Rhonda: Let's move on to the next question, which has to do with vitamin D. The question is, would you shed light on the conversation regarding vitamin D upregulating ACE2 receptors and its influence on susceptibility to COVID-19 infection? This is such an important question, and I'm so glad to get a chance to talk about vitamin D. I think it is a very important topic, and I think there's been some confusion around vitamin D. The first thing to know about vitamin D is that it is not just a vitamin, it's a steroid hormone. Underscoring its importance for health, it actually regulates more than 5% of the protein in coding human genome.

Our biology needs this hormonal signal to know how to function optimally. Period. Immune function is just one important aspect of what is actually a larger conversation around the incredibly broad role of vitamin D. It wouldn't make sense to try to talk about everything there is to know about vitamin D here today. However, some background is useful. So let's dig in. Vitamin D3 is a fat-soluble vitamin that gets converted into a steroid hormone. It is made in the skin upon UVB exposure from the sun. This fact alone leaves many of us vulnerable to at least suboptimal levels. We wear clothes. We wear sunscreen. We may work inside all day. Many aspects of modern life are in a sense at odds with our natural production of vitamin D.

Let's talk about deficiency and insufficiency, how widespread it is, who is likely to have it and how it is defined. According to N. Haynes' data, approximately 70% of the U.S. has what is called vitamin D insufficiency while a further 28.9% has low enough levels to be called deficient. According to the Endocrine Society, blood levels of 25 hydroxyvitamin D below 20 nanograms per milliliter is considered deficient, and less than 30 nanograms per is insufficient. The reason the endocrine society defines vitamin D deficiency as below 20 nanograms per milliliter, by the way, is because this is the cutoff point where parathyroid hormone levels, which are involved in calcium homeostasis start to rise outside of healthy ranges. This point at which the balance of parathyroid hormone begins to shift is the physiological definition for the beginning of vitamin D deficiency. When we look at the demographics, epidemiologists have long known where vitamin D deficiency tends to concentrate and what populations are the most affected. It is the elderly where efficiency of cutaneous biosynthesis of vitamin D declines with age.

According to N. Haynes' data older adults were 63% more likely to have vitamin D deficiency and 46% more likely to have vitamin D insufficiency than young adults while other sources have suggested a 70-year-old may produce four times less vitamin D than their former 20-year-old selves. It is also in the obese where fat-soluble vitamin D has greater difficulty being released into the bloodstream. Obese individuals have greater than 50% less bioavailability of vitamin D compared to non-obese individuals. Obese adults in the U.S. had three times higher prevalence of vitamin D deficiency and 1.9 times higher prevalence of vitamin D insufficiency than non-obese adults. It is also in those living in northern latitudes where less UVB radiation reaching the atmosphere means less of it reaching our skin to facilitate the production of vitamin D. And it is in darker skin people where synthesis of vitamin D is naturally reduced as a biological bargain made by melanin and natural sunscreen, which protects us from the damaging effects of UV radiation.

According to N. Haynes' data, non-Hispanic blacks have 24.6 times higher vitamin D deficiency and 3.7 times higher vitamin D insufficiency than non-Hispanic whites. A recent CDC study of about 1500 hospitalized COVID-19 patients in 14 U.S. States found that 48% of people hospitalized for COVID-19 were obese. COVID-19 hospitalizations were also much higher in people over the age of 65. According to the CDC, 33% of people hospitalized for COVID-19 were African-American who only constitute 13% of the U.S. population. By contrast, this report found that 45% of hospitalizations were among white people who make up 76% of the population in the United States, suggesting that African-Americans may be at a much higher risk for severe COVID-19.

But the epidemiological story doesn't end there. Another paper published in BMJ in March of 2020 reported Somali immigrants living in Stockholm, Sweden make up 40% of the COVID-19-related deaths reported at that time in Sweden. Sweden is a country that is at a northern latitude, which increases the risk of vitamin D deficiency. This rate of hospitalization is really high considering that Somalis only make up 0.84% of the Stockholm County population. Several studies have previously established that Somalis living within Sweden have extreme vitamin D deficiency due to overlapping factors of hereditary and geography. Whether these are just associations or whether vitamin D really does play a crucial role in COVID-19 defense, only time will tell. However, the authors of the study felt the connection strong enough to make the following statement. "In order to cope with the COVID-19 epidemic, preventative measures could be administration of vitamin D to high-risk population. Example, dark-skin adults with low sun exposure, and/or individuals with risk factors for respiratory tract infections. Although it may not always be helpful, it is unlikely to be harmful."

