RGD‐binding integrins and TGF‐β in SARS‐CoV‐2 infections – novel targets to treat COVID‐19 patients?

Abstract

The new coronavirus SARS‐CoV‐2 is a global pandemic and a severe public health crisis. SARS‐CoV‐2 is highly contagious and shows high mortality rates, especially in elderly and patients with pre‐existing medical conditions. At the current stage, no effective drugs are available to treat these patients. In this review, we analyse the rationale of targeting RGD‐binding integrins to potentially inhibit viral cell infection and to block TGF‐β activation, which is involved in the severity of several human pathologies, including the complications of severe COVID‐19 cases. Furthermore, we demonstrate the correlation between ACE2 and TGF‐β expression and the possible consequences for severe COVID‐19 infections. Finally, we list approved drugs or drugs in clinical trials for other diseases that also target the RGD‐binding integrins or TGF‐β. These drugs have already shown a good safety profile and, therefore, can be faster brought into a trial to treat COVID‐19 patients.

SARS‐CoV‐2 is highly contagious and shows high mortality rates especially in elderly and patients with pre‐existing medical conditions. In this review, we explain the rationale of targeting RGD‐binding integrins to potentially inhibit viral cell infection and to block TGF‐b activation, which is involved in the severity of several human pathologies, including the complications of severe COVID‐19 cases.

Introduction

The new coronovirus SARS‐CoV‐2 has become a public health challenge worldwide, declared pandemic in March 2020, with millions of affected patients. Dr Marc Lipsitch from Harvard University cautioned that 40–70% of the human population will become infected if no actions are taken. Moreover, it is estimated that about 14% of the patients will develop serious conditions requiring hospitalisation, and approximately 1.4–3.4% will die from this infection, putting an unprecedented strain on healthcare systems. ¹ , ² The leading causes of deaths are acute respiratory distress syndrome (ARDS), septic shock, haemorrhage/coagulopathy, acute heart, liver, kidney injury and secondary bacterial infections. ² Currently, there are no medications approved to treat this virus. A recent study demonstrated that the most promising drugs (remdesivir, hydroxychloroquine, lopinavir and interferon) appeared to have little or no effect on hospitalised COVID‐19 patients. ³ Some vaccines have finished phase III trials with a good safety profile and more than 90% efficacy (press release websites of Biontech/Pfizer and Moderna). However, as Dr Anthony Fauci and others have estimated, 70% of the world population will need to receive the vaccine to achieve herd immunity, and this will take time. Furthermore, it is not clear whether the vaccine will protect the most vulnerable people; for example, it has been reported that vaccines in elderly population have a lower efficacy to build an immune memory. Therefore, there is still an urgent need to treat these patients with known approved drugs or drugs that are further along the development pipeline.

SARS‐CoV‐2 infects cells via its spike protein, which binds the ACE2 receptor on target cells. The virus enters the cells after the proteolytic cleavage of the spike protein by the transmembrane protease TMPRSS2. Other receptors, for example CD147 and CD26 (DPP4), have also been proposed to be a potential entry point of the virus. ⁴ , ⁵ However, no direct binding activity of CD147 to spike has been reported. ⁶ For CD26, which is the receptor of MERS (Middle East respiratory syndrome) to infect cells, a model of docking analysis predicted a binding of CD26 to the spike protein, but experimental data still need to confirm this bioinformatic approach. ⁵ We reviewed a novel mutation (K403R) in the spike protein that does not exist in other strains of the coronavirus (Figure 1). ⁷ , ⁸ This mutation creates an RGD motif, which could be recognised by integrins. Integrins are cell adhesion receptors that play important roles during pathological processes. Eight out of 24 known integrins recognise the RGD sequence in the natural ligands. ⁹ The RGD is also a common motif for other types of viruses to infect cells (e.g. Epstein–Barr virus, rotavirus, human cytomegalovirus, Ebola). ¹⁰ Therefore, the new RGD motif in SARS‐CoV‐2 could increase the binding potency of ACE2‐positive target cells as well as infecting ACE2‐negative cells. This could also explain why the virus spreads faster and more aggressively than SARS‐CoV‐1, which belongs to the same family of coronaviruses. However, further studies are required to evaluate this hypothesis. SARS‐CoV‐1 was declared an epidemic in 2003 and showed higher fatality rates, but lower infection rates than SARS‐CoV‐2.

Figure 1.

Schematic representation of SARS‐CoV‐2 S‐protein with a focus on the receptor‐binding domain. The sequences of the spike protein of human SARS‐CoV‐2, human SARS‐CoV‐1 (75% similarity) and bat RaTG13 (96% similarity) were aligned using MAFFT. ¹⁷⁶ The receptor‐binding domain and the ACE2 receptor‐binding region are coloured in light blue. The RGD motif of SARS‐CoV‐2 is highlighted in red. Numbers refer to the SARS‐CoV‐2 spike protein sequence. FP, fusion peptide; RGD, amino acids arginine‐glycine‐aspartic acid; S1, subunit of spike; S2, subunit of spike (fusion domain); S2´, cleavage site; TM, transmembrane domain.

RGD‐binding integrins are the main regulator for the activation of transforming growth factor beta (TGF‐β). ¹¹ TGF‐β plays an important role in many of the observed complications of severe COVID‐19 patients (discussed below). Furthermore, TGF‐β is increased in patients with pre‐existing medical conditions (discussed below). In this review, we are discussing the rationale of using integrin inhibitors as potential treatment of COVID‐19 patients.

