Carbon Monoxide Induced Erythroid Differentiation of K562 Cells Mimics the Central Macrophage Milieu in Erythroblastic Islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage

Carbon Monoxide Promotes Respiratory Hemoproteins Iron Reduction Using Peroxides as Electron Donors

The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O2 via its binding to their ferrous (FeII) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (FeIII, met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage

COA induces erythroid differentiation demonstrated by GPA expression and Hb synthesis.

<p>Cells were incubated under aerobic and anaerobic conditions for 4 days. (<b>A</b>) Cell suspensions were centrifuged and pellets were photographed. Note the prominent red color in the CO-cell pellet. (<b>B</b>) FACS analysis of GPA expression. Dashed line: auto-florescence, black - air, blue - N₂A and red - COA. Note the scale differences on the Y-axis. (<b>C</b>) Average cellular Hb content under different conditions using “Hb quantification assay” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033940#s2" target="_blank">Material and Methods</a> for details). Mean ± SD from 3 independent experiments are shown.</p

COA induces morphological changes typical of different stages of erythroid maturation.

<p>Cells were incubated for 4 days under air or COA. (<b>A</b>) Cytospin slides of cells stained with Giemsa-May-Grünwald. Arrows point to COA-cells in various maturation stages. Note the nuclei positioned toward the cell periphery and their condensation. Insert: terminal differentiation as seen by enucleation. (<b>B</b>) Cells were double stained with Hoechst 33342 (blue) and α-GPA (red) and then examined by fluorescence microscopy. Arrows indicate the positive GPA and negative Hoechst stained enucleated cells. (<b>C</b>) Cell morphology by Scanning Electron Microscopy (SEM). Air panel - a typical immature cell is shown, COA panel - cell size and concave shape typical for erythrocyte. (<b>D</b>) FACS analysis of GPA expression and cell size (sample of 10,000 cells). Square-enclosed parameters typical of differentiated of erythroid cells: small diameter due to condensation and population expressing high level of GPA.</p

COA but not N₂A preserves cell viability.

<p>Cells were incubated under aerobic and two types of anaerobic atmosphere conditions, N₂A or COA for 4 days, after which viability was measured via FACS analysis using PI penetration assay. Viability measurements (mean ± SD) from 3 independent experiments are shown.</p

1% COA-cells contain a subpopulation with elevated Hb content.

<p>Cells were grown for 4 days in highly enriched medium under 1% COA and sorted by FACS for higher (H-GPA) and lower (L-GPA) GPA expression. (<b>A</b>) Grey area: GPA of total cell population. Black area: cells with H-GPA population. White area: cells with L-GPA population. (<b>B</b>) Hb content of sorted cells was measured by “Hb quantification assay” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033940#s2" target="_blank">Material and Methods</a> for details). White bar: L-GPA average Hb level. Black bar: H-GPA average Hb level.</p

Can gas replace protein function? CO abrogates the oxidative toxicity of myoglobin.

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium

COA in enriched medium induces erythrocytes Hb synthesis.

<p>Cells were incubated for 3 days in regular or enriched medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033940#s2" target="_blank">Material and Methods</a> for details). Hb content of cytosols was measured using “Hb quantification assay”. Compositions of media included: (<b>1</b>) medium only; (<b>2</b>) medium supplemented with “heme required nutrients”; (<b>3</b>) highly enriched medium; <b>RBC</b> - Hb content of mature erythrocytes.</p

Increased peroxidation in aged LDL.

<p><b>A</b> - Oxidation by hemin (1 µM) presented as derivatives of time course (λ_{268 nm}), indicating formation of conjugated dienes. Heavy line – aged LDL, narrow line – fresh LDL. <b>B</b> - Increased content of conjugated dienes in aged LDL presented as difference spectrum of aged and fresh LDL.</p

COA, but not N₂A, results in S-phase arrest of cell cycle.

<p>Cells were incubated under aerobic and anaerobic conditions, N₂A or COA, for 4 days. Insert: representative FACS analysis of cell cycle for K562 cells incubated under different atmospheres. Arrows indicate center of S-phase. Main Figure: phase distribution of viable cells. Mean ± SD from 3 independent experiments is shown. Empty bars - G₀/G₁ phase, black bars - S phase and grey bars - G₂/M phase.</p