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: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Portal pressure responses and angiotensin peptide production in rat liver are determined by relative activity of ACE and ACE2

doi: 10.1152/ajpgi.00045.2009

Figure Lengend Snippet: Angiotensin-(1-7) production in rat liver. Ang I (99 pmol) or Ang II (60 pmol) bolus was injected into the portal vein of in situ perfused healthy (○ and open bars) and fibrotic (• and solid bars) rat liver preparations with or without the ACE inhibitor lisinopril (10−6 mol/l), ACE and NEP inhibitor omapatrilat (10−6 mol/l), ACE2 inhibitor MLN4760 (10−6 mol/l), or a combination of MLN4760 and omapatrilat. Angiotensin-(1-7) production from Ang II (A) and the baseline-corrected total area under the respective curves (B), and angiotensin-(1-7) production from Ang I (C) and the baseline-corrected total area under the respective curves (D), and Ang II-induced portal pressure responses (E) and the area under pressure response curve (F) are shown. Each symbol represents the mean ± SE profile from 4–5 rats per treatment group. Note that y-axis scale is different between A and C and between B and D. Arrow indicates the time of bolus injection. ****P < 0.0005, **P < 0.01, *P < 0.05, vs. respective healthy livers. ††††P < 0.0005, between the 2 groups indicated by brackets. In E and F, *P < 0.05 vs. other groups except bile duct ligation (BDL)/ACE inhibition group (P = 0.059).

Article Snippet: MLN4760 was provided by Millennium Pharmaceuticals, Cambridge, MA.

Techniques: Injection, In Situ, Ligation, Inhibition

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Portal pressure responses and angiotensin peptide production in rat liver are determined by relative activity of ACE and ACE2

doi: 10.1152/ajpgi.00045.2009

Figure Lengend Snippet: Ang II production and portal pressure changes in response to Ang I in rat liver. Ang I bolus (99 pmol) was injected into the portal vein of in situ perfused healthy (○ and open bars) and fibrotic (• and solid bars) rat liver preparations with or without the ACE inhibitor lisinopril (10−6 mol/l), ACE and NEP inhibitor omapatrilat (10−6 mol/l), ACE2 inhibitor MLN4760 (10−6 mol/l), or a combination of MLN4760 and omapatrilat. Ang II production (A) and the area under the Ang II curve (B) and Ang I-induced portal pressure responses (C) and the area under pressure-response curve (D) are shown. Each symbol represents the mean ± SE profile from 4–5 rats per treatment group. Arrows indicate the time of bolus injection. ****P < 0.0005, **P < 0.01, *P < 0.05, vs. respective healthy livers. ††††P < 0.0005, †P < 0.05, between the 2 groups indicated by brackets.

Article Snippet: MLN4760 was provided by Millennium Pharmaceuticals, Cambridge, MA.

Techniques: Injection, In Situ

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Portal pressure responses and angiotensin peptide production in rat liver are determined by relative activity of ACE and ACE2

doi: 10.1152/ajpgi.00045.2009

Figure Lengend Snippet: ACE2 activity of the perfused rat liver. In situ perfused healthy (open bars) and fibrotic (solid bars) rat livers, incubated with or without the ACE inhibitor lisinopril (10−6 mol/l), ACE and NEP inhibitor omapatrilat (10−6 mol/l), ACE2 inhibitor MLN4760 (10−6 mol/l), or a combination of MLN4760 and omapatrilat were used for cell membrane preparations for measurement of ACE2 activity. ACE2 activity was determined by measuring nanomoles of ACE2 substrate [Mca-APK-(Dnp)-OH] cleaved by solubilized membrane fractions. Each bar represents the mean ± SE activity from 4–5 rats per treatment group. ACE2 activity was not different between different groups of healthy livers so that the mean value from all groups is shown. ****P < 0.0005, ***P < 0.005, **P < 0.01, *P < 0.05 vs. group indicated by brackets.

Article Snippet: MLN4760 was provided by Millennium Pharmaceuticals, Cambridge, MA.

Techniques: Activity Assay, In Situ, Incubation, Membrane

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Standard curves for ACE2 activity assay. The assay was performed for 16 h incubation. Graph shows the concentrations of recombinant ACE2 (rACE2) versus the ACE2 activity (RFU). ( a ) Standard curves generated for human rACE2. The ACE2 inhibitors MLN-4760 (10 −6 M) or DX600 (10 −6 M) were used. The relationship is highly linear for rACE2 concentrations between 0 and 50 ng/ml ( R 2 = 0.9998, p < 0.001 for MLN-4760, R 2 = 0.9999, p < 0.001 for DX600). ( b ) Standard curve generated for mouse rACE2. The ACE2 inhibitor MLN-4760 (10 −6 M) was used. A highly linear relationship exists for rACE2 concentrations between 0 and 50 ng/ml ( R 2 = 0.9976, p < 0.001)

Article Snippet: 2 inhibitor: MLN-4760 (10 −5 M stock in ddH 2 O, also called GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA, and commercially available from Calbiochem, San Diego, CA, USA, Catalog Number: 530616) or DX600 (linear form, 10 −5 M stock in ddH 2 O, AnaSpec, Catalog Number: 62337) .

Techniques: Activity Assay, Incubation, Recombinant, Generated

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Effect of ACE2 inhibitors MLN-4760 (10 −6 M), DX600 (10 −6 M), and ACE inhibitor captopril (10 −5 M) on ACE2 enzyme activity. The assay was performed for 16 h incubation. Graph depicts the concentrations of recombinant ACE2 versus the Relative Fluorescence Units (RFU). A background RFU value (67.5 RFU for a , 62.0 RFU for b ) of the substrate blank control was subtracted from the readings for each well. ( a ) Effect of MLN-4760, DX600, and captopril on human rACE2 activity. ( b ) Effect of MLN-4760, DX600, and captopril on mouse rACE2 activity

Article Snippet: 2 inhibitor: MLN-4760 (10 −5 M stock in ddH 2 O, also called GL1001, provided by Ore Pharmaceuticals, Cambridge, MA, USA, and commercially available from Calbiochem, San Diego, CA, USA, Catalog Number: 530616) or DX600 (linear form, 10 −5 M stock in ddH 2 O, AnaSpec, Catalog Number: 62337) .

Techniques: Activity Assay, Incubation, Recombinant, Fluorescence