Background
Persistent symptoms for more than six months following mild to moderate coronavirus disease-2019 (COVID-19) are reported in 10–30% of patients [
1‐
3]. The WHO recently defined the post-COVID-19 condition as a state persisting at least three months from the onset of COVID-19 with common symptoms such as fatigue, post-exertional malaise and cognitive dysfunction impacting everyday functioning.
In our observational longitudinal PA-COVID Fatigue study of PCS patients with persistent moderate to severe fatigue and exertion intolerance for more than six months after mild to moderate COVID-19, we found that approximately 50% of patients fulfilled the diagnostic criteria of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), but many PCS patients not fulfilling these criteria were equally impaired [
4]. ME/CFS is a complex disease frequently triggered by an infection with Epstein-Barr virus (EBV) or parvovirus B19, but several other viral and nonviral triggers have been described [
5‐
7]. Postexertional malaise (PEM), which describes a disproportional aggravation of symptoms typically lasting for more than 14 h up to several days following a mild mental or physical exertion is a key symptom of ME/CFS [
8].
The pathomechanism of ME/CFS is not well understood, but there is ample evidence for impaired perfusion and endothelial dysfunction (ED) [
7,
9‐
12]. ED is characterised by a diminished bioavailability of vasodilators, while on the other hand, endothelium-derived vasoconstrictors are increased, leading to impaired endothelium-dependent vasodilation [
13,
14]. In the context of infection with SARS-CoV-2, the potential role of ED is of particular interest, as the virus can directly infect the endothelium by engaging the Angiotensin-Converting Enzyme (ACE) 2 receptor [
15]. In acute COVID-19, there is evidence for vascular endothelial cell infection, endotheliitis and microthrombosis across multiple vascularized tissues [
16,
17].
In this study, we aimed to characterise peripheral endothelial function using postocclusive reactive hyperaemia peripheral arterial tonometry (RH-PAT) in PCS patients following mild to moderate COVID-19. In addition, we analysed several endothelial biomarkers including Endothelin-1 (ET-1), Angiopoietin-2 (Ang-2) and Endocan (ESM-1) which play an important role in inflammatory and noninflammatory diseases associated with ED [
18‐
22]. Furthermore, we assessed Interleukin 8 (IL-8), Angiotensin-Converting Enzyme (ACE) and ACE2. IL-8 is secreted not only by monocytes/macrophages but also by endothelial cells and vascular smooth muscle cells. This chemokine plays an important role in (endothelial) inflammation and regulation of leukocyte rolling as well as vascular permeability [
23]. ACE and ACE2 are crucial actors in the maintenance of blood pressure and vascular homeostasis [
2]. ACE converts angiotensin I into the vasoconstrictive angiotensin II, which activates angiotensin II receptors. Counterregulatory ACE2 cleaves angiotensin I into angiotensin 1–9 and metabolises angiotensin II to angiotensin 1–7. Peptides generated by ACE2 are ligands of the receptor Mas, which triggers protective, vasodilative signalling [
24].
Discussion
Long COVID is a poorly understood condition with multiple features and a broad range of symptoms. Endothelial infection in COVID-19 may have long-term consequences for vascular function. In our present study, we found ED and dysregulated levels of the endothelial markers ET-1 and Ang-2 in a subset of PCS patients eight months, on average, after mild to moderate COVID-19.
In acute COVID-19, there is ample evidence for ED and impaired microcirculation [
16,
34]. SARS-CoV-2 alters vascular homeostasis by directly infecting endothelial cells via ACE2 [
16]. ACE2 is expressed in arterial and venous endothelial cells [
35] and is internalized and downregulated after binding of the virus. The local angiotensin II hyperreactivity that is triggered by this action leads to the progression of inflammation and fibrosis [
36]. In addition to direct injury to the vascular endothelium by endothelial cell infection, inflammatory mediators can also contribute to endotheliitis and endothelial cell injury [
37]. There is also evidence for endothelial damage, especially in pulmonary microvascular cells, by apoptosis or autophagy in postacute COVID-19 [
16,
38].
Furthermore, there is evidence of ED occurring in patients following infection with SARS-CoV-2. A recent study analysed endothelial function using EndoPAT technology in patients during the acute infection as well as a median of 100 days post-COVID-19; the study reported impaired RHI in the postinfection stage only [
39]. However, in this study, no information about the severity of acute COVID-19 or symptom persistence was reported. Another study of patients after severe acute COVID-19 found impaired ED in half of them, which was associated with fatigue, chest pain, neurocognitive difficulties and severity of the acute COVID-19 [
40]. The patients included in our study all had mild to moderate COVID-19. Thus, from our rather homogeneous patient cohort, we cannot draw conclusions regarding the impact of acute COVID-19 severity on endothelial function. A recent study reported elevated levels of circulating endothelial cells in COVID-19 convalescents on average 34 days post-symptom onset as a biomarker for ED associated with levels of several cytokines [
41].
