Endothelial protein C receptor promotes retinal neovascularization through heme catabolism

Song, Hongyuan; Li, Qing; Gui, Xiao; Fang, Ziyu; Zhou, Wen; Wang, Mengzhu; Jiang, Yuxin; Geng, Ajun; Shen, Xi; Liu, Yongxuan; Zhang, Haorui; Nie, Zheng; Zhang, Lin; Zhu, Huimin; Zhang, Feng; Li, Xuri; Luo, Fanyan; Zhang, Hongjian; Shen, Wei; Sun, Xiaodong

doi:10.1038/s41467-025-56810-0

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Published: 13 February 2025

Endothelial protein C receptor promotes retinal neovascularization through heme catabolism

Hongyuan Song ORCID: orcid.org/0000-0001-8187-2799^1,2,3,4^na1,
Qing Li^1,5^na1,
Xiao Gui^1,6^na1,
Ziyu Fang⁷^na1,
Wen Zhou¹^na1,
Mengzhu Wang¹,
Yuxin Jiang^2,8,
Ajun Geng^9,10,
Xi Shen¹¹,
Yongxuan Liu¹,
Haorui Zhang¹,
Zheng Nie¹,
Lin Zhang¹,
Huimin Zhu¹,
Feng Zhang ORCID: orcid.org/0000-0002-6103-1498¹²,
Xuri Li ORCID: orcid.org/0000-0002-8277-0767¹²,
Fanyan Luo ORCID: orcid.org/0000-0002-1195-4585¹³,
Hongjian Zhang ORCID: orcid.org/0000-0002-1937-2766^4,14,
Wei Shen ORCID: orcid.org/0000-0001-5754-316X¹ &
…
Xiaodong Sun ORCID: orcid.org/0000-0001-5015-0945^2,3,8

Nature Communications volume 16, Article number: 1603 (2025) Cite this article

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Abstract

Pathological retinal neovascularization (RNV) is one of the leading causes of blindness worldwide; however, its underlying mechanism remains unclear. Here, we found that the expression of endothelial protein C receptor (Epcr) was increased during RNV, and its ligand was elevated in the serum or vitreous body of patients with proliferative diabetic retinopathy. Deleting endothelial Epcr or using an EPCR-neutralizing antibody ameliorated pathological retinal angiogenesis. EPCR promoted endothelial heme catabolism and carbon monoxide release through heme oxygenase 1 (HO-1). Inhibition of heme catabolism by deleting endothelial Ho-1 or using an HO-1 inhibitor suppressed pathological angiogenesis in retinopathy. Conversely, supplementation with carbon monoxide rescued the angiogenic defects after endothelial Epcr or Ho-1 deletion. Our results identified EPCR-dependent endothelial heme catabolism as an important contributor to pathological angiogenesis, which may serve as a potential target for treating vasoproliferative retinopathy.

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Introduction

Pathological retinal neovascularization (RNV) is a major cause of vision loss worldwide^1,2. The most common causes include diabetic retinopathy, retinopathy of prematurity (ROP), and retinal vein occlusion, which affect millions of people^3,4,5. The aberrant vessel growth usually leads to macular edema, fibrotic scarring, and retinal detachment^2,6. These conditions are the primary causes of blindness in working-age and pediatric populations in developed countries^6,7. Endothelial cells (ECs) rarely proliferate and remain in a quiescent state in the adult organism, whereas uncontrolled proliferation of ECs is initiated and drives aberrant vessel growth under pathological stimuli⁸. Vascular endothelial growth factor (VEGF) is extensively studied in the regulation of EC proliferation and angiogenesis^9,10. Anti-VEGF regents are first-line therapy for RNV, but resistance occurs in certain patients^11,12. Hence, it is necessary to identify novel therapeutic targets of RNV.

Endothelial protein C receptor (EPCR, also known as PROCR) is a single-pass transmembrane glycoprotein expressed in ECs and stem cells^13,14. In addition to its anti-coagulation role¹⁵, EPCR mediates intracellular signaling of activated protein C (APC)¹⁶. Activation of downstream protein kinase B (AKT) promotes cell growth, improves cardiac function, and favors tumor progression^{16,17,18,19,20}. Lineage tracing of Epcr-positive cells indicate that Epcr is specifically expressed in the ECs of the retinal vasculature²¹. Epcr transcription is activated in the retinal vessels during the angiogenic expansion stage but not during the quiescent stage²¹. Ablation of Epcr-positive cells leads to delayed retinal vascular expansion in postnatal mice, suggesting a role for EPCR in retinal angiogenesis²². However, the role of EPCR in RNV pathology remains unclear.

This study found that Epcr transcription was activated in ECs during RNV. Conditional knockout of Epcr in ECs or using an EPCR-neutralizing antibody ameliorated pathological RNV. Further data showed that EPCR affected heme catabolism by regulating heme oxygenase 1 (HMOX1, also known as HO-1). Conditional knockout of Ho-1 in ECs or treatment with an HO-1 enzymatic inhibitor suppressed pathological angiogenesis in retinopathy. Carbon monoxide-releasing molecule-3 (CORM3) partially rescued the reduced neovascularization caused by the deletion of endothelial Epcr and Ho-1, indicating a role for carbon monoxide (CO). Clinically, levels of APC, which is an EPCR ligand, were elevated in proliferative diabetic retinopathy (PDR) patients’ serum and vitreous body. These results indicate that EPCR-dependent endothelial heme catabolism is an important contributor to RNV.