RGD‐binding integrins for SARS‐CoV‐2 cell infection

Several viruses are known to use a RGD motif to bind to the surface of cells, which is crucial for a successful infection. ¹⁰ For example, the West Nile virus uses the integrins αvβ1 and αvβ3 for cell entry, ¹² , ¹³ Ebola uses the integrin α5β1, ¹⁴ and the Herpes simplex virus type 1 (HSV‐1) interacts with αvβ3. ¹⁵ A more detailed list of viruses using the RGD motif for cell entry is reviewed in Hussein et al. ¹⁰ Therefore, the RGD motif in the Spike protein of SARS‐CoV‐2 (Figure 1) could be critical in infecting cells through the RGD‐binding integrins. Others have already suggested that the RGD motif of the Spike protein may enhance infection efficiency. ⁷ , ¹⁶ , ¹⁷ A recent study further supports the hypothesis that SARS‐CoV‐2 can infect target cells via RGD‐binding integrins. ¹⁸ The group used different culture conditions to induce ACE2 expression. They observed that ACE2 was virtually absent in human small intestinal organoids (hSIOs) cells grown in expansion medium and increases dramatically when hSIOs cells were grown in differentiation medium with or without BMP (bone morphogenetic protein) stimulation; nevertheless, both showed similar infection rates. The group suggests that a low level of ACE2 might be sufficient for viral entry. However, the ACE2⁺ and TMPRSS2⁺ club cells, which are found in the airway epithelium and are the progenitor cells of the trachea and the bronchiolar region, were neither infected in vitro nor in vivo with Sars‐CoV‐2. ¹⁹ Additionally, cell lines that are used for analysing SARS‐CoV‐2 entry via ACE2, for example the monkey kidney epithelial cell line VERO E6, are also expressing RGD‐binding integrins. For VERO E6, α5β 1, αvβ 1 and αvβ 3 have been detected. ²⁰ A recent study showed that the spike protein of SARS‐CoV‐2 binds to both α5β1 and α5β1/hACE2 on VERO E6 cells. ²¹ Adding the α5β 1 integrin inhibitor ATN‐161, which is in a phase II clinical trial for renal cancer, disrupts SARS‐CoV‐2 infection. Furthermore, pretreating VERO E6 cells with ATN‐161 before adding SARS‐CoV‐2 increased cell viability and decreased cytopathic effects associated with viral infection. A non‐peer review published result by the group of Dr Jenkins (University of Nottingham, UK) showed in a solid‐phase binding assay that the spike S1 subunit potentially also binds αvβ 1, αvβ 5, αvβ 6 and αvβ 8. ²² The binding appears to be 10² times lower than the binding to ACE2. However, RGD specificity still needs to be confirmed and whether this potentially weaker interaction makes the integrins a less promising target needs further evaluation. Interestingly, it has been shown that SARS‐CoV‐2 reduces the expression of ACE2. ²³ That seems counterproductive since ACE2 appears to be the main receptor that the virus uses to infect cells. It is known that different viruses use multiple receptors to infect cells. Recently, it has been shown that neuropilin‐1 (NRP‐1) could be such a factor that increases the binding of the spike protein to ACE2 and blocking NRP‐1 reduced viral infection. ²⁴ RGD‐binding integrins could play a similar role, as it has been shown that RGD‐binding integrins α5β1, αvβ5, αvβ6 and αvβ8 are expressed on human airway epithelial cells. ²⁵ In that case, a low expression of ACE2 would still be sufficient for the virus to infect the cells. In conclusion, the acquired RGD motif in the spike protein could potentially promote viral infection in ACE2‐negative cells as well as serving as an additional factor to increase binding and infectivity in ACE2‐positive cells. ¹⁸

Involvement of TGF‐β in different pathogeneses

Activation of TGF‐β

RGD‐binding integrins are the main regulator of TGF‐β activation. TGF‐β plays an important role in several biological processes including embryogenesis, tissue regeneration, immune responses and tumorigenesis. Additionally, TGF‐β can also act as a pro‐viral factor. ²⁶ Upregulation of TGF‐β is also involved in mediating different pulmonary diseases, for example bronchial asthma, emphysema, pulmonary fibrosis and lung cancer. ²⁷ Three isoforms of TGF‐β are known (TGF‐β 1, β2 and β3). All three are highly conserved between species, and they have demonstrated similarity in functional properties. However, specific and non‐overlapping functions have been suggested for each isoform. ¹¹ TGF‐β is sequestered as an inactive protein, forming non‐covalently small‐latent complex (SLC) with the latency‐associated peptide (LAP). This complex is bound covalently by disulfide bonds to the latent TGF‐β‐binding protein (LTBP) to form the large latent complex (LLC), which is stored in the extracellular matrix. ¹¹ TGF‐β can be activated through physical processes like acidification, extreme temperature changes and oxidation, by several proteases, for example plasmin, elastase, matrix metalloproteinase (MMP)‐2 and MMP‐9, and by interactions with integrins or thrombospondin. ¹¹ The LAP of TGF‐β₁ and TGF‐β₃ contain a RGD motif that can bind to at least six αv‐containing integrins (αvβ 1, αvβ 3, αv β5, αv β6, αv β8 and α8 β1). So far, four of them have been shown to activate TGF‐β in vivo (αvb1, αvb5, αvb6 and αvb8). ²⁸ , ²⁹ For the latent TGF‐β₂, an alternative mechanism must be responsible for the activation since the LAP of TGF‐β₂ does not contain an RGD motif.

As previously mentioned, infection with SARS‐CoV‐2 reduces the expression of ACE2. ²³ That seems counterproductive since ACE2 appears to be the main receptor that the virus uses to infect cells. Nevertheless, a reduced expression of ACE2 in COVID‐19 patients could be the potential source for TGF‐β. ACE2 usually catalysed Angiotensin II (AngII) to Ang1‐7. Ang1‐7 is a biological active peptide that binds and activates the Mas receptor. This ACE2/Ang1‐7/Mas pathway counterbalances RAAS (renin, angiotensin, aldosterone system), promoting the activation of anti‐inflammatory pathways. ³⁰ Reduced ACE2 leads to an overactive RAAS, which provoked a local vascular inflammation and, through aldosterone, activates TGF‐β production. ³¹

Another potential source of TGF‐β is different immune cells. Boumaza et al. showed in vitro that SARS‐CoV‐2 efficiently infects monocytes and macrophages, which resulted in the secretion of TGF‐β. ³² There are no reports though that show that this occurs in COVID‐19 patients. In an additional study, TGF‐β1 was increased in two different subsets of CD4⁺ immune cells of the COVID‐19 patient group compared to the healthy control group. ³³ However, the activation of TGF‐β has not been studied during the complete infectious process. Therefore, it is not known whether TGF‐β activation occurs at early or late infection.

