Lower Gene Expression of Angiotensin Converting Enzyme 2 Receptor in Lung Tissues of Smokers with COVID-19 Pneumonia

Abstract

Angiotensin-converting enzyme 2 (ACE-2) is the main cell entry receptor for severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2), thus playing a critical role in causing Coronavirus disease 2019 (COVID-19). The role of smoking habit in the susceptibility to infection is still controversial. In this study we correlated lung ACE-2 gene expression with several clinical/pathological data to explore susceptibility to infection. This is a retrospective observational study on 29 consecutive COVID-19 autopsies. SARS-CoV-2 genome and ACE-2 mRNA expression were evaluated by real-time polymerase chain reaction in lung tissue samples and correlated with several data with focus on smoking habit. Smoking was less frequent in high than low ACE-2 expressors (p = 0.014). A Bayesian regression also including age, gender, hypertension, and virus quantity confirmed that smoking was the most probable risk factor associated with low ACE-2 expression in the model. A direct relation was found between viral quantity and ACE-2 expression (p = 0.028). Finally, high ACE-2 expressors more frequently showed a prevalent pattern of vascular injury than low expressors (p = 0.049). In conclusion, ACE-2 levels were decreased in the lung tissue of smokers with severe COVID-19 pneumonia. These results point out complex biological interactions between SARS-CoV-2 and ACE-2 particularly concerning the aspect of smoking habit and need larger prospective case series and translational studies.

Keywords: COVID-19; SARS-CoV-2; angiotensin converting enzyme receptor; smoking.

Figure 1

Real-Time PCR for ACE-2 expression. Typical amplification plots of GAPDH (A) and ACE-2 (B) genes. Cts of GAPDH plotted against the ACE-2 gene Cts (Rho = 0.43, p = 0.020) (C). Normalized ACE-2 Ct values (log(2^−ΔCt) = log(2 − (Ct_target − Ct_reference)) plotted against the ACE-2 gene Cts (D). The normalized Ct values are relative expression values (ratios of target gene to housekeeping gene) and are transformed to logarithmic scale for graphical representation. Shaded areas represent the 95% CI of the linear correlation line.

Figure 2

Box-violin plots of ACE-2 expression comparison in the three groups of patients. Overall comparison was implemented with the Kruskal–Wallis test. Pairwise comparisons with the Dunn post-hoc test. Only the COVID non-smokers vs. non-COVID non-smokers comparison was significant (p = 0.002). ACE-2 ΔCts (Ct_ACE-2 − Ct_GAPDH) are inversely correlated with ACE-2 expression.

Figure 3

Relation between ACE-2 expression and virus quantity. Direct linear relation between SARS-CoV-2 quantity and ACE-2 receptor expression (p = 0.028, rho = 0.44). Shaded areas represent the 95% CI of the linear correlation line.

Figure 4

Bayesian linear regression model coefficients. Data are presented as median with 95% highest density interval of the posterior distribution. Coefficients are ranked from the lowest to the highest median.

Figure 5

Histological lung sections of an index case with prevalent vascular injury (male, 82 years old, high ACE-2 expressor with ΔCt = 4.4) that shows a thrombus in a centrilobular arteriolar vessel (A, hematoxylin and eosin stain, original magnification ×100) and diffuse capillaritis in the alveolar septa (B, hematoxylin and eosin stain, original magnification ×200). Histological lung sections of an index case (male, 76 years old, low ACE-2 expressor with ΔCt = 8.2) with diffuse alveolar damage: hyaline membrane deposits along the alveolar walls (C, hematoxylin and eosin stain, original magnification ×100), foci of squamous metaplasia associated with endoalveolar exudative material including connective tissue components, fibrin, and fibroblasts (organizing pneumonia) (D, hematoxylin and eosin stain, original magnification ×100).