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Potential long-term effects of SARS-CoV-2 infection on the pulmonary vasculature: a global perspective

The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with severe hypoxia being the cause of death in the most critical cases. Coronavirus disease 2019 (COVID-19) is extremely heterogeneous in terms of severity, clinical phenotype and, importantly, global distribution. Although the majority of affected patients recover from the acute infection, many continue to suffer from late sequelae affecting various organs, including the lungs. The role of the pulmonary vascular system during the acute and chronic stages of COVID-19 has not been adequately studied. A thorough understanding of the origins and dynamic behaviour of the SARS-CoV-2 virus and the potential causes of heterogeneity in COVID-19 is essential for anticipating and treating the disease, in both the acute and the chronic stages, including the development of chronic pulmonary hypertension. Both COVID-19 and chronic pulmonary hypertension have assumed global dimensions, with potential complex interactions. In this Review, we present an update on the origins and behaviour of the SARS-CoV-2 virus and discuss the potential causes of the heterogeneity of COVID-19. In addition, we summarize the pathobiology of COVID-19, with an emphasis on the role of the pulmonary vasculature, both in the acute stage and in terms of the potential for developing chronic pulmonary hypertension. We hope that the information presented in this Review will help in the development of strategies for the prevention and treatment of the continuing COVID-19 pandemic.

