Graphical abstract graphic file with name fx1.jpg [47]Open in a new tab Highlights * • Lipo-PF4 inhibited the development of thoracic aortic aneurysm and dissection * • PF4 maintained the endothelial integrity of the aorta effectively in TAAD-mice * • PF4 improves endothelial cell function in vitro by blocking FGF-FGFR signaling __________________________________________________________________ Cardiovascular medicine; Therapeutics; Drug delivery system; Biological sciences Introduction Thoracic aortic aneurysm and dissection (TAAD) is a highly lethal cardiovascular condition that can lead to sudden death owing to aortic rupture. TAAD refers to dilation or aneurysm of the thoracic aorta, often accompanied by aortic dissection. Various risk factors contribute to the development of TAAD, including underlying genetic predisposition, hypertension, atherosclerosis, aortic diameter, and/or aneurysm.[48]^1 Currently, no pharmacological therapies have been validated as effective in mitigating or preventing the progression of TAAD, despite recent advancements in the surgical repair of TAAD.[49]^2 To date, the pathogenic mechanisms of TAAD remain elusive. TAAD is characterized by the pathological manifestation of endothelial cell dysfunction, loss of vascular smooth muscle cells (VSMCs), and degradation of elastic fibers in the aortic wall.[50]^3^,[51]^4 These modifications can result in rupture and death. Tearing of the intima and endothelial cell dysfunction are critical steps in the development of TAAD. Endothelial cell (EC), one of the vital cell types implicated in vascular pathophysiology, have attracted increasing attention in the study of aortic diseases. Recently, Yang et al. reported that endothelial tight junctions’ function was disrupted in a β-aminopropionitrile (BAPN)-induced TAAD mouse model through a new type of contrast medium. Furthermore, inhibitors targeting endothelial tight junctions have been shown to reduce the incidence of TAAD. However, the mechanism by which the molecular function of EC is disrupted during TAAD development remains unclear. Human platelet factor 4 (PF4), also known as chemokine ligand 4 (CXCL4), is a chemokine with a molecular weight of 7.8 kDa. It is primarily synthesized by megakaryocytes and released from platelet α-granules. PF4 primarily exists as a tetramer[52]^5 and has a strong affinity for negatively charged molecules, such as infectious agents[53]^6 and endothelial proteoglycans.[54]^7 It has been reported that PF4 has a significant involvement in various diseases, particularly cardiovascular diseases. Gentilini et al.[55]^8 found that PF4 inhibits human umbilical vein endothelial cell proliferation by interfering with the activity of cyclin E-cyclin-dependent kinase 2 (cdk2). Bikfalvi demonstrated the anti-angiogenic effects of PF4 through three distinct mechanisms.[56]^9 Despite the crucial role of PF4 in the inhibition of endothelial cell proliferation and angiogenesis, no previous study has explored the potential relationship between PF4 and TAAD. Our study revealed that PF4 can improve TAAD in mice. We constructed liposome-PF4 (Lipo-PF4) to explore the mechanisms by which liposomes improve drug bioavailability and effectiveness.[57]^10 Our findings validated that Lipo-PF4 significantly improved the pathological condition of TAAD. Results PF4 improves endothelial cell function under pathological conditions We treated human aortic endothelial cells (HAECs) with various concentrations of Ang II or TNF-α for 48 h to establish their effective doses. Subsequently, we assessed the proliferation ([58]Figures S1A and S1B) and adhesion abilities of the cells ([59]Figures S1C and S1D). At low concentrations, Ang II stimulated HAEC proliferation, whereas at high concentrations, it inhibited proliferation. Treatment with 1 μM Ang II decreased the cell adhesion rate by 50%. Therefore, we selected 1 μM Ang II as the pharmacological dose for subsequent experiments. Cell proliferation was not affected by low concentrations of TNF-α but was slightly inhibited at high concentrations. When treated with 5 μM TNF-α, the cell adhesion rate decreased by 50%. Therefore, 5 μM was selected as the pharmacological dose of TNF-α for subsequent experiments. We analyzed PF4 levels to address Ang II- or TNF-α-induced endothelial cell dysfunction. We found that increasing the PF4 concentration resulted in a stronger adhesion ability of HAECs ([60]Figures 1A and 1B). In addition, PF4 reduced the migration ([61]Figures 1C and 1D) and inhibited angiogenesis ([62]Figures 1E and 1F) of Ang II- or TNF-α-induced HAECs. Figure 1. [63]Figure 1 [64]Open in a new tab PF4 improves endothelial cell function in pathological conditions (A) Human aortic endothelial cells were treated with 1 μM AngII alone, 1 μM AngII plus PF4 at the indicated concentrations. Cell adhesion was measured 48 h later using CCK8 assay. (B) Human aortic endothelial cells were treated with 5 μM TNFα alone, 5 μM TNFα plus PF4 at the indicated concentrations. Cell adhesion was measured 48 h later using CCK8 assay. (C and D) Human aortic endothelial cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 at the indicated concentrations for 36 h, treated cells were seeded onto the upper chamber for 12 h, cells on the lower side of the filter were detected using an inverted microscope. (E and F) Human aortic endothelial cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 at the indicated concentrations for 36 h, treated cells were seeded onto the matrigel for 4 h in a 24-well plate. After incubating with 2 μM calcein AM for 30 min, invert the fluorescence microscope for detection. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. Liposome encapsulates PF4 and reduces its degradation rate in mice Considering the positive effect of PF4 on improving endothelial cell function in vitro, validation of its effectiveness in vivo was necessary. By expressing plasmid pET-28a in E. coli, we produced and obtained murine-derived PF4 protein. Protein purity was detected by SDS-PAGE with Coomassie brilliant blue staining ([65]Figure S2A). We utilized the well-established lipid film hydration method to encapsulate PF4 into liposomes, which are widely recognized as highly effective nanodrugs. The morphology of the liposomal nanodrugs was examined using an electronic microscope to confirm their formation. Lipo-PF4 samples displayed liposomal structures measuring approximately 100 nm in diameter ([66]Figure 2A). Dynamic light scattering (DLS) analysis revealed that the intensity averaged hydrodynamic diameter of Lipo-PF4 was 100 nm ([67]Figure 2B). Furthermore, we assessed the encapsulation efficiency of the liposomes using an ELISA assay ([68]Figure 2C). In summary, we successfully prepared Lipo-PF4, a systemic drug with a size of approximately 100 nm that can be transported in the bloodstream. Liposomes maintained a higher concentration of PF4 in the blood of mice after 48 h ([69]Figure 2D). After 100 μg/kg PF4 liposome-CY5 was injected into 8-week-old C57 mice for 12 h through the tail vein, the PF4 liposome-CY5 distribution was detected by immunofluorescence. We found that Lipo-PF4 was mainly found in endothelial cells of vascular intima, and a small part was found in smooth muscle cells of vascular media ([70]Figure 2E). Figure 2. [71]Figure 2 [72]Open in a new tab Liposome encapsulation treatment reduces the degradation rate of PF4 protein in mice (A) PF4-SOD liposome was made through the rotation-evaporation method. Its morphology and diameter were detected using an electronic microscope. (B) Detection of liposome particle size using dynamic light scattering method. (C) ELISA assay for detecting the encapsulation efficiency of liposomes. (D) 100 μg/kg PF4 or PF4 liposome was injected into 8-week-old C57 mice through the tail vein, and the concentration of PF4 in the mouse blood was detected using ELISA assay at different time points. (E) After 100 μg/kg PF4 liposome-CY5 was injected into 8-week-old C57 mice for 12 h through the tail vein, the PF4 liposome-CY5 distribution was detected by immunofluorescence. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. Additionally, we evaluated the biosafety of Lipo-PF4. H&E staining of the liver, heart, spleen, lung, and kidneys of mice after different treatments revealed no histopathological damage ([73]Figure S2B). Next, we measured routine blood indicators, including alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), creatinine (CR), and microalbumin (mALB). No statistically significant differences were observed ([74]Figures S2C–S2E). These results demonstrated the biological safety of Lipo-PF4. Lipo-PF4 nanoparticles ameliorate TAAD formation in vivo Four-week-old mice were then subjected to TAAD induction (4 weeks with BAPN for 21 days and 7 days with AngII) to investigate the potential effect of Lipo-PF4 on the progression of TAAD.