Mediator Med23 deficiency in smooth muscle cells prevents neointima formation after arterial injury

Vascular smooth muscle cells (VSMCs) that line the arteries and veins, are able to increase their proliferation rate greatly following vessel wall injury and in atherogen-esis. Changes in VSMC proliferation rate and differentiation state have been proposed as required procedures of injury repair. The regulation of the balance between proliferation and differentiation is of key importance in vascular biology. Our previous works have shown that Mediator MED23 subunit restricts smooth muscle cell lineage development and promotes growth related gene expression 1 . Recently, we investigated the roles of Med23 in VSMC proliferation and differentiation as well as injury-induced neointima formation by generating and examin-ing the smooth muscle speci ﬁ c Med23 -knockout ( Med23 sm − / − ) mice. Our results showed that Med23 reg-ulates the balance of VSMC growth and differentiation in mouse aorta. The Med23 sm − / − mice showed enlarged lumen and impaired contractility of aorta. RNA pro ﬁ ling of the aorta samples revealed that gene expression pro-grams switching between proliferation and differentiation upon the presence or absence of Med23. More impor-tantly, we found that Med23 de ﬁ ciency prevented neointima formation after vascular injuries, through repressing VSMC proliferation. Collectively, our data demonstrate that Med23 has positive effects on VSMC plasticity and plays a novel pathological role in vascular injury induced neointima formation through promoting the proliferation and growth of VSMCs, thus providing a novel mechanism understanding the VSMC plasticity and related vascular diseases. Previously we have demonstrated that Mediator

Med23 deficiency in smooth muscle cells prevents neointima formation after arterial injury. a Immunoblot of Med23 in A7R5 cells after viral-mediated siRNA knockdown of Med23. b-c EdU assay in siMed23 and control A7R5 cells. d-e Wound healing assay of siMed23 and control A7R5 cells in the presence of either 20% FBS (d) or 20 ng/ml PDGFbb (e). f Immunoblot of Med23 in vascular SMCs isolated from the aorta of Med23 sm-/mice. g Transwell assay in vascular SMCs isolated from the aorta of Med23 sm-/mice. Data are presented as mean ±SEM.

Animals and treatment
Med23 Floxed mice and smooth muscle aortic alpha-actin (Acta2)-Cre transgenic mice have been described previously 1,2 . Briefly, exons 5-7 were flanked by two loxP sites and this plasmid was delivered to the embryonic stem cells from Sv129 strain for homologous recombination mediated gene targeting. Smooth muscle specific Med23 knockout mice (SMKO, Med23 f/f ; Acta2-Cre + ) was obtained by crossing female Med23 f/+ mice with male Acta2-Cre + mice. Littermates of the genotype Med23 f/f ; Acta2-Creor Med23 +/+ ; Acta2-Cre + were used as controls. All mice were maintained in a specific pathogen-free facility in the Shanghai Laboratory Animal Center (Chinese Academy of Sciences) and were genotyped by PCR analysis of genomic DNA. For

Protein isolation and Immunoblot analyses
Total protein extracts were prepared by homogenizing tissue (IKA) or lysing in RIPA solution (50 mM Tris, 10 mM EDTA, 150 mM NaCl, 0.25% Deoxycholic acid, 0.1% SDS, 2% NP-40 substitute, 0.01% Sodium azide). Protein lysates were separated on SDS-PAGE gels and transferred for 1 hour at 4°C on to a nitrocellulose membrane (BioRad). After blocking for an hour in 5% dry milk, membranes were incubated overnight at 4°C with the primary antibody in blocking buffer. Blots were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson lab) for 1 hour at room temperature. GE ImageQuant LAS 4000 was used to detect protein signals after the incubation with an enhanced chemiluminescence reagent (Thermo Fisher Scientific). The antibodies were listed as below: Med23 antibody was from BD Biosciences. GAPDH,β-actin, γ-tublin antibodies were from Santa Cruz Biotechnology. CNN1 and Acta2 antibodies were from Lifespan. Akt, p-Akt, Erk, p-Erk and PCNA antibodies were purchased from Cell Signaling Technology.