Following the initial reports from China in December 2019 of atypical pneumonia caused by a novel corona virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 , and confirmation of human-to-human transmission 2 , coronavirus disease 2019 (COVID- 19) rapidly progressed to a global pandemic. As of September 2021, there have been >217,558,771 reported cases and >4,517,240 deaths associated with COVID-19 across all inhabited continents 3 , with heterogeneous incidence, severity and phenotype. The lungs seem to be the primary target of infection, with major involvement of the pulmonary vasculature. However, the exact pathobiology of the pulmonary vascular effects of COVID-19, in both the acute and the chronic stages, are poorly understood.
In this Review, we describe the possible virus-related and host-related factors that could be responsible for the heterogeneity of COVID-19. In addition, we discuss in detail the pathobiology of the pulmonary vascular effects of SARS-CoV-2 infection and their clinical implications for chronic pulmonary hypertension. A thorough understanding of these changes should inform the evolving global pre-emptive strategies for dealing with the COVID-19 pandemic and its sequelae.
The SARS-CoV-2 virus: an update Studying the origins and dynamic behaviour of the SARS-CoV-2 virus is essential to understanding the pathobiology of the infection and in developing strategies to manage it. SARS-CoV-2 is the seventh coronavirus known to infect humans. Four coronaviruses are endemic in the population: these viruses typically cause a mild respiratory tract infection in the winter months, although the disease can be severe in neonates, elderly 0123456789();: individuals and those with predisposing conditions. The highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) each caused outbreaks in humans, with a case fatality rate of 9% and 40%, respectively 4 . However, these two viruses do not transmit readily between humans and, therefore, containment of the outbreaks was relatively simple. By contrast, SARS-CoV-2 is intermediate in virulence between the endemic coronaviruses and the highly pathogenic viruses but its high human-to-human transmissibility has led to the current human pandemic.
Like the other coronaviruses, SARS-CoV-2 has a non-segmented, positive-sense RNA genome 4 (Fig. 1a,b). The structure of the SARS-CoV-2 virus particle (virion) is shown in Fig. 1c,d. The virion is enclosed in a lipid envelope, derived from the host cell. Projecting from the virion envelope is the spike (S) protein, producing the familiar crown-like appearance that gives the coronavirus its name. The host protease furin cleaves the S protein into two moieties: S1 and S2. S1 binds to the angio tensin-converting enzyme 2 (ACE2) receptor on the cell surface, which thereby serves as the cellular entry point for the virus 5 . S2 fuses the virion envelope with the cell membrane, resulting in infection of the cell (Fig. 1e). A second cell-surface protein, neuropilin 1, acts as a cofactor for SARS-CoV-2 infection by binding the furin-cleaved S1 protein 6,7 . The S protein is an important target of both the antibody response 8 and the T cell immune response 9 (Fig. 1f) and the majority of the existing vaccines use the S protein as the immunogen.
SARS-CoV-2 shares up to 97% nucleotide sequence identity with coronaviruses of bats (Fig. 1b) and, like the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV, is likely to have originated in bats 10,11 and spread to humans either directly or via an unidentified intermediate mammalian host 10,12 .
Coronaviruses mutate less quickly than other RNAbased viruses because they express a 'proof-reading' 3′-5′ exonuclease, which increases the fidelity of replication of the virus. Nonetheless, mutants do arise by nucleotide misincorporation and recombination and by the action of the host innate immune response 13 . On average, any two circulating SARS-CoV-2 isolates differ by about 10 nucleotides 14 . Each isolate that differs in its genome sequence from other identified viruses can be called a 'variant' , and the total number of variants of SARS-CoV-2 is therefore very large -many thousands have been identified. However, the great majority of these sequence variants do not substantially alter either the infectiousness of the virus or the severity of the disease that it causes.
The resulting sequence diversity between isolates of SARS-CoV-2 has important practical and theoretical implications. First, sequence diversity makes it possible to trace the origin and spread of the infection both at the population level and in local outbreaks, thereby providing valuable information for contact tracing, epidemiology and public health. For example, sequence analysis showed that there were >1,170 importations of the virus into the UK in February to March 2020, mostly from southern Europe, with <1% from China 15 . Second, the rapid sequence variation allowed SARS-CoV-2 to evolve quickly, raising the question of whether newly emerging variants of the virus might differ from the currently circulating strains either in virulence (pathogenicity) or transmissibility. Weak correlations have indeed been observed between some 11 sequence variants of the virus and the case fatality rate in patients with COVID-19 (reF. 16 ) but the mechanisms involved are not yet known. The rapid spread of a variant in the spike protein (D614G) might result from increased transmissibility or infectiousness of this variant 17,18 and does not seem to confer a difference in virulence (Fig. 1b).
Third, the question arises whether a mutation in the viral RNA might render the vaccines ineffective. At present, this outcome is improbable, because the vaccines that are now being given use the full-length S protein as the immunogen; given that the S protein encodes several antibody epitopes and several T cell epitopes, a small number of mutations are unlikely to abrogate the immune response to the vaccine. However, direct evidence is needed to verify the effectiveness of existing vaccines against newly emerging variants. Additionally, monoclonal antibodies are used to treat some patients with severe COVID-19. Given that monoclonal antibodies are specific to a single epitope, often in the receptor-binding domain of SARS-CoV-2, even a point mutation might confer resistance to a monoclonal antibody.
A small minority of the variant sequences that arise confer substantial changes in the behaviour of the virus, chiefly to increase its transmissibility. These variants, known as variants of concern, include the viruses designated Alpha 19 , Beta 20 and Delta 21 (Table 1), are substantially more infectious and spread more rapidly in the population than the preceding variants and, in some cases, might cause more severe disease. These variants of concern typically contain certain First, the mutations increase the binding affinity of the S protein to its receptor (ACE2) and thereby increase the efficiency of infection. Secondly, the mutation can diminish the recognition of the spike protein by some antibodies -that is, immune escape. However, this escape is only partial 22,23 , and current evidence indicates that the existing vaccines provide substantial protection against the variants of concern 24-27 , although the incidence of mild-to-moderate disease might be higher 28 .
The variant designated Delta (Table 1), first isolated in India in October 2020, similarly contains two mutations (E484Q and L452R) that seem to confer the greater infectiousness and transmissibility of this variant. Whether the Delta variant causes more severe disease than other currently circulating variants remains uncertain 29 . In addition, the immunity resulting either from previous infection with another strain of SARS-CoV-2 or from vaccination with existing vaccines seems to give an important degree of protection against serious disease 29 . The Iota variant (Table 1), first detected in New York, USA, in November 2020, carries the E484K mutation and a further mutation at amino acid 477 (S477N). In the future, modification of vaccines might be necessary, perhaps to contain more than one variant sequence of the S protein, in order to provide efficient protection against new variants that differ substantially from the currently circulating strains.
Heterogeneity of COVID-19 disease Severity COVID-19 is mild in the majority of infected individuals and is therefore associated with a substantial rate of asymptomatic and undocumented infection 30 . However, 4-5% of people develop more severe disease that requires hospitalization, thought to be related to hyperinflammation and/or a failure to resolve the initial infection. Although pneumonitis with hypoxia is a major feature, severe COVID-19 has multisystem manifestations, including systemic thrombosis, cardiac injury, renal failure and hepatic dysfunction. In the initial China-wide outbreak of COVID-19, the overall case fatality rate was 2.3%, rising to 49% in those with critical disease (defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure) 31 . However, mortality among hospitalized patients with COVID-19 has fallen to 20-25% with advances in management and the introduction of disease-modifying drugs such as dexamethasone 32 .

Determinants of severity.
Current data suggest that the severity and outcome of COVID-19 are determined mainly by host factors rather than by viral genetic variation 33 (box 1). Although the latter might affect transmissibility 17,18 , large-scale, population-based data suggest that factors that predispose patients with COVID-19 to death include increasing age (>60 years), male sex, socioeconomic deprivation, pre-existing comorbidities (notably cardiovascular disease and diabetes mellitus) and a non-white ethnic background 34 . These data represent the combined effect of factors at all points in the disease trajectory, from initial infection to the development of severe disease and life-threatening complications. However, different risk factors might have an effect at different stages of the disease. For example, analyses based on hospitalized patients with COVID-19 confirm that people with pre-existing cardiovascular comorbidities have higher mortality, but the presence of diabetes had only a minor independent effect in this study 35 . Predicting outcomes from the phenotype and severity of clinical presentation can be challenging 36,37 , but can be aided by expanding the use of clinical, haemodynamic, imaging, biochemical, immunological and genetic biomarkers [38][39][40][41][42][43][44] .

