APEX1 enhances endothelial regeneration and prevents neointima thickening by promoting cell proliferation, migration, adhesion, and junction formation

To investigate the role of APEX1 in post-injury endothelial regeneration, we generated inducible EC-specific Apex1-deletion Apex1iECKO (Apex1flox/flox, Cdh5-CreERT2+) and corresponding controls Apex1WT (Apex1wt/wt, Cdh5-CreERT2+) as we described previously [8]. Cre expression was induced by intraperitoneal injection of tamoxifen (50 mg/kg/day) for 7 consecutive days. Subsequently, guidewire-induced injury was performed on the left carotid artery, and vessels were harvested for evaluation at 5 or 28 days post-operation (Fig. 1A). Evans blue staining at 5 days post-operation revealed increased endothelial denudation in Apex1iECKO mice, with the denudated area measuring 65.8% larger compared to Apex1WT controls (Fig. 1B). Furthermore, neointima areas in Apex1iECKO mice were significantly thicker than those in Apex1WT mice 28 days post-injury (Fig. 1C).

Fig. 1

APEX1 Enhances Endothelial Regeneration and Prevents Neointimal Thickening by Promoting Cell Proliferation, Migration, Adhesion, and Junction Formation. A Schematic diagram of animal experiment. B Left: Representative images of left carotid arteries from the indicated mice at 5 days after guidewire induced injury. Red parts stand for Evans blue positive area. Right: Quantification of Evans blue positive area (%). n = 10 mice. C Right: Representative H&E staining of neointima in carotid arteries from the Apex1WT and Apex1iECKO mice at 28 days after guidewire induced injury. Right: Quantification of neointima area. n = 6 mice. D Left: Representative cell cycle analysis of PI flow cytometry of HUVECs with Ad-Null or Ad-APEX1 infection. Right: Quantification of S phase (%) of HUVECs. n = 6 biological replicates. E Up: Representative images of scratch assays of HUVECs with Ad-Null or Ad-APEX1 infection at 0, 3 and 6 h after scratching. Down: Quantification of relative migration area. n = 6 biological replicates. F Up: Representative immunofluorescence of HUVECs adhesion with Ad-Null or Ad-APEX1 infection at 0.5, 1, 2 and 4 h after adding cell suspension. Down: Quantification of relative area. n = 9 images from 3 biological replicates. G Up: Representative immunofluorescence of scratch assays of HUVECs with Ad-Null or Ad-APEX1 infection at 3 h after scratching. Down: Quantification of VE-Cadherin length per cell (pixel). n = 9 images from 3 biological replicates. H Representative en face immunofluorescence of aortic arches from the Apex1WT and Apex1iECKO. Data were all expressed as the means ± SD

As EC proliferation, migration, and adhesion are essential for endothelial regeneration [2], we examined whether APEX1 influences these processes using human umbilical vein endothelial cells (HUVECs). Overexpression of APEX1 via infecting cells with an APEX1-Flag adenovirus (Ad-APEX1) compared to control adenovirus (Ad-Null) was confirmed (Fig. S1). The fusion of the Flag motif with the APEX1 protein caused a shift in the overexpressed APEX1-Flag band. Flow cytometry following propidium iodide (PI) staining demonstrated that Ad-APEX1 markedly enhanced EC cell cycle progression (Fig. 1D). Similarly, scratch assays revealed a significant increase in migration capacity with Ad-APEX1 (Fig. 1E). Additionally, Ad-APEX1 accelerated cell adhesion and spreading (Fig. 1F).

Restoration of a functional endothelial monolayer during endothelial repair requires re-establishment of endothelial junctions [2]. Immunofluorescence staining of scratch assay samples showed that Ad-APEX1 promoted the appropriate localization and integrity of adherens junctions, as evidenced by increased VE-cadherin staining length, while the distribution of tight junctions labeled by ZO-1 seemed unaffected (Fig. 1G). Consistently, en face immunofluorescence of the aortic arch where disturbed blood flow occurs demonstrated proper localization of VE-cadherin and ZO-1 at cell–cell junctions in Apex1WT. In contrast, VE-cadherin staining was dispersed and partially translocated to the cytoplasm in Apex1iECKO mice, with ZO-1 signals remaining largely unchanged (Fig. 1H). These findings indicate that the depletion of Apex1 impairs adherens junctions but not tight junctions.

