cryopreserved human coronary artery endothelial cells (hcaecs)  (Lonza)

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: KLK (kallikrein-related peptidase) 8 knockdown alleviates high glucose-induced endothelial dysfunction and endothelial-to-mesenchymal transition in human coronary artery endothelial cells (HCAECs). HCAECs were transfected with control siRNA or KLK8 siRNAs, and were cultured with or without high glucose (HG, 25 mM) treatment for 5 days. A-C, Levels of thrombomodulin (A), von Willebrand factor (B) and E-selectin (C) as markers of endothelial damage/dysfunction and activation in the culture medium. D, MTT assay showed that KLK8 knockdown alleviates HG-induced cell injury in HCAECs. E, KLK8 knockdown alleviates HG-induced TGF-β1 release in the supernatant of HCAECs. F & G, Immunoblots of the endothelial markers (VE-cadherin, CD31) and mesenchymal markers (vimentin, α-SMA). The representative protein bands (F) and the corresponding histograms (G) showed that HG-induced loss of CD31 and VE-cadherin was largely prevented, whereas the acquisition of α-SMA and vimentin was decreased by KLK8 knockdown. Data are expressed as means ± SEM (n = 4). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Knockdown, Transfection, Control, Cell Culture, Activation Assay, MTT Assay, Western Blot

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: KLK (kallikrein-related peptidase) 8 overexpression leads to cardiac fibrosis and endothelial damage. A, Masson's trichrome staining showed collagen deposition in the perivascular and intramyocardial regions in the hearts obtained from 12-weeks-old Tg-KLK8 rats (scale bar = 50 µm). B-D, Cardiac fibrosis was also quantified by determination of collagen-I (B), hydroxyproline (C), and TGF-β (D) levels in heart tissues obtained from 6-weeks-old and 12-weeks-old control and Tg-KLK8 rats. E-G, Plasma levels of thrombomodulin (E), von Willebrand factor (F) and E-selectin (G) as markers of endothelial damage/dysfunction and activation in 6-weeks-old and 12-weeks-old control and KLK8 transgenic (Tg-KLK8) rats. Data are expressed as means ± SEM (n = 7). *** p < 0.001, **** p < 0.0001. H-K, HCAECs were infected with increasing doses of KLK8 adenovirus (Ad-KLK8) at a multiplicity of infection (MOI) of 1, 3, or 10 for 72 h. H-J, Levels of thrombomodulin (H), von Willebrand factor (I) and E-selectin (J) in the culture medium. K, MTT assay showed that Ad-KLK8 induced cell injury in a dose- and time-dependent manner in human coronary artery endothelial cells (HCAECs). L, The permeability of a confluent HCAECs monolayer measured by FITC-dextran flux assay showed that Ad-KLK8 (MOI 10) treatment for 72 h led to significantly increased permeability of HCAECs. Data are expressed as means ± SEM (n = 4). * p < 0.05, ** p < 0.01, **** p < 0.0001, ## p < 0.01, #### p < 0.0001 versus Ad-vector Day 3; $$$$ p < 0.0001 versus Ad-vector Day 5.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Over Expression, Staining, Control, Clinical Proteomics, Activation Assay, Transgenic Assay, Infection, MTT Assay, Permeability, Flux Assay, Plasmid Preparation

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: KLK (kallikrein-related peptidase) 8 overexpression induces endothelial-to-mesenchymal transition in the myocardium and human coronary artery endothelial cells (HCAECs). A, Immunofluorescent staining showed increased colocalization of α-SMA (green) and FSP-1 (green) and CD31 (red) in the hearts obtained from 12-weeks-old Tg-KLK8 rats as compared to age-matched control rats. Nuclei were counterstained with DAPI (blue), scale bar = 50 µm. B, Representative z-stack image analysis showed specific overlay of double immunostaining. CD31 + /α-SMA + and CD31 + /FSP-1 + cells in specific ordinate were analyzed in z-stack with optimal interval range of 0.8 µm. Nuclei were counterstained with DAPI (blue), scale bar = 50 µm. C & D, Immunoblots of the VE-cadherin, CD31, vimentin, and α-SMA in the hearts obtained from 12-weeks-old control and Tg-KLK8 rats. The representative protein bands (C) and the corresponding histograms (D) showed that transgenic KLK8 overexpression significantly upregulated vimentin and α-SMA expression, whereas downregulated VE-cadherin and CD31 expression in heart tissues. E & F, Immunoblots of the endothelial markers (VE-cadherin, CD31) and mesenchymal markers (vimentin, α-SMA) in HCAECs infected with increasing doses of KLK8 adenovirus (Ad-KLK8) at a multiplicity of infection (MOI) of 1, 3, or 10 for 72 h. The representative protein bands (E) and the corresponding histograms (F) showed that Ad-KLK8 dose-dependently increased protein expressions of the mesenchymal markers, whereas decreased the endothelial markers in HCAECs. G, Infection of Ad-KLK8 at an MOI of 10 for 72 h led to an increase of TGF-β1 release in the supernatant of HCAECs. H & I, Immunoblots of the endothelial markers (VE-cadherin, CD31) and mesenchymal markers (vimentin, α-SMA). The representative protein bands (H) and the corresponding histograms (I) showed that the KLK8-induced loss of CD31 and VE-cadherin was largely prevented, whereas the acquisition of α-SMA and vimentin was decreased by TGF-β1 neutralizing antibody. Data are expressed as means ± SEM (n = 4). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Over Expression, Staining, Control, Double Immunostaining, Western Blot, Transgenic Assay, Expressing, Infection

