Doxycycline modulates smooth muscle cell growth, migration, and matrix remodeling after arterial injury

Abstract

The tetracyclines function as antibiotics by inhibiting bacterial protein synthesis, but recent work has shown that they are pluripotent drugs that affect many mammalian cell functions including proliferation, migration, apoptosis, and matrix remodeling. Because all of these processes have been implicated in arterial intimal lesion development, the objective of these studies was to examine the effect of doxycycline treatment using a well-characterized model of neointimal thickening, balloon catheter denudation of the rat carotid artery. Rats were treated with 30-mg/kg/day doxycycline. Doxycycline reduced the activity of matrix metalloproteinase (MMP)-2 and MMP-9 in the arterial wall, and inhibited smooth muscle cell migration from media to intima by 77% at 4 days after balloon injury. Replication of smooth muscle cells in the intima at 7 days was reduced from 28.3 plus minus 2.5% in controls to 17.0 +/- 2.8% in doxycycline-treated rats. The synthesis of elastin and collagen was not affected, but accumulation of elastin was blocked in the doxycycline-treated rats. By contrast, collagen accumulation was not affected, which led to the formation of a more collagen-rich intima. At 28 days after injury, the intimal:medial ratio was significantly reduced from 1.67 +/- 0.09 in control rats to 1.36 +/- 0.06 in the doxycycline-treated rats. This study shows that doxycycline is an effective inhibitor of cell proliferation, migration, and MMP activity in vivo. Further study in more complicated models of atherosclerosis and restenosis is warranted.

Figures

Figure 1.

A: SMC replication in the media at various times after carotid artery injury. B: SMC replication in the intima at 7 and 14 days after carotid artery injury. C: SMC migration at 4 days after carotid artery injury. Filled bars represent values from control rats, and open bars values from doxycycline-treated rats. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, The value measured in the doxycycline group was significantly less than the control group.

Figure 2.

A: Gelatin zymogram showing activity of MMP-9 (88 kd active) and MMP-2 (70 kd latent and 62 kd active) in carotid arteries from control and doxycycline-treated rats 4 days after balloon catheter injury. B: Scanning densitometry was performed on zymogram gels, and the values for each MMP band were expressed as a percentage of the value obtained for control. Individual carotid extracts were prepared and run separately on the gel. Three samples per group were averaged to obtain these measurements.

Figure 3.

Collagen and elastin synthesis measured at 7 days after carotid artery injury in control rats (filled bars) or rats treated with doxycycline (open bars). Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar.

Figure 4.

Content of collagen per carotid segment (A) and elastin per carotid segment (B) in the injured carotid arteries of control rats (filled bars) and doxycycline-treated rats (open bars) at 7 and 21 days after injury. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, Collagen or elastin content measured at 21 days is significantly greater than content measured at 7 days.

Figure 5.

Ratio of intima:media cross-sectional areas (A), intimal cross-sectional area (B), and medial cross-sectional area (C) of carotid arteries from control (filled bars) and doxycycline-treated rats (open bars), measured at 28 days after balloon catheter injury. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, The value in the doxycycline-treated rats is significantly less than the value in controls.