[75]^11 Moreover, A BAPN-induced mouse (3 weeks with BAPN for 25 days) TAAD model[76]^12 was established to investigate the potential effect of Lipo-PF4 on the progression of TAAD. The experimental protocol is briefly described in [77]Figures 3A and [78]S3A. The increased aortic diameter of TAAD mice was reduced after treatment with Lipo-PF4 ([79]Figures 3B–3E). However, PF4 had a tendency to decrease the maximum aortic diameter of TAAD mice, but there was no significant statistical difference between PF4 and TAAD mice ([80]Figures 3D and 3E). Lipo-PF4 significantly reduced the incidence of BAPN with AngII-induced TAAD from 70% to 20% ([81]Figure 3F). In addition, Lipo-PF4 significantly reduced the incidence of BAPN-induced TAAD from 70% to 20% ([82]Figure S3B). Lipo-PF4 was more effective in reducing the incidence of TAAD in mice than PF4 (10% vs. 40% and 20% vs. 50%) in both models ([83]Figures 3F and [84]S3B). Lipo-PF4 effectively maintained the endothelial integrity of the aorta in mice ([85]Figures 3G and [86]S3C). In addition, Lipo-PF4 and PF4 decreased MMP activity in cultured supernatant of vascular wall endothelial cells as evidenced by gelatin zymography assay ([87]Figures 3H and [88]S3D). Furthermore, histological staining analysis of the aortic tissue demonstrated that both Lipo-PF4 and PF4 improved the condition of the aorta and decreased elastin disruption ([89]Figures 3I, 3J, [90]S3E, and S3F). Taken together, our data strongly suggested that Lipo-PF4 ameliorated TAAD formation in vivo, and its protective effect is obviously better than that of PF4. Figure 3. [91]Figure 3 [92]Open in a new tab Liposome-PF4 nanoparticles repress thoracic aortic dissection formation by improving endothelial function (A) Mice were treated with liposome-PF4, PF4, or empty liposome once every three days at 4 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile monofumarate) for 21 days. Subsequently, mice were treated with 1 μg/kg per minute AngII using minipump (n = 10, per group). (B and C) Representative images of mice aorta were shown. (D and E) maximum aortic diameter was measured (n = 10, per group). (F) AD incidence was statistically analyzed. (G) Representative images of mouse aorta stained with CD31 (red) and DAPI (blue). (H) Effect of PF4 on MMP activities in cultured supernatant of vascular wall endothelial cells by gelatin zymography. (I) Representative macroscopic images of aorta sections were stained with hematoxylin and eosin (H&E), Masson, and elastic-Van Gieson (EVG) staining. (J) Quantification of elastin integrity in each group of mouse aortas. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. PF4 improves endothelial cell function by combining combination with heparin sulfate We further explored the relative mechanism of PF4 on endothelial cell function to better understand its biological safety and effectiveness in TAAD treatment. Recently, Lord et al. reported that PF4 has a strong affinity for endothelial-derived heparan sulfate chains, which are crucial for signal transduction.[93]^13 We hypothesized that PF4 would improve endothelial cell function by combining with heparin sulfate. HSase has been extensively used for the removal of heparin sulfate from the cell surface, as mentioned in previous studies.[94]^14^,[95]^15 We observed that HSase addition did not affect the adhesion, invasion, and angiogenesis of Ang II- or TNF-α-stimulated HAECs ([96]Figure 4). However, HSase reversed the cell adhesion effect of PF4 on HAECs ([97]Figures 4A and 4B). PF4 inhibited HAECs invasion; however, this effect was abolished by HSase ([98]Figures 4C and 4D). The tube formation assay demonstrated that HSase reversed the angiogenic effect of PF4 on HAECs. Our findings confirmed that PF4 improved HAECs function via heparin sulfate. Figure 4. [99]Figure 4 [100]Open in a new tab PF4 improves endothelial cell function by combining with heparin sulfate (A and B) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 (10 μM) for 48 h, Cell adhesion was measured using CCK8 assay. (C and D) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4 (10 μM) for 36 h, treated cells were seeded onto the upper chamber for 12 h, cells on the lower side of the filter were detected using an inverted microscope. (E and F) After incubated with HSase (1 mU/ml, Sigma-Aldrich) for 4 h, cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4(10 μM) for 36 h, treated cells were seeded onto the matrigel for 4 h in a 24-well plate. After incubating with 2 μM calcein AM for 30 min, the vascular cyclization ability of cells is detected. Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. PF4 inhibits TAAD and improves endothelial cell function by blocking FGF-FGFR signaling Our findings verified that PF4 improved the function of HAECs via heparin sulfate. However, the precise mechanism by which PF4 interacted with heparin sulfate remained unclear. Structural and biochemical data have revealed that heparan sulfate proteoglycan is required as an additional co-receptor to induce fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) signaling.[101]^16^,[102]^17 FGF-FGFR signaling has been linked to cell adhesion, angiogenesis, and migration.[103]^18^,[104]^19^,[105]^20 Next, aortic intima was isolated from BAPN group, Lipo-PF4 + BAPN group, and FGFR inhibitor + BAPN group mice for transcriptomic analysis ([106]Figures 5A and [107]S4A). Gene Ontology (GO) pathway enrichment analysis indicated the differentially expressed genes compared between control group and Lipo-PF4 group were enriched in transmembrane receptor protein tyrosine kinase signaling pathway and ERK1 and ERK2 cascade. FGF-FGFR signaling is one of the main members of transmembrane receptor protein tyrosine kinase signaling pathway ([108]Figure 5B). GO pathway enrichment analysis indicated the differentially expressed genes compared between control group and AZD4547 group were enriched in Notch signaling pathway and transmembrane receptor protein tyrosine kinase signaling pathway ([109]Figure S4B). Thus, we inferred that PF4 improved HAECs function by inhibiting FGF-FGFR activation. However, FGF has some side effects, such as reducing blood pressure and increasing cell proliferation during treatment in vivo.[110]^21 [111]SUN11602 was synthesized to mimic the mechanism of basic FGF and overcome the limitations of FGF.[112]^22 We administered [113]SUN11602 with Lipo-PF4 to BAPN-induced mice via the tail vein ([114]Figure 5C). [115]SUN11602 reversed the protective effect of Lipo-PF4 against TAAD. The maximum diameter and incidence of TAAD increased after the addition of [116]SUN11602 compared with the Lipo-PF4 group ([117]Figures 5D–5F). [118]SUN11602 worsened the pathological condition of the aorta in BAPN-induced mice compared to that in the Lipo-PF4 group, as evidenced by the results of H&E, Masson, EVG staining, and gelatin zymography ([119]Figures 5H–5G). Additionally, immunofluorescence staining revealed that HAEC integrity was disrupted in the Lipo-PF4 + [120]SUN11602 group ([121]Figure 5J). Figure 5. [122]Figure 5 [123]Open in a new tab PF4 represses thoracic aortic dissection formation and improves endothelial cell function by inhibiting the activation of FGF-FGFR (A) Differential gene analysis between BAPN group and Lipo-PF4 group. Mice were treated with liposome-PF4 once every three days at 3 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile monofumarate) for 14 days (n = 9, per group).Mouse aortic intima were isolated, and the aortic intima of three mice were mixed into one sample for transcriptome sequencing, and performed differential gene analysis. N: BAPN group; P: lipo-PF4+BAPN group. (B) GO analysis of differential genes. (C) Mice were treated with liposome-PF4 or liposome-PF4 plus [124]SUN11602 once every three days at 3 weeks. Three days after the first administration, mice were treated with 0.25% BAPN (β-aminopropionitrile mono fumarate) for 25 days (n = 10, per group). (D) Representative images of mice aorta were shown. (E) Maximum aortic diameter was measured (n = 10, per group). (F) AD incidence was statistically analyzed. (G) Effect of PF4 and [125]SUN11602 on MMP activities in cultured supernatant of vascular wall endothelial cells by gelatin zymography. (H) Representative macroscopic images of aorta sections were stained with hematoxylin and eosin (H&E), Masson, and elastic-Van Gieson (EVG) staining. (I) Quantification of elastin integrity in each group of mouse aortas. (J) Representative images of mouse aorta stained with CD31 (red) and DAPI (blue). Data are expressed as mean ± SD. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. [126]SUN11602 decreased HAEC adherence and increased migratory and angiogenic capacity. [127]SUN11602 reversed the effect of PF4 on the adhesion, migration, and angiogenic capacity of HAECs ([128]Figures S5A–S5F). These results demonstrated that PF4 inhibited the development of TAAD and improved HAECs function by blocking the FGF-FGFR signaling pathway. PF4 blocks the downstream signal transduction induced by FGF-FGFR The FGF-FGFR signaling pathway is involved in the regulation of various cellular functions, including migration, differentiation, and proliferation.