Roles of fibronectin isoforms in neonatal vascular development and matrix integrity

Fibronectin (FN) exists in two forms—plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.

Fibronectin (FN) exists in two forms-plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.

Author summary
Fibronectin is a protein that exists in vertebrates in two distinct forms: one present in the blood and the other in blood vessel walls. In mammals, fibronectin is important for the development of blood vessels before birth, but whether it is continuously required for blood vessel homeostasis from birth to adulthood is unknown. We present important a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Fibronectin (FN) is an abundant and ubiquitously expressed protein in the extracellular matrix (ECM) of various connective tissues as well as in blood of vertebrates [1,2]. Even though coded by a single gene [3], it exists in multiple isoforms as a result of alternate splicing [4]. Each monomer of FN consists of three types of repeating units, FNI, FNII, and FNIII domains ( Fig  1A) [5]. Alternative splicing occurs within the central array of FNIII repeats, leading to either inclusion or exclusion of the FNIII extra domain-A (EDA) and extra domain-B (EDB) domains ( Fig 1A). Another region of alternative splicing occurs towards the C-terminus of this array, the V region [6]. FNs occur in two principal forms, the soluble plasma FN (pFN) circulating in the blood and the cellular FN (cFN), which polymerizes into insoluble fibers in the ECM of tissues, including blood vessels [7]. pFN is synthesized exclusively in the liver by hepatocytes [8,9] and shows a relatively simple splicing pattern lacking the EDA and EDB domains, although forms with and without the V region exist [10]. The soluble pFN circulates in blood at a high concentration of about 0.6 mg/mL in mice and about 0.3 mg/mL in humans [11,12]. The cFN consists of a much larger and more heterogeneous group of isoforms, with either EDA, EDB, or both domains present [4,13].
The functional relevance of FN in critical physiological and pathological developmental processes is validated by severe defects in mice globally lacking the FN gene (Fn) [30], including vascular, mesodermal, and neural tube defects leading to death around embryonic day 9.5. Deletion of both EDA and EDB exons (present only in cFN) from Fn in mice also results in embryonic lethality with incomplete penetrance, displaying multiple embryonic cardiovascular defects [31]. Although pFN knockout (KO) mice have a normal life expectancy [32], it is a critical player in various pathologies. pFN supports neuronal survival and reduces infarct size following cerebral ischemia [32]. It is also important in thrombosis, regulating thrombus stability and growth [33,34]. pFN worsens the course of atherosclerosis but promotes the formation of protective fibrous cap, which prevents plaque rupture [35]. In humans, dysregulation of FN   . RT-PCR of Fn from total RNA isolated from the aortic media layer, confirming deletion of Fn in the cFN iKO post-tamoxifen injection (bottom panel). Gapdh was used as a control (n = 3). (C) Genotype of the LacZ Reporter-Cre mouse strain (top panel). β-galactosidase (β-gal) staining of aortae from tamoxifen injected Reporter-control (R26(fl/fl))and Reporter-Cre (R26(fl/fl); Sma-Cre-ER T2 /+) mice (middle panel) (n = 3). Note positive blue staining in the Reporter-Cre (white arrowheads) and the absence of staining in the Reporter-control (black arrowheads) aortae. Immunostaining for α-SMA in control and in cFN iKO mice (bottom panel). Note that the expression of α-SMA in the tunica media is at the same topological location as the β-galactosidase staining in the Reporter-Cre, confirming that the induced Cre recombinase is specifically active in aortic SMCs (n = 5). Scale bar represents 50 μm and asterisk ( Ã ) denotes aortic lumen in (C). cFN, cellular fibronectin; cFN iKO, cellular fibronectin inducible knockout; CreERT2, Cre recombinase fused with the estrogen receptor tamoxifen-binding domain; FN, fibronectin; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; pFN, plasma fibronectin; RGD, Arg-Gly-Asp; RT-PCR, reverse transcriptase polymerase chain reaction; R26(fl/fl),Reporter-control; R26(fl/fl);Sma-Cre-ER T2 /+, Reporter-Cre; SMC, smooth muscle cell; α-SMA, smooth muscle α-actin; β-gal, β-galactosidase. studies highlight the importance of FN during embryonic vascular development and in postnatal diseases associated with the cardiovascular system.
The aim of this study was to elucidate the role of FN isoforms during postnatal vascular development, with a focus on the elastic fiber-rich tunica media. To specifically delete cFN in smooth muscle cells (SMCs), the most prevalent cells in the tunica media, we employed tamoxifen-dependent Cre recombinase under a smooth muscle specific promoter. To discern the role of pFN in vascular development, a liver-specific Cre mouse was used. Our study demonstrates the importance and function of FN isoforms during the development of the aorta in the early postnatal period.

