Macrophage-mediated efferocytosis is crucial for hematoma absorption and inflammation resolution in intracranial hemorrhages. We previously demonstrated that atorvastatin (ATO) expedites the absorption of subdural hematoma (SDH); however, the extent to which this therapeutic effect is associated with ATO-mediated modulation of macrophage efferocytosis remains unclear. In this study, we established a rat model of SDH through autologous blood transfusion into the subdural space. We used primary bone marrow-derived macrophages (BMDMs) to investigate the roles of heme and ATO. The results of enzyme-linked immunosorbent assay and flow cytometry demonstrated that, in addition to its proinflammatory effect, heme also inhibits macrophage efferocytosis. However, ATO reversed this inhibition of efferocytosis in both in vivo and in vitro experiments. Transcriptomic analysis of BMDMs revealed that ATO promotes Krüppel-like factor 4 (KLF4) expression and is mainly enriched in phagocytosis and lysosome-related pathways. Finally, ATO restored the heme-induced reduction in the macrophage phagocytic rate and impairment of lysosomal function. However, KLF4 knockout abolished the beneficial effects of ATO. In conclusion, this study demonstrated that ATO ameliorates heme-inhibited efferocytosis via KLF4, thereby promoting hematoma absorption. We demonstrated a novel mechanism of ATO in the treatment of SDH, providing a new therapeutic prospect for patients (IRB2022-YX-007-01).
El RahalA, BeckJ, AhlbornP, et al.Incidence, therapy, and outcome in the management of chronic subdural hematoma in Switzerland: A population-based multicenter cohort study. Front Neurol, 2023; 14:1206996; doi: 10.3389/fneur.2023.1206996
2.
HutchinsonPJ, EdlmannE, BultersD, et al.; Dex-CSDH Trial Collaborators. Trial of dexamethasone for chronic subdural hematoma. N Engl J Med, 2020; 383(27):2616–2627; doi: 10.1056/NEJMoa2020473
3.
NouriA, GondarR, SchallerK, et al.Chronic Subdural Hematoma (cSDH): A review of the current state of the art. Brain Spine, 2021; 1:100300; doi: 10.1016/j.bas.2021.100300
4.
EdlmannE, Giorgi-CollS, WhitfieldPC, et al.Pathophysiology of chronic subdural haematoma: Inflammation, angiogenesis and implications for pharmacotherapy. J Neuroinflammation, 2017; 14(1):108; doi: 10.1186/s12974-017-0881-y
5.
FeghaliJ, YangW, HuangJ. Updates in chronic subdural hematoma: Epidemiology, etiology, pathogenesis, treatment, and outcome. World Neurosurg, 2020; 141:339–345; doi: 10.1016/j.wneu.2020.06.140
6.
MehtaV, HarwardSC, SankeyEW, et al.Evidence based diagnosis and management of chronic subdural hematoma: A review of the literature. J Clin Neurosci, 2018; 50:7–15; doi: 10.1016/j.jocn.2018.01.050
7.
RauhalaM, LuotoTM, HuhtalaH, et al.The incidence of chronic subdural hematomas from 1990 to 2015 in a defined Finnish population. J Neurosurg, 2020; 132(4):1147–1157; doi: 10.3171/2018.12.jns183035
8.
CofanoF, PesceA, VercelliG, et al.Risk of recurrence of chronic subdural hematomas after surgery: A multicenter observational cohort study. Front Neurol, 2020; 11:560269; doi: 10.3389/fneur.2020.560269
9.
HusemannJ, LoikeJD, AnankovR, et al.Scavenger receptors in neurobiology and neuropathology: Their role on microglia and other cells of the nervous system. Glia, 2002; 40(2):195–205; doi: 10.1002/glia.10148
10.
GongZ, ZhanD, NieM, et al.Dexamethasone enhances the efficacy of atorvastatin in inhibiting excessively inflammation-induced abnormal angiogenesis by regulating macrophages. J Neuroinflammation, 2021; 18(1):203; doi: 10.1186/s12974-021-02257-1
11.
