Journal: Engineering

Article Title: PAI-1 Derived from Alveolar Type 2 Cells Drives Aging-Associated Pulmonary Fibrosis

doi: 10.1016/j.eng.2024.08.014

Figure Lengend Snippet: Fig. 4. Impact of the secretome from senescent epithelial cells on fibrosis and Collagen I synthesis. (a) Schematic of Collagen I biosynthesis; (b) schematic diagram of the cell culture models used for conditioned medium culture and coculture experiments in this study; (c) Western blot analysis of senescence- and cell cycle-associated proteins in primary AT2 cells treated with 30 gL1 D-galactose for 48 h (n = 3); (d–e) Western blot analysis of fibrosis, collagen synthesis, and degradation-related proteins in NIH3T3 cells cultured with conditioned medium from D-galactose-induced senescent (d) primary AT2 cells and (e) A549 cells for 48 h (n = 3). MMP1: matrix metalloproteinase.

Article Snippet: After 8 h of transfection, the NIH3T3 cells were exposed to medium containing 2 lg mL 1 PAI-1 (HY-P71133, MCE) and 0.4% serum.

Techniques: Cell Culture, Western Blot

Journal: Engineering

Article Title: PAI-1 Derived from Alveolar Type 2 Cells Drives Aging-Associated Pulmonary Fibrosis

doi: 10.1016/j.eng.2024.08.014

Figure Lengend Snippet: Fig. 5. PAI-1 secreted by senescent epithelial cells promotes collagen I synthesis via the TGF-b/Smad2/3 pathway. (a) Schematic diagram of the process used to screen for key SASP factors associated with fibrosis; (b) RT-qPCR analysis of key fibrosis-related SASP factors in 3 and 28 month old mouse lung tissues. Each group comprised six biological replicates and three technical replicates. Results are presented as mean ± standard deviation (SD); (c) expression analysis of key fibrosis-related SASP factors in primary AT2 cells from 3 and 24 month old mice (n = 3); (d) Western blot analysis of PAI-1 protein in mouse lung tissues of different ages and D-galactose-induced senescent A549 cells (n = 3); (e, f) Western blot analysis of fibrosis and collagen synthesis-related proteins in (e) NIH3T3 cells and (f) WI-38 cells stimulated with gradient concentrations of PAI-1 (0, 0.5, 1.0, and 2.0 lgmL1) for 48 h (n = 3); (g, h) activation of the TGF-b pathway in (g) NIH3T3 and (h) WI-38 cells after stimulation with 2.0 lgmL1 PAI-1 for 48 h (n = 3); (i) effect of PAI-1 on Col1a1 synthesis after TGF-b pathway inhibition with SB431542 (n = 3); (j) impact of PAI-1 on Col1a1 synthesis after silencing Smad2/3 with siSmad2/3 (n = 3).

Article Snippet: After 8 h of transfection, the NIH3T3 cells were exposed to medium containing 2 lg mL 1 PAI-1 (HY-P71133, MCE) and 0.4% serum.

Techniques: Quantitative RT-PCR, Standard Deviation, Expressing, Western Blot, Activation Assay, Inhibition

Journal: Antioxidants

Article Title: Olive Leaf Extract Added to Losartan Treatment Improved Klotho/Wnt/β-Catenin Signaling in Hypertensive Rats with Focal Segmental Glomerulosclerosis

doi: 10.3390/antiox15010146

Figure Lengend Snippet: Effects of L, L+T and L+O on 6–nitrotryptophan, Klotho, Wnt4, β–catenin, PAI–1 and fibronectin protein expressions in the kidney of SHR with ADR-induced FSGS. The analysis of 6–nitrotryptophan/actin ( A ), Klotho/actin ( B ), Wnt4/GAPDH ( C ), β–catenin/GAPDH ( D ), PAI–1/GAPDH ( E ), fibronectin/GAPDH ( F ) protein expressions and representative Western blots ( G ). Values represent mean ± SEM of three to four independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. SHC; # p < 0.05 and ### p < 0.001 vs. SHADR; $$ p < 0.01 and $$$ p < 0.001 vs. SHADR+L; and &&& p < 0.001 vs. SHADR+L+T. SHC, control group; SHADR, model group; L, losartan; T, tempol; and O, olive leaf extract.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS-Tween) and then incubated with the following primary antibodies: 6-nitrotryptophan (1:1000, ab243072, Abcam, Cambridge, UK), Klotho (1:1000, NBP1-76511, Novus biologicals, LCC, USA), Wnt-4 (1:2000, NBP2-20909, Novus biologicals, LCC, Centennial, CO, USA), β-catenin (1:300, AF1329, R&D Systems, Inc., Minneapolis, MN, USA), PAI-1 (1:1000, NBP1-19773, Novus biologicals, LCC, Centennial, CO, USA), fibronectin (1:1000, ab2413, Abcam, Cambridge, UK), actin (1:500, A5060, Sigma-Aldrich, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK).