Results

EPCR is highly expressed in retinal ECs during RNV

The mouse model of oxygen-induced retinopathy (OIR) serves as a proxy for human pathological RNV, including PDR and ROP²³. The neovascular response peaked on postnatal day 17 (P17) when the mice were returned to room air for five days (Fig. 1a). To identify the potential genes involved in pathological neovascularization, we performed transcriptomic analyses of the retinas of P17 mice of OIR (GSE241239). To minimize the RNA-seq bias, we further analyzed transcriptomic data from two other independent studies (GSE194176 and GSE158799)^24,25. A combined analysis revealed 303 upregulated genes (Fig. 1b, c). Among these candidate genes, we decided to focus on Epcr because EPCR was reported to be specifically detected on the surface of vascular ECs and stem cells^13,14. Further data confirmed that Epcr expression was upregulated in the retinas of P17 mice in the OIR (Fig. 1d, e). We sorted ECs from the mouse retina to evaluate the expression of Epcr in the retinal vasculature. Endothelial Epcr expression was much higher in the OIR group than in the control group (Fig. 1f, g).

**Fig. 1: EPCR is specifically highly expressed in retinal neovascularization.**

Next, we examined the expression pattern of EPCR in the retina. Immunofluorescence staining with an anti-EPCR antibody showed that EPCR was selectively detected in the retinal ECs of P17 mice under physiological conditions (Supplementary Fig. 1a). In the OIR model, EPCR was detected only in retinal ECs, and the fluorescence intensity of EPCR signals was much higher in the neovascular tufts (Supplementary Fig. 1b, c). Epcr-CreER^T2 (also known as Procr-CreER^T2) mice were previously used to mark proliferative ECs in the retinal vasculature²¹. Here, we used Epcr-CreER^T2 and Rosa26^tdT mice to perform genetic lineage tracing of Epcr-positive cells in the retina of P17 mice. Tamoxifen (TAM) was administered for three consecutive days at P10, P11, and P12, and the retina was analyzed at P17 (Supplementary Fig. 1d). Under physiological conditions, a very small proportion of Epcr+ cells were observed (Fig. 1h). There was a large proportion of Epcr+ cells in the retina of OIR mice, and Epcr+ cells mainly colocalized with neovascular tufts (Fig. 1h, i, Supplementary Fig. 1e). These data indicated that hypoxia significantly increased the transcription of Epcr. We also assessed the protein levels of EPCR in the retina of db/db mice with early-stage diabetes. The data showed that there was no difference in the protein levels of EPCR between db/db mice and db/m mice (Supplementary Fig. 1f–h). Hypoxia is also known to play a vital role in the retinal vascular development process²⁶. Therefore, the expression pattern of EPCR in the retinal vasculature of postnatal mice was then assessed. Immunofluorescence staining with anti-EPCR showed that the levels of EPCR were much higher in the margin of the retinal vasculature (Supplementary Fig. 1i). Consistent data were acquired using Epcr-CreER^T2 and Rosa26^tdT mice (Supplementary Fig. 1j, k). Collectively, these data suggest that EPCR is specifically detected in ECs of the retina and is upregulated in neovascularization in the OIR model and during retinal vascular expansion.

EPCR activates downstream signaling pathways by binding to its ligands. Thus, we measured the serum concentrations of APC, a major ligand for EPCR, in OIR mice. The data showed that the levels of APC in the serum of OIR mice were higher than those in control mice (Fig. 1j). To evaluate whether the findings in mice hold promise for human patients with RNV, we investigated the levels of APC in the vitreous humor and serum in patients with PDR. The concentrations of APC in the vitreous humor and serum were significantly higher in patients with PDR than in the control with a macular hole or idiopathic epiretinal membrane (Fig. 1k, l).

EPCR contributes to RNV in vivo and in vitro

To determine the role of EPCR in RNV, we intercrossed the Cdh5-CreER^T2 mouse with Epcr^flox/flox mouse to generate EC-specific Epcr KO mice (Epcr^iΔEC). The protein levels of EPCR were almost completely ablated in ECs sorted from Epcr^iΔEC mice (Supplementary Fig. 2a, b). Cre toxicity has been reported in another study²⁷, whereas our data showed that this mice strain did not cause vascular changes in the OIR model (Supplementary Fig. 2c–e). To assess the role of EPCR in pathological RNV, we examined the changes in the retinal vasculature of Epcr^iΔEC mice of OIR at P17. Our data showed that the area of neovascular tufts in the retina of Epcr^iΔEC mice was smaller than that in Epcr^WT mice (Fig. 2a, b). In contrast, the avascular area in Epcr^iΔEC mice’s retina was larger than that in Epcr^WT mice (Fig. 2a, c). Furthermore, we used 5-ethynyl-2’-deoxyuridine (EdU) to label proliferative cells in the retina and used E26 transformation-specific (ETS)-related gene (ERG) as an EC marker. The result showed that EdU⁺ ECs in the retina decreased significantly in Epcr^iΔEC mice compared to Epcr^WT mice (Fig. 2d, e). Retinal vascular leakage was also attenuated in Epcr^iΔEC mice’s retina compared to the retina of Epcr^WT mice (Supplementary Fig. 2f, g). Meanwhile, the data indicated that the deletion of endothelial Epcr did not affect the mature physiological angiogenesis of mice at P17 (Supplementary Fig. 2h, i). These in vivo results suggest that upregulated EPCR in pathological vasculature drives EC growth and angiogenesis in OIR.

