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Novel carbazole attenuates vascular remodeling through STAT3/CIAPIN1 signaling in vascular smooth muscle cells
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Joo-Hui Hana, *, Jong-Beom Heob, Hyung-Won Leec, Min-Ho Parkd, Jangmi Choid, Eun Joo Yunb, Seongpyo Leec, Gyu Yong Songb, *, Chang-Seon Myunge, *
Acta Pharmaceutica Sinica B | 2025, 15(3) : 1463 - 1479
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Acta Pharmaceutica Sinica B | 2025, 15(3): 1463-1479
• ORIGINAL ARTICLE •
Novel carbazole attenuates vascular remodeling through STAT3/CIAPIN1 signaling in vascular smooth muscle cells
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Joo-Hui Hana, *, Jong-Beom Heob, Hyung-Won Leec, Min-Ho Parkd, Jangmi Choid, Eun Joo Yunb, Seongpyo Leec, Gyu Yong Songb, *, Chang-Seon Myunge, *
Affiliations
  • aCollege of Pharmacy and Research Institute of Pharmaceutical Sciences, Woosuk University, Wanju 55338, Republic of Korea
  • bCollege of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
  • cCollege of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
  • dInstitute of Drug Research and Development, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
  • eDepartment of Pharmacology, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
About Author:

E-mail addresses: (Joo-Hui Han)

(Gyu Yong Song)

(Chang-Seon Myung).

Author contributions

Joo-Hui Han: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. Jong-Beom Heo: Validation, Formal analysis. Hyung-Won Lee: Visualization, Formal analysis. Min-Ho Park: Validation, Formal analysis. Jangmi Choi: Validation, Formal analysis. Eun Joo Yun: Validation, Formal analysis. Seongpyo Lee: Visualization, Formal analysis. Gyu Yong Song: Visualization, Validation, Supervision. Chang-Seon Myung: Writing – review & editing, Supervision, Funding acquisition.

doi: 10.1016/j.apsb.2024.12.035
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This study investigated the molecular mechanism of phenotypic switching of vascular smooth muscle cells (VSMCs), which play a crucial role in vascular remodeling using 9H-Carbazol-3-yl 4-aminobenzoate (CAB). CAB significantly attenuated platelet-derived growth factor (PDGF)-induced VSMC proliferation and migration. CAB suppressed PDGF-induced STAT3 activation by directly binding to the SH2 domain of STAT3. Downregulation of STAT3 phosphorylation by CAB attenuated CIAPIN1/JAK2/STAT3 axis through a decrease in CIAPIN1 transcription. Furthermore, abrogated CIAPIN1 decreased KLF4-mediated VSMC dedifferentiation and increased CDKN1B-induced cell cycle arrest and MMP9 suppression. CAB inhibited intimal hyperplasia in injury-induced neointima animal models by inhibition of the CIAPIN1/JAK2/STAT3 axis. However, CIAPIN1 overexpression attenuated CAB-mediated suppression of VSMC proliferation, migration, phenotypic switching, and intimal hyperplasia. Our study clarified the molecular mechanism underlying STAT3 inhibition of VSMC phenotypic switching and vascular remodeling and identified novel active CAB. These findings demonstrated that STAT3 can be a major regulator to control CIAPIN1/JAK2/STAT3 axis that may be a therapeutic target for treating vascular proliferative diseases.

Atherosclerosis  /  Vascular smooth muscle cell  /  Signal transducer and activator of transcription 3  /  Cytokine induced apoptosis inhibitor 1  /  Janus tyrosine kinase 2  /  Phenotyping switching  /  Krüppel-like factor 4  /  Carbazole
Joo-Hui Han, Jong-Beom Heo, Hyung-Won Lee, Min-Ho Park, Jangmi Choi, Eun Joo Yun, Seongpyo Lee, Gyu Yong Song, Chang-Seon Myung. Novel carbazole attenuates vascular remodeling through STAT3/CIAPIN1 signaling in vascular smooth muscle cells[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (3) : 1463 -1479 . DOI: 10.1016/j.apsb.2024.12.035
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1.　Introduction
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Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) leads to neointima formation and vascular remodeling, followed by atherosclerotic progression1,2. Most VSMCs in the arterial tunica media display a contractile phenotype, which allows them to maintain vascular tone, blood pressure, and blood flow distribution to tissues3. However, VSMCs can phenotypically switch and are considered to be a key mechanism in arterial remodeling4. VSMCs transit from a contractile phenotype to a synthetic phenotype (VSMC dedifferentiation) in response to vascular injury and stimulation of growth factors such as platelet-derived growth factors (PDGFs)5-7. Synthetic-state VSMCs actively proliferate and migrate into the arterial intima, leading to vascular proliferative diseases such as atherosclerosis, postangioplasty restenosis, and failure of arterial bypass grafts8,9. Thus, it is essential to investigate the molecular mechanisms underlying VSMC phenotypic switching to identify novel therapeutics for atherosclerosis and related cardiovascular disorders.
Signal transducer and activator of transcription 3 (STAT3) signaling pathway affects various biological processes, such as cell differentiation, proliferation, and apoptosis10. STAT3 activation is vital as a transcription factor to regulate downstream gene expression11-15. Although STAT3 is critically involved in vascular injury, the underlying mechanisms of how STAT3 regulates VSMC phenotypic switching remain poorly understood, and the functional importance of suppressing these pathways by novel inhibitors during the development of vascular proliferative diseases has not been demonstrated.