Geographical distribution
The global epidemiology of COVID-19 is striking in its heterogeneity. Particularly high case and death rates (per 100,000 population) were observed in Europe, the Americas and parts of the Middle East during 2020 but relatively low rates were observed across much of Africa 3 (Fig. 2). Countries such as India initially showed a low number of deaths relative to reported cases 45 . Although some of this disparity might reflect differences in the infections with a fatality rate substantially greater than that of SARS-CoV-2; however, of these three viruses, SARS-CoV-2 transmits most efficiently between humans. b | Phylogenetic tree depicting the relatedness between SARS-CoV (from the 2003 SARS epidemic), SARS-CoV-2, and coronaviruses isolated from bats and civets. SARS-CoV-2 has ~97% nucleotide identity with the bat coronavirus RaTG13. c | Electron micrograph of a SARS-CoV-2 particle, covered in spike (S) proteins. d | Structure of a SARS-CoV-2 particle, showing the structural proteins of the virus. The particle has a diameter of ~125 nm. The trimerized S protein, which is the receptor-binding protein, gives the virion its crown-like appearance. The membrane (M) protein is the most abundant structural protein in the virus and seems to give the virus its shape. The third protein in the viral lipid envelope is the envelope (E) protein, which has ion channel activity and facilitates the assembly and release of the virion from the infected cell. The nucleoprotein (N) binds to the single-stranded RNA genome and regulates its structure and replication. extent of testing or the intensity of suppression strategies (such as lockdowns), many other factors are likely to be involved. Differences in population age structure and the rates of prevalent comorbidities might be important. The adaptive 46 and trained innate 47-50 immune systems have also been implicated in these differences. In addition, higher vitamin D levels in sunnier climates were speculated to be protective 51 . However, 2021 has seen a reversal of some of these trends, with substantial increases in the number of cases reported in many African countries, South and Central America, and Asia. India has seen the highest case numbers of any country since the beginning of the pandemic 52 . At the time of writing, probable contributors to this resurgence seem to include the spread of new SARS-CoV-2 variants, public behaviour and government policies 53,54 .

Ethnicity and socioeconomic factors.
Numerous reports from multi-ethnic regions in the UK and the USA suggest disproportionately higher mortality from COVID-19 in non-white groups than in the majority white population 34,55 . Children of non-white ethnicity are also over-represented among those admitted to hospital with COVID-19-associated inflammatory syndromes 56 . The relationship between ethnicity and the severity of COVID-19 is likely to be complex and might involve many factors, including worse socioeconomic deprivation in minority communities, larger and intergenerational households, a higher risk of occupational exposure, poorer access to health care and a higher burden of comorbidities. UK data suggest that individuals of South Asian ethnicity have worse outcomes than other minority groups for reasons that are unclear 57 ; genetic factors 58 and/or an amplified response to cardiovascular risk factors 59 might contribute (see next section). However, the effect of socioeconomic factors might be more nuanced in developing countries. A striking example is provided by a study in Mumbai, India, in which slum dwellers had a higher rate of infection but much lower death rates than non-slum residents (0.076% versus 0.263%), indicating a higher rate of infection and/or transmission but potentially less severe disease and higher levels of antibodies to SARS-CoV-2 (reF. 60 ), possibly as a result of a more robust trained innate immunity [47][48][49][50] .
Host genetics. Host genetic background can influence the severity of the initial effects of COVID-19 in several ways [61][62][63][64] . Rare or moderately rare host genetic variants can confer a dominant trait with regard to COVID-19 infection 65 . This situation is exemplified by the familial incidence of severe disease reported in two families of Dutch and African ancestries with monogenic, rare, putative loss-of-function variants of TLR7 on the X chromosome 66 . Other studies reported monogenic inborn errors of interferon immunity in patients with life-threatening COVID-19 (reFs 67,68 ). The influence of monogenic disorders in different populations needs to be studied further.
In addition, genome-wide association studies have shown that common variants in multiple loci can induce additive effects that weaken defence mechanisms and increase COVID-19 incidence and severity 65,[69][70][71] . Some of these studies might explain the heterogeneous global incidence described above 58,72 . A study from 2021 combined multiomics and machine learning approaches to decode the effects of sequence variation identified The table lists five of the 'variants of concern' of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that spread rapidly in different populations between October 2020 and April 2021, each with its numerical lineage number and the agreed single Greek letter designation. New variants are constantly being identified. a The mutations listed are those thought to be chiefly responsible for the increased transmissibility of the respective variants. These mutations are usually present at one or more of ~8 amino acid residues in the spike protein in the variants of concern. However, the viruses typically contain several additional mutations in the spike protein, outside the receptor-binding domain; therefore, they are more accurately referred to as variant lineages. (an analogue of ACE2), TMPRSS2 (encoding a cell receptor) and FURIN (encoding a host protease)) 75,78-91 and epithelial-to-mesenchymal transformation 231 . Studying the overlap between susceptibility genes for COVID-19 and those for other vascular diseases such as diabetes, atherosclerosis and pulmonary hypertension can further our understanding of the fundamental mechanisms of severity and the development of strategies to manage related pathways. Gene-regulatory mechanisms, such as DNA methylation, histone modifications and RNA-based mechanisms, might affect viral entry, replication, transcription and protein maturation [92][93][94] . Epigenetic regulation of genes encoding cell-entry proteins such as ACE2 (reFs 87,94-96 ), genes encoding proteins with innate immune functions involved in vaccine-induced increased immunity 97 and NETosis 98 , and genes encoding proteins with adaptive immune functions such as interferons 99 might explain the increased severity of COVID-19 in some patients. Given that epigenetic alterations are reversible, they are attractive drug targets and, therefore, research is now focusing on deciphering the epigenetic landscape of COVID-19 for the development and/or repurposing of drugs 92,93,100 .