Fig. 2: Loss of endothelial Epcr suppresses pathological RNV. — **Fig. 2: Loss of endothelial *Epcr* suppresses pathological RNV.**

Next, we determined the effect of Epcr deletion on retinal vascular expansion in postnatal retinas. EC-specific deletion of Epcr led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (Supplementary Fig 3a, b). Tip cells’ filopodia and branch points were decreased in the retina of endothelial Epcr KO mice (Supplementary Fig. 3c–e). Meanwhile, EC proliferation was also significantly inhibited in Epcr^iΔEC mice (Supplementary Fig. 3f, g).

To further evaluate the impact of elevated EPCR on angiogenesis, we used an adenovirus to overexpress EPCR and assessed the angiogenic behavior of ECs in vitro. EPCR adenovirus treatment significantly increased the protein levels of EPCR in ECs, which promoted EC proliferation in an EdU staining assay (Fig. 2f, g, Supplementary Fig. 3h). A three-dimensional endothelial spheroid assay showed that the length of endothelial sprouts were increased in the EPCR adenovirus treatment group (Fig. 2h, i). Moreover, a wound scratch assay revealed that EPCR adenovirus-treated ECs were more motile than control adenovirus-treated ECs (Fig. 2j, k). Furthermore, the knockdown of EPCR using siRNA suppressed EC proliferation, migration, and sprouting (Fig. 2l–q, Supplementary Fig. 3i). Together with in vivo results, these data indicated that EPCR promotes vascular growth.

Pharmacological inhibition of EPCR ameliorates pathological retinal neovascularization

To evaluate the translational potential of targeting EPCR in anti-angiogenic therapy, we assessed the effects of a neutralizing antibody against EPCR on pathological angiogenesis in vitro and in vivo. We found that a single dose of EPCR antibody significantly reduced the area of neovascular tufts compared to the vehicle and anti-IgG groups (Fig. 3a, b). Whereas the avascular area remained unchanged after EPCR antibody treatment (Fig. 3a, c). Suppressed proliferation of ECs in retinal neovascular tufts was also observed after treatment with the EPCR antibody (Fig. 3d, e). Similar results were obtained in vitro as EPCR antibody treatment significantly inhibited EC proliferation, migration, and sprouting (Fig. 3f–k). These results were consistent with our findings using Epcr^iΔEC mice, suggesting the translational potential of anti-EPCR in the treatment of retinal neovascularization.

**Fig. 3: Pharmacological inhibition of EPCR attenuates pathological RNV.**

EPCR regulates the expression of HO-1 through NRF2/KEAP1

To reveal the possible molecular mechanisms underlying EPCR-regulated angiogenesis, we performed transcriptomic analyses of EPCR siRNA- or control siRNA-treated ECs (GSE249130, Supplementary Fig. 4a). These data were then combined and analyzed with the mice retina results with OIR (Supplementary Fig. 4b). Thirty genes were upregulated in the retina of OIR mice and downregulated in Epcr-depleted ECs (Fig. 4a). Among these genes, Ho-1 was the most significantly affected (Fig. 4a). Increased mRNA and protein levels of Ho-1 were confirmed in the retinas of OIR mice (Supplementary Fig. 4c, d). Further data showed that the expression of Ho-1 was suppressed at the mRNA and protein levels in ECs treated with EPCR siRNA (Fig. 4b, c). Additionally, the expression of Ho-1 was upregulated at the mRNA and protein levels in ECs treated with EPCR adenovirus (Fig. 4d, e). The expression of Ho-1 in retinal ECs of OIR mice was then evaluated. We sorted ECs from the retina of OIR mice using flow cytometry. The results indicated that the expression of Ho-1 in ECs was upregulated in the OIR group (Supplementary Fig. 4e, f). This was further confirmed by immunofluorescence staining. The expression of HO-1 in the physiological retina did not show an apparent pattern, while it was highly expressed in neovascular tufts of OIR retinas (Supplementary Fig. 4g, h). Further data showed that the expression of Ho-1 in ECs sorted from the retina of Epcr^iΔEC mice decreased compared with that in Epcr^wt mice (Fig. 4f, g). Collectively, these results suggested that HO-1 was a downstream molecule of EPCR in regulating angiogenesis.

AKT is recognized as a target of EPCR signaling²⁸. High EPCR expression or stimulation with APC usually leads to AKT activation^16,19. We found that overexpression of EPCR using an adenovirus promoted the activation of AKT, whereas silencing EPCR with siRNA inhibited AKT phosphorylation (Fig. 4h, i). Nuclear factor erythroid 2-related factor 2 (NRF2) is a ubiquitous transcription factor directly regulating HO-1²⁹. NRF2 is tightly regulated by Kelch-like ECH-associated protein 1 (KEAP1) through ubiquitination and proteasome-dependent degradation³⁰. Decreased expression of NRF2 and HO-1, and increased expression of KEAP1 were observed after EPCR knockdown in ECs (Fig. 4). Therefore, we assessed the effects of EPCR on the expression of KEAP1 and NRF2. Overexpression of EPCR in ECs promoted the levels of NRF2 and HO-1 and inhibited the expression of KEAP1 (Fig. 4i). Meanwhile, APC treatment stimulated the activation of AKT and increased the expression of NRF2 and HO-1 while suppressing the expression of KEAP1 (Supplementary Fig. 4i).