We previously reported that cytokine-induced apoptosis inhibitor 1 (CIAPIN1) induced VSMC dedifferentiation and promoted neointima formation increasing of Janus kinase 2 (JAK2) and STAT3 activation16. PDGF receptor-beta (PDGFR-β) pathways affect CIAPIN1 expression, but the underlying mechanisms and upstream molecules that regulate CIAPIN1 remain unclear.
Carbazole and its derivatives are an important nitrogen-containing aromatic heterocyclic compound easily introduced into the structurally rigid carbazolyl ring17. In addition, carbazole is an “advantageous scaffold” containing desirable electronic and charge-transport properties since it reacts with many receptors and can bind reversibly to proteins and enzymes, providing an opportunity to study and develop novel drugs that target one or more biological structures18,19. Through these characteristics, carbazole derivatives have been reported to have various biological activities, such as anticancer20, antimicrobial21, antioxidants22, and anti-inflammatory23 activities. In our ongoing search for novel synthesized compounds that attenuate vascular remodeling by inhibiting STAT3, we found that the 9H-Carbazole derivatives inhibited STAT3 activation.
In this study, we investigated the therapeutic effects of 9H-Carbazol-3-yl 4-aminobenzoate (CAB) on vascular remodeling by regulating VSMC phenotypic switching, proliferation, migration, and the pathogenesis of neointima formation. Our findings showed the inhibitory effects of CAB on STAT3 activation through direct binding in the Src homology 2 (SH2) domain. In addition, a decrease in STAT3 activity inhibits CIAPIN1 transcription by affecting its promoter activation. These effects suppress CIAPIN1/JAK2/STAT3 axis to inhibit Krüppel-like factor 4 (KLF4)-mediated VSMC dedifferentiation. Moreover, CAB-mediated STAT3 inhibition promoted cyclin-dependent kinase inhibitor 1B (CDKN1B) transcription as a functional cell cycle regulator24, resulting in decreased VSMC proliferation and migration. Therefore, the physiological impacts of the identified targets were corroborated in arterial tissues from neointima lesions.
2.　Materials and methods
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2.1.　 Animals and drug administration
Sprague–Dawley rats (SD rats, male, 10-weeks-old, 380–420 g) and C57BL/6 mice (male, 6-weeks-old, average 23 g) were obtained from Samtako (Osan, Korea). The animals were housed two per cage. They fed a standard maintenance diet (1314 FORTIFIED, Altromin, Lage, Germany) ad libitum in the animal facility under controlled temperature (22 ± 2 ℃), humidity (50 ± 5%), and lighting (12/12 h dark–light cycle, lights on at 7:00 a.m.) conditions. Animal experiments were approved by the Institutional Animal Care and Use Committee of Chungnam National University (202012A-CNU-192) and Woosuk University (WS-2023-10). Animals' protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. After a 1-week acclimatization period, rats were randomly divided into five groups (n = 7 in each group): sham, balloon injury (BI) and BI with 20 mg/kg CAB, rat CIAPIN1 transduction (rCIA1), rCIA1, BI with 20 mg/kg CAB. After 1 day of balloon injury to induce neointima formation, rats were orally administered CAB dissolved in 0.5% carboxymethyl cellulose (CMC, C5678, Sigma–Aldrich) daily. The body weight of the rats was recorded on the indicated days for CAB toxicity (Supporting Information Fig. S7A). Mice were randomly divided into three groups (n = 6 in each group): sham, carotid ligation (LI), and LI with 3 mg/kg CAB. The mouse dosage was converted from the rat dosage using Meeh's constant for total body surface area (TBSA) as Eq. (1)25:
TBSA=K×weight2/3
The average weight of the rats used in our study was approximately 400 g, with a K value set at 9.83 based on a previous study26. Similarly, the average weight of the mice was 23 g, with a K value of 9.82, referenced from another study27. We calculated the appropriate dosage for mice as 2.98 mg/kg, rounded to 3 mg/kg for administration, as shown in Eq. (2):
Mouseequibalentdose=9.82×(23g)2/39.83×(400g)2/3×20mg/kg
After 1 day of carotid ligation to induce neointima formation, mice were orally administered with CAB dissolved in 0.5% CMC daily.
2.2.　 Induction of neointima formation and morphometric analysis
To induce neointima formation, we carried out balloon injury in rat carotids28-30 and carotid ligation in a mouse model31, as described previously. The balloon injury in the rat carotid artery was performed using SD rats. Briefly, rats were anesthetized via an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). A balloon catheter (Fogarty 2F, Edwards Lifesciences, CA, USA) was inserted into the left common carotid artery via the external carotid artery. Then, the inflated balloon was drawn gently toward the external carotid artery. After this procedure was repeated five times, the catheter was removed, and the injured artery was washed with saline. Then, 50 μL of lentiviral vectors (2 × 109 plaque-forming units/mL) expressing control vector (Con) or rat Ciapin1 (rCIAPIN1 or rCIA1) were incubated into the injured artery for 30 min. The ligation of the mouse carotid artery was performed using the C57BL/6 mouse. Mice were anesthetized via an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). After we closed the wound using skin sutures and swabbed all sides of the closed wound with povidone-iodine, the rats and mice were allowed to recover while being kept warm and pain-free on a heated pad by treatment with 1% ketoprofen (0.2 mL/kg) subcutaneously. After 14 or 28 days, all animals were anesthetized by CO2 administration, following a gradual CO2 displacement protocol as outlined in the American Veterinary Medical Association Guidelines for the Euthanasia of Animals: 2020 Edition (typically at 30%–70% chamber volume per minute) while monitoring the animals for signs of distress, and their arteries, liver, heart, lung, and kidney tissues were collected and stored at −80 ℃ until further analysis for qPCR and Western blotting or embedded in paraffin for histological analysis.
The tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then, the tissues were cut into 4-μm-thick sections. The sections were stained with hematoxylin and eosin (H&E), images were acquired using a microscope (Nikon Eclipse Ti, Nikon Instruments, Inc., Tokyo, Japan), and histological analysis was performed using AxioVision (Version 4.8.3, Carl Zeiss Microscopy, White Plains, NY, USA). The area of the residual lumen and the areas circumscribed by the internal and external elastic lamina were measured to determine the intima and media areas. The degree of neointimal thickening was assessed using the intima-to-media area ratio and the intimal area. In vivo studies were performed in a blinded fashion at the drug administration and histological stages. For the quantification of tissue staining and pathological evaluation of animal specimens, the investigator was blinded to ensure an unbiased interpretation of the results.
2.3.　 Immunohistochemistry
Immunohistochemical assays for proliferating cell nuclear antigen (PCNA) were performed in rat carotid artery sections. The sections were subjected to antigen retrieval using a 10 mmol/L sodium citrate buffer (pH 6.0) and incubated for 10 min in a 3% hydrogen peroxide (H2O2) solution to quench endogenous peroxidase activity. After the sections were incubated in blocking buffer (5% goat serum in phosphate buffered saline) for 1 h at room temperature, they were treated with PCNA antibody diluted in antibody diluent (Dako, Glostrup, Denmark) overnight at 4 ℃, followed by incubation with biotin-conjugated secondary antibodies. Staining signals were detected using a standard avidin-biotin complex-peroxidase system (Vector Laboratories, CA, USA). Subsequently, positive antibody binding was visualized using a 3,3′-diaminobenzidine substrate (Vector Laboratories, CA, USA), followed by hematoxylin counterstaining. Immunohistochemistry staining levels were quantified using ImageJ software (Version 1.53c).
2.4.　 Statistical analyses
All data are expressed as the mean ± standard deviation (SD) of at least 3–5 independent experiments. Statistical analyses were performed with GraphPad Prism software (Version 10, San Diego, CA, USA). The normality of the data distributions was tested using the Shapiro–Wilk test. A two-sided, unpaired Student's t-test was used to analyze the difference between two data groups with normally distributed variables. Differences among three or more groups were analyzed with one-way analysis of variance (ANOVA) followed by a post hoc Bonferroni's test if F achieved statistical significance (P < 0.05) and there was no significant variance in homogeneity with Bartlett's test. Differences with P < 0.05 were considered statistically significant.
3.　Results
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3.1.　 Identification of active 9H-Carbazole derivatives to inhibit PDGF-BB-induced vascular remodeling
To validate drug-likeness and therapeutic efficacy, we designed and synthesized a library of four 9H-Carbazole derivatives by modifying the C-4 position moiety of benzoate, which was linked to the C-3 position of the 9H-Carbazole moiety (Supporting Information Fig. S1A). To assess the effects of the 9H-Carbazole derivatives on PDGF-BB-induced VSMC proliferation, we treated cells with 5 μmol/L derivatives for 2 h, followed by PDGF-BB (25 ng/mL) for 24 h. As shown in Fig. S1B, the derivatives, including CAB, 2, 3, and 4, significantly inhibited PDGF-BB-stimulated VSMC proliferation. Among them, CAB exhibited the most potent inhibitory activity on PDGF-BB-induced VSMC proliferation, suggesting that a free amino group at the C-4 position of benzoate plays an important role in the inhibitory activity.
Since CAB exhibited the most significant inhibitory activity on PDGF-BB-induced VSMC proliferation at the lowest IC50 compared to other CAB derivatives (Supporting Information Table S1), we performed further studies using CAB (Fig. 1A). CAB (1–5 μmol/L) dose-dependently inhibited PDGF-BB-induced VSMC proliferation (Fig. 1B). Exposure to the highest concentration of CAB (5 μmol/L) did not induce cytotoxic effects in VSMCs compared to H2O2 (1 mmol/L), which is a major reactive oxygen species causing cell death via apoptosis (Fig. 1C and D)32. In addition, there were no signs of cell apoptosis induction by CAB compared with the control, indicating that CAB-mediated inhibition of VSMC proliferation was not due to toxic effects. In addition, CAB treatment significantly inhibited PDGF-BB-induced proliferation of human aortic smooth muscle cells, as observed in VSMCs, without inducing cytotoxicity (Fig. S1C and S1D).
VSMC migration as well as proliferation are critical processes for vascular remodeling in response to arterial injury or mitogenic factors33. To examine the effects of CAB on VSMC migration, we performed a wound healing assay in CAB-treated VSMCs. VSMC migration occurred upon PDGF-BB stimulation, while CAB treatment significantly lowered VSMC migration compared to the control (Fig. 1E).
