Vascular remodeling in fbln-5^–/– mice after 28 days of ligation. Representative photos of transverse sections at indicated levels stained with hematoxylin/eosin. Wild-type (WT) vessels are shown in Left. Note an increased diameter of the vessel with a thickening of adventitia (a) and thinning of the medial layer (m) in the fbln-5^–/– vessel. Thrombus formation (thr) and neovascularization (nv) were observed in the fbln-5^–/– mouse. ni, neointima. (Bars, 40 μm.)

Morphometric analysis of ligated arteries after 28 days of ligation. (A) EEL perimeter of the ligated and contralateral vessels. (B) IEL perimeter of the ligated and contralateral vessels. (C) Thickness of the medial wall of ligated and contralateral vessels. (D) Intima/media area ratio. Wild-type (n = 5), fbln-5^–/– vessels (n = 6), and eln^+/– (n = 5) vessels were examined at level 400. Three measurements were taken for each parameter from each section. *, P < 0.05.

Up-regulation of fbln-5 and eln in the neointimal lesion. In situ hybridization of transverse sections of wild-type carotid arteries before ligation (A–C) and 28 days after ligation (D–F). (A and D) Bright-field images showing hematoxylin counterstaining. The area surrounded by a red line in D and a white line in E and F are the neointima (ni). Sections were hybridized with cRNA probes for fbln-5 (B and E) and eln (C and F). Note the up-regulation of fbln-5 (E) and eln (F) in the neointima. Arrow in E indicates fbln-5 expression in the adventitia. m, media; a, adventitia. (Bars, 80 μm.)

Reduced vessel extensibility in the fbln-5^–/– carotid artery. (A) Pressure-diameter curve. Three measurements were taken for each mouse. At pressures >100 mmHg, there was a significant difference in the outer diameter between wild-type (n = 9) or fbln-5^+/– vessels (n = 7) and fbln-5^–/– (n = 7) vessels. (B–D) Hart's staining of the transverse sections of ligated arteries. Neointima and vascular remodeling were compared between wild-type (B), fbln-5^–/– carotid arteries (C) and eln^+/– (D) carotid arteries after 28 days of ligation. Both fbln-5^–/– carotid arteries and eln^+/– carotid arteries exhibited decreased extensibility (see A and ref. 21); however, there was no outward remodeling or increased neointima formation in the eln^+/– vessel. Level shown is 400. (Bars, 40 μm.)

Electron micrographs of neointimal lesions and in vitro analysis of fbln-5^–/– SMCs. (A and B) Representative photos of electron micrographic observations of ligated arteries from wild-type (A) and fbln-5^–/– vessels (B). Note that elastic fibers are continuous and firm in the wild-type vessel (arrow), whereas elastic fibers are irregular and discontinuous in the mutant (double arrows). (Bars, 0.5 μm.) (C) Cell proliferation assays of wild-type (WT) and fbln-5^–/– SMCs. WT cells cultured in the serum free condition for 24 h were assigned a value of 1. A fold increase in the indicated condition was shown to allow comparison between the genotype. Data are from three independent experiments performed in triplicate. *, P < 0.05. (D) Modified Boyden chamber assays. Numbers of WT and fbln-5^–/– SMCs that migrated to the filter with or without PDGF-BB (50 ng/ml) were counted. Data are from three independent experiments performed in duplicate. n.s., not significant. (E) Cell proliferation assays of fbln-5^–/– SMCs infected with the adenovirus expressing GFP (Ad-GFP) or fbln-5 (Ad-Fbln-5). y axis indicates absorbance at OD₄₉₀ after 24 or 48 h of culture in PDGF-BB (50 ng/ml). Overexpressions of fbln-5 inhibited proliferation of SMCs. Data are from two independent experiments performed in triplicate. (F) In vitro scratch assays of fbln-5^–/– SMCs infected with the adenovirus expressing GFP or fbln-5. The distance from the left border of the scratch injury was measured after 6 h of injury. Six to eight measurements were made for each condition. Data are from two independent experiments.