ACE2 Gender Differences in Stroke With COVID-19

The new coronavirus SARS-CoV-2, causes the COVID-19 infection, which showed a form of neurovirulence involving the Central and peripheral Nervous Systems \[Baig et al, 2020\]. In a mouse model for human ACE2 expression, the virus entered the brain mainly through the olfactory bulb pathway \[Netland et al, 2008\], with an encephalic invasion uniformly lethal even with low viral doses and without lung involvement. The death of the animal was reasonably related to neuronal dysfunction/death in cardiorespiratory bone marrow centers, while the absence of ACE2 prevented severe encephalopathy.

Men has a highly frequency of severe and lethal COVID-19, and the observed gender difference could be related to the regulation of ACE2 receptor expression.

The ACE2 gene is encoded by a region of the X chromosome that escapes inactivation, so that women have an increased expression of this protein. The process of inactivation of the X chromosome includes DNA methylation with a decrease in the expression of genes that are affected by methylation. In This way an epigenetic mechanism could modulate the expression of ACE2 in a gender-specific way determining its levels and consequently its protective role.

Also in this regulatory context of ACE2 expression the role of microRNA (miRNA) could be very important. In fact, the untranslated 3' region (UTR) of ACE2 presents a binding sequence for miRNA miR-200c-3p that has been found at high levels of expression in cellular models infected with H5N1 influenza virus \[Liu et al, 2017\].

In addition, high plasma levels of miR-200c-3p were found in patients with severe pneumonia while ACE2 was reduced suggesting a regulatory role of this miRNA in ACE2 receptor expression \[Liu et al, 2017\]. Deficiency of 25 (OH)D is common among elderly and obese men (during winter and spring), highlighting the sex-specific difference observed in COVID-19 infection \[La Vignera et al, 2020\]. This vitamin, envolved in physical recovery \[Siotto et al, 2019\], and in the pathway of the renin angiotensin system, seems important to be assessed in ex-COVID-19 patients with stroke outcomes in admission and at the end of the rehabilitation process.

The study will consist in:

* Epigenetic study: evaluation of methylation of ACE2 promoter and miR-200c-3p levels.
* Biochemical analysis: the evaluation of levels of angiotensin II, ACE2 and Vitamin D.
* Correlation between rehabilitative outcome and biological markers

A new coronavirus was identified in December 2019 in Wuhan, China as the causative agent of "Severe Acute Respiratory Syndrome" (SARS-CoV-2), a viral lung infection indicated by the acronym COVID-19 (coronavirus disease 2019). By the end of January 2020, this rapidly spreading virus had already infected more than 100,000 people in several countries, leading the World Health Organization to declare a "global emergency" \[Wu et al 2020\]. The clinical manifestations of COVID-19 can vary from the common cold to more serious lung diseases such as those observed in the "Severe Acute Respiratory Syndrome" (SARS) of 2002-2003 and the "Middle East Respiratory Syndrome" (MERS) of 2011.

The Sars-Cov2 virus, like other RNA viruses, also showed a form of neurovirulence with consequent involvement in some patients of the Central Nervous System (CNS) and Peripheral Nervous System (SNP) \[Baig et al, 2020\].

Neurological symptoms in patients with COVID-19 infection fall into three categories:

1. neurological expressions of the symptoms of the underlying disease (headache, dizziness, dysfunction of consciousness, ataxia, epileptic manifestations and stroke)
2. symptoms of neuro-peripheral origin (hypo-ageusia, hyposmia, neuralgia);
3. symptoms of skeletal muscle damage, often associated with liver and kidney damage.

The first data on COVID-19 infection are in favor of neurological involvement in a variable percentage of cases with particular expression in more severe patients \[Mao et al, 2020\]. According to some authors, involvement of the nervous system may be partly responsible for respiratory impairment \[Yan-Chao et al, 2020\].

