*Contributed equally
It has been previously reported that cell division control 42 (CDC42) protein can regulate macrophage recruitment, T cell-associated inflammation and lung injury. However, its role in chronic obstructive pulmonary disease (COPD) remain poorly understood. Therefore, the present study aimed to investigate the possible association among CDC42 expression, the risk of acute exacerbation and disease features in patients with COPD. Peripheral blood mononuclear cells (PBMCs) and serum samples were collected from 60 patients with acute exacerbation COPD (AE-COPD), 60 patients with stable COPD (S-COPD) and 60 healthy control (HCs) individuals. The mRNA expression levels of CDC42 in PBMCs were then measured using reverse transcription-quantitative PCR. The serum levels of TNF-α, IL-1β, IL-6 and IL-17 were measured using ELISA. The results showed that the expression of CDC42 was dysregulated among patients with AE-COPD and S-COPD compared with that in HCs. Specifically, the expression level of CDC42 was the highest in patients with AE-COPD, followed by those with S-COPD and the lowest in HCs (P<0.001). Furthermore, receiver operating characteristic curve analysis demonstrated that CDC42 expression was associated with an increased risk of acute exacerbation in COPD with an area under curve of 0.690 (95% confidence interval=0.595-0.785). CDC42 was found to be positively associated with Global Initiative for Chronic Obstructive Lung Disease staging in patients with AE-COPD (P<0.01) and S-COPD (P<0.05). Additionally, CDC42 expression associated positively with the serum levels of TNF-α, IL-1β, IL-6 and IL-17 in patients with AE-COPD (all P<0.05). However, this association was weaker in patients with S-COPD and became negligible in HCs. In conclusion, data from the present study suggest that CDC42 is associated with an increased risk of acute exacerbation, inflammation and disease severity in patients with COPD, implicating its application as a potential biomarker for COPD.
Chronic obstructive pulmonary disease (COPD) is a serious lung condition that is characterized by persistent respiratory symptoms and progressive airflow obstruction (
Cell division cycle 42 (CDC42) is a key regulator of several cellular processes, including cell proliferation, division, migration, morphogenesis and establishment of epithelial polarity (
Based on these previous observation, it can hypothesized that CDC42 can possibly serve as a novel biomarker for COPD. Therefore, the present study aimed to measure the expression levels of CDC42 in blood samples derived from patients with AE-COPD and S-COPD in addition to healthy control (HCs) individuals. The objective was to evaluate the potential association of CDC42 expression with the risk acute exacerbation of COPD, inflammation and disease severity.
The present study was approved by the Institutional Review Board of the Central Hospital of Wuhan. A total of 60 patients with AE-COPD and 60 with S-COPD were enrolled in the Central Hospital of Wuhan (Wuhan, China) between May 2019 and November 2020. All patients, aged >18 years, were diagnosed with COPD according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (
The mean ages of the HC group, patients with S-COPD and patients with AE-COPD were 66.1±6.4, 67.3±7.1 and 66.7±7.2 years, respectively (
The demographic features and medical history of patients were recorded after enrollment. Forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC) were recorded following the pulmonary function test (PFT) (
In addition, peripheral blood samples were collected from all 180 participants in the present study. The serum samples and peripheral blood mononuclear cells (PBMCs) were collected by centrifugal separation and density gradient separation, respectively. In detail, peripheral blood samples were naturally coagulated for about 20 min at room temperature and centrifuged at 650 x g for 10 min using a refrigerated centrifuge (4˚C) (Thermo Fisher Scientific, Inc.). The supernatant serum was collected carefully and stored at -70˚C. PBMCs were separated following gradient centrifugation of the blood over Ficoll-Hypaque density gradient (Biochrom, Ltd.). PBMCs were used to measure the expression levels of CDC42 using reverse transcription-quantitative PCR (RT-qPCR). The levels of TNF-α, IL-1β, IL-6a and IL-17 secreted were assessed in the serum samples using the corresponding human ELISA kits.