Histological analysis
Hearts and vessels were dissected from the mouse and were fixed overnight in 4% PFA/PBS. The fixed samples were embedded in OCT tissue tek (Sakura) or were dehydrated and then embedded in paraffin. Sections were cut between 5 and 10 µm thickness by using Leica microtome. Sections for histology were stained with kits for hematoxylin and eosin (Sigma-Aldrich) staining following the manufacturer's instructions.

Immunofluorescence analysis
Cells were fixed overnight in 4% PFA/PBS and blocked in 10% Goat serum/PBS before incubation with antibodies. The antibodies were diluted in a 2% bovine serum albumin solution in PBS. CNN1 antibody was purchased from Lifespan.
Immunofluorescence images were acquired using Leica SP8 confocal microscope.

RNA isolation and semi-quantitative real time PCR
Total RNA was prepared using the Trizol Reagent (Sigma-Aldrich). The first strand cDNA was generated using PrimeScript RT Reagent Kit (Takara) according to the manufacturer's instructions. Real-time PCR was performed in triplicate using a SYBR Green PCR master mix in an Eppendorf Mastercycler. All values were normalized to the level of EF2 mRNA, which is constitutively expressed and not changed during the experiments. The primer sequences are listed as below: Primer sequences used for real-time PCR analysis:

Echocardiography
Adult mice were anesthetized with 5% isoflurane for 15 seconds and maintained at 0.5% isoflurane during the procedure. Echocardiography was performed by using a were determined from the M-mode tracing.

Primary mouse vascular smooth muscle cells (VSMCs) Cultures
VSMCs were isolated from 6-to 8-wk-old mice as described 3 . In brief, aortas were excised from the mouse. The fat, adhering connective tissues and endothelial cells were removed under a dissecting microscope. The vessels were cut into small pieces (1 mm 2 ) and then covered by a glass cover slip. The cells were cultured in DMEM/F-12 media (Thermo Fisher Scientific) with 20% FBS at 37°C in a humidified atmosphere containing 5% CO2. Cultured aortic smooth muscle cells were routinely used between passages 2 and 3. For PDGFbb stimulation, 20 ng/ml human recombinant PDGFbb (R&D Systems) was added to culture media for the indicated times.

Cell proliferation assay
Cells were seeded at 2 × 10 4 cells per well in 12-well plates. Cells were allowed to proliferate for an additional 4-6 days. Cell number was measured every other day after plating by a CYTORECON cell counter (GE Healthcare). Experiments were done in triplicate.

EdU incorporation assays were performed using EdU Cell Proliferation
Detection Kits (Sigma Aldrich). Briefly, the Cells were seeded at 2 × 10 4 cells/well and quiesced for 48 h. Then, the cells were incubated in EdU labeling reagent and immunofluorescence assay was used for the detection of EdU incorporated into cellular DNA following the manufacturer's instructions. Total cellular nuclei were stained with DAPI. The results were reported as a percentage of EdU-labelled cells to the total amount of cells.

Cell migration assays
For the scratch wound healing assays in VSMCs and A7R5 cells, the confluent monolayer of cells was scratched with a P200 pipette tip. Cellular progress was

Femoral artery wire injury model
Femoral arteries in mice were subjected to transluminal wire injury as described 4 .
Briefly, 12 weeks male mice were anesthetized with isoflurane during surgery. The right femoral arteries were exposed using blunt dissection. A guide wire (0.38 mm in diameter; Cook, Bloomington, IN, USA) was inserted into the arterial lumen through an arteriotomy and left in place for an additional 3 min to denude the artery. Sham surgeries were performed on the left femoral arteries of the same mouse as the control.
Mice were allowed to recover and femoral arteries were harvested 4 weeks after the injury. Sections were stained with hematoxylin and eosin (H&E). The intimal area was defined as the area encircled by the internal elastic lamina minus the lumen area.
The medial area was calculated as the area encircled by the external elastic lamina minus the intima area. The intima/media (I/M) ratio was calculated as the intimal area divided by the medial area. Restenosis (%) were defined as the intimal area divided by the area encircled by the internal elastic lamina.

Measurement of isometric force in aortic rings
Measurement of isometric force in mouse aortic rings was performed as