Journal: Diabetes

Article Title: SARS-CoV-2 Spike S1 Subunit Triggers Pericyte and Microvascular Dysfunction in Human Pancreatic Islets

doi: 10.2337/db24-0816

Figure Lengend Snippet: Vascular pericytes in human pancreatic islets express ACE2. A : Z projection of confocal images of pancreas of organ donor without diabetes (age 29 years) showing ACE2 immunostaining (green). Dashed regions indicate pancreatic islets (insulin staining shown in Supplementary Fig. 1 ). B : Z projection of confocal images of islet in human pancreatic section from organ donor without diabetes (age 15 years) showing ACE2 immunostaining (green) and insulin (white). ACE2 is present in islets but not expressed by islet β-cells. C : Z projection of confocal images of islet in tissue section from donor (age 44 years) immunostained for ACE2 (green) and pericyte marker NG2 (magenta). D and E : Confocal images of regions in pancreas of donor (age 44 years) showing ACE2 (green) and either pericyte/stellate cell marker platelet-derived growth factor receptor β (PDGFRβ; magenta ( D ) or endothelial cell marker CD31 (magenta) ( E ). F : Quantification of colocalization of ACE2 and either NG2, PDGFRβ, CD31, or insulin in confocal images of islets from donors without diabetes (ages 15–54 years; n = 9–30 islets from three donors/mix). Mander coefficients are shown reflecting fraction of ACE2 that overlaps with each marker. G : Quantification of transcript levels of Ace2 gene in different clusters of islet vascular and perivascular cells (left) and endocrine cells (right) from 27 donors without diabetes (from Network for Pancreatic Organ Donors With Diabetes and University of Pennsylvania). Data extracted from single-cell RNA sequencing database ( www.isletgenomics.org ). Mean ± SD are shown. H : Correlation between Ace2 levels in quiescent stellate cells and donor age. Simple linear regression shown in magenta. I : Quantification of colocalization of NG2 with ACE2 (Mander coefficient showing fraction of NG2 that overlaps with ACE2) in 19–26 islets from organ donors age 14–54 years. Scale bars, 100 ( A ), 50 ( B ), 20 ( C ), and 10 μm ( E ). * P < 0.0001 ( H ) and P = 0.0003 by unpaired t test ( I ). TPM, transcripts per million.

Article Snippet: We recorded changes in islet pericyte [Ca 2+ ]i and capillary diameter induced by angiotensin II (angII; 100 nmol/L; cat. no. 1158; Tocris Bioscience), angiotensin (1-7) (ang1-7; 100 nmol/L; cat. no. 1562; Tocris Bioscience), ACE2 inhibitor MLN4760 (10 μmol/L; cat. no. 3345; Tocris Bioscience), norepinephrine (20 μmol/L; cat. no. 5169; Tocris Bioscience), and endothelin-1 (10 nmol/L).

Techniques: Immunostaining, Staining, Marker, Derivative Assay, RNA Sequencing

Journal: Diabetes

Article Title: SARS-CoV-2 Spike S1 Subunit Triggers Pericyte and Microvascular Dysfunction in Human Pancreatic Islets

doi: 10.2337/db24-0816

Figure Lengend Snippet: Model of SARS-CoV-2–dependent impairment of islet function. In our model, a dysfunctional pancreatic islet microvasculature underlies some diabetogenic actions of SARS-CoV-2. In this article, we show that local RAS exists in human endocrine pancreas and is associated with the microvasculature. Pericytes in pancreatic islets express different components of local RAS, such as ACE2 and AT1 receptors. Under physiologic conditions, ACE2 is a carboxypeptidase that degrades vasoconstrictor angII into vasodilator ang1-7, preventing angII from activating AT1 receptors and keeping pericytes functional and capillaries responsive. On exposure to SARS-CoV-2 spike protein, ACE2 binds to this spike protein, and it is internalized, no longer degrading angII into ang1-7. This leads to increase in endogenous levels of angII in pancreas, which bind to AT1 receptors in pericytes, activating them and compromising vasomotive responses. Our study suggests that islet pericyte dysfunction when SARS-CoV-2 spike is present can interfere with proper islet responses and compromise glucose homeostasis. This figure was made using BioRender ( https://biorender.com ).

