ABSTRACT
Introduction
Previous studies have demonstrated that chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by excessive fibrin deposition which is related to impaired production of tissue plasminogen activator(t‐PA) by epithelial cells. This study aims to evaluate whether t‐PA expression in endothelial cells is also decreased under the inflammatory milieu of CRSwNP.
Methods
Vascularity and proangiogenic genes expression in polyp tissues from eosinophilic CRSwNP (eCRSwNP) and non‐eosinophilic CRSwNP (neCRSwNP) were assessed by immunohistochemistry and real‐time PCR. Single‐cell RNA sequencing data set of CRS, Immunohistochemistry were used. Human primary nasal endothelial cells were stimulated by IL‐13 and IFN‐γ with or without retinoic acid.
Results
We observed the increased expression of proangiogenic genes and vascularity in both eCRSwNP and neCRSwNP. Single‐cell RNA sequencing and immunostaining revealed that t‐PA expression was decreased in endothelial cells of polyp tissues. In vitro study, IL‐13 and IFN‐γ could significantly attenuate t‐PA expression in endothelial cells, which can be rescued by retinoic acid.
Conclusions
Our findings showed a significant contribution of endothelial cells in the production of t‐PA in sinonasal tissues. Furthermore, the levels of t‐PA in endothelial cells could also be impaired in the inflammatory environment of CRSwNP. Retinoic acid could restore t‐PA expression in endothelial cells impaired by inflammatory cytokines (including IL‐13 and IFN‐ γ), thus degrading the deposited fibrin in polyp tissue.
Keywords: endothelial cells, interleukin‐13, nasal polyps, retinoic acid, tissue plasminogen activator
1. Introduction
Chronic rhinosinusitis with nasal polyps (CRS) is a heterogeneous inflammatory diseases in the nasal cavity and paranasal sinuses [1]. CRSwNP in Western populations is mostly characterized by eosinophilic inflammation and the elevation of type 2 cytokines including IL‐5 and IL‐13 [2]. Whereas a higher proportion of patients in Asian countries present with non‐eosinophilic inflammation [3]. Despite the recent progress in the understanding of endotype and biologic therapy, the mechanisms of polyp formation and the relationship between inflammation and tissue remodeling remains to be clarified [4].
Nasal polyps (NP) tissues are benign and edematous masses arising from the ostiomeatal complex or paranasal sinus cavity. Excessive fibrin deposition in NP tissues leads to plasma protein retention, and further promotes the formation of intense edema and pseudocysts [5]. The level of tissue plasminogen activator (t‐PA), which could convert plasminogen to plasmin and induce fibrinolysis, was significantly decreased in NPs from patients with CRSwNP [6]. Previous studies also observed that the t‐PA expression was mainly distributed in nasal epithelial cells and significantly downregulated under the stimulation of type 2 cytokines including IL‐4 and IL‐13 [5, 7]. Recent study by Chen et al. [8] also suggested common features of fibrin deposition and t‐PA downregulation in polyp tissues from non‐eosinophilic CRSwNP patients.
Early studies revealed that t‐PA was stored and constitutively secreted in endothelial cells [9]. Angiogenesis was also an important remodeling feature for CRS [10]. Akin to t‐PA regulation in nasal epithelial cells, whether the expression of t‐PA in endothelial cells from polyp tissue was decreased remains unclear. Therefore, the aim of the current study was to evaluate the expression of t‐PA in nasal endothelial cells as well as the effect of major inflammatory cytokines such as IL‐13. In light of the potent upregulation of retinoic acid on t‐PA expression in nasal epithelial cells, we will further explore whether retinoic acid could rescue the expression of t‐PA in primary cultured nasal endothelial cells.
