Cardiac Pericytes

Corresponding Author

The association of SARS-CoV-2 infection with acute myocardial injury, as well as the risk for late-onset vascular complications, including myocardial infarction and stroke, remain major public health concerns. However, the mechanisms of these cardiovascular complications are incompletely understood. Despite injury indicated by cardiac troponin elevations, the demonstration of widespread myocarditis with viral infection of myocardial tissue is rare. In contrast, microvascular thrombosis has been reported in autopsy analyses of the heart, and thrombosis is a common feature in hospitalized patients with COVID-19. However, studies reporting that SARS-CoV-2 does not infect cultured endothelial cells suggest direct endothelial infection may not be the mechanism.

Vascular integrity, crucial for maintaining microcirculation during physiological and pathological conditions, is mediated by endothelial cells and pericytes. Whereas endothelial cells line the inner side of blood vessels, pericytes are mural cells that encapsulate endothelial cells of microcapillaries through their cytoplasmic projections. They provide structural and functional support to endothelial cells through adherens junctions and gap junctions between these 2 cell types.¹ Besides transforming growth factor-β, angiopoietin 1/angiopoietin 2, Tie-2 receptor, and vascular endothelial growth factor, platelet-derived growth factor-β is a soluble molecular mediator secreted by endothelial cells that recruits platelet-derived growth factor receptor-β–expressing pericytes to angiogenic sprouts during vascular remodeling,¹ which is required to maintain homeostasis during diseases and infections such as those caused by SARS-CoV-2.

The susceptibility of the cardiovascular system to SARS-CoV-2 relates to the presence in heart tissues of angiotensin-converting enzyme 2 (ACE-2), the main host receptor for SARS-CoV-2. ACE2 and genes important for fusion and entry of the virus are expressed in cardiomyocytes² and ventricular myocardium. Single nuclei RNA sequencing studies demonstrate high expression of ACE2 in pericytes compared with fibroblasts and ventricular cardiomyocytes in a cell- and disease-specific manner.^2,3 The vulnerability of the heart cells to SARS-CoV-2 is, thus, variable. Cardiac pericytes that robustly express ACE2 could represent prime targets of this virus that might explain its cardiac thrombotic manifestations. ACE2 is not expressed or is expressed at extremely low levels at best in endothelial cells; accordingly, there is no evidence of replication of SARS-CoV-2 in endothelial cells.^3,4 However, transcriptomic, morphological, and ultrastructural signatures reflecting a compromised permeability of the vasculature, endothelial dysfunction, and cardiac thrombi in COVID-19 hearts have been reported. Diminished pericyte encapsulation of alveolar capillaries,⁵ decreased paracrine signaling via angiopoietin,⁶ and endothelial dysfunction⁷ in in vivo and in vitro studies implicate human cardiac pericytes as a SARS-CoV-2 target cell that may mediate the endothelial damage observed in patients with COVID-19.

In this issue of JACC: Basic to Translational Science, Brumback et al⁸ demonstrate convincingly that human cardiac pericytes, and not endothelial cells, are permissive to SARS-CoV-2 infection. The authors derived primary human cardiac pericytes from organotypic cardiac slices of nonfailing human ventricles. Using fluorescent in situ hybridization and immunostaining, the authors demonstrated the localization of viral signatures in these preparations. Immunohistochemistry depicting viral presence in pericytes of 2 patients with COVID-19 afflicted with myocarditis confirmed the authors’ premise that this virus infects pericytes. A larger sample size would have helped to determine whether pericytes are the main targets of SARS-CoV-2 infection in the heart. Using flow cytometry, the authors established the pericyte-specific population, their infectivity by the Wuhan strain and other subvariants of SARS-CoV-2, and viral dose-specific death of these cells. Infection was corroborated by transcriptomic traces of the virus in the infected cardiac pericytes. Interestingly, cultured pericytes from placenta and brain harbored vastly lower ACE-2 levels than those observed in pericytes from the heart, despite prior findings of ACE-2 in murine and human brain pericytes and in brain perivascular cells of patients with COVID-19.⁹ Nonetheless, these cell-based studies in conjunction with the histopathology of patients with COVID-19 myocarditis highlights the potential specific vulnerability of pericytes in the heart of patients with COVID-19.

Brumback et al⁸ also demonstrated that their cultured human cardiac pericytes harbor substantial levels of ACE-2, cathepsin H, cathepsin L, and furin, but undetectable levels of TMPRSS2 and TMPRSS4. Similar to cardiomyocytes,¹⁰ pericytes were targeted by SARS-CoV-2 via ACE-2 and endosomal pathways without involvement of TMPRSS2 or TMPRSS4, because infection could be blocked by an ACE-2 antibody, a cysteine protease inhibitor, E-64, and bafilomycin-A1, which inhibits the acidification of cysteine proteases like cathepsins. Moreover, both cell types seem to downregulate ACE-2 after viral infection, suggesting internalization or shedding of this receptor. Further studies investigating this aspect are warranted because ACE-2 is an important component of the renin–angiotensin system, which converts Ang II to noninflammatory Ang 1–7 peptide. The latter’s role in diminishing the impact of the inflammatory cascade imposed by SARS-CoV-2 may be crucial, especially in patients with COVID-19 with underlying cardiovascular problems.

