Systematic Review and Meta-Analysis on Angiotensin Converting Enzyme 2 in Head and Neck Region

The objective of this systematic review was to investigate the expression of angiotensin converting enzyme 2 (ACE 2) in the head and neck region. We examined the evidence of the association of ACE 2 expression in oral tissues, salivary glands, and head and neck carcinoma. We searched Pub Med/Medline, Biorxiv, and Google Scholar to identify relevant literature. Studies reporting ACE 2 expression in human oral tissues and with a focus on head and neck carcinoma samples were included. From 110 studies, we extracted 15 studies analyzing the distribution and expression of ACE 2 in different head and neck tissues - olfactory mucosa and nasopharynx n=5, oral mucosa n=5, salivary gland n=5, head and neck squamous cell carcinoma patients n=3. ACE 2 was found to be expressed at a 4.43-fold increase in the head and neck region (OR, 4.43; 95% CI, 3.76-5.22; I2= 97%, Ph=<0.00001) when compared with controls (other tissues except for head and neck region). RNA expression of ACE 2 was 60% higher in head and neck squamous cell carcinoma patients than that in the normal tissues (OR=0.60, 95% CI, 0.04-9.26, Ph=0.00001). In conclusion, the meta-analysis of the studies indicated that ACE 2 is highly expressed in olfactory mucosa, nasopharynx, oral mucosa, and salivary glands. Furthermore, the results indicate that ACE 2 expression is increased in patients with head and neck cancer.


Introduction And Background
ACE 2, a homolog of angiotensin converting enzyme (ACE), is known to be of pathophysiological importance in various human tissues in both healthy and disease states [1]. Since 2019, the focus of attention is towards "ACE 2" -a key cell receptor for the SARS-CoV-2 virus and its emerging variants. ACE 2 being the functional receptor of SARS-CoV-2 has become the potential risk site for infection and pathogenesis. As a transmembrane protein, ACE 2 also serves as an entry receptor for other viruses including, SARS-CoV and HCoV-NL63. ACE 2 is the counter-modulator component of the Renin-Angiotensin-Aldosterone System (RAAS) [2][3][4][5]. The renin-angiotensin system is the critical regulator of blood volume, systemic vascular resistance, and cardiorenal function.
Studies have demonstrated the importance of ACE 2 in maintaining the balance of RAAS. Considering that SARS-CoV-2 cell entry depends on the expression of ACE 2 entry genes, we could speculate that the transmissibility and clinical manifestations of SARS-CoV-2 could be affected by the levels of ACE 2 expression on the cell surface. Multiple physiologic roles are known for ACE 2 impacting systems but perhaps most notably related to SARS-CoV-2, pulmonary. ACE 2 has been described to limit severe acute lung injury. Expression of ACE 2 is different in varied organs and their tissues. It is important to understand the distribution of ACE 2 in cells in different parts of the epithelium but also between cell-bound. In most studies, ACE 2 expressions in normal tissues were analyzed and compared to differential expression in cancer types. ACE 2 is highly expressed in alveolar epithelial cells of the lungs, myocardial cells, renal tubular cells, endothelial cells, enterocytes, cholangiocytes, and bladder urothelial cells. A study by Xu et al. [6] in the year 2020 showed that in the head and neck region, oral tissues, especially epithelial cells of the tongue, express higher ACE 2 levels. The pervasive expression of ACE 2 points us in the direction of hypothesizing its physiological plot in many organs and tissues.
Though ACE 2 is physiologically protective, it is virally conducive. The head and neck sphere at the forefront, assessing ACE 2 expression at the head and neck region, is mattering much for determining the vulnerability of these anatomical sites. Accordingly, this review focuses on ACE 2 expression in the head and neck region.

Review Methods
The methodology of Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) guidelines was followed. The criteria for considering studies for the systematic review were based on the PICOTS framework: Population, Intervention, Comparison, Outcomes, Timing, and Setting framework.