2. Materials and Methods
2.1. Patients and Samples
Patients with CRSwNP was diagnosed based on the criteria defined in the European Position Paper on Rhinosinusitis and Nasal Polyps 2012 [10, 11]. The exclusion criteria included the presence of antrochoanal polyps, cystic fibrosis, fungal sinusitis, or unilateral nasal polyps. Oral glucocorticoid and intranasal steroid sprays were discontinued 1 month before surgery. CRSwNP patients were further subdivided into eosinophilic CRS (eCRSwNP) and non‐eosinophilic CRS (neCRSwNP) according to the cutoff values of 10 eosinophils/hpf to define eosinophilic inflammation as previously recommended [12]. Control subjects were those undergoing endoscopic surgery without CRS, such as benign nasal tumor, and repair of cerebrospinal fluid rhinorrhea. Polyp tissues from CRSwNP patients and uncinate tissue (UT) from control subjects were collected during surgery. The clinical features of all patients were collected and analyzed (Table 1). The study was approved by local institutional ethics review boards. Written informed consent is obtained from all participants before enrollment in the study.
Table 1.
Subject clinical characteristics.
| Items | Control | neCRSwNP | eCRSwNP |
|---|---|---|---|
| Total no. of subjects | 23 (15 male) | 23 (16 male) | 23 (19 male) |
| Age (y), median (range) | 46 (19–74) | 37 (24–75) | 47 (26–60) |
| Atopy n (%) | 1 (4.3) | 6 (26.1) | 16 (69.6) |
| Asthma n (%) | 0 (0) | 1 (4.3) | 5 (21.7) |
| Smoking n (%) | 5 (21.7) | 6 (26.1) | 4 (17.4) |
| Methodology used | |||
| qRT‐PCR, no of subjects | 15 (8 male) | 16 (13 male) | 16 (5 male) |
| Age (y), median (range) | 52 (27–69) | 40 (24–75) | 40 (26–50) |
| IHC, no of subjects | 7 (6 male) | 7 (3 male) | 8 (5 male) |
| Age (y), median (range) | 38.5 (19–74) | 35 (26–50) | 40 (26–50) |
| IF, no of subjects | 8 (5 male) | 7 (3 male) | 8 (5 male) |
| Age (y), median (range) | 39 (18–75) | 35 (26–50) | 40 (26–50) |
| Primary endothelial cells subjects | 5 (3 male) | — | — |
| Age (y), median (range) | 35 (19–50) |
Abbreviations: eCRSwNP, eosinophilic chronic rhinosinusitis with nasal ployps; IHC, immunohistochemistry; IF, immunofluorescence, neCRSwNP, non eosinophilic chronic rhinosinusitis with nasal ployps; qRT‐PCR, quantitative real time polymerase chain reaction.
2.2. Quantitative Real‐Time PCR
Quantitative real‐time PCR (qRT‐PCR) analysis was applied to assess the mRNA expression. RNA was extracted and reverse‐transcribed as previously described [13]. The cDNA samples were subsequently subjected to qRT‐PCR using an SYBR Green I Real‐time system (Applied Biosystems, Bio Rad, USA). The primer sequences for the qRT‐PCR were presented in the supporting material Table 1. Relative gene expression was calculated by using the method.
2.3. Immunohistochemistry
Immunohistochemistry (IHC) was performed as in a previous study [14]. The detailed protocol for IHC is provided in the Methods of Supporting Information. The average number of blood vessels was measured from three randomly different fields.
2.4. Immunofluorescence Staining
Immunofluorescence co‐staining of t‐PA and CD31 in NP tissues were performed on paraffin sections. The percentage of t‐PA‐positive vessels among all vessels was calculated by dividing the number of t‐PA‐positive vessels by the number of CD31‐positive vessels (the t‐PA + /CD31+ vessel ratio) [15]. Immunofluorescence was also performed to detect CD31, COL1A1 and pan‐keratin in cultured primary endothelial cells. The detailed protocol is provided in the Supporting Information.
2.5. Human Primary Nasal Endothelial Cells Isolation and Stimulation
Briefly, the middle turbinate tissues from control subjects were collected to culture primary nasal endothelial cells. The detailed protocol is also provided in the Supporting Information. The endothelial cells were stimulated with 100 ng/mL of recombinant human interleukin (IL)‐13 (Peprotech, 200‐13), 1 μM retinoic acid and (Sigma, R2625) and 100 ng/mL IFN‐ γ (R&D Systems, 200‐13). RNA and supernatants were collected 6 h and 24 h after stimulation, respectively.
