To the Editor:
Chronic obstructive pulmonary disease (COPD), so far simply defined by persistent
airflow limitation mostly due to prolonged tobacco smoking exposure, is now clearly
depicted as a heterogeneous and complex disease, with lung but also systemic manifestations
such as sarcopenia, osteoporosis, or cardiovascular diseases
1
. Chronic exposure of lung cells to cigarette smoke can trigger the activation of
several cellular processes such as oxidative stress, cellular senescence and autophagy.
Induced in response to various stressful situations (e.g. starvation, hypoxia, DNA
damage, or infection), autophagy is a major cellular adaptive pathway that, to a certain
extent, helps maintain cellular homeostasis through phagolysosomal self-degradation
of supernumerary or defective organelles and proteins
2
. Indeed, autophagy theoretically promotes cellular survival but can also favor cell
death when this adaptive process is overwhelmed. The implication of autophagy in the
pathogenesis of COPD initially looked controversial, with some data showing autophagy
activation that favors apoptotic death of bronchial epithelial cells
3
, and some other demonstrating a defective autophagy that may promote cellular senescence
4
. However, recent studies uncovered the link between cigarette smoke-induced oxidative
stress, autophagy-flux impairment, accumulation of autophagic vacuoles/aggresome-bodies,
chronic inflammatory-apoptotic responses, premature senescence, and emphysema progression
in the context of chronic cigarette smoke exposure or in COPD patient’s lung tissue
5–7
. Although there are still areas of uncertainty, like specific steps and mechanisms
by which autophagy is impaired, strategies aiming to counteract these phenomena have
already emerged as a way to prevent or limit the consequences of chronic cigarette-smoke
exposure
8
.
Molecular pathways triggering autophagy are complex and may involve the class III
Bcl-2 interacting protein (Beclin1)/phosphoinositol-3-kinase complex, which then participates
in the induction and the initial steps of autophagy
2
. A recent study showed that healthy centenarians have increased circulating Beclin1
protein levels in comparison to a population of young healthy subjects or patients
with myocardial infarction
9
. Thus, as it has been shown that the induction of autophagy may promote longevity,
the authors have suggested a relationship between the increased level of this potential
biomarker of autophagy and the exceptional longevity of these patients.
Given that cellular senescence is involved in COPD pathophysiology and that autophagy
defect may be a trigger of this process, we hypothesized that circulating Beclin1
levels, taken as a reflect of autophagy process efficiency, are reduced in COPD patients.
We therefore tested whether circulating Beclin1 levels are reduced in COPD patients
and if so, if this reduction is linked to telomere shortening, a hallmark of senescence.
Finally, as deficient autophagy may also be implicated in many pathophysiological
processes such as cardiovascular diseases, neuromuscular disorders, or bone loss,
we also tested whether Beclin1 level is linked to systemic manifestations of COPD.
For that, we took advantage of a cohort of 301 participants recruited at the Henri
Mondor Teaching Hospital, Creteil France (COPD, n = 100; smokers n = 100 and non-smoker
patients n = 101), already thoroughly phenotyped with a special focus on aging-related
markers such as arterial stiffness (aortic pulse-wave velocity, PWV), bone mineral
density (BMD), appendicular skeletal muscle mass (ASMM), pinch and grip strengths,
insulin resistance, renal function, and telomere length in circulating leucocytes
10
. In 280 (COPD n = 92, smokers n = 93, non-smokers n = 95) of the 301 patients for
whom a serum sample was available, we performed a quantitative evaluation of circulating
Beclin1 protein level (ELISA Kit for Beclin1; cat. no. E98557Hu, Uscn Life Science
Inc., Wuhan, China). Descriptive results are given as numbers and percentages for
categorical data, and means (±standard deviation) for continuous variables. Unadjusted
comparisons of Beclin1 levels between study groups were conducted using one-way ANOVA
for overall significance, and post hoc t-tests for pairwise comparisons applying Sidak
correction for test multiplicity. Linear regression was used to compare Beclin1 levels
across the three groups while adjusting for age. Associations between Beclin1 and
biological parameters were assessed by computing Pearson correlation coefficients.
All analyses were performed using Stata 14.1 (StataCorp, USA).
Results
Clinical characteristics (except pulmonary function tests) did not differ across the
three groups, but COPD patients had a higher pack-year value compared to control smokers.
A statistically significant negative trend was found in serum Beclin1 protein level
of non-smokers, smokers, and COPD patients (p = 0.022; Fig. 1). Beclin1 protein level
was correlated to age, degree of airway obstruction, telomere length, appendicular
skeletal muscle mass index, and grip strength (Table 1). None of the 27 chemokines
and growth factors tested was correlated to Beclin1 level. After adjustment for age,
the statistically significant negative trend between serum Beclin1 protein level in
each group persisted (non-smokers: 2.31 ± 0.23, smokers: 1.66 ± 0.23, and COPD: 1.52 ± 0.24 ng/mL,
respectively; p = 0.036). In COPD patients, Beclin1 protein level was correlated to
pulse-oxygen saturation (R = 0.25; p = 0.024), telomere length (R = 0.26; p = 0.014),
and TNF-α levels (R = 0.35; p = 0.001).
Fig. 1
Beclin1 levels according to the study groups.
