Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture.

OBJECTIVE: Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1alpha. MATERIALS AND METHODS: HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O(2), and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1alpha was studied using methods of protein stabilization and gene silencing. RESULTS: When long-term FLT3(-)CD34(-) Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(Kip1) and p57(Kip2) was dependent of HIF-1alpha. Surprisingly, overexpression of constitutively active HIF-1alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability. CONCLUSIONS: Our results imply that hypoxia, in part via HIF-1alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

Activation of hypoxia-inducible factors ameliorates hypoxic distal tubular injury in the isolated perfused rat kidney.

BACKGROUND: Preconditional activation of HIF with specific prolyl-hydroxylase inhibitors (PHD-I) attenuates proximal tubular injury, induced by warm ischaemia/ reperfusion (Bernhardt, JASN, 2006). Distal tubular damage occurs in humans with acute kidney injury (AKI), in experimental contrast media-induced nephropathy (CIN), as well as in cell-free isolated perfused kidneys (IPKs). Since in the IPK distal tubular damage inversely correlates with HIF activation (Rosenberger, KI, 2005), we explored the potential of PHD-I to improve morpho-functional outcome in this model. METHODS: Male SD rats were randomly given the synthetic PHD-inhibitor FG-4497 (FibroGen, 50 mg/kg IV) or its vehicle (CTR, n = 10 per group). Six hours later, the right kidney was perfused for 90 min with cell-free oxygenated medium and subsequently perfusion-fixed for morphologic assessment. The left kidney was used for HIF immunostaining. RESULTS: As compared with CTR kidneys, at 6 h after FG-4497 HIF-alpha isoforms were markedly up-regulated in all renal zones: HIF-1alpha in tubules and in papillary interstitial cells (IC), HIF-2alpha in IC and vascular endothelial cells. FG-4497 treatment resulted in a higher perfusate flow rate (P < 0.04, ANOVA). Tubular injury to medullary thick ascending limbs (mTALs) was significantly attenuated in the treatment versus control group (38.9 +/- 7.4% versus 62.7 +/- 4.9% of mTALs in the mid-inner stripe (P < 0.02); 23.8 +/- 6.8% versus 45.6 +/- 7.4% in the innermost zone of the inner stripe (P < 0.05). CONCLUSIONS: These findings illustrate that PHD-I preconditioning attenuates hypoxic distal tubular injury produced in the IPK in the same fashion in which it protects proximal tubules. mTAL conservation may be related to the stabilization of cellular HIF, as well as to preserved endothelial function and microcirculation.

Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells and explants of primate lung.

Preterm neonates with respiratory distress syndrome (RDS) often develop a chronic form of lung disease called bronchopulmonary dysplasia (BPD), characterized by decreased alveolar and vascular development. Ventilator treatment with supraphysiological O2 concentrations (hyperoxia) contribute to the development of BPD. Hyperoxia down-regulates and hypoxia up-regulates many angiogenic factors in the developing lung. We investigated whether angiogenic responses could be augmented through enhancement of hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and -2alpha, respectively) via blockade of prolyl hydroxylase domain-containing proteins (HIF-PHDs) in human microvascular endothelial cells from developing and adult lung, in epithelial A549 cells, and in fetal baboon explants in relative or absolute hyperoxia. PHD inhibitor (FG-4095) and positive control dimethyloxaloylglycine (DMOG), selective and nonselective HIF-PHD inhibitors, respectively, enhanced HIF-1alpha and -2alpha, vascular endothelial growth factor (VEGF), and platelet-endothelial cell adhesion molecule 1 expression in vitro in 95% and 21% O2. Furthermore, VEGF receptor fms-like tyrosine kinase 1 (Flt-1) was elevated, whereas kinase insert domain-containing receptor/fetal liver kinase 1 (KDR) was diminished in endothelial, but not epithelial, cells. Intracellular Flt-1 and KDR locations were unchanged by PHD blockade. Like VEGF, FG-4095 and DMOG increased angiogenesis in vitro, both in 95% and 21% O2, an effect that could be blocked through either Flt-1 or KDR. Notably, FG-4095 was effective in stimulating HIFs and VEGF also in fetal baboon lung explants. FG-4095 or DMOG treatment appeared to stimulate the feedback loop promoting HIF degradation in that PHD-2 and/or -3, but not PHD-1, were enhanced. Through actions characterized above, FG-4095 could have desirable effects in enhancing lung growth in BPD.