The research, led by Shuhan Lv and Qian Chen and their colleagues, focused on Olfr544 for a compelling reason. Previous work had shown that this receptor is highly expressed in the cerebral cortex and hippocampus of nine-month-old 5×FAD transgenic mice, a widely used model of Alzheimer’s disease. Its expression localizes near amyloid-beta plaques, the pathological hallmark of the disease, and its mRNA levels fluctuate with both age and disease progression. That pattern alone would be intriguing, but Olfr544 also belongs to a small club of olfactory receptors that have been deorphanized, meaning scientists know their chemical activator. In this case, the trigger is azelaic acid, a naturally occurring dicarboxylic acid that has already been implicated in metabolic regulation elsewhere in the body, including glucagon secretion in pancreatic alpha cells, lipolysis, and mitochondrial biogenesis in skeletal muscle. What remained entirely unknown was the receptor’s function inside the nervous system.

To probe that function, the team turned to the mouse neuroblastoma cell line N2a, a standard workhorse for studying neuronal differentiation and neurite extension. Their first task was to confirm that these cells genuinely possess the machinery for Olfr544 signaling. Using reverse transcription polymerase chain reaction followed by agarose gel electrophoresis and DNA sequencing, they demonstrated that N2a cells endogenously express not just Olfr544 itself but also its canonical downstream partners, the olfactory G protein Gαolf and adenylyl cyclase 3. That trio constitutes the full signaling axis that olfactory receptors use in the nose, and its presence in neuronal cells suggested that Olfr544 could operate as a functional GPCR in the brain rather than sitting there as molecular decoration.

The functional experiments began with azelaic acid. After establishing with an MTT assay that concentrations up to 640 micromolar had no effect on N2a cell viability, the researchers treated serum-deprived cultures with 500 micromolar of the compound for 24 hours. Under phase-contrast microscopy, the effect was unmistakable. Roughly 30 percent of treated cells extended neurites, defined as processes longer than the cell body’s diameter, compared with only about 10 percent of serum-deprived controls. Because short-term serum deprivation itself modestly promotes neurite outgrowth, the azelaic acid effect represented a genuine, receptor-linked enhancement of neuronal differentiation. Direct morphological comparison with receptor-deficient cells proved technically awkward because Olfr544 knockout cells grow and adhere poorly even at baseline, so the team pivoted to a cleaner strategy: dissect the downstream signaling and transcriptional consequences of receptor loss.

Before those genetic experiments, the researchers sought independent confirmation that Olfr544 is sufficient, on its own, to drive CREB-dependent transcription. They cloned the full-length mouse Olfr544 cDNA into a specialized expression vector that promotes proper membrane trafficking of olfactory receptors, then co-transfected it into HEK293T cells, which lack endogenous Olfr544, together with a CREB-responsive dual-luciferase reporter. When azelaic acid was added, luciferase activity surged in receptor-expressing cells but stayed flat in empty-vector controls. Co-treatment with SQ22536, an inhibitor of adenylyl cyclase, markedly blunted the response, confirming that the signal flowed through cAMP production. This heterologous reconstitution ruled out the possibility that azelaic acid’s effects in neuronal cells depend on indirect pathways unique to the N2a environment, and it established a direct mechanistic line from receptor to transcription factor.

The genetic core of the study came from CRISPR-Cas9 editing. The team designed two single-guide RNAs targeting exon 2 of the mouse Olfr544 gene, transfected them into N2a cells, selected edited populations with puromycin, and isolated monoclonal lines by limiting dilution. Sanger sequencing confirmed a homozygous 149-base-pair deletion that introduces a frameshift and a premature stop codon, and reverse transcription PCR showed loss of the wild-type transcript, consistent with nonsense-mediated decay. The authors candidly note that no validated specific antibody existed to confirm receptor loss at the protein level, a limitation they flag explicitly. Still, the functional evidence was decisive. When wild-type N2a cells were stimulated with 50 micromolar azelaic acid for 30 minutes, levels of phosphorylated CREB, the transcription factor that sits at the crossroads of neuronal survival, plasticity, and memory formation, rose significantly. In knockout cells, that increase vanished entirely, while basal pCREB remained unchanged, proving that azelaic acid’s activation of CREB is strictly Olfr544-dependent.

