Vascular development is a pivotal aspect of embryogenesis, and its disruption can lead to developmental abnormalities or lethality. Although numerous studies have demonstrated a significant association between heme oxygenase 1 (Hmox1) and vascular biology, this link has not been reported so far during mouse embryonic development. Hmox1 is the rate-limiting enzyme that catalyzes the breakdown of heme to equimolar amounts of biliverdin, carbon monoxide, and ferrous iron. Here, we report that embryos lacking Hmox1 exhibit significant reductions in superficial blood vessel formation during mid-gestation, accompanied by organ-specific disruptions in vascular patterning. A comparative analysis of VEGF, VEGFR2, and CD31 revealed tissue-specific disruptions in angiogenic signaling and endothelial integrity in the brain, heart, and lungs of Hmox1-deficient embryos. The localization and abundance of these molecules were altered in affected organs, with isoform- and receptor subtype-specific expression changes raising the possibility of an impact on the structural integrity of developing vascular networks. These findings suggest that the absence of Hmox1 disrupts essential regulatory mechanisms required for angiogenesis, potentially contributing to the partial prenatal lethality observed in knockout embryos. Our results point to a previously unrecognized role for Hmox1 in regulating organ-specific vascular development during late gestation, with its deficiency leading to tissue-specific disruptions in angiogenesis and impaired blood vessel formation.

Heme oxygenase-1 (HO-1) deficiency manifests with severe metabolic abnormalities, yet the underlying mechanisms remain incompletely understood. We investigated metabolic adaptations in HO-1-deficient cells using patient-derived lymphoblastoid cells (LCL) and HEK293T knockout cells. Both cell types exhibited concordant metabolic signatures, characterized by a substantial reduction in intracellular ascorbic acid levels, despite compensatory upregulation of sodium-dependent vitamin C transporter 2 (SVCT2) and downregulation of GLUT1, compared to wild-type cells. Ultrastructural examination revealed abnormal mitochondrial morphology in both models. Both cell types showed higher baseline hydrogen peroxide levels compared to wild-type cells. Treatment with 2-phospho-L-ascorbic acid (AA2P) restored cell viability in both models upon hemin-induced stress, with protection requiring functional SVCT2-mediated uptake. AA2P supplementation attenuated the elevated H2O2 levels without altering glutathione redox homeostasis as measured by GSH/GSSG ratios. These findings illuminate ascorbic acid metabolism as a critical node in HO-1 deficiency pathophysiology and suggest ascorbic acid supplementation as a potential therapeutic strategy for this rare disorder.

A challenge for human immune system (HIS) mouse models has been the lack of human red blood cell (hRBC) survival after engraftment of these immune-deficient mice with human CD34+ hematopoietic stem cells (HSCs). This limits the use of HIS models for preclinical testing of targets directed at hRBC-related diseases. Although human white blood cells can develop in the peripheral blood of mice engrafted with human HSCs, peripheral hRBCs are quickly phagocytosed by murine macrophages upon egress from the bone marrow. Genetic ablation of murine myeloid cells results in severe pathology in resulting mice, rendering such an approach to increase hRBC survival in HIS mice impractical. Heme oxygenase-1 (HMOX-1)-deficient mice have reduced macrophages due to toxic buildup of intracellular heme upon engulfment of RBCs, but do not have an overall loss of myeloid cells. We took advantage of this observation and generated HMOX-1-/- mice on a humanized M-CSF/SIRPα/CD47 Rag2-/- IL-2Rγ-/- background. These mice have reduced murine macrophages but comparable levels of murine myeloid cells to HMOX-1+/+ control mice in the same background. Injected hRBCs survive longer in HMOX-1-/- mice than in HMOX-1+/+ controls. Additionally, upon human HSC engraftment, hRBCs can be observed in the peripheral blood of HMOX-1-/- humanized M-CSF/SIRPα/CD47 Rag2-/- IL-2Rγ-/- mice, and hRBC levels can be increased by treatment with human erythropoietin. Given that hRBC are present in the peripheral blood of engrafted HMOX-1-/- mice, these mice have the potential to be used for hematologic disease modeling, and for testing therapeutic treatments for hRBC diseases in vivo.

In heme protein–mediated AKI (HP-AKI), a senescence phenotype promptly occurs, and increased expression of p16Ink4a contributes to HP-AKI. Renal p16Ink4a expression is induced by hemoglobin, myoglobin, and heme in vivo and in renal epithelial cells exposed to heme in vitro. Impairing the binding or degradation of heme by hemopexin deficiency or heme oxygenase-1 deficiency, respectively, further upregulates p16Ink4a. Understanding the pathogenetic basis for AKI involves the study of ischemic and nephrotoxic models of AKI, the latter including heme protein–mediated AKI (HP-AKI). Recently, interest has grown regarding the role of senescence as a mechanism of kidney injury, including AKI. We examined whether senescence occurs in HP-AKI and potential inducers of and the role of a key driver of senescence, namely, p16Ink4a, in HP-AKI. The long-established murine glycerol model of HP-AKI was used, and indices of senescence were examined. To evaluate the interaction of heme and p16Ink4a expression, murine models of genetic deficiency of hemopexin (HPX) and heme oxygenase-1 (HO-1) were used. To determine the involvement of p16Ink4a in HP-AKI, the population of p16Ink4a-expressing cells was reduced using the INK-ATTAC model. Using multiple indices, a senescence phenotype appears in the kidney within hours after the induction of HP-AKI. This phenotype includes significant upregulation of p16Ink4a. p16Ink4a is upregulated in the kidney after the individual administration of myoglobin, hemoglobin, and heme, as well as in renal epithelial cells exposed to heme in vitro. Genetic deficiencies of HPX and HO-1, which, independently, are expected to increase heme content in the kidney, exaggerate induction of p16Ink4a in the kidney and exacerbate HP-AKI, the latter shown in the present studies involving HPX−/− mice and in previous studies involving HO-1−/− mice. Finally, reduction in the population of p16Ink4a-expressing cells in the kidney improves renal function in HP-AKI even within 24 hours. The pathogenesis of HP-AKI involves senescence and the induction of p16Ink4a, the latter driven, in part, by hemoglobin, myoglobin, and heme.

Heme oxygenase 1 (HO-1) is the pivotal catalyst for the primary and rate-determining step in heme catabolism, playing a crucial role in mitigating heme-induced oxidative damage. Pathogenic variants in the HMOX1 gene which encodes HO-1, are responsible for a severe, multisystem disease characterized by recurrent inflammatory episodes, organ failure, and an ultimately fatal course. Chronic hemolysis and abnormally low bilirubin levels are cardinal laboratory features of this disorder. In this study, we describe a patient with severe interstitial lung disease, frequent episodes of hyperinflammation non-responsive to immunosuppression, and fatal pulmonary hemorrhage. Employing exome sequencing, we identified two protein truncating variants in HMOX1, c.262_268delinsCC (p.Ala88Profs*51) and a previously unreported variant, c.55dupG (p.Glu19Glyfs*14). Functional analysis in patient-derived lymphoblastoid cells unveiled the complete absence of HO-1 protein expression and a marked reduction in cell viability upon exposure to hemin. These findings confirm the pathogenicity of the identified HMOX1 variants, further underscoring their association with severe pulmonary manifestations . This study describes the profound clinical consequences stemming from disruptions in redox metabolism.