On February 23, 2021, Dr Francis Collins, director of the National Institute of Health (NIH) of the USA, announced that a major study was being launched “to identify the causes and ultimately the means of prevention and treatment of individuals who have been sickened by Covid-19, but don’t recover fully over a period of a few weeks”. He elaborated that “large numbers of patients ...continue to experience a constellation of symptoms....often referred to as ‘Long Covid’, [that] can persist for months and can range from mild to incapacitating”. Coming at a time when several variants of the original pathogen are spreading, and a new surge of cases indicates that India is heading straight for a ‘second wave’, the sombre words of the scientist reflect the grim nature of the long struggle ahead. Of course, while fresh science will continue to illuminate our battle, studies over the last year have already provided some answers as to why Covid-19’s arsenal is far more dangerous than those of ‘regular’ flu and cold and how co-morbidities and environmental hazards aggravate its dangers.
Connecting the observations
1. It is common knowledge by now that the novel coronavirus, technically named SARS-CoV-2, enters our cells using one of our own proteins: Angiotensin Converting Enzyme 2 (ACE2). The Spike protein of the virus is a ‘key’ that snugly fits onto the ‘lock-like’ ACE2 that projects out from the membrane of cells of the nasopharynx, trachea, bronchi and the lungs. This kickstarts a process that produces many progeny viruses inside the infected cell and spreads the pathogen to a widening circle of neighbouring tissues, resulting in disease. Of course, the physiological function of ACE2 is not to facilitate the entry of the virus. So what is the function of ACE2 ? How is it related to the thousands of deaths in this pandemic?
2. Worldwide, it is clear that Covid-19 is not a mere respiratory disease. Although its primary attack is on the airways and lungs, undeniable evidence now points towards a correlation between Covid-19 and cardiovascular diseases. Pre-existing co-morbidities like hypertension (i.e. chronically high blood pressure), coronary artery disease, cardiomyopathy (i.e. damage to heart muscles) and atrial fibrillation can badly increase the severity of Covid-19. The infection has been shown to trigger myocardial injury, cardiac arrhythmia, acute heart failure and thromboembolism (i.e. clots within blood vessels) in many patients. Similarly, diabetes mellitus is a critical co-morbidity that could badly affect a patient and acute kidney injury is not uncommon. Question is: how is the virus attacking non-respiratory organs?
3. The alarming effect of air pollution. Italy was one of the first nations that was ravaged by the pandemic. Now, epidemiological studies have clearly shown that it was the highly polluted cities in the Po valley and Lombardia region of northern Italy that had the highest number of Covid-19 fatalities, correlating high levels of air pollutants like PM2.5 and NO2 to the severity of the disease. Another major study compiled data from >3,000 counties in the US and concluded, “an increase of 1 ðÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂÂg/m3 in long-term average PM2.5 is associated with a statistically significant 11% increase in the county’s Covid-19 mortality rate”. But how do the pollutants help the virus to overwhelm the body’s defences? Importantly, are all the above observations interconnected by fundamental physiological mechanisms?
The actual function of ACE2
To answer the first question, ACE2 is part of a team of biomolecules that constitute the Renin-Angiotensin-Aldosterone System (RAAS). Rather unknown beyond expert circles, the RAAS is one of the pivotal systems that work literally from head to toe. It maintains health by a variety of ways, and its dysregulation invariably leads to systemic diseases. And, at the heart of RAAS is the molecule Angiotensin II.
What is Angiotensin II? Where does it come from? What does it do?
Briefly, whenever blood pressure falls below optimum or extracellular fluid volume is decreased, special ‘baroreceptor cells’ in the kidneys sense this and initiate a biochemical process that results in production of the small protein named Angiotensin II. Although puny in size, Angiotensin II has various functions. It increases the constriction of blood vessels and stimulates the brain to suggest to the body that water intake be increased. It also makes the Adrenal Cortex secrete the hormone Aldosterone which, in turn, instructs the kidneys to reduce loss of sodium and water from the body. Taken together, the actions of Angiotensin II reduce loss of fluid, increase gain of fluid, raise the tonicity of blood vessels and optimise blood pressure.
In recent years, it has been realised that Angiotensin II has other physiological roles. It is a strong controller of inflammation, the body’s non-specific immune response against any external agent (e.g. microbes, pollutants) that can cause damage to tissues. In such situations, Angiotensin II induces several changes at the inflamed tissue and activates ‘soldier cells’ like neutrophils and macrophages to produce Reactive Oxygen Species (ROS) that will destroy the incoming ‘foreigners’.