ARDS and fibrosis

TGF‐β regulates multiple cellular processes that also play an important role in the development of acute lung injury (ALI)/ARDS; for example, TGF‐β contributes to the alveolar epithelial permeability, fibroblast activation and extracellular matrix remodelling. Increased levels of TGF‐β are associated with impaired alveolar fluid clearance incapable to remove the oedema from alveoli. ³⁴ , ³⁵ , ³⁶ , ³⁷ The overall mortality rate of ARDS is between 30 and 40%. ³⁸ A similar rate has been observed in COVID‐19 patients who develop ARDS. Furthermore, patients with severe conditions also need mechanical ventilation. Different small case series demonstrated that COVID‐19 patients who received mechanical ventilation had a mortality rate between 50 and 81.1%. ³⁹ , ⁴⁰ The current medical recommendation for these patients is to delay the mechanical ventilation if possible. Interestingly, ARDS patients with a lower TGF‐β level in the bronchoalveolar lavage fluid (BALF) were associated with fewer mechanical ventilation and less intensive care unit (ICU) days. ⁴¹ However, the difference was not statistically significant and further research is necessary to evaluate this trend. The activation of TGF‐β increases the endothelial and epithelial permeability leading to the alveolar influx of fluids and proteins that impairs pulmonary gas exchange, which leads to arterial hypoxemia and respiratory failure. ⁴² , ⁴³ TGF‐β1 is a central mediator of fibrogenesis. As discussed above, SARS‐CoV‐2 infection causes an imbalance in RAAS. A recent review suggested that this imbalance favors lung fibrosis in COVID‐19 patients. ⁴⁴ The authors also hypothesised that TGF‐β might play a critical role in this process. The release of TGF‐β from injured tissue promotes lung repair, which normally leads to the resolution of infection. However, mice or rats over‐expressing TGF‐β demonstrate severe pulmonary fibrosis. ⁴⁵ , ⁴⁶ Reports of patients with influenza A (H1N1) showed elevated levels of TGF‐β1, correlated with the development of pulmonary fibrosis. ⁴⁷ TGF‐β levels were also markedly increased in SARS patients with ARDS. ⁴⁸ Zhao et al. showed that the nucleocapsid protein of SARS‐CoV‐1 is responsible for elevated TGF‐β signalling in these patients. ⁴⁹ The nucleocapsid protein of SARS‐CoV‐2 is over 90% similar to that of SARS‐CoV‐1. ⁵⁰ Whether it also plays a role for TGF‐β signalling in COVID‐19 patients is unknown. Nevertheless, the activation of TGF‐β leads to the production of fibrin, collagen and matrix metalloproteinases (MMP), which play a critical role in ALI. ⁵¹ , ⁵² , ⁵³ TGF‐β has been also shown to regenerate hyaline cartilage, which contains a large amount of collagen as well as hyaluronan (HA), for the latter through increased expression of hyaluronan synthase 2. ⁵⁴ A pathological report of a case study of a COVID‐19 patient demonstrated the formation of hyaline membrane—a suggestive sign of early ARDS. ⁵⁵ HA can absorb a high amount of water, which could explain the accumulation of fluids in the lungs of COVID‐19 patients. ⁵⁶ However, the association between HA and fluids agglomeration still needs to be confirmed. If so, blocking TGF‐β to inhibit hyaluronan synthase might hold a great promise for COVID‐19 patients.

In response to lung injury as well as inflammation, the surface expression of the epithelial integrins α2β1, α3β1, α5β1, α6β4, αvβ5 and αvβ6 is dramatically upregulated. The upregulation of the integrins α5β1 and αvβ6 is specific to alveolar epithelial cells following injury. ⁵⁷ Furthermore, activated neutrophils contribute to the development of ventilator‐induced lung injury (VILI) caused by high‐pressure mechanical ventilation and increased TGF‐β expression. In a rat model, ventilator‐induced lung injury is associated with neutrophil infiltration, macrophage activation and upregulated TGF‐β expression. ⁵⁸ Several in vivo models have shown that blocking TGF‐β via a soluble chimeric TGF‐β receptor or via the corresponding integrins (αv β5, αv β6 and αv β8) can reduce lung injury, ⁴³ , ⁵⁹ , ⁶⁰ whereas in the bleomycin model, an overexpression of TGF‐β led to increased apoptosis of airway epithelial cells and increased lung fibrosis. ⁶¹ Furthermore, αvβ6^−/− mice are protected from LPS and ventilator‐associated lung injury ⁶² and the pretreatment with antibodies to block αv β5 and αv β6 had additive protective effects against interleukin (IL)‐1β‐induced ALI. ⁶³