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: Sp-1 mediates high glucose-induced upregulation of KLK8 in human coronary artery endothelial cells (HCAECs). A-C, HCAECs were treated with increasing concentration of glucose (15 and 25 mM) for 5 days. A, The mRNA level of KLK8 in HCAECs. B and C, Immunoblots of KLK8 (B) and Sp-1 (C). The representative protein bands of KLK8 (B) and Sp-1 (C) were presented on the top of the corresponding histograms. D and E, High glucose-induced mRNA (D) and protein (E) expressions of KLK8 were abolished by Sp-1 inhibitor plicamyin (1 nM) in HCAECs. F, HCAECs were transfected with human KLK8 promoter-luciferase reporter plasmid pGL3-KLK8 or mutated plasmid pGL3-KLK8 △ Sp1, and then were exposed to high glucose (25 mM) for 48 h. Promoter activity was analyzed using a dual-luciferase reporter assay. Data are expressed as means ± SEM (n = 4). ** p < 0.01, **** p < 0.0001.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Concentration Assay, Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Reporter Assay

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: KLK (kallikrein-related peptidase) 8 degrades VE-cadherin, thus promoting plakoglobin nuclear translocation. A and B, Association of plakoglobin with VE-cadherin and KLK8 was tested by reciprocal immunoprecipitations in rat myocardium and human coronary artery endothelial cells (HCAECs). IgG was controlled for nonspecific interaction. C, HCAECs were treated with Ad-vector or Ad-KLK8 in serum-free medium for 72 h. Immunoblots showed the appearance of a ~30 kDa N-terminal VE-cadherin fragment in the medium. WB, western blot. D, The N-terminal sequence of the ~30 kDa protein band was determined by Edman assay. The sequence as indicated was belonging to VE-cadherin sequence. E, Schematic representation of the extracellular part of VE-cadherin cleaved by KLK8. VE-cadherin extracellular domain is constituted by 5 cadherin domains numbered 1-5 from the N-terminus. KLK8 cleaved VE-cadherin before amino-acid 266 in the extracellular cadherin domain 3. F, Immunofluorescent staining showed that infection of Ad-KLK8 for 72 h resulted in significant loss of both plakoglobin (red) and VE-cadherin (green) in the plasma membrane, whereas caused nuclear translocation of plakoglobin in HCAECs. Lentivirus-mediated VE-cadherin (Lv-VE-cadherin) overexpression reduced nuclear whereas increased membrane and cytosol plakoglobin levels. Nuclei were counterstained with DAPI (blue), scale bar = 100 µm. G, Immunoblots of cellular fractionations of plasma membrane, cytosol and nucleus for VE-cadherin and plakoglobin in HCAECs infected with or without Ad-KLK8 and Lv-VE-cadherin for 72 h. The representative protein bands (H) and the corresponding histograms (I) were presented. Data are expressed as means ± SEM (n = 4). ** p < 0.01, **** p < 0.0001.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Translocation Assay, Plasmid Preparation, Western Blot, Sequencing, Staining, Infection, Clinical Proteomics, Membrane, Over Expression