There are mechanistic reasons why vitamin D may be an important part of this COVID-19 story. There is very strong evidence to suggest that vitamin D is protective against respiratory tract infections. There is data from 25 different randomized control trials from around the world showing that daily or weekly supplementation of vitamin D reduced the risk of acute respiratory infection by more than 50% in people with the lowest vitamin D levels at baseline. People that had higher baseline vitamin D levels also benefited. They had a 10% lower risk of acquiring an acute respiratory tract infection. We know that vitamin D plays a very important role in the innate immune response and people with low vitamin D may have a weaker innate immune defense.

So let's talk a little bit about everything from how the ACE2 receptor is used by the SARS-CoV-2 virus to gain entry into the cell to how it is actually very important for protecting against acute lung injury and acute respiratory distress syndrome, which are sort of interchangeably used terms that describe this lung dysfunction, and that's a severe complication associated with COVID-19. So the angiotensin-converting enzyme 2 or ACE2 two acts alongside the angiotensin-converting enzyme ACE to regulate blood pressure, inflammation, and body fluid homeostasis. It's important to keep these two arms, the ACE and the ACE2 of the renin-angiotensin system in balance for it to perform its functions. ACE2 plays a crucial role in the renin-angiotensin system. In preclinical studies, a renin-angiotensin system in balance with higher ACE and lower ACE2 results in atherosclerosis, hypertension, heart failure, chronic kidney disease, serious lung injury. Conditions where ACE2 increases seem to be protective.

So balance is critical in the long where renin-angiotensin system activity and ACE2 expression levels are high. If renin-angiotensin system activity is greatly imbalanced, more severe events may occur. And this happens when ACE2 levels decrease. So the loss of ACE2 function can lead to increased neutrophil infiltration, exaggerated inflammation, and lung injury. Once lung infection leads to hypoxia, renin is released and sets up this vicious cycle for decreasing ACE2 and causing more damage. Acute respiratory distress syndrome, and which is a complication of severe COVID-19 illness and was also a complication of the original SARS is the most severe form of a wide spectrum of pathological processes designated as acute lung injury. So acute respiratory distress syndrome is characterized by pulmonary edema due to increased vascular permeability, the accumulation of inflammatory cells, and severe hypoxia. Acute lung injury results in a very strong downregulation of ACE2 which then causes more severe injury.

So the SARS-CoV-2 virus enters human cells via the ACE2 receptor. Viral particles bind to the ACE2 receptor and together they are internalized into the cell. Numerous viral particles can then bind to ACE2 molecules and sequester the ACE2 molecules from the cell surface. This suggests that a loss of ACE2 function may occur during SARS-CoV-2 infection and can have serious health consequences because of the key role of ACE2 in the rennin-angiotensin system.

This has been shown to occur in the original SARS-1 virus. So SARS-CoV-1, which also enters the cell through the ACE2 receptor, down-regulates or decreases cellular ACE2 expression levels, and this has been shown to cause worst disease severity in the SARS-1 virus. So SARS-CoV-1 also, like I said, bind to the ACE2 receptors and this results in the downregulation of the receptors through binding of the SARS-CoV-1 spike protein to the ACE2. So ACE2 was a key negative regulator for severity of lung edema and acute lung failure. So SARS-CoV-1 spike protein-mediated ACE2 downregulation then contributes to the severity of lung pathologies. So as the SARS-CoV-1 spike protein is binding to the ACE2 receptor, it's resulting in downregulation, and this then makes the lungs pathologies more severe.

So one might look at the isolated fact that SARS-CoV-2 docs on this ACE2 receptor to enter the cell and conclude that it's best to have less ACE2 receptors. But as I just explained, the opposite is true. ACE2 is really key for protection against acute lung injury and acute respiratory distress syndrome. And this has even been shown with the original SARS virus. This is really important because when you draw too broad of conclusions from a very isolated interaction that you kind of pull out from a biological system, it can lead to what seems to be a logically consistent conclusion, but it's actually the wrong interpretation. It's very common and super easy to make that kind of mistake. There's always sort of, you know, lots of compensating factors which is always the case in biology, so you always have to look at the whole context and the interaction of biological systems.

So, again, just as a quick summary, the SARS-CoV-1, and SARS-CoV-2 virus both gain entry to human cells by binding to the ACE2 receptor. It's been shown in the SARS-CoV-1 that the binding of it to the ACE2 receptor results in a downregulation in ACE2 receptors, it results in decreased levels of ACE2, and this exacerbates along injury and leads to worse outcomes. So, again, it's not as simple as more ACE2 receptor bad, less ACE2 receptor good. In fact, it seems to be quite the opposite in terms of this SARS-CoV-1 virus, at least when it's binding to the ACE2 receptor, it's decreasing the ACE2 receptor, it's also decreasing the ACE2 levels, and that is causing worst injury in the lungs because it's dysregulating the rennin-angiotensin system.

