Fig 1: Pharmacological inhibition of S1pr2 by JTE013 improves post-ischemic angiogenesis and enhances blood flow perfusion in hindlimbs. A, Representative laser Doppler images show improved blood flow perfusion in JTE013-treated mice compared with control mice. B, Cumulative results for control mice (n = 6) and JTE-treated mice (n = 6) are shown graphically as the ratio of blood flow in the ischemic limb to that in the non-ischemic limb at each time point. C-D, Representative images of isolectin-B4 staining of gastrocnemius muscles in control mice and JTE013-treated mice, with quantification of capillary density in gastrocnemius muscles after sham or HLI operation (n = 5). E-F, Representative images of α-SMA staining of gastrocnemius muscles in control mice and JTE013-treated mice (E), with quantification of arteriole density in gastrocnemius muscles after sham or HLI operation (F) (n = 5). G-H, Representative images of H&E staining of gastrocnemius muscles in control mice and JTE013-treated mice (G), with quantification of muscle fiber area after sham or HLI operation (H) (n = 5). I-J, Representative images of Sirius red-stained of gastrocnemius muscles in control mice and JTE013-treated mice (I), with quantification of the percentage of fibrotic tissue in muscle (J) (n = 5). K-M, Functional assessment of ischemic muscle over follow-up. Cumulative results for control mice and JTE013-treated mice are shown graphically as Tarlov score (K), ischemia score (L), and ambulatory impairment score (M) (n = 6). N-P, Western blotting of AKT or eNOS activation status in hindlimbs of mice treated with JTE013 or DMSO and quantification in the indicated groups (n = 4). Scale Bars: C and E, 50 μm; G and I,100 μm. Data are mean ± SEM. n.s indicates not significant. *P < 0.05; **P < 0.01.
Fig 2: Insulin‐mediated activation of eNOS (endothelial NO synthase) and AKT (protein Kinase B) in response to siRNA‐mediated knockdown of FSP27 in the subcutaneous depot. A, Representative immunoblot demonstrating insulin‐mediated activation of eNOS and AKT in subcutaneous fat under scrambled siRNA (small interfering RNA) conditions and after knockdown of FSP27 by siRNA. B, Quantification of percent change in insulin‐mediated activation of eNOS at baseline and after siRNA‐mediated knockdown of FSP27 in the subcutaneous fat depot. C, Quantification of percent change in insulin‐mediated activation of AKT at baseline and after siRNA‐mediated knockdown of FSP27 in subcutaneous fat depot (n=10, P<0.05). Data are presented as arbitrary units and as mean±SEM. FSP27 indicates fat‐specific protein 27. *indicates statistical significance, such as P < 0.05.
Fig 3: Scheme depicting proposed mechanisms involved in empagliflozin-offered protection against microvasculature damage in diabetes. Empagliflozin activates AMPK pathways through regulation of the AMP/ATP ratio. Activated AMPK pathways regulates Drp1 posttranscriptional phosphorylation modifications at Ser616 and Ser637, leading to the inability of Drp1 to translocate onto mitochondria and mitochondrial fission impairment. The loss of mitochondrial fission retards cellular senescence and preserves endothelial barrier/permeability by suppressing superfluous ROS. In consequence, endothelial migration and vascularization are improved by balanced F-actin degradation. Moreover, empagliflozin reduces CMEC apoptosis, increases cardiac microvessel density, promotes eNOS phosphorylation and alleviates vascular collagen deposition, leading to improved endothelial function and preserved vascular remodeling, ultimately lower levels of inflammatory cell penetration and better vascular relaxation. Through these aforementioned mechanisms, empagliflozin eventually facilitates diabetic myocardial perfusion and protects the heart against hyperglycemic injury.
Fig 4: Metformin alleviates aortic calcification by activating the PI3K/AKT signaling pathway in a concentration-dependent manner in vitro. a Metformin inhibits inflammatory factor secretion including IL6, IL8, and MCP-1 in cell supernatant, after treatment with phosphate medium (PM) with or without metformin for 72 h as determined by ELISA; b ROS production was detected and quantified after PM treatment with or without metformin for 72 h; c Cell viability was detected by CCK8 after treatment with PM with or without metformin for 72 h; d Immunofluorescence staining images of p-AKT and OPN expression in AVICs after PM treatment with or without 100 μM metformin for 72 h, Scale bar, 50 μm; e The protein expression of p-AMPK (Thr172), p-AKT (Ser473), PI3K, p-eNOS (Ser1177), BMP2, and OPN in AVICs after PM treatment with or without metformin for 72 h as determined by WB; f Calcium deposition were detected by ARS Staining after various treatments for 7 days, Scale bar, 200 μm. n = 6 per group. CTL, control; Met, metformin; *p < 0.05 versus PM group (one-way ANOVA with Bonferroni post hoc test)
Fig 5: Insulin‐mediated activation of eNOS (endothelial NO synthase) and AKT (protein Kinase B) in response to recombinant FSP27 in visceral depot. A, Representative visceral adipose tissue immunoblot demonstrating severe impairment in insulin‐mediated activation of eNOS and AKT in visceral fat. After 24 hours of rFSP27 (recombinant FSP27) exposure, insulin‐mediated activation is restored. B, Quantification of percent change in insulin‐mediated activation of eNOS at baseline and after 24 hours of treatment with rFSP27 in the visceral depot. C, Quantification of percent change in insulin‐mediated activation of AKT at baseline and after 24 hours of treatment with rFSP27 in the visceral depot (n=10, P<0.05). Data are presented as arbitrary units (au) and as mean±SEM. rFSP27 indicates recombinant fat‐specific protein 27. *indicates statistical significance, such as P < 0.05.
Supplier Page from Abcam for Anti-eNOS (phospho S1177) antibody