Search strategy
A systematic search was performed using PubMed/Medline, BioRxiv, and Google Scholar to identify relevant literature. The searches were limited to articles published since 1990. The electronic searches were supplemented with a manual search of citations from key review articles. The articles with database abstracts and available full texts, implying they met the criteria, were screened. The following search terms are useful:

Type of studies
The review included studies on human tissues. The searches were limited to English articles. Preprint articles were also taken into consideration derived over a few months. Studies assessing ACE 2 expression by RNA sequencing profiling, immunohistochemistry, immunoblotting, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) were included.

Participants and inclusion criteria
Papers were included in the review if they had clearly reported the expression of ACE 2 in the head and neck sites in human subjects. Animal studies, pilot studies, and systematic reviews were not included in the review.

Literature search and study characteristics
The detailed steps of the systematized search and selection are shown in the PRISMA Study Selection Flow Diagram of the literature search process (Figure 1). We extracted 14 studies from 110 studies (excluding duplicates and studies that did not meet our selection criteria) for qualitative analysis. Fourteen studies analyzed ACE 2 expression in public databases (human tissue datasets) on bioinformatic analysis. Four database studies pertained to head and neck carcinoma patients (Oral Squamous Cell Carcinoma n=1, Head and Neck Squamous Cell Carcinoma n=3). One study explored smoking-mediated ACE 2 expression. The characteristics of the study include anatomical districts (in the head and neck region), types of cells, and methods used for analyzing ACE 2 expression.

Intervention
As the rationale of this review is to collect evidence of the ACE 2 expression in the head and neck region, we did not look into an intervention in the human samples.

Outcome measure
The outcome measure for this review was the expression levels of ACE 2 in the head and neck region.

Statistical analysis
The statistical analysis was done using Revman Manager 5 (software version 5.2). Heterogeneity was estimated together with the I 2 test. Separate subgroup analyses of the studies that assessed ACE 2 in the head and neck tissues and head and neck carcinoma were assessed. We conducted a random effect metaanalysis and estimated pooled OR (Odds Ratio) along with their 95% CI (Confidence Interval). Heterogeneity was estimated together with the I 2 test. All the studies that were included in the review were not counted in the meta-analysis. Studies that did not report the OR (n=7) for ACE 2 expression in the head and neck region were excluded.

Data synthesis
When valid and relevant data were collected, we undertook a meta-analysis of the data. We grouped and analyzed studies based on the expression of ACE 2 in the normal head and neck tissues and carcinoma tissues. We conducted meta-analyzes in Review Manager software, using the Mantel-Haenszel method. Overall analysis was conducted with a fixed effect model.

Results
Of the 110 unique citations screened, we identified 14 studies relevant to ACE 2 expression in olfactory mucosa, nasopharynx, and oral mucosa n = 5, salivary gland n = 5, in head and neck carcinoma patients n = 4. Several included studies were relevant to more than one outcome of interest. Out of 14 studies, seven (n=7) studies met the criteria for meta-analysis.

Subgroup analysis
Subgroup analysis was conducted for ACE 2 expression in the head and neck tissues and in carcinoma patients separately.

Summarizing findings
We constructed a summary of findings for each included study to produce our review outcomes in Table 1 Table 2 provides an interpretation of the study's findings. 2023   Four studies with a total of 183 patients, showed 49% of increased ACE 2 expression in the head and neck tissues than that of the other tissues (OR = 0.49, 95% CI, 0.05-5.05). Heterogeneity was evinced between the studies analyzing ACE 2 in the head and neck region: I 2 = 74%, P h ≤ 0.0008 (Figure 2). In a total of three articles with 18980 head and neck carcinoma patients (10953 Oral Squamous Cell Carcinoma patients and 8027 Head and Neck Squamous Cell Carcinoma patients), the ACE 2 expression is 60% when compared to that of the normal tissues (OR = 0.60, 95% CI, 0.04-9.26). There was evidence of heterogeneity between the studies analyzing ACE 2 in head and neck carcinoma patients with I 2 = 99% and P h ≤ 0.00001 ( Figure 3). (Xu J et al. 2020 [6], Dai et al. 2020 [14], Raivoli et al. 2020 [18])