2.6. ELISA
The levels of t‐PA protein expression were measured by enzyme‐linked immunosorbent assay (ELISA) using the Human t‐PA ELISA Kit (Boster Biologic Technology, EK0897), according to the manufacturer's protocol. The t‐PA concentration was measured as picogram per milliliter.
2.7. Single Cell RNA Sequencing Analysis
The single cell RNA sequencing (scRNA‐seq) data of CRS was obtained from previous study by Jose Ordovas‐Montanes et al. [16]. The detailed protocol of further analysis for the sequencing data is also described in the Supporting Information. The correlation was derived from a pseudo‐bulk RNA‐seq analysis of endothelial cells. Single‐cell RNA sequencing data were aggregated by sample (n = 12) to generate sample‐level expression values for t‐PA and IL‐13/IFN‐γ. Spearman's correlation coefficient was calculated to assess the association.
2.8. Statistical Analysis
Statistical analyses were performed using GraphPad Prism version 8 statistical software. The data were expressed as the mean ± standard error of the mean (SEM) for continuous variables. Differences between groups were analyzed with the Kruskal–Wallis ANOVA with Dunnett's post hoc test and the Mann–Whitney U test. Significant difference was accepted at p < 0.05.
3. Results
3.1. Increased Vascularity in Both eCRSwNP and neCRSwNP
Recent studies found increased angiogenesis in CRS by using CD31 as a marker of vascularity [10]. We also evaluated the mRNA expression of CD31 in both eCRSwNP and neCRSwNP. Significantly elevated CD31 mRNA expression was found in both subtypes of CRSwNP compared to that in uncinated tissues (UT) from control patients (Figure 1A). Whereas no significant difference of CD31 expression could be observed between eosinophilic and non‐eosinophilic group. Similarly, the expression of other angiogenesis and vascular signaling pathway–associated genes, including Sox17, intracellular adhesion molecule 2 (ICAM2) and intercellular adhesion molecule 3 (ICAM3) were significantly increased in eosinophilic as well as non‐eosinophilic CRSwNP (Figure 1B–D).
Figure 1.
Sinonasal vascularity was increased significantly in CRSwNP. (A–D), The expression of CD31, Sox17, ICAM2, ICAM3 mRNA were analyzed from UT in control (CD31/ICAM2: n = 15; SOX17/ICAM3: n = 16) and polyps tissue in CRSwNP(n = 16). (E–F), CD31‐positive blood vessels (black arrows) were increased significantly in neCRSwNP (n = 7) and eCRSwNP (n = 8) patients compared with controls (n = 7), as demonstrated by immunohistochemistry and blood vessel semi‐quantification. Data were presented as mean ± SEM of three random subepithelial tissue areas 200x magnification for each patient group. UT, uncinate tissue; NeCRSwNP, non‐eosinophilic chronic rhinosinusitis with nasal polyps; eCRSwNP, eosinophilic chronic rhinosinusitis with nasal polyps. a p < 0.05, b p < 0.01, c p < 0.001.
To evaluate the extent of angiogenesis, we also performed IHC staining for CD31 in polyp and UT samples. Low vascular density in UTs from control subjects, and moderate to high density in the submucosa of nasal polyps (NP) were observed. (Figure 1E). The semi‐quantitative analysis showed that the number of vessels was markedly increased in NPs compared to the UTs from control subjects (Figure 1F). However, there was no significant difference between the eosinophilic and non‐eosinophilic CRSwNP. Taken together, these findings indicated that increased vascularity is common feature in both endotypes of CRSwNP.
