Provasculon Inc., Cambridge, MA 02142, USA.
Circulation. 2011 Mar 29;123(12):1306-15. doi: 10.1161/CIRCULATIONAHA.110.991786. Epub 2011 Mar 14.
Peripheral artery disease is a potentially incapacitating disease for which pharmacological options are limited. Stromal cell-derived factor-1 (SDF-1) is a chemokine that attracts endothelial progenitor cells and promotes angiogenesis. Therapeutic use of SDF-1 in hindlimb ischemia may be challenged by proteolytic degradation. We hypothesized that protease-resistant variants of SDF-1 can increase blood flow in an experimental model of hindlimb ischemia.
We screened a peptide library for mutations in SDF-1 that provide resistance to matrix metalloproteinase cleavage. Recombinant SDF-1 proteins carrying the mutations were designed, expressed, and purified, and activity of mutant proteins was tested with receptor activation assays and in vivo Matrigel plug assays. SSDF-1(S4V), which is resistant to both dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage, was active in vitro and induced angiogenesis in vivo. We then designed and purified fusion proteins of SSDF-1 and SSDF-1(S4V) with the sequence of self-assembling peptide nanofibers for incorporation into nanofibers. In a blinded and randomized hindlimb ischemia mouse study, SSDF-1(S4V) delivery by nanofibers improved blood flow as measured by laser Doppler from 23.1±1.9% (untreated control) to 55.1±5.7% 6 weeks after surgery (P<0.001). Nanofibers alone or SSDF-1 delivered by nanofibers did not improve blood flow. Furthermore, SSDF-1(S4V) delivered by nanofibers increased formation of new arterioles. In vitro, SSDF-1(S4V) attracts smooth muscle cells but does not induce mitosis.
SDF-1 engineered to be resistant to dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage and delivered by nanofibers improves blood flow in a model of peripheral artery disease.
外周动脉疾病是一种可能使人丧失能力的疾病，其药物选择有限。基质细胞衍生因子-1（SDF-1）是一种趋化因子，可吸引内皮祖细胞并促进血管生成。SDF-1 在下肢缺血中的治疗用途可能受到蛋白水解降解的挑战。我们假设 SDF-1 的蛋白酶抗性变体可以增加下肢缺血实验模型中的血流量。
我们从 SDF-1 中筛选出对基质金属蛋白酶切割具有抗性的突变，以构建肽库。设计、表达和纯化携带突变的重组 SDF-1 蛋白，并通过受体激活测定和体内 Matrigel 塞子测定测试突变蛋白的活性。SSDF-1(S4V) 对二肽基肽酶 IV/CD26 和基质金属蛋白酶-2 的切割均具有抗性，具有体外活性，并诱导体内血管生成。然后，我们设计并纯化了 SSDF-1 和 SSDF-1(S4V) 与自组装肽纳米纤维序列的融合蛋白，以纳入纳米纤维。在一项盲法和随机的下肢缺血小鼠研究中，通过纳米纤维递送 SSDF-1(S4V) 将手术后 6 周时的激光多普勒测量的血流量从 23.1±1.9%（未治疗对照组）提高到 55.1±5.7%（P<0.001）。单独的纳米纤维或通过纳米纤维递送的 SSDF-1 均不能改善血流量。此外，通过纳米纤维递送的 SSDF-1(S4V) 增加了新小动脉的形成。体外，SSDF-1(S4V) 吸引平滑肌细胞，但不诱导有丝分裂。
设计为对二肽基肽酶 IV/CD26 和基质金属蛋白酶-2 切割具有抗性并通过纳米纤维递送的 SDF-1 可改善外周动脉疾病模型中的血流量。
