FHL2 deficiency enhances SMC proliferation via activation of ERK1/2.

<p><b>A</b>, Serum-starved SMCs were stimulated with or without FCS and treated with or without PD98059 (ERK1/2 inhibitor, 25µM). Cells were pulse-labeled with BrdU to measure DNA synthesis. <b>B</b>, SMCs from WT and FHL2-KO were seeded at equal density. 1 day after seeding, cells were treated with or without PD98059 and cells were counted manually. <b>C–D</b>, Western blot analysis (C) and quantification (D) for pERK1/2 in serum-starved SMCs after overexpression with or with out FHL2 followed by FCS stimulation for the indicated time periods, showing enhanced and prolonged activation of ERK1/2 in FHL2-defeicient SMCs and reduced activation of ERK1/2 in FHL2-KO SMCs after overexpression of FHL2. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p

Deficiency of FHL2 accelerates neointima formation after carotid artery ligation.

<p><b>A, C</b> and <b>E</b>; Representative cross sections of hematoxylin/eosin-stained carotid arteries from WT and FHL2-KO mice ligated for 1 (A), 2 (C) and 4 weeks (E). <b>B, D</b> and <b>F</b>; Quantitative analysis of neointima/media ratio and neointimal area in histological sections from WT and FHL2-KO mice ligated for 1 (B), 2 (D) and 4 weeks (F), revealed increased lesion formation in FHL2-KO mice. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. Three consecutive sections per mouse at each location were employed in the analysis. Lesions were characterized at 1.7, 2.0 and 2.3 mm from the reference point at 1, 2 and 4 weeks, respectively. Values are mean±SEM. *<i>P</i><0.05 for FHL2-KO versus WT mice.</p

FHL2-KO SMCs migrate faster.

<p><b>A</b>, A scratch was made in a confluent layer of serum-starved SMCs that were stimulated with PDGF (20 ng/ml). Images were captured every 10 min using a live cell microscope and representative images at 0, 16 and 32 h are shown. Movies of the movement are in the online supplement. <b>B</b>, Quantitative analysis of SMC migration in the scratch wound assay showing that FHL2-KO SMCs migrated 1.8 fold faster than WT SMCs. <b>C</b>, SMCs were treated with or without PD98059 and cell migration was evaluated using a trans-well assay. Cells were labeled with a fluorescent dye and seeded in the upper chamber. Cell migration was measured as fluorescence after 3 h. <b>D</b>, SMC migration was evaluated using a trans-well assay after knock-down of FHL2 using lentiviral particles encoding shCtrl, shFHL2#1 and shFHL2#2 in WT SMCs. Cell migration was measured as fluorescence after 3 h. Data represent means±SD. *<i>P</i><0.05 for shCtrl versus shFHL2. <b>E</b>, Schematic representation of FHL2 function in the modulation of SMC-rich lesion formation. FHL2 modulates SMC-rich lesion formation by inhibiting proliferation and migration of SMCs via the ERK1/2-CyclinD1signaling pathway.</p

FHL2 deficiency enhances cell proliferation <i>in vivo</i>.

<p><b>A</b>, To assess the extent of proliferation in the vascular lesions, representative sections of injured carotid arteries from WT and FHL2-KO mice ligated for 1, 2 and 4 weeks were immunostained for Ki67. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. <b>B–C</b>, qRT-PCR was performed to assess mRNA expression of Ki67 (B) and PCNA (C) in the ligated vessels from WT and FHL2-KO mice for the indicated time periods. Data are means±SD. *<i>P</i><0.05 for FHL2-KO versus WT mice.</p

FHL2 regulates cell proliferation via modulation of CycinD1 expression.

<p><b>A</b>, SMCs were transduced with lentiviral particles encoding FHL2 and assayed for CyclinD1 mRNA expression, showing that FHL2 inhibits its expression. <b>B</b>, Serum-starved WT SMCs were transduced with lentiviral particles encoding shCtrl, shCyclinD1 #1 and shCyclinD1 #2 and were pulse-labeled with BrdU to measure DNA synthesis. <b>C</b>, The CyclinD1 promoter-reporter plasmid showed higher induction in FHL2-KO SMCs stimulated with FCS than in WT SMCs. The ERK1/2 inhibitor PD98059 partly reduces this induction. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p