Pulmonary vascular pathobiology in COVID-19
The major cause of death in patients with COVID-19 is hypoxic respiratory failure due to a combination of parenchymal and vascular changes, similar to those encountered in acute respiratory distress syndrome (ARDS) due to other causes 101 . However, several studies have suggested possibly specific features in ARDS caused by COVID-19 (reFs 38,102 ). In addition to lung parenchymal changes, such as diffuse alveolar damage with hyaline membranes and acute fibrinous and organizing pneumonia, important vascular changes have been observed in patients with COVID-19.

ARDS caused by COVID-19 versus other causes
Unlike in 'typical' ARDS, lung mechanics in patients with COVID-19 are preserved regardless of the severity of hypoxaemia 38 . This preservation was thought to be due to the presence of large intrapulmonary shunts and, importantly, the loss of regulation of lung perfusion by hypoxia, as suggested by the presence of hyperperfusion in areas with markedly reduced ventilation (gasless tissue) 38,103 . These changes could have important therapeutic implications for the management of these patients, such as using the lowest positive end-expiratory pressure to prevent further lung damage 38 .

Functional and structural changes in the arterial wall
Vascular changes affect the entire pulmonary vascular tree, from large-calibre vessels 104 to capillaries [38][39][40][41][42]104,105 . In patients with severe COVID-19, enlargement of the large-calibre vessels proximal to the opacities on pulmonary angiograms was observed during the acute phase of the disease 39,40 (Fig. 3a,c). In a study from the USA using CT pulmonary angiography, 41 out of 48 patients with COVID-19 had dilated lobar and subsegmental vessels within parenchymal radiological opacities 39 . In a study from the UK also using CT pulmonary angiography, 21 out of 33 patients with COVID-19 and at least two assessable lobes with no evidence of acute pulmonary emboli had dilated peripheral vessels, with 100% of the patients showing perfusion defects on dual-energy CT, suggesting microvascular dysfunction 41 (Fig. 3b,d).