It should be noted that in the case of SARS-CoV infection, in a mouse model for human ACE2 expression, the virus had entered the brain mainly through the olfactory bulb pathway \[Netland et al, 2008\]. Encephalic invasion was uniformly lethal with further evidence that brain inoculation with low viral doses could be lethal even without lung involvement. The death of the animal was reasonably related to neuronal dysfunction/death in cardiorespiratory bone marrow centers and the histopathological picture was characterized by a minimal cellular infiltrate in the brain supporting the hypothesis of a transsynaptic viral dissemination. The absence of ACE2 prevented severe encephalopathy in the animal model. The structures selectively affected by neuronal death were the dorsal vagal complex (nucleus of the solitary tract, postremactic area, dorsal motor nucleus of the vagus).

On the other hand, trans-nasal invasion selectively interfered with thalamic, hypothalamic, amygdala nuclei. Some affected nuclei had no explanation in the connection (e.g. cochlear nuclei). The invoked mechanism of neuronal loss was that of a "flock" of cytokines (IL-6).

CNS/SNP and muscle involvement is present in COVID-19 patients and a careful interpretation of them is desirable. The hyposmia reported suggests a nasal infection route with direct access to the CNS. This pathway could be alternative to the respiratory and intestinal pathways and theoretically it could occur, as in some cases of SARS-COV, with mainly neurological symptoms.

Recently it has been reported that symptoms of corticospinal tract impairment have been observed in 67% of patients \[Helms et al, 2020\].

The epidemiological data collected so far indicate a substantial difference between men and women in clinical manifestations and SARS-CoV-2 infections. Specifically, a mortality rate among men has been found to be 73% in China \[Chen et al, 2020\], 59% in South Korea \[Korean society of infectious disease, 2020\] and 70% in Italy as reported by the Higher Institute of Health (ISS). In addition, the mortality rate is very dependent on the presence of comorbidities. In fact, in 45000 Chinese patients positive for COVID-19 the mortality rate went from 0.9% in those patients without comorbidity to 10.5%, 7.3% and 6.3% in those with cardiovascular disease, diabetes mellitus and hypertension, respectively \[Novel Coronavirus Pneumonia Emergency Response Epidemiology Team, 2020\].

In Italy, data provided by the ISS have documented a percentage of deaths around 2.1% in patients without comorbidity, a percentage that increases to 21.3%, 25.9% and 50.7% in those patients with one, two and three comorbidities, respectively.

Thus the gender and presence of comorbidities have been identified as key factors in the evolution of COVID-19.

In addition to pre-existing comorbidities, which, as already reported, are almost always present in patients with severe and lethal COVID-19 with greater frequency in men, the biological mechanisms are to be considered the main responsible for the observed gender difference.

A hypothesis that attempts to explain all these epidiemological data is based on the regulation of ACE2 receptor expression (Angiotensin Converting Enzyme 2).

ACE2 is an enzyme that degrades angiontensin II by generating angiotensin (1-7) which plays a protective role against damage caused by infection, inflammation and stress \[Vickers et al, 2002; Zisman et al, 2003\].

The SARS-CoV-2 virus penetrates the target cells of the respiratory system through the binding of its surface S protein (spike protein) to the ACE2 receptor reducing its expression.

In this way there is also a decrease in angiotensin levels (1-7) resulting in increased hypertension and lung failure \[Gurwitz et al, 2020\].

Therefore, it is important to consider the expression of ACE2 in those patients with hypertension, heart disease or diabetes when evaluating the different mortality rate in patients with these comorbidities.

In addition, gender-specific mortality could be precisely related to modulation of ACE2 expression. In fact, estrogens induce an increase in ACE2 receptor expression, suggesting that, at least in women of childbearing age, even after infection, this enzyme is able to perform its protective function, particularly towards the lungs. In men, it seems that androgenic hormones play a pathogenetic role in modulating the expression of cellular enzymes such as serine protease TMPSSR2, involved in the phases following the attack of the virus on the receptor, i.e. in the viral entry, promoting the spread of the infection in lung cells.

The ACE2 gene is encoded by a region of the X chromosome that escapes inactivation, thus supporting the hypothesis of an increased expression of this protein in women who would have the advantage of being protected from the complications and fatalities of COVID-19 infection.

The process of inactivation of the X chromosome includes DNA methylation and as a result there is a decrease in the expression of those genes that are affected by methylation. In This way an epigenetic mechanism could modulate the expression of ACE2 in a gender-specific way determining its levels and consequently its protective role.

