CCL2-mediated endothelial injury drives cardiac dysfunction in long COVID, 2024, Dilip Thomas et al

[Mij]

Abstract
Evidence linking the endothelium to cardiac injury in long coronavirus disease (COVID) is well documented, but the underlying mechanisms remain unknown.

Here we show that cytokines released by endothelial cells (ECs) contribute to long-COVID-associated cardiac dysfunction. Using thrombotic vascular tissues from patients with long COVID and induced pluripotent stem cell-derived ECs (iPSC-ECs), we modeled endotheliitis and observed similar dysfunction and cytokine upregulation, notably CCL2. Cardiac organoids comprising iPSC-ECs and iPSC-derived cardiomyocytes showed cardiac dysfunction after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, driven by CCL2. Profiling of chromatin accessibility and gene expression at a single-cell resolution linked CCL2 to ‘phenotype switching’ and cardiac dysfunction, validated by high-throughput proteomics. Disease modeling of cardiac organoids and exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins revealed that CCL2-induced oxidative stress promoted post-translational modification of cardiac proteins, leading to cardiac dysfunction.

These findings suggest that EC-released cytokines contribute to cardiac dysfunction in long COVID, highlighting the importance of early vascular health monitoring in patients with long COVID.
LINK

 

[Dakota15]

Copied from News from the USA

10/14/24, Stanford Medicine: "Unraveling the Heart of Long COVID"

'To explore how long COVID impacts heart health, researchers in this study used induced pluripotent stem cells (iPSCs) differentiated into heart muscle cells, or cardiomyocytes (iPSC-CMs).’

"Emerging research about long COVID suggests that influence of the virus extends to the heart through an intricate interplay of immune response, inflammation, and endothelial cell (EC) dysfunction.”

"Endothelial cells have emerged as key players in cardiovascular complications of long COVID, and understanding how they contribute is crucial for developing treatments and preventative strategies.”

"These cells are important for maintaining healthy blood flow and heart function. In long COVID, endothelial cells become inflamed and dysfunctional. They also release too many CCLs, a chemokine that attracts immune cells and promotes inflammation.”

"This important study shows the potential of targeting endothelial dysfunction and the associated inflammatory responses for treatment of existing heart issues or to prevent further cardiovascular complications in long COVID patients.”

"The cutting-edge work presented in this manuscript highlights the importance of continued research to understand the full scope of the effect of long COVID on the body. As health challenges posed by long COVID continue to evolve, it becomes more important to move closer to finding ways to treat or prevent the long-term impact.”

 

[SNT Gatchaman]

I read this paper yesterday and it looks like an important study, but unfortunately paywalled.

There's also commentary in Endothelial cells as paracrine mediators of long COVID (2024, Nature Cardiovascular Research)

I'll quote liberally from that commentary to give people a summary of the main paper. I don't know how much of this might relate to ME/CFS-LC — or non-Covid ME/CFS. I assume this mechanism might explain post-vaccination myocarditis, given they also induced effects via a "non-replicative baculovirus that is pseudotyped with spike protein".

Pre-clinical and clinical data suggest that SARS-CoV-2 causes myocarditis and pericarditis in a subset of individuals. However, several other pathophysiological clinical associations have also been reported in long COVID. These differ across individuals and this heterogeneity may also underlie the diversity in clinical symptoms reported.

There is a strong association between long COVID and inflammation, and between microvascular blood clotting and endothelial dysfunction. To this end […] Thomas et al. have set out to directly assess the role of endothelial cells in long COVID and cardiac dysfunction.

Their experiments demonstrate long-term changes in endothelial cells, and identify sustained inflammation and CCL2 as drivers of altered calcium handling and cardiac dysfunction.

The researchers began by obtaining tibial arteries from individuals with long COVID, alongside those from individuals with peripheral artery disease (PAD) and healthy individuals as positive and negative controls, respectively. Vascular reactivity assessments using wire myography indicated persistent endothelial dysfunction in arteries from individuals with long COVID.

Complementary bulk and single-cell RNA sequencing of the arteries demonstrated that long COVID was specifically associated with increased expression of inflammatory cytokine and chemokine genes, including IL6, CXCL8 and CCL2, which are associated with the COVID-19 cytokine storm. This was also underpinned by increased endothelial-to-mesenchymal transition, a process previously shown to have a fundamental role in heart diseases. There were also increased levels of fibrosis-related transcripts in mesenchymal and smooth muscle cells in samples from individuals with long COVID. Importantly, these changes were not observed in individuals with diabetes or atherosclerosis who had pre-existing PAD.

To investigate endothelial dysfunction in greater detail […] next generated induced pluripotent stem (iPS) cell derived endothelial cells (iPSC-ECs) from individuals with long COVID and healthy controls.

both live SARS-CoV-2 and a non-replicative baculovirus that is pseudotyped with spike protein were able to infect iPSC-ECs. Indeed, iPSC-ECs derived from both individuals with long COVID and healthy controls could be infected, arguing against a genetic predisposition to infection.