Boada-RomeroE, MartinezJ, HeckmannBL, et al.The clearance of dead cells by efferocytosis. Nat Rev Mol Cell Biol, 2020; 21(7):398–414; doi: 10.1038/s41580-020-0232-1
12.
ZhaoJ, ZhangW, WuT, et al.Efferocytosis in the central nervous system. Front Cell Dev Biol, 2021; 9:773344; doi: 10.3389/fcell.2021.773344
13.
MoriokaS, MaueröderC, RavichandranKS. Living on the edge: Efferocytosis at the interface of homeostasis and pathology. Immunity, 2019; 50(5):1149–1162; doi: 10.1016/j.immuni.2019.04.018
14.
DoranAC, YurdagulAJr, TabasI. Efferocytosis in health and disease. Nat Rev Immunol, 2020; 20(4):254–267; doi: 10.1038/s41577-019-0240-6
15.
YanX, HeM, HuangH, et al.Endogenous H(2)S targets mitochondria to promote continual phagocytosis of erythrocytes by microglia after intracerebral hemorrhage. Redox Biol, 2022; 56:102442; doi: 10.1016/j.redox.2022.102442
16.
ZhangW, ZhaoJ, WangR, et al.Macrophages reprogram after ischemic stroke and promote efferocytosis and inflammation resolution in the mouse brain. CNS Neurosci Ther, 2019; 25(12):1329–1342; doi: 10.1111/cns.13256
17.
SolimanE, Gudenschwager BassoEK, JuJ, et al.Skull bone marrow-derived immune cells infiltrate the injured cerebral cortex and exhibit anti-inflammatory properties. Brain Behav Immun, 2025; 123:244–253; doi: 10.1016/j.bbi.2024.09.023
18.
KojimaY, VolkmerJP, McKennaK, et al.CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature, 2016; 536(7614):86–90; doi: 10.1038/nature18935
19.
HanR, LanX, HanZ, et al.Improving outcomes in intracerebral hemorrhage through microglia/macrophage-targeted IL-10 delivery with phosphatidylserine liposomes. Biomaterials, 2023; 301:122277; doi: 10.1016/j.biomaterials.2023.122277
20.
WilkinsonDA, KeepRF, HuaY, et al.Hematoma clearance as a therapeutic target in intracerebral hemorrhage: From macro to micro. J Cereb Blood Flow Metab, 2018; 38(4):741–745; doi: 10.1177/0271678x17753590
21.
WagnerKR, SharpFR, ArdizzoneTD, et al.Heme and iron metabolism: Role in cerebral hemorrhage. J Cereb Blood Flow Metab, 2003; 23(6):629–652; doi: 10.1097/01.wcb.0000073905.87928.6d
22.
XiG, WagnerKR, KeepRF, et al.Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke, 1998; 29(12):2580–2586; doi: 10.1161/01.str.29.12.2580
23.
VasconcellosLRC, MartimianoL, DantasDP, et al.Intracerebral injection of Heme induces lipid peroxidation, neuroinflammation, and sensorimotor deficits. Stroke, 2021; 52(5):1788–1797; doi: 10.1161/strokeaha.120.031911
24.
JingC, BianL, WangM, et al.Enhancement of hematoma clearance with CD47 blocking antibody in experimental intracerebral hemorrhage. Stroke, 2019; 50(6):1539–1547; doi: 10.1161/strokeaha.118.024578
25.
BuzziRM, AkeretK, SchwendingerN, et al.Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain. Free Radic Biol Med, 2022; 179:277–287; doi: 10.1016/j.freeradbiomed.2021.11.011
VasconcellosL, Pimentel-CoelhoPM. Heme as an inducer of cerebral damage in hemorrhagic stroke: Potential therapeutic implications. Neural Regen Res, 2022; 17(9):1961–1962; doi: 10.4103/1673-5374.335148
28.
BaigentC, BlackwellL, EmbersonJ, et al.Efficacy and safety of more intensive lowering of LDL cholesterol: A meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet (London, England), 2010; 376(9753):1670–1681; doi: 10.1016/s0140-6736(10)61350-5
29.
