Fibronectin is an extracellular matrix protein found only in vertebrate organisms containing endothelium-lined vasculature and is required for cardiovascular development in fish and mice. Fibronectin and its splice variants containing EIIIA and EIIIB domains are highly upregulated around newly developing vasculature during embryogenesis and in pathological conditions including atherosclerosis, cardiac hypertrophy and tumorigenesis, however, their molecular roles in these processes are not entirely understood. We review genetic studies examining functions of fibronectin and its splice variants during embryonic cardiovascular development, and discuss potential roles of fibronectins in vascular disease and tumor angiogenesis.Keywords alternative splicing; angiogenesis; cardiovascular development; endothelium; integrins; pericyte; vascular smooth muscle Blood vessel formation is an essential developmental process required for the survival of all vertebrate organisms [1]. Formation of vascular networks involves extensive interactions of endothelial cells with their environment. These interactions are positively and negatively modulated through the binding of endothelial cell integrins to extracellular matrix proteins, regulating vessel number, size, permeability, vessel sprouting and regression [2,3]. Timely activation and cessation of vessel formation is necessary to enable vascular networks to grow and change with the developing organism during embryogenesis as well as to respond to physiological and pathological instances when formation of new blood vessels is needed in the adult. One of the key steps in the formation of a functional vascular tree is the recruitment and association of endothelial and perivascular cells (also called pericytes and/or vascular smooth muscle cells) [4][5][6]. This review will highlight the emerging role of fibronectin in mediating the crosstalk between endothelial and perivascular cells and will discuss functions of fibronectins gleaned from genetic knockout studies. Functions of fibronectin in vascular morphogenesisFibronectin (FN) is an extracellular matrix protein, essential for blood vessel morphogenesis; it is incorporated between endothelial and perivascular cells. Genetic studies demonstrate the requirement for FN in cardiovascular development: absence of FN leads to embryonic lethal vascular defects [7][8][9]. These defects vary in severity depending on the genetic background indicating the existence of genetic modifier(s) acting in concert with FN during vascular morphogenesis [7]. In FN-null embryos derived from 129S4 strain of mice, endothelial cells are present within the embryo-proper but are not assembled into dorsal aortae, indicating the NIH Public Access

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA and EIIIB are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, we deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, we observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. Our studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.

Fibronectin splice variants containing the EIIIA and/or EIIIB exons are prominently expressed in the vasculature of a variety of human tumors but not in normal adult tissues. To understand the functions of these splice variants in physiological and tumor angiogenesis, we used EIIIB-null and EIIIA-null strains of mice to examine neovascularization of mouse retinas, pancreatic tumors in Rip-Tag transgenic mice, and transplanted melanomas. Contrary to expectations, physiological and tumor angiogenesis was not significantly affected by the absence of either EIIIA or EIIIB splice variants. Tumor growth was also not affected. In addition, the expression levels of smooth muscle alpha actin, believed to be modulated by EIIIA-containing fibronectins, were not affected either. Our experiments show that despite their tight regulation during angiogenesis, the presence of EIIIA or EIIIB splice variants individually is not essential for neovascularization.Angiogenesis is a process whereby new blood vessels develop from the preexisting vasculature (5). This process is crucial to provide oxygen and nutrients to a growing tumor mass; without a vascular supply, tumors fail to grow beyond 1.5 mm in diameter (15,25). Inhibition of tumor angiogenesis leads to inhibition or retardation of tumor growth in animal tumor models (7). The extracellular milieu plays a prominent role in tumor development by supplying factors that can either enhance (e.g., matrix metalloproteinases [12] or vascular endothelial growth factor [2, 28]) or inhibit (e.g., thrombospondin [48] or tumstatin [23]) tumor growth. Extracellular matrix proteins, including fibronectin (FN) and its splice variants, are prominently expressed in and around tumors (10,32,47), but their roles in tumorigenesis are poorly understood. This study investigated the potential functions of FN splice variants, EIIIA and EIIIB, in physiological and tumor angiogenesis.FN is a large modular glycoprotein composed of type I, type II, and type III FN repeats and implicated in numerous cellular processes from cell migration to hemostasis (27, 39). FN-null embryos and embryoid bodies have very low numbers of endothelial cells and develop defective vessels (16, 18), and FNnull embryos die very early in utero from cardiovascular defects (18-20). These observations underscore the importance of FN in vascular development.FN RNA is alternatively spliced at three conserved regions, EIIIA (EDA), EIIIB (EDB), and V (CS-1). Although EIIIA and EIIIB sequences are only 29% identical within a species, interspecies comparisons show that amino acid sequences of EIIIB and EIIIA are highly conserved. For example, the mouse and human EIIIB and EIIIA segments are 100% and 96% identical, respectively. The patterns of expression of these splice variants are also conserved among species. In vivo, EIIIA and EIIIB FN splice variants are expressed around developing blood vessels during embryonic growth (14, 21, 44) when vessels are actively forming and being remodeled, but they are markedly downregulated in adult tiss...