Protective effect of hypoxia inducible factor-1α gene therapy using recombinant adenovirus in cerebral ischaemia-reperfusion injuries in rats.

Context: Hypoxia-inducible factor-1α (HIF-1α)-induced genes can improve blood circulation.Objective: To investigate brain protective effect of recombinant adenovirus-mediated HIF-1α (AdHIF-1α) expression and its mechanism.Materials and methods: Male SD rats were used to establish focal cerebral ischaemia-reperfusion (CIR) injury models and randomly divided into normal, sham, CIR, Ad and AdHIF-1α groups. Ad or AdHIF-1α (108 pfu/10 µL) were administered into lateral ventricle of rats in Ad and AdHIF-1α groups. Modified neurological severity score (mNSS), brain water content (BWC) and cerebral infarct volumes (CIVs) were analyzed, and HE staining was performed using the brain tissues. Furthermore, the expression of caspase-3 and HSP90 was analyzed using qRT-PCR and Western blotting.Results: Compared to CIR (mNSS, 8.52 ± 0.52; CIV, 0.22 ± 0.01) and Ad groups (mNSS, 8.83 ± 0.41; CIV, 0.22 ± 0.02), mNSS and CIV were significantly decreased in AdHIF-1α group (mNSS, 6.03 ± 0.61; CIV, 0.11 ± 0.01) at 72 h (p < 0.05). With prolonged reperfusion time (6 h to 72 h), BWC of all rats increased gradually, although the increase was markedly less in AdHIF-1α group (78.15 ± 0.16 to 87.01 ± 0.31) compared to that in CIR (78.77 ± 0.60 to 89.74 ± 0.34) and Ad groups (78.77 ± 0.35 to 89.71 ± 0.27) (p < 0.01). There were significantly greater pathological changes in the neurons in AdHIF-1α group at 72 h following CIR. Furthermore, expression of caspase-3 (p < 0.01) down-regulated and HSP90 up-regulated (p < 0.05) at mRNA and protein levels in AdHIF-1α group.Discussion and conclusions: HIF­1α gene therapy is neuroprotective towards the CIR rat model. HIF-1α may be a candidate gene for the treatment of ischaemic brain injury.

Effects of HIF-1α on diabetic retinopathy angiogenesis and VEGF expression.

OBJECTIVE: To investigate the effect of hypoxia inducing factor (HIF)-1α on the expression of vascular endothelial growth factor (VEGF) and angiogenesis in diabetic retinopathy. PATIENTS AND METHODS: 8-week healthy SD rats were used for the experiments. Under systemic anesthesia condition, control rats received a saline injection into the left ocular body (control A group), and 2 µl antisense oligonucleotides (ASODN) (10 µmol/L) into right eye (control B group). Model rats received a saline injection into the left eye (model A group), and 2 µl ASODN (10 µmol/L) into the right eye (model B group). Rats received an intraocular injection of HIF-1α ASODN for 2, 4, and 6 weeks (A1, A2, A3, B1, B2, B3, respectively). Retinal vessel development was observed by ADP staining. Vascular endothelial cells penetrating retinal inner membrane were counted. Immunohistochemistry was used to detect expressions of VEGF and HIF-1α proteins in the retina. RESULTS: Prominent angiogenesis and hyperplasia were found in model A group. Relatively fewer newly formed vessels were shown in model group B. However, no significant change of retinal vascular morphology was presented in control group. Of note, the vascular endothelial cell counts, VEGF and HIF-1α contents were significantly increased in model group (p < 0.05). After treatment with HIF-1α ASODN, lower endothelial cell counts was found in model B group (p < 0.05 comparing to model A). VEGF expression in model B group was significantly decreased, among which, model B3 was observed with lower cell counts than model B1 or B2 (p < 0.05 comparing to model A). Injection of HIF-1α ASODN significantly suppressed HIF-1α level in model B in a time-dependent manner. CONCLUSIONS: Retinal angiogenesis is closely related with increasing level of HIF-1α. Inhibition of HIF-1α suppressed VEGF expression and deterred angiogenesis in a time-dependent manner. This provided novel insights for treating diabetic retinopathy.

Nano-microcapsule basic fibroblast growth factor combined with hypoxia-inducible factor-1 improves random skin flap survival in rats.

The present study aimed to investigate the effect of nano-microcapsule-basic fibroblast growth factor (bFGF) combined with hypoxia-inducible factor-1 (HIF-1) on the random skin flap survival of rats. Male Sprague-Dawley rats were used to establish the McFarlane flap model and subsequently, all model rats were randomly divided into four groups: Control, bFGF, HIF-1 and bFGF combined with HIF-1. The model rats were treated with 2.5 µg/day bFGF and 1.0 µg/day HIF-1 for 5 days by intraperitoneal injection. On day 5 following treatment, the boundaries between necrotic and surviving regions were significantly inhibited by bFGF combined with HIF-1. bFGF combined with HIF-1 inhibited oxidative stresses and inflammatory factors in random skin flap survival of rats. bFGF combined with HIF-1 also activated the protein expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) in the random skin flap survival of rats. In conclusion, nano-microcapsule bFGF combined with HIF-1 prevented random skin flap survival in rats through antioxidative, anti-inflammatory and activation of the protein expression levels of COX-2 and VEGF.

