Journal: Investigative and Clinical Urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Ablation of IGFBP5 expression improves erectile function in CNI-induced ED mice. (A) Schematic diagram of the mouse CNI model and experimental procedures. CNI was performed 3 days after intracavernous injection of PBS (20 µL), shCon-lentivirus, or shIGFBP5-lentivirus (5×10 4 infection units [IFU]/mouse for shRNA lentiviral particles). Erectile function measurements and additional studies were conducted 7 days later. (B) Representative ICP responses for sham operation and CNI-induced ED mice. Electrical stimulation of the cavernous nerve was performed 1 week after CNI. The stimulus interval is indicated by the solid bar. (C, D) The ratio of the mean maximal and total ICP (area under the curve) to MSBP was evaluated for each group (n=10, *** p<0.001). IGFBP5, insulin-like growth factor-binding protein 5; CNI, cavernous nerve injury; ED, erectile dysfunction; PBS, phosphate-buffered saline; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; MSBP, mean systolic blood pressure; ns, not significant; AUC, area under the curve.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×10 4 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×10 4 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Injection, Infection, shRNA, Binding Assay, Saline, Control

Journal: Investigative and Clinical Urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Ablation of IGFBP5 expression increases cavernous endothelial-mural cell and neuronal cell contents in CNI-induced ED mice. (A, B) Immunofluorescence staining of CD31 (green), NG2 (red), nNOS (green), and neurofilament (NF, red) in the corpus cavernosum (CC) and dorsal nerve bundle (DNB) tissues from sham operation and CNI-induced ED mice, which received intracavernous injection of PBS (20 µL), shCon-lentivirus, or shIGFBP5-lentivirus (5×10 4 infection units [IFU]/mouse for shRNA lentiviral particles). Nuclei are labeled with DAPI (blue). Scale bars=100 µm (CC) and 25 µm (DNBs). (C–F) Quantitative analysis of CD31-, NG2-, nNOS-, and NF-immunopositive areas in CC and DNB tissues was performed using an image analyzer system (n=4, *** p<0.001). Data are presented as mean±standard error of the mean, with the sham operation group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; CNI, cavernous nerve injury; ED, erectile dysfunction; PBS, phosphate-buffered saline; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×10 4 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×10 4 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Staining, Injection, Infection, shRNA, Labeling, Binding Assay, Saline, Control

Journal: Investigative and Clinical Urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Ablation of IGFBP5 expression induces endothelial proliferation and reduced apoptosis in CNI-induced ED mice. (A) Representative immunofluorescence images staining of corpus cavernosum (CC) tissue showing CD31 (green) and phospho-histone H3 (pH3; red) after ICP studies. Scale bar=100 µm. (B) Immunofluorescence staining of CC tissue for CD31 (red) and TUNEL assay (green) after ICP studies. Scale bar=100 µm. Nuclei are labeled with DAPI (blue). (C, D) The number of pH3-positive or TUNEL-positive endothelial cells was quantified using an image analyzer system (n=4, *** p<0.001). Data are presented as mean±standard error of the mean values, with the sham operation group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; CNI, cavernous nerve injury; ED, erectile dysfunction; PBS, phosphate-buffered saline; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×10 4 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×10 4 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Staining, TUNEL Assay, Labeling, Binding Assay, Saline, shRNA, Control, End Labeling

Journal: Investigative and Clinical Urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Ablation of IGFBP5 expression reduces ROS production in CNI-induced ED mice. (A) Representative images of immunofluorescence staining of corpus cavernosum (CC) tissue for nitrotyrosine (a marker for peroxynitrite generation, green) and hydroethidine (an oxidative fluorescent dye detecting superoxide anions, red) after ICP studies. Scale bar=100 µm. Nuclei are labeled with DAPI (blue). (B, C) The nitrotyrosine- and hydroethidine-immunopositive areas were quantified using an image analyzer system (n=4, ** p<0.01; *** p<0.001). (D) Representative western blot showing p47 phox , iNOS, and IGFBP5 levels in the mouse cavernous tissue after ICP studies. (E–G) Normalized band intensity values for p47 phox , iNOS, and IGFBP5 (n=4, *** p<0.001). Data are presented as mean±standard error of the mean, with the sham operation group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; ROS, reactive oxygen species; CNI, cavernous nerve injury; ED, erectile dysfunction; PBS, phosphate-buffered saline; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; iNOS, inducible nitric oxide synthase.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×10 4 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×10 4 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Staining, Marker, Labeling, Western Blot, Binding Assay, Saline, shRNA, Control