The team then traced the pathway upstream of CREB. Canonical GPCR signaling proceeds through Gαs proteins to activate adenylyl cyclase, elevate intracellular cAMP, and stimulate protein kinase A, which in turn phosphorylates CREB. Enzyme-linked immunosorbent assays showed that azelaic acid triggered a rapid rise in intracellular cAMP in wild-type cells, though a smaller one than forskolin, a direct adenylyl cyclase activator, could produce. In knockout cells, the cAMP response to azelaic acid was completely abolished, and PKA activity followed the same pattern, rising with drug treatment in wild-type cells but not in receptor-deficient ones. Together, these measurements delineate a clean linear cascade: Olfr544 activation, cAMP elevation, PKA activation, CREB phosphorylation. It is the textbook GPCR framework, operating in a neuronal context and converging on a transcription factor that governs synaptic plasticity.

To see what genes this cascade controls, the researchers performed RNA sequencing on wild-type and Olfr544 knockout cells, generating more than 41 gigabytes of clean data with mapping rates between 86 and 95 percent. Differential expression analysis identified 1,727 genes whose expression changed, 1,397 upregulated and 330 downregulated. Enrichment analysis revealed a striking asymmetry. Upregulated genes clustered in the neuroactive ligand-receptor interaction pathway, involving 41 genes encoding GPCRs and ion channels, which the authors interpret as possible compensatory mechanisms. Downregulated genes were concentrated in the motor protein pathway, including members of the myosin, actin, and kinesin families. That downregulation is precisely what one would expect if Olfr544 supports neurite dynamics, because motor proteins are essential for the cytoskeletal remodeling that drives process extension and neuronal morphogenesis. The authors caution that the transcriptomic comparison used a single monoclonal knockout line, so the results should be read as hypothesis-generating candidate pathways rather than a definitive catalog of receptor targets.

From the downregulated genes, computational prediction of CREB binding sites flagged three high-confidence candidates: Igf2bp3, Sorbs2, and Kctd14. Independent validation in the unedited parental cell line strengthened the case, particularly for IGF2BP3, an mRNA-binding protein implicated in axon guidance and synaptic plasticity. Its protein level rose significantly with azelaic acid treatment in wild-type cells and was markedly reduced in knockout cells, with no rescue by the agonist, making it the most robust Olfr544-dependent effector identified. SORBS2, which maintains dendritic complexity and synaptic integrity, and KCTD14, a member of a family linked to neurodevelopmental disorders, showed consistent downward trends in knockout cells, though not every pairwise comparison reached statistical significance. The authors are careful to note that the link between CREB activation and these targets remains correlative and that chromatin immunoprecipitation studies will be needed to confirm direct transcriptional regulation.

The bigger picture is what makes this work resonate beyond the cell culture hood. Olfactory receptors account for roughly 60 percent of human GPCRs, and mounting evidence ties their brain expression to Alzheimer’s disease, Parkinson’s disease, and schizophrenia, with some receptors modulating microglial phagocytosis or tau phosphorylation. This study establishes, for the first time, a direct linear signaling axis connecting a brain-expressed olfactory receptor to the structural machinery of the neuron itself. It also hints at a possible bridge to tau biology, since a homolog of IGF2BP3 binds tau mRNA and IGF2BP3 variants correlate with tau burden in Alzheimer’s patients. The authors are appropriately restrained about the disease connection, emphasizing that their findings are in vitro and that the relevance to Alzheimer’s pathology remains speculative. Future work, they argue, should test Olfr544 in knockout and transgenic mouse models, identify the receptor’s endogenous brain ligands, and determine whether modulating the pathway affects synaptic integrity, neurodegeneration, or cognition. If those experiments pan out, a receptor once thought to exist solely to smell the world may turn out to be a lever for protecting the brain from one of its most devastating diseases.

Subject of Research: The role of the extra-olfactory olfactory receptor Olfr544 in neuronal neurite outgrowth and CREB signaling

Article Title: Role of olfactory receptor Olfr544 in neuronal cells: Insights into neurite outgrowth and CREB signaling

Article References: Lv, S., Liu, J., Zhou, Y., Yang, R., Zhang, T., Deng, Y., Chen, Y., Lai, C., & Chen, Q. (2026). Role of olfactory receptor Olfr544 in neuronal cells: Insights into neurite outgrowth and CREB signaling. iScience, 29(11), Article 117757. https://doi.org/10.1016/j.isci.2026.117757

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117757

Keywords: olfactory receptors, Olfr544, azelaic acid, CREB signaling, neurite outgrowth, N2a cells, cAMP, PKA, CRISPR-Cas9, Alzheimer's disease, GPCR, IGF2BP3

