Korean J Physiol Pharmacol.  2009 Apr;13(2):123-129. 10.4196/kjpp.2009.13.2.123.

Aprotinin Inhibits Vascular Smooth Muscle Cell Inflammation and Proliferation via Induction of HO-1

• Lee DH
• Choi HC
• Lee KY
• Kang YJ

• KMID: 2071661
• DOI: http://doi.org/10.4196/kjpp.2009.13.2.123

Abstract

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1beta plus TNF-alpha), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.

Keyword

Aprotinin; Inflammation; Vascular smooth muscle cell; Proliferation; HO-1; iNOS

MeSH Terms

Animals
Aprotinin
Cardiopulmonary Bypass
Cell Proliferation
Inflammation
Metalloporphyrins
Muscle, Smooth, Vascular
Nitric Oxide Synthase Type II
Phosphorylation
Phosphotransferases
Protoporphyrins
Rats
Reactive Oxygen Species
Tin
Aprotinin
Metalloporphyrins
Nitric Oxide Synthase Type II
Phosphotransferases
Protoporphyrins
Reactive Oxygen Species
Tin

Figure

• 

  Fig. 1. Aprotinin induces HO-1 on cytokine-stimulated VSMCs. VSMCs were pre-treated with aprotinin (1~100 μM) for 1 hr and treated with cytokine (10 ng/ml IL-1β plus 25 ng/ml TNF-α, I + T) for 12 hrs prior to measurement of HO-1 proteins by Western blotting (A, C). Cells were pre-treated with aprotinin (10 μM) and SnPPIX (1 μM) for 1 hr, and were treated with cytokine for 12 hrs prior to measurement of HO-1 mRNA by RT-PCR (B, D). Results represent three independent experiments. ∗p value<0.001 compared with control, ∗∗p value<0.001 compared with I + T plus aprotinin.

• 

  Fig. 2. Aprotinin inhibits cytokine-induced iNOS expression in VSMCs through HO-1. VSMCs were pre-treated with aprotinin (1~100 μM), were treated with cytokine (I + T) for 12 hrs prior to measurement of iNOS protein expression (A), and for 24 hrs prior to measurement of nitrite production (B). Cells were pre-treated with aprotinin (10 μM) and SnPPIX (1 μM), and were treated with cytokine for 12 hrs prior to measurement of iNOS protein expression (C) and nitrite production (D). Data represent mean±SD values of four independent experiments. ∗p value<0.001 compared with control, ∗∗p value<0.001 compared with I + T, †p value <0.001 compared with aprotinin.

• 

  Fig. 3. Aprotinin inhibits cytokine-induced VSMC proliferation through HO-1. VSMCs were pretreated with aprotinin (1~100 μM) for 1 hr, and were treated with cytokine (I+T) for 48 hrs prior to measurement of cell proliferation by the MTT assay (A) and for 4 days prior to counting cell numbers (B). Cells were pre-treated with aprotinin (10 μM) and SnPPIX (1 μM) for 1 hr, and were treated with cytokines for 48 hrs prior to measurement of cell proliferation by the MTT assay (C) and for 4 days prior to counting cell numbers (D). Data represent the mean±SD values of four independent experiments.

• 

  Fig. 4. Aprotinin inhibits cytokine-induced ROS generation in VSMCs through HO-1. VSMCs were pre-treated with aprotinin (10 μM) and SnPPIX (1 μM) for 1 hr, and were treated with cytokine for 12 hrs prior to measurement of ROS generation by live image microscopy (A) and FACs analysis (B). ROS was detected using H2DCFHDA. Data represent the mean±SD values of four independent experiments.

• 

  Fig. 5. JNK plays a role in the prevention of cytokine-induced iNOS expression and proliferation. VSMCs were pre-treated with the MEK inhibitor (PD98059, 2.5 μM), p38 inhibitor (SB203580, 10 μM), and JNK inhibitor (10 μM) for 1 hr, and were treated with cytokines for 12 hrs prior to measurement of iNOS protein expression (A), for 24 hrs prior to measurement of nitrite production (B), for 48 hrs prior to measurement of cell proliferation by MTT assay (C), and for 4 days prior to measurement of cell proliferation by cell counts (D). Data represent the mean±SD values of four independent experiments. ∗p value<0.0001 compared with control, ∗∗p value < 0.0001 compared with I + T.

• 

  Fig. 6. JNK plays a role in the prevention of cytokine-induced ROS generation in VSMCs. VSMCs were pre-treated with MEK inhibitor (PD98059, 2.5 μM), p38 inhibitor (SB203580, 10 μM), and JNK inhibitor (10 μM) for 1 hr, and were treated with cytokine for 12 hrs prior to measurement of ROS generation by live image microscopy (A) and by FACs analysis (B). Data represent the mean±SD values of four independent experiments.

• 

  Fig. 7. Aprotinin inhibits cytokine-induced JNK phosphorylation through HO-1. Phosphorylated JNK was measured in cells treated with cytokines (I+T) for 1 hr by Western blotting (A). Cells were pre-treated with aprotinin (10 μM) and SnPPIX (1 μM) for 1 hr, and were treated with cytokines for 5 minutes prior to measurement of phosphorylated JNK by Western blotting (B). Results are representative of three experiments.

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