Experimental animal transplantation
Cell models: Endothelium
Apoptosis in Endothelial Cells by Cyclosporine

https://doi.org/10.1016/j.transproceed.2012.01.089Get rights and content

Abstract

Objectives

The immunosuppressive drug cyclosporine (CsA) is a potent agent widely used after organ transplantations and to treat various autoimmune disorders. After using CsA, some patients suffer severe complications including renal and vascular toxicity, which are influenced by the degree of the endothelial damage. Several studies have demonstrated CsA treatment to directly induce apoptosis in several cell types. Thus, CsA may induce endothelial damage via activation of proapoptotic proteins. The present study was undertaken to investigate the effects of CsA on apoptosis of endothelial cells using human umbilical vein endothelial cells.

Methods

Proliferation was measured by using the Cell Counting Assay Kit after cells were exposed to CsA (0 L, 10 L, 30 L, 50 L or 100 μg/mL). Apoptotic cells were identified by fluorescence microscopy of 4′, 6-diamidino-2-phenylidole-stained nuclei. Western blot analysis was done for poly(ADP-ribose) polymerase (PARP), p27, p53 and caspase.

Results

Cell viability decreased dependent on the CsA concentration. CsA treatment group showed chromatin condensation and nuclear fragmentation. CsA produced a dose-dependent induction of p27 and reduction of procasapase-3. CsA treatment induced the degradation of 116-kDa PARP into an 89-kDa fragment.

Conclusions

CsA induced apoptosis of endothelial cells.

Section snippets

Cell Culture

HUVECs were isolated from umbilical cords by collagenase digestion. In brief, after clamping both ends of the umbilical cord, the vein was cannulated with blunt needles, flushed first with air, and then filled with 37°C prewarmed 0.25% trypsin solution for incubation in a humified 37°C incubator for 15 minutes. The collected effluent was centrifuged for 10 minutes and the pellet resuspended in Endothelial Basal Medium-2 Lonza Biologics, Slough, UK) containing 2% fetal bovine serum and

Results

The cell viability in the presence of CsA, was 100.0% ± 0.0% for 0 (controls; 92.7% ± 0.1% for 10 (P < .05 vs control); 84.1% ± 0.1% for 30 (P < .01 vs control); 22.7% ± 0.0% (P < .001 vs control); and 6.5% ± 0.0% for 100 μg (P < .001 vs control).

CsA concentrations of 50 μg/mL showed chromatic condensation of nucleotides and nuclear fragmentation (Fig 1).

Western blot analysis detected p27, p53, procaspase-3, and PARP proteins (Fig 2). CsA unduced p27, reduced procasapase-3, and caused

Discussion

Apoptosis, programmed cell death, is an essential process in development and tissue homeostasis of most multicellular organisms. Deregulation of apoptosis has been implicated in the pathogenesis of many diseases.5, 6, 9, 10 Apoptosis can be induced by stress, radiation, heat, infection, and days nutrition.5, 6, 9, 10 Apoptosis produces characteristic morphological changes including DNA fragmentation, nuclear fragmentation, and chromatin condensation.5, 6, 9, 10 Apoptosis must be controlled,5, 6

References (10)

There are more references available in the full text version of this article.

Cited by (9)

  • Secondary thrombotic microangiopathy and eculizumab: A reasonable therapeutic option

    2017, Nefrologia
    Citation Excerpt :

    This is observed in 4–15% of patients treated with cyclosporine and in 1–4% of those treated with tacrolimus.75,79,90 CNIs have a direct toxic effect on the endothelium which is mediated by the formation of microparticles stimulating the alternative C pathway and by tissue ischaemia (reduction of prostacyclin and nitric oxide vasodilators), formation of reactive O2 species and increased platelet aggregation.90–97 The mTOR inhibitors enhance post-transplant TMA by inhibiting VEGF synthesis by podocytes, enhancing endothelial damage and reducing endothelial regeneration capacity.

  • TMA in Kidney Transplantation

    2023, Transplantation
View all citing articles on Scopus

The present research has been conducted by the Research Grant of Dongsan Kidney Institute, Keimyung University in 2009.

View full text