Collectively, these results demonstrate that APEX1 promotes post-injury endothelial regeneration by regulating EC functions, including proliferation, migration, adhesion, and the re-establishment of adherens junctions.

APEX1 interacts with the transcription factor STAT3

APEX1 has been shown to regulate the activity of transcription factors [26]. To investigate whether APEX1 modulates endothelial regeneration via transcription factors, we performed RNA sequencing and transcription factor enrichment analysis of differentially expressed genes (DEGs) in HUVECs infected with either Ad-Null or Ad-APEX1. Concurrently, APEX1 was immunoprecipitated from HUVECs, and its interacting proteome was identified through mass spectrometry. The transcription factor enrichment results were integrated with the mass spectrometry data to identify potential transcription factors interacting with APEX1 (Fig. 2A).

Fig. 2

APEX1 Interacts with the Transcription Factor STAT3. A Schematic diagram of RNA-seq and mass spectrometry. B KEGG analysis of genes significantly up-regulated (P-value < 0.05 & P-adj ≠ 1 & log2FC > 0.275) in Ad-APEX1 vs Ad-Null. C GO biological process analysis of genes significantly up-regulated (P-value < 0.05 & P-adj ≠ 1 & log2FC > 0.275) in Ad-APEX1 vs Ad-Null. D TF enrichment analysis from ChEA3 of genes significantly up-regulated (P-value < 0.05 & P-adj ≠ 1 & log2FC > 0.275) in Ad-APEX1 vs Ad-Null. E STRING analysis of significantly enriched (Fold Change > 2) TF of mass spectrometry. F Western blots of APEX1 co-IP in HUVECs. Co-IP using an anti-APEX1 antibody was conducted in the cell lysates. G Western blots of APEX1 co-IP in HUVECs with or without IL-6 treatment (10 ng/mL, 15 min). Co-IP using an anti-APEX1 antibody was conducted in the cell lysates. H Heatmap of selected DEGs. Blue and red indicate low or high expression, respectively. I RT-qPCR of DEGs in HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h). n = 6 biological replicates. Data were all expressed as the means ± SD

RNA-sequencing analysis revealed 1,648 DEGs between Ad-APEX1 and Ad-Null, with 774 upregulated and 874 downregulated genes (P < 0.05) (Fig. S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that upregulated DEGs were enriched in pathways related to adherens junctions, cell cycle, and gap junctions, among others (Fig. 2B). Gene Ontology Biological Process (GO-BP) analysis revealed enrichment in processes such as cell division, positive regulation of blood vessel endothelial cell migration, cell–cell junction organization, and positive regulation of endothelial cell proliferation (Fig. 2C). These findings suggest that APEX1 plays a role in regulating endothelial regeneration-related cellular functions.

The transcription factor enrichment analysis results are shown (Fig. 2D). APEX1 immunoprecipitation efficiency was validated by Western blotting (Fig. S3), and the identified interactome from mass spectrometry is presented in Table S1. Transcription factors identified through mass spectrometry were subjected to STRING network analysis (Fig. 2E). The intersection of transcription factor enrichment analysis and mass spectrometry data (Table S2) highlighted STAT3 as a top candidate, prompting further validation. Co-immunoprecipitation confirmed that APEX1 interacted with STAT3 in HUVECs (Fig. 2F). Given that interleukin-6 (IL-6), a key proinflammatory cytokine released during tissue injury, is known to activate STAT3 [27], we tested whether IL-6 influences the APEX1-STAT3 interaction. Co-immunoprecipitation assays revealed that IL-6 enhanced the interaction between APEX1 and STAT3 (Fig. 2G), demonstrating both constitutive and inducible binding of APEX1 to STAT3.