Results are shown as boxplots, with each box representing the interquartile range
(1st to 3rd quartile, IQR), the line within the box indicating the mean, and the whiskers
extending to 1.5 times the IQR above and below the box; the dots represent individual
values for each patient
Table 1
Main subjects characteristics and Pearson correlation coefficients with Beclin1 level
N
Mean (±SD)
Correlation coefficient with Beclin1 level
p-value*
Age, years
279
59.02 (±7.76)
−0.13
0.027
BMI, kg/m2
279
26.03 (±4.69)
0.08
0.182
Pack-years
276
27.61 (±27.62)
−0.10
0.088
Pulmonary function parameters
FEV1, % predicted
279
86.56 (±29.20)
0.12
0.039
FVC, % predicted
278
93.59 (±21.49)
0.07
0.263
DLCO, % predicted
227
76.12 (±19.19)
−0.01
0.906
PaO2, mmHg
91
77.47 (±10.32)
0.18
0.086
SpO2, %
239
96.59 (±1.35)
0.09
0.178
6-min walking distance, m
239
556.37 (±100.56)
0.05
0.475
Systemic manifestations
Pulse wave velocity, m/s
271
11.55 (±2.32)
−0.04
0.488
ASMMI, kg/m2
270
7.49 (±1.34)
0.21
0.001
BMD, total lumbar, g/cm2
275
1.11 (±0.18)
0.03
0.619
BMD, hips, g/cm2
275
0.98 (±0.14)
0.10
0.108
Grip test, kg
244
37.47 (±12.26)
0.13
0.046
Glomerular flow rate, mL/min
261
93.34 (±48.78)
0.02
0.802
HOMA-IR
265
2.65 (±2.81)
0.05
0.465
Biological parameters
Telomere length (T/S) ratio
274
0.42 (±0.11)
0.17
0.005
IL-6, pg/mL
271
18.07 (±15.97)
−0.01
0.905
IL-8, pg/mL
271
46.94 (±9.88)
−0.09
0.155
TNF-α, pg/mL
271
89.52 (±134.53)
0.07
0.228
MCP-1, pg/mL
271
41.66 (±22.44)
−0.02
0.800
N
N (%)
Mean (±SD)
p-value
Gender
Men
279
185 (66.31%)
1.80 (±1.45)
0.230
Women
94 (33.69%)
1.58 (±1.40)
FEV1, % predicted
≥50
279
241 (86.38%)
1.80 (±1.50)
0.035
<50
38 (13.62%)
1.27 (±0.73)
*p-values for Pearson correlation coefficients; bolded results are statistically significant
at the p < 0.05 level
BMI body mass index, FEV1 forced expiratory volume in 1 s, % predicted percentage
of the predicted value, FVC forced vital capacity, SpO
2
oxygen saturation by pulse oximetry, ASMMI appendicular skeletal muscle mass index,
BMD bone mineral density, HOMA-IR homeostatic model assessment of insulin resistance,
IL interleukin, TNF-α tumor necrosis factor alpha, MCP-1 monocyte chemotactic protein-1,
SD standard deviation
Discussion
We show a significant decrease in serum Beclin1 protein level, a key regulator of
autophagy, in smokers and even more in COPD patients compared to healthy controls.
Autophagy induction and apoptosis have been widely described in bronchial and alveolar
epithelial cells exposed to cigarette smoke extracts in vitro and in lung tissues
of COPD patients
3
. However, evidence on the implication of Beclin1 in the cellular consequences of
cigarette smoke exposure are limited to its capacity to permit autophagic-dependant
apoptosis induced by acute cigarette smoke exposure
11
. Our results suggest another potential role of Beclin1 in the regulation of autophagy
in the specific context of chronic cigarette-smoke exposure. Indeed, accumulation
of autophagic vacuoles/agresomes observed in the lungs of patients with severe COPD
3
already reflects an acquired defect in autophagic process during COPD, attributed
to an autophagy-flux impairment that may be accessible to corrective therapies to
prevent cellular senescence and emphysema
8
. Thus, Beclin1 circulating levels decline might also participate to autophagy impairment
during COPD.
Furthermore, we show a correlation between the level of circulating Beclin1 and the
degree of airflow obstruction, which is consistent with a progressive defect in autophagy
during COPD, and also with two features of accelerated aging already associated to
COPD, cellular senescence
12
and muscle wasting
10
, respectively evaluated by telomere length, ASMM and grip strength. Of course, the
exact relationship between autophagy and cellular senescence remains unclear
13
, especially in the specific context of COPD, and the real implication of autophagy
in skeletal muscle dysfunction is still debated
14
. Beclin1 has a pivotal role in the regulation of cell survival, apoptosis, and autophagy,
especially via its interaction with anti-apoptotic proteins of the Bcl-2 family (Bcl-2,
Bcl-XL, Bcl-w, and Mcl-1), the BH3 domain of Beclin1 important for the binding to
the BH3 domain of the pro-apoptotic factors Bak, Bad, and Bim to Bcl-XL, and the activation
of the Beclin1-interacting complex that generates phosphatidylinositol-3-phosphate,
which promotes autophagosomal membrane nucleation and is indispensable for autophagy
15
. It now seems necessary to further investigate its potential regulatory role in the
process of accelerated aging associated to chronic cigarette smoke exposure and COPD,
at lung but also at systemic level.
In conclusion, although our results need to be confirmed in other cohorts of COPD
patients or with other potential biomarkers of autophagy, they support the hypothesis
of a relationship between autophagy deficiency and COPD pathogenesis. They also incite
to refine our knowledge on the complex mechanisms linking defective autophagy and
cellular senescence in the progressive pathogenesis of COPD and of its systemic manifestations,
with a special focus on Beclin1.