Korean J Physiol Pharmacol.  2009 Aug;13(4):309-313. 10.4196/kjpp.2009.13.4.309.

Heme Oxygenase-1 Induced by Aprotinin Inhibits Vascular Smooth Muscle Cell Proliferation Through Cell Cycle Arrest in Hypertensive Rats

• Choi HC
• Lee KY
• Lee DH
• Kang YJ

• KMID: 2071686
• DOI: http://doi.org/10.4196/kjpp.2009.13.4.309

Abstract

Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.

Keyword

Aprotinin; Hypertension; Proliferation; Heme oxygenase-1; Cell cycle arrest

MeSH Terms

Animals
Aprotinin
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Cyclin D
G1 Phase
Heme
Heme Oxygenase (Decyclizing)
Heme Oxygenase-1
Hypertension
Metalloporphyrins
Muscle, Smooth, Vascular
Phosphotransferases
Protoporphyrins
Rats
Tin
Transfection
Aprotinin
Cyclin D
Heme
Heme Oxygenase (Decyclizing)
Heme Oxygenase-1
Metalloporphyrins
Phosphotransferases
Protoporphyrins
Tin

Figure

• 

  Fig. 1. Aprotinin treatment inhibits VSMC proliferation of SHR. VSMC were treated with aprotinin (1~10 μM) for 24 h, and cell proliferation determined by MTT assay (A). Bars represent the mean±SEM of four different experiments. ∗p<0.01 compared with WKY control, †p<0.01 compared with SHR control. Cell were treated with or without 10 μM and cell numbers determined after 0, 1, 2 and 3 days by cell counts (B). Bars represent the mean±SEM of three independent experiments.

• 

  Fig. 2. Aprotinin treatment inhibits cell cycle progression of VSMC from SHR through HO-1. VSMC were treated with 10 μM aprotinin or 10 μM aprotinin plus 1 μM SnppIX for 24 h, and then analyzed for DNA content by flow cytometry. The percentages of cells in G1 phase are graphically represented (A). The data are representative of three separate experiments (B). ∗p<0.01 compared with WKY control, †P<0.001 compared with SHR control, ¶p<0.001 compared with 10 μM aprotinin-treated SHR. Bars represents the mean±SEM of four independent experiments.

• 

  Fig. 3. Aprotinin increases HO-1 and p21 but decreases cyclin D levels. Quiescent VSMC were stimulated with 10% FBS then serum starved for the indicated time, and cell lysates were processed for the detection of cyclin D, CDK4, and p21 by Western blot (A). Cells were treated with aprotinin (1~10 μM) for 48 h, and cell lysates were processed for the detection of HO-1, cyclin D, and p21 by western blot (B). The blots are representative of three independent experiments.

• 

  Fig. 4. Aprotinin treatment inhibits VSMC proliferation of SHR through HO-1. VSMC were treated with 10 μM aprotinin or 10 μM aprotinin plus 1 μM SnppIX for 24 h, and cell proliferation determined by MTT assay (A). ∗p<0.01 compared with WKY control, †p<0.001 compared with SHR control, ¶p<0.001 compared with 10 μM aprotinin-treated SHR. Cells were transfected with mouse HO-1 DNA for 3 h and cell lysates were processed for the detection of HO-1 by Western blot (B). The blots are representative of three individual experiments. Cells were transfected with mouse HO-1 DNA for 3 h, incubated with 10% FBS for 24 h, and then cell proliferation was determined by MTT assay (C). †p<0.001 compared with SHR control. Bars represent the mean±SEM of four independent experiments.

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