The study, published in Scientific Reports, was led by Professor Tamiki Komatsuzaki of Hokkaido University and focused on one of biology’s classic model organisms: the soil-dwelling amoeba Dictyostelium discoideum. This single-celled organism has long fascinated scientists because of what it does when food runs out. When bacteria are scarce and starvation sets in, the amoebas begin releasing a chemical signal called cyclic AMP, or cAMP. Neighboring cells detect the signal, respond by releasing more of it, and begin crawling toward its source. The result is a spreading chemical wave that sweeps across the colony, and the cells ride that wave toward one another, eventually assembling into a multicellular organism. This dramatic transformation helps the amoebas survive hostile conditions, and it recapitulates, in miniature, the kind of collective cell behavior that underlies development, immunity, and disease in animals.

For decades, researchers studying this aggregation process have faced a practical dilemma. One approach is to track individual cells, following each amoeba’s path frame by frame to see how it moves. That works reasonably well when cells are sparse, but it becomes nearly impossible once the cells crowd together during aggregation, when one cell’s trail blurs into its neighbor’s. A second approach is to measure the cAMP wave itself, determining the direction in which the chemical signal is traveling across the colony. But these two measurements have traditionally been carried out separately, leaving a crucial gap: no direct, simultaneous view of how the wave’s dynamics relate to what each individual cell is actually doing at that instant. “How individual cells read passing waves of chemical signals and translate them into coordinated group migration has been hard to pin down,” Komatsuzaki explained.

The new study closes that gap with an ingenious imaging strategy. The team used fluorescent microscopy to record both cell movement and cAMP levels at the same time, frame by frame, throughout the aggregation process. Then came the clever part. The researchers took their sharp images and deliberately blurred them by different amounts, creating a series of versions of the same footage at different spatial scales. To each blurred version, they applied a computational technique called particle image velocimetry, or PIV, a method originally developed in fluid dynamics to trace the flow of liquids and gases by tracking patterns of motion between successive frames.

The logic behind the multiscale blurring is elegant. In the sharpest images, the analysis picks out the trajectories of individual cells, revealing each amoeba’s personal path across the colony floor. But as the images are blurred more heavily, the small, jittery movements of individual cells average out, and what remains is the larger, smoother pattern of motion embedded in the colony as a whole — the signature of the cAMP wave itself. By analyzing the same footage at multiple scales simultaneously, the researchers could directly compare the dynamics of the chemical wave with the movements of individual cells throughout the entire experiment, without ever having to separate the cells physically or measure the wave by an independent method. The team describes the result as a multiscale Eulerian velocity vector field: a map of motion that captures both the fine-grained behavior of single cells and the coarse-grained behavior of the collective at every moment.

Armed with this technique, the researchers tracked the amoebas continuously from two to seventeen hours after the onset of starvation, covering the full arc of the aggregation process. What they saw was a strikingly consistent pattern of behavior. As a cAMP wave approaches a cell, the amoeba surges forward to meet it almost head-on, racing toward the incoming chemical signal. But when the wave crests and begins to recede, something unexpected happens: the cell does not reverse direction to chase the departing wave. Instead, its motion stays locked in the same direction it was already traveling, carrying it forward even as the signal moves away. Only after the wave has passed does the cell settle down, resting directionless in the trough of the wave until the next one arrives — at which point the entire cycle repeats.

Komatsuzaki offered a vivid analogy for this behavior. “It’s like watching a crowd of surfers paddle hard to catch a wave, ride it together, and then bob around waiting for the next one,” he said. The image is apt in more than spirit. Each amoeba behaves like a surfer who paddles furiously toward an incoming swell, gains momentum as the wave lifts, coasts in the same direction even after the wave has passed beneath, and then drifts idly until the next swell appears. No individual surfer needs to know where the shore is or what the other surfers are doing; the collective pattern emerges from each cell responding to the same passing wave at the same time.

The directional locking that the researchers observed is particularly significant for understanding the physics of chemotaxis, the process by which cells move toward chemical attractants. A naive expectation might be that a cell responding to a chemical gradient would simply follow the gradient, reversing course whenever the gradient reverses. The amoebas do something subtly different: their response to the wave is temporally asymmetric, with a strong forward surge during the rising phase of the wave and a persistence of motion during the falling phase. This asymmetry, repeated wave after wave across thousands of cells, is what allows the colony to funnel itself steadily inward toward the aggregation center. The large-scale cAMP waves, visualized in the team’s images as smooth ripples spreading across the colony, were found to move in a direction precisely opposite to the average direction of individual cell movement — a 180-degree reversal that becomes visible only when single-cell motion and wave motion are compared side by side.

