Blood, Vol. 112, Issue 8, 3205-3216, October 15, 2008

Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells
Blood Chen et al. 112: 3205

Supplemental materials for: Chen et al

Files in this Data Supplement:

• Document 1. Supplemental materials and methods (PDF, 2.04 MB)
  
  
• Table S1. Sequence details of individual pairs of primers (PDF, 398 KB)
  
  
• Table S2. 95 miRNAs profiling for sCD40L or TNF-α treatment in HCAECs (PDF, 5.33 MB)
  
  
• Table S3. Protein sequence homology of CD40L and CD40 between human and porcine (PDF, 128 KB)
  
  
• Figure S1. Effects of sCD40L and TNF-α on 95 miRNA gene expression (JPG, 57.5 KB) -
  HCAECs were treated with sCD40L (5 µg/mL) or TNF-α (2 ng/mL) for 24 hours. The expression of 95 miRNAs was determined by real time PCR with a commercial 95 miRNA array kit. Expression levels of mature miRNAs were analyzed by Ct values and normalization to U6 miRNA levels (2^–ΔCT). Treated and control cells were separately calculated. Fold changes of each miRNA in treated cells were calculated in relation to that in untreated control cells.
  
  
  
  
  
  
• Figure S2. Effect of sCD40L and SeMet on HCAEC proliferation (JPG, 55.9 KB) -
  HCAECs were cultured in the presence of sCD40L (5 µg/mL) or SeMet (20 mM) for different times (1, 2, 3 and 4 days). Cell proliferation was determined by a commercial CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation Assay (MTS) Kit. Data were standardized with initial seeding density and presented as % of initial seeding density. n = 10.
  
  
  
  
  
  
• Figure S3. Effects of sCD40L on contraction and endothelium-independent vasorelaxation of porcine coronary artery rings (JPG, 29 KB) -
  Porcine coronary artery rings were cut and cultured with sCD40L (5 µg/mL) for 24 hours. Vasomotor reactivity was studied with a myograph device. (A) Vessel contraction was recorded when thromboxane A2 analogue U46619 (3 × 10⁻⁸ M) was administered. (B) Precontracted vessels were exposed to sodium nitroprusside (10⁻⁶ M) and endothelium-independent vasorelaxation was recorded. n =10.
  
  
  
  
  
  
• Figure S4. NOX4, SOD and CAT mRNA levels in Ad-NOX4 DN-, Ad-SOD- and Ad-CAT-infected cells (JPG, 35.2 KB) -
  Recombinant adenovirus (Ad-NOX4 DN, Ad-SOD or Ad-CAT) infected HCAECs for 48 hours. The mRNA levels of NOX4, SOD and CAT in these cells were determined by real time PCR to confirm the active gene delivery and expression via recombinant adenoviruses. (A) NOX4 mRNA levels. (B) SOD mRNA levels. (C). CAT mRNA levels. **P t-test, n = 3.
  
  
  
  
  
  
• Figure S5. CD40L and CD40 immunoreactivity of human atherosclerotic and non-atherosclerotic aortas (JPG, 97.4 KB) -
  Human aorta were fixed in formalin and embedded in paraffin. Immunostaining was performed. Atherosclerotic aorta had stronger immunoreactivity for both CD40L and CD40 than non-atherosclerotic aorta. Brown color represents positive staining of CD40L or CD40.
  
  
  
  
  
  
• Figure S6. Summary of major findings and potential mechanisms of sCD40L-induced eNOS downregulation and endothelial dysfunction (JPG, 62.5 KB) -
  sCD40L specifically interacts with its membrane receptor CD40 on endothelial cells and increases superoxide anion production through reduction of mitochondrial membrane potential, activation of NADPH oxidase and inhibition of CAT and SOD activities. Dysfunctional mitochondria are also demonstrated by reduction of ATP production. Superoxide anion could trigger activation of p38 and ERK1/2, which may lead to the phosphorylation of IκBα and transcriptional factor NF-κB activation. Superoxide anion may be able to directly activate the IκBα and NF-κB system. Superoxide anion and activation of p38, ERK1/2 and NF-κB could decrease eNOS mRNA stability and/or eNOS transcriptional activity, resulting in decrease in eNOS levels and NO bioavailability. Superoxide anion could directly interact with NO and reduce NO bioavailability, which causes endothelial dysfunction.