Results
It is well documented that FN is crucial in the development of blood vessels during embryogenesis [30,43,44]. The vascular wall further undergoes extensive development during the early postnatal period, including an expansion in total wall volume and wall thickness accompanied by a doubling of SMC number and an increase in matrix volume [45]. The role of FNs during this phase is not known, and thus we addressed it in this study with a focus on cellular and matrix integrity of the aortic vascular media.

Efficacy and specificity of the inducible cellular FN deletion in the aortic media
We have generated a conditional cFN inducible KO (cFN iKO) mouse model that allows postnatal, tamoxifen-inducible deletion of cFN in SMC, the major cell type in the aortic media. Mice containing Fn flanked by loxP sites (Fn(fl/fl)) were crossed with an established transgenic mouse line (Sma-Cre-ER T2 /+), in which the expression of the tamoxifen-dependent Cre-ER T2 recombinase is under the control of the mouse smooth muscle α-actin (α-SMA) promoter. After several breeding rounds, the cFN iKO genotype (Fn(fl/fl); Sma-Cre-ER T2 /+) was produced ( Fig 1B; top panel) [32,46]. The deletion of Fn, induced by intragastric tamoxifen injection (every day from P1-P3), was confirmed at P8 by a Fn-specific reverse transcriptase polymerase chain reaction (RT-PCR) of RNA isolated from the aortic media of the cFN iKO compared to control mice ( Fig 1B; bottom panel). To further validate the efficiency and specificity of Cre recombinase expression in the Sma-Cre-ER T2 /+ mice following early postnatal tamoxifen injection (P1-P3), a Reporter-Cre mouse strain (R26(fl/fl);Sma-Cre-ER T2 /+) was generated ( Fig 1C; top panel). In this mouse, the lacZ reporter gene coding for β-galactosidase is only transcribed in cells and tissues where Cre recombinase is expressed. Intense β-galactosidase staining of the aortic media harvested from tamoxifen-induced Reporter-Cre mice, but not of the tamoxifen-induced Cre negative Reporter-Control (R26(fl/fl))mice, confirmed an efficient and specific expression of the Cre recombinase in the tunica media, identified by α-SMA staining (Fig 1C; middle and bottom panel).

Postnatal deletion of cellular FN in aortic media leads to a mild phenotype
Unexpectedly, tamoxifen-injected cFN iKO mice demonstrated a normal life span in the absence of any gross phenotype. To evaluate aortic tissue integrity of these mice, we performed histological analyses of the aortic wall (Fig 2A-2H). FN guides assembly of several ECM proteins in cell culture models, but whether FN plays a similar role in vivo and what the contributions of the individual FN isoforms are have not been determined. Therefore, one major focus of this study was to analyze the contributions of FN to ECM assembly in the postnatal mouse aorta. FN acts as a scaffold for the assembly of collagen in cell culture [15][16][17][18][19]. However, deleting Fn in the tunica media of the cFN iKO mice led to an unexpected increase of collagen  (Fig 2A-2C). Collagen deposition reverted back to control levels at 8 months of age (Fig 2E-2G). Despite the fact that cFN was deleted only in the tunica media, we observed differences in the adventitial collagen organization based on Masson's trichrome staining and quantification of picrosirius red staining. Up to 3 months of age, the tamoxifen-treated cFN iKO showed a loose collagen deposition in the adventitia (Fig 2B; triangles and Fig 2D), whereas at 8 months, the pattern reverted to a denser collagen matrix as compared to the control (Fig 2F; triangles and Fig 2H). FN also promotes the assembly of fibrillin-1-containing microfibrils in cell culture [20,21]. Immunostaining of aortic sections for fibrillin-1 showed no changes in the aorta of cFN iKO as compared to the control (S1A and S1B Fig). Next, we investigated if postnatal deletion of cFN affects the formation of elastic lamellae in the aortic wall. The elastic lamellae were relatively normal, although they appeared to have more disruptions than the control mice (S1C and S1D Fig). However, quantification of breaks and forks normalized to the area did not show statistically significant differences (S1E and S1F Fig). Overall, postnatal deletion of cFN in medial SMCs led only to minor changes in the matrix organization that did not affect the overall health of the mice.