RT-qPCR was performed for the quantitative analysis of CDC42 expression in PBMCs. Total RNA was extracted from PBMCs using PureZOL RNA isolation reagent (Bio-Rad Laboratories, Inc.). Subsequently, total RNA was reverse transcribed into complementary DNA using the iScript™ Reverse Transcription Supermix kit (denaturation at 65˚C for 5 min, reverse transcriptase at 37˚C for 5 min and inactivation at 85˚C for 5 sec) (Bio-Rad Laboratories, Inc.). qPCR was performed using the TB Green Premix DimerEraser™ kit (Takara Bio, Inc.). The primer sequences used for CDC42 detection were the same as those previously reported (
Commercial ELISA kits (cat. nos. DTA00D, DLB50, D6050 and D1700; R&D Systems Inc.) were purchased to detect the serum levels of TNF-α, IL-1β, IL-6 and IL-17. All procedures, including sample and reagent preparation, assay procedure and calculation of the results were implemented according to the protocols of the kits.
Data were presented as N (%), the mean ± standard deviation (SD) or median (interquartile range); P-value represents the significance of the results; r represents linear coefficient; H and Z represent the statistic of nonparametric rank sum test; F represents the statistic of ANOVA; χ2 represents the statistic of chi-square test. Data analysis and graph plotting were performed using SPSS 26.0 (IBM Corp.) and GraphPad Prism 7.01 (GraphPad Software Inc.) software, respectively. Wilcoxon rank sum test, χ2 test, one-way ANOVA, Kruskal-Wallis and Dunn's tests were used to compare the differences amongst groups. Multiple comparisons were corrected by Bonferroni's post hoc test. Spearman's rank correlation test was performed to evaluate the association between the variables. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the variables in distinguishing different subjects. Based on ROC analysis, the best statistical cut-off value of CDC42 expression level was calculated, which corresponded to the point at which the sum of false-positives and false-negatives was less than any other point. Sensitivity and specificity for selected cut-off points were then assessed. P<0.05 was considered to indicate a statistically significant difference.
There were no differences in terms of age and sex among the three groups (
The expression levels of CDC42 varied significantly among the HC, S-COPD and AE-COPD groups (P<0.001;
Additionally, ROC curve analysis was performed to assess the viability of using CDC42 expression to differentiate patients with AE-COPD to those with S-COPD. According to ROC analysis, CDC42 exhibited potential for distinguishing patients with AE-COPD from S-COPD, yielding an area under the curve (AUC) value of 0.690 (95% confidence interval=0.595-0.785;
Subsequently, the potential association between CDC42 expression and COPD severity was evaluated. The results showed that CDC42 expression was highest in GOLD stage III, followed by GOLD stage II, and lowest in GOLD stage I AE COPD patients (P=0.003;
The expression of CDC42 was found to be positively correlated with the serum levels of TNF-α (r=0.331; P<0.05), IL-1β (r=0.259; P<0.05), IL-6 (r=0.397; P<0.01) and IL-17 (r=0.472; P<0.001) in patients with AE-COPD (
The present study demonstrated that CDC42 expression recovered phagocytosis of alveolar macrophages and is proliferated in a mouse model of COPD (
To date, studies on the possible association between CDC42 expression and COPD severity remain limited. In the present study, CDC42 expression in patients with AE-COPD was found to be associated with increased GOLD staging in both patients with S-COPD and those with AE-COPD. This could be due to the fact that CDC42 upregulation is associated with enhanced inflammation as a result of increased macrophage activation and promotion of Th cell differentiation (
Emerging evidence has suggested that inflammation can aggravate lung injury and is therefore positively associated with GOLD stage (
Previous studies revealed that the expression levels of CDC42 were positively associated with the levels of inflammatory cytokines in lung injury (
However, the present study has several limitations. The present study only included 120 patients with COPD, which is a relatively small sample size. In addition, since the present study was a single-center study, the possibility of selection bias could not be excluded. Furthermore, the molecular mechanism underlying the effects of CDC42 on the regulation of physiological changes in macrophages, recruitment of inflammation-related T cells and lung injury in COPD should be investigated further. In the present study, the expression levels of CDC42 were only assessed at one single time point. Therefore, changes in the expression levels of CDC42 for longer periods in association with COPD progression require further investigation. The present study was designed to explore the preliminary clinical value of CDC42 in patients with COPD. To translate this into clinical practice a series of further studies in the real-world setting are required. The following types of studies should be applied: i) Multicenter studies with larger sample sizes should be performed to verify its potential clinical value; ii) a study aiming to build models for predicting AE-COPD should be performed; and iii) multi-timepoint monitoring should be conducted in future studies to reveal the value of CDC42 in monitoring COPD progression and treatment response. The present study is a case-controlled study, which lacked scheduled follow-up data in the protocol. Therefore, the potential accuracy of CDC42 as a prognostic marker require further investigation.