Cellular changes in the arterial wall
All components of the vascular wall from the lumen to the perivascular regions can be affected and contribute to the overall pathobiology of COVID-19 (reFs 104,[106][107][108][109][110] ). The lumen can contain fresh microthrombi as well as chronic thrombotic lesions with acute or chronic inflammatory cells and fibroblasts, displaying varying degrees of organization (Fig. 4). However, the frequency of the microthrombi in patients with COVID-19 is strikingly varied in different case series. This observation is exemplified in one study in which fibrin thrombi were observed in only one out of eight patients with COVID-19 studied (perhaps because this patient cohort was recruited from a long-term care facility, with a high baseline prevalence of anticoagulation therapy) 108 . By contrast, fibrin thrombi were found in the alveolar capillaries in a series of seven patients who died from COVID-19 (reF. 104 ). The true incidence could be in between, as suggested by a study in which microthrombi were found in five out of 14 patients with COVID-19 (reF. 107  (reFs 104,106,111,112 ) (Fig. 5a). Changes in morphology include disruption of intercellular junctions, cell swelling and a loss of contact with the basal membrane 107 . These changes are commonly associated with the induction of inflammatory and pro-coagulant responses 111,113 . Another finding in pulmonary endothelial cells of patients with COVID-19 is the presence of higher levels of intussusceptive proliferation than in the pulmonary endothelium of patients with ARDS caused by infection with influenza A virus subtype H1N1 (reF. 104 ) (Fig. 5a).
Pericytes are mural cells present in the abluminal surface of blood vessels and have a pivotal role in blood vessel maturation and stability 114 . Studies performed using pericytes isolated from the lungs of patients with pulmonary hypertension as well as from a transgenic mouse model of pulmonary hypertension demonstrate note the immediate vicinity of lymphocytes (dark blue, round nuclei) and endothelial cells (arrows). This inflammatory pattern is not frequently encountered and seems also to involve post-capillary vessels, as shown. h | Elastic-type artery (>500 μm in diameter) containing a wall-adherent, organized thrombotic lesion with endothelium-lined, cushion-like intimal fibrosis protruding into the vascular lumen.
a dynamic contribution of pericytes to blood vessel remodelling in pulmonary hypertension 115,116 . ACE2, together with cathepsin B and cathepsin L, is highly expressed in microvascular pericytes in the heart, making them targets for SARS-CoV-2 infection 117 .
In addition, a single-cell and spatial atlas of autopsy tissues from patients with COVID-19 showed that pericytes from the lungs are positive for viral RNA, indicating that SARS-CoV-2 had entered these pericytes 118 .
Interestingly, post-mortem histological analysis of lung biopsy samples from patients with COVID-19 revealed pericyte loss or detachment from the pulmonary vasculature, which might lead to decreased ACE2 expression in the vascular microenvironment 119,120 (Fig. 5b). Similar findings in post-mortem studies have been reported by others 121 . The loss of pericytes in these patients is associated with a reduction in ACE2 receptors and could shift the humoral balance so that generation of angiotensin II by ACE would increase locally, leading to vessel constriction. Furthermore, pericyte loss or detachment would damage the endothelial barrier, leading to increased microvascular permeability, and would facilitate the entry of pro-inflammatory cells, which promote the 'cytokine storm' (Fig. 5b). Although doubts exist about the presence of pericytes on the capillaries of lung alveoli, a systematic electron microscopy study showed that pericytes were present in human lungs as well as in the lungs of dogs, guinea pigs and rats, whereas they could not be found in the lungs of smaller mammals 122 . The media in SARS-CoV-2 infection shows varying degrees of pulmonary arterial smooth muscle cell proliferation and de-differentiation 123 (Fig. 5c). These changes might be due to either direct or indirect effects of the SARS-CoV-2 virus. Patients who died from COVID-19 had increased pulmonary arterial medial wall thickness and reduced lumen area compared with pulmonary arteries from patients who died from influenza A virus subtype H1N1 infection 124,125 . Unpublished observations (S.S.P. and P.D.) in several autopsies and in three explanted lungs from patients with COVID-19 who underwent lung transplantation showed moderate muscular remodelling of the arterial wall (Fig. 5c). A single-cell, spatial landscape study of lung pathology in SARS-CoV-2 infection using high-parameter imaging mass cytometry showed a thickened medial layer with an increased number of cells positive for α-smooth muscle actin 125 . Patients with COVID-19 also have striking anomalies in the distribution of blood volume within the pulmonary vascular tree, similar to that in patients with pulmonary hypertension 126 . Of note, patients with COVID-19 have a substantial reduction in the pulmonary blood volume contained in blood vessels with a cross-sectional area of <5 mm 2 and a substantial increase in the pulmonary blood volume contained in vessels with a cross-sectional area of 5-10 mm 2 or >10 mm 2 (reFs [126][127][128] ). All these changes could predispose patients to chronic pulmonary hypertension in the future 123 .
The adventitia and perivascular region have been shown to have a major regulatory effect on the function of the vascular wall 129,130 . Interestingly, in specimens from patients with COVID-19 who underwent lung transplantation, we have observed massive and uniform interstitial fibrosis of the lung parenchyma involving the pulmonary vasculature, including the adventitia (S.S.P. and P.D., unpublished observations). Moreover, we have noted the contribution of the pulmonary arterial adventitia to the fibrotic process, which suggests that the interstitial fibrosis observed in various patients with COVID-19 might be caused by diffuse pathological remodelling of fibroblasts or myofibroblasts in response to SARS-CoV-2 infection (Fig. 5d). Perivascular inflammation is a common finding in patients with advanced COVID-19 (reF. 104 ). This finding is in accordance with unpublished observations (S.S.P. and P.D.) in patients with COVID-19 who died at the end of the first or during the second wave of infection, all of whom received anticoagulant therapy. These patients all showed intravascular neutrophil aggregations (Fig. 4a) and moderate perivascular lymphocytic infiltrates (Fig. 4b,c) at the microvascular level (arterioles and/or venules <100 μm in diameter). Phenotyping of these infiltrates demonstrated a clear T cell predominance, with the presence of both CD4 + and CD8 + cells (Fig. 4d-f). Only some patients with abundant interstitial and microvascular inflammation showed a transmural inflammatory cell infiltrate involving the intima, which led to variable endothelialitis (Fig. 4g,h).

Mechanisms of vascular lung injury
Following entry of the SARS-CoV-2 virus into different cells 131,132 , the exact mechanisms of injury remain controversial 44,111,112 . Several mechanisms have been suggested, including the induction of apoptosis or autophagy 133 , and involve several pathways.

Virus-cell interaction
With regard to SARS-CoV-2 virus entry into pulmonary vascular cells, most reports indicate that endothelial cells are a primary target of the virus 118 . However, this proposal has been questioned for two main reasons: the size of SARS-CoV-2 viral particles shown in the electron microscopic images of earlier studies are four times larger than that of the expected virion size 120 and the lack of detectable expression of two important receptors (ACE2 and TMPRSS2) 112 in endothelial cells through which SARS-CoV-2 gains entry. ACE2 receptors have been shown to be essential for the entry of SARS-CoV-2 into cells, and soluble recombinant ACE2 can inhibit viral infection in animal models and tissue-engineered organoids 134 . In addition, in vitro infection studies using endothelial cells also provided no evidence that the virus could directly infect these cells 112 . Therefore, whether SARS-CoV-2-induced lung endothelialitis is caused by direct viral infection of endothelial cells or by indirect perivascular inflammation and complement activation is uncertain. Understanding how SARS-CoV-2 causes endothelial dysfunction and pulmonary vascular abnormalities is of paramount importance.
An alternative route is that the SARS-CoV-2 virus enters smooth muscle cells and/or fibroblasts. The human ACE2 receptor is widely expressed in arterial smooth muscle cells across multiple organs, including the lungs 131 . Lung fibroblasts express both ACE2 and TMPRSS2 (reF. 132 ), and data from single-cell sequencing studies showed high levels of ACE2 expression in pericytes 117,122,135 , suggesting that the alteration of adventitial fibroblasts and pericytes might be linked to direct or indirect SARS-CoV-2 infection 122 . Furthermore, a study has demonstrated that hypoxia upregulates the expression of ACE2 in human and rat pulmonary arterial smooth muscle cells 136 , which lends support to the theory that SARS-CoV-2-induced hypoxia might further exacerbate the disease. As in pulmonary hypertension, sex hormones and age can also influence ACE2 expression (levels are lower in women 137 and increase with age 138 ), which might explain the observed sex-specific differences in COVID-19 (reF. 137 ). The SARS-CoV-2 spike protein alone, without additional viral components, is enough to elicit aberrant growth signalling (increased MEK phosphorylation) in human cultured pulmonary arterial smooth muscle cells 124 . MEK phosphorylation is associated with de-differentiation, nuclear activation and proliferation of pulmonary arterial smooth muscle cells in pulmonary hypertension 139 . More detailed studies to define the exact route of entry of SARS-CoV-2 into the arterial wall as well as the long-term effects of SARS-CoV-2 infection on the pulmonary microvasculature are urgently needed.

Pathways involved in vascular lung injury
Several pathways (hypoxia, inflammation and complement activation) have been proposed as possible drivers of pulmonary vascular injury mediated by SARS-CoV-2 infection.
Hypoxia. Hypoxia pathways have important roles in health and disease as highlighted by the recipients of the Nobel Prize in Physiology or Medicine in 2019 (reFs [140][141][142] ). In addition, hypoxia has been shown to have an important role in virus-host interactions, including in COVID-19 (reFs 143,144 ). Silent hypoxaemia, despite an apparent lack of dyspnoea, is observed in patients with COVID-19 (Fig. 6). Several reports revealed that the prevalence of silent hypoxia in patients with COVID-19 ranges from 20% to 40% 145,146 . However, the pathophysiology of silent hypoxaemia in COVID-19 lung injury and its influence on the pulmonary vasculature is still poorly understood. Hypoxia itself is known to have substantial deleterious effects on the cells of the lungs, in particular endothelial cells lining the pulmonary arteries and capillaries 147,148 , caused by various mediators, including activation of the NF-κB transcription factor 149,150 . Moreover, hypoxia can result in the overexpression of ACE2 receptors, thereby increasing the risk of damage through SARS-CoV-2 infection 136 . In addition, hypoxia can contribute to the mechanisms of cytokine storm 142 and complement activation 151 .
Inflammation. Several inflammatory pathways are involved in the severe vascular injury associated with COVID-19 (reFs 104,[152][153][154] in the pulmonary and extrapulmonary vasculature, affecting both the macrovasculature and the microvasculature 104 (Fig. 7). The underlying molecular mechanisms might include direct endothelial cell infection by SARS-CoV-2, www.nature.com/nrcardio which subsequently leads to endothelial damage and a shift to a pro-coagulant state 104 ; cytokine storm (a sudden and large increase in the circulating levels of classical pro-inflammatory cytokines, such as IL-1β, IL-6 and tumour necrosis factor, which can induce inflammation of the alveolar space and immunothrombosis of the adjoining pulmonary vasculature 155 ); or a type 3 hypersensitivity reaction as reported in some patients with COVID-19, which leads to a procoagulant environment in the vasculature 156 . Coagulation is closely linked to endothelial inflammation signalling; inflammatory moieties on the endothelium increase leukocyte infiltration and alter coagulation control, promoting a pro-coagulant status 157 . Together, these inflammatory and immunological changes have a major role in COVID-19-induced pulmonary vascular pathobiology and chronic pulmonary hypertension 158,159 .