Given that CAB attenuates VSMC proliferation and migration, we examined whether CAB can be intracellularly enriched in VSMCs to affect cellular function. In the VSMCs treated with the highest concentration of CAB (5 μmol/L), CAB was extracted by acetonitrile, and liquid chromatography–quadrupole time-of-flight tandem mass spectrometry measured the cellular CAB concentration. The intracellular CAB amounts estimated from a standard curve were 1.83 ± 0.66 and 1.82 ± 0.62 nmol/105 cells after treatment for 2 and 26 h, respectively, indicating that CAB was promptly taken up by the cells, and the concentration was maintained until 26 h (Fig. 1F). Given the average volume of a single cell (≈5 pL)34, the actual CAB concentration per cell reached ≈3.67 mmol/L, implying that intracellular pharmacokinetics (PK) of CAB may affect the disease phenotype. Taken together, these results identify a role of CAB in VSMC proliferation and migration without cytotoxicity.
3.2.　 CAB specifically inhibits STAT3 phosphorylation by binding to SH2 domain in PDGF-BB-induced VSMCs
In the early stages of intima hyperplasia, PDGF-BB binds to its receptor, PDGFR-β, to trigger phosphorylation of downstream pathways, including protein kinase B (AKT/PKB), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, JAK2, and STAT335-38. To explore the underlying molecular mechanism of CAB activity, we determined whether CAB functioned by affecting PDGFR-β pathways. The results of Fig. 2A show that CAB did not affect PDGF-BB-stimulated PDGFR-β, AKT, ERK1/2, and p38 phosphorylation. However, CAB significantly inhibited PDGF-BB-induced STAT3 tyrosine (Tyr) 705 phosphorylation without affecting the serine (Ser) 727 residue, which is a downstream active MAPK signaling residue (Fig. 2B and Supporting Information Fig. S2A)39,40. Since angiotensin II (Ang II) or IL-6 induce VSMC proliferation by activating STAT341,42, we found CAB (3–5 μmol/L) inhibited Ang II or IL-6 induced VSMC proliferation (Fig. S2B).
Since STAT3 Tyr705 residue is regulated by JAK2 or Src proto-oncogene (SRC)43,44, we examined the effect of CAB on JAK2 and SRC phosphorylation in PDGF-BB-induced VSMCs using AG490 (JAK2 inhibitor) or SU6656 (SRC inhibitor). As shown in Fig. 2C and Fig. S2C, CAB did not inhibit PDGF-BB-induced JAK2 and SRC phosphorylation unlike AG490 and SU6656, implying a direct function to STAT3.
Given that CAB inhibits STAT3 phosphorylation without affecting upstream molecules, we hypothesized that CAB regulated STAT3 by direct inhibition. We performed a ligand-docking assay using crystal structure to detect the docking possibility of CAB in the STAT3. Through the docking study, CAB showed interactions with STAT3 at residues Ile589, Arg609, and Glu612 in the SH2 domain, which is necessary for receptor association and tyrosine phosphodimer formation (STAT3 docking score: −3.25 kcal/mol) (Fig. 2D and E)45. His-tag pull-down assays were performed to further elucidate the interaction between CAB and the SH2 of STAT3. Briefly, a series of His-STAT3-fusion protein-expressing vectors with different domains, as well as Ile589, Arg609, and Glu612 mutant (Fig. 2F), were constructed and used to investigate whether SH2 is the specific binding site for CAB. The result showed that CAB-conjugated beads did not pull down STAT3 fragments lacking SH2, whereas full-length was successfully pulled down by CAB-conjugated beads (Fig. 2G). In addition, surface plasmon resonance (SPR) analysis demonstrated that CAB binds to the STAT3 protein (amino acids 127–688) in a concentration-dependent manner (Fig. 2H). The sensorgram displayed distinct association and dissociation phases for each concentration of CAB (1.5625, 3.125, 6.25, 25, and 100 μmol/L) or stattic (Fig. 2I) tested (1.5625, 6.25, 12.5, 50, and 100 μmol/L). As the concentration of CAB or stattic increased, the response units (RU) also increased, indicating a higher binding affinity at higher concentrations. The binding interaction of CAB was quantified with an association rate constant (Ka) of 1.602 × 105/mol/L/s, a dissociation rate constant (Kd) of 5.964 × 10−4/s, and an equilibrium constant (KD) of 3.722 × 10−9 mol/L. In contrast, the binding interaction of stattic was quantified with an association rate constant of 1.002 × 103/mol/L/s, a dissociation rate constant of 9.083 × 10−3/s, and an equilibrium constant of 9.068 × 10−6 mol/L (Table 1). Thus, the interaction between CAB and the STAT3 protein was more substantial than that of stattic, which is previously known as a STAT3 inhibitor46, indicating the potential of CAB as a more potent STAT3 inhibitor.
These findings suggested that CAB could directly bind to STAT3, and its binding site was likely located in the SH2 of STAT3. Furthermore, CAB did not bind to STAT3 strongly compared to full-length when Ile59, Arg609, and Glu612 in the SH2 were mutated, suggesting that those residues in SH2 are critical for direct binding between CAB and STAT3.