Article Snippet: We recorded changes in islet pericyte [Ca 2+ ]i and capillary diameter induced by angiotensin II (angII; 100 nmol/L; cat. no. 1158; Tocris Bioscience), angiotensin (1-7) (ang1-7; 100 nmol/L; cat. no. 1562; Tocris Bioscience), ACE2 inhibitor MLN4760 (10 μmol/L; cat. no. 3345; Tocris Bioscience), norepinephrine (20 μmol/L; cat. no. 5169; Tocris Bioscience), and endothelin-1 (10 nmol/L).

Techniques: Functional Assay

Journal: Diabetes

Article Title: SARS-CoV-2 Spike S1 Subunit Triggers Pericyte and Microvascular Dysfunction in Human Pancreatic Islets

doi: 10.2337/db24-0816

Figure Lengend Snippet: SARS-CoV-2 spike affects ACE2 surface expression, activity, and angII levels. A : Traces showing changes in Fluo4 fluorescence in islet pericytes on application of ACE2 inhibitor MLN4760 (10 μmol/L; in 3G) in living human pancreas slices incubated previously for 1 h with either SARS or HCoV spike. B : Violin plots showing quantification of total area under curve (AUC) of fluorescence traces as in A showing pericyte [Ca 2+ ]i responses to MLN4760 ( n = 24–40 islet pericytes per four donors). C and D : Confocal images of pericytes in islets in slices treated with either HCoV spike ( C ) or SARS spike ( D ) immunostained for NG2 (magenta), ACE2 (green), and somatostatin (to identify islet; not shown). Images of islet pericytes from two different donors are shown. Scale bar, 10 μm. E : Dashed lines in C and D indicate where lines were drawn to generate line plot profiles. Each line corresponds to pericytes from different donors. F : Quantification of fold change in angII concentration in supernatant of slices treated for 1 h with either spike protein. Each well contained two slices from same donor ( n = 13–16 wells; from eight organ donors; P = 0.14 by one-sample t test compared with theoretic mean of 1 for HCoV incubated). * P = 0.02 by ; unpaired t test ( B ) and P = 0.02 by one-sample t test compared with theoretic mean of 1 for SARS incubated ( F ). ns, not significant.

Article Snippet: We recorded changes in islet pericyte [Ca 2+ ]i and capillary diameter induced by angiotensin II (angII; 100 nmol/L; cat. no. 1158; Tocris Bioscience), angiotensin (1-7) (ang1-7; 100 nmol/L; cat. no. 1562; Tocris Bioscience), ACE2 inhibitor MLN4760 (10 μmol/L; cat. no. 3345; Tocris Bioscience), norepinephrine (20 μmol/L; cat. no. 5169; Tocris Bioscience), and endothelin-1 (10 nmol/L).

Techniques: Expressing, Activity Assay, Fluorescence, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: A time-dependent increase in ACE2 activity occurred in the cell media. Results are means ± SE, corrected for total cell protein from culture dishes set up in parallel to those where media was analyzed for ACE2 activity on three successive days. *P<0.001 vs day 4 and day 5, and **P<0.01 vs day 4, n = 4.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: (A) Representative immunoblot for ACE2 protein in concentrated media (Lanes 1–3) and cell lysates (Lanes 4–6) from mouse PT cells. Lanes 1 and 4: wildtype cells, Lanes 2 and 5: ACE2 knockout (KO) cells, Lanes 3 and 6: ACE2 KO cells transfected with a human ACE2 expression vector, Lane 7: mouse kidney cortex showing a band at ∼100 kDa, used as a positive control. Lane 1 shows two bands in the media at ∼90 kDa and ∼70 kDa for mouse ACE2. Lane 3 shows two bands in the media for human ACE2 in transfected cells, at ∼110 kDa and ∼95 kDa. Lanes 4 and 6 show a single band in cell lysates at ∼100 kDa for mouse ACE2, and ∼120 kDa for human ACE2, respectively. Lanes 2 and 5 show no ACE2 bands detected on immunoblots of both media and cell lysates from untransfected ACE2 KO cells. (B) Increased ACE2 activity in the media from ACE2 KO cells transfected with a human ACE2 expression vector (HA-hACE2, 3.75 µg on 35 mm culture dishes). Untransfected cells and cells transfected with an empty pcDNA3 vector had no detectable ACE2 activity in the media. Numbers in parentheses represent mean values for ACE2 activity. *P<0.001 vs untransfected control or empty pcDNA3 vector, n = 4.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Western Blot, Knock-Out, Transfection, Expressing, Plasmid Preparation, Positive Control, Activity Assay, Control