Brumbach et al⁸ showed that viral entry into cardiac pericytes upregulated inflammatory signaling (type I interferon), innate immunogenic responses, and vasoactive genes. Strikingly, interleukin-6 was robustly enhanced, as has been shown in COVID-19 cases. Chemically induced inhibition of interferon (IFN) signaling by TPCA-1 led to decreased cardiac pericyte death by virus, suggesting that this pathway may be a promising target to explore for treatment of SARS-CoV-2–induced damage in the heart. Interestingly, the nuclear factor-κB pathway, although not involved independently, worked in concert with IFN signaling to lead to cell death in virally infected pericytes. Endothelial cell–pericyte cross-talk was demonstrated using conditioned media from infected cardiac pericytes, which led to the upregulation of chemokines (CXCl2 and CXCL5) in endothelial cells. The decrease in the expression of thrombomodulin, a natural anticoagulant expressed by endothelial cells due to SARS-CoV-2, or SARS-CoV-2–induced paracrine effects from cardiac pericytes, may contribute to the thrombosis that has been observed.

The myocardium is populated by various cell types—cardiomyocytes, pericytes, endothelial cells, fibroblasts, and macrophages (resident and infiltrating). Work by Bailey et al¹⁰ and the findings in the current study support that it is mostly cardiomyocytes and cardiac pericytes that are permissive to SARS-CoV-2 infection. However, the potential contribution of the other cell types through the secretion of soluble molecular mediators and cytokines cannot be ignored. Most of the studies have focused on cell tropism and the pathways that are involved in SARS-CoV-2 infection in specific cell types. This study underscores the cross-talk between pericytes and endothelial cells that can be critical to the maintenance of homeostasis of microvascular, coagulatory, and immunogenic systems. Although it now seems established that it is the pericytes and not endothelial cells that are targets of SARS-CoV-2, the consequences of this targeting impact both pericyte and endothelial cell biology. Moreover, high expression of ACE-2 in cardiac pericytes suggests an important role in the regulation of the renin angiotensin system.

Illuminating the potential contribution of pericytes to cardiovascular sequelae raises many questions for future studies. How the cytokines and vasoactive and molecular mediators released from these diverse cell types converge to a common pathway, like that of type I IFN, to elicit the virus mediated responses will be critical to dissect. Ligand binding of receptors involved in viral entry and fusion versus paracrine signaling by cell types needs to be distinguished and may help to target novel therapeutics. The effect of pericyte infection on angiogenesis, the microcirculation, and continued predisposition to microthrombosis during the recovery phase of infection will also be of interest. Paracrine effects on other cell types are intriguing possibilities raised by the current study. Although macrophages and fibroblasts may not be directly infected by SARS-CoV-2,¹⁰ they may be impacted by the effects on pericytes and cardiomyocytes. A novel strategy could be to coculture cardiomyocytes, pericytes, and endothelial cells, and potentially macrophages and fibroblasts, then monitor angiogenesis, pericyte migration, and functional effects in thrombotic pathways using primary cell cultures and organoid models.

In future studies, it will be important to determine whether the downstream effects of pericyte infection and cross-talk with endothelial cells may lead to the development of microthrombosis, including the effects of platelet-derived growth factor-β, which is an important mediator of endothelial and pericyte cross-talk. Type I IFN-γ signaling seems to be the common pathway elicited in both human cardiac pericytes⁸ and cardiomyocytes¹⁰ upon infection by SARS-CoV-2. Both cardiomyocytes and cardiac pericytes are susceptible to SARS-CoV-2 infection in an ACE-2 and endosomal pathway dependent manner, but cells may have differing levels of ACE-2. The determination of which cell type the virus preferentially targets may enhance our understanding of whether vascular dysfunction occurs before any direct cardiomyocyte effects. Increasing pericyte coverage or function by enhancing endothelial cell health may be a promising avenue in this infection.

Focused investigations that reveal the underlying molecular mechanisms responsible for the cardiovascular complications of COVID-19 remain critically important. The study by Brumbach et al⁸ in this issue of the JACC: Basic to Translational Science places human cardiac pericytes in the limelight so that they may be considered in the context of vascular dysfunction and myocarditis that is observed in patients with COVID-19. The findings in the present study implicate pericytes as a key cell type that mediates the cardiovascular and thrombotic complications of COVID-19. The contribution of infected pericytes to microthrombosis, endothelial damage, and future risk of cardiovascular complications after recovery will be critical questions for the scientific community to tackle. Answers to these questions may lead to novel therapeutic strategies for not only acute COVID infection, but also potentially its longer-term sequelae.

Funding Support and Author Disclosures

This work was funded by 810959 AHA Covid-19 Rapid Response Research Coordinating Center grant, 814633 AHA Covid-19 Rapid Response Research grant, NIH/NHLBI R01HL111314, and P01HL158502. The authors have reported that they have no relationships relevant to the contents of this paper to disclose.