3.2. Decreased Expression of t‐PA in NPs and Endothelial Cells
In the integrated scRNA‐seq analysis, we included samples from patients with CRS and identified 7 subclusters, which were presented in two‐dimension uniform manifold approximation projection (UMAP) plot (Figure 2A). After initial quality control, a total of 18,895 cells were acquired from 12 CRS samples and epithelial cells accounted for highest fraction in sinonasal tissue (Figure 2B). Further we found that the epithelium and endothelium had nearly equivalent contribution of t‐PA in the sinonasal tissue (Figure 2C). The average expression of t‐PA was remarkably higher in endothelial cells compared to epithelial cells (Figure 2D and Supporting Information S1: Figure 1B). Immunofluorescence double‐staining indicated that t‐PA was expressed in both nasal endothelial cells and epithelial cells (Figure 2E). The violin plots using scRNA‐seq data showed that the expression levels of t‐PA were significantly decreased, while inhibitors SERPINE1 (PAI‐1) and SERPINB2 were significant upregulated in CRSwNP in comparison to chronic rhinosinusitis without nasal polyps (CRSsNP) (Figure 3A, Supporting Information S1: Figure 1A), indicating systemic fibrinolytic dysregulation. Likewise, the t‐PA mRNA levels were significantly decreased in both eCRSwNP and neCRSwNP compared to control subjects (Figure 3B). Furthermore, we calculated the percentage of t‐PA‐expressing vessels among all vessels (the t‐PA + /CD31+ vessel ratio) using immunofluorescence co‐staining (Figure 3C,D). We found that the percentages of t‐PA‐expressing vessels were lower in both subtypes of CRSwNP than control group. However, no significant difference could be observed between neCRSwNP and eCRSwNP group. Hence, these findings confirmed the downregulation of t‐PA in endothelial cells in CRSwNP compared to control.
Figure 2.
Expression of t‐PA in endothelial cells of nasal tissues. scRNA‐seq data of CRS was obtained from previous study by Jose Ordovas‐Montanes et al. (A) UMAP displaying 18,895 cells from 12 ethmoid sinus tissues separated into 7 cell types. The red dashed lines indicated endothelial cells. (B) Boxplots showing the proportions of endothelial cells and epithelial cells in 12 ethmoid sinus tissues from scRNA‐seq datasets. Statistical significances were assessed using a two‐side Wilcoxon rank‐sum test. (C) Pie chart showing the proportion of t‐PA expression by different cell types in 12 ethmoid sinus tissues. (D) Dot plot representing the average expression of t‐PA in each cell type. (E) Immunofluorescence was performed with CD31 as a marker for endothelial cells (green fluorescence) and t‐PA (red fluorescence) in nasal polyp from a patient with CRSwNP. The dashed box indicates the area of enlargement shown in the inset in the top right corner. Each field was observed at 200× magnification (scale bar: 50 am).
Figure 3.
The expression levels of t‐PA were decreased in endothelial cells from nasal polyp tissues. (A) Violin plots using scRNA‐seq data from Jose Ordovas‐Montanes' study showed that the expression of t‐PA in CRSwNP and CRSsNP (n = 6 for each group). (B) t‐PA mRNA levels were measured by qRT‐PCR in nasal tissues (Control: n = 16; NeCRSwNP: n = 15; ECRSwNP: n = 15). (C) Representative immunofluorescence images of t‐PA (red) and CD31 (green) in sinonasal tissues. Nuclei counterstained with DAPI (blue). White arrows represented the positive blood vessels. (D) Quantification of t‐PA + /CD31+ vessel ratio between three groups. (Control: n = 8; NeCRSwNP: n = 7; ECRSwNP: n = 7). Data shown are mean ± SEM of independent experiments. a p < 0.05, b p < 0.01, c p < 0.001.
3.3. IL‐13 and IFN‐γ Attenuates t‐PA Expression in Endothelial Cells
IL‐13 was a pivotal cytokine in type 2 CRSwNP, whereas IFN‐ γ, a key cytokine in type 1 CRSwNP, is associated with t‐PA downregulation in nasal epithelial cells [7]. We performed correlation analysis using scRNA data expression and found that t‐PA levels in endothelial cells were significantly negative correlated with IL‐13 and IFN‐γ levels in ethmoid sinus tissues (Figure 4A,C). Moreover, we analyzed the target cells of IL‐13 using scRNA‐seq data and found IL‐13 receptors (IL4R and IL13RA1) were also located in sinonasal endothelial cells (Figure 4B). In vitro, we successfully isolated and cultured primary endothelial cells from normal middle turbinates (Figure 4D). Further stimulation with IL‐13 revealed that the mRNA expression of t‐PA in endothelial cells could also be attenuated by IL‐13 and IFN‐γ (Figure 4E,F).
