Objective— Small-molecule hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) inhibitors are being explored in clinical studies for the treatment of anemia. HIF-P4H-2 (also known as PHD2 or EglN1) inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. We studied here whether HIF-P4H-2 inhibition could also protect against atherosclerosis. Approach and Results— Atherosclerosis development was studied in low-density lipoprotein (LDL) receptor–deficient mice treated with an oral HIF-P4H inhibitor, FG-4497, and in HIF-P4H-2-hypomorphic/C699Y-LDL receptor–mutant mice, all mice being fed a high-fat diet. FG-4497 administration to LDL receptor–deficient mice reduced the area of atherosclerotic plaques by ≈50% when compared with vehicle-treated controls and also reduced their weight gain, insulin resistance, liver and white adipose tissue (WAT) weights, adipocyte size, number of inflammation-associated WAT macrophage aggregates and the high-fat diet–induced increases in serum cholesterol levels. The levels of atherosclerosis-protecting circulating autoantibodies against copper-oxidized LDL were increased. The decrease in atherosclerotic plaque areas correlated with the reductions in weight, serum cholesterol levels, and WAT macrophage aggregates and the autoantibody increase. FG-4497 treatment stabilized HIF-1α and HIF-2α and altered the expression of glucose and lipid metabolism and inflammation-associated genes in liver and WAT. The HIF-P4H-2-hypomorphic/C699Y-LDL receptor–mutant mice likewise had a ≈50% reduction in atherosclerotic plaque areas, reduced WAT macrophage aggregate numbers, and increased autoantibodies against oxidized LDL, but did not have reduced serum cholesterol levels. Conclusions— HIF-P4H-2 inhibition may be a novel strategy for protecting against the development of atherosclerosis. The mechanisms involve beneficial modulation of the serum lipid profile and innate immune system and reduced inflammation.

Hypoxia of residence at high altitude (>2500 m) decreases birth weight. Lower birth weight associates with infant mortality and morbidity and increased susceptibility to later-in-life cardiovascular and metabolic diseases. We sought to determine the effects of hypoxia on maternal glucose and lipid metabolism and their contributions to fetal weight. C57BL6/NCrl mice, housed throughout gestation in normobaric hypoxia (15% oxygen) or normoxia, were studied at mid (E9.5) or late gestation (E17.5). Fetal weight at E17.5 was 7% lower under hypoxia than normoxia. The hypoxic compared with normoxic dams had ~20% less gonadal white adipose tissue at mid and late gestation. The hypoxic dams had better glucose tolerance and insulin sensitivity compared with normoxic dams and failed to develop insulin resistance in late gestation. They also had increased glucagon levels. Glucose uptake to most maternal tissues was ~2-fold greater in the hypoxic than normoxic dams. The alterations in maternal metabolism in hypoxia were associated with upregulation of hypoxia-inducible factor (HIF) target genes that serve, in turn, to increase glycolytic metabolism. We conclude that environmental hypoxia alters maternal metabolism by upregulating the HIF-pathway, and suggest that interventions that antagonize such changes in metabolism in high-altitude pregnancy may be helpful for preserving fetal growth.

Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIF␣ subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hifp4h-2 gt/gt ) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1␣ and HIF-2␣ were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2␣ dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIF␣ stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.KEYWORDS erythrocytosis, hypoxia, hypoxia-inducible factor prolyl-4-hydroxylase 2, spleen E rythrocytosis, defined as an absolute increase in red cell mass, is associated with increased hematocrit and hemoglobin values. Erythropoietin (EPO) stimulates erythropoiesis by binding to the EPO receptor (EPOR) on hematopoietic progenitors to promote erythroid differentiation, survival, and proliferation (1, 2). Classically, erythrocytosis is classified as either primary, caused by intrinsic defects in erythroid progenitor cells in the presence of normal or low serum EPO levels, or secondary. The causes of primary erythrocytosis include mutations in the Janus kinase 2 (JAK2) and EPOR genes, which can lead to EPO-independent proliferation of erythroid precursors or hypersensitivity to EPO (3). Secondary erythrocytosis is due to defects in the oxygen-sensing pathway, including mutations in the genes for hypoxia-inducible factor (HIF) prolyl 4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), HIF-2␣, and von Hippel Lindau (VHL) protein, and impaired oxygen delivery or tissue hypoxia, all associated with the activation of the EPO pathway and elevated serum EPO levels (3-5).During human development, the bone marrow becomes a functional site for hematopoiesis in the fetus at 4 to 5 months, whereas in mice, bone marrow hematopoiesis becomes active after birth (6). To supply oxygen for the needs of the developing fetus, nonmarrow tissues, such as the spleen and liver, serve as sites of extramedullary

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Objective: Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases). Approach and Results: We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor ( P4h-tm −/− /Ldlr −/− ) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm +/+ /Ldlr −/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher lipoprotein lipase protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm −/− / Ldlr −/− livers as compared with P4h-tm +/+ / Ldlr −/− . Conclusions: Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it.