According to the authors, the findings offer the first systematic map of how the amoebas’ collective behavior emerges from individual action. That map matters beyond the world of social amoebae. Because the imaging technique separates single-cell motion from collective motion in the same dataset, the team believes it could be applied to far more complex systems, including populations of mammalian cells. One tantalizing possibility is identifying which cells act as “leaders” — initiating and shaping the waves — and which act as “followers,” responding to signals generated by others. In immune responses, where swarming neutrophils and other cells coordinate their pursuit of pathogens, and in cancer biology, where collectively invading tumor cells move through tissue in coordinated groups, such leader-follower dynamics are thought to play a decisive role but are notoriously hard to disentangle.

The study also demonstrates the value of borrowing tools from other disciplines. Particle image velocimetry was developed to measure fluid flow in wind tunnels and rivers, not to watch amoebas crawl across a petri dish. By adapting it to multiscale biological imaging, the researchers have created a method that treats a cell colony like a flowing medium with structure at every scale — a perspective that may prove broadly useful for studying active matter, tissue mechanics, and developmental biology. For now, the work provides something biologists have long lacked: a direct, quantitative picture of the moment when solitary cells stop being solitary. In the starved amoeba’s simple act of paddling toward a chemical wave and riding it together with its neighbors lies a principle that echoes through wound healing, immune defense, and cancer spread — the principle that a crowd, given the right signal, can behave like a single living thing.

Subject of Research: Collective cell migration in Dictyostelium discoideum via cAMP chemotactic wave signaling

Article Title: Study reveals how individual cells ‘surf’ chemical waves to form a collective

Article References: Study reveals how individual cells ‘surf’ chemical waves to form a collective. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: Dictyostelium discoideum, cAMP, chemotaxis, collective cell migration, particle image velocimetry, fluorescent imaging, Hokkaido University, multicellularity, cell signaling, immune response, cancer biology, Scientific Reports

Myelination is one of the most elegant tricks in biology. In the peripheral nervous system, Schwann cells wrap themselves around large-caliber axons in multiple spiral layers of membrane, forming an insulating sheath that allows electrical impulses to leap between gaps called the nodes of Ranvier, where clusters of sodium channels reignite the signal. Without this insulation, impulses crawl; with it, they race. The process depends on precise timing: Schwann cells must proliferate, sort individual axons from bundles, differentiate, and only then begin wrapping. Decades of in vitro work had suggested that cAMP, a classic second messenger, drives Schwann cell proliferation and differentiation, but the identity of the enzyme that actually produces cAMP in living animals, and its full role in nerve development, had remained stubbornly unknown.

To find that missing regulator, the researchers combined pharmacological, genetic, molecular, and live-imaging approaches in two vertebrate systems: zebrafish and mice. Zebrafish larvae are transparent, which allowed the team to watch peripheral nerves develop in real time under the microscope, while mice provided a mammalian counterpart for physiological validation. The answer that emerged was Adcy6, one of nine membrane-bound adenyl cyclase isoforms in vertebrates, each of which converts ATP into cAMP in response to different upstream signals. In zebrafish, loss of Adcy6 disrupted the timely radial sorting of axons and delayed myelination in the peripheral nervous system, without altering the distribution or proliferation of Schwann cells along the nerves. That distinction is critical: the enzyme is not needed to put Schwann cells in the right place, but to tell them when to start building myelin.

The study also clarified where Adcy6 sits in a well-known signaling cascade. A G-protein-coupled receptor called Gpr126 has long been recognized as essential for Schwann cell myelination, but the steps between receptor activation and myelin gene expression were incompletely mapped. The new data show that Adcy6 is the main driver of early Gpr126 signaling, positioned upstream of protein kinase A, or PKA, the enzyme that cAMP activates and that ultimately switches on myelin genes. In other words, when Gpr126 fires during development, it is Adcy6 that translates that signal into the cAMP burst that PKA needs to initiate the myelination program on schedule. This places a specific adenyl cyclase isoform, rather than a generic pool of cAMP, at the head of one of the most important developmental pathways in the peripheral nervous system.