Not surprisingly, Angiotensin II’s supreme importance also means that any unregulated increase of its activity is akin to ‘friendly fire’. Indeed, chronically high levels of Angiotensin II leads to sustained hypertension, excessive inflammation, coronary artery disease, arrhythmias, fibrosis (i.e. build-up of collagen fibers between cardiac muscle cells) and increased chances of thrombosis (i.e. clot formation within blood vessels). Excess of Angiotensin II can cause edema in the lungs and acute respiratory distress syndrome (ARDS). Similarly, dysregulated RAAS is seen in diabetes mellitus and can lead to problems of the intestine. Drugs like Losartan control blood pressure by reducing the effect of Angiotensin II.
Now, ACE2 is a counter-balance to Angiotensin II within the RAAS system. How does it work ACE2 is a protein present on the membranes of cells of the lungs, the cells that line the airways from nasopharynx to the lower bronchioles, the endothelial cells that line small and big blood vessels, several cells of the heart, kidneys, testes, and the enterocytes of the small intestine. Its function is to cut Angiotensin II to produce an even smaller molecule called Ang(1-7). This has two major effects. One, the levels of Angiotensin II in blood get reduced. Two, most actions of Ang(1-7) are the opposite of Angiotensin II. Ang(1-7) induces vasodilation and urination, thus helping lower blood pressure. It is anti-fibrotic, anti-thrombotic, anti-arrhythmogenic—all of which protects cardiac tissue from damage. The kidneys too are protected from the stress of unbridled Angiotensin II by Ang(1-7). Additionally, Ang(1-7) reduces the severity of inflammation and thus protects cells of the pulmonary alveoli. In experimental animals, Ang(1-7), which as we noted is produced via the actions of ACE2, shields against acute lung injury. And that’s why a fall of ACE2 activity promotes Chronic Obstructive Pulmonary Disease (COPD) and ARDS. In summary, the fine balance between Angiotensin II and ACE2 is a crucial parameter for healthy life.
The ups and downs of ACE2
Now, ACE2 levels are not constant; it can vary. Notably, the number of ACE2 molecules increases in the respiratory tract cells of COPD patients. Also, cigarette smoke induces inflammation in the lungs as a protective response against the irritants and pollutants present in the smoke. However, since unrestricted inflammation can damage the lungs, the cells also “increase the expression of ACE2 in the Mammalian Respiratory Tract” to prevent ‘same-side goals’.
In the laboratory, scientists have induced inflammation in the airways of animals by exposing them to air pollutants like PM1 and PM2.5 . This increased Angiotensin II activity which, in turn, raised the levels of pro-inflammatory cytokines like IL-6, TNF-α and TGF-β (cytokines are signalling molecules that help to communicate and coordinate between various cells). Of course, excessive chronic inflammation led to lung damage and also caused “upregulation of its protective mechanisms, such as ACE2”. Thus, the animals suffered nasty lung damage but recovered. However, when a genetic engineering technique called knockout was used to remove the ACE2 gene (ACE2 is a protein and like every protein it is coded by a gene present in one of our chromosomes), then the pulmonary damage was far more severe. Such studies highlight the protective role of ACE2.
ACE2 also protects another organ anatomically close to the lungs—the heart. Chronically high Angiotensin II triggers faulty activation of the immune system, causing inflammation, sustained high blood pressure, cardiac hypertrophy and fibrosis. The hyper-activation of ROS pathways damages cellular proteins, membranes and DNA leading to high levels of fats in blood and atherosclerosis. Moreover, since blood from lungs directly travels to the heart, air pollutants can trigger unsolicited inflammation and damage in cardiac tissue.
The anti-inflammatory roles of ACE2 provide a big antidote. Several cardiac cells, such as the pericytes and the endothelial cells lining the cardiac arteries and capillaries, produce a lot of ACE2 and its level is significantly elevated in patients of cardiomyopathy and myocardial infarction—certainly to protect them from the deleterious fluctuations of Angiotensin II. Ang(1-7), the product of ACE2, maintains proper coronary blood flow and reduces cardiac arrhythmia. This explains why inactivation of ACE2 in laboratory animals leads to severe systolic impairment while increasing ACE2 levels protects against excessive rise of blood pressure. ACE2 also protects the kidneys by regulating the levels of Angiotensin II. To sum it up, the cardio-respiratory system boosts its ACE2 regiment to protect against pre-existing co-morbidities as well as external pollutants.
