SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

doi:10.1016/j.cmet.2020.11.006

. 2020 Dec 1;32(6):1028-1040.e4.

doi: 10.1016/j.cmet.2020.11.006. Epub 2020 Nov 13.

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

Katie C Coate¹, Jeeyeon Cha¹, Shristi Shrestha¹, Wenliang Wang², Luciana Mateus Gonçalves³, Joana Almaça³, Meghan E Kapp⁴, Maria Fasolino², Ashleigh Morgan², Chunhua Dai¹, Diane C Saunders¹, Rita Bottino⁵, Radhika Aramandla¹, Regina Jenkins¹, Roland Stein⁶, Klaus H Kaestner², Golnaz Vahedi²; HPAP Consortium⁷; Marcela Brissova⁸, Alvin C Powers⁹

Affiliations

¹ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
² Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
³ Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
⁴ Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
⁵ Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA 15212, USA; Imagine Pharma, Devon, PA 19333, USA.
⁶ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
⁷ The Human Pancreas Analysis Program.
⁸ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address: marcela.brissova@vumc.org.
⁹ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address: al.powers@vumc.org.

PMID: 33207245
PMCID: PMC7664344
DOI: 10.1016/j.cmet.2020.11.006

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

Katie C Coate et al. Cell Metab. 2020.

. 2020 Dec 1;32(6):1028-1040.e4.

doi: 10.1016/j.cmet.2020.11.006. Epub 2020 Nov 13.

Authors

Affiliations

¹ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
² Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
³ Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
⁴ Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
⁵ Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA 15212, USA; Imagine Pharma, Devon, PA 19333, USA.
⁶ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
⁷ The Human Pancreas Analysis Program.
⁸ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address: marcela.brissova@vumc.org.
⁹ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address: al.powers@vumc.org.

PMID: 33207245
PMCID: PMC7664344
DOI: 10.1016/j.cmet.2020.11.006

Abstract

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2.

Keywords: ACE2; COVID-19; SARS-CoV-2; TMPRSS2; beta cell; duct; islet; microvasculature; pancreas; pericyte.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

**Figure 1**
ACE2 and TMPRSS2 Expression in Isolated Human Islet Cells and Juvenile Pancreas (A) Relative expression of *ACE2* and *TMPRSS2* compared with select α (white bars) and β (green bars) cell-type-enriched genes in sorted human islet α and β cells from previously published bulk RNA-seq datasets, reported as transcript per million mapped reads (TPM; n = 7; Blodgett et al., 2015) or reads per kilobase of transcript per million mapped reads (RPKM; n = 8; Arda et al., 2016). Mean expression values are presented as log₂ (TPM+1) or log₂ (RPKM+1) to account for negative values. Dotted line highlights *ACE2* and *TMPRSS2* expression. (B) Dot plots of *ACE2*, *TMPRSS2*, *CTSL*, *ADAM17*, *FURIN*, *TMPRSS4*, and *DPP4* expression compared with cell-type-enriched genes from three single-cell (sc) RNA-seq datasets (Baron et al., 2016; Kaestner et al., 2019; Segerstolpe et al., 2016). Dot size indicates percentage of cells in a given population expressing the gene; dot color represents scaled average expression. Dotted lines highlight expression of putative SARS-CoV-2 entry machinery. Percentages of β cells expressing and co-expressing these genes are available in Table S1. (C) Representative images of an isolated islet from an adult human donor (top panels; n = 6 total images analyzed) and a pancreatic section from a juvenile human donor (bottom panels; n = 10 total images analyzed) stained for ACE2 (red; antibody ab15348), insulin (INS; green), glucagon (GCG; white), and DAPI (blue). Dotted yellow line denotes islet area. Human pancreatic donor information is available in Table S2 (donors I1–I3 and J1–J5). See also Figures S1, S2, and S4. Scale bar, 50 μm.