3.3.　 CAB inhibits STAT3 activation as a transcription factor in PDGF-BB-induced VSMCs
In accordance with the docking results, CAB (3–5 μmol/L) inhibited STAT3 dimerization, and this inhibitory effect was similar to AG490 and stattic (STAT3 inhibitor) (Fig. 3A and Supporting Information Fig. S3). Since tyrosine-phosphorylated and dimerized STAT3 triggers nuclear translocation to function as a transcription factor47, we examined the effect of CAB on PDGF-BB-induced STAT3 nuclear translocation using cellular fractions and immunofluorescence. PDGF-BB increased STAT3 phosphorylation and nuclear translocation; however, CAB blocked these phenomena (Fig. 3B and C). To assess transcriptional activity, we transfected VSMCs with the luciferase reporter, which consisted of four copies of the STAT3 binding site in a minimal promoter. We observed that CAB inhibited PDGF-BB-induced STAT3 DNA binding activity, similar to JAK2 inhibition (AG490) (Fig. 3D).
These results led us to hypothesize that CAB regulated the transcription of STAT3 downstream genes to regulate VSMC proliferation, migration, and phenotypic switching. Thus, we measured the mRNA levels of Ciapin1, Jak2, Stat3, Klf4, secreted phosphoprotein 1 (Spp1), Transgelin (Tagln), and Desmin and observed that CAB significantly restored the alteration of mRNA levels (Fig. 3E). These findings, together with those shown above, reinforce the idea that CAB suppresses PDGF-BB-induced activation of STAT3 as a transcription factor.
3.4.　 CAB inhibits PDGF-BB-induced CIAPIN1/JAK2/STAT3 activation to regulate VSMC phenotypic switching
Next, we examined a series of proteins involved in the STAT3 pathways using the STRING database. In the protein network analysis, CIAPIN1 and KLF4 were found to be interconnected with a subset of proteins (Fig. 4A). Of those linked to the core network, the proteins were associated with VSMC phenotypic switching16,48. Interestingly, we found STAT3 phosphorylation was increased at the early time (15 min–1 h) and subsequently late time (24 h) point, indicating early time activated STAT3 functions to increase in CIAPIN1 expression as a transcription factor (Fig. 4B and Supporting Information Fig. S4), and CIAPIN1 may affect STAT3 phosphorylation through JAK2 upregulation16.
To verify the effect of CAB on associated protein regulation, we first examined the levels of CIAPIN1, JAK2, and STAT3 activation in PDGF-BB-induced VSMCs to examine the alteration of CIAPIN1/JAK2/STAT3 axis. As shown in Fig. 4C and Supporting Information Fig. S5A, CAB significantly inhibited PDGF-BB-stimulated CIAPIN1 and JAK2 expression. Owing to the alteration of the total JAK2 level, phosphorylation of JAK2 and STAT3, the JAK2 downstream molecule, was attenuated by CAB treatment. To determine whether STAT3 regulates CIAPIN1/JAK2/STAT3 signaling pathway via an autoregulatory circuit, we introduced stattic and STAT3 Tyr705 mutant (Y705F). As shown in Fig. 4D and Fig. S5B, stattic significantly inhibited PDGF-BB-induced CIAPIN1 and JAK2 expression, and JAK2 and STAT3 phosphorylation. Furthermore, STAT3 Y705F abrogated PDGF-BB-induced CIAPIN1 and JAK2 expression, and JAK2 and STAT3 phosphorylation, indicating that STAT3 plays a pivotal role in the CIAPIN1/JAK2/STAT3 autoregulatory circuit (Fig. 4E and Fig. S5C).
In response to vascular injury and other stimuli, such as growth factors, including PDGF, VSMCs actively proliferate and migrate into the arterial intima by VSMC dedifferentiation, switching from a contractile to a synthetic phenotype that results in neointima formation4. Since CIAPIN1 and STAT3 regulate VSMC dedifferentiation16,49, we examined the effect of CAB on PDGF-BB-induced VSMC phenotypic switching. KLF4 is one of the corepressors that competes with myocardin activity to increase contractile genes for enhancing VSMC differentiation50. As shown in Fig. 4C and Fig. S5A, CAB decreased PDGF-BB-induced expression of KLF4 and SPP1, one of the synthetic phenotype markers. Similar to CAB, stattic and STAT3 Y705F inhibited KLF4 and SPP1 expression, indicating that STAT3 affects KLF4 to regulate VSMC phenotypic switching (Fig. 4D and E, Fig. S5B and S5C). In addition, PDGF-BB increased STAT3 phosphorylation mainly in the nucleus, but decreased the expression of transgelin, one of the contractile phenotype markers51,52, indicating phenotypic transition from a contractile to a synthetic state (Fig. 4F and Fig. S5D). However, CAB treatment restored PDGF-BB-mediated alterations in STAT3 phosphorylation and transgelin by blocking VSMC dedifferentiation.
