ACE2 knockout cell line | high-efficiency and low off-target rate

Angiotensin-converting enzyme (ACE) is a kind of metalloproteinase encoding 805 amino acids, located in X chromosome (Xp22.2). ACE is a type I transmembrane glycoprotein with a single extracellular catalytic domain that plays an important regulatory role in the renin-angiotensin system (RAS). ACE2, a homolog of ACE, can split Ang II into Ang (1-7) polypeptide, which has anti-inflammatory functions: protecting cardiomyocytes, relaxing blood vessels, anti-proliferation, and can enhance the activity of bradykinin (an inflammatory mediator).

Angiotensin-converting enzyme 2 (ACE2) is a type 1 integral membrane glycoprotein that is expressed and active in most tissues. Also, it shares some homology with angiotensin-converting enzyme (ACE) but is not inhibited by ACE inhibitors.

 

ACE2 expression restrict virus replication in human cells

It was isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells that efficiently binds the S1 domain of the SARS-CoV S protein. It has been reported that ACE2 is the main host cell receptor of 2019-nCoV and plays a crucial role in the entry of virus into the cell to cause the final infection. It was recently shown that murine ACE2 does not allow for efficient SARS-CoV replication, raising the possibility of alternate receptor use in non-primate cells.

Cell lines were infected with the pseudotyped retrovirus expressing ACE2. At 24 to 48 h after lipofection or 72 h after retrovirus infection, cells were infected with SARS-CoV. ACE2 expression from the pseudotyped retrovirus resulted in SARS-CoV replication in all cell lines examined, including those still refractory following plasmid ACE2 expression. Based on these results, the in vitro host range of SARS-CoV is primarily determined by the presence of its receptor, ACE2.

The 293T cells were unable to support virus replication after plasmid ACE2 expression despite high levels of ACE2 expression. However, ACE2 expression from the pseudotyped retrovirus resulted in efficient virus replication in 293T cells. Similar observations were made in A549 and AK-D cells. Therefore, restriction of SARS-CoV replication in these cells was recently shown to be overcome by expression of human ACE2.

 

The importance of ACE2 in maintaining the balance of the RAS system

The main role of ACE2 is the degradation of Ang II resulting in the formation of angiotensin 1–7 (Ang 1–7) which opposes the actions of Ang II. Increased Ang II levels are thought to upregulate ACE2 activity, and in ACE2 deficient mice Ang II levels are approximately double that of wild-type mice, whilst Ang 1–7 levels are almost undetectable. Thus, ACE2 plays a crucial role in the RAS because it opposes the actions of Ang II. Consequently, it has a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease where its expression is decreased.  Current therapeutic strategies for ACE2 involve augmenting its expression using ACE2 adenoviruses, recombinant ACE2 or compounds in these diseases thereby affording some organ protection.

 

ACE2 helps research in Kidney Disease

Kidney injury is largely mediated by Ang II. Some studies have shown that ACE2 gene knockout can lead to an increase in blood pressure, glomerular damage, and renal fibrosis in diabetic mice. Exogenous human recombinant ACE2 (hACE2) can slow down the progress of diabetic nephropathy (DKD) by reducing albumin excretion. ACE2 was highly expressed in kidney, mainly in brush border cells of proximal renal tubules, endothelial cells, smooth muscle cells of renal vessels, and podocytes. It has been reported that in damaged renal tubules, the increase of Ang II may be a possible mediator for further renal damage in human renal diseases. Therefore, the imbalance between ACE and ACE2 in the kidney followed by high level of Ang II may lead to renal damage. Hypertensive nephropathy is a common complication of hypertension, with its main mechanisms being inflammation associated with Ang II, oxidative stress, and renal fibrosis. It is worth noting that in April 2019, the school of medicine of Jilin University found that Ginsenoside Rg3 can alleviate the Ang II-mediated renal injury in rats and mice by upregulating ACE2 in renal tissue. In addition to anti-tumor activity, Rg3 can also protect the cardiovascular system through various mechanisms, including anti-inflammatory, anti-oxidative stress, and anti-fibrosis.

 

ACE2 deletion resulted in modest elevations in systolic blood pressure levels

ACE2 is widely expressed in cardiomyocytes, cardiac fibroblasts, and coronary artery endothelial cells. ACE2 is an important regulatory protein in RAS, the system which regulates the balance of body fluid and blood pressure and maintains the tension of blood vessels. The overactivation (increase of vasoconstriction) or depletion (decrease of vasodilation) of RAS will lead to vascular dysfunction, which is the main cause of atherosclerosis and cardiovascular disease (CVD). 

The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys. Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. 

 

ACE2 knockout mouse

The first description of an ACE2 knockout mouse line by Crackower and associates suggested that ACE2 plays an essential role in regulating normal cardiac function in vivo. In this mouse line, a null mutation was generated by replacing portions of exons 7–9 of the Ace2 gene with a neomycin cassette in the antisense orientation; exon 9 encodes the zinc‐binding (HEMGH) motif of the enzyme (Donoghue et al. 2000; Tipnis et al. 2000). The dominant phenotype of these ACE2‐deficient mice was a marked defect in cardiac contractility. This decrease in left ventricular systolic function, documented by echocardiography in anaesthetized mice, was more severe in older, male ACE2‐deficient mice and was accompanied by reduced blood pressures. In 6‐month‐old male mice, left ventricular fractional shortening was reduced by as much as 40%. Additionally, cardiac structure was abnormal, with wall thinning and enlarged cardiac chambers. However, there was no indication of cardiac hypertrophy or fibrosis in the ACE2‐deficient mice, and overall heart weights were very similar between ACE2 knockout and wild‐type animals.

 

References:

1.      Mossel EC, Huang C, Narayanan K, Makino S, Tesh RB, Peters CJ. Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication. J Virol. 2005;79(6):3846‐3850.

2.      Li, W., Moore, M., Vasilieva, N. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).

3.      Xu, H., Zhong, L., Deng, J. et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 12, 8 (2020).

4.      Tikellis C, Thomas MC. Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease. Int J Pept. 2012;2012:256294.

5.      Angiotensin‐converting enzyme 2 gene targeting studies in mice: mixed messages. Susan B. Gurley  Thomas M. Coffman. Experimental PhysiologyVolume 93, Issue 5.