Figure 4.
IL‐13 and IFN‐γ attenuates t‐PA expression in nasal endothelial cells. (A) Scatterplot using scRNA‐seq data showed the negative correlation between expression of IL‐13 in ethmoid sinus tissues and t‐PA expression in endothelial cells of each sample. Dashed line represented the linear regression curve. The gray band represented the 95% confidence interval of the regression line. Two‐sided Spearman correlation coefficient and p value were indicated (n = 12). (B) Dot plot showing the average expression of IL‐4 and IL‐13 receptor genes and percentages of expressed cells in each subset. (C) Scatterplot using scRNA‐seq data showed the negative correlation between expression of IFN‐γ in ethmoid sinus tissues and t‐PA expression in endothelial cells of each sample. (D) The purity of the primary endothelial cells was identified by immunofluorescence staining. HUVEC cells were used as positive controls. Primary endothelial and HUVEC cells stained positive for CD31 as a marker of endothelial cells and negative for COL1A1 (marker for fibroblasts) and pan‐keratin (marker for epithelial cells). Each field was observed at 400× magnification (scale bar: 20 μm). HUVEC, human umbilical vein endothelial cells; PEC, primary endothelial cells. (E–F) Submerged primary nasal endothelial cells were stimulated with IL‐13 (100 ng/mlL) and IFN‐γ (100 ng/mL) for 6 h, followed by extraction of mRNA and analyzed by qRT‐PCR (n = 10). Data shown are mean ± SEM of independent experiments. a p < 0.01 and b p < 0.0001.
3.4. RA Restores t‐PA Production in Nasal Endothelial Cells
Recent study further demonstrated that retinoic acid could rescue decreased production of t‐PA by IL‐13 in human nasal primary epithelial cells (HNEC) [7]. To determine whether RA could restore IL‐13‐mediated attenuation of t‐PA levels in primary nasal endothelial cells, we stimulated primary endothelial cells by using IL‐13 with or without RA and found that RA could also significantly restore t‐PA production in nasal endothelial cells (Figure 5A,B). IFN‐γ also possessed the negative effect of t‐PA expression on the airway epithelial cells in recent report [8]. Similarly, we found IFN‐γ could significantly inhibit the secretion of t‐PA in nasal endothelial cells. Moreover, RA could also restore the decreased production of t‐PA by IFN‐γ (Figure 5C,D).
Figure 5.
RA restores IL‐13‐mediated attenuation of t‐PA expression in primary nasal endothelial cells. (A and B) Primary nasal endothelial cells were incubated with IL‐13 (100 ng/mL) with or without RA and RA alone (1 μM) for 6 h and 24 h, respectively, and then collect cell lysates and supernatants to measure t‐PA mRNA and protein levels (n = 9). (C and D) Primary nasal endothelial cells were incubated with IFN‐γ (100 ng/mL) with or without RA and RA alone (1 μM) for 6 h and 24 h, respectively, to measure t‐PA mRNA and protein levels using qRT‐PCR and ELISA (n = 9). Data shown are mean ± SEM of independent experiments. a p < 0.05, b p < 0.01, c p < 0.001, d p < 0.0001.
4. Discussion
Vascular dysfunction and the role of endothelial cells in the pathogenesis of CRSwNP were less clarified in the literature. In the current study, we firstly demonstrated increased expression of proangiogenic gene and vessels in polyp tissues from both subendotypes of CRSwNP. Interestingly, we also demonstrated significant contribution of endothelial cells into the production of t‐PA in sinonasal tissues using scRNA‐seq data. Furthermore, we demonstrated that t‐PA produced by endothelial cells was downregulated by IL‐13 and IFN‐γ in CRSwNP, which implicated the plasticity of endothelial cells in the inflammatory milieu of CRSwNP. Finally, we demonstrated that RA could also restore t‐PA expression in endothelial cells which was akin to the effect in airway epithelial cells, as previously reported [7].