To elucidate the underlying mechanism of CIAPIN1 inhibition by CAB-mediated STAT3 suppression, we hypothesized that STAT3, as a transcriptional factor, contributes to the CIAPIN1 gene transactivation. We postulated that a putative DNA-binding site would be present in the gene promoter. To elucidate this interaction in situ, we found a putative STAT3-binding site in the Ciapin1 gene promoter (STAT3-RE1) using JASPAR 2024 (https://jaspar.elixir.no/). Then, we sought to evaluate the interaction with STAT3 and Ciapin1 promoter by the chromatin immunoprecipitation (ChIP)-PCR assay. Using anti-STAT3 antibody, we immunoprecipitated protein/DNA complexes from VSMCs treated with CAB for 2 h, followed by stimulation with PDGF-BB for 6 h and amplified the putative interacting region of the endogenous rat Ciapin1 promoter via PCR. While the binding of STAT3 to the Ciapin1 promoter region significantly increased after stimulation with PDGF-BB, CAB treatment inhibited protein/DNA binding (Fig. 4G). The specificity of STAT3 binding was confirmed using franking primers targeting irrelevant regions in the promoter (STAT3-RE2 and -RE3). qPCR analysis verified STAT3 and STAT3-RE1 binding (Fig. 4H). Treatment of VSMCs with PDGF-BB significantly promoted luciferase expression from a reporter construct containing −1.6 kb Ciapin1 gene, but CAB inhibited PDGF-BB-induced CIAPIN1 luciferase expression (Fig. 4I). However, the mutation of STAT3-RE1 abolished the upregulation of PDGF-BB-induced CIAPIN1 luciferase expression.
Taken together, these results demonstrate that STAT3, activated by PDGF-BB, transactivates the Ciapin1 gene, which promotes VSMC dedifferentiation from contractile to synthetic phenotype. However, CAB blocks this phenotype.
3.5.　 Suppression of STAT3 by CAB leads to CDKN1B-mediated cell cycle arrest and MMP9 suppression
VSMC proliferation and migration are regulated by cell cycle progression and matrix metalloproteinase (MMP) activation53. STAT3 regulates the transcription of cell cycle-arresting genes14,15. Among cyclin-dependent kinase (CDK) inhibitors, CDKN1B suppresses cell cycle progression and MMP9 activation54,55.
To verify the effect of CAB on PDGF-BB-induced CDKN1B expression, we examined the level of CDKN1B. As shown in Fig. 5A, CAB dose-dependently restored PDGF-BB-mediated inhibition of CDKN1B expression. In addition, stattic and STAT3 Y705F increased CDKN1B expression in the PDGF-BB-induced VSMCs, indicating that inhibition of STAT3 by CAB increased CDKN1B through transcriptional regulation (Fig. 5B–D, Supporting Information Fig. S6A and S6B).
Next, we investigated whether the CAB-mediated reduction in proliferation was directly linked to cell cycle arrest. PDGF-BB treatment increased cell fraction in the S and G2/M phases (Fig. 5E). An increase in the G0/G1 phase proportion of the CAB-treated VSMCs versus the PDGF-BB-induced VSMCs was observed, indicating that CAB-mediated cell cycle arrest occurred due to disturbed progression from G0/G1 to S phase.
As CDKN1B effectors, cyclin and CDK trigger cell cycle progression by sequential intramolecular processes that block retinoblastoma protein (pRb) and activate PCNA in cells entering the G1 phase1,56,57. In addition, MMPs are endopeptidases that regulate vascular structure and remodeling, and MMP9 plays a critical role in VSMC migration and neointima formation1,4. As shown in Fig. 5F and G, CAB significantly inhibited PDGF-BB-induced cyclin E1, CDK2, cyclin D1, CDK4, phospho-pRb (p-pRb), PCNA and MMP9 expression, and MMP9 proteolytic activity. Taken together, these results reinforce the understanding that CAB regulates VSMC proliferation and migration via the STAT3/CDKN1B pathways.
3.6.　 CIAPIN1 overexpression counteracts the inhibitory effect of CAB on STAT3-mediated VSMC dedifferentiation, proliferation, and migration
Since CIAPIN1/JAK2/STAT3/KLF4 axis regulated by STAT3 cause alteration of VSMCs proliferation, migration, and dedifferentiation (Fig. 4), we transduced CIAPIN1 [human CIAPIN1 (hCIA1) at a MOI of 15] into VSMCs. As shown in Fig. 6A and Fig. S6C, hCIAPIN1 and rCIAPIN1 expression was increased by PDGF-BB treatment. Since CIAPIN1 nuclear accumulation was detected in an abnormal proliferative state58, we examined the CAB-mediated alteration of CIAPIN1 localization. As shown in Fig. 6B, the PDGF-BB-induced increase in CIAPIN1 expression was mainly located in the nucleus. CAB and stattic treatment significantly reduced CIAPIN1 nuclear accumulation, indicating STAT3 regulates CIAPIN1 expression and translocation. Using immunofluorescence, we found that CIAPIN1 significantly accumulated in the nucleus and that transgelin was decreased by PDGF-BB stimulation. In addition, CIAPIN1 overexpression, mainly located in the nucleus, diminished CAB-mediated transgelin restoration, indicating blockade of CAB-induced VSMC differentiation (Fig. 6C). Furthermore, CIAPIN1 overexpression attenuated the inhibitory effect of CAB on PDGF-BB-stimulated VSMC proliferation and migration (Fig. 6D and E). These findings, together with those shown above, reinforce the idea that CIAPIN1 is one of the regulators for STAT3-mediated VSMC dedifferentiation, proliferation, and migration.
3.7.　 CIAPIN1 overexpression suppresses the inhibitory effect of CAB on neointima formation by affecting identified target alteration
To investigate the physiological effect of CIAPIN1 and CAB on neointima formation after vascular injury, we performed balloon injury and CIAPIN1 overexpression in rat common carotid arteries and administered CAB for 14 days.
