Fibulin-2 and Fibulin-5 Cooperatively Function to Form the Internal Elastic Lamina and Protect From Vascular Injury

Abstract

Objective-Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall. Methods and Results-Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury. Conclusions-Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults. Key Words: internal elastic lamina Ⅲ vascular remodeling Ⅲ development Ⅲ injury T he internal elastic lamina (IEL) is located beneath the endothelium of blood vessels and forms the innermost elastic lamina. The IEL provides elasticity and recoil to the vessel wall, as well as functions as a physical barrier against chemical/mechanical stresses, preventing direct contact of plasma components to smooth muscle cells (SMCs). Several pathological processes have been shown to bring about disruption of the IEL. For example, increased mechanical forces and shear stress associated with angioplasty, enzymatic activation of matrix degrading enzymes in atherosclerosis, and abdominal aneurysms are all associated with disruption of the IEL and progression of vascular disease. [1][2][3] Molecular mechanisms of elastic fiber assembly have begun to be explored by the identification of elastic fiber-associated proteins and their biochemical interactions with elastin or the microfibrillar scaffold. 4 Members of the fibulin family of extracellular matrix (ECM) proteins, particularly fibulin-4 and fibulin-5, play essential roles in elastic fiber development. [5][6][7] We demonstrated that fibulin-5 preferentially binds the monomeric form of elastin, but not polymerized elastin. 8 Others have shown that fibulin-5 accelerates the self-aggregation process of elastin, called coacervation, 9 and fibulin-5 limits maturation of the coacervated elastin. 10 Among 5 of the known fibulins tested, fibulin-2 and fibulin-5 exhibit the highest binding affinity to elastin. 11 Biochemical interaction assays showed that fibulin-2 also binds numerous basement membrane (BM) proteins including nidogen, laminin, and fibronectin. 12 Recently,...

Expression of Versican V3 by Arterial Smooth Muscle Cells Alters Tumor Growth Factor β (TGFβ)-, Epidermal Growth Factor (EGF)-, and Nuclear Factor κB (NFκB)-dependent Signaling Pathways, Creating a Microenvironment That Resists Monocyte Adhesion

“…Consistent with our previous studies (19,28), V3 expression by ASMCs significantly increased mRNA levels of tropoelastin as well as fibulin 5, a component of microfibrils critical in elastic fiber formation (Fig. 1, A and B) (22)(23)(24). Fibulin 1 and fibrillin 1 mRNA levels did not differ between V3-expressing ASMCs and control ASMCs (data not shown).…”

“…Previously, we have demonstrated that expression of the V3 isoform of versican in ASMCs generates an ECM enriched in elastic fibers and that this ECM had reduced capacity to bind monocytes (4,18,19,28). We now show a causal relationship between changes in elastin accumulation and monocyte adhesion such that knocking down tropoelastin or fibulin 5, a component of microfibrils critical for elastic fiber formation (22)(23)(24), partially reverses the inhibition of monocyte adhesion caused by V3 expression. in control and V3-expressing ASMCs cultured for 7 days was detected at mRNA (A) and protein (B) levels using real-time PCR and Western blotting as described under "Materials and Methods."…”

“…Elastic laminae extracted from rat aorta are resistant to leukocyte adhesion and transmigration in vivo and in vitro compared with the collagen-enriched adventitia extracted from the same aorta (46,47). Deficiency in fibulin 2 and fibulin 5 in animals results in disrupted elastic laminae accompanied by an increase in vascular adhesion molecules and tissue factor expression as well as thrombus formation after carotid artery ligation injury (23), suggesting that the elastin-enriched ECM present in healthy aortas protects against vascular injury. Previously, we have demonstrated that expression of the V3 isoform of versican in ASMCs generates an ECM enriched in elastic fibers and that this ECM had reduced capacity to bind monocytes (4,18,19,28).…”

“…Tropoelastin and Fibulin 5 Knockdown-To determine whether monocyte adhesion is dependent on elastic fiber accumulation in ECM deposited by ASMCs, tropoelastin, a soluble monomeric subunit of elastic fiber, or fibulin 5, a scaffold protein of microfibrils critical in elastic fiber formation (22)(23)(24), was knocked down using lentiviral shRNA (Santa Cruz Biotechnology, Inc.). Briefly, control or V3-expressing ASMCs were infected with lentiviral particles containing tropoelastin shRNA sequences (CUG-CAUCCAAAGCUGCUAA, CCACUAUCAACCUGGUUCA, and CACAUGCAGUACUGUAAUC) or fibulin 5 (CCUAUUC-CUGUACGUGUAA, GGUCUUGCCAAGAUGUGAA, and CUCUAUCUCUUGCUGCAUU) at a multiplicity of infection of 0.5.…”

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

“…These fibulins are microfibrillarscaffolding proteins that can promote self-aggregation (coacervation) and cross-linking of tropoelastin. 39,40 Fibulin-2 is increased in neointimal lesions in an atherosclerosis model, and in the dermis of sun-exposed skin, related to elastolysis. 41,42 This suggests that a high proportion of fibulin in elastic laminae may be evidence of elastic fibers that have undergone degradation and repair.…”