Also in this regulatory context of ACE2 expression the role of microRNA (miRNA) could be very important. In fact, the untranslated 3' region (UTR) of ACE2 presents a binding sequence for miRNA miR-200c-3p that has been found at high levels of expression in cellular models infected with H5N1 influenza virus \[Liu et al, 2017\].

In addition, high plasma levels of miR-200c-3p were found in patients with severe pneumonia while ACE2 was reduced suggesting a regulatory role of this miRNA in ACE2 receptor expression \[Liu et al, 2017\].

Vitamin D reduces the risk of viral infections, especially respiratory infections as described in literature \[Martineau et al, 2016; Gruber-Bzura et al, 2018; Gombart et al, 2020; Grant et al, 2020\]. In fact, Vitamin D increases cellular immunity by reducing circulating cytokines induced by the innate immune system in response to viral infections \[Huang et al, 2020\].

Vitamin D deficiency or deficiency contributes to acute respiratory syndrome in which mortality increases with age and chronic comorbidities \[Vásárhelyi et al, 2011\]. This vitamin is a prohormone that has been shown to attenuate acute lipopolysaccharide-induced lung damage in mice by regulating the expression of components of the renin angiotensin system including ACE and ACE2, renin and angiotensin III \[Xu J, 2017; Tsujino et al, 2019\]. In these two months different research groups strongly suggested the need for an analysis on the correlations between vitamin D levels and COVID-19 infections \[Tian et al, 2020; Panarese et al, 2020; Marik et al, 2020\]. Serum concentrations of Vitamin D (25 (OH)D) tend to decrease with age, which may be determinant in COVID-19 infection due to case fatality rates (CFR) that increase with age.

Reasons include less time spent in the sun and reduced vitamin D production as a result of lower levels of 7-dehydrocholesterol in the skin \[Siotto et al, 2019\]. In addition, it has been pointed out that deficiency of 25 (OH)D is particularly common among elderly and obese men (post-menopausal women tend to control levels through Vitamin D supplements) especially during winter and spring, highlighting the sex-specific difference observed in COVID-19 infection \[La Vignera et al, 2020\].

Considering the importance of this vitamin also in physical recovery \[Siotto et al, 2019\], in addition to its role in the pathway of the renin angiotensin system, it seems important to assess serum levels in ex-COVID-19 patients with stroke outcomes in admission and at the end of the rehabilitation process.

In summary, epidemiological data collected in recent months in different countries around the world have shown how gender differences and the presence of comorbidities affect the mortality rate due to COVID-19. Our hypothesis is that biological factors could play an important role in determining the severity of the disease, in particular the ACE2 receptor could be the key element in the development of the differences in gender-related immune response.

Study objectives

Main objectives:

To study the molecular mechanism of regulation of ACE2 in relation to gender, in patients with NeuroCovid19 outcomes and in particular with stroke outcomes in Covid19, hospitalized in 3 rehabilitation facilities.

In particular they will be performed:

* Epigenetic study: evaluation of methylation levels of ACE2 promoter and miR-200c-3p levels.
* Biochemical analysis: the evaluation of serum levels of angiotensin II, ACE2 and Vitamin D.
* Correlation between rehabilitative outcome and biological markers (epigenetic and biochemical) This project will study the molecular mechanisms underlying the regulation of ACE2 related to gender differences in patients post stroke in NeuroCOVID-19 (stroke) hospitalized in Rehabilitation facilities and the relationship between these variables and the rehabilitation outcome.

If the study showed the presence of molecular mechanisms capable of influencing recovery, we could identify rehabilitation pathways more tailored to the characteristics of the patient.

Eligibility Criteria:
Inclusion Criteria:

* stroke patients (hemorrhagic or ischemic) documented through Magnetic Resonance Imaging (MRI) or Computed Tomography (CT);
* NeuroCOVID19 stroke patients with double nasopharyngeal swab negative after 24 hours for SARS-Cov2.
* latency time within 6 months after stroke event;
* sufficient cognitive and language skills to understand the instructions related to the administration of the assessment scales and to sign informed consent;

Exclusion Criteria:

* behavioral and cognitive disorders that may interfere with the therapeutic activity;
* other orthopaedic or neurological complications that may interfere with the rehabilitation protocol;
* inability to understand and sign informed consent;