A heatmap of common DEGs involved in endothelial regeneration-related pathways (KEGG and GO-BP) showed upregulation of endothelial functional genes related to cell division (ANAPC1, CDCA5, KNL1, NDC80, RAD21, and RB1), junction formation (GJA1, TJP1, and HEG1), proliferation (PDGFB and PRKCA), and migration (TGFBR1) upon APEX1 overexpression (Fig. 2H). To link the regulation of these genes by APEX1 to STAT3, we treated APEX1-overexpressing cells with stattic, a STAT3 inhibitor. The inhibitory efficiency of stattic was confirmed by Western blotting (Fig. S4). Quantitative RT-PCR results demonstrated that STAT3 inhibition suppressed the APEX1-induced upregulation of GJA1, TJP1, HEG1, PDGFB and TGFBR1 (Fig. 2I), suggesting that STAT3 inhibition counteracts the promotive effects of APEX1 on junction formation, proliferation, and migration. However, STAT3 inhibition did not affect the APEX1-induced upregulation of ANAPC1, CDCA5, KNL1, NDC80, PRKCA, RAD21, and RB1 (Fig. S5). Notably, several of these genes (ANAPC1, CDCA5, KNL1, NDC80, and RAD21) are associated with the mitotic pathway, suggesting that APEX1 may regulate mitosis independently of STAT3.

Taken together, these findings suggest that APEX1 physically interacts with STAT3 to regulate the expression of genes critical for endothelial regeneration.

STAT3 mediates the functional regulation of APEX1 in ECs

We next explored whether STAT3 mediates the functional effects of APEX1 in ECs. Flow cytometry following PI staining and EdU incorporation assay demonstrated that inhibition of STAT3 with stattic suppressed the proliferation of HUVECs induced by Ad-APEX1 (Fig. 3A and Fig. 3B). Similarly, STAT3 inhibition reduced migration (Fig. 3C), decelerated cell adhesion (Fig. 3D), and impaired adherens junction formation during scratch wound assays (Fig. 3E), effectively reversing the positive effects of Ad-APEX1. These findings suggest that APEX1 regulates endothelial function via STAT3.

Fig. 3

STAT3 Mediates the Functional Regulation of APEX1 in ECs. A Left: Representative cell cycle analysis of PI flow cytometry of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h). Right: Quantification of S phase (%) of HUVECs. n = 6 biological replicates. B Left: Representative images of EdU incorporation assay of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h). Right: Quantification of EdU positive cell (%). n = 9 images from 3 biological replicates. C Left: Representative images of scratch assays of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h) at 0 and 3 h after scratching. Right: Quantification of relative migration area. n = 6 biological replicates. D Left: Representative immunofluorescence of HUVECs adhesion with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h) at 0.5, 1, 2 and 4 h after adding cell suspension. Right: Quantification of relative area. n = 9 images from 3 biological replicates. The p-value is a comparison between group Ad-APEX1 + DMSO and group Ad-APEX1 + stattic. E Left: Representative immunofluorescence of scratch assays of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and stattic treatment (2.5 μmol/L, 24 h) at 3 h after scratching. Right: Quantification of VE-Cadherin length per cell (pixel). n = 9 images from 3 biological replicates. F Left: Representative Western blots of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. G Detection of STAT3 luciferase activity in HEK293T cells with APEX1 overexpression. n = 6 biological replicates. H Schematic diagram of APEX1 mutants. I and J Detection of STAT3 luciferase activity in HEK293T cells with corresponding APEX1 mutations overexpression. n = 6 biological replicates. K Left: Representative immunofluorescence of scratch assays of HUVECs at 3 h after scratching. Right: Quantification of APEX1 and STAT3 localization. n = 9 images from 3 biological replicates. Data were all expressed as the means ± SD