Journal: Investigative and Clinical Urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Ablation of IGFBP5 expression modulates PI3K/AKT and JNK/c-Jun signaling and enhances the expression of neurotrophic factors in CNI-induced ED mice. (A) Representative western blot showing levels of p-c-Jun, c-Jun, p-JNK, JNK, p-AKT, AKT, p-PI3K, PI3K, neurotrophic factors (BDNF, NGF, and NT-3), p-IGF1R, IGF1R, Ang-1, and VEGF in the mouse cavernous tissue after ICP studies. (B–K) Normalized band intensity values for the indicated targets (n=4, * p<0.05; ** p<0.01; *** p<0.001). Data are presented as mean±standard error of the mean, with the sham operation group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; CNI, cavernous nerve injury; ED, erectile dysfunction; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; IGF1R, insulin like growth factor 1 receptor; VEGF, vascular endothelial growth factor; ns, not significant.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×10 4 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×10 4 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Binding Assay, shRNA, Control, Derivative Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Protein expression levels of Cav1.2, Piezo1, CaM, and Src in human LAA tissues. (A) Representative western blots and densitometric analysis of Cav1.2 and Piezo1 proteins in LA tissues of AF patients and those with SR. (B) Representative western blots and densitometric analysis of CaM and Src protein in LA tissues of AF patients and those with SR. GAPDH was the internal control. ** p < 0.01. Values are presented as the mean ± standard error of the mean (SEM).

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effects of hypertension on the incidence of AF, I Ca,L , and Piezo1 expression in Wistar rats and SHRs with and without Val treatment. (A) Representative baseline surface ECG and intra-atrial electrocardiogram (IAEG); (B) The incidence AF in Wistar rats and SHRs treated with and without Val treatment ( n = 8). ** p < 0.01 vs. Wistar rat; ## p < < 0.01 vs. SHRs. (C) Typical surface ECG recordings of rats with AF that spontaneously reverted to SR and typical disorganized amplification of atrial waves (f wave). (D) Representative traces of AP in atrial myocytes from Wistar rats, SHRs, and SHR + Val groups and a histogram of APD in atrial myocytes from each group ( n = 12–15 myocytes from 3–4 rats). * p < 0.05 vs. Wistar rat; # p < 0.05 vs. SHRs. (E) Representative traces of I Ca,L (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in atrial myocytes of each group ( n = 8–14 myocytes from 3–4 rats). (F) Representative examples of immunohistochemical analysis of LA tissues from Wistar rats and SHRs treated with and without Val using Ab against Piezo1. Scale bar, 20 μm. (G) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in LA tissues of Wistar rats and SHRs. GAPDH was the internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Amplification, Activation Assay, Immunohistochemical staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of HHP on the depression of I Ca,L in HL-1 cells. (A) Representative traces of AP in HL-1 cells under various hydrostatic pressures (0, 20, and 40 mmHg) for 24 h. APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 10, and 7 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (B) Representative traces (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L ( n = 8–18 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (C) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in HL-1 cells under various hydrostatic pressure (0, 20, and 40 mmHg) for 24 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: The effects of Piezo1 on perceiving HHP and mediating the decrease of I Ca,L . (A,B) Representative Ca 2+ traces and Δ Ca i 2 + (ΔF/F) are shown. Ca 2+ entry was evoked by 10 μm Yoda1 in HL-1 cells stimulated by HHP in the presence or absence of the Piezo1 inhibitor GsmTx4 ( n = 50) or siRNA specifically knockdown Piezo1 ( n = 53–58). si-C, scrambled (control) siRNA; si-P, siRNA directed against Piezo1. (C) Representative traces of AP in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment (3.0 μM) and APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 7, and 11 at 0, 40, and 40 mmHg + GsmTx4). * p < 0.05, ** p < 0.01 vs. 0 mmHg; # p < 0.05 vs. 40 mmHg. (D) Representative traces (pulse protocol, inset), corresponding current–voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment ( n = 9–15). ** p < 0.01 vs. 0 mmHg; ## p < 0.01 vs. 40 mmHg. (E) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (F) Representative blots and densitometry analysis of Cav1.2 in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of CaM/Src on the decrease of I Ca,L induced by HHP or Yoda1 stimulation. (A) Representative blots and densitometry analysis of CaM and Src in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (B) Representative blots and densitometry analysis of CaM and Src in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. (C) Representative blots and densitometry analysis of Src and p-Src ( n = 4) in HL-1 cells transfected with scrambled (control) siRNA or siRNA directed against Piezo1 for 48 h, then treated with Yoda1 at different dosages (0, 1, and 3 μM) for 15 min. (D) Representative traces of AP and histogram of APD in HL-1 cells ( n = 7–8). * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (E) Current–voltage relationship for I Ca,L ( n = 9–17) in HL-1 cells stimulated by 40 mmHg pressure treated with 15 μM PP1 or W7. * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (F) Representative blots and densitometry analysis of Cav1.2 and Src in HL-1 cells stimulated by 40 mmHg pressure treated with W7 under different concentrations (5, 10, 15, and 20 μM). (G) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure treated with PP1 (15 μM). (H) Current-voltage relationship for I Ca,L ( n = 8–10) in HL-1 cells stimulated by Yoda1(3 μM) treated with PP1. * p < 0.05, ** p < 0.01 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Yoda1. (I) Representative blots and densitometry analysis of Cav1.2 in Yoda1(3μM) -stimulated HL-1 cells treated with PP1 (15 μM). GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Schematic representation of the mechanism for the decrease of I Ca,L induced by HHP. Piezo1 activated by HHP depressed I Ca,L contributing to increased AF susceptibility through the CaM/Src/Pitx2 pathway.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: