Chronic vascular toxicity of doxorubicin in an organ-cultured artery

Abstract

1. We investigated the chronic effects of doxorubicin (DXR) on morphological and functional changes in the rabbit mesenteric artery using an organ culture system. 2. In arteries cultured with 0.3 microM DXR for 7 days, the contractions induced by noradrenaline, but not those induced by endothelin-1 or high K(+), were strongly inhibited. This reaction was followed by a decrease in the induction of the alpha(1A)-adrenoceptor without any change in the mRNA level. Inhibition of noradrenaline-induced contractions by DXR was attenuated by superoxide dismutase, and alpha(1A)-adrenoceptor protein expression recovered. 3. In the arteries cultured with 1 microM DXR for 7 days, contractions induced by endothelin-1 or high K(+) and absolute force in the permeabilized muscles were also inhibited. Morphological examinations revealed the existence of concentrated nuclei and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive smooth muscle cells, and internucleosomal DNA fragmentation was also detected, indicating the induction of apoptosis. 4. In the arteries cultured with 10 microM DXR for 7 days, nuclear swelling, karyolysis and random DNA fragmentation indicative of necrosis were observed, and muscle contractility was abolished. 5. These results suggest that 0.3 microM DXR selectively down-regulates the alpha(1A)-adrenoceptor protein expression, resulting in a decrease in the noradrenaline-induced contraction. This down-regulation may be at least partly due to the production of a superoxide radical. DXR also caused a decrease in muscle contractility followed by apoptotic changes at 1 microM and necrotic changes at 10 microM. These changes might be responsible for the disturbance of the circulatory system that is often observed during the course of repetitive chemotherapy.

Figure 1

Time-dependent changes in contractions induced by noradrenaline (A) and endothelin-1 (B) after cultivation in DMEM with or without 1 μM DXR. Noradrenaline (10 μM) or endothelin-1 (100 nM) was added to rabbit mesenteric arteries cultured in DMEM for 1, 3, 5, and 7 days in the absence (control) or presence of 1 μM DXR. Results are expressed as means±s.e.mean (n=5 – 15). ^**Significantly different from the results in control arteries at P<0.01. ^§Significantly different from the results in control arteries after cultivation for 1 day at P<0.01.

Figure 2

Chronic effect of various concentrations of DXR on contractions induced by noradrenaline (A), endothelin-1 (B), and high K⁺ (C). Noradrenaline (0.01 – 100 μM), endothelin-1 (0.1 – 100 nM), or high K⁺ (15.4 – 65.4 mM) was added cumulatively. Results are expressed as means±s.e.mean (n=5 – 15). ^**Significantly different from results in control arteries at P<0.01.

Figure 3

Chronic effect of 0.3 μM DXR on α_1A-adrenoceptor (α_1A-AR) protein and mRNA expression. (A) Shows a typical trace of the 4 – 5 experiments in Western blot analysis for ET_A-receptor (ET_A-R) and α_1A-adrenoceptor (α_1A-AR) protein expression. (B) Shows quantitative RT – PCR for the determination of mRNA of the ET_A-receptor (ET_A-R) and the α_1A-adrenoceptor (α_1A-AR). The results were expressed as the ratio of GAPDH of each artery at 30 cycles.

Figure 4

The effects of SOD (100 u ml⁻¹) on DXR (0.3 μM)-induced inhibition of noradrenaline-induced contractions (A) and α_1A-adrenoceptor (α_1A-AR) protein expression (B). Results are expressed as means±s.e.mean (n=5 – 7). (B) Shows a typical result of the five experiments in Western blot analysis for α_1A-adrenoceptor (α_1A-AR) protein expression. ^**Significantly different from results in DXR treated arteries at P<0.01.

Figure 5

Chronic effect of DXR on muscle contractility in the permeabilized rabbit mesenteric artery. (A) Shows the maximum contractile capability induced by adding 30 μM Ca²⁺ with 1 μM calyculin-A. (B) Shows the pCa²⁺-tension relationship. 100% represents the maximum force induced by 30 μM Ca²⁺ with 1 μM calyculin-A. ^**Significantly different from results in control arteries at P<0.01.

Figure 6

Representative light micrographs of sections stained with hematoxylin and eosin in rabbit mesenteric arteries chronically treated with DXR for 7 days. Arrow heads indicate typical apoptotic smooth muscle cells. Magnification:×184. Bar, 85 μm.

Figure 7

Detection of apoptosis induced by chronic treatment with DXR for 7 days. (A) Shows the results of TUNEL assay. Arrow heads indicate TUNEL-positive smooth muscle cells. Magnification:×304. Bar, 50 μm. (B) Shows the results of the DNA ladder formation assay using gel electrophoresis.