Phosphorylation of STAT3 at tyrosine 705 (Y705) is a key hallmark of its activation [28]. However, whether APEX1 influences STAT3 phosphorylation remains controversial [29,30,31]. To address this, we assessed STAT3 phosphorylation in HUVECs. Western blotting revealed that Ad-APEX1 significantly elevated the STAT3 Y705 phosphorylation levels (Fig. 3F). APEX1 has been reported to promote STAT3 transcriptional activity through its redox activity [29,30,31]. In partial agreement with these findings, our results showed that overexpression of APEX1 enhanced STAT3 transcriptional activity, as evidenced by a luciferase promoter activity assay (Fig. 3G). However, neither inhibition of APEX1 redox activity using E3330 (Fig. S6) nor the introduction of redox defective mutants of APEX1 (65-cysteine to alanine and 93-cysteine to alanine, C65A and C93A) (Fig. 3H) [32] abrogated this effect (Fig. 3I). These results suggest that APEX1 promotes STAT3 transcriptional activity independent of its redox function.

It has been suggested that APEX1 and STAT3 exist in the same transcriptional complex [33], indicating a potential nuclear role for APEX1 in STAT3 activation. Western blotting analysis of isolated cytoplasmic and nuclear fractions revealed that both endogenous and exogenous APEX1 were distributed in the cytoplasm and nucleus (Fig. S7). To further explore this, we generated APEX1 nuclear location (NLS-APEX1) and nuclear export (NES-APEX1) mutants (Fig. 3H). The localization of these mutants was confirmed by Western blotting and immunofluorescence (Fig. S8). Luciferase reporter assays showed that both mutants promoted STAT3 transcriptional activity, indicating that APEX1 regulates STAT3 activation in both the cytoplasm and the nucleus (Fig. 3J). STAT3 translocate to the nucleus in a phosphorylation-dependent manner [3]. We examined STAT3 nuclear translocation during endothelial regeneration in vitro. Immunofluorescence staining of scratch assays showed that STAT3 underwent nuclear translocation during cell migration and junction reannealing (Fig. 3K).

Collectively, these results demonstrated that STAT3 mediated the functional regulation of APEX1 in ECs, with APEX1 enhancing STAT3 activity through mechanisms involving both its cytoplasmic and nuclear functions.

APEX1 promotes endothelial proliferation, adhesion, and junction formation regulated by vascular wall and matrix stiffness

Previous study in rodents have shown that vascular wall stiffness increases two weeks after intimal injury, primarily due to neointima formation [15]. However, how vascular stiffness evolves during the early stages of endothelial repair post-injury remains unclear. To address this, we performed guidewire-induced injury on the left carotid artery of wild-type mice and evaluated vascular stiffness using nanoindentation at 5 and 14 days post-surgery (Fig. 4A). The results demonstrated a transient decrease in vascular stiffness at 5 days post-injury (7.83 \(\pm\) 3.49 kPa and 1.69 \(\pm\) 0.66 kPa for uninjured vs. injured), followed by an increase at 14 days post-injury (9.24 \(\pm\) 2.94 kPa and 23.43 \(\pm\) 5.92 kPa for uninjured vs. injured) (Fig. 4B).

Fig. 4

APEX1 Promotes Endothelial Proliferation, Adhesion, and Junction Formation Regulated by Vascular Wall and Matrix Stiffness. A Schematic diagram of animal experiment. B Quantification of Young's Modulus measured by nanoindentation of carotid arteries from mice at 5 or 14 days after guidewire induced injury; n = 6–7 mice. C Up: Representative en face immunofluorescence of carotid arteries from indicated mice at 5 days after guidewire induced injury. Down: Quantification of APEX1 and STAT3 localization. n = 9 images from 3 biological replicates. D Schematic diagram of producing GelMA hydrogel. E Quantification of Young's Modulus measured by nanoindentation of GelMA hydrogel. F Left: Representative cell cycle analysis of PI flow cytometry of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection. Right: Quantification of S phase (%) of HUVECs. n = 6 biological replicates. G Up: Representative images of EdU incorporation assay of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection. Down: Quantification of EdU positive cell (%). n = 9 images from 3 biological replicates. H Up: Representative immunofluorescence of HUVECs adhesion on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection at 0.5, 1, 2 and 4 h after adding cell suspension. Down: Quantification of relative area. n = 9 images from 3 biological replicates. I Representative immunofluorescence of cell junctions of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection. Data were all expressed as the means ± SD