**Figure 2**
ACE2 and TMPRSS2 Protein in Human Islets and Exocrine Tissue of Adult Pancreas (A–F″) Immunostaining of SARS-CoV-2 cell entry markers ACE2 (red; antibody AF933; A, C, C,′ D, F, and F′) or TMPRSS2 (green; B, C, C″, E, F, and F″) in islet β cells (insulin, blue; C and C′) and islet α cells (glucagon, blue; F and F′) in native pancreatic sections from adult normal donors without diabetes (n = 14 total images analyzed). (G–L″) Immunostaining of SARS-CoV-2 cell entry markers ACE2 (red; antibody AF933; G, I, I′, J, L, and L′) or TMPRSS2 (green; H, I, I″, K, L, and L″) in islet β cells (insulin, blue; I and I′) and islet α cells (glucagon, blue; L and L′) in native pancreatic sections from adult donors with type 2 diabetes (n = 8 total images analyzed). (M–R″) Immunostaining of SARS-CoV-2 cell entry markers ACE2 (red; antibody AF933; M, O, O′, P, R, and R′) or TMPRSS2 (green; N, O, O″, Q, R, and R″) in islet β cells (insulin, blue; O and O′) and islet α cells (glucagon, blue; R and R′) in native pancreatic sections from adult donors with type 1 diabetes (n = 4 total images analyzed). Dotted yellow lines denote islets. Islet (i) and exocrine (e) inset areas are marked by yellow boxes in MERGE column with DAPI counterstain. Pancreatic ducts, identified structurally by rosette pattern of DAPI-labeled nuclei (white), are shown within dotted yellow lines in exocrine INSET column. Human pancreatic donor information is available in Table S2 (A–F″, donor N9; G–L″, donor 2H; M–R″, donor 1C). See also Figures S2–S4. Scale bars, 50 μm (A–R) and 25 μm (insets, C′–R″).

**Figure 3**
ACE2 Protein Localization with Islet and Exocrine Capillaries in Adult and Juvenile Human Pancreas (A–C') Representative images of endothelial cells (CD31, white) and ACE2-positive perivascular cells (red; antibody ab15348) in the adult islet microvasculature (n = 14 total images analyzed). (D and D′) ACE2-positive perivascular cells (red; antibody ab15348) and the extracellular matrix marker collagen-IV (COL4, green) within the vascular basement membrane; DAPI counterstain (blue). (E–G') Representative images of endothelial cells (CD31, white) and ACE2-positive perivascular cells (red; antibody ab15348) in the exocrine tissue microvasculature (n = 14 total images analyzed). (H and H′) ACE2-positive perivascular cells (red; antibody ab15348) and the extracellular matrix marker collagen-IV (COL4, green) within the vascular basement membrane; DAPI counterstain (blue). (I–L′) Representative images of endothelial cells (CD31, white) and ACE2-positive perivascular cells (red; antibody ab15348) in juvenile human islet microvasculature. Islet endocrine cells expressing insulin and glucagon (ENDO, green; L and L′) are shown with DAPI counterstain (blue) (n = 10 total images analyzed). Yellow arrowheads point to CD31-positive endothelial cells while magenta arrowheads point to perivascular ACE2-positive cells. Inset areas (A′–L′) are marked by yellow boxes in (A)–(L). Human pancreatic donor information is available in Table S2 (A–H′, donor N2; I–L′, donor J4). See also Figure S5. Scale bars, 50 μm (A–L) and 10 μm (insets, A′–L′).

**Figure 4**
ACE2 Colocalization with Islet Pericytes (A–C) Representative images of an adult human islet labeled for platelet-derived growth factor receptor β-positive (PDGFRβ, red; A and C) and ACE2 (green; antibody ab15348; B and C) in perivascular cells within a normal donor. DAPI (blue) (n = 20 total images analyzed). (D) Image of the same islet shown in (A)–(C) with insulin labeling (INS, blue). Inset areas in (A)–(C) are marked by the yellow box. (E–G′) Representative images of an adult human islet labeled for neuron-glial antigen 2 (NG2, red; E and G), ACE2 (green; antibody ab15348; F and G), and insulin (INS, blue; G) in perivascular cells within a normal donor. DAPI (blue; E, F, and G′). Inset areas in (G′) are marked by yellow boxes in (G) and represent a subset of ACE2-expressing islet pericytes (n = 36 total images analyzed). (H) Colocalization of ACE2 with NG2- or PDGFRβ-positive cells. (I) Quantification of ACE2-positive cells expressing pericyte markers PDGFRβ and NG2. Shown are average values obtained for 3 confocal planes per islet for a minimum of 5 islets per each donor of a given age. Human pancreatic donor information is available in Table S2 (donors HP1754, HP2041, and HP2091). See also Figure S5. Scale bars, 20 μm (A–C), 40 μm (D–G), and 10 μm (G′).