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: Representative immunoblot for ACE2 treated without (−) or with (+) deglycosylation with PNGase F in the media (Lanes 1–2) and cell lysates (Lanes 3–4). Lanes 1 and 3: wildtype PT cells, Lanes 2 and 4: ACE2 knockout (KO) PT cells transfected with a human ACE2 vector, Lane 5: mouse kidney cortex. Lanes 1+ and 2+ show a reduction in the sizes of ACE2 fragments in media fractions to ∼75 kDa and ∼60 kDa for mouse ACE2, and to ∼80 kDa and ∼65 kDa for human ACE2, respectively. Lanes 3+ and 4+ show a reduction in the sizes of ACE2 in cell lysates to ∼85 kDa for both mouse and human ACE2 treated with the PNGase F, respectively. Lane 5+ shows a reduction in size of ACE2 in mouse cortex from ∼100 kDa to ∼85 kDa after treatment with PNGase F.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Western Blot, Knock-Out, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: (A) Mass spectrometry identified peptides matched with the mouse ACE2 sequence (SWISS-PROT database no. Q8R0I0) at positions 18-706. The identified peptide sequences significantly matched with mouse ACE2 precursor in the database are underlined and shown in bold. The peptides matched with mouse ACE2 sequence, but not statistically significant, are underlined only (see for detailed analyses of peptides). The overall matched sequences cover 32% of mouse ACE2 sequences. (B) Tandem mass spectrum of the C -terminal peptide (SEVEDAIRM 698–706). The b and y ions result from the cleavage of peptide bonds and correspond to N -terminal and C -terminal fragments of the peptide, respectively. The detected b and y ions are consistent with the peptide sequence shown on the top part of the panel. Ions score: 50, Observed ion: 533.25, Mr(expt): 1064.48, Mr(calc): 1064.48. Expect value: 0.002 (see for details). The amino acid Met 706 detected is from non-tryptic cleavage, suggesting a cleavage site for the ectodomain shedding of mouse ACE2 into the media.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Mass Spectrometry, Sequencing

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: (A) Mass spectrometry identified peptides matched with the mouse ACE2 sequence (SWISS-PROT database no. Q8R0I0) at positions 18-577. The identified peptides significantly matched with mouse ACE2 sequences are underlined and shown in bold. The peptides matched with mouse ACE2 sequence, but not statistically significant, are underlined only (see for detailed analyses of peptides). The overall sequence coverage is 24%. (B) Tandem mass spectrum of the C -terminal peptide (ALENVVGAR 569-577). The b and y ions result from the cleavage of peptide bonds and correspond to N -terminal and C -terminal fragments of the peptide, respectively. The detected b and y ions are consistent with the peptide sequence shown on the top part of the panel. Ions score: 59, Observed ion: 464.77, Mr(expt): 927.52, Mr(calc): 927.51. Expect value: 0.0002 (see for details).

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Mass Spectrometry, Sequencing

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: A) Partial amino acid sequence of 75 kDa shed ACE2 fragment (deglycosylated) is shown, beginning with N -terminal amino acid Gln 18 (Q). The first and last amino acids of the C-terminal and N-terminal fragments are underlined, where numbers below the line indicate their amino acid positions. The dashed line represents amino acids between position 40 and 701 (sequence not shown). The most C-terminal amino acid observed is shown in bold. Vertical black bars represent sites of tryptic cleavage. Red vertical bar is non-tryptic cleavage site at Met 706 (M), a putative ACE2 cleavage site. B) Partial amino acid sequence of 60 kDa shed ACE2 fragment (deglycosylated) is shown, beginning with N -terminal amino acid Gln 18 (Q). The first and last amino acids of the C-terminal and N-terminal fragments are underlined, where numbers below the line indicate their amino acid positions. The dashed line represents amino acids between position 40 and 571 (sequence not shown). The most C-terminal amino acid observed is shown in bold. Vertical black bars indicate sites of tryptic cleavage, and include Arg 577 (R) and Lys 596 (K). Cleavage site for this fragment may occur at or C -terminal to Arg 577 .