AKT activates NRF2 in multiple cells^31,32. Our data showed that inactivating AKT with LY294002 (a PI3K inhibitor) blocked the effect of EPCR overexpression on NRF2, KEAP1, and HO-1 (Fig. 4j). Consistent results were observed when ECs were treated with APC (Supplementary Fig. 4j). These results suggested that AKT plays an important role in mediating EPCR signaling. Furthermore, we observed that depleting the expression of KEAP1 using siRNA rescued the downregulation of NRF2 and HO-1 caused by EPCR siRNA (Fig. 4k, Supplementary Fig. 4k). Consistent results were obtained after treatment with Ki696 (a KEAP1 inhibitor) (Fig. 4l). These data suggested that EPCR controlled HO-1 expression via the AKT-KEAP1-NRF2 pathway.

Considering the critical role of VEGF in promoting RNV, we assessed the effect of EPCR signaling on VEGF. The results showed that neither knockdown nor overexpressing EPCR affected the protein levels of VEGFA (Supplementary Fig. 5a, b). The data suggested that endothelial EPCR signaling might be VEGF-independent. BTB and CNC Homology 1 (BACH1) is a transcription factor that regulates oxidative stress response and heme metabolism²⁹. Stromal Cell-Derived Factor 1 (SDF1), also known as CXCL12, has been shown to promote angiogenesis through HO-1³³. Thus, we further evaluated the role of BACH1 and SDF1 in EPCR signaling. The results showed that depletion of BACH1 in ECs could increase the protein levels of HO-1 (Supplementary Fig. 5c). However, the protein levels of BACH1 did not change in the retina of OIR mice compared to control mice (Supplementary Fig. 5d), and neither knockdown nor overexpressing EPCR affected the protein levels of BACH1 (Supplementary Fig. 5e, f). The data indicated that BACH1 did not participate in the endothelial EPCR signaling pathway. Consistent with a previous report^34,35, the results showed that the protein levels of SDF1 increased significantly in the retinal neo-vasculature in OIR mice (Supplementary Fig. 6a, b), and knockdown of SDF-1 decreased the expression of HO-1 (Supplementary Fig. 6c). SDF-1 usually interacts with C-X-C motif chemokine receptor 4 and activates downstream AKT signaling pathways to promote angiogenesis³³. Knockdown of EPCR in ECs could suppress the expression of SDF1, while overexpression of EPCR could increase the expression of SDF1 (Supplementary Fig. 6d, e). These data suggested that EPCR might regulate the expression of SDF-1 through AKT signaling. This was further confirmed as ECs treated with LY294002 exhibited reduced SDF-1 expression (Supplementary Fig. 6f). However, the knockdown of SDF1 did not reverse the increased expression of HO-1 induced by EPCR overexpression (Supplementary Fig. 6g). All these data suggested that EPCR regulated the expression of SDF-1 through of AKT signaling, and SDF-1 did not contribute to the expression of HO-1 regulated by EPCR.

Genetic ablation of endothelial Ho-1 attenuates angiogenesis

To evaluate the role of HO-1 in retinal angiogenesis, we bred Cdh5-CreER^T2 mice with Ho-1^flox/flox mice to generate EC-specific Ho-1 KO mice (Ho-1^iΔEC). The deletion efficiency was determined using flow cytometry-sorted ECs, and the protein levels of HO-1 in ECs sorted from Ho-1^iΔEC mice were significantly decreased (Supplementary Fig. 7a). The role of HO-1 in pathological retinal angiogenesis was then determined in the OIR mice. Our data showed that the area of neovascular tufts in the retina of Ho-1^iΔEC mice was smaller, while the avascular area in the retina of Ho-1^iΔEC mice was larger than that in Ho-1^WT mice (Fig. 5a–c). Furthermore, our results showed that EdU⁺ ECs decreased significantly in the retina of Ho-1^iΔEC mice than in Ho-1^WT mice (Fig. 5d, e).

Fig. 5: Endothelial deletion of Ho-1 inhibits pathological RNV. — **Fig. 5: Endothelial deletion of *Ho-1* inhibits pathological RNV.**

Physiological retinal vascular development was also affected in Ho-1^iΔEC mice. Deleting endothelial Ho-1 reduced radial expansion of the superficial retinal vascular plexus (Supplementary Fig. 7b, c). Meanwhile, the tip cells’ filopodia and branch points were decreased in the retina of Ho-1^iΔEC mice (Supplementary Fig. 7d–f). Additionally, EdU⁺ ECs decreased significantly in the retina of Ho-1^iΔEC mice than in Ho-1^WT mice (Supplementary Fig. 7g, h).

The role of HO-1 in angiogenesis was further assessed in vitro. First, the knockdown efficiency of HO-1 siRNA was evaluated (Supplementary Fig. 7i). The EdU assay showed that silencing HO-1 inhibited EC proliferation induced by the EPCR adenovirus (Fig. 5f, g). A three-dimensional endothelial spheroid assay was used to evaluate the angiogenic potential of ECs treated with EPCR adenovirus and HO-1 siRNAs. Fewer and shorter sprouts were observed in HO-1 siRNA-treated ECs than in EPCR adenovirus-treated ECs (Fig. 5h, i). Besides, ECs treated with HO-1 siRNAs exhibited impaired migration ability compared to the EPCR adenovirus treatment group (Fig. 5j, k). We also assessed the effects of HO-1 knockdown on ECs. The results showed that silencing HO-1 suppressed EC proliferation, migration, and sprouting (Supplementary Fig. 7j–o). These results collectively revealed the vital role of HO-1 in developmental angiogenesis and RNV.

