ACE2 (angiotensin-converting enzyme 2) degrades Ang (angiotensin) I and II and is a cellular receptor for severe acute respiratory syndrome coronavirus 2, the virus that causes coronavirus disease 2019 (COVID-19). Viral entry into host cells occurs through binding of the viral spike (S) protein and ACE2.¹ Preclinical data suggest that renin-angiotensin system (RAS) blockers upregulate ACE2.^2,3 As a consequence, RAS blockers have been suggested to increase the risk of developing severe acute respiratory syndrome coronavirus 2 infection. However, recent large retrospective studies strongly argue against this hypothesis and rather suggest that RAS blockers be protective in such patients.⁴ Since the findings on RAS blocker-induced ACE2 upregulation are inconsistent, and differed not only per type of RAS inhibitors (ACE inhibitors versus ARB [angiotensin receptor blockers]),³ between blockers of a certain type (ie, between various ARBs), but also per organ, and required high doses, one further option is that this ACE2 upregulation is not the unavoidable consequence of RAS suppression, but rather reflects the nonspecific effects of a certain RAS blocker when applied at a high dose. Applying antisense oligonucleotides (ASO) as a tool to suppress the RAS would circumvent the latter. In the present study, we determined effects of an ACE inhibitor (enalapril) and an ARB (losartan) as well as AGT (angiotensinogen) ASO on tissue ACE2 in male C57BL/6J mice.

After 14 days of infusion, both enalapril and losartan increased plasma renin, measured as direct concentrations, by over 100-fold, confirming effective RAS inhibition (Figure [A]). ACE2 mRNA abundance was determined by quantitative PCR in lung, ileum, kidney, and heart tissues. Neither enalapril nor losartan changed the abundance of ACE2 mRNA in any of the tissues (Figure [B]). Ferrario et al³ reported that administration of lisinopril (an ACE inhibitor) or losartan for 12 days increases ACE2 mRNA abundance ≈3- to 5-fold in male rat heart. It is worth noting that ACE2 mRNA is much less abundant in heart compared with lung, ileum, and kidney (data not shown).

Figure. Effects of enalapril, losartan, and AGT (angiotensinogen) antisense oligonucleotides (ASO) on ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane protease serine 2) mRNA abundance. A–C, Male C57BL/6J mice were infused subcutaneously with either enalapril (3 mg/kg per day) or losartan (15 mg/kg per day) for 14 days. D–F, Male C57BL/6J mice were administered AGT ASO (80 mg/kg at day 1 and 4, 40 mg/kg at day 8 and 15) or vehicle (PBS) subcutaneously. Plasma renin (A and D) were measured by ELISA. B, C, E, and F, ACE2 and TMPRSS2 mRNA abundance were quantified by qPCR with TaqMan probes (ID: Mm01159003_m1 and ID: Mm00443687_m1, respectively) and normalized to the geomean of three reference genes: ACTB, GAPDH, and PPIA. Genes with a cycle threshold (Ct) >35 were considered undetectable. Error bars denote SEM; n=5/group. *P<0.001 vs vehicle, 1-way ANOVA with Holm-Sidak method. **P<0.05 vs vehicle, Student t test. ACTB indicates beta-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; and PPIA, peptidylprolyl isomerase A.

Viral entry also depends on TMPRSS2 (transmembrane protease serine 2) to prime S protein.¹ TMPRSS2 mRNA was highly abundant in lung and ileum, moderately in kidney, while barely detectable in heart (data not shown). Thus, ACE2 and TMPRSS2 are co-expressed most abundantly in lung and ileum, consistent with their roles in severe acute respiratory syndrome coronavirus 2 infection. As with ACE2, TMPRSS2 mRNA abundance was not altered by either enalapril or losartan (Figure [C]).

Next, we determined whether depletion of AGT, the unique substrate of the RAS, changes ACE2 and TMPRSS2 mRNA abundance. AGT ASO led to elevation of plasma renin concentrations, an immediate consequence of RAS blockade (Figure [D]). However, abundance of ACE2 mRNA was unchanged in all tissues (Figure [E]). Interestingly, AGT ASO significantly decreased TMPRSS2 mRNA abundance in lung (Figure [F]).

In summary, RAS inhibition does not affect mRNA abundance of ACE2 in male C57BL/6J mice administered enalapril, losartan, or AGT ASO. AGT ASO reduces TMPRSS2 mRNA expression in lungs, which is potentially protective against viral entry. These data support that RAS inhibition per se does not regulate ACE2 and hence is unlikely to increase the risk for COVID-19. In agreement with this concept, Sama et al⁵ recently were unable to detect changes in circulating ACE2 in patients taking RAS inhibitors.

C. Wu is an National Institutes of Health (NIH)/NHLBI K99 awardee. This work was supported by NIH grants K99HL145117 and R01HL139748.

A. Daugherty and H.S. Lu filed a patent application for use of AGT (angiotensinogen) antisense oligonucleotides (ASO) in aortic aneurysmal disease. A.E. Mullick is an employee in Ionis Pharmaceuticals, Inc, who provided the AGT ASO. The other authors report no conflicts.

*C. Wu and D. Ye are joint first authors.