Specific uptake of pFN in the vessel wall
Because FN is crucial for vascular development during embryogenesis [30], a much stronger phenotype was expected for the deletion of FN in the aortic wall during early postnatal development. To exclude the possibility that these minor phenotypes in cFN iKO mice were potentially a consequence of incomplete deletion of cFN in the aortic media layer, we immunostained aortic sections using a cFN-specific antibody that recognizes the alternatively spliced EDA domain, which is absent in pFN. This analysis clearly demonstrated that the EDA-containing cFN levels were very low, as expected, from an effective Fn deletion (see Fig 1B bottom panel) for the cFN iKO, as compared to the controls (Fig 2I-2K). However, staining with an anti-mouse FN antiserum that recognizes total FN (pFN and cFN) surprisingly revealed the presence of FN in the aortic media of the tamoxifen-induced cFN iKO, similar to the control (Fig 2L-2N). These data led to the hypothesis that pFN from blood can transfer to the aortic wall, which in turn rescues a potential severe phenotype upon cFN deletion in the cFN iKO mice.
To test this hypothesis, exogenous pFN labeled with a fluorophore (Oyster-680), was injected intraperitoneally daily from P8 to P10 into tamoxifen-treated (P1-P3) control and cFN iKO mice, and mice were sacrificed and analyzed at P11. A strong fluorescent red signal in the ascending as well as in the descending aorta of both the control (Fig 3A and 3C) and cFN iKO mice (Fig 3B and 3D) demonstrated an efficient transfer of Oyster-680-labeled pFN into the vessel wall. Within the vessel wall, the Oyster-680-labeled pFN localized to the elastic fibers (merged images in Fig 3A-3D), which were visualized label-free by detection of the elastin autofluorescence. To test if the transfer of intraperitoneally injected pFN to the vessel wall iKO and control mice (n = 4-8). Parameters quantified are total collagen intensity (Total), mean collagen pixel intensity (Mean), and area of collagen distribution in μm 2 (Area) in the tunica media/adventitia. Note that the changes in collagen deposition observed at 3 months (A-D) revert back at 8 months (E-H). Underlying data are provided in S1 Data. (I and J) Immunofluorescence staining of the aortic wall with anti-EDA FN antibody (specific for cFN) shows an effective deletion and reduction of cFN in cFN iKO mice (I) as compared to control mice (J) (arrowheads) (n = 3). is specific for pFN, an Oyster-680-labeled 250 kDa polysaccharide dextran (similar molecular mass as a pFN monomer) was injected in an identical regimen as used for the fluorophorelabeled pFN. Contrary to the findings with injected pFN, no labeled dextran was observed in the aortic media of ascending and descending aortae of control (Fig 3E and 3G) and cFN iKO (Fig 3F and 3H) mice. As an additional control to exclude the possibility of vascular leakiness, injected Oyster-680 alone did also not transfer into the vessel wall ( Fig 3I-3L), despite a much smaller molecular mass (approximately 2 kDa) compared to pFN. These results clearly show that pFN efficiently translocates to the aortic wall in the absence of vascular leakiness.

Deletion of pFN does not affect the vascular wall
To assess if pFN then plays a more important role in maintaining the cellular and matrix integrity of the vasculature, we generated pFN KO mice by crossing the Fn(fl/fl) mice with transgenic mice expressing the Cre recombinase under the control of the albumin promoter (Alb-Cre) active in hepatocytes [32,47], the exclusive site for pFN synthesis (Fig 4A). The Alb-Cre/+ strain has been used successfully by others to generate liver-specific KO mice [32,47,48]. The resulting Fn(fl/fl); Alb-Cre/+ strain lacks expression of pFN from P3 onwards [49], and the absence was confirmed by immunoblotting of plasma obtained from pFN KO and control mice at P30 (Fig 4B). No gross or histological phenotypes were observed in the aorta of pFN KO mice that would affect cellular or matrix integrity of the vessel wall (S2 Fig). These results together with the observation of pFN transfer into the vascular wall suggested that pFN has an important role in safeguarding the vasculature in the absence of cFN; however, pFN is not needed for vascular integrity and function in the presence of cFN.