Angiogenesis and coagulation

It has been suggested that SARS‐CoV‐2 is not only a respiratory infection but also a haematologic disease because of its significant impact on the haematopoietic system. ⁶⁴ For instance, the findings of blood clots in COVID‐19 patients and their involvement from deep venous thrombosis in lower extremities to blocked arteries in the brain and lungs, resulting in strokes and pulmonary embolism, are of great concern. ⁶⁵ , ⁶⁶ The source of these blood clots has not been defined; however, TGF‐β is known to stimulate the production of Factor XII (FXII). ⁶⁷ The zymogen FXII stands at the beginning of the coagulation signalling of the intrinsic cascade. ⁶⁸ Thrombin, which is the last step of the coagulation cascade, mediates the cleavage of fibrinogen to fibrin. The fibrin monomers polymerise and form a fibrin clot that stop the bleeding. FXII knockout mice are largely defective for thrombus formation and are protected from experimental cerebral ischaemia and pulmonary embolism. ⁶⁹ FXII, thrombin and fibrinogen have also been linked to inflammatory disorders like multiple sclerosis, sepsis, rheumatoid arthritis and colitis. ⁷⁰ Thrombin cleaves GARP (glycoprotein A repetitions predominant) that is found on the surface of platelets, and the cleaved GARP in cooperation with αvβ8 leads to the release of mature TGF‐β1, creating a positive feedback loop. Blocking αvβ8 on dendritic cells and fibroblasts impairs TGF‐β‐dependent generation of Th17 cells and reduces the inflammatory disease in the lungs. ⁷¹ It has been postulated that FXII potentially mediates the development of neuroinflammation via upregulation of neutrophil functions, contributing to macrophage polarisation, and inducing T‐cell differentiation. ⁷² FXII signalling is also involved in activating pro‐inflammatory pathways that induce chemotaxis of leukocytes and increases vascular permeability. ⁷⁰

The expression of plasminogen activator inhibitor‐1 (PAI‐1), another important factor in the coagulation process, is induced by TGF‐β. ⁷³ Elevated PAI‐1 has been observed in myocardial infarction and cardiac failure, and the latter is one of the death causes for COVID‐19 patients. ¹ , ² , ⁷⁴ , ⁷⁵ Furthermore, Ackermann et al. showed that the expression of the gene for PAI‐1, SERPINE1, is upregulated in COVID‐19 patients. ⁷⁶ PAI‐1 is a serine‐protease inhibitor and the main inhibitor of the tissue‐type (tPA) and the urinary‐type plasminogen activator (uPA). Both are able to activate plasminogen and, hence, fibrinolysis. Besides the elevated levels of fibrin in COVID‐19 patients and their resulting blood clots, increased levels of the fibrin‐degradation product D‐dimer have been detected. ⁷⁷ A reduced fibrinolysis can cause thrombosis, pulmonary embolus, and it increases the risk of stroke and heart attack. A case series was performed to treat blood clots with tPA in COVID‐19 patients. However, this treatment only showed temporary improvements. ⁷⁸

Soluble urokinase‐type plasminogen activator receptor (suPAR) is another factor that is elevated in COVID‐19 patients and may serve as an early predictor of severe respiratory failure in patients with COVID‐19 pneumonia. ⁷⁹ Elevated suPAR is associated with increased inflammation, disease progression and risk of mortality in several infectious diseases. ⁸⁰ It is also associated with acute kidney injury in various clinical and experimental contexts. ⁸¹ Chang et al. showed that TGF‐β stimulates PAI‐1 and suPAR secretion in a system of repair/regeneration activities of stem cells from apical papilla (SCAP). ⁸²

Besides a widespread thrombosis in patients with COVID‐19, the examination of the lung during an autopsy of 7 deceased COVID‐19 patients also demonstrated increased angiogenesis. ⁷⁶ Recent evidence has revealed higher vascular endothelial growth factor (VEGF) levels in COVID‐19 patients than in healthy controls. VEGF plays a major role in the formation of new blood vessels, and TGF‐β has been shown to induce angiogenesis through VEGF‐mediated apoptosis. ⁸³ Furthermore, several studies have shown VEGF’s potential key role in the pathogenesis of ALI/ARDS. ⁸⁴ Additionally, hypoxia‐inducible factor 1 alpha (HIF‐1α) that is also increased in COVID‐19 patients ⁸⁵ regulates VEGF and, in turn, TGF‐β induces HIF‐1α stabilisation. ⁸⁶ Blocking RGD‐binding integrins reduces VEGF expression. ⁸⁷ A clinical trial is evaluating the capacity of anti‐VEGF antibody bevacizumab to inhibit ALI/ARDS as well as to reduce the mortality in severe COVID‐19 patients through the suppression of pulmonary oedema (NCT04275414). In conclusion, blocking TGF‐β can potentially inhibit FXII, PAI‐1, suPAR and VEGF. In conclusion, blocking TGF‐β can potentially inhibit FXII, PAI‐1, suPAR and VEGF, which might prevent and resolve blood clotting and reduce inflammation and vascular permeability.

Acute kidney injury, cytokines, Th17 cells and complement

In addition to the above‐mentioned common complications of severe COVID‐19 cases, other common problems are as follows: pneumonia, sepsis and septic shock, cardiomyopathy and arrhythmia, acute kidney injury (AKI), and several additional complications derived from prolonged hospitalisation, including secondary bacterial infections, gastrointestinal bleeding and critical illness polyneuropathy/myopathy. ² Special interest of this list of medical complications falls to AKI. Recent work demonstrated that TGF‐β/SMAD2 (homologues of the Drosophila protein, Mothers against decapentaplegic (Mad) and the C. elegans protein Sma) plays a pivotal role in AKI. ⁸⁸ Liu et al. showed in a mouse model of obstructive nephropathy that the loss of ACE2 enhances renal fibrosis, which is mediated by increased TGF‐β/SMAD signalling. ⁸⁹ Reduced expression of ACE2 has also been described for SARS‐CoV‐2 infections (see sections above). Furthermore, Breuss et al. observed beta 6 integrin expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. ⁹⁰ Blocking the integrin αvβ5 protected rats from AKI. ⁹¹ Therefore, TGF‐β might play a pivotal role in the development of AKI in COVID‐19 patients. Moreover, blocking RGD‐binding integrins could prevent AKI.

