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Figure S1. A. B. n.s. n.s. EC apoptosis (OD 450 nm). SMC apoptosis (OD 450 nm). pre-miR-92a. pre neg. pre-miR-92a. pre neg.

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Pre neg

Figure S1

A

B

n.s.

n.s.

EC apoptosis(OD 450 nm)

SMC apoptosis(OD 450 nm)

pre-miR-92a

pre neg.

pre-miR-92a

pre neg.

Figure S1: pre-miR-92a does not influence vascular cell apoptosis in vitro. (A+B) EC and SMC were treated with pre-miR-92a or a pre-miR control (pre neg.; 20nM), and apoptosis was assessed using a TUNEL based ELISA (n=4; P=n.s.)


Pre neg

Figure S2

*

*

*

Figure S2.Downregulation of Itga5, Sirt1 and eNOS by pre-miR-92a. Transfection of EC with pre-miR-92a induced a significant down-regulation of Itga5, Sirt1 or eNOS mRNA levels 24 hours post transfection, as determined by pPCR.


Pre neg

Figure S3

B

A

Aorta

Lung

miR-92a expression

(normalised to sno202)

miR-92a expression

(normalised to sno202)

*

*

Heart

C

*

*

*

D

Integrinα5DAPI

Integrinα5DAPI

SIRT1DAPI

SIRT1 DAPI

50µm

LNA-Control

LNA-92a

Figure S3.Systemic application of LNA-92a leads to down-regulation of miR-92a. (A-C) LNA-92a or an unspecific LNA-control (LNA-Co) were injected into the tail vein of mice at 5mg (A+B) or the indicated concentrations (C), and the expression of miR-92a was determined in the Aorta, the lung or the heart at 7 days after injection. (D) At 14 days after vascular injury, immunohistochemical analysis indicates an increased expression of Itga5 and Sirt1 following treatment with LNA-92a compared to controls. The arrow indicates the luminal side of the neointimal lesion which is partially covered by endothelial cells expressing Sirt1.


Pre neg

Figure S4

A

B

fl-Genotyping

Cre-Genotyping

fl allele(362 bp)

WT allele(247 bp)

IL-2(internal control)(324 bp)

Cre(100 bp)

300 bp

400 bp

300 bp

200 bp

200 bp

100 bp

100 bp

Cre -

Cre +

miR-92a fl/fl

miR-92a fl/+

C

Figure S4: Genotyping of Tie2-Cre;miR-92a(fl/fl) mice. (A, B) Genotypes of miR-92a(fl/+) mice and miR-92a(fl/fl) mice with or without Cre recombinase expression were confirmed by PCR and gel-electrophoresis. IL-2 was used as an internal control. (C) Primers used for genotyping are indicated above.


Pre neg

Figure S5

A

B

*

*

C

Figure S5:miR-92a and miR-17~92a cluster expression in conditional miR-92a KO mice. (A) In total heart tissue, miR-92a expression was slightly reduced as confirmed by qPCR (n=10, *P<0.05) (B) miR-92a expression was abolished in sorted vascular endothelial (VE)-cadherin positive lung EC (n=6, *P<0.05). (C) The expression of the other cluster members miR-17, miR-18a, miR-19a, miR-20a, and miR-19b was not affected within VE cadherin positive lung EC (n=6, P=n.s.).


Pre neg

Figure S6

Figure S6.Overview on the miR-92a-expression following vascular injury. (A) In quiescent vessels, miR-92a is predominantly expressed in EC. (B) During re-endothelialization following vascular injury, miR-92a is highly expressed in activated EC adjacent to the injury and EC that repopulate the injury site during the healing process, thus limiting the regenerative capacity of EC. (C) LNA-based knock down of miR-92a enhances the regenerative capacity of EC, accelerates re-endothelialization of the injured area and thus limits the inflammatory response and subsequent SMC proliferation and vessel narrowing.


Pre neg

Table S1

Table S1: Analysis of peripheral blood after injection of LNA-92a, LNA-control, or saline. Blood samples were collected at 14 days after injury in mice treated with LNA-92a, control LNA, or saline (n=6). The analysis was performed by the department of clinical chemistry at the Justus-Liebig-University, Giessen, based on a biochemical panel and a full blood count.


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