FN3K expression in COPD: a potential comorbidity factor for cardiovascular disease

Abstract

Introduction: Cigarette smoking and oxidative stress are common risk factors for the multi-morbidities associated with chronic obstructive pulmonary disease (COPD). Elevated levels of advanced glycation endproducts (AGE) increase the risk of cardiovascular disease (CVD) comorbidity and mortality. The enzyme fructosamine-3-kinase (FN3K) reduces this risk by lowering AGE levels.

Methods: The distribution and expression of FN3K protein in lung tissues from stable COPD and control subjects, as well as an animal model of COPD, was assessed by immunohistochemistry. Serum FN3K protein and AGE levels were assessed by ELISA in patients with COPD exacerbations receiving metformin. Genetic variants within the FN3K and FN3K-RP genes were evaluated for associations with cardiorespiratory function in the Subpopulations and Intermediate Outcome Measures in COPD Study cohort.

Results: This pilot study demonstrates that FN3K expression in the blood and human lung epithelium is distributed at either high or low levels irrespective of disease status. The percentage of lung epithelial cells expressing FN3K was higher in control smokers with normal lung function, but this induction was not observed in COPD patients nor in a smoking model of COPD. The top five nominal FN3K polymorphisms with possible association to decreased cardiorespiratory function (p<0.008-0.02), all failed to reach the threshold (p<0.0028) to be considered highly significant following multi-comparison analysis. Metformin enhanced systemic levels of FN3K in COPD subjects independent of their high-expression or low-expression status.

Discussion: The data highlight that low and high FN3K expressors exist within our study cohort and metformin induces FN3K levels, highlighting a potential mechanism to reduce the risk of CVD comorbidity and mortality.

Keywords: COPD ÀÜ mechanisms; systemic disease and lungs.

Figure 1

Representative immunohistochemical staining for fructosamine-3-kinase (FN3K) in bronchiolar epithelial cells of stable chronic obstructive pulmonary disease (COPD) patients and control subjects (A: non-smoking control; B: control smoker with normal lung function; C: patients with stable COPD; 100× magnification; bar: 20 µm). (D) The percentage of FN3K immunopositive epithelial cells from peripheral lung sections following immunohistochemical staining. Each data point is the average score of immunopositive epithelial cells counted over several random fields of view for each subject. The mean±SEM is overlaid on top of the scatter plot for each cohort sub grouping. Each cohort (non-smoker, smoker with normal lung function (smoker) and COPD) is split into two subgroups, those exhibiting a low frequency of FN3K expression (low) and those exhibiting a high frequency of FN3K expression (high). Non-parametric Kruskal-Wallis test was employed, where applicable, to determine significant differences within each cohort subgrouping (**p<0.01). (E) The percentage of subjects exhibiting immunopositive FN3K expression in >15% of lung epithelial cells, as illustrated in(D), between non-smokers (NS), smokers with normal lung function (smokers) and stable COPD patients (COPD). The numbers in brackets represent the mean percentage of FN3K immunopositive epithelial cells in each cohort exhibiting high FN3K expression. A χ² test was used to check for independence. The probability of independence (p) between the three groups is illustrated.

Figure 2

Impact of metformin (A) versus placebo (B) on serum fructosamine-3-kinase (FN3K) levels in chronic obstructive pulmonary disease (COPD) patients with an acute exacerbation. Data are plotted as the mean±SEM for FN3K protein expression as assessed by ELISA at each of the sampling points. Entry was at day 1, with a median discharge at day 7 (days 4–10) and follow-up at day 35. Data are expressed as ng/mL FN3K (A and B) and per cent change from baseline entry (C). In (A) and (B) one way analysis of variance (ANOVA) and Tukey’s multiple comparison test was used to determine significant changes relative to entry within each group (either placebo or metformin. A p<0.05 was considered significant (†††p<0.001, ††††p<0.0001). (C) Statistical analysis was performed using two way ANOVA and Sidak’s multiple comparison test across both placebo and metformin groups to test for significant differences between placebo (∎) and metformin (●) at each sampling point. A p<0.05 was considered significant (*p<0.05).

Figure 3

Impact of metformin (A) versus placebo (B) on serum protein carbonyl levels in chronic obstructive pulmonary disease (COPD) patients with an acute exacerbation as measured by ELISA. Data are plotted as a scatter plot with mean±SEM (A and B) and per cent change from baseline entry (C). Placebo is represented by solid squares and metformin treatment by solid circles in (C). Entry was at day 1, with a median discharge at day 7 (days 4–10) and follow-up at day 35. To test for significant difference, one way repeated measures analysis of variance was performed with Tukey’s post hoc analysis. A p<0.05 was considered significant (****p<0.0001).

Figure 4

Fructosamine-3-kinase (FN3K) expression is reduced in murine airway epithelial cells exposed to cigarette smoke for 6 months. C57BL/6J wild type mice were exposed to cigarette smoke (CS) or ambient air (Air) for 6 months the percentage of airway epithelial cells staining positive for FN3K expression in peripheral lung sections following immunohistochemical staining was recorded over several random fields of view for each mouse and the data expressed as the mean±SEM (N=8 for each group). Further details are in the Materials and methods section. Statistical significance was determined using Mann-Whitney U test (*p<0.05).

Figure 5

Fructosamine-3-kinase (FN3K) as a potential gatekeeper to prevent the accumulation of damaging AGEs that may predispose subjects to increased risk of cardiovascular disease. Accumulation of AGEs and carbonyl stress may come directly through raised uncontrolled blood glucose levels in diabetes or indirectly through chronic oxidative stress from cigarette smoking in COPD resulting in mitochondrial dysfunction, an altered metabolic profile and elevated cellular ribose levels and increased carbonyl stress. AGE, advanced glycation endproduct; COPD, chronic obstructive pulmonary disease; CVD, cardiovasculardisease.