Sandra L. Holzhauer

L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease

Background—Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)–dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2·−) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor–null (Ldlr−/−) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2·− generation. Methods and Results—Arterioles were isolated from hypercholesterolemic Ldlr−/− mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2·− generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase–inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr−/− mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2·− in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. Conclusions—These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Effects of Experimental Asthma on Inflammation and Lung Mechanics in Sickle Cell Mice

Experimental asthma increases eosinophil and collagen deposition in the lungs of sickle cell disease (SCD) mice to a greater extent than in control mice. However, the effects of asthma on inflammation and airway physiology remain unclear. To determine effects of asthma on pulmonary inflammation and airway mechanics in SCD mice, hematopoietic stem cell transplantation was used to generate chimeric SCD and hemoglobin A mice. Experimental asthma was induced by sensitizing mice with ovalbumin (OVA). Airway mechanics were assessed using forced oscillation techniques. Mouse lungs were examined histologically and physiologically. Cytokine, chemokine, and growth factors in bronchoalveolar lavage fluid were determined by multiplex. IgE was quantified by ELISA. LDH was quantified using a colorimetric enzymatic assay. At baseline (nonsensitized), chimeric SCD mice developed hemolytic anemia with sickled red blood cells, mild leukocytosis, and increased vascular endothelial growth factor and IL-13 compared with chimeric hemoglobin A mice. Experimental asthma increased perialveolar eosinophils, plasma IgE, and bronchoalveolar lavage fluid IL-1β, IL-4, IL-6, and monocyte chemotactic protein 1 in chimeric hemoglobin A and SCD mice. IFN-γ levels were reduced in both groups. IL-5 was preferentially increased in chimeric SCD mice but not in hemoglobin A mice. Positive end-expiratory pressures and methacholine studies revealed that chimeric SCD mice had greater resistance in large and small airways compared with hemoglobin A mice at baseline and after OVA sensitization. SCD alone induces a baseline lung pathology that increases large and small airway resistance and primes the lungs to increased inflammation and airway hyperresponsiveness after OVA sensitization.

Sickle cell disease increases high mobility group box 1: a novel mechanism of inflammation

High mobility group box 1 (HMGB1) is a chromatin-binding protein that maintains DNA structure. On cellular activation or injury, HMGB1 is released from activated immune cells or necrotic tissues and acts as a damage-associated molecular pattern to activate Toll-like receptor 4 (TLR4). Little is known concerning HMGB1 release and TLR4 activity and their role in the pathology of inflammation of sickle cell disease (SCD). Circulating HMGB1 levels were increased in both humans and mice with SCD compared with controls. Furthermore, sickle plasma increased HMGB1-dependent TLR4 activity compared with control plasma. HMGB1 levels were further increased during acute sickling events (vasoocclusive crises in humans or hypoxia/reoxygenation injury in mice). Anti-HMGB1 neutralizing antibodies reduced the majority of sickle plasma-induced TLR4 activity both in vitro and in vivo. These findings show that HMGB1 is the major TLR4 ligand in SCD and likely plays a critical role in SCD-mediated inflammation.

A novel hemoglobin-binding peptide reduces cell-free hemoglobin in murine hemolytic anemia

Hemolysis can saturate the hemoglobin (Hb)/heme scavenging system, resulting in increased circulating cell-free Hb (CF-Hb) in hereditary and acquired hemolytic disease. While recent studies have suggested a central role for intravascular hemolysis and CF-Hb in the development of vascular dysfunction, this concept has stimulated considerable debate. This highlights the importance of determining the contribution of CF-Hb to vascular complications associated with hemolysis. Therefore, a novel Hb-binding peptide was synthesized and linked to a small fragment of apolipoprotein E (amino acids 141-150) to facilitate endocytic clearance. Plasma clearance of hE-Hb-b10 displayed a rapid phase t(1/2) of 16 min and slow phase t(1/2) of 10 h, trafficking primarily through the liver. Peptide hE-Hb-B10 decreased CF-Hb in mice treated with phenylhydrazine, a model of acute hemolysis. Administration of hE-Hb-B10 also attenuated CF-Hb in two models of chronic hemolysis: Berkeley sickle cell disease (SS) mice and mice with severe hereditary spherocytosis (HS). The hemolytic rate was unaltered in either chronic hemolysis model, supporting the conclusion that hE-Hb-B10 promotes CF-Hb clearance without affecting erythrocyte lysis. Interestingly, hE-Hb-B10 also decreased plasma ALT activity in SS and HS mice. Although acetylcholine-mediated facialis artery vasodilation was not improved by hE-Hb-B10 treatment, the peptide shifted vascular response in favor of NO-dependent vasodilation in SS mice. Taken together, these data demonstrate that hE-Hb-B10 decreases CF-Hb with a concomitant reduction in liver injury and changes in vascular response. Therefore, hE-Hb-B10 can be used to investigate the different roles of CF-Hb in hemolytic pathology and may have therapeutic benefit in the treatment of CF-Hb-mediated tissue damage.

T Cells Deficient in Diacylglycerol Kinase ζ Are Resistant to PD-1 Inhibition and Help Create Persistent Host Immunity to Leukemia

Efforts to improve the efficacy of adoptive T-cell therapies and immune checkpoint therapies in myelogenous leukemia are desired. In this study, we evaluated the antileukemia activity of adoptively transferred polyclonal cancer antigen-reactive T cells deficient in the regulator diacylglycerol kinase zeta (DGKζ) with or without PD-1/PD-L1 blockade. In the C1498 mouse model of myeloid leukemia, we showed that leukemia was eradicated more effectively in DGKζ-deficient (DGKζ-/-) mice than wild-type mice. T cells transferred from DGKζ-deficient mice to wild-type tumor-bearing recipients conferred this benefit. Leukemia clearance was similar to mice treated with anti-PD-L1. Strikingly, we found that the activity of adoptively transferred DGKζ-/- T cells relied partly on induction of sustainable host T-cell immunity. Transferring DGKζ-deficient T cells increased the levels of IFNγ and other cytokines in recipient mice, especially with coadministration of anti-PD-L1. Overall, our results offered evidence that targeting DGKζ may leverage the efficacy of adoptive T-cell and immune checkpoint therapies in leukemia treatment. Furthermore, they suggest that DGKζ targeting might decrease risks of antigen escape or resistance to immune checkpoint blockade. Cancer Res; 77(20); 5676-86. ©2017 AACR.