In conclusion, the present study revealed that CDC42 expression was associated with an increased risk of acute exacerbation, inflammation and COPD severity, which provides a potential novel biomarker for COPD.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by XM, FY and HZ. XM and HZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was performed in line with the principles of the Declaration of Helsinki. Ethics approval was granted by the Institutional Review Board of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). All individuals in the present study signed the informed consents.
Not applicable.
The authors declare that they have no competing interests.
Comparison of CDC42 expression among HCs, patients with AD-COPD and S-COPD. CDC42, cell division cycle 42; COPD, chronic obstructive pulmonary disease; S-COPD, stable COPD; AE-COPD, acute exacerbation COPD; HCs, health controls. *P<0.05 AE-COPD vs. HCs; #P<0.05, AE-COPD vs. S-COPD; $P <0.05, S-COPD vs. HCs.
Receiver operating characteristic curve for the ability of differentiating patients with AE-COPD from patients with S-COPD using CDC42 expression. CDC42, cell division cycle 42; COPD, chronic obstructive pulmonary disease; AE-COPD, acute exacerbation COPD; S-COPD, stable COPD; AUC, area under curve; CI, confidence interval.
Association between CDC42 expression and GOLD staging. Association of CDC42 expression with GOLD staging in (A) patients with AE-COPD and (B) patients with S-COPD. CDC42, cell division cycle 42; COPD, chronic obstructive pulmonary disease; AE-COPD, acute exacerbation COPD; S-COPD, stable COPD; GOLD, Global Initiative for Chronic Obstructive Lung Disease. *P<0.05 Stage III vs. Stage I, #P<0.05 Stage II vs. Stage I.
Correlation between CDC42 expression and each of the inflammatory cytokines. Correlation analysis of CDC42 expression with (A) TNF-α, (B) IL-1β, (C) IL-6 and (D) IL-17 in patients with acute exacerbation chronic obstructive pulmonary disease. CDC42: cell division cycle 42.
Characteristics of patients with COPD and HCs.
Parameter | HCs (N=60) | Stable-COPD (N=60) | Acute exacerbation-COPD (N=60) | Statistic ( |
P-value |
---|---|---|---|---|---|
Age, years | 66.1±6.4 | 67.3±7.1 | 66.7±7.2 | 0.394 | 0.675 |
Sex | 2.646 | 0.266 | |||
Female | 24 (40.0) | 18 (30.0) | 16 (26.7) | ||
Male | 36 (60.0) | 42 (70.0) | 44 (73.3) | ||
BMI, kg/m2 | 22.8±2.6 | 22.1±2.6 | 22.5±3.0 | 0.999 | 0.370 |
Family history of COPD | 10 (16.7) | 20 (33.3) | 17 (28.3) | 4.550 | 0.103 |
History of smoking | 17 (28.3) | 33 (55.0) | 28 (46.7) | 9.095 | 0.011 |
History of hypertension | 24 (40.0) | 30 (50.0) | 35 (58.3) | 4.045 | 0.132 |
History of hyperlipidemia | 16 (26.7) | 14 (23.3) | 15 (25.0) | 0.178 | 0.915 |
History of diabetes mellitus | 10 (16.7) | 11 (18.3) | 14 (23.3) | 0.922 | 0.631 |
FEV1/forced volume vital capacity, % | 82.2 (79.8-84.2) | 61.3 (57.6-65.2) | 60.7 (54.9-65.8) | 119.447 | <0.001 |
FEV1, predicted % | 99.2 (95.7-100.6) | 74.6 (57.4-82.5) | 57.1 (46.6-81.4) | 121.708 | <0.001 |
Global Initiative for Chronic | -1.980 | 0.048 | |||
Obstructive Lung Disease stage | |||||
Stage I | - | 27 (45.0) | 16 (26.7) | ||
Stage II | - | 22 (36.7) | 28 (46.6) | ||
Stage III | - | 11 (18.3) | 16 (26.7) |
Data were presented as N (%), the mean ± SD or median (interquartile range). COPD, chronic obstructive pulmonary disease; HCs, health controls; FEV1, forced expiratory volume in 1 second; FVC, forced volume vital capacity.