Using in silico toxicology analysis for favorable drug-like properties, we found that the CAB had no predicted toxicities (Supporting Information Table S2). In addition, CAB administration did not show any side effects, such as diarrhea and weight loss (Supporting Information Fig. S7A). As shown in Fig. S7B, there were no significant changes in the histological morphology of major organs, such as the liver, heart, lung, and kidney. Moreover, there were no alterations in glutamic pyruvic transaminase (GPT/ALT, soluble) or glutamic-oxaloacetic transaminase 1, (GOT1/AST, soluble) levels following CAB administration (Fig. S7C). Since VSMC apoptosis is closely associated with neointima formation, determining plaque stability59, we also examined the influence of CAB on VSMC apoptosis using TUNEL staining. There were no signs of cell death induction by CAB administration compared with that of the control group, supporting drug safety (Fig. S7D).
CAB administration significantly attenuated balloon injury-induced neointima formation compared to the BI group (Fig. 7A and B). The arterial intimal areas and intima/media ratios were lower in the BI + CAB group than in the BI group, while no differences were noted in the media area and circumference of the external elastic lamina (EEL). Furthermore, we investigated the effect of CAB in mouse carotid ligation model, which induced neointima formation by disturbing flow or increasing shear stress, thus stimulating growth factor secretion and VSMC activation, to determine the therapeutic effect of CAB in vascular dysfunction60. The group administrated with CAB in ligated arteries of mouse (LI + CAB) significantly reduced neointima formation by lowering intimal areas and intima/media ratios from ligation-induced carotid artery in comparison with control group (LI + Con) (Fig. S7E and S7F). In addition, CAB administration significantly decreased the expression of PCNA, a proliferative marker, compared with that in the BI group, indicating inhibition of VSMC hyperplasia (Fig. 7C).
Given that CAB regulates CIAPIN1, we performed CIAPIN1 overexpression in arteries and examined the effect of CAB on neointima formation. As shown in Fig. 7A and B, CIAPIN1 overexpression enhanced neointima formation and attenuated the inhibitory effect of CAB on neointima formation, validated by intima area and PCNA staining (Fig. 7C).
Since vascular remodeling causes dysfunction in several cell types, we investigated the localization and expression of CIAPIN1 in arteries. The immunofluorescence results of arteries showed abundant CIAPIN1 expression with smooth muscle (SM) α-actin in the neointima region. CAB administration remarkably decreased CIAPIN1 expression, but CIAPIN1 overexpression induced CIAPIN1 expression in the neointima region, indicating CIAPIN1 functions to intima hyperplasia mostly in VSMCs (Fig. 7D).
In VSMCs, CIAPIN1 overexpression attenuated CAB-mediated suppression of JAK2 expression and JAK2 and STAT3 phosphorylation (Fig. 7E and Supporting Information Fig. S8). In addition, CIAPIN1 overexpression attenuated the inhibition of KLF4 and SPP1 expression and the increase in CDKN1B expression by CAB treatment, indicating that CAB inhibits VSMC dedifferentiation, proliferation, and migration via CIAPIN1-associated downstream pathways.
Taken together, our results support the conclusion that CAB inhibits STAT3 activation and subsequently attenuates the CIAPIN1/JAK2/STAT3 pathways to affect KLF4-mediated VSMC phenotypic switching and CDKN1B-mediated proliferation and migration.
4.　Discussion
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VSMC phenotypic switching leads to abnormal cell proliferation and migration and contributes to the development of neointima formation, followed by atherosclerotic progression and vascular remodeling5. In the present study, we demonstrated the therapeutic relevance of novel 9H-carbazol-3-yl 4-aminobenzoate (CAB) based on the following: (1) CAB suppressed PDGF-BB-induced VSMC dedifferentiation, proliferation and migration; (2) CAB inhibited PDGF-BB-induced STAT3 activation and impeded CIAPIN1/JAK2/STAT3 autoregulatory circuits, affecting their transcription; (3) STAT3 inhibition via CAB is critical for KLF4-mediated phenotypic switching and CDKN1B-mediated cell cycle arrest and MMP9 suppression; and (4) STAT3 inhibition by CAB blocked VSMC neointima formation through suppressing the CIAPIN1/JAK2/STAT3 pathways, which was consistent with the in vitro results. These findings demonstrate the role of STAT3 on CIAPIN1 regulation in vascular remodeling.
Carbazole is a tricycle compound with two benzene rings fused on either side of a pyrrole core61. Although studies initially focused on the fluorescent properties, naturally occurring bioactive carbazole alkaloids were isolated mainly from taxonomically similar plants of the genera Murraya, Flycosmis, and Clausena in the family Rutaceae62,63. Since then, carbazole has been considered an important privileged scaffold in drug discovery. Many derivatives with a carbazolic core have been developed, and some of them have shown biological activities due to electrophilic aromatic substitution, oxidative reactions, and alkylation reactions upon binding to the target proteins64. Their pharmaceutical potential provides an opportunity to discover and develop drug candidates with antioxidant65, antiviral66, antimicrobial67, and anti-inflammatory properties68. We previously reported that murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin, inhibited PDGF-BB-induced VSMC cell cycle progression38. LCY-2-CHO ([9-(2-chlorobenzyl)-9H-Carbazole-3-carbaldehyde]) inhibited inflammatory gene expression inducing heme oxygenase-1 gene expression in VSMCs68. Moreover, 3-N- or 3-O-cinnamoyl carbazole derivatives protected against high-mobility group box 1-mediated vascular disruptive responses in human umbilical vein endothelial cells23. In this study, CAB selectively inhibited STAT3 activation by PDGF-BB and suppressed VSMC-mediated neointimal hyperplasia (Figs. 2 and 7, Figs. S2, S5, and S7). Furthermore, we validated the drug-likeness of CAB using PK and toxicity analysis. From the perspective that intracellular PK is converted to pharmacodynamics, the effect of CAB in VSMCs appeared immediately and was maintained for a long time; thus, the PK results provided further evidence of a CAB-mediated therapeutic effect (Fig. 1F). Moreover, toxicity prediction results and in vitro and in vivo toxicity analysis supported CAB as a safe drug candidate and scaffold (Table S2, Fig. 1C and D, Fig. S7).