KhuranaS, GuptaS, BhallaH, et al.Comparison of anti-inflammatory effect of atorvastatin with rosuvastatin in patients of acute coronary syndrome. J Pharmacol Pharmacother, 2015; 6(3):130–135; doi: 10.4103/0976-500x.162011
YuD, LiaoJK. Emerging views of statin pleiotropy and cholesterol lowering. Cardiovasc Res, 2022; 118(2):413–423; doi: 10.1093/cvr/cvab032
32.
JiangR, ZhaoS, WangR, et al.Safety and efficacy of atorvastatin for chronic subdural hematoma in Chinese patients: A Randomized ClinicalTrial. JAMA Neurol, 2018; 75(11):1338–1346; doi: 10.1001/jamaneurol.2018.2030
33.
TangR, ShiJ, LiX, et al.Effects of atorvastatin on surgical treatments of chronic subdural hematoma. World Neurosurg, 2018; 117:e425–e429; doi: 10.1016/j.wneu.2018.06.047
34.
YuanJ, LiuX, NieM, et al.Inactivation of ERK1/2 signaling mediates dysfunction of basal meningeal lymphatic vessels in experimental subdural hematoma. Theranostics, 2024; 14(1):304–323; doi: 10.7150/thno.87633
35.
JarrKU, YeJ, KojimaY, et al.The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nat Cardiovasc Res, 2022; 1(3):253–262; doi: 10.1038/s44161-022-00023-x
36.
ManegaCM, FiorelliS, PorroB, et al.12(S)-Hydroxyeicosatetraenoic acid downregulates monocyte-derived macrophage efferocytosis: New insights in atherosclerosis. Pharmacol Res, 2019; 144:336–342; doi: 10.1016/j.phrs.2019.03.012
37.
TajbakhshA, GheibihayatSM, AskariH, et al.Statin-regulated phagocytosis and efferocytosis in physiological and pathological conditions. Pharmacol Ther, 2022; 238:108282; doi: 10.1016/j.pharmthera.2022.108282
38.
LiuX, GaoC, YuanJ, et al.Subdural haematomas drain into the extracranial lymphatic system through the meningeal lymphatic vessels. Acta Neuropathol Commun, 2020; 8(1):16; doi: 10.1186/s40478-020-0888-y
39.
QuanW, ZhangZ, LiP, et al.Role of regulatory T cells in atorvastatin induced absorption of chronic subdural hematoma in rats. Aging Dis, 2019; 10(5):992–1002; doi:10.14336/ad.2018.0926
40.
LiuL, ChenF, LiuK, et al.The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury. Int J Biol Sci, 2025; 21(13):5802–5820; doi: 10.7150/ijbs.110790
41.
ZhangZC, YangYL, LiB, et al.Low-intensity pulsed ultrasound promotes spinal fusion by regulating macrophage polarization. Biomed Pharmacother, 2019; 120:109499; doi: 10.1016/j.biopha.2019.109499
42.
ChangCF, GoodsBA, AskenaseMH, et al.Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage. J Clin Invest, 2018; 128(2):607–624; doi: 10.1172/jci95612
43.
ZhangY, XuL, ZhangY, et al.Discovery of novel MIF inhibitors that attenuate microglial inflammatory activation by structures-based virtual screening and in vitro bioassays. Acta Pharmacol Sin, 2022; 43(6):1508–1520; doi: 10.1038/s41401-021-00753-x
44.
YangM, AntoineDJ, WeemhoffJL, et al.Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice. Liver Transpl, 2014; 20(11):1372–1382; doi: 10.1002/lt.23958
45.
WuQ, ZhangD, DaiS, et al.Desflurane attenuates renal ischemia-reperfusion injury by modulating ITGB1/CD9 and reducing oxidative stress in tubular epithelial cells. Redox Biol, 2025; 80:103490; doi: 10.1016/j.redox.2025.103490
46.
RaymondMH, DavidsonAJ, ShenY, et al.Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo. Science, 2022; 375(6585):1182–1187; doi: 10.1126/science.abl4430
47.
WangD, LiT, WeiH, et al.Atorvastatin enhances angiogenesis to reduce subdural hematoma in a rat model. J Neurol Sci, 2016; 362:91–99; doi: 10.1016/j.jns.2016.01.017
48.
