**2. ACE2 and TMPRSS2 expression profiles in ocular tissues**

In 2020, various cases of positive conjunctival swabs and conjunctivitis were reported as COVID-19 symptoms. Therefore, several researchers have investigated the ocular surfaces as a potential infection route for SARS-CoV-2 [18, 19]. To this end, intensive research focused on the presence of ACE2 and TMPRSS2 receptors in various ocular tissues since both receptors play important roles in the entry of SARS-CoV-2 to the host cells [20, 21].

ACE2 is an important component of Renin-Angiotensin System (RAS). The circulatory RAS is composed of certain enzymes and active-inactive peptides and plays crucial roles in human body including the regulation of blood pressure, fluid volumes and electrolyte homeostasis [22–27]. These regulations are controlled through the digestion of Angiotensinogen by Renin to generate Angiotensin I and transformation of Angiotensin I to the active form Angiotensin II by angiotensinconverting enzyme (ACE). Recently, Renin and ACE independent generation of Angiotensin II has also been reported [27]. In addition to the circulatory system, RAS is also found locally in some tissues and two separate research groups, Fischer-Ferraro *et al.* and Ganten *et al.* discovered the first clues on local RAS and its tissuespecific roles in 1971 [28, 29]. To date, local RAS has been reported in various organs such as brain, heart, intestine, kidney, and the eye [30, 31]. The presence of RAS in the eye suggested its involvement in various ocular diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and glaucoma [22].

ACE2 is a carboxypeptidase found in circulatory system and in some tissues and regulates RAS negatively by cleaving angiotensin II [21]. This carboxypeptidase is structurally similar to angiotensin converting enzyme (ACE) with a 42% sequence similarity [32]. ACE2 was first discovered and cloned in 2000 as a counter-regulator of ACE, which generates Angiotensin (1–7) by cleaving a single residue from Angiotensin II or Angiotensin (1–9) by removing single residue from Angiotensin I [32–35]. In the eye, ACE2 expression has been demonstrated in a wide variety of ocular tissues, including aqueous humor, retina, corneal epithelium, conjunctival epithelium, and limbal epithelium [6, 7, 22]. ACE2 is found to decrease intraocular pressure (IOP) upon activation with chemical inducers [36].

In addition to ACE2, recent studies showed that TMPRSS2 receptor was also contributing to the cell entry of SARS-CoV-2 by cleaving the spike protein after its' binding to ACE2 receptor [6, 37, 38]. TMPRSS2 is one of the serine proteases, involved in various physiological and pathological processes, including protein catabolism, blood coagulation and tissue rearrangement [39, 40]. As a homologous to enterokinase, the

#### *Potency of SARS-CoV-2 on Ocular Tissues DOI: http://dx.doi.org/10.5772/intechopen.97055*

function of TMPRSS2 is suggested to be similar to enterokinase that cleaves acidic pro-peptide from trypsinogen to generate active enzyme. However, exact physiological functions of TMPRSS2 are still not clear [40, 41]. Many ocular surfaces express TMPRSS2 receptor such as conjunctiva and corneal stroma [42].

Due to the important roles of ACE2 and TMPRSS2 in SARS-CoV-2 infection, their individual and co-expression in ocular tissues is investigated [43]. In an early study, local ACE2 expression in rodent retina was evaluated by using immunoblotting, immunohistochemistry analyses and mRNA levels. Expression of ACE2 was broadly localized in the inner nuclear layer and photoreceptors of rodent retinas [44]. Similarly, TMPRSS2 expression was also shown in the retina [45]. One of the most comprehensive studies on this subject was the investigation of coronavirus-2 (CoV-2) tropism in ocular tissues [6]. Here, co-expression of ACE2 receptor and TMPRSS2 protease was shown in human adult conjunctival, limbal and corneal epithelium but not in embryonic and fetal ocular tissues [6]. On the other hand, comparative RNA expression levels of ACE2 and TMPRSS2 in various tissues suggested ACE2 being the limiting factor for infection because TMPRSS2 expression showed a broader tissue distribution [46]. Similarly, expression of ACE2 and TMPRSS2 in post-mortem eyes of non-diabetic and diabetic retinopathy specimens revealed significantly strong expression of ACE2 in corneal and conjunctival epithelium while broad expression of TMPRSS2 in all ocular surfaces [42]. A comparable expression pattern with post-mortem eyes was found in five surgical conjunctival specimens as well, only with higher ACE2 staining intensity in the surgical specimen [42].

Co-expression profile of ACE2 and TMPRSS2 genes shows some contradicting expression profiles between human primary conjunctival and pterygium cells and different cell lines including ARPE-19, HUVEC, HaCaT, HepG2, and A549 [37]. For instance, persistent expression of ACE2 and TMPRSS were observed in conjunctival and pterygium cells of some patients, which was concluded not to be enough for SARS-CoV-2 cell entry [37]. In contrast, a significantly higher gene expression of TMPRSS2 and a lower but notable ACE2 gene expression in studied ocular (ARPE-19, HUVEC) and lung cell (A549) lines were observed [37]. Investigation of healthy and diseased conjunctival samples for mRNA expression levels of ACE2 and TMPRRS2, and ACE2 protein expression by immunostaining revealed ACE2 expression in conjunctival samples [47]. However, protein

#### **Figure 1.**

*Representative schematic of the relative expression profile of ACE2 and TMPRSS2 in ocular surfaces. (This schematic was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com).*

expression of ACE2 and other SARS-CoV-2 mediators of cell entry found not significant enough for the infection [47]. On the other hand, the expression of ACE2 and TMPRSS2 in ocular epithelium such as corneal epithelial cells, conjunctival epithelial cells and corneal endothelial cells is also reported. Herein, co-expression of ACE2 and TMPRSS2 in corneal epithelium and endothelium suggested the susceptibility of cornea for a potential SARS-CoV-2 infection site (**Figure 1**) [48].