Endothelial heme catabolism impacts angiogenesis

HO-1 catalyzes heme degradation into CO, biliverdin, and iron³⁶. Catabolism of cytotoxic labile heme and the generation of CO exert cytoprotective effects³⁷. Thus, we further assessed whether the angiogenic role of HO-1 was mediated by its enzymatic function. Here, a heme oxygenase (both HO-1 and HO-2) enzymatic inhibitor, zinc protoporphyrin IX (ZnPPIX), was used to determine its effect on pathological RNV. The data showed that ZnPPIX treatment decreased the area of neovascular tufts in the retina and increased the avascular area (Fig. 6a–c). Besides, ZnPPIX suppressed EC proliferation as EdU+ retinal ECs decreased significantly in mice treated with ZnPPIX (Fig. 6d, e).

**Fig. 6: Endothelial heme catabolism inhibition ameliorates RNV.**

Next, we determined the effect of ZnPPIX on retinal vascular expansion. ZnPPIX treatment led to a sparse vascular network and reduced radial expansion of the superficial retinal vascular plexus (Supplementary Fig. 8a, b). The tip cells’ filopodia and branch points were decreased in the retina of mice treated with ZnPPIX (Supplementary Fig. 8c–e). Meanwhile, ZnPPIX significantly suppressed ECs proliferation in the retina (Supplementary Fig. 8f, g).

Further data showed that EPCR-induced cell proliferation, migration, and sprouting of ECs in vitro were inhibited by ZnPPIX (Fig. 6f–k). Additionally, we evaluated the effect of ZnPPIX on ECs treated with HO-1 siRNA. The result showed that ZnPPIX did not affect EC proliferation, migration, and sprouting after HO-1 knockdown (Supplementary Fig. 8h–m). These data indicated that endothelial heme catabolism contributes to EPCR-mediated RNV.

Heme catabolism-derived CO contributes to pathological angiogenesis

Endogenous CO is a signaling molecule that promotes tumor growth and angiogenesis^38,39. Heme catabolism in macrophages increases tumor angiogenesis through CO production, suggesting a role for CO in regulating angiogenesis³⁷. Here, we showed that pathological retinal neovascular tufts in carbon monoxide-releasing molecule-3 (CORM3)-treated Ho-1^iΔEC mice significantly increased compared with that in Ho-1^iΔEC mice, while the avascular area was decreased (Fig. 7a–c). Furthermore, the data showed that EdU⁺ ECs also increased significantly in the CORM3-treated retina of Ho-1^iΔEC mice than that in Ho-1^iΔEC mice (Fig. 7d, e). Pathological retinal neovascular tufts in CORM3-treated Epcr^iΔEC mice also significantly increased, and avascular area decreased compared with that in Epcr^iΔEC mice (Supplementary Fig. 9a–c). Consistent data were acquired for the number of EdU⁺ ECs (Supplementary Fig. 9d, e). Meanwhile, the role of other byproducts of heme catabolism was evaluated. The data showed that bilirubin and iron supplements did not reverse the vascular changes caused by endothelial Epcr deletion (Supplementary Fig. 10).

Fig. 7: CO rescued the phenotype of Ho-1 deletion in vitro and in vivo. — **Fig. 7: CO rescued the phenotype of *Ho-1* deletion in vitro and in vivo.**

We then assessed the effect of CORM3 on postnatal retinal vasculature in Ho-1^iΔEC and Epcr^iΔEC mice. Treatment with CORM3 could rescue the sparse vascular network and increase the radial expansion of superficial retinal vascular plexus in Ho-1^iΔEC mice (Supplementary Fig. 11a, b). Compared with Ho-1^iΔEC mice, tip cells’ filopodia and branch points increased in the retina of Ho-1^iΔEC mice treated with CORM3 (Supplementary Fig. 11c–e). Consistent results were acquired in CORM3-treated Epcr^iΔEC mice (Supplementary Fig. 11f–j).

Furthermore, CORM3 rescued HO-1 silence-caused inhibition of EC proliferation, migration, and sprouting (Fig. 7f–k). Additionally, the effect of CORM3 on EPCR-deleted ECs was evaluated. The results showed that EPCR silencing inhibited EC proliferation, migration, and sprouting, which was partially reversed after treatment with CORM3 (Supplementary Fig. 12a–f). These results suggested that CO was an important contributor to EPCR-dependent endothelial heme catabolism-regulated angiogenesis.

Discussion

Therapeutic strategies for RNV are effective, but off-target effect and resistance limit their efficacy^12,40. The reason is that these therapies cannot distinguish between healthy and pathological vessels. Our current study showed that EPCR was selectively detected in ECs of the retina, and EPCR transcription was activated under angiogenic stimulus. Conditional deletion of Epcr in ECs or anti-EPCR neutralizing antibody treatment attenuated pathological retinopathy. Mechanistically, EPCR-controlled endothelial heme catabolism through HO-1 and heme catabolism-derived CO plays a vital role. Notably, the EPCR ligand levels were elevated in the vitreous body or serum of patients with PDR. These observations revealed the potential therapeutic value of targeting EPCR for suppressing pathological RNV (Fig. 8).

**Fig. 8: Illustration showing the expression pattern and role of EPCR during RNV.**

Here, we observed that the expression of EPCR was detected specifically in the retinal ECs and was upregulated in neovascular tufts using anti-EPCR staining. EPCR was not detected in retinal pigment epithelia, photoreceptor cells, or ganglion cells. Consistent with our previous work²¹, we found that Epcr transcription is activated during the vessel expansion stage in the retina. Meanwhile, Epcr transcription was activated in ECs during RNV using Epcr-CreER^T2; Rosa26^tdT mice. These findings revealed that EPCR expression was selectively detected on retinal ECs and was upregulated under angiogenic stimulus. Vascular endothelial growth factor receptor 2 (VEGFR2) is the major receptor for VEGF guiding angiogenesis⁴¹. However, VEGFR2 is widely detected in the retina, including ECs and neurons⁴². Sustained suppression of VEGF would damage the photoreceptors and lead to loss of vision^43,44. It is observed in the clinic that retinal atrophy occurs in patients who received anti-VEGF treatment⁴⁵. The specific expression pattern of EPCR in the retina indicates that targeting EPCR is unlikely to induce off-target effects.

