Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19), predominantly affecting the lungs. The SARS-CoV-2 virus infects cells by using the membrane bound angiotensin converting enzyme 2 (ACE2) as a site of entry into the cell. These have been found in the lungs to be mainly situated on the membranes of type II pneumocytes, the cells responsible for producing pulmonary surfactant. It is possible that COVID-19 patients will have a shortage of effective endogenous pulmonary surfactant (the natural lipoprotein complex which lines the alveoli to primarily reduce surface tension) because the type II pneumocytes are going to be the most compromised by the virus. The administration of exogenous surfactant could help in the management of COVID-19 by replacing the now depleted, endogenous pulmonary surfactant. This review aims to analyse the pathophysiology of COVID-19, its similarities to infant respiratory distress syndrome (IRDS) and the openings for potential exogenous surfactant use in the management of COVID-19 patients.
Importance of surfactant within the lung
A surfactant is a substance which reduces the surface tension of a liquid in which it is dissolved. These are found throughout our everyday lives in soaps, lubricants and emulsifiers and their common goal is normally to reduce the surface tension of water. Pulmonary surfactant is a vital lipoprotein complex produced by type II pneumocytes, found in the lungs on the surface of alveoli. Its main role is to reduce the surface tension at the air-liquid interface. Its composition is very complex and has evolved to have the properties required to be an effective surfactant. The phospholipids, mainly dipalmitoylphosphatidylcholine (DPPC), reduce the surface tension of the air-liquid interface. Not all the phospholipids in the alveolar fluid are selected to enter the air-liquid interface due to the fact that they have a lower compaction capacity as their apolar tails are less bent. This deselection is down to a protein SP-C (surfactant protein C). DPPC may be an incredibly effective surface-tension reducing agent however, without the other components of pulmonary surfactant, it wouldn’t be able to adsorb into the air-liquid interface. This is because the phase transition temperature between gel to liquid of pure DPPC is 41.5ºC which is higher than the human body temperature of 37ºC. The surfactant proteins are responsible for a decrease in this critical temperature to below 37ºC, particularly SP-B (surfactant protein B), which allows for improved adsorption and spreading velocity once it has entered the gas-liquid interface .
The role of surfactant is to reduce the surface tension in the alveoli but it is important to understand why this is necessary. Firstly, it prevents atelectasis (collapsing of the alveoli), particularly at low lung volumes such as at end expiration. This is vital as it maintains a large surface area for the diffusion of gases into and out of the blood. To prevent atelectasis there must be an opposing pressure force acting outwards against the inwards surface tension forces trying to collapse the alveoli. This is expressed by the law of Laplace : , where P is the pressure needed to prevent collapse, Ɣ is the collapsing force of surface tension and r is the radius of the alveoli . In addition to preventing collapse a reduction in surface tension increases lung compliance, the volume change per unit of pressure change. Therefore it allows for the lungs to inflate a lot more easily, reducing the work required for breathing and the pressure differences required to ventilate the lungs . This is significant as when the body becomes weaker and the lungs become fibrotic, the surfactant helps to keep ventilation as easy as possible, although this can’t always be the case. Not only does surface tension attempt to collapse the lung but also it draws in fluid from the capillaries to the alveoli spaces. By reducing surface tension the surfactant reduces fluid accumulation so preventing pulmonary oedema occurring .
Finally, SP-A and SP-D (surfactant proteins A and D) play a major role in the immune function of the lungs. They are collectins which have a carbohydrate recognition domain which binds preferentially to sugars on the surface of pathogens and therefore opsonizes them for uptake by alveolar macrophages. Furthermore, these proteins have been found to regulate inflammatory responses, so by having a decrease in SP-A and SP-D a lung is potentially more susceptible to inflammation and infection .
SARS-CoV-2, COVID-19 and ARDS
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of virus which causes coronavirus disease 2019 (COVID-19). It comes from the coronaviridae family which are characterised by the visible surface projections on an electron micrograph which are reminiscent of a corona (Latin for ‘crown’) . These crowns, called spike (S) glycoproteins, are vital to their survival as they allow binding to host cells which in turn allows for entry into the cells. The viruses first enter our bodies via our mouths, noses and eyes after being in contact with an infected person (usually less than 2 metres) or we pick them up from an infected surface and then touch our mouths, noses or eyes. Even though they can spread throughout the body I will concentrate on their effects in the lungs.