FIGURE 3: Angiotensin converting enzyme 2 expression in head and neck carcinoma cells than normal tissues
Of the total seven studies (n=7) included for the meta-analysis shows that ACE 2 is 4.43-fold times increased in the head and neck tissues (OR = 4.43, 95% CI, 3.76-5.22) with overall heterogeneity I 2 = 97% and P h ≤ 0.00001 (Figure 4). This evinces that ACE 2 is widely expressed all over the head and neck region, which acts as a portal for infectious disease. Our systematic review and meta-analysis included studies analyzing ACE 2 in the head and neck region. In n = 42,458 tissue samples, a subgroup meta-analysis was performed based on the pathological condition of the tissues (normal and carcinoma tissues). Of the total 14 studies from the review, seven studies were included in the meta-analysis (Figure 2-4). The quality of the evidence for each included study was assessed using the Oxford Levels of Medicine (Oxford Centre of Evidence-Based Medicine-Levels of Evidence (March 2009)) (  The overall analysis of studies addressing ACE 2 in the head and neck tissues showed that ACE 2 expression is 4.43 times more highly expressed in the head and neck region (OR = 4.43, 95% CI, 3.76-5.22) (Figure 4).
mucosa. Goblet cells and epithelial cells lining the nasal turbinates, ethmoid sinus, and unicate sinus express the ACE 2 receptors. According to the literature, ACE 2 is primarily expressed in non-neuronal cells, as well as other cells of the olfactory epithelium. Secretory goblet cells of the nasal mucosa did not express ACE 2. ACE 2 expression is distributed in the basal layer of the non-keratinizing squamous epithelium in the nasal, oral mucosa, and nasopharynx [6]. ACE 2 expressions were demonstrated in ciliated epithelial cells, glial cells, non-neuronal cells, sustentatorial cells, and stem cells of the olfactory epithelium [8,19]. ACE 2 and transmembrane serine protease 2 (TMPRSS 2) were co-expressed by secretory cells of the nasal epithelium.
ACE 2 expressions gradually increase from the oral cavity to the esophagus, stomach, and then colon. ACE 2 positive cell ratio in digestive tract organs was significantly higher than in the lung [9]. The nuclei and cytoplasm of the spinous and basal cell layers of the oral epithelium show increased ACE 2 expression. Masticatory mucosa expresses higher levels of ACE 2 [10]. Based on the analysis in mice, ACE 2 was displayed in tongue epithelial cells in non-gustatory papillae but not in taste buds. Contrastingly, a study by Sakaguchi et al. 2020 shows that ACE 2 and TMPRSS 2 are co-expressed in the taste buds of the tongue [7]. The expression of ACE 2 in the taste buds remains unclear, although taste impairment has received particular attention as a symptom of COVID-19. Also, the literature suggests that the squamous epithelium of the vocal cords exhibited extensive staining for ACE 2 [11].
ACE 2 positive cells in salivary glands may be SARS-CoV-2 virus target cells, as RNA expression of ACE 2 genes was found to be high in human salivary glands. ACE 2 was moderately expressed by the serous, glandular cells, ducts, and acini of the salivary glands. Song et al. discovered that the level of the ACE 2 receptor is positively correlated with the level of TMPRSS 2 expression in most organs, including the salivary gland [12]. Intriguingly, higher expression of ACE 2 was recognized in the minor salivary glands than in the lung tissues [12][13][14].
Our subgroup analysis addressing ACE 2 expression levels in head and neck squamous cell carcinoma patients found that ACE 2 expression was at 60% (OR = 0.60, 95% CI, 0.04-9.26) ( Figure 3) shows that it is elevated in carcinoma cells than normal tissues. mRNA expression of ACE 2 was higher in head and neck squamous cell carcinoma patients. ACE 2 expression levels in tumor cells were found to be higher than in normal healthy cells. ACE 2 expression is correlated with the differentiation state of epithelia [6]. Undifferentiated cells express less ACE 2 and well-differentiated cells express more ACE 2.
ACE 2 expression in tissues gradually increased from inflammation to metaplasia and then to cancer. In the tumor microenvironment, ACE 2 hydrolyzes Ang-II to Ang-(1-7), where Ang-(1-7) inhibits tumor growth. ACE 2/Ang-(1-7)/Mas axis counteracts the profibrotic effects of the ACE/Ang II/AT 1R. ACE 2 expression plays a regulating role in the signal transducer and activator of the transcription 1 (STAT 1) pathway. STAT 1 could promote the T-cell immune response and inhibit myeloid-derived suppressed cell aggregation, thus mediating an anti-tumor response. The overall expression of ACE 2 in cancer patients was comparable to that in the normal population, and ACE 2 was slightly upregulated in female and older HNSCC patients [13]. Expression of ACE 2 was positively correlated with the level of immune cell infiltration. ACE 2 positively correlates with the state of epithelial differentiation [14,15]. ACE 2 expression with human leukocyte antigens (HLA), immune in cancer and normal tissues and found that ACE 2 expression was positively correlated with interferon-stimulated gene (ISG) in tumor samples and negatively correlated with angiogenesis and TGF-β [1]. ACE 2 was expressed directly in the immune cells of patients with head and neck cancer. Tobacco smoking induces dose-dependent effects on ACE 2 [16,17]. An experimental study by Wang Z et al. [3] shows that tobacco carcinogens cause degradation, ubiquitination of ACE 2 expression at the protein level, and upregulation of ACE 2 at the mRNA level. This discrepancy in mRNA and protein levels of ACE 2 due to tobacco usage should be investigated [18,19]. In oral epithelial tissue, ACE 2 levels were higher in smokers than in non-smokers, which could indicate the increased susceptibility of smokers to COVID-19 [19]. However, the association between tobacco carcinogens and ACE 2 expression remains controversial.

