Abstract-The heterogeneous nature of the cell populations involved in vascular repair remains a major hurdle for the assessment of the cellular events that take place in injured arteries. The present experiments were designed to estimate the proportions and cell cycle progression of infiltrating leukocytes versus resident vascular cells after balloon injury of the rat common carotid artery. After tissue disaggregation, cell suspension samples from each artery were analyzed by flow cytometry. Cells were stained with anti-CD45 or anti-␣-smooth muscle actin antibodies to identify leukocytes and smooth muscle cells, respectively. A day after injury, a 12-fold increase in CD45ϩ leukocytes was found. Double labeling with CD45 and CD-3, ED-1, or granulocyte markers revealed that most infiltrating cells were monocytes and granulocytes. Approximately 14% of infiltrating leukocytes were found to enter apoptosis at day 1, and 17% entered S phase at day 3. In contrast, the highest proliferation rate of resident ␣-smooth muscle actin-positive cells was observed at day 7 (19% Key Words: restenosis Ⅲ vascular remodeling Ⅲ inflammation Ⅲ flow cytometry Ⅲ apoptosis Q uiescent vascular smooth muscle cells (SMCs) respond to acute vascular injury with an array of phenotypic changes, including proliferation, migration, programmed cell death, and modulation to a less differentiated state. 1-3 Deendothelialization of the carotid artery is followed by proliferation of medial SMCs, with subsequent migration of these cells to the intima, where they proliferate and secrete extracellular matrix. 4 Inflammation is associated with the development of atherosclerotic lesions and is thought to play a critical role in vascular SMC proliferation, migration, and matrix production in response to injury. 5 Interleukin (IL)-1 and tumor necrosis factor-␣ may induce the expression of monocyte chemoattractant protein (MCP)-1, IL-8, intercellular adhesion molecule-1, and vascular adhesion molecule (VCAM)-1 to assist the recruitment of leukocytes into a mechanically injured tissue or an atherosclerotic lesion. 6 -9 MCP-1 and MCP-3 have been shown to be upregulated in the neointima, 10,11 and inhibition of intimal proliferation by antibodies (Abs) against MCP-1 supports the role of these molecules in vascular lesion formation. 12 To further evaluate the contribution of proliferating leukocytes to the vascular repair process, we examined the cell cycle status of infiltrating leukocytes and vascular SMCs in vivo by using a well-characterized model of balloon injury. By use of a double-labeling technique, the proliferative and DNA content profiles of vascular SMCs and infiltrating leukocytes were analyzed by flow cytometry.
Methods
Carotid InjuryBalloon injury of the common carotid artery was performed as described by Clowes et al. 13 To identify proliferating cells, bromodeoxyuridine (BrdU, 50 mg/kg IP, Sigma Chemical Co) was administered 18 hours before tissue harvesting. For the time-course flow cytometric analysis, rats (nϭ4 per time point) were euthaniz...
