The authors from the United States investigated whether SARS-CoV-2 directly infects atherosclerotic plaques in the coronary vasculature. The results demonstrated that SARS-CoV-2 is able to infect and replicate in macrophages in the coronary atherosclerotic lesions, promoting plaque inflammation.
The authors have noted that the clinical complications of COVID-19 encompass, among others, ischemic cardiovascular events such as acute myocardial infarction and stroke due to the disruption of chronically inflamed atherosclerotic plaques. They hypothesized that a potent activation of macrophages that infiltrate arterial vessels, in response to SARS-CoV-2 virus, could boost plaque inflammation. Consequently, this increases the risk for acute myocardial infarction and stroke in COVID-19 patients.
About the study
Researchers conducted an analysis of autopsy specimens of coronary arteries from eight deceased individuals who were diagnosed with COVID-19, as confirmed by RT-PCR. The mean age of individuals was 69.6 (59-84) and 75% of patients were male (6/8). Coronary artery disease (8/8), myocardial infarction (1/8), and ischemic stroke (1/8) were registered in this cohort, along with numerous cardiovascular risk factors, such as hypertension (8/8), overweight or obesity (7/8), hyperlipidemia (7/8), type 2 diabetes (6/8), and chronic kidney disease (4/8).
A clinical cardiovascular pathologist classified sections of coronary arteries from all autopsies as adaptive intimal thickening, pathological intimal thickening, fibrocalcific plaque, and fibroatheroma. The macrophage infiltration was identified using immunohistochemical staining for CD68. A neural network artificial intelligence approach was employed to distinguish the coronary arterial wall from perivascular fat in each sample.
The results showed that SARS-CoV-2 was detectable and replicated in atherosclerotic lesions from coronary artery specimens obtained from patients who died from severe COVID-19. In the samples of the coronary arterial wall from all patients, SARS-CoV-2 viral RNA encoding the spike protein was found in different lesions, including adaptive intimal thickening, pathological intimal thickening, fibrocalcific plaque, and fibroatheroma. However, the SARS-CoV-2 replication was highest in the pathological intima thickening, which is early-stage lesion that progress to more advanced atherosclerotic plaque.
Perivascular fat contained a significantly lower amount of the spike protein than the corresponding arterial wall across all samples. It is noteworthy that the vascular and perivascular fat tissue of COVID-19 patients who had acute cardiovascular manifestations accumulated more SARS-CoV-2 viral RNA encoding S protein compared to patients who did not experience cardiovascular complications.
The analysis of the main SARS-CoV-2 receptors and co-factors in the human vasculature revealed that the aorta, coronary, and tibial arteries displayed a similar expression pattern for ACE2, neuropilins (NRP1, NRP2), FURIN, Cathepsin B , and Cathepsin L, compared to the lung. The expression of ACE2 was low in the aorta and tibial artery, but higher in the coronary artery, comparable to the expression of ACE2 in the lung. This suggests that the coronary vasculature may be more susceptible to SARS-CoV-2 viral infection than other vascular beds.
Further analysis showed that SARS-CoV-2 was capable to infect and replicate in macrophages in the coronary vasculature of patients with COVID-19. CD68+ cells expressing the SARS-CoV-2 S protein were significantly higher in the pathological intimal thickening of coronary arteries than in the associated perivascular fat. In addition, pathological intimal thickening contained significantly more cells than other types of lesions and 4.8-fold more cells than corresponding perivascular tissue.
The SARS-CoV-2 infection of human primary macrophages in vitro revealed that cholesterol-loaded macrophages (foam cells), were more susceptible to infection than other macrophages, with a significant accumulation of viral nucleoprotein in these cells. Also, the SARS-CoV-2 preferentially replicated in foam cells compared to other macrophages, leading to accumulation of SARS-CoV-2 viral RNA and viral proteins. This suggests that foam cells are more permissive and susceptible to SARS-CoV-2. It should be noted that atherosclerosis is characterized by the accumulation of foam cells at all stages of the disease, from early pathological intimal thickening to late fibroatheroma lesions.