To investigate whether this early-stage decrease in vascular stiffness affects APEX1 function, we conducted guidewire-induced injury on Apex1WT and Apex1iECKO mice. The left carotid arteries were harvested for en face immunofluorescence staining 5 days post-operation (Fig. 4A). In the presence of APEX1, STAT3 predominantly localized to the nucleus in uninjured but remained in the cytoplasm at injured sites; however, APEX1 depletion abolished STAT3 nuclear localization, resulting in similar cytoplasmic localization of STAT3 in both uninjured and injured regions (Fig. 4C). These findings suggest that vascular wall stiffness, largely determined by ECM stiffness, might regulate the endothelial STAT3 activity through APEX1.

To further test whether the ECM mechanics regulate endothelial regeneration via APEX1, we used Gelatin Methacryloyl (GelMA) hydrogel (Fig. 4D), which are widely employed in bioengineering modeling as ECM stiffness models [34], to mimic the stiffness of injured and uninjured vascular regions. We selected GelMA hydrogel with a substitution degree of 30% and concentration of 5% for (low stiffness: ~ 1.4 kPa, mimicking injured regions) and substitution degree of 30% and concentration of 10% (high stiffness ~ 7.8 kPa, mimicking uninjured regions) for further experiments (Fig. 4E). HUVECs adhered and spread effectively on both soft and stiff GelMA hydrogel (Fig. S9). We next examined whether APEX1 modulates endothelial function in response to ECM stiffness. Flow cytometry following PI staining and EdU incorporation assay showed that high stiffness promoted cell cycle progression and proliferation in HUVECs; overexpression of APEX1 via Ad-APEX1 rescued cell proliferation on soft hydrogels and further enhanced cell proliferation on stiff hydrogels (Fig. 4F and Fig. 4G). Similarly, high ECM stiffness facilitated cell adhesion and junction formation, and overexpression of APEX1 restored endothelial functions on soft hydrogels (Fig. 4H and I).

These findings indicated that APEX1 plays a critical role in regulating endothelial regeneration in response to ECM stiffness, promoting EC proliferation, adhesion, and junction formation under varying biomechanical conditions.

Matrix stiffness induces STAT3 phosphorylation and nuclear translocation through its action on APEX1

Previous studies have shown that high ECM stiffness promotes STAT3 activation in various cell types [35,36,37]. Our prior work demonstrated that APEX1 is a mechanoresponsive molecule that responds to sheer stress and shuttles between the nucleus and cytoplasm [8]. To further investigate whether ECM stiffness regulated STAT3 activation through APEX1, we first confirmed that ECM stiffness did not alter the protein levels of APEX1 and STAT3 in HUVECs (Fig. S10). Western blotting analysis of HUVECs cultured on hydrogels with different stiffness revealed that high stiffness promoted STAT3 phosphorylation (Fig. 5A). Similarly, high ECM stiffness enhanced STAT3 transcriptional activity (Fig. 5B).

Fig. 5

Matrix Stiffness Induces STAT3 Phosphorylation and Nuclear Translocation through Its Action on APEX1. A Left: Representative Western blots of HUVECs on hydrogels with different stiffness. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. B Detection of STAT3 luciferase activity in NIH3T3 and Hela cells on hydrogels with different stiffness. C Left: Representative Western blots of HUVECs on hydrogels with different stiffness with or without APEX1 knockdown. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. D Left: Representative Western blots of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection. Right: Quantification of STAT3 phosphorylation level of total. n = 7 biological replicates. E Detection of STAT3 luciferase activity in Hela cells on hydrogels with different stiffness with or without APEX1 knockdown. F Detection of STAT3 luciferase activity in Hela cells on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection. G Up: Representative immunofluorescence of HUVECs on hydrogels with different stiffness. Down: Quantification of APEX1 and STAT3 localization. n = 9 images from 3 biological replicates. H Western blots of APEX1 co-IP in HUVECs on hydrogels with different stiffness. Co-IP using an anti-APEX1 antibody was conducted in the cell lysates. Data were all expressed as the means ± SD