Mammals added a twist. In mice, the researchers found that Adcy6 works in synergy with a related isoform, Adcy5, to fulfill the same function. Mice engineered to lack both Adcy6 and Adcy5 displayed excess mortality, severe tremor, reduced nerve size, defective nerve conduction, and motor dysfunction. Electrophysiological and anatomical analyses revealed both axonal and glial defects, indicating that the consequences of losing these enzymes extend beyond the wrapping cells themselves to the neurons whose axons they insulate. The tremor and motor impairment are exactly what one would expect from peripheral nerves that fail to transmit signals efficiently, and the reduced survival underscores how fundamental this pathway is to viable nerve function.

Perhaps the most surprising part of the story is that Adcy6 is not merely a glial enzyme. The team provided evidence that Adcy6 regulates neuronal numbers, mediates axonal transport, and, acting autonomously within neurons, influences the clustering of sodium channels at the nodes of Ranvier. Node formation is a cooperative feat between axons and myelinating glia, and the new results suggest that the cAMP-generating machinery inside neurons helps determine where and how the ion channels that regenerate the action potential assemble. A single adenyl cyclase isoform thus touches nearly every layer of peripheral nerve construction: the survival of neurons, the movement of materials along axons, the differentiation of Schwann cells, and the electrical architecture of the mature nerve.

From an evolutionary standpoint, the conservation of this mechanism is striking. Zebrafish and mice diverged hundreds of millions of years ago, yet in both species the same enzyme family member governs the timing of peripheral myelination. This deep conservation implies that the Adcy6-centered cAMP pathway was already embedded in the vertebrate developmental toolkit before the major lineages split, and that evolution has preserved it because the timing of myelination is too important to leave to redundant systems. The partial redundancy observed in mice, where Adcy5 can compensate alongside Adcy6, suggests a layered system in which one isoform dominates early signaling while a close relative provides backup, a configuration that may buffer development against genetic or environmental perturbation.

The technical achievement behind these conclusions deserves emphasis. Live imaging in zebrafish allowed the authors to distinguish between defects in Schwann cell migration, proliferation, and differentiation, disentangling processes that are usually conflated in fixed tissue. Pharmacological manipulation of cAMP levels, combined with genetic loss-of-function models and a constitutively active PKA construct, helped establish the ordering of the pathway from Gpr126 through Adcy6 to PKA and finally to myelin gene expression. In mice, the availability of Adcy5 and Adcy6 knockout animals, acknowledged from collaborators at Oslo University Hospital, enabled the double-mutant analysis that revealed the synergistic requirement and the dramatic physiological consequences of removing both enzymes.

The clinical implications are tantalizing, even if the study is fundamentally basic science. Congenital and inherited peripheral neuropathies often involve defective myelination, and understanding the master timing switch for the myelination program could eventually point to therapeutic targets. If the cAMP-PKA axis controlled by Adcy6 could be modulated pharmacologically, it might in principle be possible to nudge Schwann cells toward myelination in disorders where the process is delayed or stalled. The finding that Adcy6 also affects neuronal survival and axonal transport adds further relevance, since many neuropathies involve secondary axonal degeneration. The authors note no competing interests, and the work was funded by the AFM, Inserm, and Université Paris-Saclay, with the article published under a Creative Commons open access license.

For now, the study’s chief contribution is conceptual: it replaces a vague notion of diffuse cAMP signaling with a concrete, named molecular actor at the top of the peripheral myelination cascade. Adenyl cyclase 6, long one of nine relatively anonymous isoforms, is now positioned as the chief regulator that vertebrates rely on to build fast, properly insulated peripheral nerves on schedule. As the authors conclude, their findings identify Adcy6 as a primary driver of early and timely cAMP activity in vivo during peripheral nervous system development. Future work will likely explore how Gpr126 activates Adcy6 specifically, why Adcy5 can only partially substitute in mammals, and whether the same pathway can be harnessed to repair nerves after injury, questions that could keep this enzyme at the center of neurobiology for years to come.

Subject of Research: The role of adenyl cyclase 6 in cAMP signaling during peripheral nerve development and myelination

Article Title: Adenyl cyclase 6 is an evolutionary conserved chief regulator of peripheral nerve development and timely myelination in vertebrates

Article References: El-Hage, O., Boueid, M.-J., Magné, F., Echaniz-Laguna, A., Melki, J., Degerny, C., & Tawk, M. (2026). Adenyl cyclase 6 is an evolutionary conserved chief regulator of peripheral nerve development and timely myelination in vertebrates. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06443-3

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06443-3

Keywords: Adcy6, Adcy5, cAMP, Schwann cells, myelination, peripheral nervous system, Gpr126, PKA, zebrafish, mice, nodes of Ranvier, nerve development

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