What happens when the coronavirus finds individuals whose lungs and heart have higher levels of ACE2 because of existing diseases and chronic exposure to air pollutants?
It is quite possible that SARS-CoV-2 finds it easier to infect such people because their cells have more ACE2 ‘docking sites’. This could (at least partially) explain why hypertension, cardiovascular ailments, COPD, smoking—all of which can raise ACE2 levels—are recognised risk factors in Covid-19. It also partially explains why so many deaths and hospitalisations have happened in the most polluted of cities. Remarkably, a recent study (spearheaded by two Indian scientists) actually shows that respiratory airway cells pre-exposed to even low doses of cigarette smoke are more susceptible to infection by SARS-CoV-2, providing a good explanation about why smokers are more likely to develop severe Covid-19. Taken together, the scientific evidence indicates that while heightened levels of ACE2 helps in combating several cardio-respiratory co-morbidities and air pollution, it becomes a Trojan horse facilitating the entry of SARS-CoV-2 into airway cells. In other words, the coronavirus is using our own shield against us!
Making the lungs gasp
Unfortunately, this is not the end of our molecular miseries. When the viral spike protein latches onto the ACE2 structure, it activates a few other proteins. This, in turn, leads to receptor-mediated endocytosis, a cellular process where the cell membrane neighbouring the ACE2+virus complex buds inwards (i.e. into the cell) and then pinches off, forming a virus-containing vesicle inside the new cell! This transports the virus into the cell, but it’s not only the virus that enters this way; the ACE2 also comes inside. Moreover, endocytosis also switches on a molecular process that further reduces the presence of ACE2 on the cell membrane. So, at one level, the presence of more ACE2 on cell membranes favours the easier entry of SARS-CoV-2. And then, as the infection spreads, ACE2 is increasingly lost from the respiratory system’s surface, which probably decreases the protection ACE2 usually provides.
The body does try to fight back the viral invasion and Covid-19 seems to be a mild disease in most infected people. However, the loss of ACE2 shifts the balance towards higher and uncontrolled Angiotensin II and can make things go haywire. Indeed, higher levels of Angiotensin II increases vascular permeability in the lungs of many Covid-19 patients. The unbridled inflammation and oxidative stress (i.e. damage due to uncontrolled production of ROS) induced by hyperactive Angiotensin II can also fuel a vicious loop of damage to alveolar cells and pulmonary capillaries, hyperplasia (i.e. pathological increase of cell size) of the alveolar cells, formation of aberrant hyaline membranes and thick fibrosis in the damaged zones. WBCs crowd at these damaged sites, leading to more inflammation and further tissue damage. The clinical result is pulmonary edema and ARDS, a common state in Covid-19 emergencies.
The matter of the heart
Cardiac tissue can be subjected to a similar battering. Up to 28% of hospitalised cases suffer cardiac injury. SARS-CoV-2 could invade pericytes and the endothelial cells of capillaries in the heart, leading to micro-circulation disorders. Increased myocardial fibrosis, myocarditis (i.e. inflammation of heart muscles), cardiac arrhythmias, acute cardiac injury and cell death are not uncommon. Furthermore, the downregulation of ACE2 unleashes inflammation which, in turn, raises blood pressure—an extra load on the already-weakened cardiac system. Heart diseases that were under control can suddenly become unmanageable post-Covid. As the infection radiates out, the virus directly targets the kidney, especially the specialised cells of the renal tubular epithelium. The renal blood vessels are also attacked, and this can induce Acute Kidney Injury. There is also growing evidence that the testes are affected and a link with brain damage is being reported. Unless stopped, Covid-19 rages towards multi-organ damage.
Blockade of the blood vessels
Perhaps the most baffling and spooky thing about Covid-19 is that patients recover from the infection but then succumb to a heart attack, stroke or renal failure. Even young people, with little symptoms of the infection, have died rather inexplicably. Autopsy reports show that many of the deceased had thrombosis inside smaller blood vessels. The clot (thrombus) blocks blood supply, resulting in tissue damage and death. In addition, clots formed in deep veins of the lower limbs can dislodge from their original site, travel in the bloodstream and occlude a vessel supplying blood to the lungs.
Besides the lungs, such thrombi have been observed in the heart, kidneys and liver of Covid-19 patients. Over 20-30% of critically ill Covid-19 patients have thrombosis during the infection and such Deep Vein Thrombosis, coupled with Pulmonary Embolism, seems to be a ‘serial killer’ in Long Covid complications.
