Low-oxygen conditioning of transplant recipients extends heart graft survival in mice

Heart transplants save lives, but the transplanted organs rarely last as long as patients and their doctors would like. Acute and chronic rejection of the donor heart remains a major barrier to long-term survival, and the immunosuppressive drugs that patients take for life carry their own burdens. Now a study in mice, published in EMBO Molecular Medicine, reports that a surprisingly simple environmental manipulation—keeping the transplant recipient at high altitude before surgery—markedly prolongs graft survival, and that the benefit depends on a specific molecular pathway in the recipient’s immune cells rather than on any change to the donor heart itself.

The research team, led by investigators at the University of Colorado Anschutz Medical Campus, used a fully mismatched heterotopic heart transplantation model in which hearts from C57BL/6J donor mice were transplanted into BALB/c recipients receiving low-dose CTLA4-Ig immunosuppression. When both donor and recipient animals were housed under chronic hypoxic conditions at Denver elevation—5280 feet, roughly 630 millimeters of mercury barometric pressure—graft survival more than doubled compared with pairs maintained under simulated sea-level normoxia in hyperbaric chambers. Median survival was 62 days in the hypoxia-conditioned pairs versus 25 days in normoxic controls, a difference the authors report as statistically significant with a log-rank P value of 0.0015.

What makes the finding clinically intriguing is that the protective effect did not require continued exposure to thin air after the operation. When the researchers exposed donor–recipient pairs to hypoxia for three weeks before transplantation and then returned them to normoxic conditions immediately after surgery, pre-transplant conditioning alone conferred a survival benefit comparable to continuous hypoxic exposure and remained significantly protective relative to normoxic controls. The overall comparison across groups yielded a P value of 0.0002, and the difference between continuous and pre-transplant-only hypoxia was not statistically significant (P = 0.78). Blinded histopathologic grading by a transplant pathologist using International Society for Heart and Lung Transplantation criteria showed comparable rejection severity among groups at the time of graft failure, suggesting that hypoxic conditioning delays the onset of rejection rather than fundamentally altering its character.

To determine whether the benefit came from the donor organ or the recipient’s body, the team separated the two exposures. In one group, donor mice lived under hypoxia for three weeks before donation while recipients stayed at sea-level conditions; in the mirror-image group, donors were kept under normoxia while recipients were hypoxia-conditioned for at least three weeks before transplantation and remained so afterward. Only recipient hypoxic conditioning consistently conferred protection. Recipient-conditioned grafts survived a median of 64 days versus 27 days in normoxic controls (P = 0.0048), whereas donor hypoxia produced only a trend toward longer survival that did not reach statistical significance (P = 0.51). The authors note that several donor-hypoxia grafts did survive beyond 60 days, but the effect was inconsistent across the cohort, localizing the protective mechanism primarily to the recipient compartment.

Because hypoxia acts on cells largely through hypoxia-inducible factors—transcription factors that coordinate adaptation to reduced oxygen availability—the researchers next asked which HIF isoform mattered. Single-cell RNA sequencing of peripheral blood mononuclear cells from non-transplanted mice conditioned for three weeks under hypoxia or normoxia revealed a striking transcriptional shift. In normoxic animals, circulating immune cells preferentially enriched inflammatory pathways, including antigen presentation, lymphocyte activation, and immune effector processes. In hypoxia-conditioned animals, the dominant programs involved protein homeostasis, protein folding, biosynthetic processes, and cellular adaptation. Module scores built from canonical antigen-presentation genes such as H2-Aa, H2-Ab1, Cd74, and Cd86 were consistently higher in normoxic animals, while a tolerance-associated signature comprising genes including Cd209a, Cd274, Ido1, Hmox1, and the TGF-beta family was preferentially enriched in hypoxia-conditioned cells, with the strongest signal within myeloid populations.

Within the myeloid compartment, the compositional changes were equally telling. Chronic hypoxia expanded non-classical monocytes—a population generally associated with patrolling, reparative, and immunoregulatory functions—while reducing classical monocytes, which are typically linked to inflammatory recruitment and antigen presentation. Flow cytometry independently confirmed the shift. Notably, HIF1-associated transcriptional activity remained largely unchanged across myeloid populations despite hypoxic conditioning, whereas HIF2-associated programs, built from genes including Vegfc, Flt1, Epo, and Epas1, were enriched in hypoxia-conditioned myeloid cells, with the most prominent enrichment within non-classical monocytes. These transcriptomic findings were associative on their own, so the team moved to a direct functional test.