Next, we manipulated APEX1 expression in cells on hydrogels of varying stiffness using siRNA and adenovirus. The RNAi efficiency was verified by Western blotting (Fig. S11). Knockdown of APEX1 suppressed the increase in STAT3 phosphorylation induced by stiff matrix (Fig. 5C), while overexpression of APEX1 via Ad-APEX1 enhanced STAT3 phosphorylation on both soft and stiff substrates (Fig. 5D). These findings were corroborated by luciferase reporter assays measuring STAT3 transcriptional activity: knockdown of APEX1 impaired ECM stiffness-induced STAT3 activation (Fig. 5E), whereas overexpression of APEX1 promoted STAT3 activation on soft and stiff gels. (Fig. 5F).

Immunofluorescence staining further confirmed that high stiffness promoted the nuclear translocation of both APEX1 and STAT3 (Fig. 5G). Given that STAT3 is an interacting protein of APEX1, we next explored whether the interaction between STAT3 and APEX1 could be modulated by ECM stiffness. Co-immunoprecipitation assays of HUVECs cultured on hydrogels with different stiffness revealed that high ECM stiffness enhanced the interaction between STAT3 and APEX1 (Fig. 5H).

These results collectively demonstrate that ECM stiffness induced STAT3 activation through APEX1, implicating APEX1 as a key mediator in the cellular response to mechanical cues in the ECM.

APEX1 facilitates stiffness-induced STAT3 activation by enhancing the interaction between ROCK2 and STAT3

Matrix stiffness has been shown to modulate the activity of kinases such as JAK1, JAK2 and ROCK1/2 that are involved in STAT3 phosphorylation [37, 38]. To investigate whether APEX1 promotes STAT3 phosphorylation through these kinases, we treated HUVECs overexpressing APEX1 with inhibitors of JAK1, JAK2 and ROCK1/2 (upadacitinib, fedratinib, and Y27632, respectively). Inhibition of all these kinases reversed the enhanced STAT3 phosphorylation induced by APEX1 (Fig. 6A). However, only ROCK1/2 inhibitor Y27632 effectively eliminated the increased STAT3 phosphorylation induced by stiffness (Fig. 6B). Similarly, the stimulatory effect of APEX1 on STAT3 phosphorylation under low ECM stiffness was completely abolished by Y27632 (Fig. 6C). These results suggest that ROCK1/2 plays a pivotal role in mediating STAT3 phosphorylation promoted by high ECM stiffness through APEX1.

Fig. 6

APEX1 Facilitates Stiffness-Induced STAT3 Activation by Enhancing the Interaction between ROCK2 and STAT3. A Left: Representative Western blots of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection and Upadacitinib (1 μmol/L, 24 h), Fedratinib (1 μmol/L, 24 h) or Y27632 (5 μmol/L, 24 h) treatment. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. B Left: Representative Western blots of HUVECs on hydrogels with different stiffness with or without and Upadacitinib (1 μmol/L, 24 h), Fedratinib (1 μmol/L, 24 h) or Y27632 (5 μmol/L, 24 h) treatment. Right: Quantification of STAT3 phosphorylation level of total. n = 7 biological replicates. C Left: Representative Western blots of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection and Upadacitinib (1 μmol/L, 24 hh), Fedratinib (1 μmol/L, 24 h) or Y27632 (5 μmol/L, 24 h) treatment. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. D Left: Representative Western blots of HUVECs with or without ROCK1 knockdown. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. E Left: Representative Western blots of HUVECs with or without ROCK2 knockdown. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. F Left: Representative Western blots of HUVECs on hydrogels with different stiffness with Ad-Null or Ad-APEX1 adenovirus infection and ROCK2 knockdown. Right: Quantification of STAT3 phosphorylation level of total. n = 6 biological replicates. G Western blots of STAT3 co-IP in HUVECs with Ad-Null or Ad-APEX1 adenovirus infection. Co-IP using an anti-STAT3 antibody was conducted in the cell lysates. Data were all expressed as the means ± SD. H Western blots of STAT3 co-IP in HUVECs on hydrogels with different stiffness with or without APEX1 knockdown. Co-IP using an anti-STAT3 antibody was conducted in the cell lysates. Data were all expressed as the means ± SD