HIF-regulated HO-1 gene transfer improves the post-ischemic limb recovery and diminishes TLR-triggered immune responses - Effects modified by concomitant VEGF overexpression.

Heme oxygenase-1 (HO-1) mitigates cellular injury by antioxidant, anti-apoptotic, anti-inflammatory and proangiogenic effects. Vascular endothelial growth factor (VEGF) is a critical regulator of blood vessel growth. Their coordinated action was analyzed in a model of femoral artery ligation (FAL) in mice lacking HO-1 gene (HO-1 KO). Gastrocnemius skeletal muscles of HO-1 KO mice were preemptively injected with plasmids containing hypoxia-response element (HRE) driving the expression of only HO-1 (pHRE-HO1) or both HO-1 and VEGF (pHRE-HO1-VEGF). At day 14th the pHRE-HO1 vector increased an impaired post-ischemic blood flow recovery in HO-1 KO mice to the level observed in wild-type (WT) mice subjected to FAL and pHRE-HO1-VEGF restored it already at day 7. The pHRE-HO1 gene therapy diminished, when compared to control pHRE-empty-treated HO-1 KO mice, the expression of toll-like receptors (TLR4 and TLR9) and inflammatory cytokines (IL-1ß, IL-6 and TNFα) at day 3, whereas opposite effects were observed following concomitant HO-1 and VEGF gene transfer. Moreover, HO-1 diminished ischemia-induced expression of MyoD involved in satellite cell differentiation in HO-1 KO mice. Our results confirm the therapeutic potential of HO-1 and VEGF against critical limb ischemia although, their concomitant delivery may have contradictory actions on the resolution of inflammation.

The effect of HIF stabilizer on distraction osteogenesis.

OBJECTIVE: The aim of this study was to investigate the effect of an orally applicable hypoxia-inducible factor (HIF) stabilizer on distraction osteogenesis (DO) in a rat model. METHODS: The study included 24 Wistar albino rats undergoing osteotomy of the left tibia diaphysis. Rats were divided equally into experiment and control groups. Tibias were fixed using an external fixator. HIF stabilizer was administered to the experiment group. On the 5th postoperative day, distraction with increased rate (0.4 mm twice a day) was commenced and continued for 10 days. Histological and immunohistochemical evaluation was performed. RESULTS: Vascular endothelial growth factor levels of the experiment group were higher than those of the control group (p<0.05). The experiment group had slightly better intramembranous ossification quality than the control group on both Day 16 and 30. Endochondral ossification rates were better in the experiment group on Day 16. CONCLUSION: Vascular endothelial growth factor levels increased and stimulated angiogenesis in the presence of HIF pathway activation by oral administration of HIF stabilizer during DO. The biomechanical features of the distraction and angiogenesis should be coupled to achieve adequate bone homeostasis.

Injectable system for spatio-temporally controlled delivery of hypoxia-induced angiogenic signalling.

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O2). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5 °C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37 °C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol-gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3 months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions.

BACKGROUND: Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype. CONCLUSIONS/SIGNIFICANCE: These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.

Effect of hypoxia-inducible factor-1alpha gene therapy on walking performance in patients with intermittent claudication.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulatory factor that orchestrates cellular responses to hypoxia. It increases collateral vessel growth and blood flow in models of hind-limb ischemia. This study tested whether intramuscular administration of Ad2/HIF-1α/VP16, an engineered recombinant type 2 adenovirus vector encoding constitutively active HIF-1α, improves walking time in patients with peripheral artery disease and intermittent claudication. METHODS AND RESULTS: Two hundred eighty-nine patients with claudication were randomized in a double-blind manner to 1 of 3 doses of Ad2/HIF-1α/VP16 (2×10(9), 2×10(10), or 2×10(11) viral particles) or placebo, administered by 20 intramuscular injections to each leg. Graded treadmill tests were performed at baseline and then 3, 6, and 12 months after treatment. The primary end point was the change in peak walking time from baseline to 6 months. The secondary end point was change in claudication onset time, and tertiary end points included changes in ankle-brachial index and quality-of-life assessments. Median peak walking time increased by 0.82 minutes (interquartile range, -0.05-1.93 minutes) in the placebo group and by 0.82 minutes (interquartile range, -0.07-2.12 minutes), 0.28 minutes (interquartile range, -0.37-1.70 minutes), and 0.78 minutes (interquartile range, -0.02-2.10 minutes) in the HIF-1α 2×10(9), 2×10(10), and 2×10(11) viral particle groups, respectively (P=NS between placebo and each HIF-1α treatment group). There were no significant differences in claudication onset time, ankle-brachial index, or quality-of-life measurements between the placebo and each HIF-1α group. CONCLUSIONS: Gene therapy with intramuscular administration of Ad2/HIF-1α/VP16 is not an effective treatment for patients with intermittent claudication. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00117650.