[129]^23 The classical downstream signaling pathways of FGF-FGFR include PI3K/AKT, Ras/Raf-MEK-MAPK, and STAT signaling pathways.[130]^24 Our findings confirmed that PF4 inhibited TAAD in mice and improved endothelial cell function by blocking FGF-FGFR signaling. However, the effect of PF4 on the FGF-FGFR downstream signaling pathway in HAECs remained to be determined. Western blot analysis revealed that PF4 decreased the expression of phosphorylated (p)-ERK1/2, phosphorylated (p)-P38, and phosphorylated (p)-AKT (Ser473) in Ang II- or TNF-α-stimulated HAECs ([131]Figures 6A and 6B). However, these inhibitory effects were abolished by [132]SUN11602 ([133]Figures 6C and 6D) and HSase ([134]Figures S6A and S6B). Collectively, these findings revealed that PF4 blocked the downstream signaling pathways of FGF-FGFR. Figure 6. [135]Figure 6 [136]Open in a new tab PF4 represses MAPK signal transduction by blocking FGFs-FGFR (A and B) Human aortic endothelial cells were treated with 1 μM AngII or 5 μM TNFα alone, 1 μM AngII or 5 μM TNFα plus PF4 at the indicated concentrations for 24 h. The phosphorylated and total levels of Erk1/2, P38, Akt (Ser 473), and Akt (Thr 308) were detected by western blotting analysis (n = 3). (C and D) Cells were treated with 1 μM AngII or 5 μM TNFα alone, AngII or TNFα plus PF4(10 μM) and [137]SUN11602(10 μM) for 24 h. The phosphorylated and total levels of Erk1/2, P38, and Akt (Ser 473) were detected by western blotting analysis (n = 3). Density ratios of phosphorylated proteins to total proteins were presented as the mean ± standard deviation. ∗p < 0.05, ∗ ∗p < 0.01, ∗ ∗ ∗p < 0.001. Discussion Our study provides the evidence that PF4 attenuates TAAD development in a mouse model. Elevated circulating levels of PF4 injected into the tail vein significantly reduced the incidence and dilatation of TAAD. Additionally, it maintained the integrity of the endothelial cells and improved the condition of the aorta. Treatment of HAECs with appropriate concentrations of PF4 mitigated Ang II- or TNF-α-induced reduction in cellular adhesion and increased cell migration and angiogenesis by blocking FGF-FGFR signaling. These findings suggest that PF4 may play a role in reducing the development of TAAD, at least in part, through maintaining endothelial cell integrity and improving endothelial function. PF4, the first isolated chemokine with biological function, is involved in various diseases, including immune thrombocytopenia,[138]^25 age-related cognitive decline,[139]^26 cancer,[140]^27 and atherosclerosis.[141]^28 A recent proteomics study[142]^29 identified PF4 as a potential biomarker for the pathogenesis of aortic dissection. In addition, Xavier et al.[143]^30 revealed the involvement of platelet-derived PF4 and neutrophil-derived IL-8 in the formation of intraluminal thrombus in abdominal aortic aneurysm. Ample evidence suggests a potential association between PF4 and aortic disease; however, no study has been conducted to explore this relationship. The present study demonstrated that PF4 attenuates TAAD formation by improving endothelial cell function. Moreover, the constructed nanoparticle drug containing PF4 exhibited a remarkable protective effect in vivo, offering a perspective for the application of PF4. The nanoparticle drug decreased the biodegradability rate of PF4 and was safe in vivo, as previously demonstrated for other nanoparticle drugs.[144]^31^,[145]^32^,[146]^33 Recently, many studies have revealed that endothelial cell barrier dysfunction is a critical characteristic of TAAD progression. Endothelial tight junction disruption, vascular inflammation, and edema have been observed in the aorta of BAPN-induced TAAD.[147]^34 Shanshan et al. reported that the endothelial HDAC1-ZEB2-NuRD complex drives aortic aneurysm[148]^35 and dissection by regulating protein S-sulfhydration.[149]^36 Our findings revealed that PF4 maintains endothelial cell integrity, to alleviate the formation of TAAD in vivo. This finding further confirms the significance of endothelial cells in the development of TAAD.[150]^36 Cell migration and tube formation have been identified as characteristic features of aortic dissection. PF4 inhibited the migration and tube formation of HAECs but strengthened cell adhesion. We used a cell adhesion assay to evaluate endothelial cell function in TAAD, which provides an approach for validating cellular functions in TAAD. Previous studies have discovered relevant molecular mechanisms of PF4 in cardiovascular diseases. Sachais et al. found that PF4 could promote atherosclerosis in mice.