基质细胞衍生因子-1（SDF-1/CXCL12）变体的体外血管生成作用及人类严重下肢缺血中 CXCL12 变体和 CXCR4 的体内表达。
Angiogenic effects of stromal cell-derived factor-1 (SDF-1/CXCL12) variants in vitro and the in vivo expressions of CXCL12 variants and CXCR4 in human critical leg ischemia.
机构信息
Vascular Unit, University Department of Surgery, The Royal Free and University College Medical School, University College London (Hampstead Campus), London, United Kingdom.
出版信息
J Vasc Surg. 2010 Mar;51(3):689-99. doi: 10.1016/j.jvs.2009.10.044.
PURPOSE
Critical leg ischemia (CLI) is associated with a high morbidity and mortality. Therapeutic angiogenesis is still being investigated as a possible alternative treatment option for CLI. CXCL12, a chemokine, is known to have two spliced variants, CXCL12alpha and CXCL12beta, but the significance remains unknown. The study investigated the angiogenic effects of CXCL12, protein expressions of CXCL12, and the receptor CXCR4 in human CLI.
METHODS
In vitro, human microvascular endothelial cells (HMEC-1) were used. Cell proliferation was assessed using methylene blue assay and cell count method. Apoptosis was determined by counting the pyknotic nuclei after 4'-6-diamidino-2-phenylindole staining and confirmed by caspase-3 assay. We employed matrigel as capillary tube formation assay. The activity of signaling pathways was measured using Western blotting. In vivo, gastrocnemius biopsies were obtained from the lower limbs of patients with CLI and controls (n = 12 each). Immunohistochemistry, double immunofluorescence labeling, and Western blotting were then performed.
RESULTS
CXCL12 attenuated HMEC-1 apoptosis (P < .01), stimulated cell proliferation (P < .05) and capillary tube formation (P < .01). Compared with CXCL12alpha, CXCL12beta has a greater effect on apoptosis and cell proliferation (P < .01). Treatment with both variants resulted in time-dependent activation of PI3K/Akt and p44/42 but not p38 MAP kinase. In CLI, CXCL12alpha was expressed by skeletal muscle fibers with minimal expression of CXCL12beta. CXCR4 was extensively expressed and colocalized to microvessels. A significant 2.6-fold increase in CXCL12alpha and CXCR4 expressions (P < .01) were noted in CLI but not for CXCL12beta (P > .05).
CONCLUSIONS
The study showed that CXCL12beta had more potent angiogenic properties but was not elevated in human CLI biopsies. This provided an interesting finding on the role of CXCL12 variants in pathophysiologic angiogenic response in CLI.
目的
严重肢体缺血（CLI）与高发病率和死亡率相关。治疗性血管生成仍在被研究作为 CLI 的一种可能的治疗选择。趋化因子 CXCL12 有两种剪接变体，CXCL12alpha 和 CXCL12beta，但意义尚不清楚。该研究调查了 CXCL12、CXCL12 蛋白表达和受体 CXCR4 在人类 CLI 中的血管生成作用。
方法
在体外，使用人微血管内皮细胞（HMEC-1）。通过亚甲蓝测定法和细胞计数法评估细胞增殖。通过 4'-6-二脒基-2-苯基吲哚染色后计数固缩核来确定细胞凋亡，并通过 caspase-3 测定法确认。我们采用基质胶作为毛细血管管形成测定法。通过 Western blot 测量信号通路的活性。在体内，从 CLI 患者和对照者（每组 12 例）的下肢获得比目鱼肌活检。然后进行免疫组织化学、双免疫荧光标记和 Western blot。
结果
CXCL12 减轻了 HMEC-1 细胞凋亡（P <.01），刺激了细胞增殖（P <.05）和毛细血管管形成（P <.01）。与 CXCL12alpha 相比，CXCL12beta 对细胞凋亡和细胞增殖的影响更大（P <.01）。两种变体的处理均导致 PI3K/Akt 和 p44/42 的时间依赖性激活，但不导致 p38 MAP 激酶的激活。在 CLI 中，CXCL12alpha 由骨骼肌纤维表达，CXCL12beta 表达很少。CXCR4 广泛表达并与微血管共定位。在 CLI 中，CXCL12alpha 和 CXCR4 的表达显著增加了 2.6 倍（P <.01），但 CXCL12beta 的表达没有增加（P >.05）。
结论
该研究表明，CXCL12beta 具有更强的血管生成特性，但在人类 CLI 活检中没有升高。这为 CXCL12 变体在 CLI 病理生理血管生成反应中的作用提供了一个有趣的发现。
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