Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

Abstract

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS) −/− mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS −/− mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS −/− mice, despite comparable reductions in tissue PO 2 . Paradoxically, nNOS −/− mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S -nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S -nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Plasma methemoglobin as a potential biomarker of anemic stress in humans

“…The proposed mechanism for increased MetHb may include NO-mediated Hb oxidation or reduction of nitrite (NO 2 -) by DeoxyHb, as suggested by experimental and clinical studies. 3,[18][19][20][21][22] It will be important to assess these changes in subsequent studies to determine if the increase in MetHb is reflective of activated adaptive mechanisms (nNOS) 7 and/ or increased OxyHb desaturation to DeoxyHb and tissue hypoxia [18][19][20][21][22] during acute hemodilutional anemia.…”

“…4,5 Experimental studies have shown that both hypoxic and nonhypoxic mechanisms may contribute to anemia-induced organ injury and mortality. 3,6,7 The complex nature of the cellular mechanisms involved may explain why anemiainduced mortality is not necessarily improved by therapies which increase blood oxygen content, including red blood cell (RBC) transfusions, 8,9 use of erythropoiesis stimulating agents (ESAs), 10 or hemoglobin-based oxygen carriers (HBOCs). 11 Each of these strategies is capable of increasing blood oxygen content but may not increase tissue oxygen delivery.…”

“…3,13,14 We have shown that deficiency of neuronal NOS (nNOS) is associated with increased mortality in a model of hemodilutional anemia, such that mice lacking nNOS die at a mean hemoglobin (Hb) value of 35 gÁL -1 , a level that is significantly higher than nNOS replete mice or humans (*25 gÁL -1 ). 7,15 In search of a clinical biomarker of NOS activation during anemia, we have hypothesized that increased nNOS-derived NO promotes a tissue-plasma NO gradient that favours the oxidation of Hb to MetHb, as shown by the negative correlation between increasing MetHb and decreasing Hb in experimental models of anemia ( Fig. 1).…”

“…23,24 For example, in the brain, acute anemia increases Hb O 2 extraction from *30% (Hb * 140 gÁL -1 ) to *50% (Hb * 50 gÁL -1 ), 23 while brain microvascular PO 2 decreases from *70 to *50 mmHg. 6,7 As such, methemoglobin (MetHb) production may be favoured in anemic vascular beds. Fig.…”

“…Anemia increases NOSderived nitric oxide (NO), which can stabilize hypoxia inducible factor (HIF) in tissues by S-nitrosylation (SNO) of the von HippelLindau protein (pVHL) to activate adaptive cellular mechanisms. 7 Increased tissue NOS activity generates tissue to blood nitric oxide (NO) gradient, which promotes NO diffusion into the vascular lumen to cause local vasodilation and oxidize hemoglobin (Hb) to methemoglobin (MetHb). In addition, local conversion of nitrite (NO 2 -) to NO by deoxyhemoglobin (DeoxyHb) can promote vasodilation via a blood to tissue NO gradient In attempting to identify a link between anemia and MetHb levels in patients, we observed that anemia was a common clinical finding ([84%) in two of the largest reported case series of acquired methemoglobinemia.…”