human primary coronary artery smc culture  (PromoCell)

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Human cardiovascular tissue contained immunoreactive retinoic acid receptor. (A) Human carotid artery and (B) aortic valve RARα immunohistochemistry (red color); scale bars, 50 μm. IgG control antibody immunohistochemistry staining for matching donor adjacent sections used as negative controls. RARα (red color) immunofluorescence showing localization with α-smooth muscle actin (α-SMA) positive cells (green color) in human carotid artery (white arrow) and with vimentin-positive cells (green color) in human aortic valve (white arrow); scale bars, 20 μm. One of five carotid artery and aortic valve donors shown, with representative RARα immunohistochemistry shown for two additional donors in Figures I and II in the online-only Data Supplement. (C) Human carotid artery, SMC, (D) aortic valve, and VIC RARα mRNA levels in calcified and non-calcified tissues (n = 5 donors) and CM, OM or PM treated cells after two weeks in culture (n = 3 donors). Error bars indicate STDEV of mean. Data analyzed by Student’s t-test.

Article Snippet: Cell culture Human coronary artery SMC (PromoCell, Heidelberg, Germany) were expanded in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with epidermal growth factor (0.5ng/mL), insulin (5μg/mL) basic fibroblast growth factor- B (2ng/mL), and 5% fetal bovine serum, and cells were used between passages 3 and 8.

Techniques: Immunohistochemistry, Staining, Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Retinoic acid attenuated human SMC calcification. (A) RAR siRNA knockdown validation by mRNA level quantification, and Alizarin red stain and relative quantification for primary human SMC treated for two weeks in OM with negative control siRNA, RARα siRNA, RARβ siRNA, or RARγ siRNA. (B) Alizarin red stain and relative quantification for primary human artery SMC treated for three weeks in CM or OM with DMSO vehicle (0.1%), 0.1 μmol/L or 1 μmol/L ATRA, 0.1 μmol/L or 1 μmol/L AGN 193109 (AGN), or (C) 1 μmol/L 9-cis retinoic acid (9-cis RA). (D) Von Kossa and (E) OsteoSense 680 stain for SMC treated for three weeks; scale bars, 100 μm. (F) Cellular ALPL mRNA levels and corresponding TNAP enzyme activity (G) for SMC treated for two weeks. Error bars indicate STDEV of mean. aP < 0.0001 vs OM, bP < 0.01 vs OM, cP < 0.0001 vs OM + 0.1 μmol/L AGN, dP < 0.0001 vs OM + 1 μmol/L AGN, *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001 analyzed by ANOVA or Student’s t-test; n = 3 donors, except for TNAP activity in which n = 6 donors.

Article Snippet: Cell culture Human coronary artery SMC (PromoCell, Heidelberg, Germany) were expanded in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with epidermal growth factor (0.5ng/mL), insulin (5μg/mL) basic fibroblast growth factor- B (2ng/mL), and 5% fetal bovine serum, and cells were used between passages 3 and 8.

Techniques: Staining, Negative Control, Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Retinoic acid suppressed SMC EV TNAP activity. (A) EV TNAP activity from primary human coronary artery SMC treated for two weeks in CM or OM with DMSO vehicle (0.1%) or 1 μmol/L ATRA. (B) SMC EV abundance and (C) diameter from SMC treated for two weeks. Error bars indicate STDEV of mean. ****P < 0.0001 analyzed by ANOVA; n = 9 donors.

Article Snippet: Cell culture Human coronary artery SMC (PromoCell, Heidelberg, Germany) were expanded in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with epidermal growth factor (0.5ng/mL), insulin (5μg/mL) basic fibroblast growth factor- B (2ng/mL), and 5% fetal bovine serum, and cells were used between passages 3 and 8.

Techniques: Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Retinoic acid increased SOX9 and MGP in human SMC. (A) RARα, (B) RUNX2, and (C) MSX2 mRNA levels from primary human coronary artery SMC treated for two weeks in CM or OM with DMSO vehicle (0.1%) or 1 μmol/L ATRA. (D) SOX9 mRNA levels and representative SOX9 and 4’,6-diamidino-2-phenylindole (DAPI) immunofluorescence from SMC treated for two weeks; white arrow depicts an example of SOX9 nuclear localization. (E) MGP mRNA levels, representative MGP and DAPI immunofluorescence, and secreted MGP protein abundance from SMC treated for two weeks. Error bars indicate STDEV of mean. **P < 0.01, ***P < 0.001, ****P < 0.0001 analyzed by ANOVA; n = 3 donors. Scale bars, 50 μm.

Article Snippet: Cell culture Human coronary artery SMC (PromoCell, Heidelberg, Germany) were expanded in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with epidermal growth factor (0.5ng/mL), insulin (5μg/mL) basic fibroblast growth factor- B (2ng/mL), and 5% fetal bovine serum, and cells were used between passages 3 and 8.

Techniques: Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Peretinoin inhibits human cardiovascular cell calcification without inducing liver cell APOC3 secretion. (A) APOC3 and (B) APOA1 mRNA levels and secreted protein in HepG2 treated for 24 hours with 0.1% DMSO vehicle (control), 1 μmol/L ATRA, 1 μmol/L 9-cis retinoic acid (9-cis RA), 1 μmol/L peretinoin, 13-cis retinoic acid (13-cis RA), or 1 μmol/L AGN 193109 (AGN). (C) CYP7A1 and (D) HNF4α, PPARGC1α mRNA levels in HepG2 cells treated for 24 hours. Alizarin red stain and relative quantification for (E) primary human coronary artery SMC and (F) VIC treated for three weeks, and (G) primary human femur osteoblasts treated for one three weeks in CM, OM, or PM with DMSO vehicle (0.01%), 1 μmol/L peretinoin, or 1 μmol/L ATRA. (H) RAR siRNA knockdown validation by mRNA level quantification, and Alizarin red stain and relative quantification for primary human femur osteoblasts treated for two weeks in OM with negative control, RARα, RARβ, or RARγ siRNA. Error bars indicate STDEV of mean. aP < 0.0001 vs control, bP < 0.0001 vs peretinoin, cP < 0.0001 vs AGN, dP < 0.001 vs control, eP < 0.001 vs peretinoin, fP < 0.01 vs peretinoin, gP < 0.01 vs control, hP < 0.01 vs AGN, iP < 0.001 vs AGN, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 analyzed by ANOVA or Student’s t-test; n = 6 independent experiments for HepG2 and n = 3 donors for primary human VIC, SMC, and osteoblasts.

Article Snippet: Cell culture Human coronary artery SMC (PromoCell, Heidelberg, Germany) were expanded in Smooth Muscle Cell Growth Medium 2 (PromoCell) supplemented with epidermal growth factor (0.5ng/mL), insulin (5μg/mL) basic fibroblast growth factor- B (2ng/mL), and 5% fetal bovine serum, and cells were used between passages 3 and 8.

Techniques: Staining, Negative Control