WynnTA, VannellaKM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity, 2016; 44(3):450–462; doi: 10.1016/j.immuni.2016.02.015
49.
LuYZ, NayerB, SinghSK, et al.CGRP sensory neurons promote tissue healing via neutrophils and macrophages. Nature, 2024; 628(8008):604–611; doi: 10.1038/s41586-024-07237-y
50.
BlacherE, TsaiC, LitichevskiyL, et al.Aging disrupts circadian gene regulation and function in macrophages. Nat Immunol, 2022; 23(2):229–236; doi: 10.1038/s41590-021-01083-0
51.
ChakrabortyS, SinghA, WangL, et al.Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis. J Exp Med, 2023; 220(11):e20221388; doi: 10.1084/jem.20221388
52.
ShiJ, WuX, WangZ, et al.A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis. Nat Commun, 2022; 13(1):7929; doi: 10.1038/s41467-022-35604-8
53.
PerryJSA, MoriokaS, MedinaCB, et al.Interpreting an apoptotic corpse as anti-inflammatory involves a chloride sensing pathway. Nat Cell Biol, 2019; 21(12):1532–1543; doi: 10.1038/s41556-019-0431-1
54.
SchlamD, BagshawRD, FreemanSA, et al.Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins. Nat Commun, 2015; 6:8623; doi: 10.1038/ncomms9623
55.
WalpoleGFW, PlumbJD, ChungD, et al.Inactivation of Rho GTPases by Burkholderia cenocepacia Induces a WASH-Mediated Actin Polymerization that Delays Phagosome Maturation. Cell Rep, 2020; 31(9):107721; doi: 10.1016/j.celrep.2020.107721
56.
RidleyAJ. Life at the leading edge. Cell, 2011; 145(7):1012–1022; doi: 10.1016/j.cell.2011.06.010
57.
MartinsR, MaierJ, GorkiAD, et al.Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions. Nat Immunol, 2016; 17(12):1361–1372; doi: 10.1038/ni.3590
58.
LermM, BrodinVP, RuishalmeI, et al.Inactivation of Cdc42 is necessary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation. J Immunol, 2007; 178(11):7357–7365; doi: 10.4049/jimmunol.178.11.7357
59.
AikemuB, ShaoY, YangG, et al.NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity. Int J Biol Sci, 2021; 17(7):1716–1730; doi: 10.7150/ijbs.56694
60.
TetreaultMP, WeinblattD, ShaverdashviliK, et al.KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification. Sci Rep, 2016; 6:26130; doi: 10.1038/srep26130
61.
ZhouN, ChenJ, HuM, et al.SLC7A11 is an unconventional H(+) transporter in lysosomes. Cell, 2025; 188(13):3441–3458.e25; doi: 10.1016/j.cell.2025.04.004
62.
AitsS, KrickerJ, LiuB, et al.Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy, 2015; 11(8):1408–1424; doi: 10.1080/15548627.2015.1063871
63.
AiS, TaoJ, WangY, et al.Paeonol ameliorates diabetic nephropathy by promoting TFEB-mediated lysosome biogenesis and lipophagy. Phytomedicine, 2025; 143:156901; doi: 10.1016/j.phymed.2025.156901
64.
Guixé-MuntetS, de MesquitaFC, VilaS, et al.Cross-talk between autophagy and KLF2 determines endothelial cell phenotype and microvascular function in acute liver injury. J Hepatol, 2017; 66(1):86–94; doi: 10.1016/j.jhep.2016.07.051
65.
BonacinaF, ZhangX, ManelN, et al.Lysosomes in the immunometabolic reprogramming of immune cells in atherosclerosis. Nat Rev Cardiol, 2025; 22(3):149–164; doi: 10.1038/s41569-024-01072-4
66.
WellingLC, WellingMS, TeixeiraMJ, et al.Chronic subdural hematoma: So common and so neglected. World Neurosurg, 2018; 111:393–394; doi: 10.1016/j.wneu.2018.01.131
67.
ChenC, XiongY, HuangX, et al.Subperiosteal/subgaleal drainage vs. subdural drainage for chronic subdural hematoma: A meta-analysis of postoperative outcomes. PLoS One, 2023; 18(8):e0288872; doi: 10.1371/journal.pone.0288872
68.
