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)• 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- 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

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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

组织浴槽系统

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Figure 1. (EDR). 4乙酰胆碱引起的内皮依赖性舒张 内膜暴露于血栓下 小时可显, EDR .著降低动脉的舒张效应 单纯缺血不引起 降低

内皮依赖性舒张与乙酰胆碱关系

(B)(A)

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

血栓形成 vs. EDR溶栓治疗的

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L- 精氨酸对 EDR和 NO 的影响

Kashyap et al., JVS 2001

Western Blot

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.

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内皮依赖性舒张 (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

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使用特异性精氨酸抑制酶后 EDR

人体试验

EDR 测量

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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 )

– 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

• 体外试验 (啮齿动物模型 )– 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• 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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Tissue Bath system

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Figure 1. Endothelial dependent relaxation (EDR) response to acetylcholine. Exposure of endothelium to thrombus for 4 hours significantly decreases vasorelaxation of the arterial segment. Ischemia alone does not decrease EDR. Relaxation is expressed as mean percent relaxation after contraction to 75% F-max with 10-5 mol/L phenylephrine.

Endothelial Dependent Relaxation to Acetylcholine

(B)(A)

Reil, et al EJVES 2000

Luminal Thrombus

SEM- Post-thrombosis

Factor VIII Immunohistochemistry

Normal Post-thrombosis

Thrombolysis Experiments

EDR for Thrombosis vs. Thrombolytic Regimens

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Effect of L-arginine supplementation on EDR and NO Production

Kashyap et al., JVS 2001

Western Blot

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.

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Endothelial Dependent Relaxation (EDR)

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.

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EDR after specific arginase inhibition

Human Studies

EDR measurement

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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?