The team began by isolating so-called red blood cell ghosts, the hemoglobin-free membrane remnants left behind after erythrocytes are lysed under controlled laboratory conditions. When human cardiac microvascular endothelial cells were exposed to these ghosts for just two hours, bulk RNA sequencing revealed 113 differentially expressed genes, the vast majority of them upregulated. Among the most enriched biological pathways were inflammatory response and blood vessel morphogenesis, with prominent induction of genes such as VEGF, TNFA, PTGS2 and NFKBIA. Bioinformatic mapping also linked the altered genes to disease pathways including ischemia, peripheral vascular disease, arteriosclerosis and diabetic retinopathy, hinting that the response is directly relevant to cardiovascular pathology.

Chromatin immunoprecipitation sequencing added an epigenetic dimension to the picture. The researchers detected 77 sites of altered histone 3 lysine 27 acetylation, a mark of active enhancers, with the overwhelming majority gaining acetylation after ghost treatment. Motif analysis of these newly opened genomic regions showed a striking predominance of binding sites for the activator protein-1, or AP-1, family of transcription factors, which accounted for 55 percent of the target sequences. Immunofluorescence and cellular fractionation confirmed that JUNB, a key AP-1 member, accumulated in the nuclei of treated endothelial cells, indicating that the ghost signal rewires the transcriptional landscape rather than merely nudging existing programs.

On the inflammatory side, the membrane fraction triggered robust activation of the transcription factor NFκB. Within one to two hours, endothelial cells showed increased phosphorylation of the NFκB p65 subunit at serine 536, followed by phosphorylation at serine 276, a site targeted by cyclic AMP-dependent protein kinase and critical for transcriptional activity. The inflammatory enzyme COX2 rose in parallel. Blocking NFκB activation with the IκBα inhibitor BAY11-7082 abolished both the COX2 increase and, crucially, the angiogenic response, establishing NFκB as the central gatekeeper of the ghost-induced program.

Functionally, the treated endothelial cells behaved like cells primed for vessel building. In spheroid angiogenesis assays, exposure to red blood cell ghosts increased the number of sprouts per spheroid and the total endothelial network length, an effect comparable to recombinant VEGF. The cells also proliferated more readily, as measured by Ki-67 staining, and migrated faster in scratch-wound assays. Notably, the hemoglobin-free membrane fraction alone was sufficient to drive these effects, while isolated heme had no impact, ruling out the iron-containing pigment as the responsible agent and pointing instead to components retained in the membrane itself.

Metabolic profiling revealed the engine behind this angiogenic conversion. Using Seahorse extracellular flux analysis, the researchers observed that ghost addition boosted endothelial glycolysis within minutes, with elevated proton efflux and extracellular acidification sustained for roughly five hours. Mass spectrometry-based metabolomics confirmed accumulation of glycolytic intermediates, activation of the pentose phosphate pathway, and increased acetyl-CoA and TCA cycle metabolites. Isotope tracing showed glucose-derived ribose feeding purine synthesis and glutamine carbon flowing into pyrimidine synthesis, a classic anabolic profile. The glycolytic regulator PFKFB3, previously established as a hub of vessel sprouting metabolism, rose significantly at the protein level, and blocking glycolysis with the glucose analog 2-deoxy-D-glucose or inhibiting glutaminolysis with BPTES abolished the sprouting response.

The hunt for the transcriptional conductor of this program led to NR4A1, an orphan nuclear receptor also known as NUR77, which was among the most strongly induced genes in both the RNA and ChIP sequencing datasets. NR4A1 protein, nearly undetectable in resting cells, surged after two hours of ghost exposure and accumulated in the nucleus in an NFκB-dependent manner. Silencing NR4A1 with siRNA or blocking it pharmacologically prevented the induction of JUNB, PFKFB3 and VEGF and significantly reduced sprout formation. Intriguingly, NR4A1 knockdown also increased basal NFκB phosphorylation, revealing a built-in negative feedback loop: the receptor dampens inflammation while simultaneously switching on the metabolic and angiogenic machinery.

The causal trigger turned out to be adenosine triphosphate stored within the erythrocyte membrane. Fluorescent ATP analog labeling confirmed ATP binding sites on red cell membranes, and extracellular ATP at micromolar concentrations reproduced the full ghost response, from NR4A1 induction to sprouting. Degrading extracellular ATP with apyrase blunted the angiogenic effect. Pharmacological and genetic experiments identified two purinergic receptors as essential: the fast-acting ionotropic P2X7 receptor, which drives the inflammatory NFκB activation, and the metabotropic P2Y11 receptor, which couples to adenylyl cyclase and cyclic AMP production. Real-time FRET imaging with a genetically encoded cAMP biosensor captured robust cytosolic cAMP surges in single endothelial cells exposed to either ATP or ghosts, and inhibiting membrane-bound adenylyl cyclase or preventing cAMP degradation with PDE4 inhibitors modulated the response accordingly.

The translational payoff came from patients with severe peripheral artery disease, a condition defined by defective vessel growth. Immunostaining of ischemic muscle from mice and patients confirmed that erythrocyte extravasation accompanies ischemia in vivo. When endothelial cells were treated with ghosts prepared from PAD patients, the inflammatory activation persisted, but the upregulation of PFKFB3, VEGF and the cAMP sensor EPAC1 was significantly blunted, and the sprouting response was markedly weaker than with ghosts from age- and sex-matched healthy controls. Remarkably, inhibiting phosphodiesterase 4 with rolipram to prevent cAMP degradation restored the patient ghost response to healthy levels, suggesting that the ATP–P2Y11–cAMP axis fails in disease and can be pharmacologically rescued.

The authors caution that their patient cohort was small and exploratory, that P2Y11 receptors are absent in mice, limiting animal validation, and that other red cell membrane factors may contribute alongside ATP. Even so, the study fundamentally repositions erythrolysis as an instructive event in vascular repair. The same mechanism that helps restore perfusion after hemorrhage could be exploited therapeutically to boost angiogenesis in ischemic limbs, or conversely suppressed in settings such as cancer or retinal disease, where erythrocyte leakage and pathological vessel growth feed each other. Targeting endothelial cAMP metabolism, the work suggests, may be a promising lever for tipping that balance in either direction.

Subject of Research: How lysed erythrocyte membranes and their ATP content metabolically prime endothelial cells for angiogenesis

Article Title: Lysed erythrocyte membranes metabolically prime endothelial cells for angiogenesis

Article References: Gogiraju, R., Moiko, K., Bochenek, M. L., Greulich, F., Witzler, C., Schmitz, W., Zifkos, K., Derieux, C., Ghasemi, I., Guliani, P., Sun, B., Espinola-Klein, C., Uhlenhaut, H. N., Bock, A., Ruf, W., Madhusudhan, T., Lurz, P., & Schäfer, K. (2026). Lysed erythrocyte membranes metabolically prime endothelial cells for angiogenesis. Angiogenesis, 29(4), Article 74. https://doi.org/10.1007/s10456-026-10096-y

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10096-y

Keywords: angiogenesis, endothelial cells, erythrocytes, hemolysis, ATP signaling, NFκB, NR4A1, PFKFB3, cAMP, P2X7 receptor, P2Y11 receptor, peripheral artery disease