LiuT, ZhaoZ, LiuM, et al.The pharmacological landscape of chronic subdural hematoma: A systematic review and network meta-analysis of randomized and non-randomized controlled studies. Burns Trauma, 2024; 12:tkae034; doi: 10.1093/burnst/tkae034
69.
MorimotoK, JanssenWJ, FesslerMB, et al.Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for chronic obstructive pulmonary disease. J Immunol, 2006; 176(12):7657–7665; doi: 10.4049/jimmunol.176.12.7657
70.
TetreaultMP, YangY, TravisJ, et al.Esophageal squamous cell dysplasia and delayed differentiation with deletion of krüppel-like factor 4 in murine esophagus. Gastroenterology, 2010; 139(1):171–181.e9; doi: 10.1053/j.gastro.2010.03.048
71.
KharasMG, YusufI, ScarfoneVM, et al.KLF4 suppresses transformation of pre-B cells by ABL oncogenes. Blood, 2007; 109(2):747–755; doi: 10.1182/blood-2006-03-011106
72.
ZhangX, WangL, HanZ, et al.KLF4 alleviates cerebral vascular injury by ameliorating vascular endothelial inflammation and regulating tight junction protein expression following ischemic stroke. J Neuroinflammation, 2020; 17(1):107; doi: 10.1186/s12974-020-01780-x
73.
ZhuH, WangW, ZhuJ, et al.Methylglyoxal deteriorates macrophage efferocytosis in diabetic wounds through ROS-induced ubiquitination degradation of KLF4. Free Radic Biol Med, 2025; 231:23–37; doi: 10.1016/j.freeradbiomed.2025.02.030
74.
HodgeRG, RidleyAJ. Regulating Rho GTPases and their regulators. Nat Rev Mol Cell Biol, 2016; 17(8):496–510; doi: 10.1038/nrm.2016.67
75.
HaradaY, TanakaY, TerasawaM, et al.DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses. Blood, 2012; 119(19):4451–4461; doi: 10.1182/blood-2012-01-407098
76.
SarantisH, GrinsteinS. Subversion of phagocytosis for pathogen survival. Cell Host Microbe, 2012; 12(4):419–431; doi: 10.1016/j.chom.2012.09.001
77.
BealesILP, OgunwobiOO. Leptin activates Akt in oesophageal cancer cells via multiple atorvastatin-sensitive small GTPases. Mol Cell Biochem, 2021; 476(6):2307–2316; doi: 10.1007/s11010-021-04067-8
78.
CichaI, Schneiderhan-MarraN, YilmazA, et al.Monitoring the cellular effects of HMG-CoA reductase inhibitors in vitro and ex vivo. Arterioscler Thromb Vasc Biol, 2004; 24(11):2046–2050; doi: 10.1161/01.ATV.0000145943.19099.a3
79.
BeemillerP, ZhangY, MohanS, et al.A Cdc42 activation cycle coordinated by PI 3-kinase during Fc receptor-mediated phagocytosis. Mol Biol Cell, 2010; 21(3):470–480; doi: 10.1091/mbc.e08-05-0494
80.
FujiiM, KawaiK, EgamiY, et al.Dissecting the roles of Rac1 activation and deactivation in macropinocytosis using microscopic photo-manipulation. Sci Rep, 2013; 3:2385; doi: 10.1038/srep02385
81.
AstutiY, RaymantM, QuarantaV, et al.Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis. Nat Cancer, 2024; 5(5):774–790; doi: 10.1038/s43018-024-00731-2
82.
FanPL, LaiHQ, WangHY, et al.CKLF1 disrupts microglial efferocytosis following acute ischemic stroke by binding to phosphatidylserine. Cell Death Differ, 2025; 32(8):1499–1517; doi: 10.1038/s41418-025-01475-x
WangB, Martini-StoicaH, QiC, et al.TFEB-vacuolar ATPase signaling regulates lysosomal function and microglial activation in tauopathy. Nat Neurosci, 2024; 27(1):48–62; doi: 10.1038/s41593-023-01494-2
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
