Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: Panel A: Box-plot showing the differences in the median (horizontal lines), interquartile range (boxes) and 95% of the observed values (whiskers) for sACE2 activity between controls and STEMI patients. Panel B: Scatter-plot detailing individual changes in sACE2 activity among patients. *: p<0.001 for comparison between baseline measurements in patients and controls. †: p<0.01 for comparison between baseline and 7 days measurements by paired analysis. ACE2: Angiotensin Converting Enzyme 2; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: A weak but significant correlation was observed between infarct size and sACE2 activity at 7 days (panel A) and at 6 months follow-up (panel B). FU: Follow-up. sACE2: Serum Angiotensin Converting Enzyme 2; LV: left ventricular; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: Scatter-plots showing a statistically significant correlation between ACE2 activity at 7 days and EF during admission (panel A) and at 6 months follow-up (panel B). sACE2: Serum Angiotensin Converting Enzyme 2; EF: ejection fraction; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: Concentration-dependent stimulation of ACE2 shedding by D -glucose in mouse proximal tubular cells. (A) Graph depicts effect of varying concentrations of D - or L -glucose on ACE2 activity recovered from cell culture media. ∗ P < 0.01 vs. 0, 2, 4, and 7.8 mM D -glucose. ∗∗ P < 0.01 vs. 0 mM D -glucose; n = 4–5. (B) Graph depicts effect of varying concentrations of D -glucose on ACE2 shedding into culture media, determined by immunoblot analysis. Representative immunoblot is depicted above graph, showing ACE2 fragments at 90 and 70 kDa, with mouse kidney cortex ACE2 at 100 kDa. ∗ P < 0.01 vs. 0, 2, 4, and 7.8 mM; ∗∗ P < 0.05 vs. 0 and 2 mM; † P < 0.05 vs. 0 mM; n = 5.

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Concentration Assay, Activity Assay, Cell Culture, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: The pan-PKC inhibitor sotrastaurin reduces ACE2 shedding in mouse proximal tubular cells. (A) Graph depicts effect of sotrastaurin (10-5-10-8 M) on ACE2 activity recovered from cell culture media. ∗ P < 0.005 vs. all other groups (C, vehicle-treated control); ∗∗ P < 0.05 vs. C and 10 -8 M; n = 6. (B) Graph depicts effect of varying concentrations of sotrastaurin on ACE2 shedding into culture media, determined by immunoblot analysis. Representative immunoblot is depicted above graph, showing ACE2 fragments at 90 and 70 kDa, with mouse kidney cortex ACE2 at 100 kDa. ∗ P < 0.01 vs. all other groups; ∗∗ P < 0.05 vs. 10 -7 M and vehicle-treated control (C); n = 5. (C) Graph depicts effect of sotrastaurin (10 -5 M), with or without the ADAM17 inhibitor TAPI-1 (10 -5 M), on ACE2 activity in the culture media, in basal conditions ( D -glucose 7.8 mM) or high D -glucose (28 mM). ∗ P < 0.01 vs. Control and TAPI-1 (7.8 mM D -glucose); In D -glucose 28 mM, † P < 0.001 vs. Control and TAPI-1, †† P < 0.05 vs. Control; ‡ P < 0.001 vs. Control ( D -glucose 7.8 mM); n = 4–5.

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Activity Assay, Cell Culture, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: Lack of inhibitory effect of the PKC-α and -β1 inhibitor Go6976 (A) and the PKC-β1 and β-2 inhibitor ruboxistaurin (B) on constitutive ACE2 shedding in mouse proximal tubular cells. Graphs depict dose-dependent effects of Go6976 and ruboxistaurin on ACE2 activity in the culture media, compared to vehicle-treated cells (C); n = 4 experiments each for (A,B) .

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: Inhibition of ADAM10 or matrix metalloproteinases (MMPs)-2, -8, and-9 has no effect on constitutive ACE2 shedding in mouse proximal tubular cells. Graphs depict dose-dependent effects of the ADAM10 inhibitor GI250423X (A) , MMP-2 inhibitor (B) , MMP-8 inhibitor (C) , and MMP-9 inhibitor (D) on ACE2 activity in the culture media, compared to vehicle-treated cells (C); n = 4–5 experiments each for (A–D) .

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Inhibition, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: The PKC-δ inhibitor rottlerin reduces constitutive and high D -glucose-induced ACE2 shedding in mouse proximal tubular cells. (A) Graph depicts effect of rottlerin (0.01–1 μM) on ACE2 activity recovered from cell culture media, in D -glucose 7.8 mM. ∗ P < 0.001 vs. all other groups (C, vehicle-treated control); ∗∗ P < 0.005 vs. C, 0.01 μM, 0.05 μM, and 0.1 μM; n = 5–6. (B) Graph depicts effect of rottlerin (1 μM) on ACE2 shedding into culture media, determined by immunoblot analysis. Representative immunoblot is depicted above graph, showing ACE2 fragments at 90 and 70 kDa, with mouse kidney cortex ACE2 at 100 kDa. ∗ P < 0.05 vs. vehicle treated control (C); n = 4. (C) Graph depicts effect of rottlerin (1 μM), with or without the ADAM17 inhibitor TAPI-1 (10 -5 M), on ACE2 activity in the culture media, in basal conditions ( D -glucose 7.8 mM) or high D -glucose (28 mM); ∗ P < 0.001 vs. Control and TAPI-1, in D -glucose 7.8 mM; † P < 0.001 vs. Control, in D -glucose 28 mM; ∗∗ P < 0.001 vs. Control (7.8 mM D -glucose); n = 9.

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Activity Assay, Cell Culture, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells

doi: 10.3389/fphar.2016.00146

Figure Lengend Snippet: siRNA knockdown of PKC-δ inhibits constitutive ACE2 shedding in mouse proximal tubular cells. (A) Graph depicts effect of transfection of siRNA against PKC-δ (100 nM) on protein expression of PKC-δ in mouse proximal tubular cells. Results are depicted as PKC-δ protein relative to cells transfected with scrambled siRNA sequence. Representative immunoblot is shown above graph, with PKC-δ at 78 kDa, and α-tubulin loading control at 50 kDa. ∗ P < 0.05; n = 4. (B) Graph depicts effect of transfection of siRNA against PKC-δ (100 nM) on ACE2 activity in cell culture media. Results are depicted as ACE2 activity relative to cells transfected with a scrambled siRNA sequence. ∗ P < 0.05; n = 4. (C) Graph depicts effect of transfection of siRNA against PKC-ε (30 nM) on protein expression of PKC-ε in mouse proximal tubular cells. Results are depicted as PKC-ε protein relative to cells transfected with scrambled siRNA sequence. Representative immunoblot is shown above graph, with PKC-ε at 82 kDa, and α-tubulin loading control at 50 kDa. ∗ P < 0.02; n = 6. (D) Graph depicts effect of transfection of siRNA against PKC-ε (30 nM) on ACE2 activity in cell culture media. Results are depicted as ACE2 activity relative to cells transfected with a scrambled siRNA sequence; n = 7.

Article Snippet: Angiotensin-converting enzyme 2 activity in cell culture media was measured using a commercially available synthetic fluorogenic substrate for ACE2 [Mca-Ala-Pro-Lys(Dnp)-OH] (AnaSpec, San Jose, CA, USA), as we have described ( , ).

Techniques: Knockdown, Transfection, Expressing, Sequencing, Western Blot, Control, Activity Assay, Cell Culture