The S glycoproteins of SARS-CoV-2 have a high affinity to angiotensin converting enzyme 2 (ACE2). These enzymes are responsible for lowering blood pressure by catalysing the hydrolysis of angiotensin II to angiotensin-(1-7). Angiotensin-(1-7) is a vasodilator protein compared to angiotensin II which is a vasoconstrictor protein. Angiotensin converting enzyme (ACE) has the opposite effect and provides a target for hypertension treatment in the form of ACE inhibitors. The S proteins of SARS-CoV-2 bind to the ACE2 enzymes, which are membrane bound to the lung/alveolar epithelial cells, due to this affinity to them . Their affinity to the enzymes is estimated to be 10-20 fold greater than SARS-CoV which could provide an explanation for the increased infectivity of SARS-CoV-2 compared to SARS-CoV . Once they have bound to the enzymes they are endocytosed and their single-stranded RNA is injected into the cells, where they ‘hijack’ the cells’ replication systems to reproduce their component proteins and RNA.
Finally, these components are exocytosed as new viral particles . This rapid viral replication can cause massive epithelial and endothelial cell apoptosis and vascular leakage, triggering the release of inflammatory cytokines . This mass, rapid, inflammatory response is very characteristic of acute respiratory distress syndrome (ARDS), the pathological process associated with COVID-19. In addition, non-cardiogenic pulmonary oedema (a buildup of fluid in the alveoli) can occur as a result of the damage to the alveoli and capillary epithelial and endothelial cells, due to the fact that they have become leaky or have died. This further contributes to the severity of the ARDS and combined they contribute to the lack of oxygen that the blood can uptake, leading to hypoxemia .
ACE2 Enzymes : the significance of their distribution
As ACE2 enzymes provide the entry point for the virus to enter our cells it is important when thinking about potential therapeutics to know which cells ACE2 enzymes are situated on and where they are most abundant. This will be the most likely place the virus will ‘attack’ due to the probability being higher that their spike proteins will come into contact with the ACE2 enzymes. To gain an insight into the distribution of ACE2 enzymes in the lung tissues, state-of-the-art single-cell RNA sequencing was used in a study by Tongji University in Shanghai . This allows for analysis of which cells produce the RNA which codes for ACE2 enzymes. They used this technique on 43,134 cells derived from normal lung tissues of 8 adult donors. Even though the number of ACE2 expressing cells was very small, just 0.64%, the majority of these cells (83%) were type II pneumocytes. Other cells which showed a significant amount of expression were, type 1 pneumocytes, endothelial cells, fibroblasts and macrophages. However, the ratio of ACE2 expressing versions of these cells to non ACE2 expressing cells is relatively low compared to type II pneumocytes. After discovering this they went on to perform gene ontology enrichment analysis of ACE2-expressing type II pneumocytes which led them to learn that multiple viral life cycle-related genes are upregulated significantly compared to non ACE2-expressing type II pneumocytes. A couple of these were the endosomal sorting complexes required for transport (ESCRT) machinery genes such as CHMP3 and VPS37B which are related to virus budding and release.
Overall, what we can learn from this study is that type II pneumocytes are the cells that are the most susceptible to infection from SARS-CoV-2 viruses and so are going to be the cells which are affected the most by them. This could provide us with an explanation to why COVID-19 causes severe alveoli damage but minimal upper airway symptoms.
Why exogenous surfactant could have a role in therapeutics
The significance of this distribution in potential therapeutics is huge as now the pharmaceutical companies know what is likely to be the target for the viruses. They can go about either blocking the mechanism of action of entry, or partially replacing the function of the affected cells to mitigate the effects of the loss of function. The role of pulmonary surfactant in potential therapeutics will provide the patient with the latter of the two options. This is because type II pneumocytes are the cells in the alveolar walls that synthesize and secrete pulmonary surfactant. If these are affected severely by the viruses then there will likely be a shortage of pulmonary surfactant. The result of the lack of surfactant may cause atelectasis due to the increased surface tension, a decrease in lung compliance making it a lot harder to ventilate the alveoli properly and an increase in fluid will be drawn into the alveoli (pulmonary oedema) due to the increase in surface tension and damage to the alveolar wall. All these pathophysiological processes have been associated in parts with COVID-19 patients and are characteristic of ARDS.