It is reported that Epcr + ECs are highly proliferative and major contributors to vessel development²¹. Conditional expression of diphtheria toxin (DTA) in Epcr+ cells delays retinal vessel extension in postnatal mice, indicating a role for EPCR in angiogenesis²². Consistent data was acquired in our study that conditional deletion of Epcr in ECs suppressed the expansion of the retinal vascular plexus. Using the OIR mice model, we showed that endothelial deletion of Epcr or EPCR neutralization antibody reduced ECs proliferation and attenuated pathological RNV. The angiogenic role of EPCR in neovascularization is also observed in hindlimb ischemia mice⁴⁶. Deletion of Epcr in ECs using Tie2-Cre; Epcr^flox/flox mice suppresses new vessel formation⁴⁶. Given that Epcr transcription is specifically activated in proliferating ECs²¹, EPCR blockade could be a novel option for treating RNV with minimal effects on quiescent ECs. However, it is also noteworthy that endothelial deletion of Epcr or Ho-1 could impair the physiological vascular regeneration during OIR, as evidenced by increased vaso-obliteration observed at P17. Consistent data were acquired that inhibition of EPCR or HO-1 suppresses retinal physiological vascular development at P7. Therefore, the EPCR blocking strategy could be specific to proliferating ECs but not to pathological angiogenesis.

The mechanism by which EPCR contributes to RNV remains to be revealed. We found that the deletion of EPCR in ECs reduced the expression of HO-1 through the AKT/KEAP1/NRF2 pathway. In comparison, EPCR ligand (APC) treatment upregulated protein levels of HO-1 via AKT/KEAP1/NRF2. The data were consistent with studies showing that EPCR activates AKT signaling in the heart and multiple cells^16,17,18,19. The levels of the EPCR ligand were elevated in the serum or vitreous body of patients with PDR, suggesting that it activates EPCR signaling to favor retinal neovascularization. KEAP1 is a negative regulator of NRF2, affecting the expression of NRF2 in ECs⁴⁷. NRF2 is known to directly regulate the expression of HO-1 during tumor growth and angiogenesis^29,48. However, its role in retinal angiogenesis is complex. NRF2 is detected in retinal ganglion cells, astrocytes, müller glial cells, and ECs, with prominent expression in retinal ganglion cells and müller glial cells during retinopathy^49,50. Enhancing NRF2 activity in retinal neurons can attenuate diabetic retinopathy and inhibit RNV⁴⁹. Meanwhile, endothelial deletion of Nrf2 delays vascular expansion and decreases vascular density⁵¹. Our data showed that the deletion of EPCR in ECs decreased the protein levels of NRF2 and HO-1 and inhibited the angiogenic function of ECs. These results indicate a notable difference in the cellular context of NRF2 action, and NRF2 plays a vital role in regulating HO-1 after endothelial deletion of EPCR.

Here, we showed that EC-specific deletion of Ho-1 suppressed retinal neovascularization in OIR and postnatal mice’s retina. HO-1 catalyzes heme degradation to CO, ferrous iron, and biliverdin⁵². While initially considered a waste product, CO is increasingly recognized as a cytoprotective and homeostatic molecule⁵³. Heme catabolism-derived CO is a major source of endogenous CO⁵⁴, which contributes to tumor metastasis and angiogenesis³⁷. In ECs, CO is reported to increase cell growth in vitro, whereas its role in vivo is unknown³⁸. Here, our data indicate that heme catabolism-derived CO promoted retinal angiogenesis, revealing a vital role of CO in angiogenesis. Clinically, the levels of exhaled CO significantly increase in patients with diabetes and correlate with blood glucose levels and disease duration^55,56. PDR, characterized by uncontrolled RNV, is a late-stage microvascular complication of diabetes³. Elevated levels of APC in the serum or vitreous body of patients with PDR could activate EPCR signaling. This data suggests that this upregulated EPCR signaling may enhance heme catabolism and increase CO production, potentially contributing to RNV. However, the precise roles of EPCR and heme catabolism in the retinas of patients with PDR require further investigation.

In summary, we determined that EPCR is highly expressed in retinal ECs and that blocking EPCR attenuates pathological retinal angiogenesis. Mechanistically, EPCR controls heme catabolism via the KEAP1/NRF2/HO-1 pathway, and CO derived from heme catabolism plays a vital role in RNV. Hence, our findings elucidate a novel mechanism of RNV and suggest that EPCR may be a promising therapeutic target for translational application. However, these mouse models do not accurately represent the pathological progression of retinopathy in primates. Therefore, determining the therapeutic efficiency of anti-EPCR for pathological retinal angiogenesis in non-human primates is indispensable in the future. Moreover, the expression pattern of EPCR in the human retina is unknown. For translational significance, it is important to determine the expression pattern of EPCR in post-mortem retinas under diverse pathophysiological conditions.

