Male bias in ACE2 basic science research: missed opportunity for discovery in the time of COVID-19

Abstract

Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

Keywords: angiotensin; gonadectomy; heart; kidney; ovariectomy.

Figure 1.

The renin-angiotensin system (RAS). The decapeptide angiotensin (Ang)-[1–10] is cleaved from the protein angiotensinogen by renin. Angiotensin-converting enzyme (ACE) removes two amino acids from the carboxy terminus of the precursor peptide Ang-[1–10] to form the octapeptide hormone Ang-[1–8] (also known as ANG II). Ang-[1–8] exerts its physiological effects by binding to the angiotensin type 1 and type 2 receptors (AT₁R and AT₂R). Overactivity of the Ang-[1–8]-AT₁R pathway leads to hypertension, inflammation, and tissue fibrosis, which is counter regulated by the actions of the AT₂R. Angiotensin-converting enzyme 2 (ACE2) attenuates Ang-[1–8] activity primarily by removing one amino acid from the carboxy terminal to form Ang-[1–7].

Figure 2.

Literature search on angiotensin-converting enzyme 2 (ACE2). A database search of Ovid MEDLINE and Embase led to the identification of 2,131 published articles on ACE2 through December 31, 2020. Inclusion and exclusion criteria led to the identification of 853 original research articles on nonhuman mammalian primary cells, tissues, or whole animals; studies conducted solely in cells transfected with ACE2 cDNAs were excluded. Note the original paper describing the discovery of ACE2 in 2000 (34) was not included because it did not meet our inclusion criterion. The article reported on ACE2 expression in human tissues and in cells transfected with ACE2 cDNAs. Only 79 of the 853 original articles included data in both sexes and of those, only 25 compared the data by sex. The number of articles as a function of species studied is also shown.

Figure 3.

Sex of nonhuman mammals reported in angiotensin-converting enzyme 2 (ACE2) publications over the past two decades. Shown is the proportion of original research articles on nonhuman mammalian primary cells, tissues or whole animals that are conducted in male only, female only, both sexes or studies in which the sex is unknown (i.e., not reported) as a function of the total number of articles as a pie chart (A) or number of articles published each year since ACE2 was discovered (B).

Figure 4.

Substrate dependence of angiotensin-converting enzyme 2 (ACE2) activity in the hamster lung and heart. Substrate dependence of ACE2 enzyme activity in 0.5 µg/µL of lung (n = 2) (A) and heart (n = 4) (B) tissue from male 11-wk-old hamsters. The data are presented as the means ± SE.

Figure 5.

Commercial angiotensin-converting enzyme 2 (ACE2) antibody sensitivity and specificity. Shown are representative Western blots from two gels with 16 wells each. After protein transfer, the PVDF membranes contained protein markers (M; lanes 1, 6, and 13), mouse recombinant ACE2 protein (RA; lanes 2, 8, and 14), and renal tissue from ACE2 knock out (KO; lanes 3, 9, and 15) and wild-type (WT; lanes 4, 10, and 16) female C57BL/6 mice. Membranes were cut into three pieces each and blotted with either anti-mouse ACE2 antibodies from: R&D (A), Abcam (B), and Santa Cruz (C) or anti-human ACE2 antibodies from: Cell Signaling (E), Novus (F), and, Proteintech (G). Super Signal West Pico Plus substrate was used to detect ACE2 immunoreactive protein in A–C and E–G. D shows membrane exposed to the Santa Cruz antibody (C) re-probed with the Femto maximum sensitivity substrate. After probing with ACE2 antibodies, all membranes were stripped and incubated with antibodies to β-actin to control for differences in protein loading.

Figure 6.

Impact of biological sex on angiotensin-converting enzyme 2 (ACE2) activity in the rat kidney and lung. Shown is ACE2 enzyme activity in kidney (A) and lung tissue (B) from female (circle) and male (square) 2–4 mo-old Long-Evans rats at a substrate concentration of 50 µM. The data are presented as a scatter plot showing individual data points (n = 5–6) with the means and SE indicated by horizontal and vertical bars, respectively. The data were analyzed by an unpaired t test with Welch’s correction, *P < 0.05 vs. male.

Figure 7.

Impact of biological sex on angiotensin-converting enzyme 2 (ACE2) activity in the hamster kidney, lung, and heart. Shown is ACE2 enzyme activity from 10 ng/µL of kidney tissue (A) and 50 ng/µl of lung (B) and heart (C) from female (circle) and male (square) 11-wk-old Syrian golden hamsters at 30 µM substrate concentration. The data are presented as a scatter plot showing individual data points (n = 6) with the means and SE indicated by horizontal and vertical bars, respectively. The data were analyzed by an unpaired t test with Welch’s correction, *P < 0.05 vs. male.