**Figure 5**
ACE2 and TMPRSS2 Protein in Pancreatic Ducts and Pancreas Histology from an Individual Infected with SARS-CoV-2 (A–D′) TMPRSS2 (green), ACE2 (red; antibody R&D AF933), and KRT19 (blue) immunostaining of the exocrine compartment. DAPI (white). Yellow arrowheads point to TMPRSS2 on the apical surface of ductal cells (KRT19-positive). Magenta arrowheads point to ACE2-positive non-ductal cells. Inset areas in (A′)–(D′) are marked by yellow boxes in (A)–(D) (n = 44 total images analyzed). (E–H′) TMPRSS2 (green) and ACE2 (red; antibody R&D AF933) immunostaining of ductal epithelial cells (KRT19, blue). Yellow and white arrowheads point to TMPRSS2 and ACE2 labeling, respectively, localized to the apical surface of larger ducts (KRT19-positive cells). Inset areas in (E′)–(H′) are marked by yellow boxes in (E)–(H) (n = 44 total images analyzed). (I) Representative H&E image of human pancreas upon autopsy after COVID-19 disease. BV, blood vessel; D, duct (n = 7 total images analyzed). Human pancreatic donor information is available in Table S2 (A–D′, donor N8; E–H′, donor N9; I, COVID-19 donor 1). See also Figure S3. Scale bars, 50 μm (A–H), 10 μm (A′–H′), and 100 μm (I).

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Update of

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but are Not Enriched in Islet Endocrine Cells.
Coate KC, Cha J, Shrestha S, Wang W, Gonçalves LM, Almaça J, Kapp ME, Fasolino M, Morgan A, Dai C, Saunders DC, Bottino R, Aramandla R, Jenkins R, Stein R, Kaestner KH, Vahedi G, Consortium H, Brissova M, Powers AC. Coate KC, et al. bioRxiv [Preprint]. 2020 Oct 20:2020.08.31.275719. doi: 10.1101/2020.08.31.275719. bioRxiv. 2020. Update in: Cell Metab. 2020 Dec 1;32(6):1028-1040.e4. doi: 10.1016/j.cmet.2020.11.006. PMID: 33106804 Free PMC article. Updated. Preprint.

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References

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[1] Aid M., Busman-Sahay K., Vidal S.J., Maliga Z., Bondoc S., Starke C., Terry M., Jacobson C.A., Wrijil L., Ducat S. Vascular disease and thrombosis in SARS-CoV-2-infected Rhesus macaques. Cell. 2020 doi: 10.1016/j.cell.2020.10.005. Published online October 9, 2020. - DOI - PMC - PubMed

[2] Aid M., Busman-Sahay K., Vidal S.J., Maliga Z., Bondoc S., Starke C., Terry M., Jacobson C.A., Wrijil L., Ducat S. Vascular disease and thrombosis in SARS-CoV-2-infected Rhesus macaques. Cell. 2020 doi: 10.1016/j.cell.2020.10.005. Published online October 9, 2020. - DOI - PMC - PubMed

[3] Almaça J., Weitz J., Rodriguez-Diaz R., Pereira E., Caicedo A. The pericyte of the pancreatic islet regulates capillary diameter and local blood flow. Cell Metab. 2018;27:630–644.e4. - PMC - PubMed

[4] Almaça J., Weitz J., Rodriguez-Diaz R., Pereira E., Caicedo A. The pericyte of the pancreatic islet regulates capillary diameter and local blood flow. Cell Metab. 2018;27:630–644.e4. - PMC - PubMed

[5] Almaça J., Caicedo A., Landsman L. Beta cell dysfunction in diabetes: the islet microenvironment as an unusual suspect. Diabetologia. 2020;63:2076–2085. - PMC - PubMed

[6] Almaça J., Caicedo A., Landsman L. Beta cell dysfunction in diabetes: the islet microenvironment as an unusual suspect. Diabetologia. 2020;63:2076–2085. - PMC - PubMed

[7] Apicella M., Campopiano M.C., Mantuano M., Mazoni L., Coppelli A., Del Prato S. COVID-19 in people with diabetes: understanding the reasons for worse outcomes. Lancet Diabetes Endocrinol. 2020;8:782–792. - PMC - PubMed

[8] Apicella M., Campopiano M.C., Mantuano M., Mazoni L., Coppelli A., Del Prato S. COVID-19 in people with diabetes: understanding the reasons for worse outcomes. Lancet Diabetes Endocrinol. 2020;8:782–792. - PMC - PubMed

[9] Arda H.E., Li L., Tsai J., Torre E.A., Rosli Y., Peiris H., Spitale R.C., Dai C., Gu X., Qu K. Age-dependent pancreatic gene regulation reveals mechanisms governing human β cell function. Cell Metab. 2016;23:909–920. - PMC - PubMed

[10] Arda H.E., Li L., Tsai J., Torre E.A., Rosli Y., Peiris H., Spitale R.C., Dai C., Gu X., Qu K. Age-dependent pancreatic gene regulation reveals mechanisms governing human β cell function. Cell Metab. 2016;23:909–920. - PMC - PubMed

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SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

Affiliations

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

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Abstract

Conflict of interest statement

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