Double knockout of cFN in SMC and pFN causes postnatal lethality
To further study the role of pFN and its functional interaction with cFN in the vasculature, we produced double knockout (dKO) mice deficient in pFN and cFN with the Fn(fl/fl); Sma-Cre-ER T2 /+; Alb-Cre/+ genotype ( Fig 4C). These mice lack pFN from P3 onwards and are additionally inducible by tamoxifen to delete cFN in α-SMA expressing tissues. When both pFN and cFN are deleted in the tamoxifen-injected dKO (P1-P3 tamoxifen injection), the mice die postnatally between P3 and P20 (median age of death, P11.5). The Mendelian ratio was significantly reduced from the expected 25% to 4.23% by P30 ( Table 1). Necropsy of dead dKO mice showed hemorrhages in the thoracic cavity (Fig 4D), indicating a severe phenotype in the vasculature due to combined loss of pFN and cFN in SMCs. Absence of any phenotypes in nontamoxifen-injected Fn(fl/fl); Sma-Cre-ER T2 /+; Alb-Cre/+ mice eliminated the possibility of Cre toxicity. These mice had a normal life span and were used as breeders to maintain the mouse colony. Tamoxifen-injected Cre-negative Fn(fl/fl) mice also survived normally, demonstrating the absence of tamoxifen side effects. All other analyses throughout this study were performed on mice dissected immediately after cardiac perfusion to optimally preserve the tissue structure. Immunostaining on dissected dKO aortic wall tissues using a FN antibody that recognizes both isoforms showed a complete absence of pFN and cFN in the media layer, as compared to the control (Fig 4E; triangles). Similarly, immunostaining with an EDA-specific antibody confirmed an effective deletion of cFN in the dKO (Fig 4F; triangles). These results validated that both pFN and cFN are normally present in the aortic wall. The postnatal lethality observed in the dKO suggested that at least one of the two FN isoforms is required for maintaining vessel wall integrity and function.

FN isoforms are important for maintaining cellular and matrix integrity of the vasculature
We further investigated the dKO mice for the consequences of complete FN deficiency on aortic wall development in the early postnatal period. We choose P8 for this analysis, because most dKO mice survive until this time point (median age of death, P11.5). To determine cell integrity, we stained aortic cross sections from all four tamoxifen-injected mouse strains with hematoxylin and eosin. SMCs in the aortic walls from dKO mice appeared to be disorganized,  (Fig 5A; triangles). Frequently, but not always, we observed thinning of the aortic wall in the dKOs, as compared to the other strains analyzed. For a more detailed analysis, we performed transmission electron microscopy on aortic sections. The tunica media of dKO mice appeared disorganized, with curled and irregular elastic lamellae (Fig 5B; S3 Fig; yellow triangles) and irregular shaped cell nuclei (Fig 5B; S3 Fig; red triangles) compared to control and single KO mice.
The affected elastic lamellae in the dKO led us to further investigate the consequences of Fn deletion on matrix integrity of the vascular wall, with a focus on ECM proteins important for elastogenesis. We analyzed FBN-1 in the vessel walls of the KO mice, because it constitutes the backbone of microfibrils [50], which serve as a scaffold for elastin deposition and it is dependent on FN in cell culture studies [20,21]. Immunostaining clearly showed a reduction of FBN-1 in both the single KOs (cFN iKO and the pFN KO) and the dKO, as compared to the control, demonstrating that both pFN and cFN contribute to the deposition/assembly of FBN-1 in vivo (Fig 6A). Quantitative analysis revealed that the levels of deposited/assembled FBN-1 are lowest in the dKO and that cFN supports more FBN-1 deposition/assembly than pFN ( Fig  6E). pFN and cFN were similarly important for the deposition/assembly of FBLN-4, albeit it was reduced to the same level in all strains (Fig 6B and 6E). For LTBP-4, which is also known to be dependent on FN for its assembly and is important in elastogenesis [25,51], cFN was important for its deposition/assembly but not pFN (Fig 6C and 6E). To determine if loss of FN affects tropoelastin deposition, either directly or indirectly by affecting other ECM proteins, sections were immunostained with a tropoelastin antibody and mature elastic lamellae were visualized by autofluorescence in aortic sections of all mouse strains analyzed. Even though the mature elastic lamellae appeared relatively normal at this magnification (whereas electron microscopy shows disorganized elastic lamellae in Fig 5B and S3 Fig), immunostaining with tropoelastin antibody was much reduced in sections from cFN iKO and dKO mice, compared to the control and the pFN KO mice (Fig 6D and 6E). Altogether, these data suggest a role of cFN in new tropoelastin deposition and in elastin assembly in vivo. To analyze if this immunostained images of aortic walls from P1-P3 tamoxifen-injected control mice and dKO mice at P8 with antibodies against total FN (pFN and cFN) (E) and against EDA FN (specific for cFN) n = 4 (F). Blue staining represents nuclei counterstaining with DAPI. Quantification of the red FN signals is shown at the bottom of each figure and the underlying data are provided in S1 Data. Note the significantly reduced levels of both total FN and EDA FN in the media (arrowheads). Lumen is indicated with an asterisk, and scale bar represents 50 μm in (E) and (F   Fig 6A-D (n = 3). Underlying data are provided in S1 Data. (F) Biochemical analysis was performed after NaOH extraction of aortae of all four mouse strains dissected at P8 (n = 6). Note the decrease in elastin content without changes in elastin cross-links in the dKO. Underlying data are provided in S1 Data. Ã represents a p-value of 0.05, ÃÃ a tropoelastin reduction affects the total elastin deposited in the aorta, we biochemically determined the elastin content and cross-links after NaOH extraction of P8 aortae. Surprisingly, the elastin per total protein was significantly reduced in dKO mice, whereas no significant changes occurred in the cFN iKO and pFN KO mice, as compared to the control (Fig 6F). The crosslinks desmosine (Des) and isodesmosine (Isodes) per total elastin content and their ratio were not altered between the mouse strains ( Fig 6F). mRNA expression analysis of the analyzed ECM components using quantitative polymerase chain reaction (qPCR) demonstrated elevated Fbn1 mRNA levels in pFN KO aortae, which might represent a mechanism of the tissue to rescue reduced FBN-1 deposition/assembly (S4 Fig). mRNA expression of Fbln4, Ltbp4, and Eln was not altered, demonstrating that FN isoforms exclusively impact the deposition/ assembly of these proteins (S4 Fig). In summary, these data show that the complete loss of FN impacts early postnatal vascular development by affecting both cellular and matrix integrity of the vessel wall. The results also demonstrate for the first time that FN is important for the deposition of various elastogenic proteins in vivo and its isoforms have both common and distinct roles in the organization of elastogenic proteins in the aortic media.