[151]^37 Yu et al. discovered that PF4 could increase the expression of E-selectin in endothelial cells through activation of transcriptional activity of nuclear factor-kappa B.[152]^38 Aidoudi and Bikfalvi summarized the proatherogenic role of PF4. PF4 deposited on impaired endothelial cells, recruited peripheral monocytes, and facilitated its differentiation into macrophages, leading to localized inflammation and plaque formation.[153]^39 TAA shares most of the pathogenic mechanisms with atherosclerosis. However, we demonstrated that PF4 protects TAAD formation through maintaining the integrity of endothelial cells in vivo. TAAD, as distinguished from atherosclerosis, is characterized by intimal tearing, and false lumen formation. It had been proved that endothelial cell loss and subsequent exposure of sub-endothelial basement occurred before intimal tearing.[154]^40^,[155]^41 We surprisingly discovered that PF4 could maintain the integrity of endothelial cells in vivo and enhance the adhesion of endothelial cells in vitro. We inferred that the effect of PF4 on endothelial cells prevents intimal tearing and protects TAAD formation. Further in-depth studies should be conducted to investigate the converse effect of PF4 between TAAD and atherosclerosis. The multimerized form of PF4 exerts its pharmacological effects through several mechanisms. PF4 has been proven in studies to effectively inhibit FGF2-induced proliferation of capillary endothelial cells in the adrenal cortex.[156]^42 PF4 inhibits FGF-2 dimerization, contributing to its anti-angiogenic properties in murine microvascular endothelial cells.[157]^43 Our findings validated that PF4 effectively inhibits the migration and angiogenesis of HAECs, while simultaneously improving cell adhesion by blocking FGF-FGFR signaling. We also assessed the effects of PF4 on the downstream signaling of FGF-FGFR. A previous study indicated that PF4 significantly inhibits ERK phosphorylation, with no effect on Akt phosphorylation.[158]^42 PF4 increases the expression of phosphorylated-P38, as previously reported.[159]^44^,[160]^45 However, our findings revealed that PF4 decreased the expression of phosphorylated (p)-ERK1/2, phosphorylated (p)-P38, and phosphorylated (p)-AKT (Ser473) in Ang II- or TNF-α-stimulated HAECs. We inferred that these discrepancies could be attributed to the different stimulations and cell types, implying that further in-depth investigations into the mechanism of PF4 and its signaling pathways are necessary. In conclusion, the present study demonstrated that PF4 has a protective effect against the development of TAAD. PF4 improves endothelial cell adhesion and inhibits cell migration and angiogenesis. These effects were attributed, at least partially, to the inhibition of the FGF-FGFR signaling pathway and that it ameliorated TAAD development. Therefore, PF4 may serve as promising therapeutic approach for the prevention and treatment of TAAD. Limitations of the study Despite these significant and findings, the present study has several limitations. In this study, we used two types of TAAD model, and the scope of application of PF4 should be clarified through further investigation. Clinical data on the association between PF4 and TAAD prevalence are lacking. Further clinical studies are required to explore the relationship between PF4 and TAAD. Furthermore, the present in vivo study was validated by the systemic administration of PF4. Therefore, loss-of-function experiments are required to elucidate the role of endogenous PF4 in TAAD. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr Jianfang Luo (jianfangluo@sina.com). Materials availability Materials used in this study are available from the [161]lead contact upon reasonable request. Data and code availability * • The deposited data of this study are presented in [162]key resources table. The data of the RNA-seq (SRA code PRJNA112618061) was deposited to the NIH SRA database. All data reported in this paper will be shared by the [163]lead contact upon reasonable request. * • This paper does not report original code. * • Any additional information required to reanalyze the data reported in this paper is available from the [164]lead contact upon reasonable request. Acknowledgments