ACE2（血管紧张素转换酶2）降解Ang（血管紧张素）I和II，是严重急性呼吸系统综合症冠状病毒2的细胞受体，冠状病毒2是导致冠状病毒疾病2019（COVID-19）的病毒。病毒进入宿主细胞是通过病毒刺突（S）蛋白和ACE2的结合而发生的。¹临床前数据表明，肾素-血管紧张素系统（RAS）阻断剂可上调ACE2。^2,3因此，有人建议使用RAS阻滞剂增加发生严重急性呼吸系统综合症冠状病毒2感染的风险。但是，最近的大型回顾性研究强烈反对这一假设，而是暗示RAS阻滞剂对此类患者具有保护作用。⁴由于在RAS阻断剂诱导的ACE2表达上调的结果是不一致的，并且不仅每类型RAS抑制剂（ACE抑制剂与ARB [血管紧张素受体阻断剂）表示的在不同³某种类型的阻断剂之间（即，各种的ARB之间），但同样对于每个器官，以及所需的高剂量，另一种选择是，这种ACE2上调不是RAS抑制的必然结果，而是反映了以高剂量使用时某些RAS阻滞剂的非特异性作用。应用反义寡核苷酸（ASO）作为抑制RAS的工具将绕过后者。在本研究中，我们确定了ACE抑制剂（依那普利）和ARB（氯沙坦）以及AGT（血管紧张素原）ASO对雄性C57BL / 6J小鼠组织ACE2的影响。

输注14天后，依那普利和氯沙坦都将血浆肾素（以直接浓度计）增加了100倍以上，从而证实了有效的RAS抑制作用（图[A]）。ACE2 mRNA的丰度通过定量PCR在肺，回肠，肾脏和心脏组织中确定。依那普利和氯沙坦均未改变任何组织中ACE2 mRNA的丰度（图[B]）。Ferrario等[ ^3]报告说，在雄性大鼠心脏中，赖诺普利（一种ACE抑制剂）或氯沙坦给药12天可使ACE2 mRNA的丰度提高约3-5倍。值得注意的是，与肺，回肠和肾脏相比，心脏中的ACE2 mRNA含量要低得多（数据未显示）。

数字。依那普利，氯沙坦和AGT（血管紧张素原）反义寡核苷酸（ASO）对ACE2（血管紧张素转换酶2）和TMPRSS2（跨膜蛋白酶丝氨酸2）mRNA丰度的影响。A–C雄性C57BL / 6J小鼠皮下注射依那普利（依那普利（每天3 mg / kg）或氯沙坦（每天15 mg / kg））14天。D–F，雄性C57BL / 6J小鼠皮下注射AGT ASO（第1和第4天为80 mg / kg，第8和15天为40 mg / kg）或溶媒（PBS）。用ELISA法测定血浆肾素（A和D）。B，C，E和F，使用TaqMan探针（分别为ID：Mm01159003_m1和ID：Mm00443687_m1）通过qPCR对ACE2和TMPRSS2 mRNA的丰度进行定量，并针对三个参考基因ACTB，GAPDH和PPIA的几何平均值进行标准化。周期阈值（Ct）> 35的基因被认为是不可检测的。误差线表示SEM。n = 5 /组。* P <0.001，相对于车辆，采用Holm-Sidak方法的1路ANOVA。**与车辆相比，P <0.05，学生t检验。ACTB表示β-肌动蛋白；GAPDH，3-磷酸甘油醛脱氢酶；和PPIA，肽基脯氨酰异构酶A。

病毒进入还依赖于TMPRSS2（跨膜蛋白酶丝氨酸2）来引发S蛋白。¹ TMPRSS2 mRNA在肺和回肠中高度丰富，在肾脏中中等，而在心脏中几乎检测不到（数据未显示）。因此，ACE2和TMPRSS2在肺和回肠中最丰富地共表达，这与它们在严重急性呼吸综合征冠状病毒2感染中的作用一致。与ACE2一样，依那普利或氯沙坦也不会改变TMPRSS2 mRNA的丰度（图[C]）。

接下来，我们确定RAS唯一底物AGT的消耗是否会改变ACE2和TMPRSS2 mRNA的丰度。AGT ASO导致血浆肾素浓度升高，这是RAS阻断的直接结果（图[D]）。但是，ACE2 mRNA的丰度在所有组织中均未改变（图[E]）。有趣的是，AGT ASO显着降低了肺部TMPRSS2 mRNA的丰度（图[F]）。

总之，在给予依那普利，氯沙坦或AGT ASO的雄性C57BL / 6J小鼠中，RAS抑制作用不会影响ACE2的mRNA丰度。AGT ASO降低肺中TMPRSS2 mRNA的表达，这可能对病毒进入具有保护作用。这些数据支持RAS抑制本身并不调节ACE2，因此不太可能增加COVID-19的风险。与这一概念一致的是，Sama等人⁵最近未能检测到服用RAS抑制剂的患者循环中ACE2的变化。

C. Wu是美国国立卫生研究院（NIH）/ NHLBI K99获奖者。NIH赠款K99HL145117和R01HL139748支持了这项工作。

A. Daugherty和HS Lu提出了AGT（血管紧张素原）反义寡核苷酸（ASO）在主动脉瘤病中的使用的专利申请。AE Mullick是Ionis Pharmaceuticals，Inc的员工，他提供了AGT ASO。其他作者报告没有冲突。

*C。吴和叶烨是第一作者。