So let's talk a little bit about relations that are at risk for decreased ACE2 levels. People with chronic diseases. Age, ACE2 levels decrease with age. And males also have decreased levels of ACE2 compared to females. So the ACE2 gene is located on the X chromosome. And so, women have two X chromosomes, and so they have more copies of the ACE2 gene and therefore higher levels. Interestingly, these seem to be the same populations of people that are at increased risk for COVID-19, people with chronic diseases, elderly people, and even in some cases, some data suggests that males may be more susceptible to severe COVID-19.

So let's talk a little bit about vitamin D and the rennin-angiotensin system. It's very important to reduce the SARS-CoV-2 unbalanced rennin-angiotensin system. SARS-CoV-2 can unbalance the rennin-angiotensin system in the lung. And this may occur the same way that has been shown for SARS-1 via ACE to downregulation, which is followed by inflammation and hypoxia-induced renin release. During a cytokine storm, the renin-angiotensin system is severely disturbed. The purpose of the rennin and angiotensin system is to regulate blood pressure. It does so through a series of enzymes, including ACE2 that involved the kidneys, adrenal glands, lungs, heart, and brain. Vitamin D deficiency leads to overexpression of renin and thus activation of the rennin-angiotensin system, causing renal and cardiovascular injuries. So vitamin D acts as an endocrine repressor of the renin-angiotensin system by down-regulating the expression of renin, the limiting enzyme in the renin-angiotensin cascade.

So in a preclinical model for acute lung injury, when the active form of vitamin D called calcitriol was administered before lung injury in animals, it protected them from acute lung injury by helping to balance the renin-angiotensin system. It did this by increasing ACE2 levels and down-regulating rennin. The increased ACE2 resulted in protection against acute lung injury. It is important to point out that the acute lung injury itself led to a decrease in ACE2, which again, I mentioned has been shown to occur with SARS-CoV-1 infection, and this resulted in worse disease outcome.

The vitamin D increased ACE2 receptor levels only in conditions of acute lung injury, where ACE2 levels decreased. When vitamin D was given to control animals that did not have an induced lung injury, it did not cause or increase ACE2 receptor levels. This means that vitamin D is normalizing the ACE2 receptor levels in situations where it is downregulated or dysregulated. In another preclinical study, it showed that vitamin D increased ACE2 levels in another situation where the rennin-angiotensin system was again dysregulated in rats with diabetic kidney disease. Vitamin D increased ACE2 levels in diabetic rats, and this improved kidney function. It is worth noting that the increase in ACE2 levels in this study was actually soluble ACE2 in the bloodstream and not ACE2 in the lungs. It's possible that ACE2 in the blood could actually be protective against COVID-19 infection because it could bind and sequester viral particles.

In fact, a new study found that human recombinant soluble ACE2 reduce the SARS-CoV-2 infection in engineered human organoid tissues. This is because the soluble ACE2 is thought to bind to the SARS-CoV-2 virus and prevent it from infecting the cells. This recombinant soluble ACE2 is soon to be tested in clinical trials by European biotech company at APEIRON Biologics. This data suggests that vitamin D may play a very important role in protecting against acute respiratory distress syndrome, acute lung injury, and it may do so by regulating the rennin-angiotensin system. In cases where the ACE2 levels are decreased, which has been shown to happen in acute lung injury, it's been shown to happen with SARS-CoV-1, it may then help rebalance that ACE2 level by increasing the levels in that situation.

Let's briefly talk about vitamin D supplementation. Approximately 1,000 IUs of vitamin D generally increases blood levels of 25 hydroxyvitamin D, which is the precursor to the active hormone by around 5 nanograms per mil. There are genetic factors that contribute to this, and some people do it better than others. while the best way to determine how much vitamin D to supplement with is a blood test done at baseline and a month after supplementation, these are unprecedented times in going to a health care setting to try to get a vitamin D blood test can potentially put a person at risk for COVID-19 infection. I currently supplement with 4,000 IUs per day, and my levels are consistently 50 nanograms per milliliter. I do not have any vitamin D related snips known to hamper the production of vitamin D steroid hormone. Vitamin D insufficiency is common, and the more time people spend indoors as during these times, when many are sheltering in place, the more vitamin D insufficiency and deficiency may occur. But it is important to note that it is not good to take too much vitamin D. The Food and Nutrition Board of the Institute of Medicine conservatively set the tolerable upper intake of vitamin D at 4,000 IUs a day for all adults, and that is currently what I am taking.