were harvested and the mRNA levels of HIF-1α, VEGF, PLGF, PAI-1, PDGF, Ang-1, Ang-2, LEP, ACTB and HPRT-1 were measured by the QuantiGene analysis 2.0 (QGP-2) detection kit according to the manufacturer’s instructions. These luminescent signals were detected in an LMax (Molecular Devices, Mountain View, CA), which was previously described in detail. (2) Protein Level:HMVEC-Ls (5.0×103 cells/cm2 in 150-cm2 flask) maintained in lOmL EBM supplemented with 2% FBS (without SingleQuots) were treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip or Ad-Null (MOI-200) for 72 h. The cells were harvested and analyzed by Western blotting for HIF-1αexpression. The cell-culture supernatant was collected and assessed by ELISA for the protein levels of VEGF, PLGF, PDGF and Ang-2 according to the manufacturer’s instructions.4) The half-life of HIF-1αand the HIF-1α-induced angiogenic factor (VEGF, PLGF, PDGF and PAI-1) mRNA was determined by treating HMVEC-Ls with actinomycin D as described previously. Briefly, HMVEC-Ls (8.0×103 cells/well in 96-well plate) maintained in EBM supplemented with 2% FBS (without SingleQuots) were treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip or Ad-Null (MOI200) for 48 h, and then treated with actinomycin D (10μmol/L). Cells were collected sequentially for QGP-2 analysis at 0,1,2,4 or 6 hours after addition of actinomycin D, respectively. Data from QGP-2 were normalized and expressed as a percentage of the mRNA levels before actinomycin D was added.5) In vitro angiogenic study:(1) HMVEC-Ls proliferation was measured by MTS assay:HMVEC-Ls (2.0×103 cells/well in 96-well plate) maintained in EBM supplemented with 2% FBS (without SingleQuots) were treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803 or Ad-HIF-1α-Trip (MOI-200) or Ad-Null (the empty vector, MOI=200) for 7 consecutive days. Proliferation was measured at 490 nm by using CellTiter96 Cell Proliferation Assay kit, according to the manufacturer’s manual. (2) HMVEC-Ls migration was detected by using a wound-healing migration assay:HMVEC-Ls (1.0×104 cells/dish) maintained in EBM supplemented with 4% FBS and SingleQuots were cultured on 35-mm dishes (Corning Co.) for 72 h to form the fully confluent HMVEC-L monolayers. The monolayers were then scratched with a pipette tip and washed with phosphate-buffered saline to remove floating cellular debris, and then EBM without FBS was added into the wells that were treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip or Ad-Null (MOI=200) for 12 hours. Cell migration was photographed and measured using Image Pro Plus version 6.1(IPP 6.1). (3) Capillary tube formation was assessed by Matrigel assay:Growth factor-reduced Matrigel (50μL Matrigel/well, BD Bioscience) was pipetted into pre-chilled 96-well plates and polymerized for 45 min at 37℃. HMVEC-Ls (1.0×104 cells/well) were placed onto the layer of Matrigel and cultured in EBM without FBS under normoxic conditions, or subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip (MOI=200) with or without the specific inhibitor (anti-VEGF antibody,1μg/mL), or Ad-Null (MOI=200). Furthemore, The tube structures were visualized by light microscopy 8 hours later and analyzed using IPP 6.1. (4) The maturity of neovascularization was estimated by Permeability Assay:HMVEC-Ls (1.0×104 cells/well) maintained in EBM supplemented with 2% FBS (without SingleQuots) were cultured on fibronectin-coated 12-well transwell (0.4-μm pore size, Corning Co.) under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564, Ad-HIF-1α-564/402, Ad-HIF-1α-564/803, Ad-HIF-1α-Trip or Ad-Null (MOI=200) for 72 h to form full confluent HMVEC-L monolayers. Then FITC-dextran (1 mg/mL, average molecular weight 100 000; Sigma) was added into the upper compartment of the transwell cultures, followed by stimulation with thrombin (1 U/mL, Sigma) or not. The amount of FITC-dextran in the culture medium (25μL) taken from the lower compartment at 0,1,2, 4,8,12,24,48,72 or 96 h, indicative of the permeability of the HMVEC-L monolayer, was then determined using a fluorometer (excitation at 492 nm and emission at 520 nm).6) The role of CBP/p300 or/and HADC on HIF-1αand HIF-1α-induced angiogenic genes transcription:HMVEC-Ls (8.0×103 cells/well in 96-well plate) maintained