Patients with severe COVID‐19 infections may present a high level of cytokines (cytokine storm), especially interferon gamma (IFN‐y), tumor necrosis factor alpha (TNF‐a), IL‐17, IL‐8, IL‐1β and IL‐6 are increased. ⁹² The largest studies so far that analysed 11, 27, 48 and 76 cytokines, respectively, did not measure protein expression of TGF‐β in the serum or BALF. ⁹³ , ⁹⁴ , ⁹⁵ , ⁹⁶ However, in a small study of three patients, mRNA expression of TGF‐β2 was elevated in BALF samples compared to healthy controls. ⁹⁷ TGF‐β1 was also increased in two different subsets of CD4⁺ immune cells of the COVID‐19 patient group compared to healthy control. ³³ A new study also showed that TGF‐β is increased in the serum of COVID‐19 patients compared to healthy controls. ⁹⁸ In the case of ALI induced by IL‐1β, pretreating mice with antibodies to block αvβ5 and αvβ6, which inhibits TGF‐β activation, can prevent ALI development, ⁶³ . Type I interferons (IFN) are key cytokines to challenge viral infections. However, in severe COVID‐19 patients IFNs expression are impaired. ⁹⁹ , ¹⁰⁰ In cancer, IFNs can be potent anti‐tumoral agents. Yet, abundant TGF‐β in tumors can limit this IFN‐induced tumor regression. ¹⁰¹ This has also been reported in respiratory viral infections like rhinovirus infections ¹⁰² , ¹⁰³ and respiratory syncytial virus (RSV) infections. ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ Another interesting cytokine is IL‐17 because of its broad pro‐inflammatory effect on the induction of cytokines. Mouse experiments with Neisseria gonorrhoeae indicate that IL‐17 suppresses Th1/Th2 immune responses through TGF‐β. Treating mice with anti‐TGF‐antibodies resulted in increased Th1 and Th2 responses and diminished Th17 response, following by an accelerated clearance of N. gonorrhoeae. ¹⁰⁷ The main producer of IL‐17 is Th17 cells. ¹⁰⁸ Pathologic findings showed an increased concentration of pro‐inflammatory Th17 cells in COVID‐19 patients. ⁵⁵ , ¹⁰⁹ In mice, Th17 cells are induced through TGF‐β and IL‐6, ¹¹⁰ although in humans, it is less clear. It seems that IL‐6 and IL‐1β are the main inducers of human Th17 cells. However, other groups also demonstrated the importance of TGF‐β–inducing Th17 cells. ¹¹¹ , ¹¹²

The complement activation is a key player in the fight against pathogens. However, excessive or unregulated complement activation might be involved in the pathogenesis of ALI and ARDS in COVID‐19 patients. ¹¹³ Gu et al. showed that a crosstalk between TGF‐β1 and complement activation augments epithelial injury in pulmonary fibrosis. ¹¹⁴ They suggest that increased TGF‐β levels may crosstalk with the complement cleavage products C3a and C5a and downregulates complement‐inhibitory proteins, which links complement activation to epithelial injury in IPF. Furthermore, C5a triggers the formation of neutrophil extracellular traps (NETs) that are capable of activating platelets to release TGF‐β. NETs are released by neutrophils to halt an infection. They are extracellular webs containing chromatin, microbicidal proteins and oxidant enzymes. However, when NETs are not properly regulated, they have the potential to propagate inflammation and microvascular thrombosis. Recent studies have shown elevated NET release in the serum of COVID‐19 patients and provided clinical evidence of its fundamental role in the pathogenesis of COVID‐19‐related ALI/ARDS and coagulopathy. ¹¹⁵ , ¹¹⁶ Therefore, blocking TGF‐β could be beneficial to prevent complement‐induced ALI as well as coagulopathy.

Kawasaki‐like syndrome in children

Until recently, children were thought to have less severe SARS‐CoV‐2 infections. However, new reports showed that there is an increase of Kawasaki‐like syndrome in infected children. ¹¹⁷ The Kawasaki disease (KD) is an acute and usually self‐limiting vasculitis, which usually affects children < 5 years of age. ¹¹⁸ It has been reported that genetic variants in the TGF‐β pathway genes influence the susceptibility to KD and that TGF‐β may contribute to aneurysm formation. ¹¹⁹ Targeting TGF‐β could therefore be beneficial in preventing KD. However, caution must be taken, because a mouse model of KD using a neutralising pan‐TGF‐β antibody worsened inflammatory‐induced coronary artery lesions. ¹²⁰ It is possible that using an antibody was not an appropriate strategy to block TGF‐β because it can induce inflammation via Fc receptors (FcR), which are expressed in different types of innate immune cells. Blocking TGF‐β via RGD‐binding integrins or using a specific drug to block particularly one TGF‐β isoform may show a better outcome.

TGF‐β and the potential reason why patients with pre‐existing conditions have a higher risk of developing severe COVID‐19 symptoms