Techniques: Western Blot, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Angiogenic Potential of Tissue Engineered Cartilage From Human Mesenchymal Stem Cells Is Modulated by Indian Hedgehog and Serpin E1

doi: 10.3389/fbioe.2020.00327

Figure Lengend Snippet: Blocking IHH or Serpin E1 increased angiogenic potential of CM of chondrogenically differentiated BMSC in vitro and showed no effect in vivo CAM assay. (A) Quantification of endothelial cell migration assay comparing the impact of pre-incubation of CM with blocking antibodies for factors of interest. Values are normalized to the BMSC-CM condition. UCM depicts the un-conditioned medium. (B) Quantification of endothelial cell proliferation assay comparing the impact of pre-incubation of CM with blocking antibodies for factors of interest. Values are normalized to the BMSC-CM condition. UCM depicts the un-conditioned medium. (C) Images of chick chorioallantoic membrane incubated for 72 h with CM (blocked for 30 min) soaked 5 mm filter disks. Filter circles were placed on a vessel free area on day 7 of the chick development. (D) Boxplot depicting the ranking of the angiogenic potential (lowest = 1 highest = 45: average of 3 independent observers: box = interquartile range, whiskers + 1–99%). N = 5 per condition. A & B were performed in two batches each pooling 3 donors; n = 3. Significance was determined by mixed-linear model with Bonferroni post hoc test.

Article Snippet: Blocking antibodies against VEGF-A (Cat. # AF-293-SP) and Serpin E1 (Cat.# MAB1786-SP, Clone # 242816) were obtained from R&D-Systems (Minneapolis, MN, United States) and utilized at 0.5 μg/mL according to the manufacturers in vitro testing protocol.

Techniques: Blocking Assay, In Vitro, In Vivo, Chick Chorioallantoic Membrane Assay, Cell Migration Assay, Incubation, Proliferation Assay, Membrane

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 3 PAI-1 derived from endometrial stromal cells promotes senescence of ECs. A Analysis of interactions between various cells. B Analysis of interactions between PAI-1 + ESC and other various cells. C Immunofluorescence staining of PAI-1 (green) and vessels (red) in IUA patients (n = 5). scale bar = 100 μm. The supernatant from PAI-1+ ESCs was collected to stimulate HUVECs. D–J. Subsequently, the mRNA levels of P16 and P21 were analyzed by qPCR (n = 12) (D); the protein level of P21 was assessed by western blotting (n = 3) (E); the mRNA and protein levels of IL-6 were examined by qPCR (n = 6) and western blotting (n = 3), respectively (F); and the protein level of eNOS was determined by western blotting (n = 3) (G). SA-β-gal staining was observed (n = 3). scale bar = 100 μm (H), HUVECs migration was assayed (n = 3). scale bar = 100 μm (I), and tube formation was examined (n = 3) (J). scale bar = 100 μm.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Staining, Western Blot, Migration

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 4 PAI-1 promotes endothelial cell senescence through uPAR. A Immunofluorescence staining of uPAR (green) and CD31 (red) in endometria from IUA patients (n = 12) and controls (n = 12). Quantized co-expression area (%) was showed at right. scale bar = 100 μm. B, C The mRNA (n = 12) and protein levels (n = 3) of uPA in PAI-1+ESCs. D, E The mRNA (n = 11) and protein levels (n = 3) of uPAR in HUVECs treated with PAI-1+ESCs supernatant. F, G The mRNA and protein levels of P21 (n = 3) in HUVECs treated with Dox and transfected with si- PLAUR.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Expressing, Transfection

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 5 TGF-β upregulates PAI-1 in SMAD dependent manner. A The mRNA levels of PAI-1, ACTA2, and COL1A1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β for 3, 6, 12, 24 h respectively (n = 3). B The protein levels of PAI-1, ACTA2, and COL1A1 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β for 6, 12, 24 h respectively (n = 3). C The mRNA level of PAI-1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3). D The protein levels of PAI-1, p-SMAD2, and SMAD2 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3).

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Western Blot

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 6 PAI-1 inhibitors can inhibit endothelial senescence and endometrial fibrosis in mice. A Masson’s trichrome staining and immunohistochemical staining for COL1A1, α-SMA, PAI-1, uPA, and uPAR were performed on endometrial samples from normal controls, both with and without TPX, as well as in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. B Immunofluorescence staining was conducted to detect P21 (green) and CD31 (red) in endometrial samples from normal controls, both with and without TPX, and in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. The quantized results analyzed by ImageJ showed on right, each bar represents the mean ± SEM. ANOVA test, **p < 0.001, ***p < 0.0001.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Immunohistochemical staining

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 7 Schematic diagram illustrating how PAI-1 derived from ESCs promotes endothelial aging, leading to endometrial fibrosis. Trauma or infection elevates TGF-β in the endometrial microenvironment. TGF-β promotes the expression of PAI-1 in ESCs by activating SMAD2. PAI-1 synergizes with uPA to act on the uPAR receptor on endothelial cells, accelerating cellular senescence. Following endothelial cell senescence, the cells’ angiogenic capacity is inhibited, and they contribute to endometrial fibrosis by releasing factors such as SASP.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Infection, Expressing