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: Plakoglobin is required for KLK (kallikrein-related peptidase) 8-induced endothelial-to-mesenchymal transition by cooperating with p53. A & B, Immunoblots of plakoglobin-knockdown human coronary artery endothelial cells (HCAECs) under KLK8 adenovirus (Ad-KLK8) treatment for 72 h. The representative protein bands (A) and the corresponding histograms (B) were presented. C, Association of plakoglobin with TCF-4 and p53 was observed by immunoprecipitation in HCAECs, whereas Ad-KLK8 treatment enhanced the interactions. IgG was controlled for nonspecific interaction. D, The KLK8 overexpression-induced mRNA expression of TGF-β1 was reduced by plakoglobin knockdown or HIF-1α inhibitor echinomycin (20 nM) in HCAECs. E, ChIP assay showed that the KLK8 overexpression-induced HIF-1α binding to TGF-β1 promoter was blocked by plakoglobin knockdown or p53 inhibitor pifithrin-α (20 μM). F, Immunoprecipitation assay showed that Ad-KLK8 treatment enhanced the association of p53 with HIF-1α and Smad3 in HCAECs, which was reduced by plakoglobin knockdown. G and H, The KLK8 overexpression-induced mRNA levels of the pro-EndMT target genes of TGF-β1/Smad pathway (Snail, Slug, Zeb1, Zeb2 and Twist) were reduced by plakoglobin knockdown (G) or p53 inhibitor pifithrin-α (H, 20 μM). Data are expressed as means ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Over Expression, Expressing, Binding Assay

Journal: Theranostics

Article Title: A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis

doi: 10.7150/thno.48530

Figure Lengend Snippet: High glucose promotes plakoglobin-dependent cooperation of p53 with HIF-1α and Smad, subsequently increasing the expression of TGF-β1 and its pro-EndMT target genes in a KLK (kallikrein-related peptidase) 8-dependent manner. A, Immunoprecipitation assay showed that high glucose (HG, 25 mM glucose) treatment enhanced the association of p53 with plakoglobin, HIF-1α and Smad3 in HCAECs, which was reduced by KLK8 knockdown. B, Immunoprecipitation assay showed that HG-induced association of p53 with HIF-1α and Smad3 was reduced by plakoglobin knockdown in HCAECs. C and D, ChIP assay showed that the HG-induced HIF-1α binding to TGF-β1 promoter was blocked by knockdown of KLK8 or plakoglobin (C) or p53 inhibitor pifithrin-α (D, 20 μM). Data are expressed as means ± SEM (n = 4). NG indicates normal glucose. E-G, Heart tissues were obtained from the KLK8-deficient (KLK8 -/- ) and KLK8 +/+ mice after 24 weeks of diabetes. The mRNA levels of the pro-EndMT target genes of TGF-β1/Smad pathway (Snail, Slug, Zeb1, Zeb2 and Twist) were decreased in the hearts obtained from KLK8 -/- diabetic mice, as compared to KLK8 +/+ diabetic mice (E). F, Immunoprecipitation assay showed that the association of p53 with plakoglobin, HIF-1α and Smad3 was reduced in KLK8 -/- diabetic mice as compared to KLK8 +/+ diabetic mice. G, ChIP assay showed that KLK8 deficiency suppressed HIF-1α binding to TGF-β1 promoter in diabetic myocardium. Data are expressed as means ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. H, Schematic diagram of the mechanism by which KLK8 promotes EndMT and cardiac fibrosis in diabetic cardiomyopathy. Hyperglycemia upregulates KLK8 expression in endothelial cells, which cleavages the VE-cadherin extracellular domain and promotes plakoglobin nuclear translocation and its cooperation with p53. The plakoglobin-dependent cooperation of p53 with HIF-1α and Smad3 subsequently increased the expression of TGF-β1 and the pro-EndMT target genes of TGF-β1/Smad pathway, which finally promotes the differentiation of endothelial cells into mesenchymal cells and the pathogenesis of cardiac fibrosis.

Article Snippet: Cryopreserved human coronary artery endothelial cells (HCAECs) were purchased from Lonza (Walkersville, MD).

Techniques: Expressing, Immunoprecipitation, Knockdown, Binding Assay, Translocation Assay

Journal: Molecular cancer research : MCR

Article Title: Differential Expression of Stress and Immune Response Pathway Transcripts and miRNAs in Normal Human Endothelial Cells Subjected to Fractionated or Single-Dose Radiation

doi: 10.1158/1541-7786.MCR-13-0623

Figure Lengend Snippet: Table 1

Article Snippet: Cells Cryopreserved human coronary artery endothelial cells (HCAEC) and the media were purchased from Lonza Walkersville Inc (Walkersville, MD).

Techniques: Irradiation