Since no selective inhibitor for ROCK1/2 is available, we performed follow-up experiments using siRNA to knock down ROCK1 and ROCK2, verifying RNAi efficiency via Western blotting (Fig. S12). Knockdown of ROCK1 and ROCK2 revealed that only ROCK2 knockdown reversed the enhanced STAT3 phosphorylation induced by high ECM stiffness (Fig. 6D and E). Western blotting confirmed that ECM stiffness did not alter ROCK2 protein levels (Fig. S13). Furthermore, the reversal of STAT3 phosphorylation on low matrix stiffness by APEX1 was also abolished by ROCK2 knockdown (Fig. 6F). These findings suggest that the stimulatory effect of APEX1 on STAT3 phosphorylation under high ECM stiffness is primarily mediated through ROCK2.

Notably, co-immunoprecipitation assays revealed that overexpression of APEX1 enhanced the interaction between STAT3 and ROCK2 (Fig. 6G); high ECM stiffness also promoted the interaction between STAT3 and ROCK2, and this enhancement was reversed by APEX1 knockdown (Fig. 6H).

Taken together, these results demonstrated that APEX1 facilitates stiffness-induced STAT3 activation by promoting the interaction between ROCK2 and STAT3.

APEX1 inhibits cytoplasmic condensation of STAT3, which impedes STAT3 activation

Immunofluorescence analysis revealed that STAT3 in the unwounded sites or under low ECM stiffness exhibited a punctate distribution in the cytoplasm (Fig. 7A and B), leading we to hypothesize that inactivated STAT3 may condense within the cytoplasm. In determine the optimal conditions for STAT3 activation, we conductions pre-experiments examining serum concentration and its effects on STAT3 activation. The results showed that a higher serum concentration (10%) stimulated STAT3 activation more effectively than a lower serum concentration (2%), although the lower concentration maintained the sensitivity of STAT3 to external stimuli (Fig. S14). Consequently, we chose the lower serum concentration for subsequent experiments.

Fig. 7

APEX1 Inhibits Cytoplasmic Condensation of STAT3, Which Impedes STAT3 Activation. A Left: Representative immunofluorescence of scratch assays of HUVECs at 3 h after scratching. Right: Quantification of number of condensations per cell. n = 9 images from 3 biological replicates. B Left: Representative immunofluorescence of HUVECs on hydrogels with different stiffness. Right: Quantification of number of condensations per cell. n = 9 images from 3 biological replicates. C Left: Representative immunofluorescence of HUVECs with or without IL-6 treatment (10 ng/mL, 15 min). Right: Quantification of STAT3 localization and number of condensations per cell. n = 9 images from 3 biological replicates. D Up: Representative immunofluorescence of HUVECs with Ad-Null or Ad-APEX1 adenovirus infection. Down: Quantification of STAT3 localization and number of condensations per cell. n = 9 images from 3 biological replicates. E Schematic diagram of OptoDroplet system. F Images of HEK293T cells expressing OptoSTAT3 upon blue light exposure. G FRAP analysis of cherry-STAT3 condensation in HEK293T. n = 8 biological replicates. Data were all expressed as the means ± SD

Immunofluorescence staining revealed that IL-6 promoted STAT3 nuclear translocation and disrupted the condensed cytoplasmic distribution of STAT3 (Fig. 7C). Overexpression of APEX1 via Ad-APEX1 yielded similar results (Fig. 7D), further supporting the notion that activated STAT3 exhibits nuclear localization, whereas inactivated STAT3 forms cytoplasmic condensates.

Extensive research has shown that protein condensation can indicate phase separation [23,