in EBM supplemented with 2% FBS (without SingleQuots) were pre-treated with 40μmol/L curcumin or/and 100 ng/mL trichostatin A (TSA) for 1 h, and were then treated under normoxic conditions, subjected to hypoxia, deferoxamine (100μmol/L), or infected with Ad-HIF-1α-native, Ad-HIF-1α-564/402 Ad-HIF-1α-Trip (MOI=200) or Ad-Null (MOI=200) for a total 24 h. Cells were harvested sequentially for QGP-2 analysis. Results1) The morphologic characteristics of HMVEC-Ls have been shown to be highly variable, exhibit a typical "cobblestone" monolayer at confluent cell densities. HMVEC-Ls also test double-positive for von Willebrand Factor VIII and DC31. endothelium-specific organelles (Weibel-Palade bodies) were observed by a transmission electron microscope.2) The Plaque titration of the purified Ad-HIF-1α-Trip on HEK293 cells showed titers of 1.99x1012 pfu/ml by End-point Dilution Assay. The DNAs of the purified recombinant adenoviruses and the HIF-la gene were extracted to confirm the presence of recombinant adenoviruses by PCR. The sizes of PCR products were 287bp,380 bp,460bp and 214bp in excellent accord with expectation, respectively. The DNA sequence alignment revealed that the codons of Pro402 (CCA), Pro564 (CCC) and Asn803 (AAT) in the triple mutation of HIF-1αsequence were mutated to the codons of alanine (GCA, GCC and GCT, respectively). The Mycoplasma and the Chlamydia were not observed by electronmicroscope. The optimal multiplicity of infection (MOI) was 200 pfu/cell by X-gal staining.3) Although there is no significant difference between the mRNA level of the triple-mutated HIF-1αand that of the double Pro402-and Pro564-mutated HIF-1α(P=0.104), the mRNA level of the triple-mutated HIF-1αwas significantly higher than the wild-type and all other mutants of HIF-1α(all P<0.01). Nevertheless, the protein level of the triple-mutated HIF-1αwas significantly higher than the wild-type and all other mutants of HIF-1αincluding the double Pro402-and Pro564-mutated HIF-1α(all P<0.001). Simultaneously, compared with the expression of the wild-type and all other mutants of HIF-1αinduced angiogenic genes at both mRNA and protein levels, the triple-mutated HIF-1αsignificantly promoted the expression of these genes (all P<0.01).4) Regardless of whether the Asn803 site was mutanted or not, there was no significant difference between the mRNA half-life of the single Pro564 HIF-1αmutants (Ad-HIF-1α-564 vs Ad-HIF-1α-564/803, P=0.082) as well as between that of the double Pro402 and Pro564 HIF-1αmutants (Ad-HIF-la-564/402 vs Ad-HIF-la-Trip P= 0.199), but the mRNA half-life of the double Pro402 and Pro564 HIF-la mutants were significantly longer than that of the single Pro564 HIF-la mutants(all P<0.01). Simultaneously, compared with the mRNA half-life of the wild-type and all other mutants of HIF-la induced angiogenic genes, the triple-mutated HIF-la significantly extend the mRNA half-life of these genes (all P<0.01).5) Compared with the angiogenic potential of the wild-type and all other mutants of HIF-la induced angiogenic genes at both mRNA and protein levels, the triple-mutated HIF-la significantly promoted HMVEC-L proliferation (all P<0.001), migration(all P<0.001) and tube formation(all P<0.05), and decreased vascular permeability(all P<0.001).6) Inhibition the function of CBP/p300 using its inhibitor, curcumin, significantly prevented HIF-la-induced angiogenic genes transcription (all P<0.001). Suppression the function of HDAC with TS A, had similar effects on these genes (all P<0.001). Moreover, the expression of these genes got significantly worse by simultaneously inhibiting the CBP/p300 and HDAC (all P<0.001). During these inhibition processes of CBP/p300 or/and HDAC, the mRNA levels of HIF-1α-induced angiogenic genes induced by triple-mutated HIF-la were significantly higher than the wild-type and all other mutants of HIF-la (all P<0.01).Conclusion1) The triple-mutated HIF-la is more stable and effectively transcriptional, increase the mRNA stability of itself induced angiogenic genes and induce functional mature neovascularization. Hence, the triple-mutated HIF-la has a unique angiogenic potency.2) CBP/p300-and HDAC-regulative pathway are non-independent and synergistic for HIF-la-induced angiogenic genes transcription.