Cytokines or growth factors, such as PDGF, bind to their corresponding cell surface receptors69. The formation of a dimer complex by these bound receptors initiates the recruitment of JAKs, thereby activating the JAK/STAT signaling pathway through a phosphorylation cascade. The cytoplasmic phosphorylated tyrosine residues of these receptors create a dock for the STAT3 SH2 domain70. STAT3 is activated through phosphorylation of Tyr705 located in the SH2 domain. Once activated, phospho-STAT3 monomers interact via their SH2 domain to form a homodimer of phospho-STAT3, which then dissociates from cytoplasmic partners and translocates to the nucleus, where it binds to DNA elements within target genes to regulate transcription71. Blockade of the SH2 domain of STAT3 using a small-molecule inhibitor can be a potentially promising therapeutic approach in the development of molecularly STAT3 targeted therapies for the treatment of abnormal proliferative disease. Our study showed that CAB interacted with the STAT3 SH2 domains using docking experiments, His-tag pull-down assay, and SPR assay. In addition, CAB inhibited PDGF-BB-induced STAT3 Tyr705 phosphorylation, dimerization, nuclear translocation, and transcriptional activity (Figs. 2 and 3). We investigated the inhibitory effects of CAB in PDGF signaling and STAT3 activation of VSMCs, employing multiple treatment time points with PDGF-BB. Initially, phosphorylation of PDGFR-β, AKT, ERK1/2, p38, and STAT3 has observed around the 15-min time point72. Dimerization of the receptor or cytosolic proteins occurred approximately 1 h after phosphorylation73,74. Dimerized STAT3 then functioned as a transcription factor in the DNA binding region around 6 h75. Subsequently, through STAT3 activation and the downstream gene expression, events such as cell cycle progression, migration, and phenotype switching took place, with related target proteins detected around the 24-h mark76.
VSMCs can be classified into two major phenotypes: fully differentiated contractile cells responsible for vasocontraction and dedifferentiated synthetic cells consisting of proliferative and migratory cells activated during growth and injury77. Notably, switching of the two phenotypes from contractile to synthetic is pivotal for stable plaque formation. Thus, reducing the excessive proliferation and migration of VSMCs via phenotypic switching is a therapeutic strategy for atherosclerosis. Our previous study reported that CIAPIN1 accelerates VSMC dedifferentiation through contractile and synthetic gene regulation under injury conditions16. STAT3 inhibition using CAB, stattic and the Tyr705 mutant suppressed CIAPIN1 expression, indicating STAT3 as an upstream molecule and showing that STAT3 regulated CIAPIN1 transcription (Figs. 3, 4). Interestingly, CIAPIN1 has been reported to be a JAK2 regulator78, and the CIAPIN1/JAK2/STAT3 pathway can be an autoregulatory circuit from STAT3 activation. In addition, we found that KLF4 is a downstream molecule of CIAPIN1/JAK2/STAT3. Since KLF4 acts as a repressor of myocardin activity and subsequent contractile gene transcription50, CIAPIN1-mediated KLF4 activation is important evidence of VSMC proliferation and migration (Fig. 7E). While our qPCR and transcriptional data suggest that CAB, as a novel compound, potentially suppresses the CIAPIN1 and KLF4 pathways, the overall transcriptional landscape requires further characterization through bulk RNA-seq to fully profile its effects.
5.　Conclusions
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In conclusion, this study demonstrated that the compound CAB inhibited vascular intima hyperplasia by affecting VSMC phenotypic switching, proliferation, and migration. In addition, CAB suppressed PDGF-induced STAT3 activation by directly binding to the SH2 domain of STAT3. Moreover, STAT3, target molecules of CAB, play essential roles in CIAPIN1/JAK2/STAT3-mediated KLF4 and CDKN1B regulation in VSMCs. These findings provide new insight into the mechanism by which STAT3 regulates vascular remodeling and molecular links to therapeutic targets for the treatment of atherosclerosis, restenosis, and other vascular proliferative diseases.
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Year 2025 volume 15 Issue 3
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doi: 10.1016/j.apsb.2024.12.035
  • Receive Date：2024-07-19
  • Online Date：2026-09-18
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  • Received：2024-07-19
  • Revised：2024-09-24
  • Accepted：2024-10-22
Affiliations
    aCollege of Pharmacy and Research Institute of Pharmaceutical Sciences, Woosuk University, Wanju 55338, Republic of Korea
    bCollege of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
    cCollege of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
    dInstitute of Drug Research and Development, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
    eDepartment of Pharmacology, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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