Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: ApoA1 production following AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction assayed by ELISA and immunofluorescence. ( A ) Human embryonic kidney cells (HEK-293), rat smooth muscle cells (SMC), rat blood outgrowth endothelial cells (BOEC), and murine macrophages (Raw 264.7) were transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF). ApoA1 production was determined by ELISA of the media conditioned by transduced cells 3 days post-transduction. ( B ) Rat endothelial cells (a-c) and rat smooth muscle cells (d-f) were transduced with either AAV2-apoA1(WT) (a and d) or AAV2-apoA1(4WF) (b, e and f) for 3 days, methanol-fixed and reacted with anti-apoA1 antibody except (c), followed by either Cy 3.5- (a, b and c) or AlexaFluor488-labeled (d, e and f) secondary antibodies. Original magnification is 100×.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: ApoA1(4WF)-, but not apoA1(WT)-mediated efflux following transduction with the respective AAV2 vectors demonstrates oxidative resistance. Cell culture media conditioned by HEK 293 cells transduced with AAV2-apoA1(WT), AAV2-apoA1(4WF), both containing 10 µg/ml of AAV2-encoded apoA1 variants, and the conditioned media from the non-transduced HEK-293 cells supplemented with 10 µg/ml of recombinant human apoA1 (rhapoA1) were incubated with increasing concentrations of HOCl. Cholesterol efflux assays were then performed on 3 H-cholesterol-loaded Raw 264.7 cells incubated with differently oxidized conditioned media from HEK-293 cells for 4 h. *P < 0.05 4WF vs WT. ✞P < 0.05 4WF vs rhapoA1.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Cell Culture, Recombinant, Incubation

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: Impact of AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction on the proliferation and migration of SMC and BOEC, reactive oxygen species (ROS) production, and anti-inflammatory responses in endothelial cells. WST-8 assay was used to evaluate the effect of apoA1(WT) and apoA1(4WF) overexpression on proliferation of ( A ) TNFα (20 ng/ml)-stimulated rat aortic SMC and ( B ) non-stimulated rat BOEC. The results (both A and B ) were normalized by WST-8 assay values produced in confluent cultures of rat SMC ( A ) and rat BOEC ( B ) and expressed as the percent of a monolayer confluency. A monolayer scratch injury assay was used to assess migratory capacity of apoA1(WT)- and apoA1(4WF)-transduced rat SMC ( C ) and rat BOEC ( D ) compared with non-transduced counterparts. A closure of the gap inflicted by pipette tip was quantified 24 h after the scratch injury. ( E ) CM-H2DCFDA assay in non-transduced, apoA1(WT)- and apoA1(4WF)-transduced RAEC with/without TNFα stimulation. ( F ) Attachment of PKH26-labeled rat monocytes to TNFα-stimulated RAEC.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Migration, Over Expression, Produced, Transferring, Labeling

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: Long-term expression of the therapeutic transgene, the anti-AAV2 neutralizing antibodies dynamics, and the morphometric results in hypercholesterolemic diabetic pigs. ( A ) The percent of retained AAV2eGFP transduction capacity after incubation with 1:20 diluted serum from experimental pigs at different time points throughout the study. NAB positivity was denoted at 50% reduction of transducibility of sera-incubated compared to the not incubated AAV2 vectors. ( B ) Representative images of Verhoeff-van Gieson – stained sections of a normal pig coronary artery and pig coronary arteries treated with BMS, AAV2-eGFP- and AAV2-apoA1(4WF)-eluting stents. ( C ) Morphometric measurements of the stented segments using 3 different calculation methods (percent of luminal stenosis, neointimal thickness, and the ratio of the neointimal area to the medial area).

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Transduction, Incubation, Staining