Mesenchymal stem cells (MSCs) engrafted into the lung parenchyma and expressed secreted Klotho (SKL). A, Green fluorescent protein (GFP)-positive cells in the lungs of monocrotaline (MCT)+MSC-SKL-GFP and MCT+MSC-GFP groups. B, Western blot analysis of SKL protein levels in the lung lysates. *P<0.05 vs saline. n=6. Data=means±SEM.
Mesenchymal stem cells (MSCs) overexpressing secreted Klotho (SKL) attenuated monocrotaline (MCT)-induced inflammation in pulmonary arteries (PAs) and abolished downregulation of SIRT1 expression in the lungs. A, Immunohistochemical analysis of macrophages around small PAs (dark brown staining, CD68 marker). B, Quantification of CD68 staining. C, Western blot analysis of SIRT1 expression in lung lysates. *P<0.05, ***P<0.001 vs saline; #P<0.05 vs MCT. n=6. Data=means±SEM. GFP indicates green fluorescent protein; and SIRT1, Sirtuin 1.
Serum Klotho was decreased in patients with arterial stiffness and hypertension. A, Serum levels of Klotho. B, Brachial-ankle pulse wave velocity in hypertensive patients (HP) and normal controls (NC). C, Systolic blood pressure. D, Diastolic blood pressure in hypertensive patients and normal controls. n=13 to 14 participants/group. Data=means±SEM. *P<0.05, ***P<0.001 vs NC.
Klotho deficiency caused arterial stiffness and spontaneous hypertension, which were associated with decreased expression and activity of SIRT1 in aorta. A, Time course of pulse wave velocity and (B) systolic blood pressure in wild-type (WT) and KL+/– mice, n=10 to 12 mice/group. C, Representative Western blot bands and quantitative analysis of SIRT1 protein expression in aortas. D, Representative Western blot bands and quantitative analysis of Acetyl-p53 and total p53 in aortas. SIRT1 activity is expressed as the relative ratio of Acetyl-p53/total P53. E, Representative western blot bands and quantitative analysis of plasma Klotho. Total protein with Ponceau staining served as loading control. Protein expression was normalized to the loading control, and the relative expression calculated as the fold change relative to the experimental control (WT). n=6 animals/group for Western blot analysis. Data=means±SEM.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs WT.
Activation of SIRT1 attenuated arterial stiffening and hypertension in KL+/– mice without affecting levels of circulatory Klotho. A, Pulse wave velocity and (B) systolic blood pressure in KL+/– mice. C, Direct BP measured by intra-arterial cannulation under anesthesia. D, Representative Western blot bands and quantitative analysis of plasma Klotho. Protein expression was normalized to the loading control, and the relative expression calculated as the fold change relative to the experimental control (wild type [WT]+saline). n=10 to 12 animals/group. Data=means±SEM. *P<0.05, **P<0.01, ***P<0.001 vs WT+saline; +P<0.05, ++P<0.01, +++P<0.001 vs KL+/–+saline.
SRT1720 rescued the downregulation of SIRT1 activity and protein expression in the aortas of KL+/– mice. A, Representative Western blot bands of Acetyl-P53 and total P53 and the quantification of SIRT1 activity. Data were expressed as the ratio of acetyl-p53/total P53. B, Representative Western blot bands and quantitative analysis of SIRT1 protein expression in aortas. Protein expression was normalized to β-actin, and the relative expression calculated as the fold change relative to the control (wild type [WT]+saline). C, Representative photomicrographs of immunohistochemical analysis of SIRT1 expression in aortic sections. The negative control was sample in the absence of the primary antibody. Brown staining indicates positive SIRT1 staining, arrows indicate positively stained endothelial cells, and arrowheads indicate positively stained smooth muscle cells. Semi-quantitative analysis of SIRT1-positive staining in the intima and media of the aortic wall (data were calculated as fold changes of the WT+saline group). n=6 animals/group. Data=means±SEM. *P<0.05, **P<0.01 vs WT+saline; +P<0.05, ++P<0.01 vs KL+/–+saline.
Activation of SIRT1 by SRT1720 enhanced AMP-activated protein kinase α (AMPKα) activity in aortas. A, Representative Western blot bands and quantitative analysis of phosphorylated AMPKα (p-AMPKα) and AMPKα in aortas. Protein expression was normalized to β-actin, and the relative expression was calculated as the fold change relative to the control (wild type [WT]+saline). B, Representative photomicrographs of immunohistochemical analysis of p-AMPKα and AMPKα in aortic sections. Brown staining indicates positive staining, arrows indicate positively stained endothelial cells, and arrowheads indicate positively stained smooth muscle cells. Semi-quantitative analysis of p-AMPKα–positive and AMPKα-positive staining in the intima and media of aortas (data were calculated as fold changes of the WT+saline group). n=6 animals/group. Data=means±SEM. *P<0.05, **P<0.01 vs WT+saline; +P<0.05, ++P<0.01 vs KL+/–+saline.
Activation of SIRT1 by SRT1720 enhanced endothelial NO synthase (eNOS) activity in aortas. A, Representative Western blot bands and quantitative analysis of phosphorylated eNOS (p-eNOS) and eNOS in aortas. Protein expression was normalized to β-actin, and the relative expression calculated as the fold change relative to the control (wild type [WT]+saline). B, Representative photomicrographs of immunohistochemical analysis of p-eNOS and eNOS in aortic sections. Brown staining indicates positive staining, and arrows indicate positively stained endothelial cells. Semi-quantitative analysis of p-eNOS–positive and eNOS-positive staining in aortic intima (data were calculated as fold change of the WT+saline group). n=6 mice/group. Data=means±SEM. *P<0.05, **P<0.01 vs WT+saline; +P<0.05, ++P<0.01 vs KL+/–+saline.