Complement.
Several studies have shown that the complement system is activated in an uncontrolled manner in response to SARS-CoV-2 infection 44,104,160,161 . Strong immunohistochemical signals for many different complement components were detected in the lung tissue of patients with COVID-19, and complement deposition is also observed in the damaged lung microvasculature 44,162 . Complement components have also been found to be deposited in type 1 and type 2 alveolar epithelial cells as well as in inflammatory cells and their exudates (hyaline membranes) in the alveolar spaces filled with necrotic debris 163 . This deposition in turn triggers the activation of the membrane attack complex, causing microvascular endothelial injury and activation of the clotting pathways. High levels of d-dimer have also been measured in samples in which complement activation has been documented, which might in part explain COVID-19-associated coagulopathies 160 .  -19). The figure shows the receptor-mediated transduction pathways from receptors to second messengers and ultimately to the nucleus and/or mitochondria. Targeting these pathways using selective anti-inflammatory drugs (pink boxes) might be therapeutically beneficial in patients with COVID-19. AAK1, AP2 associated protein kinase 1; ADAM17 , a disintegrin and metalloproteinase 17; AP1, activator protein 1; ARB, angiotensin II receptor blocker; AT2R, angiotensin II receptor; COX2, cyclooxygenase 2; GHR, growth hormone receptor; ICAM, intercellular adhesion molecule; IFNγ, interferonγ; JAK, Janus kinase; IκB, inhibitor of NF-κB; mTOR, mechanistic target of rapamycin; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MyD88, myeloid differentiation primary response protein MyD88; NF-κB, nuclear factor-κB; NSAID, non-steroidal anti-inflammatory drug; PI3K, phosphoinositol 3-kinase; PPAR, peroxisome proliferative activator receptor; RAF, RAF protooncogene serine/threonine-protein kinase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; STAT, signal transducer and activator of transcription; sTNF, soluble tumour necrosis factor; TAK1, nuclear receptor subfamily 2 group C member 2; TGFβ, transforming growth factorβ; TNF, tumour necrosis factor; TLR, Toll-like receptor; TRADD, TNF receptor type 1-association DEATH domain protein; TRAF, TNF receptor-associated factor; TZD, thiazolidinedione; VCAM, vascular cell adhesion molecule. Adapted with permission from reF. 152 , Elsevier.
Obliterating endothelialitis was associated with an accumulation of complement component 5a receptor 1 (C5aR1)-positive macrophages around the arteries and in the thrombus 164 (Fig. 8a,b). C5a can also promote the secretion of CCL2, leading indirectly to the recruitment of inflammatory cells. The levels of soluble C5a increased proportionately to the severity of COVID-19, and high levels of C5aR1 expression were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of ARDS 164 . Complement activation in plasma from patients with COVID-19 was greater than in patients with influenza-associated lung injury 165 . The alternative pathway is activated in patients with COVID-19 and is associated with worse outcomes. Higher complement levels identified those patients at increased risk of worse outcomes 165 . These observations support the hypothesis that complement activation and C5a production lead to the chemoattraction and activation of myeloid cells in the lungs and contribute to the excessive release of inflammatory cytokines in SARS-CoV-2-infected lungs 164 (Fig. 8c).
Complement component C3 exacerbates disease in SARS-CoV-associated ARDS 166 . A study in C3-deficient mice showed that they were protected against SARS-CoV lung injury despite a high viral load and that fewer neutrophils and inflammatory monocytes were present in their lungs 166 . This finding has raised interest in using anti-complement regimens for the treatment of COVID-19. C3 activation is upstream of most anaphylatoxin signalling (C3a, C5a) and the formation of membrane attack complex-C5b-9. C3 is implicated as an initial effector mechanism that can exacerbate injury. These observations have led to the initiation of several small clinical trials using various anti-complement strategies. Complement C3 inhibitor AMY101 was safely and successfully used to treat a severe case of ARDS in a patient with COVID-19-related pneumonia 167 . In another study, four patients with COVID-19 were treated with the C5-blocking monoclonal antibody eculizumab, which reduced the levels of inflammatory biomarkers 168   www.nature.com/nrcardio not severely ill and had only moderately elevated levels of C-reactive protein, which nevertheless decreased with eculizumab treatment from 14.6 mg/dl to 3.5 mg/dl. In a third study, treatment with the monoclonal antibody LFG316 blocked C5 activity and complement activation for ≥4 days 169 . Four out of five patients with severe COVID-19 showed a sustained improvement in their clinical state, which persisted beyond the duration of C5 blockade 169 . These results suggest that transient blockade of C5 is sufficient to interrupt the hyperinflammatory cycle in COVID-19 and allow recovery. The findings also suggest that prolonged complement blockade might not only be unnecessary for patient benefit but might actually be harmful by increasing the risk of infection.
Therefore, accumulating evidence supports the idea that interrupting activated complement signalling might be beneficial in patients with severe COVID-19. However, to date, the number of patients tested has been small and, importantly, no randomized trials have been performed. Much work needs to be done to understand how and when to target the complement cascade to ameliorate SARS-CoV-2-related lung disease.

Chronic sequelae of COVID-19
Following the acute phase, many patients with COVID-19 continue to have symptoms due to persistent damage in several organs, including the lung vasculature 170,171 , and the sequelae of this damage such as chronic lung fibrosis and possibly chronic pulmonary hypertension 170 . Given that the COVID-19 pandemic started only in 2019, no long-term data similar to those reported in SARS and MERS or in ARDS due to other causes 172,173 are so far available. However, several specific pathological changes during the acute phase could be used to predict chronic changes.