Lack of surfactant has been seen before in other disease processes, most notably in infant respiratory distress syndrome (IRDS). This occurs in premature neonates who are born before 35 weeks of gestation who can’t ventilate their lungs by themselves enough due to collapsed alveoli and they can’t overcome the surface tension forces, leading to hypoxia. This is because type 2 pneumocytes develop quite late, 26 weeks, and only begin to produce sufficient pulmonary surfactant at 35 weeks. In the treatment of IRDS in neonates, surfactant replacement therapy either from natural sources (lung lavage of other animals) or synthetic surfactants has become routine and successful, so much so that pulmonary surfactant is in the World Health Organization’s List of Essential Medicines.
Comparisons between IRDS and COVID-19/ARDS may lead to the use of similar treatment strategies such as the administration of exogenous surfactant. The pathophysiology in parts is very similar, the type II pneumocytes are compromised leading to insufficient surfactant production. Also, the imbalance in the number of boys and girls who suffer from IRDS is reciprocated in the number of males and females that suffer severely from COVID-19 due to this insufficient surfactant production. In IRDS this is because the lecithin-sphingomyelin ratio, a measure of lung maturity by assessing the ratio of certain components of surfactants, in girls reaches 2:1 (a mature enough level) on average at 33.7 weeks which is 1.4 weeks earlier than in boys . This has been shown to be because androgens inhibit the maturity process and oestrogen stimulates the process of maturation and surfactant production [14,15,16,17]. In comparison to this it has been noted that deaths due to COVID-19 are more common in males, the death rate in confirmed cases at the time of writing this is 4.7% compared to 2.8% in females, a ratio of 1.68:1. In a study by the Federal University of Espirito Santo, it was shown that sex hormones played a major role in ACE2 expression . When the female rats in question underwent an ovariectomy there was a significant increase in ACE2 expression, from 0.06±0.002 AFU/µg to 0.09±0.001 AFU/µg. Conversely, an orchiectomy in male rats significantly reduced the expression of ACE2, from 0.14±0.002 AFU/µg before to 0.01±0.003 AFU/µg after. This shows that oestrogen has a down regulating effect on the expression of ACE2 compared to testosterone which had an upregulating effect. This could provide an explanation to the reason for men suffering worse from COVID-19 because there are more entry points for the virus so more cells are going to be affected. There are going to be more type II pneumocytes affected so the production of pulmonary surfactant is going to be reduced more and there will likely be more inflammation and damage to the alveolar walls.
Advancements in surfactant technology and the potential for novel uses in therapy
Even with their success in neonates, there are limitations to the administration of exogenous surfactants, some of which have been eliminated over time but some are still relevant today. Even though lung lavage surfactant from animals provides the surfactant with the most resemblance to endogenous surfactant, its scale-up ability, purity and quality control are all lacking. However, they can be used on a local scale in neonates. To get around this issue pharmaceutical companies designed synthetic surfactants with lipids that resembled those in the human lungs. These were protein free at first (ALEC and colfosceril palmitate), so their efficacy was quite limited leading to them no longer being used clinically . Therefore they set about engineering proteins that had a strong resemblance to SP-B, the hydrophobic protein that is responsible for the improved adsorption of the lipids into the air-liquid interface .
They engineered a protein called KL4 which is a protein only 21 amino acids long but had the necessary properties to mimic the C-terminal helical domain (the active part) of SP-B. The complete primary structure of the protein was : KLLLLKLLLLKLLLLKLLLLK, where K represents lysine and L represents leucine. Lysine is positively charged so the overall protein charge is positive and leucine is hydrophobic so gives the protein its periodic hydrophobicity, both of which are important characteristics of SP-B [20,21]. When the KL4 protein was added to DPPC, other phospholipids and palmitic acid, a much more successful drug came about, lucinactant (Surfaxin). It is administered as an intratracheal suspension when treating IRDS and has shown considerable success. The mortality rate of a trial in which 124 infants born between 24 and 28 weeks, when treated with Surfaxin, was 11.8% through day 28 and at 36 weeks gestational age was 16% . This is considerably lower than between 1987 and 1993, when pulmonary surfactant therapy wasn’t widely available, in a study in Karachi where there was a 40.5% mortality rate in neonates with IRDS . So if there has been a significant decrease in mortality in IRDS why can’t surfactant therapy be used in ARDS or ARDS induced by COVID-19?