Limitations
The overall methodology quality of the studies was poor with no single study having a low risk of bias. The included studies were mostly bioinformatic analyzes (n = 10 studies) of ACE 2 using the Human Protein Atlas consortium. An important distinction between the studies is that the techniques used to detect ACE 2 differ significantly, potentially resulting in identification bias between the studies. The detection methods of ACE 2 expression used were as follows: five of the summarized studies (n = 5) used an immunohistochemistry staining technique to detect ACE 2. Ten studies (n = 10) analyzed ACE 2 expression using the RNA sequencing method; of those, four studies (n = 4) used single-cell RNA sequencing and one study (n = 1) used the bulk sequencing method for detecting ACE 2 expression. Two studies (n = 2) used the RT-PCR technique as an add-on analysis. This heterogeneity among the studies can lead to identification bias in analyzing ACE 2 expression, as some methods are more sensitive than others. In addition, the sample size taken in individual studies was limited, and statistical heterogeneity was found between the case and control groups. In the studies, there was a lack of information about demographic variables such as the gender and age of the patients. There were concerns regarding the applicability of the results of individual studies to address the review question. The findings cannot be generalized to the wider population because the studies did not include geographical diversity. Further, more studies should focus on the expression of ACE 2 in the head and neck tissues in a diverse population.

Conclusions
Our review looks at the ACE 2 expression in the head and neck region. The meta-analysis of the studies evinced that ACE 2 is highly expressed in olfactory mucosa, nasopharynx, oral mucosa, salivary glands, and in patients with head and neck cancer. However, the currently available evidence is insufficient to conclude that ACE 2 expression is markedly present all over the head and neck tissues. This emphasizes that succeeding studies should be extended to comprehend the role of ACE 2's molecular mechanism in head and neck-specific tissues and in tumor types. A deeper understanding of the role of ACE 2 in disease and health conditions may provide novel therapeutic targets.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.