血栓形成后增加内皮功能 Vikram S. Kashyap, M. D. , F. A. C. S 血栓形成后增加内皮功能 Vikram S. Kashyap, M.D., F.A.C.S. Associate Professor of Surgery Department of Vascular Surgery The Cleveland Clinic New Horizons in Cardiovascular Treatment (Angiogenesis and other) December 11, 2008

病 例 53 岁、女性、右下肢急性疼痛4天 右下肢无波动，股动脉无明显Doppler 信号 (Rutherford 缺血分级 IIA) 病 例 53 岁、女性、右下肢急性疼痛4天 右下肢无波动，股动脉无明显Doppler 信号 (Rutherford 缺血分级 IIA) 既往史—90年代早期行主动脉重建，2002年右髂动脉植入支架 1998年因缺血/感染行左侧髋关节离断术 肝素过敏—本病例使用比伐卢定抗凝

主动脉造影 左肱动脉途径 主动脉造影

使用Angiojet行脉冲式溶栓治疗 20 cm输注导管, tPA at 0.5 mg/hr

每隔12小时行动脉造影, 反复 PMT 10 cm 输注导管, 持续 tPA

本病例技术—24小时检查示血栓消退 血栓溶解后暴露病变 使用7mm球囊行PTA 动脉造影

病变

完成后动脉造影

溶栓治疗的益处 能够显示引起急性闭塞的潜在“病变” 降低再灌注损伤 可选择更多治疗手段 - 腔内治疗: 血管成形+/- 支架植入 - 腔内治疗: 血管成形+/- 支架植入 - 有限的外科手术 (补片成形,动脉内膜切除) - 在完善检查后行新的旁路手术 (心功能,静脉检查, 等.)

尽管初见成效，但有时…

尽管初见成效，但有时… 临床上急性动脉血栓形成仍然是一个棘手的疾病 临床常见血栓切除或溶栓后继发血管痉挛或再发血栓形成 近20% 病例发生再发血栓形成 (易于血栓形成内膜表面—考虑可引起血小板聚集形成凝血) 内皮血栓形成效应还没有完全阐明

在成功除去左肱动脉内急性栓子后的动脉造影 血栓-内皮相互作用的临床关联 在成功除去左肱动脉内急性栓子后的动脉造影

血管痉挛= 栓子引起的内皮功能异常? 血管痉挛可通过NO供体来缓解 如果有持续血凝块，血管痉挛难以处理 在多个动物模型中，血栓形成可引起内皮功能异常 NO水平减低可导致内皮功能异常

组织浴槽系统

Reil, et al EJVES 2000

腔内血栓

血栓形成后扫描电镜观察

VIII因子免疫组化 正 常 血栓形成后

溶栓试验

p=0.007 * p=0.003 *

L-精氨酸对EDR和NO的影响 Kashyap et al., JVS 2001

Western Blot

内皮依赖性舒张(EDR) ** ** ** ** * 在鼠肾下腹主动脉（IRA），血栓形成对EDR的影响。 对照组EDR（无血栓形成IRA n=5) 与实验组EDR比较（血栓形成后IRA n=4). *P<0.05, **P<0.001 by two way ANOVA.

使用特异性精氨酸抑制酶后 EDR ** ** ** ** ## ## ** ## # 血栓形成IRA使用特异性精氨酸酶抑制剂对EDR的影响.在血栓形成前以精氨酸酶抑制剂预处理可以恢复EDR

人体试验

EDR 测量 N >3

精氨酸酶 II

血栓怎样引起内皮功能异常 ? 机制 造成内皮细胞的丢失-No 清除 NO--Perhaps 降低NO的分泌或弥散-No 通过影响eNOS减低 NO 的产量 --No 通过Y+ 通道影响L-精氨酸运输- No 增加内源性血管收缩剂—No 降低L-精氨酸的可用性—Yes 凝血酶增加精氨酸酶活力

L-精氨酸代谢途径

未来研究方向 体外试验 (内皮细胞) 体外试验 (啮齿动物模型) 人体试验 凝血酶、纤维蛋白暴露于精氨酸酶阻断剂 活化中性粒细胞效应 在血栓形成的肾下腹主动脉使用精氨酸酶阻断剂 基因敲除鼠模型 人体试验 人动脉标本—内皮细胞功能 使用溶栓治疗与L-精氨酸行RCT研究

结 论 暴露于凝血酶的内皮细胞增加了精氨酸酶的活性 NO 产生能力不受影响 局部使用L-精氨酸和精氨酸酶阻断剂可以调整内皮细胞异常功能 结 论 暴露于凝血酶的内皮细胞增加了精氨酸酶的活性 NO 产生能力不受影响 局部使用L-精氨酸和精氨酸酶阻断剂可以调整内皮细胞异常功能 目的性增加NO可能是一个有前途的治疗方法

Questions?

