Granulocyte-colony stimulating factor for repair of injured arteries in rats

Jun WU; Shaoliang CHEN; Zhizhong LIU; Xiaobo LI; Xiaoye SU

ABSTRACT

BACKGROUND:

It has been reported that treatment with granulocyte-colony stimulating factor (G-CSF) increases the abundance of circulating CD34+ cells in rats. Data from the study, more important, suggested that mobilized by G-CSF may enhance rapid reendothelization and reduce neointimal formation after vascular injury.

OBJECTIVE:

To evaluate whether BM-derived CD34+ cells could enhance rapid reendothelization and reduce neointimal formation after balloon-injured carotid artery in an intact rat model.

DESIGN:

Randomized control animal study.

SETTING:

Nanjing First Hospital Affiliated to Nanjing Medical University.MATERIALS The experiment was carried out in Nanjing First Hospital from December 2005 to April 2006. A total of 40 male Sprague-Dawley rats, weighing 200-250 g and of SPF grade, were purchased from National Rodent Laboratory Animal Resources, Shanghai Branch. The recombinant human G-CSF was purchased from Qilu Pharmaceutical. The 2F Fogarty arterial embolectomy catheters were purchased from Edwards Lifesciences. Anti-human CD34 and anti-human CD45 were purchased from Multi Sciences.

METHODS:

SD rats were divided randomly into treated group (n =20) and control group (n =20). Subcutaneous injection of recombinant human G-CSF (100 μg/kg/day) once daily for 8 days for treated group. Control group as treated with subcutaneous injection of saline. Five days after initiation of G-CSF treatment or saline, the rats were anesthetized by intraperitoneal injection with ketamine. The left common carotid artery was exposed through a midline incision of the ventral side of the neck. A 2F Fogarty arterial embolectomy catheter was inserted through the external carotid artery,inflated with 200 μL air, and passed 3 times along the length of the segment, which was defined proximally by the carotid bifurcation and distally by the edge of the omohyoid muscle. After removal of the catheter, the proximal ligature of the external carotid artery was tied off. ① An average of 1 mL venous blood per rat was collected for enumeration of the white blood wells (WBCs) and CD34+ cells before and 5 days after initiating G-CSF or saline treatment. ② Ten rats in each group were killed with overdose ketamine at 14 and 28 days after balloon injury and left common carotid arteries were harvested. The luminal surface of carotid arteries (n =5, each group) was exposed to calculate the reendothelialized area, which was manually traced with software (Image ProPlus). Reendothelialized area = non-stained with Evans blue area/the total area of balloon-injuried. The cross sections of carotid arteries (n =5, each group) were stained with hematoxylin and eosin (HE) and calculated intima-to-media area ratio (I/M) with software (Image ProPlus) to assess the extent of neointimal thickening. ③ To evaluate the extent of reendothelialization of arteries injury, sections were stained with CD31 and vWF by immunohistochemistry analysis.MAIN OUTCOME

MEASURES:

① The number of WBCs and CD34+ cells; ② the extent of reendothelialization of arteries injury; ③ the extent of neointimal hyperplasia (I/M); ④ CD31 + and vWF+ endothelial cells.

RESULTS:

A total of 40 rats were involved in the final analysis. ① The number of WBCs and CD34+ cells After 5 days of treatment, the number of WBCs in the treated rats increased more than 2.7-fold compared with control group [(27.60±2.45) ×109 L-1, (10.11±1.81) ×109 L-1, P ＜ 0.01], CD34+ cells increased more than 12.2-fold compared with control group (38.31×107 L-1, 3.14×107 L-1, P ＜ 0.01). ② The extent of reendothelialization At 14 and 28 days after balloon injury,carotid artery of reendothelialization in the treated group were (68.3±8.3)％ and (97.6±4.1)％, superior than the control group (33.8±6.3)％ and (76.1±5.2)％ (P ＜ 0.01). ③ The extent of neointimal hyperplasia At 14 and 28 days after balloon injury, the neointima-media (I/M) ratios in the treated rats were 0.39±0.11 and 0.45±0.09, less than the control group 0.87±0.15,1.26±0.16 (P ＜ 0.01). A highly significant inhibition of neointimal hyperplasia was observed in the treated group. ④ CD31+ and vWF+ endothelial cells At 28 days after injury, sections from G-CSF treated group showed almost complete and continuous monolayer of CD31 and vWF positive cells.In contrast, a patchy and interrupted CD31 and vWF positive cells were found lining the lumen of control group.

CONCLUSION:

Treatment with G-CSF significantly increases the number of CD34+ cells and accelerates the rate of reendothelialization of injured vessels, leading to marked inhibition of neointimal formation after vascular injury in rats.