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: Primary cultures of mouse PT cells were incubated for 24, 48 and 72(C, 7.8 mM D-glucose), with Ang II (10 −7 M) or in high D-glucose (D-G, 25 mM) media. As a control for osmolality, some cells were incubated with L-glucose (L-G, 25 mM). ACE2 activity in the media was assayed at each time point. *p<0.001 vs L-G, p<0.004 vs C, p<0.025 vs Ang II, all at 48 hrs, n = 7–9. **p<0.001 vs all 3 other groups at 72 hrs, n = 14–18. # p< 0.04 vs C at 72 hrs, n = 14–18.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Incubation, Control, Activity Assay

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: (A) Effect of MMP inhibitor GM6001 on ACE2 activity in the media. Mouse PT cells were incubated for 72 hrs in high D-glucose (D–G, 25 mM) media in the presence or absence of GM6001 (GM, 5×10 −5 M). *p<0.001 vs C and GM, **p<0.025 vs C and GM, n = 5. (B) Effect of the ADAM17 inhibitor TAPI-1 on high glucose-stimulated ACE2 activity in the media. Mouse PT cells were incubated for 72 hrs in high D-glucose (D–G, 25 mM) media in the presence or absence of TAPI-1 (10 −7 –10 −5 M). *p<0.001 vs C, TAPI-1 10 −5 M, and D-G+TAPI-1 10 −5 M. **p<0.001 vs C and TAPI-1 10 −5 M, p>0.05 vs D-G,D-G+TAPI-1 10 −7 M and D-G+TAPI-1 10 −5 M. n = 4–9. (C) Effect of TAPI-1 on Ang II-stimulated ACE2 activity in the media. Mouse PT cells were incubated for 72 hrs with Ang II (10 −7 M) in the presence or absence of TAPI-1 (10 −5 M). *p<0.015 vs C, p<0.03 vs TAPI-1, n = 8. (D) Effect of TAPI-2 on high D-glucose-stimulated ACE2 activity in the media. Mouse PT cells were incubated for 72 hrs in high D-glucose (D-G, 25 mM) media in the presence or absence of TAPI-2 (10 −6 – 5×10 −5 M). *p<0.001 vs C and TAPI-2 (5×10 −5 M), p<0.015 vs D-G+TAPI-2 (5×10 −5 M). **p<0.005 vs C, p<0.015 vs TAPI-2 (5×10 −5 M), p>0.05 vs D-G and D-G+TAPI-2 (5×10 −5 M). n = 4.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

doi: 10.1371/journal.pone.0085958

Figure Lengend Snippet: (A) Mouse PT cells were incubated for 72 hrs in normal media (C, 7.8 mM D-glucose), with or without Ang II (10 −7 M), high D-glucose (D-G, 25 mM), or high L-glucose (25 mM). Above graph is representative immunoblot for ACE2 in the media, showing bands at ∼90 kDa and ∼70 kDa. (B) Graphical representation of densitometry analysis of two ACE2 bands on immunoblots. For the ∼90 kDa band, *p<0.05 vs C, **p<0.001 vs C, **p<0.003 vs L-G; n = 5. For the ∼70 kDa band, *p<0.04 vs C; **p<0.001 vs C or L-G, **p<0.03 vs Ang II; n = 5.

Article Snippet: Cell media (15 μL) or membrane fractions (1-2 μg) were then added to the wells of a 96-well plate (total volume 100 μL/well) in a solution containing 37.5 mM MES, 225 mM NaCl, 7.5 μM ZnCl 2 , 0.75 mM N -ethylmaleimide (NEM), 0.75 mM phenylmethylsulfonyl fluoride (PMSF), 11.25 μM ACE2 substrate, with or without 1 μM of the ACE2 inhibitor MLN-4760 (GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA).

Techniques: Incubation, Western Blot