Increased SARS-CoV-2 entry in cholesterol-loaded macrophages was dependent on neuropilin-1 (NRP-1). NRP-1 blocking reduced the infection of foam cells and other macrophages in vitro, and this effect was twofold greater in foam cells. Furthermore, the viral replication was reduced. This suggests that NRP-1 plays a key role in mediating the SARS-CoV-2 infection of these cells.
Myeloid cells were then divided into two clusters of dendritic cells, three clusters of monocytes/macrophages, one cluster of mixed myeloid cells, and four clusters of macrophages/foam cells. CD16+ monocytes, inflammatory monocyte/macrophages, IL1B dendritic cells, and CD36+ mixed myeloid cells were exclusively present in carotid samples, whereas LYVE1+ macrophages were present in coronary tissue. SPP1+ macrophages were found in both tissues. TREM2 high macrophages were enriched in coronary tissue, while VCAN+ monocytes/macrophages and CD1c+ dendritic cells were enriched in carotid samples.
The plaque assay confirmed a progressive decrease in viral titer in conditioned media from infected macrophages and foam cells over 48 hours. The observed decrease in the levels of SARS-CoV-2 viral RNA showed that even though foam cells and other macrophages were susceptible to SARS-CoV-2 infection, neither macrophages nor foam cells were capable of sustaining a productive viral infection.
Although viral replication was abortive in macrophages and foam cells, it promoted a strong inflammatory response characterized by release of cytokines implicated in the pathogenesis of atherosclerosis and increased risk of cardiovascular events. The most upregulated genes were those involved with antiviral response including interferon (IFN) signaling pathways and antiviral processes by type I and II IFN signaling, the OAS antiviral response, negative regulation of viral replication and viral life cycle, as well as complement activation and cytokine signaling. The ISG15 antiviral signaling, which reduces IFN signaling and is implicated in the hyperinflammatory response associated with COVID-19 severity, was also upregulated in both cell types. Both types of infected macrophages significantly upregulated several pro-inflammatory and proatherogenic cytokine and chemokine genes, including CCL7, TNFSF10, CXCL10, CCL7, and CCL3. The upregulation of genes involved in lipid metabolism was also observed, indicating a reprogramming of lipid metabolism in macrophages in response to the virus.
Researchers also quantified the secretion of cytokines and chemokines that were released in conditioned media. Following infection of foam cells and other macrophages, numerous pro-inflammatory and pro-atherogenic cytokines were released, including IL-6, CCL7, IL-1β, β-NGF, IL-3, LIF, MIF, CXCL-9, IFN-α, and IFN-γ. The proatherogenic cytokine IL-18 was significantly released by infected macrophages but not foam cells, whereas IFN-α2, a type I IFN response cytokine that inhibits viral replication, was significantly released by infected foam cells but not macrophages. The production of IL-6 is associated with ischemic cardiovascular events, whereas the inhibition of IL-1β reduces secondary cardiovascular events in high-risk individuals following myocardial infarction.
Lastly, an ex vivo model of viral infection of human vascular explants demonstrated that atherosclerotic plaques could be directly infected by SARS-CoV-2. The infection of vascular tissue by SARS-CoV-2 triggered an inflammatory response and induced the release of key pro-atherogenic cytokines IL-6 and IL-1β, as seen in cultured macrophages and foam cells. The viral titer decreased over time, with no detectable infectious particles. This suggests abortive replication in the vascular explants, similar to the results observed with foam cells and macrophages in vitro.
The authors said that this study represents the first evidence that SARS-CoV-2 replicates in macrophages within atherosclerotic plaques in human coronaries. Because plaque inflammation promotes disease progression and contributes to plaque rupture, these findings provide the molecular basis for how SARS-CoV-2 infection of coronary lesions may contribute to the acute cardiovascular manifestations of COVID-19. A hyperactivated immune response could increase the long-term risk of ischemic cardiovascular events in COVID-19 patients with underlying atherosclerosis.
The authors emphasized that the study focused on a small group of older individuals with COVID-19, preexisting atherosclerosis and numerous cardiovascular risk factors, so, these observations cannot be extrapolated to younger, healthy individuals.
Eberhardt N, et al. SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels.
The results of the study have been published on a preprint server and are currently being peer-reviewed. https://doi.org/10.1101/2023.08.14.553245