Chronic in vivo hypoxia in various organs: hypoxia-inducible factor-1alpha and apoptosis

We studied the in vivo persistence of hypoxia-inducible factor-1alpha (HIF-1alpha), main transducer of hypoxia, the differential response in organs exposed to the same degree of hypoxemia and the relationship with apoptosis. We measured HIF-1alpha (immunohistochemistry peroxidase and Western blot) and apoptosis (TUNEL) in heart, liver, kidney, gastrocnemius, and brain of rats exposed to chronic normobaric hypoxia (10% O2) or normoxia (21% O2) for 2 weeks. Despite same arterial O2 pressure and increased hemoglobin concentration (219 +/- 5 vs. 124 +/- 4 g/L), the organs responded differently. While marked in brain, muscle, and kidney cortex, HIF-1alpha was undetectable in heart and liver. In kidney medulla, HIF-1alpha was high in both normoxia and hypoxia. By contrast, apoptosis was marked in heart, slight in kidney medulla, and undetectable in other organs. We conclude that the HIF-1alpha response to chronic hypoxia can be a sustained phenomenon, but not in all organs, and that apoptosis responds differently from HIF-1alpha.     

HIF-1beta determines ABCA1 expression under hypoxia in human macrophages

ATP-binding cassette transporter A1 plays (ABCA1) a major role in reverse cholesterol transport, a process closely related to atherogenesis. In the thickening atherosclerotic lesions lipid loaded macrophages are exposed to regions of local hypoxia that may influence reverse cholesterol transport. Here we studied the effect of hypoxia on ABCA1 regulation and cholesterol efflux in human macrophages.We found that the hypoxia-inducible factor 1 (HIF-1) specifically binds to the HIF-1 response element of the ABCA1 promoter and the HIF-1 complex increases ABCA1 promoter activity along with ABCA1 expression. Primary human macrophages exposed to hypoxia or expressing constitutively active HIF-1alpha responded with a potent change in ABCA1 expression, which showed a strong correlation with HIF-1beta expression (r: 0.95–0.91). Moreover, ABCA1-mediated cholesterol efflux was also found to be regulated by HIF-1beta under hypoxia. In vivo, in macrophages prepared from human atherosclerotic lesions ABCA1 levels showed a strong correlation with HIF-1beta expression. This in vivo regulatory mechanism was confirmed in human pre-eclamptic placentas, a clinical condition with severe local hypoxia.These results demonstrate that HIF-1beta availability determines ABCA1 expression and cholesterol efflux in macrophages under hypoxia and may contribute to the interpersonal variability of atherosclerotic lesion progression.     

Hypoxia regulates avian cardiac Arnt and HIF-1alpha mRNA expression

The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-1alpha mediate cellular responses to hypoxia. We investigated the ability of hypoxia to regulate Arnt and HIF-1alpha mRNA in the heart in vivo. We cloned avian Arnt, developed an in vivo model of chronic cardiac hypoxia, and measured expression of cardiac Arnt and HIF-1alpha mRNA by quantitative RT-PCR. Chronic hypoxic exposure (24 h to 15% O(2)) of day 9 chick embryos resulted in a 30-fold increase in covalent binding of (3)H-misonidazole, a hypoxic tissue marker, to cardiac tissue, and a 2-fold induction of cardiac inducible nitric oxide synthase mRNA, compared to normoxic controls. In this same model, cardiac Arnt mRNA expression decreased by 35%, while HIF-1alpha mRNA expression increased 400%. These data suggest that regulation of Arnt and HIF-1alpha mRNA expression may contribute to the physiological responses of the heart during prolonged hypoxia.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Hypoxia-driven proliferation of embryonic neural stem/progenitor cells--role of hypoxia-inducible transcription factor-1alpha

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.