Patients with pre‐existing medical conditions have an increased risk of developing severe COVID‐19 infections. ⁴⁰ , ¹²¹ , ¹²² The specific reasons are currently unknown. We hypothesise that TGF‐β could be the potential link. TGF‐β plays an important role in the progress of chronic conditions such as diabetes, ¹²³ hypertension, ¹²⁴ kidney injuries, ¹²⁵ heart disease, ¹²⁶ lung diseases (asthma, ¹²⁷ chronic obstructive pulmonary disease (COPD) ¹²⁸ and fibrosis ¹²⁹ ), obese patients ¹³⁰ and immunocompromised patients (cancer, ¹³¹ transplantation ¹³² , ¹³³ ) as well as in complications associated with elderly patients. ¹³⁴ , ¹³⁵ Blocking TGF‐β in animal models was associated with an improved outcome in obesity, ¹³⁶ diabetes, ¹³⁶ kidney injuries ⁹¹ , ¹³⁷ and hypertension. ¹³⁸ , ¹³⁹ In the case of lung diseases, it has been shown that elevated TGF‐β plays an important role in an exaggerated inflammatory response as well as disease exacerbation. ¹⁰² , ¹⁰⁴ , ¹⁰⁵ , ¹⁴⁰ Furthermore, the infection with SARS‐CoV‐2 between men and women seems to be equivalent. Nevertheless, men developed more severe symptoms with higher mortality rates. A new study analysing ACE2 concentrations in the plasma of non‐infected men and women with heart failure demonstrated a higher expression of soluble ACE2 in men, suggesting that the higher soluble ACE2 expression is responsible for the severe symptoms. ¹⁴¹ On the contrary, soluble recombinant human ACE2 is suggested to prevent binding of viral particles to the cell surface of ACE2‐positive cells. Recent in vitro studies have shown that an ACE2 Fc‐fusion protein neutralises SARS‐CoV‐2 and prevents viral cell infection. ¹⁴² Another study analysing GTEx and other public data in 30 tissues across thousands of individuals demonstrated that Asian women have a higher ACE2 cell surface expression than Asian men. ¹⁴³ This study also found an age‐dependent ACE2 decrease as well as a significant ACE2 decrease in type II diabetic patients. Furthermore, they found that ACE2 expression is upregulated by oestrogen. In conclusion, their data suggest that ACE2 might play a protective role in developing severe conditions for COVID‐19 patients. Additionally, the study showed that ACE2 expression is negatively correlated with severe outcome in patients with pre‐existing conditions. The same relationship is observed following age and gender of the patients. Another possible reason for less severe cases observed in women could be that women have a potential more efficient immune system than men. ¹⁴⁴ However, we hypothesise that TGF‐β might be the important factor for the difference. Loss of ACE2 expression increases TGF‐β expression in a mouse model, and men show a higher expression of TGF‐β at baseline than women. ⁸⁹ , ¹⁴⁵ Furthermore, TGF‐β induces the expression of αv, α5, β1, β3 and β5 integrins, ¹⁴⁶ , ¹⁴⁷ creating a positive feedback loop on TGF‐β activation and potentially increases the hypothesised infectivity via RGD‐binding integrins. This hypothesis is further supported by the findings that in severe patients cases, vital tissues with little ACE2 expression are also severely damaged by the SARS‐CoV‐2 infection. ¹⁴⁸ Additionally, the infection with SARS‐CoV‐2 reduces ACE2 expression, which, therefore, can increase TGF‐β expression. The reason why women show less severe COVID‐19 infections might be that the female oestrogen suppresses TGF‐β‐induced gene expression, for example type IV collagen. ¹⁴⁹ Collagens play an important role in the development of fibrosis. ¹⁵⁰ In a mouse kidney injury model, it was also shown that oestradiol can reverse renal injury. ¹⁵¹ A clinical trial just started to use oestrogen as treatment for COVID‐19 patients to evaluate whether oestrogen can reduce the damaging effects of the virus on the lung and other severe symptoms (NCT04359329).

In conclusion, based on the aforementioned evidence, we hypothesise that blocking TGF‐β might be a possible treatment opportunity for COVID‐19 patients with pre‐existing medical conditions to reduce the risk of developing a more severe disease that could potentially lead to death (Figure 2).

Figure 2.

Multiple conditions lead to higher levels TGF‐β. Representation of medical conditions that are associated to high levels of TGF‐β. These can be related to the severe cases of COVID‐19. Specific inhibition of TGF‐β mechanism of action could prevent severe COVID‐19 symptoms. COPD, chronic obstructive pulmonary disease; IPF, idiopathic pulmonary fibrosis; CF, cystic fibrosis.

RGD‐binding integrins

RGD‐binding integrins are heterodimeric proteins composed of two membrane‐spanning subunits. They are part of the superfamily of cell adhesion receptors that recognise their ligand via the RGD motif. Below, we summarise each of the 8 known RGD‐binding integrins on their potential role for SARS‐CoV‐2.

αvβ1

The integrin αvβ1 is highly expressed on activated fibroblasts in the lung and directly binds to the latency‐associated peptide of TGFβ1 to mediate TGFβ1 activation. αv and β1 integrins can also be induced by TGF‐β, creating a positive feedback loop. ¹⁴⁶ A therapeutic delivery of an αvβ1 inhibitor has been shown to attenuate bleomycin‐induced pulmonary fibrosis. ²⁹ Furthermore, inhibiting this integrin in a kidney injury‐induced mouse model ameliorates renal failure and fibrosis. ¹⁵² These results make αvβ1 a potential target in the treatment for severe COVID‐19 infections where these complications have also been observed.

αvβ3

αvβ3 plays a fundamental role in neovascularisation. This integrin is elevated on endothelial cells during wound angiogenesis, tumor angiogenesis and inflammation. ¹⁵³ , ¹⁵⁴ αvβ3 is also upregulated on epithelial cells and can activate TGF‐β in scleroderma fibroblasts, even though it binds weaker to latent TGF‐β than to αvβ6 and αvβ8. ¹⁵⁵ , ¹⁵⁶ Furthermore, TGF‐β increases the expression of αv and β3 integrins. ¹⁴⁶ Of note, excessive blood clot formation was also demonstrated in COVID‐19 patients. ⁶⁵ Furthermore, Ackermann et al. demonstrated increased angiogenesis in the lung of deceased COVID‐19 patients. ⁷⁶ There is evidence that some COVID‐19 patients show an increased level of VEGF compared to healthy controls. For this reason, a clinical trial using bevacizumab, an anti‐VEGF‐A antibody, was initiated for COVID‐19 patients (NCT04275414 and NCT04305106). As mentioned above, a small case series was started to treat the blood clots with tPA. ⁷⁸

αvβ5

αvβ5 plays a role in several biologic processes, for example tumor angiogenesis, phagocytosis, fatty acid uptake and retinal pigment epithelium homeostasis. ¹⁵⁷ , ¹⁵⁸ , ¹⁵⁹ αvβ5 is also involved in vascular permeability, and blocking αvβ5 inhibits vascular leakage in mouse models of ALI, sepsis and AKI. ⁶³ , ⁹¹ , ¹⁶⁰ Similar to αvβ3, αvβ5 is also upregulated in the dermal epithelium of patients with systemic sclerosis and activates TGF‐β in scleroderma fibroblasts. ¹⁵⁵ Both subunits of αvβ5 can be upregulated through TGF‐β. ¹⁴⁶

