Molecular mechanisms underlying synergistic adhesion of sickle red blood cells by hypoxia and low nitric oxide bioavailability

Abstract

Key Points• Adhesion of sRBCs is synergistically regulated by hypoxia and low NO bioavailability.• P-selectin and p38 kinase pathways play a role in the synergistic adhesion of sRBCs.The molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD. (Blood. 2014; 123(12):1917-1926 IntroductionAlthough sickle cell disease (SCD) arises from a single mutation of b-globin, clinical severity varies significantly.1 A well-known clinical modifier is fetal hemoglobin, which is expressed at variable levels in patients and inhibits the polymerization of sickle hemoglobin. 2 The frequency of vaso-occlusive crisis was also used to evaluate disease severity of SCD patients.3 Vaso-occlusive crisis is likely triggered by multiple physiological insults such as infection and cytokine-mediated inflammation that may cause tissue hypoxia, and mediated by multistep cell adhesion mechanisms involving sickle red blood cells (sRBCs). 4 The degree of sRBC adhesion to endothelial cells was shown to correlate with SCD severity. 1 Nitric oxide (NO) bioavailability is another physiological factor capable of modulating clinical severity.5 NO bioavailability decreases during vaso-occlusive crisis, primarily because of NO scavenging by cell-free hemoglobin 6 as well as other mechanisms including arginase, 7 reactive oxygen species, 8 and NO synthases.9 NO metabolite levels vary among patients even at steady state and may inversely correlate with the frequency of vaso-occlusive crisis. 10 We showed that inhalation of low-dose NO permitted SCD mice to survive hypoxic stress. 11 The gender difference in the clinical severity of SCD may result in part from distinct levels of NO bioavailability.12 Thus, heterogeneity of the clinical expression in SCD appears ...

The sickle cell mouse lung: proinflammatory and primed for allergic inflammation

“…It is also possible that the dysfunction involves endothelial cells but is not directly related to their lack of function. Multiple reports have noted that erythrocytes in SCD are more adherent 56–59 , particularly to activated endothelium. In the presence of a multitude of adherent erythrocytes, competition for adhesion sites on endothelial cells may be too great for the relatively smaller number of lymphocytes to successfully adhere and pass through the vascular wall.…”

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“…In addition, hypoxia results in increased reticulocyte egress from the bone marrow, and these reticulocytes exhibit greater adhesive properties to the endothelium when compared to mature erythrocytes, and thus, may further aggravate vaso-occlusion [16,17]. Tissue ischemia is the net result of all these pathological cascades, resulting in pain episodes, vaso-occlusive attacks and acute chest syndrome [18,19]. As most SCD patients are not constantly hypoxemic, the blood hypoxemia and the tissue hypoxia they experience are presumably of intermittent nature, a phenomenon that has been well documented in murine models of SCD [20].…”

“…At the cellular level, decreased NO bioavailability results in endothelial dysfunction, manifested by increased adhesion of sickled RBCs to endothelial cells [19]. The mechanism for reduced NO bioavailability is through both increased NO consumption and decreased NO synthesis.…”

Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells

“…Nitric oxide (NO) and its derivatives can influence RBC-EC adhesion, reminiscent of the ability of NO to prevent or reverse leukocyte adhesion to endothelial cells (1). Specifically, inhibition of NOS production promoted endothelial adhesion of RBCs, and NO donors attenuated the excess adhesivity of RBCs in diabetes, malarial infection, sickle cell disease, and blood storage (2-6). The mechanistic basis of the antiadhesive effect of NO or its derivatives is uncertain, but could reflect inhibitory protein S-nitrosylation or downregulation (6) of either an adhesion receptor or a downstream element of signal transduction.…”