ED has been described in non-COVID-19 postinfectious ME/CFS [
9‐
12]. In our previous study of 35 ME/CFS patients, peripheral ED assessed by RH-PAT was observed in half of the patients and was associated with disease severity [
9]. A recent study of postinfectious ME/CFS confirmed these findings by demonstrating ED through both RH assessment and flow-mediated dilatation [
11]. ED resulting in muscle and cerebral hypoperfusion are considered key pathomechanisms in ME/CFS [
7,
12,
42].
The RHI in healthy individuals is usually inversely associated with age, blood pressure, BMI and further known cardiovascular risk factors [
43], which we also observed for RHI and age in our HC group. Surprisingly, we found a paradoxical positive correlation of the RHI with age, blood pressure and BMI in PCS which may suggest that ED develops independently of classical cardiovascular risk factors in these patients and that vascular stiffness may even help to stabilise the vascular diameter. Further comparative studies of patients with PCS and of post-COVID reconvalescents are required to provide evidence whether a diminished RHI might be associated with PCS.
Remarkably, PCS patients with and without ME/CFS showed elevated levels of the endothelial biomarker ET-1, while reconvalescents had normal levels. Endothelins are the most important, potent vasoconstrictors and are produced by endothelial cells [
18,
19]. ET-1 mediates vasoconstriction via the ETA receptor, which is mainly located on vascular smooth muscle cells. Thus, our findings may indicate hypoperfusion in PCS, which is in line with a recent study in PCS patients with exertion intolerance. Data from cardiopulmonary exercise testing (CPET) provide evidence for a marked reduction in oxygen consumption during exercise, which is attributed primarily to reduced oxygen diffusion in the peripheral microcirculation [
44]. Targeting the ETA and ETB receptors via an antagonist improved the peripheral endothelial function defined by RHI in patients with type 2 diabetes [
45]. Thus, ET-1 may be both a biomarker of endothelial involvement and a therapeutic target in PCS.
Ang-2 belongs to the angiopoietin/tie-2 pathway that regulates endothelial homeostasis and angiogenesis. In the present study, non-ME/CFS PCS patients unexpectedly showed reduced serum Ang-2 levels compared to those in HCs. Ang-2 expression is increased during COVID-19 infection presumably due to endothelial inflammation [
20]. Endothelial cells were shown to downregulate Ang-2 expression under high flow and shear stress [
46]. Thus, a possible explanation may be the occurrence of high shear stress in PCS due to chronic inflammation or endothelial damage [
47]. Remarkably, Ang-2 was also diminished in reconvalescent PCHC, which may also indicate a longer lasting change in vascular perfusion in asymptomatic individuals. With increased levels of ET-1 in both patient groups, the finding of decreased Ang-2 levels exclusively in PCS could provide a starting point for differentiation between PCS and ME/CFS in terms of biomarker profiles.
As reported in our previous study [
4], we found elevated levels of the pro-inflammatory mediator IL-8 in approximately 60% of PCS patients. As IL-8 is rapidly degraded in serum, its concentration was determined in lysed erythrocytes, which bind IL-8 via a Duffy antigen receptor [
48,
49]. Endothelial cells and monocytes are the main producers of IL-8 [
23,
49]. IL-8 promotes endothelial cell migration and proliferation as well as survival and endothelial permeability [
23,
50]. Elevated IL-8 levels were described in patients with severe as well as mild COVID-19 and correlated with disease prognosis [
51]. Therefore, IL-8 might indicate ongoing endothelial inflammation in our PCS patients.
Conclusion
In conclusion, our study found that a subset of PCS patients had a diminished RHI, indicating peripheral ED. A limitation of our study is the lack of a reconvalescent cohort for RHI assessment; thus, we do not know whether a diminished RHI is associated with PCS symptoms. Elevated ET-1 levels were, however, found in PCS patients only, indicating that endothelial hypoperfusion plays a role in PCS and providing a rationale for therapeutic targeting. The paradoxical association of RHI with age, blood pressure and BMI as well as diminished Ang-2 may indicate a distinct pathomechanism in the non-ME/CFS PCS subgroup.
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