Human Cardiac Pericytes Are Susceptible to SARS-CoV-2 Infection

Abstract

COVID-19 is associated with serious cardiovascular complications, with incompletely understood mechanism(s). Pericytes have key functions in supporting endothelial cells and maintaining vascular integrity. We demonstrate that human cardiac pericytes are permissive to SARS-CoV-2 infection in organotypic slice and primary cell cultures. Viral entry into pericytes is mediated by endosomal proteases, and infection leads to up-regulation of inflammatory markers, vasoactive mediators, and nuclear factor kappa-B-dependent cell death. Furthermore, we present evidence of cardiac pericyte infection in COVID-19 myocarditis patients. These data demonstrate that human cardiac pericytes are susceptible to SARS-CoV-2 infection and suggest a role for pericyte infection in COVID-19.

Keywords: ACE2, angiotensin-converting enzyme 2; COVID-19; IFN, interferon; MOI, multiplicity of infection; PDGFRβ, platelet-derived growth factor receptor beta; SARS-CoV-2; cardiovascular disease; pericytes.

Graphical abstract

Figure 1

Detection of SARS-CoV-2 in Pericytes of Inoculated Ventricular Organotypic Slices (A) Representative immunostaining for angiotensin-converting enzyme 2 (ACE2) (green) and platelet-derived growth factor receptor beta (PDGFRꞵ) (red) in human left ventricular tissue (n = 3 independent donor hearts). (B to D) Cardiac slices were inoculated with 5 × 10⁵ focus forming units of SARS-CoV-2 WA1/2020 (approximately MOI of 1) and harvested 48 hours later (n = 3). (B) Representative RNA in situ hybridization of SARS-CoV-2 S sense (S-S) (green) and SARS-CoV-2 ORF1ab antisense (O-AS) (red) at 48 hours postinoculation. (C) Representative RNA in situ hybridization of SARS-CoV-2 S sense (S-S) (green) and pericyte marker PDGFRB sense (red), and (D) immunostaining for the SARS-CoV-2 NP (green) with pericyte marker PDGFRꞵ (red). All scale bars: 10 μm. All images representative from 3 technical slice replicates from each of the 3 independent donor hearts. DAPI = 4′,6-diamidino-2-phenylindole.

Figure 2

SARS-CoV-2 Productively Infects Human Primary Cardiac Pericytes (A) Immunostaining for ACE2 (green) and PDGFRꞵ (red) of cardiac pericytes isolated from left ventricle (n = 3 donors). (B) Representative flow cytometry histogram and quantification of PDGFRꞵ in pericytes (n = 3). (C and D) Focus-forming assay time course of pericyte infection with SARS-CoV-2 WA1/2020 and SARS-CoV-2 mNeonGreen (n = 3) (C) and Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) SARS-CoV-2 variants (representative of n = 2) (D); dotted line indicates limit of detection. (E) Immunostaining for the SARS-CoV-2 nucleocapsid protein (NP) (green) and PDGFRꞵ (red) (n = 3). (F) Representative flow cytometry histogram and quantification of cardiac pericyte infection (mNeonGreen) at day 1 postinoculation with multiplicity of infection (MOI) of 20 (n = 3). (G) Flow cytometry-based cell viability (ZombieViolet+ = dead) time course of inoculated pericytes. Statistical comparison to mock at corresponding time point. (H to J) Pericytes were pretreated with vehicle (unt), 10 μg/mL of human anti-ACE2 antibody (α-hACE2), 100 μmol/L of serine protease inhibitor camostat methylate (camo), 100 μmol/L of cysteine protease inhibitor E-64 (E64), or a combination of camostat methylate and E-64 (camo-E64) for 1 hour, inoculated with mock or SARS-CoV-2 mNeonGreen at MOI of 20 for 20 hours (n = 2). Quantification of infection (mNeonGreen+) (D), pericyte identity (PDGFRꞵ+) (E), and cell viability (Zombie-Violet-) (F). (D to F) Points represent separate wells; statistical comparison to untreated inoculated group. Repeated in 2 donors. 2-way analysis of variance with Dunnet’s post-test: ∗∗P < 0.01; ∗∗∗P < 0.001. Scale bars: 20 μm. Abbreviations as in Figure 1.

Figure 3

SARS-CoV-2 Infection Upregulates Type I IFN Signaling, Inflammatory Pathways, and Vasoactive Genes in Cardiac Pericytes (A to G) RNA sequencing of pericytes at 4 dpi. Cardiac pericytes from 3 donors were inoculated with SARS-CoV-2 mNeonGreen (5 technical replicates/donor) or mock (3 technical replicates/donor). (A) Multidimensional scaling plot with mock (red) and SARS-CoV-2 infected (CoV2) (green) samples; dots represent technical replicates. (B) Volcano plot showing differentially expressed genes between mock and infected samples. Each dot represents a gene, with blue denoting down-regulated genes, black denoting genes nonsignificant changes, and red denoting up-regulated genes. (C) Gene ontology pathway analysis with top 10 up-regulated pathways in infected pericytes. (D to G) Heat maps of selected differentially expressed genes of viral origin (D), pericyte genes involved in inflammatory response (E); response to IFN signaling (F); and vascular development, homeostasis, and function (G). (H to J) Reverse transcription quantitative polymerase chain reaction analyses of genes encoding inflammatory cytokines and chemokines (H), type I interferon signaling pathway components (I), and vasoconstriction and angiogenesis mediators (J); data points denote separate wells (3 technical replicates/donor) with statistical comparison to mock group for each donor; an equal or unequal variance Student's t-test, as determined by an F test for equality of 2 variances: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, nonsignificant comparisons not shown. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; other abbreviations as in Figure 1.

Figure 4

Detection of SARS-CoV-2 in Pericytes of COVID-19 Cardiac Autopsy Specimens (A) RNA in situ hybridization of SARS-CoV-2 S sense (S-S) (green) with pericyte marker PDGFRB sense (PDGFRB-S) (red) in the perivascular region of autopsy control (n = 1) and COVID-19–positive formalin-fixed paraffin-embedded specimens (n = 2). (B) Immunostaining for the SARS-CoV-2 NP (green) and pericyte marker PDGFRꞵ (red) within the perivascular region of autopsy control (n = 1) and COVID-19–positive formalin-fixed paraffin-embedded specimens (n = 2). Abbreviations as in Figure 1.