【新文速递】小鼠压力负荷分泌的exosome含有具有功能的AT1R

惜名

[size=11.0045003890991px]Circulation.[size=11.0045003890991px] 2015 Jun 16;131(24):2120-30
Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1Receptors.
AbstractBACKGROUND:

Whether biomechanical force on the heart can induce exosome secretion to modulate cardiovascular function is not known. We investigated the secretion and activity of exosomes containing a key receptor in cardiovascular function, the angiotensin II type I receptor (AT1R).

METHODS AND RESULTS:

Exosomes containing AT1Rs were isolated from the media overlying AT1R-overexpressing cells exposed to osmotic stretch and from sera of mice undergoing cardiac pressure overload. The presence of AT1Rs in exosomes was confirmed by both electron microscopy and radioligand receptor binding assays and shown to require β-arrestin2, a multifunctional adaptor protein essential for receptor trafficking. We show that functional AT1Rs are transferred via exosomes in an in vitro model of cellular stretch. Using mice with global and cardiomyocyte conditional deletion of β-arrestin2, we show that under conditions of in vivo pressure overload the cellular source of the exocytosis of exosomes containing AT1R is the cardiomyocyte. Exogenously administered AT1R-enriched exosomes target cardiomyocytes, skeletal myocytes, and mesenteric resistance vessels and are sufficient to confer blood pressure responsiveness to angiotensin II infusion in AT1R knockout mice.

CONCLUSIONS:

AT1R-enriched exosomes are released from the heart under conditions of in vivo cellular stress to likely modulate vascular responses to neurohormonal stimulation. In the context of the whole organism, the concept of G protein-coupled receptor trafficking should consider circulating exosomes as part of the reservoir of functional AT1Rs.

第一步：
体外发现牵张模型或AngII可以增加HEK293T分泌exosome，阿米洛利可以减少这个分泌。
体内发现，TAC模型后7天，小鼠血清中exosome含量显著增加。
exo的浓度评价采用Nano的方法。
下面做了exo的电镜、western等鉴定exo。

第二步：
发现exosome含有AT1R，并且体外牵张及体内TAC均可以导致AT1R表达升高。

第三步：
用YFP和mCherry等荧光方法证实exosome携带的AT1R可以在细胞见转导，并且具有功能，影响ERK途径。

第四步：
体内实验。首先尾静脉注射普通exo，发现对血压的影响很小，猜测是容量变化导致的；而注射体外牵张exo或TAC血清exo后，小鼠血压升高30%左右，而且在心肌内发现ERK受体被激活。

第五步：
发现体内注射exo后，被心肌及骨骼肌细胞摄取，成纤维细胞并不会摄取。
肠系膜血管也可以摄取exo。

第六步：
证实β-Arrestin参与exo对AT1R的装载过程

第七步：
在β-Arrestin KO小鼠上，发现其血清中的exosome含量无变化，但是AT1R含量显著下降，提示β-Arrestin对体内exo装载AT1R也很重要。
最后，证实在循环中的exo主要来源于心肌细胞。

【一些方法和细节】：
exosome分离用超速离心法，在最后一步离心之前还过了一下0.22um的柱子。
小鼠尾静脉注射exosome数量为1-2.3*10^11（细胞来源）或4.6-5.6*10^13（血清来源），注射24小时后评价小鼠血流动力学。
用western鉴定exosome：和cell相比，exosome高表达Alix、CD63、CD9，不表达Calnexin

华猪猪2005

好文章学习了

惜名

Johnny 发表于 2015-6-22 13:38
有劳啦，惜名兄。相关帖子：http://www.exosome.com.cn/forum.php?mod=viewthread&tid=223&extra=page%3D1& ...

刚才去翻了我的邮箱，的确，NCBI在5月23号online的时候就给了我这篇文献，当时我没注意
昨天晚上一个师弟告诉我这篇文献的，我才注意起来。。。

惜名

Johnny 发表于 2015-6-22 13:38
有劳啦，惜名兄。相关帖子：http://www.exosome.com.cn/forum.php?mod=viewthread&tid=223&extra=page%3D1& ...

啊   你已经写过啦？
我竟然还评论了
这记性真是老年人的节奏啊