Elevated plasma levels of pigment epithelium-derived factor correlated with inflammation and lung function in COPD patients

Figures & data

Figure 1 Cigarette smoke (CS)-induced pigment epithelium-derived factor (PEDF) expression both in vitro and in vivo.

Notes: (A) Human H292 cell lines were cultured and labeled with a stable isotope. Cells were incubated with or without the presence of cigarette smoke extract (CSE) for 24 hours. Then, cells were harvested to measure molecular species compositions of the cell protein using mass spectra. CSE significantly induced PEDF protein expression as much as 16.2-fold when compared with untreated controls. The experiment was repeated three times and a representative result is shown here. (B) Wild-type rats were exposed to CS for 4 weeks (2×30 minutes/day and 6 days/week) using a mechanical smoking chamber. Sham rats were exposed to ambient air as control. Afterwards, rat lung tissues were homogenized to extract protein. Samples were detected using Western Blotting with specific PEDF and β-actin antibodies. Band densities were quantified and bars showed fold changes normalized to β-actin as control. Each group included five rats and the experiment was repeated three times.

Table 1 Characteristics of healthy volunteers and COPD patients in stable condition

	NS (n=20)	HS (n=10)	COPD (n=50)	P-value
Age, years	60.25±1.62	60.70±1.23	63.78±1.37	0.242
Sex, male %	50	100§	72	0.016
Body mass index, kg/m^2	23.54±0.64	24.46±1.08	23.65±0.48	0.739
Physiological parameters
Systolic blood pressure, mmHg	145.05±5.02	129.90±4.88	137.35±2.88	0.137
Diastolic blood pressure, mmHg	86.95±2.98	78.70±2.99	80.65±1.68	0.094
PaO2, mmHg	98.55±0.14	98.10±0.35	97.21±0.34*	0.028
Risk factors
Pack-years smoking index	0	33.00±5.37§	21.48±2.46*	0.073
Metabolism parameters
Total cholesterol, mg/dL	4.94±0.19	4.59±0.30	4.92±0.14	0.556
Fasting glucose, mg/dL	6.18±0.78	5.68±0.46	5.36±0.09	0.273

Notes: Values are presented as the means ± standard error, unless otherwise indicated. The normal distribution of the data has been checked. Analysis of variance was used to analyze the difference between groups and followed by least significant difference test to compare any of the two groups.

* Represents significance for COPD versus NS;

§ for HS versus NS. Bold denotes a value of P<0.05, which was considered to be statistically significant.

Abbreviations: COPD, chronic obstructive pulmonary disease; NS, healthy nonsmokers; HS, healthy smokers.

Figure 2 Levels of pigment epithelium-derived factor (PEDF) in the plasma from the healthy nonsmokers group, healthy smokers group, and chronic obstructive pulmonary disease (COPD) group. Circulating PEDF concentrations were measured using multiplex enzyme-linked immunosorbent assay. COPD patients had a significantly elevated PEDF level when compared with those of the healthy nonsmoker and smoking subjects (P=0.0001 and P=0.0477, respectively). There was no difference between the healthy nonsmokers and smokers (P=0.2712). The expression of PEDF is presented as the median (interquartile range) and compared by one-way analysis of variance. A value of P<0.05 was considered to be statistically significant.

Figure 3 Circulating pigment epithelium-derived factor (PEDF) levels were correlated with established chronic obstructive pulmonary disease (COPD) biomarkers. PEDF concentration was positively correlated with (A) serum C-reactive protein (CRP) (r=0.4294; P<0.0001); (B) interleukin (IL)-8 (r=0.2947; P=0.0080); (C) tumor necrosis factor (TNF)-α (r=0.2665; P=0.0169); and (D) IL-6 (r=0.3433; P=0.018). (E, F) The absolute value of PEDF was also correlated with either neutrophil number or neutrophil percentage (r=0.3086, P=0.0054; r=0.2202, P=0.0497, respectively). The solid line denotes the line of best fit, and Pearson’s correlation coefficient is presented as an r-value.

Table 2 Spirometry results of healthy volunteers and COPD patients in stable condition

Lung function	NS (n=20)	HS (n=10)	COPD (n=50)	P-value
FEV1, L	2.71±0.16	2.79±0.15	1.67±0.09*,#	<0.0001
FVC, L	3.47±0.19	3.59±0.17	3.21±0.12	0.27
FEV1/FVC ratio	84.75±1.03	82.03±1.00	54.06±1.75*,#	<0.0001
FEV1% pred	118.80±3.77	107.80±3.58	70.28±2.42*,#	<0.0001
VC max	3.40±0.20	3.58±0.19	3.27±0.13	0.55
IC	2.33±0.11	2.31±0.20	2.21±0.10	0.75
MMEF 75/25	2.74±0.25	2.41±0.22	0.72±0.08*,#	<0.0001
PEF	7.04±0.49	7.19±0.37	4.00±0.29*,#	<0.0001
FEF25	6.43±0.44	6.51±0.28	2.26±0.22*,#	<0.0001
FEF50	4.07±0.31	3.74±0.37	0.92±0.09*,#	<0.0001
FEF75	1.21±0.10	1.09±0.12	0.28±0.03*,#	<0.0001

Notes: Values are presented as the means ± standard error, unless otherwise stated. Analysis of variance was used to analyze the difference between groups and was followed by the least significant difference test to compare any of the two groups.

* Represents significance for COPD versus NS;

# for COPD versus HS. Bold denotes a value of P<0.05 that was considered to be statistically significant.

Abbreviations: FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; FEV₁/FVC, ratio of FEV₁ to FVC; FEV₁% pred, FEV₁ percent predicted; VC max, vital capacity; IC, inspiratory capacity; MMEF 75/25, maximum mid-expiratory flow; PEF, peak expiratory flow; FEF25, forced inspiratory flow 25%; FEF50, forced inspiratory flow 50%; FEF75, forced inspiratory flow 75%; COPD, chronic obstructive pulmonary disease; NS, healthy nonsmokers; HS, healthy smokers.

Figure 4 Correlation analysis between PEDF levels and pulmonary function.

Notes: (A) PEDF in the plasma was negatively correlated with FEV₁% predicted (r=−0.3303; P=0.0028). (B) A similar correlation was observed between PEDF and FEV₁/FVC (r=−0.3116; P=0.0049). The solid line denotes the line of best fit. Pearson’s correlation coefficient is presented as an r-value.
Abbreviations: PEDF, pigment epithelium-derived factor; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; COPD, chronic obstructive pulmonary disease.

Table 3 Multivariable linear regression analysis for circulating PEDF in volunteers and COPD patients

Parameter	β	P-value
Age	−0.071	0.51
Sex	0.011	0.91
BMI	0.046	0.65
Pack-years	0.002	0.99
IL-6	0.28	0.01
IL-8	0.115	0.29
TNF-α	0.108	0.310
CRP	0.34	<0.0001
FEV1/FVC	−0.122	0.26
FEV1% pred	−0.188	0.080

Notes: The value of β represents standard regression coefficient. Bold denotes a value of P<0.05.

Abbreviations: PEDF, pigment epithelium-derived factor; COPD, chronic obstructive pulmonary disease; BMI, body mass index; IL, interleukin; TNF, tumor necrosis factor; CRP, C-reactive protein; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; FEV₁/FVC, ratio of FEV₁ to FVC; FEV₁% pred, FEV₁ percent predicted.