αvβ6

The integrin αvβ6 is highly expressed at high levels during embryogenesis in epithelial cells of the developing lung, but downregulated in healthy adults. ⁹⁰ , ¹⁶¹ However, during epithelial injuries, αvβ6 is highly upregulated. ⁹⁰ , ¹⁶² As mentioned earlier, blocking avb6 interleukin can reduce lung injury and IL‐1β‐induced ALI. ⁴³ , ⁵⁹ , ⁶⁰ , ⁶³

αvβ8

αvβ8 is expressed in the lung by epithelial cells and fibroblasts, and its expression is increased in airway fibroblasts of COPD patients. ¹⁶³ Compared to the other RGD‐binding integrins that activate TGF‐β through a conformational change in the LAP protein, αvβ8 activates TGF‐β through a proteolytic cleavage of LAP. ¹⁵⁶ Asthmatic children demonstrate a higher expression of epithelial αvβ8 than control children ¹⁶⁴ Kitamura et al. demonstrated that αvβ8‐mediated TGF‐β activation is important for the development of airway fibrosis and inflammation and that blocking αvβ8 represents a strategy to treat fibroinflammatory airway diseases. ¹²⁸ , ¹⁶⁵

αIIbβ3

αIIbβ3 is the major integrin expressed on the surface of platelets where it plays a critical role in platelet aggregation and blood clotting. ¹⁶⁶ αIIbβ3 plays also a role in stroke and myocardial ischaemia. ¹⁶⁷ , ¹⁶⁸ Several drugs are approved to prevent platelet aggregation and thrombus formation (Table 1), incidences that are also reported in COVID‐19 patients. There are no reports showing that αIIbβ3 binds or activates latent TGF‐β.

Table 1.

Drugs against RGD‐binding integrins and against TGF‐β

Drug	Target	Disease	Clinical Stage	Company
A: Chemical or peptide/protein based drugs
Abciximab ( a )	α IIbβ3 ( b )	Thrombosis	approved	Janssen Biologics
Tirofiban	α IIbβ3	Thrombosis	approved	Medicure Pharma
Intrifiban	α IIbβ3	Thrombosis	approved	Millennium Pharmaceuticals
GSK3008348	α vβ6	idiopathic pulmonary fibrosis (IPF)	Phase I	GSK
PLN‐74809	α vβ1 and α vβ6	IPF	Phase IIa	Pliant
THR‐687	pan RGD integrin inhibitor	Diabetic macular edema	Phase II planned	Oxurion NV
GLPG0187	α vβ1, α vβ3, α vβ5, α vβ6, and α 5β1	Solid tumors	Phase I	Galapagos NV
IDL2965	α vβ1, α vβ3, and α vβ6	IPF	Phase IIa	Indalo Therapeutics
Risuteganib	α 5β1, α vβ1, α vβ3, and α vβ5	Dry age‐related macular degeneration, diabetic macular edema	Phase IIa	Allegro
MK‐0429	αvβ1, but has also been shown to block α 5β1, α vβ3, α vβ5, α vβ6, α vβ8, α 5β1139	Prostate cancer, Post‐Menopausal Osteoporosis	Phase I, II	Merck Sharp & Dohme
Cilengitide	α 5β1, α vβ3 and α vβ5	Solid tumors	Phase II/III	Merck KGaA
ATN161	α 5β1	Renal cancer, glioma	Phase II	Attenuon
B: Antibodies against RGD‐binding integrins
Intetumumab	α vβ1, α vβ3, α vβ5, and α vβ6	Prostate cancer, melanoma	Phase II	Centocor, Inc.
BG00011 (STX‐100)	α vβ6	IPF	Phase II	Biogen
Abituzumab (EMD525797)	α vβ1, α vβ3, α vβ5, α vβ6, and α vβ8	Prostate cancer, colorectal cancer	Phase II	Merck KGaA
C: TGF‐β inhibitors
OT‐101 (Trabedersen)	TGF‐β2	Solid cancer/ COVID‐19	Phase III/IND filed for phase II study	Mateon Therapeutics
Galunisertib	TGF‐beta receptor type‐1 (TGF‐βR1)	Myelodysplastic syndrome and solid tumors	Phase II/III	Eli Lilly & Company (Lilly)
TEW‐7197	TGF‐βR1	Solid tumors	Phase I	MedPacto Inc.
LY3022859	TGF‐βR2	Advanced solid tumors	Phase I	Lilly
LY2157299	TGF‐βR2	Hepatocellular carcinoma	Phase II	Lilly
LY2382770	TGF‐β1	Diabetic kidney disease, diabetic nephropathy, diabetic glomerulosclerosis	Phase II	Lilly
Fresolimumab (GC‐1008)	Pan TGF‐β	Systemic sclerosis, focal segmental glomerulosclerosis, myelofibrosis, and solid tumors	Phase II	Genzyme
Luspartecept	TGF‐β superfamily inhibitor	β‐thalassaemia; anaemia in patients with MDS	Phase II	Acceleron Pharma
NIS793	Pan TGF‐β	Solid tumors	Phase I	Novartis
LY2382770	TGF‐β1	Diabetic kidney disease (fibrosis)	Phase II	Lilly &
CAT‐192	TGF‐β1	Systemic sclerosis scleroderma	Phase I/II	Genzyme
AVID200	TGF‐β1 & ‐β3	Scleroderma, myelofibrosis, solid tumors	Phase I	Icahn School of Medicine at Mount Sinai

IND, Investigational New Drug.

^aAbciximab is a Fab fragment. We listed it in this table because it is not a full length antibody.

^bWe only show approved drugs against αIIbβ3. There are more inhibitors in different stages of clinical trials.

