Post-COVID syndrome is associated with capillary alterations

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A new German study has found that capillary alterations, distinct transcriptomic signatures in skeletal muscle and macrophage infiltration are features of Long COVID.

The study findings were published on a preprint server and is currently being peer reviewed.
https://www.medrxiv.org/content/10.1101/2023.02.15.23285584v1

The individual and socioeconomic impact of post-COVID syndrome and other post-infectious syndromes such as ME/CSF is considerable (21, 47, 48). Affected patients often face a double challenge, the one of the direct physical and mental suffering, and the one of the psychological burden of being affected by a disease with no clear biomarkers and absence of clear-cut, easily observable structural alterations.

Therefore, studies on the tissue level as well as translational integrative studies combining clinical observations with histopathological and molecular findings are urgently needed. With all the tragedies related to the SARS-CoV-2 pandemic, it also offers the unique opportunity for studying post-viral syndromes in a more homogeneous manner than previously.

To our knowledge, this is the first case-control study examining skeletal muscle tissue obtained from patients with persisting post-infectious fatigue and exercise intolerance that newly occurred after an infection with SARS-CoV-2. In one descriptive case series lacking controls, histological changes and capillary alterations were described in deltoid muscles of patients with post-COVID syndrome (49).

In the present interdisciplinary case-control cohort study, comparing patients with post-COVID syndrome to two distinct age-matched historical control cohorts, we found, on the morphological

level, capillary alterations consisting of a decreased capillary-to-fiber ratio and an increased capillary basement membrane thickness. Patients with PCS had smaller muscle fibers and increased numbers of CD169+ macrophages in close vicinity to skeletal muscle capillaries, but no evidence of overt myositis.

Upper leg MRI also did not reveal signs of myositis, which is consistent with recently published radiological findings (50). However, biopsies were taken almost a year after acute infection and several case reports of biopsy-proven myositis after SARS-CoV-2 have been published (51-53), and we ourselves histologically diagnosed non-specific myositis in some patients in the subacute aftermath of mild or moderate COVID (Fig. S3F).

Furthermore, immune-mediated myositis in severe COVID has been well documented by two independent autopsy studies (29, 30). We can therefore not exclude that some individuals in our cohort suffered from a self-limiting acute myositis which had resolved by the time of the biopsy.

No SARS-CoV-2 specific RNA could be detected in any of the muscle samples by ultra-sensitive qPCR, strongly arguing against an unresolved infection of skeletal muscle tissues as the cause for the patients’ symptoms.

The mere fact that the number of CD169+ macrophages is increased in mildly altered skeletal muscle tissue is remarkable. CD169+ macrophages are increased in idiopathic inflammatory myopathies (54), indicating a prominent role in type I Interferon-related immune processes (55), and recent studies emphasized their highly specific functional programmes and important roles as border-associated cells at blood vessel/parenchymal interfaces (56, 57).

CD169+ macrophages have further been implicated in antiviral defense, being the primary cell infected and able to capture viral particles in the blood and subsequently presenting them to B cells (58). As numbers of circulating CD169+ monocytes are increased in acute stages of mild COVID-19 (59), we hypothesize that they could play a key role at the muscle/capillary interface in our cohort of patients with PCS.

With regards to the fact that most patients from the PCS cohort displayed a selective atrophy of type-2b-fibers – which constitutes a non-specific finding observed in diverse settings leading to disuse or deconditioning of a muscle – we considered the hypothesis that any of our observations could also be the consequence of reduced mobility in these patients due to their exercise intolerance, pain or to coincidental phenomena (lockdowns, stress, mood disorders), instead of

being directly related to the infection or to post-infectious mechanisms. We therefore added another historical control cohort to the study, consisting of vastus lateralis samples obtained from patients that displayed a selective atrophy of type-2b-fibers.

On the protein level, mass spectrometry of the vastus lateralis muscle samples revealed an upregulation of basement membrane and other extracellular matrix components only when comparing the PCS samples to the HDC samples but not in comparison to the ones with type-2b- fiber atrophy.

Overall, proteome differences were rather subtle, as none of the proteins was increased more than 1.5-fold. This is however not surprising, regarding the fact that histologically little pathology could be noted in the PCS samples. Also, the vastus lateralis muscle samples we used for the HDC cohort came from symptomatic individuals where biopsies were performed for diagnostic purposes, but for which routine histological examination had revealed no morphological abnormalities.

