A B

pre neg. pre-miR-92apre neg.pre-miR-92a

n.s.

SM

C a

popt

osis

(OD

450

nm

)

EC

apo

ptos

is(O

D 4

50 n

m)

n.s.

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.)

Figure S1

*

*

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.

*

Figure S2

* * *

C

Integrin α5 DAPI Integrin α5 DAPI

D

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.

LNA-Control LNA-92a

SIRT1 DAPISIRT1 DAPI 50µm

Figure S3Aorta Lung

*

*

miR

-92a

exp

ress

ion

(no

rmal

ised

to s

no20

2)

miR

-92a

exp

ress

ion

(no

rmal

ised

to s

no20

2)

Heart

BA

Cre +miR-92a fl/fl

fl-Genotyping Cre-Genotyping

Primer Primersequence (5‘-3‘) Length Usage

Cre 1084 GCGGTCTGGCAGTAAAAACTATC

100 bpgenotyping of the Cre-

recombinaseCre 1085 GTGAAACAGCATTGCTGTCACTT

IL-2 42 CTAGGCCACAGAATTGAAAGATCT

324 bpinternal control for

genotyping of the Cre-recombinaseIL-2 43 GTAGGTGGAAATTCTAGCATCATCC

28614flp-DIM1 AATGTGTGTCTTAGAGGCCTAGTAGTGAAGAGG fl allele: 362 bp

WT allele: 247 bp

genotyping of the miR-92a flox mice28615flp-DIM1 CACCCCCATTCCTGAAAGCTTATAGC

fl allele(362 bp)WT allele(247 bp)

400 bp

300 bp

200 bp

100 bp

miR-92a fl/+

300 bp

200 bp

100 bp

IL-2(internal control)(324 bp)

Cre(100 bp)

Cre -

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.

A

C

B

Figure S4

*

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.).

*

A

C

B

Figure S5

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.

saline LNA-control LNA-92a P value

Leukocytes (109/L) 4.2±1.1 4.3±0.7 3.9±1.1 n.s.

Platelets (109/L) 440±225 588±88 344±97 n.s.

Segmented Neutrophils (109/L) 0.59±0.23 0.48±0.09 0.32±0.11 n.s.

Monocytes (109/L) 0.13±0.21 0.13±0.06 0.14±0.14 n.s.

Lymphocytes (109/L) 3.53±0.73 3.67±0.59 3.51±1.01 n.s.

Erythrocytes (1012/L) 9.38±0.80 9.48±0.24 9.82±0.41 n.s.

Hemoglobine (g/L) 139±9.4 140±3.8 145±7.6 n.s.

Lactate dehydrogenase (U/L) 594±163 665±138 589±151 n.s.

Cholesterol (mmol/L) 2.33±0.54 1.97±0.05 1.79±0.16 n.s.

Creatinine (µmol/L) 8.8±0 8.8±0 8.8±0 n.s.

Aspartate transaminase (U/L) 163±101 135±68 119±48 n.s.

Alanine transaminase (U/L) 35±15 28±4 34±5 n.s.

Albumin (g/L) 22±3 23±1 20±3 n.s.

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.   

Table S1