Alterations in microcirculatory oxygenation in long-COVID

The COVID 19 pandemic has had a profound impact, and the past years research
has led to an accelerated effort towards understanding the consequences of
SARS-CoV-2 infection. It is now known that COVID-19 is a multi-system disease
affecting various organs in the body, characterized by a dysregulated immune
response resulting in a cytokine release storm (1). Systemic inflammation as
well as direct invasion of endothelial cells by the virus leads to endothelial
dysfunction, which disrupt vasomotor function and can impair tissue perfusion
and thus oxygenation (2) (3).

Microcirculatory tissue oxygenation
A substantial part of patients with COVID-19 disease develop long-COVID. This
is defined as lasting symptoms, sequelae or abnormal clinical parameters
existing twelve weeks or more after infection with SARS-CoV-2 onset (4).
Symptoms include dyspnea, fatigue, and mental and cognitive disorders (5). A
large group of patients are severely limited in physical exercise and report
functional impairment, despite having normal cardiopulmonary function (6) (7),
especially post-exertional malaise (PEM). PEM is the delayed onset of new
symptoms or worsening of symptoms 12-48 hours after physical or cognitive
exertion (8). Furthermore, as opposed to the systemic hypoxia in acute COVID19
infection, these patients have normal systemic oxygen saturation values (6)
(7). Previous studies showed that symptoms of fatigue and subjective dyspnea
were not related to impaired pulmonary gas exchange, with normal systemic pO2
and pCO2 during exercise exhaustion (9). However, peak VO2 was impaired in
these patients during cardiopulmonary exercise testing, suggesting alterations
in oxygen uptake and/or utilization in tissues. This was accompanied by
decreased oxygen pulse, which is the ratio of oxygen consumption to heart rate
and reflects maximal aerobic capacity. Given adequate systemic O2 levels, these
data are pointing towards impaired oxygen regulation on the level of the
microvasculature. Recent data support this by showing promising results of
treatment with hyperbaric oxygen therapy and exercise with oxygen therapy (10)
(11).

The microcirculation is a key player in regulation of oxygen delivery to
tissue, and in healthy people recruitment of the microvasculature occurs in
response to metabolic demands such as during exercise. Interestingly,
alterations found in the microvasculature of acute COVID-19 patients could
reflect adaptations to the systemic hypoxia. A previous study from our group
using direct observation and quantification of microcirculatory red and white
blood cells sublingually using handheld vital microscopy (HVM), found increased
capillary density and red blood cell availability, indicating recruitment of
the microcirculation (12). Capillary hematocrit was increased, with a shift
from red blood cells from the systemic- to the microcirculation. Possibly,
capillary recruitment and recruitment of red blood cells to the
microcirculation are adaptations to systemic hypoxia, in order to maintain
tissue oxygen availability and extraction in the hypoxic state. Similar
compensatory mechanisms are observed in experimental hypoxia (13) (14) but also
in high-altitude studies (15) (16). These adaptations allow for toleration of
low systemic arterial oxygen pressures. Interestingly, the most severely ill
patients with COVID-19, as indicated by a SOFA score>10 points, did not show
the increase in capillary density, nor the increase in capillary hematocrit.
Possibly, the most severely ill patients lack this adaptive response to
maintain tissue oxygenation.

In resting patients with long-COVID, a decreased density of small capillaries
was found. This was accompanied by increased red blood cell velocity, possibly
compensating for the loss of capillary density. However red blood cell velocity
in the microcirculation was shown to be dependent of flow in feed vessels,
which may suggest flow is dependent on these feed vessels (17). The flow being
dependent on feed vessels could indicate impaired capillary recruitment to
tissue demands. Another study showed that post-occlusion reactive hyperemia was
altered after COVID-19 (18).

Mitochondrial dysfunction
A recent study demonstrated alterations in mitochondrial function in muscles
from patients with long COVID who experience post-exertional malaise (PEM).
This could imply that there are also defect on the level of O2 utilization,
which contributes to tissue hypoxia and symptoms.

Angiogenesis
A consequence of tissue hypoxia on a microvascular level is induction of
angiogenesis pathways. These encompass cellular responses triggered by cellular
hypoxia, that eventually lead to the formation of new blood vessels. Vascular
endothelial growth factor-A (VEGF-A) other angiogenesis markers angiopoeietin-1
(ANGPT1) and P-selectin were shown to be increased in patients with long COVID
and predictive of development of long COVID syndrome (19). In line with this,
the hypoxia inducible factor 1 (HIF-1) pathway was associated with COVID-19
that progressed to long-COVID which progressed proportionally with disease
severity (20). So-called pro-angiogenic cells (PAC) (a subtype of monocytes)
play important roles in the regulation of angiogenesis and neovascularization,
primarily by the production of cytokines and other mediators (21).