α5β1

α5β1 is expressed in the foetal lung mesenchyme, but not lung epithelium. Several studies with α5β1 antagonists suggest that it plays a critical role in wound repair, inflammation and tumor angiogenesis. ¹⁶⁹ , ¹⁷⁰ Furthermore, TGF‐β treatment can increase the expression of both subunits. ¹⁴⁶ , ¹⁴⁷ A recent publication showed that the gene of α5β1 is upregulated in COVID‐19 patients. ⁷⁶ Of particular interest concerning COVID‐19 is the study showing that blocking α5β1 can also reduce severe airway hyperresponsiveness in a mouse model of asthma. ¹⁷¹

α8β1

The α8β1 integrin is expressed on human intestinal epithelial crypt cells. ¹⁷² It is also present on alveolar interstitial cells and smooth muscle cells in the lung parenchyma and is upregulated during pulmonary and hepatic fibrosis. ¹⁷³ Furthermore, T regulatory cells express high amounts of the α8β1 integrin, which enables them to activate latent TGF‐β. ¹⁷⁴

Potential drugs to block TGF‐β directly or via RGD‐binding integrins

A variety of preclinical studies have demonstrated that blocking integrins can, for example, prevent pulmonary fibrosis and protect against ALI. There are several medications for different diseases available that target different RGD‐binding integrins and that have been approved or are in clinical trials (Table 1a). From this group of potential drugs, GSK3008348 is particularly interesting. It is the first inhaled inhibitor of the integrin αvβ6. A phase I trial for idiopathic pulmonary fibrosis (IPF) has been shown to be safe. Another interesting drug is the α5β1 inhibitor ATN161, which has shown in vitro that it can inhibit SARS‐CoV‐2 infections (see section above). Our literature review supports the idea that full‐length antibodies’ treatment should be taken carefully because of their potential to further stimulate inflammation through binding to FcRs. However, due to the urgency to find a treatment, they are also listed here (Table 1b).

Targeting TGF‐β systemically bears some risks as explained below. However, considering the severity of some COVID‐19 cases these drugs could be beneficial for this group of patients. An Investigational New Drug (IND) has been filed for a phase II clinical trial using a TGF‐β2 antisense drug (OT‐101) to treat COVID‐19 patients. The rationale for this trial was based on the significant negative correlation between TGF‐β levels in BALF samples from ARDS patients and ventilator‐free days and ICU‐free days and that lower TGF‐β levels correlated with better survival outcome in ARDS patients. ¹⁷⁵ Here, we present several TGF‐β inhibitors that are in clinical trial for different diseases, for example receptor kinase inhibitors, neutralising antibodies and ligand traps (Table 1c).

Potential risks

Blocking TGF‐β has a great potential to treat COVID‐19 patients. However, a systemic anti‐TGF‐β treatment could increase severe adverse events because of its involvement in several biological processes. A partial block might be preferred. This can be accomplished through blocking RGD‐binding integrins. This would resemble the body’s natural mechanism of locally TGF‐β activation, and it has, therefore, the potential to return to a homeostatic state without the systemic side effects that have been seen with a systemic blockage of activated TGF‐β.

As mentioned above, anti‐integrin antibodies are not the first choice because latent TGF‐β can be also stored at the surface of cells. Therefore, an anti‐TGF‐β antibody could activate the complement system against the targeted cell as well as to stimulate further inflammation through the interaction with the FcR‐expressing immune cells. The option of using novel antibody formats, such as Fc‐less or Fc‐silent antibodies, is promising, and it deserves to be explored. Systemic blockade of TGF‐β through, for example a pan integrin inhibitor, also could increase the risk of adverse events similar to a systemic inhibition of activated TGF‐β. Therefore, blocking just the specific RGD‐integrins or a local delivery via an inhaler (see GSK3008348) might be preferred. The inhalation delivery has the advantage that the drug is directly delivered to the infection area in the lungs. Furthermore, part of the drug can still enter the bloodstream to reach other parts of the body that are damaged through elevated TGF‐β, for example the kidneys.

Open questions

Only three studies so far have showed elevated TGF‐β in COVID‐19 patients. One study measured mRNA expression in BALF, another study revealed elevated TGF‐β in two different types of immune cells (see section above), and the third and most recent study showed increased TGF‐β in the serum of COVID‐19 patients. ⁹⁸ However, it is not clear whether TGF‐β is the driver of the severity of COVID‐19 patients or whether it is a consequence of it. Further studies are necessary to analyse these alternatives. Nevertheless, blocking TGF‐β could still be beneficial to reduce or prevent complications associated with severe COVID‐19 infection and it deserves further investigation.

Conclusion

Considering that high levels of TGF‐β expression are involved in several pathologies including the complications of severe COVID‐19 outcome and that two studies revealed increased TGF‐β in COVID‐19 patients, we highly promote to include RGD‐binding integrin inhibitors in clinical trials as potential treatment for COVID‐19 patients (Figure 3). Blocking RGD‐binding integrins could also be beneficial for COVID‐19 patients with pre‐existing conditions since these patients have already increased TGF‐β at basal levels and blocking further TGF‐β activation could potentially reduce the risk of developing a more severe disease, including death (Figure 2).

Figure 3.

Scheme representing the potential effect of RGD‐binding integrin inhibitors to avoid the common complications derived from SARS‐CoV‐2 infection.

Furthermore, taking in consideration that there are active clinical trials using the mentioned drugs that inhibit TGF‐β directly or via the RGD‐binding integrins, it would be important to analyse whether these patients have a milder COVID‐19 disease course than comparable patients who take other medications.

Conflicts of Interest

The authors declare no competing interests.

Author Contribution

Ingrid Carvacho: Validation; Visualization; Writing‐review & editing. Matthias Piesche: Conceptualization; Formal analysis; Resources; Supervision; Validation; Visualization; Writing‐original draft; Writing‐review & editing.