Enhancing Endothelial Function After Thrombosis Vikram S. Kashyap, M.D., F.A.C.S. Associate Professor of Surgery Department of Vascular Surgery The Cleveland Clinic New Horizons in Cardiovascular Treatment (Angiogenesis and other) December 11, 2008

Case 53 y.o. female with acute R leg pain for 4 days. No R leg pulses, faint femoral Doppler signal (Rutherford Class IIA ischemia) PMH—Aortic reconstruction in the early 1990s, R iliac stent 2002 Previous L hip disarticulation secondary to ischemia/infection in the 1998 Heparin allergy—bivalirudin (Angiomax) used for this case

Aortogram L brachial approach Aortography

Power-pulse thrombolysis with Angiojet 20 cm infusion catheter, tPA at 0.5 mg/hr

Lysis check angiogram at 12 hours, repeat PMT 10 cm infusion catheter, Continue tPA

Technique in this case—lysis check 24 hours Thrombolysis uncovers the culprit lesion PTA with a 7 mm balloon Completion arteriography

Culprit lesion

Completion arteriogram

Benefits of Thrombolytic Therapy A “culprit lesion” can be identified—the underlying reason for the acute occlusion Reperfusion injury may be attenuated More options are available - endovascular only: angioplasty+/- stenting - a limited surgical operation (patch angioplasty, endarterectomy) - elective placement of a new bypass can be performed after full work-up (cardiac, vein mapping, etc.)

Despite initial success, sometimes…

Despite initial success, sometimes… Acute arterial thrombosis remains a vexing clinical entity Clinical outcomes following thrombectomy or thrombolysis are occasionally suboptimal secondary to vasospasm or rethrombosis Rethrombosis occurs in up to 20% of patients (thrombogenic intimal surface—thought to cause platelet dependent clot) The effects of thrombus on endothelium have not been completely elucidated

Clinical correlation of thrombus-endothelial interaction Digital subtraction angiogram of the R brachial artery after successful removal of an acute embolus.

Vasospasm = Thrombus-induced Endothelial Dysfunction? Vasospasm relieved by NO donors Vasospasm is hard to treat if there is persistent clot Thrombosis causes endothelial dysfunction in multiple animal models Depressed Nitric oxide (NO) levels are implicated as the source of the endothelial dysfunction

Tissue Bath system

Reil, et al EJVES 2000

Luminal Thrombus

SEM- Post-thrombosis

Factor VIII Immunohistochemistry Normal Post-thrombosis

Thrombolysis Experiments

p=0.007 * p=0.003 *

Effect of L-arginine supplementation on EDR and NO Production Kashyap et al., JVS 2001

Western Blot

Endothelial Dependent Relaxation (EDR) ** ** ** ** * Effect of thrombosis on EDR of rat infrarenal aortic ring segments (IRA). EDR of control (nonthrombosed) rat IRA (n=5) is compared with IRA’s after 1 hr of thrombosis (n=4). *P<0.05, **P<0.001 by two way ANOVA.

EDR after specific arginase inhibition ** ** ** ## ## ** ## # Effect of specific arginase inhibition on EDR of thrombosed IRA. Pretreatment with arginase inhibitor restores the EDR in thrombosed IRA. n=3 in all the three groups. **P<0.001 BEC vs thrombosed IRA analysis of variance. #P<0.01, ##P<0.001control vs thrombosed IRA by two way ANOVA.

Human Studies

EDR measurement N >3

Arginase II

How does thrombus cause endothelial dysfunction? Mechanistic insights Causes endothelial cell loss--No Scavenges NO--Perhaps Decreases NO secretion or diffusion--No Decreases NO production by affecting eNOS--No Affects L-arginine transport via the Y+ channel-- No Increases endogenous vasoconstrictor—No Decreases L-arginine availability—Yes Thrombin increases arginase activity

The Fate of L-arginine

Future Studies In vitro studies (endothelial cells) Thrombin, fibrin exposure with arginase blockade Activated neutrophil effects In vivo studies (rodent models) Arginase blockade in thrombosed infrarenal arterial segments Mouse model, knockouts Human studies Explanted human arteries—endothelial function RCT using thrombolysis and L-arginine supplementation

Conclusions Endothelial cells exposed to thrombin have increased arginase enzymatic activity NO generation capability is unaffected Local administration of L-Arginine or arginase blockade may ameliorate endothelial dysfunction Targeted NO augmentation may prove to be an attractive therapeutic alternative

Questions?