Angiogenesis is a typical feature of tissue remodeling in several chronic inflammatory diseases, which is promoted by various proangiogenic molecules [17, 18]. Until recently, a gene meta‐analysis revealed that the levels of proangiogenic genes including CD31 and L‐selecting were significantly overexpressed in patients with CRS [19]. Khurana et al. also observed increased expression of proangiogenic genes such as CD31, and increased vascularity assessed by CD31 immunostaining in polyp tissues from patients with CRSwNP. The fenestration sizes between endothelial cells were larger in CRS patients which would result in increased permeability and edema in CRS tissue [10]. In current study, we further demonstrated increased vascularity and proangiogenic gene expression in both subtypes of CRSwNP. Hence, vascular dysregulation and permeability were common events in the pathogenesis of eCRSwNP and neCRSwNP.
Endothelial cells have a critical role in allergic diseases through recruiting circulating immune cells into local inflammation sites [20]. The percentage of positive vessels for peripheral lymph node addressin (PNAd) was significantly correlated with disease burden as well as eosinophils infiltration in polyp tissues from eosinophilic CRS patients [15]. Sox17 is known as a specific transcriptional regulator of endothelial cells that plays a crucial role promoting endothelial cell proliferation and vascular development [21]. Sox17 expression in endothelial cells could be upregulated by IL‐33 and promoted the allergic inflammation in the asthma model [22]. Our study firstly observed upregulated expression of Sox17 in eosinophilic CRSwNP, which implied its potential role in eosinophilic CRSwNP.
Previous studies have suggested that t‐PA was mainly produced by nasal epithelial cells, and t‐PA expression in the mucosal epithelium of nasal polyps was decreased in comparison with normal uncinate process [6]. Early study by Huber et al. [9] has also found expression of t‐PA in human umbilical vein endothelial cells (HUVECs) by immunofluorescence. We demonstrated the expression of t‐PA in endothelial cells from sinonasal tissue using previous shared scRNA‐seq data and double‐staining immunofluorescence. Additionally, we found nearly equal contribution of sinonasal endothelium to the expression of t‐PA compared to sinonasal epithelium. We first identified vascular endothelium as the primary t‐PA source in sinonasal tissue, with critically impaired endothelial t‐PA function across CRSwNP endotypes. Whereas prior mechanisms focused on epithelial cells, Yang et al. [23] revealed that eosinophil‐derived CHI3L1 in Th2‐mediated fibrinolytic impairment via suppressed t‐PA and activated PAI‐1 in epithelial and fibroblastic cells, revealing eosinophils‐epithelial crosstalk in nasal polyp remodeling. Our data strengthened the effect of endothelial cells in the pathogenesis of CRSwNP under the research background with less studies regarding endothelial cells.
In vitro data also showed the average t‐PA secretion in sinonasal endothelial cells constitutively was around 0.6 ng/mL in each well of 12‐well culture plate which is approximately six‐fold secretion than nasal epithelial cells(average 0.1 ng/mL) as previous reported [7]. These results indicated that sinonasal endothelium may also play an unignorable role in the process of polyp formation in terms of regulating t‐PA production.
Although increased vascularity has been confirmed in the nasal polyp tissues, crucially, t‐PA expression levels in endothelium from polyp tissues were oppositely decreased. Further analysis using scRNA‐seq data set revealed negative association between IL‐13 and t‐PA expression in endothelium. We also observed significant reduced expression of t‐PA in endothelium by IL‐13 stimulation. Therefore, the regulatory role of IL‐13 on endothelial cells is similar to airway epithelial cells due to the expression of IL‐13RA1 and IL‐4R in endothelial cells. Recent study found impaired fibrin degradation and reduced t‐PA expression were common features in both subendotypes of nasal polyps from Chinese patients [8]. In addition to IL‐13, IFN‐ γ could also downregulate the expression of t‐PA in airway epithelial cells [7]. Likewise, our study also showed the downregulation effect of IFN‐γ on t‐PA expression in sinonasal endothelial cells. However, the exact mechanism of IL‐13 and IFN‐γ on the expression of t‐PA in sinonasal endothelial cells remains to be clarified. Collectively, our findings implied that the t‐PA expression in sinonasal endothelium as well as epithelium was precisely controlled by key cytokines including IL‐13 and IFN‐γ, thereby regulating the fibrin deposition and polyp formation.
