Journal: JVS-Vascular Science

Article Title: The role of 6-phosphogluconate dehydrogenase in vascular smooth muscle cell phenotypic switching and angioplasty-induced intimal hyperplasia

doi: 10.1016/j.jvssci.2024.100214

Figure Lengend Snippet: Pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase ( 6PGD ) is upregulated aberrantly in the neointimal layer of diseased human coronary arteries with extensive intimal hyperplasia (IH). (A) Representative immunohistochemical staining of 6PGD in cross-sections from human coronary arteries with or without severe IH lesions. Negative control staining followed the same procedures, with the exception that the primary antibody was omitted. Dashed lines highlight the internal and external elastic laminae (EEL). Tunica intima ( Int ), media ( M ), and adventitia ( Adv ) are annotated, respectively. (B) The quantification of 6PGD expression level in tunica intima area and tunica media area. Positive staining was assessed based on colorimetric intensity per image field, quantified by averaging three sections from each sample. The resulting averages from all four unique donors (each including healthy vs diseased arterial segments with severe IH) were then further averaged, as illustrated in the scatter plots. (C) Representative confocal images of 6PGD (red channel) and vascular smooth muscle cell (VSMC) marker α smooth muscle actin ( α-SMA ) (green channel) costaining in healthy vs diseased (with IH lesions) human coronary arteries. Student t test: ∗∗ P < .01, ∗∗∗ P < .001. Mag , magnification.

Article Snippet: Human aortic smooth muscle cells (AoSMCs, CC-2571), smooth muscle cell basal medium (SmBM, CC-3181), and SmBM plus SingleQuots of supplements (CC-3182) were purchased from Lonza (Basel, Switzerland).

Techniques: Immunohistochemical staining, Staining, Negative Control, Expressing, Marker

Journal: JVS-Vascular Science

Article Title: The role of 6-phosphogluconate dehydrogenase in vascular smooth muscle cell phenotypic switching and angioplasty-induced intimal hyperplasia

doi: 10.1016/j.jvssci.2024.100214

Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase ( 6PGD ) silencing diminishes 6PGD enzyme activity and blocks vascular smooth muscle cell (VSMC) phenotypic switching and ERK/MEK signaling in VSMCs in vitro. Primary culture of human aortic smooth muscle cells (AoSMCs) were subjected to serum starvation for 24 hours before the treatment with 6PGD-specific small interfering RNA ( si-6PGD ) or scramble control ( SCR ). After 24 hours of treatment, platelet-derived growth factor (PDGF-BB) was introduced to induce AoSMC dysfunction for an additional 24 hours. (A and B) Representative Western blot images and protein-level quantitation of phosphorylated-ERK ( P-ERK ), total-ERK ( T-ERK ), phosphorylated-MEK ( P-MEK ), total-MEK ( T-MEK ), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Enzymatic activity of 6PGD was assayed following the manufacturer's instructions. In brief, cell samples were lysed using assay buffer, mixed with the reaction mix, and absorbance was measured at 460 nm in kinetic mode for 60 minutes at 37°C. (D) Representative images of VSMC migration in the transwell assays and quantitation. (E) Quantitation of VSMC proliferation after si-6PGD or SCR treatment, with or without PDGF-BB stimulation. (F) Quantitative polymerase chain reaction analysis of messenger RNA levels of VSMC differentiation marker α smooth muscle actin ( α-SMA ), dedifferentiation marker ALDH1A3, and inflammation marker ICAM1. The data are presented as mean ± standard deviation (n = 3-8 replicates as represented by the scatter plots), and statistical analyses were performed using one-way analysis of variance with post hoc Tukey test. ns , not significant. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Human aortic smooth muscle cells (AoSMCs, CC-2571), smooth muscle cell basal medium (SmBM, CC-3181), and SmBM plus SingleQuots of supplements (CC-3182) were purchased from Lonza (Basel, Switzerland).

Techniques: Activity Assay, In Vitro, Small Interfering RNA, Control, Derivative Assay, Western Blot, Quantitation Assay, Migration, Real-time Polymerase Chain Reaction, Marker, Standard Deviation

Journal: JVS-Vascular Science

Article Title: The role of 6-phosphogluconate dehydrogenase in vascular smooth muscle cell phenotypic switching and angioplasty-induced intimal hyperplasia

doi: 10.1016/j.jvssci.2024.100214

Figure Lengend Snippet: Small molecular inhibitor Physcion treatment phenocopies 6-phosphogluconate dehydrogenase ( 6PGD ) loss-of-function against vascular smooth muscle cell (VSMC) phenotypic switching in vitro. Human aortic smooth muscle cells (AoSMCs) underwent serum starvation for 24 hours before treatment with Physcion or DMSO vehicle control. After 24 hours of treatment with Physcion (100 nM) or control, platelet-derived growth factor ( PDGF-BB ) was introduced to induce VSMC's hyperproliferative and migratory phenotypic switching for an additional 24 hours. (A ) Representative Western blot images of phosphorylated-ERK ( P-ERK ), total-ERK ( T-ERK ), phosphorylated-MEK ( P-MEK ), and total-MEK ( T-MEK ). ( B ) Enzymatic activity of 6PGD. (C) Protein quantitation of P-ERK and P-MEK (normalized to T-ERK and T-MEK, respectively). (D) Representative images of VSMC migration in the Transwell assays and quantitation. (E) Quantitation of VSMC proliferation following Physcion or vehicle control treatment, with or without PDGF-BB stimulation. The data is presented as mean ± standard deviation (n ≥ 3 replicates), and statistical analysis was performed using one-way analysis of variance with post hoc Tukey test. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Human aortic smooth muscle cells (AoSMCs, CC-2571), smooth muscle cell basal medium (SmBM, CC-3181), and SmBM plus SingleQuots of supplements (CC-3182) were purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, Control, Derivative Assay, Western Blot, Activity Assay, Protein Quantitation, Migration, Quantitation Assay, Standard Deviation