Methods

Patient samples

The patients diagnosed with proliferative diabetic retinopathy (PDR), idiopathic epiretinal membrane (ERM), and idiopathic macular hole (MH) were included. Vitreous samples were obtained from patients who underwent vitrectomy surgery by the same surgeon. Details of the patient information are shown in Supplementary Table 1. The collected vitreous samples were placed on ice immediately and centrifuged to exclude the debris. The supernatants were aliquoted into sterile tubes and stored in liquid nitrogen. Serum samples were obtained from patients who were diagnosed with PDR. The control serum samples were obtained from patients who were diagnosed with ERM and MH. Details of the patient information are shown in Supplementary Table 2. The obtained serum samples were centrifuged at 4 °C, and the supernatants were stored at −80 °C. All surgeries were performed, and human samples were harvested in accordance with the principles in the Declaration of Helsinki. This study was approved by the institutional research ethics committee of Shanghai General Hospital ([2022]-109). Individuals provided written informed consent prior to their participation in the study.

Mice

All the experimental animals were accommodated in animal facilities, where they were subjected to 12-hour cycles of light and darkness. These animals were provided with unrestricted access to standard chow and water. The C57BL/6 J mice were purchased from Jihui Laboratory Animal Care Co., Ltd. Epcr ^flox/flox mice (the Cyagen Biotechnology Co., Ltd.) and Ho-1^flox/flox mice (the Cyagen Biotechnology Co., Ltd.) were crossed with Cdh5 CreER^T2 mice⁵⁷ respectively to generate conditional knockout mice. For the lineage tracing of Epcr+ cells, the Epcr CreER^T2 mice²¹ were crossed to Rosa26^tdTomato reporter mice (the Cyagen Biotechnology Co., Ltd.) The body weight measurement and body weight analysis of mice were shown in Supplementary Table 3, 4. All animal studies were approved by the Institutional Animal Care and Use Committee at Shanghai Changhai Hospital (CHEC (A.E)2023-024).

Oxygen-induced retinopathy (OIR)

Oxygen-induced retinopathy (OIR) was performed as previously reported²⁵. In brief, the pups were exposed to 75% oxygen with their nursing mothers for five days from P7 to P12. Then, the pups were returned to room air at P12. When the mice were returned to room air, the hypoxia-induced neovascularization was initiated and peaked on P17. Animals were euthanized, and the retinas were harvested at P17. To activate CreER^T2, 50 μl of tamoxifen (2 mg/ml) was injected intraperitoneally from P10 to P12. To evaluate the anti-angiogenic role of EPCR neutralizing antibody, mice were randomly divided into three groups and injected intravitreally with 1 μl anti-EPCR antibody, PBS, and anti-IgG, respectively, at P12. Ocular surface anesthesia was performed using one drop of 0.5% proparacaine hydrochloride (ALCON-COUVREUR N.V.) before injection. ZnPPIX (25 mg/kg) and CORM3 (40 mg/kg) were injected intraperitoneally twice at P12 and P13. To detect proliferating cells, 50 μl EdU (50 mg/kg) was administered intraperitoneally 6 h before euthanasia. The neovascular tuft and avascular areas were measured using the Freehand selection and Measurement tools in Image J software and then divided by the total retinal area. Mice (P17) with body weight lower than 6 g were excluded from the analysis.

RNA-Seq sample preparation and sequencing

Total RNA from retinas and cells was extracted with RNA Easy Fast Tissue/Cell Kit (TIANGEN). Agilent 2100 Bioanalyzer and RNA nano 6000 assay kit (Agilent Technologies) were used to evaluate the quality of RNA samples. The transcriptome sequencing library was constructed through 1 ng/µl RNA randomly fragmentation, cDNA strand 1 / strand 2 synthesis, end repair, A-tailing, ligation of sequencing adapters, size selection, and library PCR enrichment. The integrity of cDNA was evaluated by Agilent 2100 Bioanalyzer. The library preparations were sequenced on an Illumina HiSeq 2500 platform (Illumina, USA). The raw data of sequences were uploaded to the NCBI Sequence Read Archive (SRA) database (GSE241239 for the retinas of OIR mice; GSE249130 for HUVECs with EPCR deletion).

Isolation of retinal ECs

Isolation of retinal ECs was performed according to protocols we previously described with some modifications²¹. The retinas were separated from eyeballs then minced into fine fragments and digested in Iscove’s Modified Dulbecco’s MediumIs (IMDM, LI1090-500, BioAgrio) with 1.5 mg/ml Collagenase H (11087789001, Roche), 1.25 mM CaCl₂, 0.4 mM MgCl₂, 1% penicillin/streptomycin and 3.5 μg/ml DNaseI (D4263, Sigma) at 37 °C for 45 min. The samples were pipetted every 10 min using a 1 ml pipette tip to ensure even digestion. The digestion mix was passed through a 40 μm nylon mesh to prepare single-cell suspensions. Cells were then incubated for 20 min with FITC-conjugated anti-CD31 (11-0317-82, Invitrogen, diluted 1:100) and PE-Cy7 conjugated anti-CD45 (25-0451-82, Invitrogen, diluted 1:200) in PBS with 5% FBS. Then, the single-cell suspensions were subjected to FACS using SONY ID7000 or LSR Fortessa (BD Biosciences). FACS data were analyzed by FlowJo software. The purity of sorted ECs was routinely checked and ensured to be more than 95%.

Real-time PCR analysis

Retinas were homogenized completely through a tissue grinding apparatus (Bioprep-24R, ALLSHENG). Total RNA was extracted from homogenized retina and HUVECs using MolPure® TRIeasy Plus Total RNA Kit (19211ES60, YEASEN). cDNA was prepared using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (11141ES, YEASEN) following manufacturer instructions. Quantitative PCR performed with Hieff UNICON® Universal Blue qPCR AYBR Green Master Mix (11184ES, YEASEN) in Bio-Rad CFX Manager 2.1 Detection system (Bio-Rad, Hercules, CA, USA). Relative gene expression was calculated with the ΔΔCT method and normalized to the expression of β-actin. Primer sequences are provided in Supplementary Table 5.