Hypothesis and aims
These data suggest a key role for alterations in microvascular function and
microvascular - not systemic - tissue oxygenation in the development of
long-COVID syndrome. The endothelium has a vital role in maintaining
microvascular function and allows the microcirculation to respond to tissue
demands and increase flow when needed. Therefore we hypothesize that
inflammatory-induced alterations in endothelial function lead to loss of normal
anti-inflammatory and anti-thrombotic function of endothelial cells, which then
contribute to impaired microcirculatory flow and oxygen delivery to tissues in
long-COVID.

In this study we aim to unravel how symptoms of long-COVID patients are related
to alterations in microcirculatory function and oxygen delivery, and the role
of inflammation, endothelial activation and coagulation.

Endothelial activation
SARS-CoV-2 infection via ACE2 receptors leads to a severe inflammatory
response, which helps to control and limit viral replication (1). However, the
immune response can become dysregulated, leading to an excessive and prolonged
inflammatory response, the "cytokine storm", which is the main contribution to
tissue damage rather than direct infection with the virus. The cytokine storm
involves the overproduction of pro-inflammatory cytokines, such as
interleukin-1* (IL-1*), interleukin-6 (IL-6), interleukin-8 (IL-8). The
pro-inflammatory cytokines cause endothelial activation with resulting loss of
vasomotor function, but also of normal anti-inflammatory and antithrombotic
function of endothelial cells.

Inflammation
IL-6 enhances the expression of adhesion molecules vascular adhesion molecule-1
(VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on
endothelial cells, which promote recruitment of leukocytes into the vascular
wall (22) (23). Indeed, our group demonstrated there was an increased number of
leukocyte aggregation visible in the microcirculatory vessels of COVID-19
patients (12). The level of leukocytes in the microcirculation was associated
with higher disease severity (SOFA score). Previous studies have also shown
that pro-inflammatory cytokine levels remain elevated after initial COVID-19
infection (24). In addition, the inflammatory response triggers formation of
neutrophilic extracellular traps (NETs) which are formed upon neutrophil
activation. They contain DNA and histones and form matrix networks of
extracellular fibers that stimulate the cytokine storm further. Increased
NETosis was demonstrated in patients with long-C

Primary Objective:
To study alterations in microcirculatory oxygenation during exercise in
patients with long-COVID in relation to clinical symptoms.

Secondary Objectives:
To study in patients with long-COVID compared to convalescent controls
• Extensive disease characterization (symptoms, quality of life, PEM)
• Systemic oxygenation at baseline and after exercise
• Microcirculatory tissue oxygenation and oxygen consumption at baseline and
after
exercise
• Microcirculatory function and recruitment at baseline and after exercise
• Angiogenesis pathways (serum levels of angiogenesis markers)
• Endothelial activation (soluble markers of endothelial activation)
• Persistent inflammation (leukocytes, serum inflammatory markers, monocyte
activation, NETs)
• Pro-coagulation (serum coagulation parameters, circulating coagulation
complexes, platelet activation, thrombus formation potential, pro-angiogenic
cells (PACs), the presence of microthrombi, vascular transformation markers of
angiogenesis)
- In vivo and in vitro mitochondrial function

This study is a comparative, non-randomized, observational study. We will
include a total of 54 long-COVID patients and 52 controls. For this study
questionnaires will be taken online to characterize clinical symptoms in all
patients. During a study visit, sublingual microcirculation and oxygenation
measurements, as well as NIRS will be performed before and after exercise by a
one-minute sit-to-stand test at the post-COVID outpatient department for 27
long-COVID patients and 26 controls. In addition, blood samples will be drawn
to determine markers of angiogenesis, endothelial activation, inflammation and
coagulation in all 54 long-COVID patients and all 52 controls.

The 27 long-COVID patients and 26 controls that underwent the entire protocol
will be asked to return for a second study visit after six months. Then, the
study protocol will be repeated and noninvasive in vivo mitochondrial function
measurements will be performed.

The individual patients participating in this research will not benefit
directly from the results of our research. Sublingual measurements of the
microcirculation and tissue oxygenation are noninvasive and have no risk. They
can however cause some discomfort, especially during the vascular occlusion
test. Patients are asked to perform physical exercise with a one minute
sit-to-stand test and this may cause fatigue. Blood withdrawal can cause
discomfort. The noninvasive mitochondrial pO2 measurements are performed using
a plaster which can cause skin irritation and pruritis, and hyperpigmentation
when exposed to the sun.

The results could lead to new insights, that are highly relevant in
understanding the pathophysiology of long COVID in general. The population of
long COVID syndrome patients in the outpatient clinic is highly motivated to
contribute to research aiming to unravel the pathophysiology of their disease.