Viral infection resulted in decreased endothelial cell function underpinned by decreased eNOS and tube formation, and increased production of inflammatory factors. Importantly, in long COVID the inflammatory response slowly declined but persisted for over two weeks after washout.

Experiments in primary arterial and venous endothelial cells led to similar results, except that it was only the endothelial cells derived from individuals with long COVID that produced an inflammatory response.

next constructed cardiac organoids consisting of iPSC-ECs and purified iPS cell-derived cardiomyocytes (iPSC-CMs).

Only the iPSC-EC-containing cardiac organoids were infected, and increased production of inflammatory factors ultimately led to the induction of fibrotic genes. This also resulted in acute calcium transient dysfunction, including decreased contraction and relaxation velocity in iPSC-CMs. This dysfunction was sustained even two weeks after washout indicating that remodeling processes had occurred.

sophisticated single-nuclei RNA sequencing (snRNA-seq) and single-nuclei assay for transposase accessible chromatin sequencing (snATAC-seq) analyses showed that SARS-CoV-2 infection induces epigenetic changes in iPSC-ECs — converging on a pro-fibrotic and inflammatory gene program in which CCL2 and COL22A1 featured prominently.

also show that CCL2 is increased in individuals with long COVID

Therefore, SARS-CoV-2 infection in iPSC-ECs drives cardiomyocyte dysfunction via paracrine signaling mechanisms. This is consistent with a recent publication in cardiac organoids in which vascular cells drive inflammation-induced cardiac dysfunction via paracrine endothelin signaling.

next addressed the mechanisms of long COVID iPSC-ECs-induced cardiac dysfunction. They focused on reactive oxygen species (ROS) given the existing links between inflammation, ROS and cardiac dysfunction

Another major regulator of contraction force and kinetics in iPSC-CMs is calcium cycling via the intracellular sarcoplasmic reticulum. During a contractile cycle, Ca 2+stored in the sarcoplasmic reticulum is released by ryanodine (RYR2) and taken back up via sarcoplasmic/endoplasmic reticulum Ca2+-ATPase […] its dysregulation is centrally implicated in heart failure and arrhythmia. […] found that SARS-CoV-2 infection increased the phosphorylation of RYR2 (Ser2808), a key signature associated with cardiac dysfunction and heart failure.

knockdown of CCL2 in iPSC-ECs reduced the induction of other inflammatory cytokines including IL1B and extracellular matrix components such as COL22A1. It also attenuated the induction of ROS, prevented the increase in RYR2 phosphorylation and partially rescued the reduction in calcium kinetics in the cardiac organoids

SARS-CoV-2 infection of iPSC-ECs but not iPSC-CMs is noteworthy, as previous reports have been conflicting.

The observation that long COVID primary endothelial cells responded with stronger inflammatory responses than control healthy venous or arterial endothelial cells indicates that crucial priming events may have a key role in the response.

SARS-CoV-2 infection of iPSC-ECs increased concentrations of IFNγ more than 80-fold, and knockdown of CCL2 resulted in a reduction of IL-1β, two critical inflammatory factors we have found to drive cardiac dysfunction.

How CCL2 leads to ROS and altered calcium handling is an intriguing question as it has important therapeutic implications that remain incompletely understood despite many years of collective efforts.

discuss a key role for CCL2 signaling to the immune system that potentially contributes to cardiac pathology […] also show […] that inflammatory cytokines are capable of driving dysfunction in the absence of the immune system itself, indicating that these factors also have crucial roles in non-immune cell types

The canonical effector for CCL2 is the chemokine receptor CCR2, and this is predominantly expressed in monocytes rather than cardiomyocytes or fibroblasts.

Thomas et al. have contributed to a body of work implicating endothelial-derived chemokines in cardiac dysfunction. This has clear implications for our mechanistic understanding of long COVID-related cardiovascular complications, as well as chronic inflammatory cardiovascular diseases more broadly

 

[Nightsong]

Interesting methodology. Very small numbers of patients & controls, though, and worth noting how LC was classified - this is a very different study population to "LC-ME/CFS" cases:

To investigate the duration of these thrombotic events after recovery, particularly regarding ECs, we recruited five healthy controls (HCs) and eight patients with long COVID with symptoms lasting at least 12 weeks (Fig. 1a and Supplementary Tables 1 and 2). Patients with long COVID were classified as mild (symptoms of fatigue or headaches) or severe (having arterial or venous thrombosis requiring surgery). Computed tomography angiography (CTA) of these severe cases showed occlusive thrombus in peripheral arteries, including popliteal arteries (Fig. 1b and Extended Data Fig. 1a), with no calcification or narrowing elsewhere, indicating CAC-induced thrombosis rather than progressive atherosclerosis.

 

[Mij]

LINK