Retinal dissection and whole-mount staining

Eyes were fixed in 4% paraformaldehyde (PFA) for 0.5 h at RT. After dissection, retinas were blocked in a blocking buffer (1% BSA, 5% FBS, and 0.5% Triton-X-100 in PBS) for 1 h at RT. Primary antibodies were diluted in PBS containing 1% BSA and incubated with the retinas at 4 °C overnight. After being washed twice with PBS, retinas were incubated with secondary antibodies at RT for 2 h. Then, the retinas were washed and flat-mounted with Fluoromount-G (SouthernBiotech). For the labeling of EdU, an additional step was performed to detect EdU-labeled proliferative cells using the Click-It EdU kit (C10338, Invitrogen) prior to mounting. Images were acquired with a Mica confocal microscope (Leica Microsystems). For the quantification of proliferating ECs in the OIR model, high-resolution confocal images (20x magnification) of IB4, ERG, and EdU labeled retinal vasculature were acquired. Four fields were taken for quantification per retina, and the total number of EdU and ERG double-positive cells were counted by image-J software per field. Then, the mean of each retina was calculated subsequently. The mean from each group was statistically compared using GraphPad Prism, and a student’s t-test was performed to calculate the p-value.

Quantification and statistical analysis

All calculations were performed using GraphPad Prism (GraphPad software 9.0, GraphPad, Bethesda, MD, USA). Statistical analysis was performed using the two-tailed Student’s t-test and one-way analysis of variance (ANOVA), where appropriate, to compare different groups. For all bar graphs, data were presented as mean ± SD. All experiments were repeated at least three times, and P values of <0.05 were considered as statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The datasets generated during the current study are available in the GEO repository, GSE 241239 and GSE 249130. Source data are provided with this paper. The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE 241239 and GSE 249130. Source data are provided with this paper.

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Acknowledgements

Thanks to Yi Arial Zeng (State Key Laboratory of Cell Biology, Chinese Academy of Sciences) for kindly providing Epcr-CreER^T2 mice and technical assistance; to Youheng Wei (State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China) for providing technical assistance. This work was supported by grants from National Natural Science Foundation of China (82171081, 82271106, 82388101, U22A20311, 82171076), National Key R&D Program (2022YFC2502800), Shanghai Municipal Education Commission (2023ZKZD18), Shanghai Science and Technology Committee (22ZR1478200), Shanghai Changhai Hospital (2023YQ01). Figure 1a, Fig. 8, and Supplementary Fig. 1d, j were created using BioRender.com.

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These authors contributed equally: Hongyuan Song, Qing Li, Xiao Gui, Ziyu Fang, Wen Zhou.

Authors and Affiliations

Department of Ophthalmology, Shanghai Changhai Hospital, Shanghai, China
Hongyuan Song, Qing Li, Xiao Gui, Wen Zhou, Mengzhu Wang, Yongxuan Liu, Haorui Zhang, Zheng Nie, Lin Zhang, Huimin Zhu & Wei Shen
Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Hongyuan Song, Yuxin Jiang & Xiaodong Sun
National Clinical Research Center for Ophthalmic Diseases, Shanghai, China
Hongyuan Song & Xiaodong Sun
Oriental Pan-Vascular Devices Innovation College, University of Shanghai for Science and Technology, Shanghai, China
Hongyuan Song & Hongjian Zhang
The Affiliated Eye Hospital of Nanjing Medical University, Nanjing, China
Qing Li
Department of Ophthalmology, Yuanwang Hospital, Wuxi, China
Xiao Gui
Department of Urology, Shanghai Changhai Hospital, Shanghai, China
Ziyu Fang
Shanghai Key Laboratory of Fundus Diseases, Shanghai, China
Yuxin Jiang & Xiaodong Sun
Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, Hangzhou, China
Ajun Geng
New Cornerstone Science Laboratory, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
Ajun Geng
Department of Ophthalmology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Xi Shen
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
Feng Zhang & Xuri Li
Department of Cardiac Surgery, Xiangya Hospital, Central South University, Changsha, China
Fanyan Luo
Shidong Hospital Affiliated to University of Shanghai for Science and Technology, 999 Shiguang Road, Shanghai, China
Hongjian Zhang

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Contributions

Conceptualization, H.S., X.D.S., W.S., H.J.Z., and F.L.; Methodology, Q.L., X.S., and A.G.; investigation, H.S., Q.L., X.G., Z.F., W.Z., M.W., Y.L., H.R.Z., Z.N., L.Z., H.M.Z., and Y.J.; Funding acquisition, X.D.S., H.S. and W.S.; Writing-original draft, H.S and Q.L.; Writing-review & editing, X.D.S., F.Z., F.L., and X.L.; Supervision, H.S., X.D.S., X.L. and F.Z. All authors approved the final version of the manuscript.

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Correspondence to Hongyuan Song, Fanyan Luo, Hongjian Zhang, Wei Shen or Xiaodong Sun.

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Song, H., Li, Q., Gui, X. et al. Endothelial protein C receptor promotes retinal neovascularization through heme catabolism. Nat Commun 16, 1603 (2025). https://doi.org/10.1038/s41467-025-56810-0

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Received: 29 March 2024
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Published: 13 February 2025
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DOI: https://doi.org/10.1038/s41467-025-56810-0

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