While the muscle samples from the 2BA cohort showed a wider distribution of CBM thickness with some individuals showing similar enlargements as the PCS cohort and higher expressions of CBM-proteins compared to the healthy control group, the mean CBM thickness was still lower in that cohort.

We speculate that the 2BA cohort was more heterogeneous than the HDC one, with some of the patients possibly being affected by yet undiagnosed musculoskeletal or systemic diseases that also affected the extracellular matrix. Alternatively, increased matricellular proteins could also be a direct consequence of the selective atrophy of type-2b-fibers, for which little molecular pathomechanistic knowledge exists as of today.

The fact that type-2b-fiber atrophy was more pronounced in the 2BA cohort than in the PCS cohort (Fig. 2B) is however an argument against a correlation between type-2b-fiber atrophy and CBM thickness. Moreover, we screened three independent bulkRNA datasets obtained from M. vastus lateralis biopsies performed before and after so-called bed rest studies.

Even in this extreme form of immobility with weeklong bedrest, it appears that there is no increased expression of CBM components (collagen IV, laminins, nidogens, heparan sulfate proteoglycans) (60-62). Another study, with only a 48h immobilization, revealed a decreased expression of Collagen IV constituents (63).

These studies however did not include ultrastructural assessment of capillaries, limiting the comparison. Another bed rest study revealed a preserved C/F ratio and increased capillary density due to decreased cross-sectional fiber size (64). Of note, macrophage infiltration was associated with the muscular growth phase in a rehabilitation context, rather than the hypotrophic phase of resting (65).

On the transcriptional level, unbiased analysis revealed a certain heterogeneity within the PCS cohort, but clustering based on immune cell markers, complement pathway and type I and III interferon related genes allowed a clear separation of the patient’s samples from the controls.

PCS samples not only showed an upregulation of immune regulatory genes such as tumor necrosis factor alpha (TNFA), but also of genes related to extracellular matrix organization and cell-cell adhesion, while pathways related to oxidative phosphorylation, mitochondria and cell respiration were downregulated. This invites for the speculation that the observed morphological alterations of the capillaries (reduced C/F; thickened CBM) are indeed responsible for metabolic disturbances, possibly explaining the exercise-dependent symptomatology.

From a theoretical, physiological perspective, an increased CBM thickness results in a reduced diffusion of oxygen according to Fick’s Lawii. Impaired oxygen delivery to skeletal muscles has been previously described in patients with ME/CFS (66), consisting mainly of a reduced peak oxygen uptake during physical activity (67) and oxygen therapy has been shown to improve the symptoms (68).

On the other hand, endothelial damage and capillary pathology have been extensively described in acute as well as in post-acute sequelae of SARS-CoV-2 infection in both human and animal studies (69-74). It therefore appears plausible that a capillaropathy contributes or even might be the cause for the described symptoms in a subset of patients with post-COVID syndrome.

Viral infections are well-known triggers for a multitude of autoimmune processes (75, 76). Our findings suggest persistent local immune responses in subsets of patients with PCS even one year after initial infection, which in the absence of evidence for an unresolved infection and the presence of autoantibodies in some individuals from our cohort, may point towards immune system dysregulations or an autoreactivity, consistent with multiple observations in patients with acute and post-acute COVID-19 (77-83).

To conclude, we hypothesize that acute infection may have caused persistent structural changes of the microvasculature in skeletal muscles in some patients, potentially explaining the exercise- dependent symptoms. The increased presence of CD169+ macrophages and the above-mentioned transcriptomic changes at the tissue level approximately one year after infection, together with the absence of SARS-CoV-2-specific RNA suggest that a sustainably dysregulated immune response could be responsible for the microvascular alterations in skeletal muscles of affected patients. Larger studies may allow to identify a “capillaropathy subset” among patients suffering from PCS, ME/CFS or other post-infectious syndromes, thus opening new doors for differential diagnosis and personalized therapies.

The fact that both of our control cohorts consisted of clinically symptomatic patients can be seen both as a strength and a weakness of the study: The weakness lies in the fact that as opposed to non-symptomatic, healthy subjects certain non-specific but pathological changes could be missed on the molecular level in our PCS cohort. The strength is that the observed differences can be attributed to the studied condition with a higher probability, as bystander effects of impaired health status resulting in a less active lifestyle will be leveled out to some degree.

Other limitations are the relatively small cohort of affected individuals, the lack of a control group of asymptomatic patients after a SARS-CoV-2 infection or of a control group with people with similar symptoms but which were not infected with SARS-CoV-2.

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