Background/Purpose: Increased expression of CD40 in SSc- Microvascular Endothelial Cells (MVEC) was noted on a gene expression array and increased concentrations of soluble CD40 ligand (sCD40L) was reported in scleroderma. In this study we investigated the effect of CD40 ligation on MVEC apoptosis, activation and dysfunction. 

Methods: MVEC were isolated from involved SSc skin and from matched healthy control subjects. MVEC apoptosis was assessed by flow cytometry, caspases3/7 activity and cell viability. MVEC genes expression was determined by real-time-PCR and results were confirmed by western blot analysis. Endothelial permeability was assessed by FITC-Dextran permeability assay.

Results:

Significant increase in CD40 expression of was noted in SSc-MVEC (3.2 folds ± 0.3 in SSc vs. control MVEC). Similar increase in expression level was noted in SS skin biopsies (2.4 folds ± 0.3 in SSc skin vs. control skin biopsies, n=3 each).

The addition of CD40 ligand to MVEC resulted in the following observations:

1. Reduction in nitric oxide synthase (NOS3) expression (80%±6 reduction in control-MVEC and 82%± 8 in SSc-MVEC, mean 4 cell lines ±SD) and in prostacyclin synthase (PTGS1), 64% ± 3.0 reduction in control-MVEC and 79% ± 4.5 reduction in SSc- MVEC, mean 4 cell lines ±SD ). While Endothelin 1 expression was significantly increased by 2.2± 0.12 folds in control MVEC and by 2.8 ±0.31 folds in SSc MVEC.
2. Increase EC permeability (2.2± 0.3 in control-MVEC and 2.8 ± 0.34 in SSc-MVEC, folds±SD)      
3. Significant Increase expression of the CC and CXC chemokines including CCL2, interleukin-8, CXCL3, 5, 6 and 9.
4. Increase expression of IL1B, hepatocyte growth factor and adhesion molecules particularly ICAM1.
5. Significant down regulation of c-fos induced growth factor (FGIF, vascular endothelial growth factor D), epidermal growth factor, Insulin-like growth factor 1 and metallopeptidase inhibitor TIMP2.
6. The addition of CD40L to MVEC cultured in 0.5% serum resulted in a dose dependent apoptosis of MVEC. SSc-MVEC were more susceptible to apoptosis than control MVEC, thus at 10 μg concentration of CD40L , MVEC apoptosis was 45% ± 5 and 20% ± 3 in SSc vs. control MVEC respectively (mean ± SD of 4 cell lines). Apoptosis was confirmed by increase caspase 3/7 activity 2.2 folds in control and 3.0 folds in SSc-MVEC.

Conclusion:

The study demonstrates increase expression of CD40 in SSc skin and in SSc-MVEC. CD40 ligation led to reduce expression of vasodilatory and increase expression of vasoconstrictive genes. Moreover, addition of CD40L increased endothelial permeability and the acquisition of an activated/ dysfunctional phenotype in association with increase MVEC apoptosis. In all instances, SSc MVEC were more susceptible to CD40 signaling effects than control MVEC. The results suggest that the blockade of CD40/CD40 ligand interaction could be an effective therapeutic strategy in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd40-signaling-results-in-microvascular-endothelial-dysfunction-a-possible-clue-to-the-pathogenesis-of-scleroderma-vasculopathy/