Recently, endogenous retinoid deficiency has been found significantly correlated with reduced t‐PA levels in nasal tissue from CRSwNP and AERD patients [7]. Moreover, retinoic acid (RA) could strongly promote t‐PA expression in airway epithelial cells and rescued the t‐PA expression suppressed by IL‐13. Although early reports found RA could induce t‐PA expression in HUVECs, we firstly observed the effect of RA restoring t‐PA expression in primary human nasal endothelial cells by IL‐13 or IFN‐γ in vitro. RA could also efficiently attenuate airway inflammation by reducing Th2 and Th17‐related cytokines and inflammatory cells infiltration [24]. Hence, RA supplementation might be able to induce t‐PA expression in both epithelial cells and endothelial cells, thus promoting degradation of fibrin mesh of polyp tissues as well as decreasing sinonasal inflammation.
In summary, our findings suggested that sinonasal endothelium were an important contributor of t‐PA expression in sinonasal tissue. Although endothelium was increased in nasal polyps, the capacity to synthesize t‐PA in nasal endothelial cells was blunt under the type 1 or type 2 inflammatory stimuli. Our findings further implied the potential utility of RA in CRSwNP treatment.
Author Contributions
Qian‐Qian Zhang: study conception and design, analysis and interpretation of data, drafting and revising the manuscript. Chen Zhang: Acquisition, analysis and interpretation of transcriptomic data. Jia‐Ni Chen and Fu‐Ying Cheng: acquisition of samples, critical revision of the manuscript. Yi‐Zhang Wang and Shi‐Rui Xue: literature search and analysis. Huan Wang and Li Hu: study design, critical revision of the manuscript. Xi‐Cai Sun and De‐Hui Wang: study design, supervision, funding acquisition, critical revision of the manuscript. All authors reviewed the results and approved the final manuscript, agreeing to be accountable for all aspects of the work.
Ethics Statement
Our research complies with all relevant ethical regulations. The study was approved by the institutional review board of EENT Hospital affiliated with Fudan University and written informed consent was obtained from each participant.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
for typesetting‐Supporting figure 1.
Table 1: Primers used for quantitative PCR analysis. Figure 1: A,Violin plots using scRNA‐seq data from Jose Ordovas‐Montanes'study showed that the expression of SERPINE1(PAI‐1), PLAU, PLAUR and SERPINB2 in CRSwNP and CRSsNP（n=6 for each group).
Acknowledgments
We sincerely appreciate the support from the Medical Science Data Center at Shanghai Medical College of Fudan University. This study was financially supported by the following programs and organizations: Natural Science Foundation of China (82460220); Natural Science Foundation of Shigatse RKZ2023ZR‐008(Z); Natural Science Foundation of Xizang Autonomous Region (XZ202401ZR0046); Science and Technology Commission of Xuhui District, Shanghai (23XHYD‐08); Clinical Scientist Training Program of Shanghai Medical College, Fudan University (DGF828019‐2/042).
Contributor Information
Xi‐Cai Sun, Email: laryngeal@163.com.
Li Hu, Email: hl318ent@163.com.
Data Availability Statement
This study did not generate new primary sequencing data. The single‐cell RNA sequencing data analyzed in this study were derived from the previously published and publicly available dataset reported by Ordovas‐Montanes et al. [16]. All data supporting the findings presented here are contained within the cited source publication. All other data relevant to this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
for typesetting‐Supporting figure 1.
Table 1: Primers used for quantitative PCR analysis. Figure 1: A,Violin plots using scRNA‐seq data from Jose Ordovas‐Montanes'study showed that the expression of SERPINE1(PAI‐1), PLAU, PLAUR and SERPINB2 in CRSwNP and CRSsNP（n=6 for each group).
Data Availability Statement
This study did not generate new primary sequencing data. The single‐cell RNA sequencing data analyzed in this study were derived from the previously published and publicly available dataset reported by Ordovas‐Montanes et al. [16]. All data supporting the findings presented here are contained within the cited source publication. All other data relevant to this study are available from the corresponding author upon reasonable request.