First implantable device for hypoxia-mediated angiogenic induction.

Delayed or inadequate vascularisation is one of the major factors leading to tissue infarction and poor graft survival. Current vascularisation strategies that rely on delivering single growth factors have proved ineffective or hard to control in practise. An alternative approach has been identified by this group that relies on stimulation of physiological angiogenic factor cascades by engineering local cell-hypoxia, within a nano-fibrillar collagen material. Here we report on a novel, practical and effective implantable device for delivering engineered angiogenic signalling, on demand. Human dermal fibroblast-seeded dense-collagen depots were pre-conditioned under physiological cell-generated hypoxia to up-regulate production of key angiogenic factors, including HIF1α and VEGF(165). The level of VEGF(165) protein retained within depots (indicating general angiogenic factor production) was directly correlated to the duration of pre-conditioning. Angiogenic factor delivery from pre-conditioned, non-viable depots rapidly induced an angiogenic response within endothelial cell-seeded constructs in vitro, while implanted acellular 3D constructs incorporating such angiogenic depots in their core were infiltrated with perfused vessels by 1 week in vivo, at which stage non-angiogenic implants were minimally perfused. Depot stability, tuneability of cell/matrix composition with long clinical experience of the collagen material, together with cost effectiveness, make this angiogenic therapy a promising addition to a clinician's tool kit for improving local tissue perfusion.

[Effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease].

OBJECTIVE: To observe the effect of intracerebroventricular injection of rAAV-HIF-1α on hippocampal neuronal apoptosis in a rat model of Alzheimer disease (AD). METHODS: Thirty-two male SD rats (250-300 g) were randomized into 4 groups (n=8), including the normal control group without any treatment, AD model group with right intracerebroventricular injection of 2 µl Aß25-35 (10 mg/m1), sham-operated group with right intracerebroventricular injection of 2 µl normal saline, and AD+ rAAV-HIF-1α group with right intracerebroventricular injection of 10 µl rAAV-HIF-1a (1×10¹² v.g./m1) one week after Aß25-35 injection. The rats were sacrificed to detect the expression of HIF-1α and apoptosis of hippocampal neurons 5 weeks after Aß25-35 or saline injection. RESULTS: Western blotting showed that the expression of HIF-1α was significantly higher in AD+rAAV-HIF-1α group (451.59±34.39) than in normal control group (229.05±41.28) and sham-operated group (216.29±37.08) (P<0.05) without significant difference between the latter two groups. The apoptotic ratio of the hippocampal neurons was significantly higher in AD model group ([19.49±2.59]%) than in normal control group ([5.41±0.75]%) and sham-operated group ([5.28±0.66]%) in (P<0.05), and intracerebroventricular injection of rAAV-HIF-1α resulted in a significant reduction of the apoptotic ratio in the AD rats ([12.07±2.06]%) (P<0.05). CONCLUSION: Intracerebroventricular injection of rAAV-HIF-1α can inhibit hippocampal neuronal apoptosis in the rat model of AD.

Synergistic effect of HIF-1alpha 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)-1alpha. In this study, we analyzed the effect of augmenting HIF-1alpha levels in ischemic limb by intramuscular injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, 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 beta(2) 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 beta(2) integrin-blocking antibody. Transduction of BMDACs with lentivirus LvCA5 induced effects similar to DMOG treatment. Thus, HIF-1alpha 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.

Effect of artificial oxygen carrier with chemotherapy on tumor hypoxia and neovascularization.

UNLABELLED: In addition to transfusion alternatives, artificial oxygen carriers are a benefit in ischemia disorders. This study aimed at evaluating the possible effects of PEG-conjugated hemoglobin (PEG-Hb) plus cisplatin on tumor hypoxia and neovasculature. METHODS: HeLa cells were injected into submucosa of golden hamster cheek pouch to build tumor model. Animals were randomly assigned to 4 groups (n=10) and treated respectively: group 1, saline; group 2, cisplatin (5mg/kg); group 3, cisplatin (5mg/kg) plus PEG-Hb (0.3g/kg); group 4, cisplatin (5mg/kg) plus PEG-Hb (0.6g/kg). Tumor neovascularization morphological variation and tissue hypoxia were detected by intravital microscopy and immunostaining, respectively. RESULTS: Microvessel tortuosity and area capillary density in peritumoral areas were notably depressed in group 4 compared with group 2 (p<0.05). Hypoxia markers pimonidazole and HIF-1alpha expression were decreased significantly in group 4. CONCLUSION: PEG-Hb in high concentration can notably improve tumor tissue oxygenation and normalize neovasculature; it may be a potential adjuvant to chemotherapy in cancer.

Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer.

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide 'degron' from hypoxia-inducible factor-1alpha, which binds to the Von-Hippel-Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-alpha (ERalpha; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERalpha in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERalpha-dependent breast cancer cells by inducing G(1) arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERalpha expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G(1) arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERalpha and AR, respectively.