That test came from belzutifan, a selective inhibitor of HIF2α. In a three-arm experiment with all donors kept under normoxia, hypoxia-conditioned recipients again showed significantly prolonged graft survival compared with normoxic controls. But when hypoxia-conditioned recipients were also treated with belzutifan—5 milligrams per kilogram twice daily by oral gavage, starting three weeks before transplantation and continuing until rejection—the survival advantage was abolished. Belzutifan-treated hypoxic recipients survived about as long as normoxic controls, providing in vivo loss-of-function evidence that HIF2 activity is required for the protective response. Histopathology again showed comparable acute cellular rejection features among all three groups at graft failure, indicating that HIF2 inhibition accelerates rejection without changing its histologic character. Because belzutifan was given systemically, the authors caution that the experiments do not identify which specific cell compartment requires HIF2 activity; cell-type-specific genetic manipulation will be needed to answer that question.

The team also tested whether pharmacologic HIF stabilization could substitute for environmental hypoxia using roxadustat, a prolyl hydroxylase inhibitor that stabilizes HIF signaling. It could not. Roxadustat-treated recipients showed only a trend toward prolonged graft survival that did not reach statistical significance, and treatment was accompanied by a high incidence of thrombotic complications—graft-limiting thrombotic events occurred in 6 of 10 recipient-treated animals and 2 of 8 donor-treated animals—that limited interpretation of the survival analysis. The authors suggest that the relatively high dose chosen for this proof-of-principle study may have contributed to the toxicity, and that lower-dose strategies were not tested. Their interpretation is that environmental hypoxia engages a broader adaptive program encompassing metabolic, epigenetic, and immunologic remodeling that indiscriminate systemic HIF stabilization does not faithfully reproduce.

A further layer of nuance comes from post-transplant analysis. The researchers integrated single-cell datasets from pre-transplant normoxic and hypoxic PBMCs with PBMCs isolated from a long-term surviving hypoxia-conditioned recipient at day 74 after transplantation. Before transplantation, chronic hypoxia had reduced the CM1 classical monocyte population and expanded the NCM1 non-classical population. After transplantation, that distribution was only partially maintained, with increased CM1 and reduced NCM1 relative to the pre-transplant hypoxic state, alongside re-emergence of inflammatory classical monocytes driven by Ccr2 and Ccl24 expression. Selected adaptive stress programs, including heat shock protein–associated transcriptional activity involving Hspa8, Hsp90aa1, Hsp90ab1, and Hspa5, remained detectable even as the monocyte composition shifted back. The authors interpret this as evidence that hypoxia induces a transient form of innate immune adaptation rather than durable tolerance, which helps explain why hypoxic conditioning substantially prolongs graft survival but ultimately fails to prevent rejection indefinitely.

The study has clear limitations that the authors themselves enumerate. The post-transplant single-cell analysis represents a limited late time point from a long-term surviving recipient and cannot establish the temporal trajectory of monocyte remodeling; serial sampling and lineage-tracing studies will be needed. Systemic belzutifan establishes a functional requirement for HIF2 but not the responsible cell type. And because the tolerance-associated signatures are transcriptional correlations rather than direct functional assays of immune tolerance, the authors prefer to describe the expanded populations as tolerance-associated rather than intrinsically tolerogenic. Direct functional studies will be required to determine whether non-classical monocytes are necessary or sufficient for graft protection. Still, the work provides a mechanistic framework for earlier clinical observations that heart transplant recipients transplanted at moderate altitude have been reported to show improved survival compared with recipients transplanted at lower elevations—observations that had remained largely correlative. By identifying recipient innate immune remodeling, rather than donor conditioning, as the dominant mediator, and by pinning that remodeling to HIF2-dependent signaling within a broader chronic adaptive program, the study suggests a therapeutic direction: selectively inducing or sustaining a protective recipient monocyte state, without the broad systemic effects of chronic hypoxia or nonspecific HIF stabilization, as a potential route to improving heart transplant outcomes.

Subject of Research: Recipient hypoxic preconditioning prolongs heart allograft survival in mice through HIF2-dependent monocyte remodeling

Article Title: Hypoxic preconditioning promotes recipient monocyte adaptation to improve heart transplant outcomes

Article References: Dun, H., Sticco-Ivins, M., Bonnici, G., Berning, A., Nelson, S. L., Hyacinth, S., Countess, J., Ju, S., Renz, J., Grijalva, A., Mackenzie, M., Beatty, H., Vagnozzi, R. J., Arenz, D., Peña, B., Jonas, E., Kuppe, C., Cornwell, W., Amrute, J., … Kopecky, B. J. (2026). Hypoxic preconditioning promotes recipient monocyte adaptation to improve heart transplant outcomes. EMBO Molecular Medicine. https://doi.org/10.1038/s44321-026-00543-0

Image Credits: AI Generated

DOI: 10.1038/s44321-026-00543-0

Keywords: heart transplantation, hypoxia, HIF2, monocytes, allograft rejection, belzutifan, roxadustat, single-cell RNA sequencing, immunosuppression, innate immunity, preclinical study, altitude