Lung fibrosis during or after acute COVID-19
Lung fibrosis is increasingly being recognized as one of the important sequelae of COVID-19 (reFs [174][175][176][177][178] ). The exact incidence depends on the radiographic criteria used for diagnosis. Using artificial intelligence with the development of a lung fibrosis score, researchers analysed a series of 284 patients discharged from hospital after COVID-19 and reported lung fibrosis in 100% of patients who had had severe disease and in 78.9% of those who had had moderate disease 179 . Lung fibrosis is attributable to chronic vascular ischaemia as well as parenchymal inflammation 179 . Probable risk factors for developing this complication include air pollution, smoking, exposure to toxins, infections and other factors that cause lung injury 176 . Certain radiological features of the lung opacities, age, IL-6 levels and other markers of the severity of inflammation have been shown to be independent risk factors for the development of lung fibrosis 179 (Fig. 6a). The rate of progression and the influence of lung fibrosis after COVID-19 on long-term prognosis are still unknown.

Chronic pulmonary hypertension after COVID-19
The pathobiology of the pulmonary vasculature in patients with COVID-19 shares many features with that of the pulmonary vasculature in patients with pulmonary hypertension 159,[180][181][182][183] . Medial hypertrophy and smooth muscle cell proliferation, essential features of pulmonary hypertension, have been reported in acute COVID-19 (reF. 123 ) (Fig. 5c). In addition, the reported intravascular clotting in the lung vessels of patients with COVID-19 during the acute phase 39,41,170,[184][185][186][187] could predispose these individuals to chronic thromboembolic pulmonary hypertension 171,185 in the future.
Furthermore, identified genetic drivers of chronic pulmonary hypertension [188][189][190] are similar to the putative genetic factors that influence the severity of COVID-19 (described above). These similarities could have important implications for the incidence and severity of chronic pulmonary hypertension following SARS-CoV-2 infection and therefore need to be studied further.
The prevalence of pulmonary hypertension during the acute phase of COVID-19 is fairly high. In 200 consecutive patients with COVID-19 disease who were admitted to non-intensive care units in Milan, Italy, the prevalence of pulmonary hypertension (defined as a systolic pulmonary artery pressure >35 mmHg) and right ventricular dysfunction was 12.0% and 14.5%, respectively 191 . Another study from Sweden published in 2021 reported that 26 out of 67 patients (39%) with severe COVID-19 had acute pulmonary hypertension 192 . Taken together, the available evidence suggests that the pulmonary vascular pathology during the acute stage of COVID-19 can predispose these patients to develop chronic pulmonary hypertension.

Interaction between COVID-19 and pulmonary hypertension: a global perspective
Pulmonary hypertension is increasingly considered to be a global disease [193][194][195] . Similarly, COVID-19 has affected all continents, including remote areas. The two diseases interact at both 'individual' and global levels.

The 'individual' level.
Evidence on the effect of pulmonary hypertension PAH on the severity and outcomes of COVID-19 is conflicting 196,197 . An observational study from a large pulmonary hypertension centre in New York City, USA, showed that pre-existing chronic pulmonary hypertension has major adverse effects on both the clinical course and mortality associated with COVID-19 (reF. 198 ). By contrast, another study on data from 32 pulmonary hypertension centres in the USA reported a paucity of hospitalized patients with both pulmonary hypertension and COVID-19, and those who developed COVID-19 had mild symptoms and tended to recover quickly 199 . In a large, international survey from 47 pulmonary hypertension centres, including patients with pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension, the findings were mixed in terms of the number of reported cases per centre in different countries (ranging from 0 to 19) and the number of deaths (ranging from 0 to 6) 200 , and we agree with Farha and Heresi 201 that the interaction between COVID-19 and chronic pulmonary hypertension at the individual level requires further intensive investigation.
Nearly 140 million people live in high-altitude locations worldwide in a hypoxic environment with an increased prevalence (5-18%) of pulmonary hypertension 214,215 . As discussed above, hypoxia has a major and varied role in the pathophysiology of COVID-19 (reFs 103,202,[216][217][218] and might therefore influence the phenotype and outcome of COVID-19 disease in these high-altitude locations. However, evidence on the incidence and progression of COVID-19 at high altitudes is incomplete and contradictory 202,219-221 , with one study reporting that high altitude was protective 222 . The presence of endemic infections and polyparasitism has been identified as an important driver of pulmonary hypertension at the global level 193,194,223 . Many infectious diseases, in particular schistosomiasis, HIV infection and, potentially, tuberculosis, can induce pulmonary vascular diseases 207-210 by altering the Zinnate and adaptive immune response to COVID-19 (reF. 224 ) and by inducing herd immunity 225 .
Pulmonary hypertension due to toxins and the use of illicit drugs is a major problem of global dimensions 226 . The effect of these substances on COVID-19 infection is the subject of ongoing studies 227,228 . For example, in a retrospective study of the electronic health records of 73,099,850 patients in the USA, patients with substance use disorder had an increased risk of COVID-19 (adjusted OR 8.699, P <10 -30 ) 227 .
Taken together, the emerging picture of an intense interaction between COVID-19 and chronic pulmonary hypertension at the global level is anticipated to have an important effect on the future of both diseases.

Conclusions
In this Review, we have presented a detailed description of the pathobiology of the pulmonary vasculature in COVID-19 and its potential interactions with pulmonary hypertension and other sequelae. These observations could be of value in developing novel strategies to deal with the ongoing COVID-19 global pandemic.
Published online 6 December 2021