There have been many trials on the use of surfactants in treatment of ARDS, however many of these have proven disappointing, showing no significant decrease in ventilator free days and similarly no significant decrease in mortality rates [24,25]. Although this seems discouraging some have hypothesized that there are plausible reasons for these results in the treatment of ARDS. The main reason for this is thought to be that it isn’t administered in high enough quantities and therefore doesn’t reach all the necessary parts of the lung, particularly the terminal bronchioles and alveoli . The disparity between infants and adults is easily explained as adult lungs are vastly larger, they have double the alveoli (300 million roughly) and twenty times the surface area of infant lungs . Therefore a more efficient method of delivery is necessary to reach all parts of the lungs, this is where Aerosurf comes in.
Aerosurf is an aerosolized KL4-surfactant (Lucinactant) which promises to be efficient at spreading throughout the lungs. It is currently undergoing clinical trials in neonates and has been encouraging so far . A big benefit in neonates is that it can be administered non-invasively, reducing the potential complications which arise from intubation. Windtree Therapeutics, the manufacturers of Aerosurf, have suggested that it would like to trial it for use in patients suffering from COVID-19 . They believe that it may help because the mechanism of injury leading to COVID-19 induced ARDS is different from other known aetiologies of ARDS. They believe that their surfactant therapy could decrease lung inflammation and improve lung function such that it will reduce the need for mechanical ventilation and hopefully a reduction in mortality.
Exogenous pulmonary surfactant may not be the ultimate cure for COVID-19, but it could potentially be used to mitigate the impact of the disease and reduce the need for invasive ventilation. This is because it could increase lung compliance such that patients could ventilate their lungs by themselves. Exogenous pulmonary surfactant may also have potential benefits for patients who require mechanical ventilation. When someone is ventilated all the work of breathing is taken up by the ventilator meaning you are reliant on it. This means that if the pressures produced are too high or too low there can be serious consequences in the form of ventilator induced lung injury (VILI). If the positive end-expiratory pressure (PEEP) is too high in an attempt to reduce atelectasis there is a risk of bursting the alveoli, they are already weak so this is a very real possibility. Then the air can leak into the pleural cavity, causing a pneumothorax which can lead to the collapse of a lung. With surfactant therapy, the need for such high PEEPs could be reduced as the surfactant will reduce the pressure needed to prevent collapse, Laplace’s law .
At the other end of the spectrum a too low PEEP can also cause VILI. This is due to the terminal bronchioles and alveoli collapsing at end expiration due to the PEEP being below that determined by Laplace’s law and then during inspiration the airways being forced open again. During the reopening, it has been shown in a study from Tulane University in New Orleans that the epithelial cells undergo a stress cycle as the air bubble moves past them causing in some cases irreparable damage to the cell membranes . This was done by recording the pressures felt by the cells during one cycle of reopening, then the cells were stained with Eth-1. The Eth-1 excludes cells with an intact cell membrane as it travels to the nucleus of those without an intact cell membrane and binds to the DNA, producing a fluorescent nucleus. Cellular injury was defined by a fluorescent, red nucleus. When the epithelial cells where in a saline solution the number of injured cells was 39.80±8.76✕103 injured cells/cm3 which reduced to 0.80±0.17✕103 injured cells/cm3 when 1mg/ml Infasurf (an FDA approved cow lavage surfactant) was added to the solution. This shows the extent to which surfactant could reduce cellular damage during ventilation, which could further complicate the situation due to the increased inflammation. Therefore lower PEEPs could be used with more confidence that VILI or atelectasis won’t occur.
Pulmonary surfactant deficiency or inactivity has proven to play a role in the pathophysiology of COVID-19 and IRDS. The increased mortality in males may be explained by the higher levels of ACE2 expression seen in males, which may lead to a higher susceptibility to infection from SARS-CoV-2. Even though surfactant therapy has proven disappointing in treatment of ARDS, promising advancements in the method of delivery could potentially improve its efficacy. The mechanism of injury of COVID-19 differentiates it from other known aetiologies of ARDS, leading to the potential role for surfactant therapy. Even if it does not act as a cure, its role as a therapy to mitigate the effects of mechanical ventilation and possible VILI shouldn’t be overlooked, possibly providing more ventilator free days. Exogenous pulmonary surfactant therapy may have a potential role in the management of COVID-19 which needs further investigation.
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Robbie is a 16 year old pupil at The Portsmouth Grammar School who is interested in medicine and the science surrounding it. He is currently working towards his A-Levels and hopes to study medicine at university in the future.