But how is a respiratory virus messing up with the physiological mechanism of blood coagulation?
Before venturing to understand this pathology, a primer to blood clotting is necessary. Briefly, the clotting of blood is a ‘cascade pathway’ that involves a set of at least 15 proteins present in the blood plasma, inside platelets and on the endothelial cells that make up the walls of our blood vessels—called ‘clotting factors’, these include fibrinogen and thrombin. Ideally, blood clotting happens only when a blood vessel is damaged. But, some diseased conditions such as atherosclerosis can initiate aberrant clotting inside blood vessels that supply key organs, leading to heart attack or stroke. SARS-CoV-2 does the same. How? The crosstalk between our biomolecular pathways can overwhelm even professional scientists and we run the risk of over-simplifying a complex scenario. But again, the dysregulated RAAS seems to be the major mischief-maker.
Earlier studies have established that clots (thrombi) are formed not only by the clotting factors and platelets. Inflammation has decisive roles; unbridled inflammation of venous walls launches many thrombus formations. This is where Angiotensin II steps in. ACE2 is anti-thrombotic. But when SARS-CoV-2 infection lowers the ACE2 level of endothelial cells, the balance gets tipped in favour of Angiotensin II. An increase in Angiotensin II levels not only accelerates the clotting cascade, its actions on the endothelial cells and smooth muscle cells that line the blood vessels promote inflammation. WBCs are recruited to the inflammation zone, and a web of pro-inflammatory cytokines, damaged alveolar and endothelial cells and the activated WBCs keeps adding to the danger. The platelets get hyperactivated here and, recent findings show that they can further activate the WBCs. The net result is thromboinflammation—a massive inflammation and hypercoagulable state that predisposes patients towards one or more thromboses. Consequently, many emergency cases show a parallel rise of inflammatory molecules (e.g., CRP, IL-6) and abnormal clotting parameters (such as the D-dimer). This makes thromboinflammation “a major cause of morbidity and mortality in patients with Covid-19”.
Notably, our platelets seem to have a pivotal role in this physiological tragedy. Thrombocytopathy (i.e. dysfunction of platelets) is now recognised as a prominent feature of Covid-19. Both thrombocytopenia (i.e. fall in platelet numbers) and hyperactivity of platelets has been noted in patients. For example, endothelial cells and platelets do not interact in healthy people. But, during SARS-CoV-2 infection, hyperactivated platelets and damaged endothelial cells do bind to each other from the early stages of thromboinflammation onward.
What activates the platelets to this extent is still a mystery, but hypoxia (i.e. reduced oxygen supply) and oxidative stress could be factors. It is known that Covid-19 can cause hypoxia with oxygen saturation level falling to <94% (aren't oxymeters in high demand nowadays?) and that critical cases suffer from ARDS and need ventilator support. Is it hypoxia that triggers the platelets towards thrombus formation? There are other factors too. For instance, many patients generate antiphospholipid autoantibodies which target not the virus but platelets and endothelial cells, and thus predispose the patient towards clot formations—another dangerous ‘same-side goal’!
The danger of co-morbidities. The natural wear ’n tear of aging reduces the efficiency of our endothelial cells. This leads to failure of cellular antioxidant mechanisms, and a consequent rise of ROS and tissue damage and inflammation. Both diabetes and obesity are now known to involve excess inflammation and oxidative damage. When the virus infects a body and spreads, the lowered activity of ACE2 and increased level of Angiotensin-II dangerously adds to this already-risky situation, setting up disastrous thromboses. If unchecked, blood supply to vital organs begins to get cut off. The result could be fatal multi-organ failure or the crippling damages of Long Covid.
We, the people... What should particularly bother us is that a large number of Indians, irrespective of region, age, gender and class, suffer from diabetes and hypertension. Obesity is also on the rise. There are millions of Indians over 65 years old and the air of our densely packed cities is full of choking pollutants. Is it any wonder that the elderly S.P. Balasubrahmanyam, Soumitra Chatterjee, Tarun Gogoi are among the 1,57,000-odd Indians who have succumbed? And that for every one of them lost to Covid-19, there are several others trying to make a slow and exhausting recovery? As positive cases wax and wane in a seemingly patternless way and some cities are under stringent regulations, it is clear that the battle of the pandemic will extend well into this year.
(Anirban Mitra is a teacher of molecular biology and biotechnology, based in Calcutta.)