Similar roles of FN isoforms in ECM assembly
The in vivo data indicated a functional relationship between pFN and cFN in matrix organization. To further study how the two isoforms interact with each other and contribute to its own assembly and deposition of other ECM proteins, a cellular model was developed. vSMCs from Fn(fl/fl); Sma-Cre-ER T2 /+ mouse aortae were treated with 4-hydroxytamoxifen (4-OH Tamox) for 3 days to induce cFN deletion. As controls, the cells were treated with ethanol (EtOH; solvent of 4-OH Tamox). RT-PCR showed an effective deletion of Fn upon treatment with 4-OH Tamox as compared to the EtOH-treated control (Fig 7A). Immunoblotting of the serum-free conditioned medium collected from these cells using specific FN antibodies showed an effective reduction of cFN in 4-OH Tamox-treated cells, compared to the EtOHtreated cells (Fig 7B). Indirect immunofluorescence of SMCs cultivated in FN-depleted medium additionally demonstrated the absence of a FN network in 4-OH Tamox-treated cells versus a fully developed network in the EtOH-treated cells (Fig 7C). This result was further confirmed by the absence of FN in deoxycholate (DOC)-soluble and -insoluble fractions extracted from 4-OH Tamox-treated cells (S5 Fig). Deletion of Fn prevented the assembly of FBN-1 ( Fig 7D) and of FBLN-4 ( Fig 7E). This experimental model was then expanded by combining it with plasma obtained from control and pFN KO mice to test the functional relationship between pFN and cFN in matrix organization. The cells were tested under the following conditions: (1) cFN+/pFN+; (2) cFN+/pFN−; (3) cFN−/pFN+; and (4) cFN−/pFN− (for details, see Materials and methods). Indirect immunofluorescence under these conditions and fiber length quantification showed that either cFN or pFN alone (Fig 7F; arrowheads) can form fibers, corroborating the functional in vivo data that showed that either one of the individual FN isoforms is sufficient for maintaining the vascular integrity. FN assembly is also enhanced in the presence of both isoforms (Fig 7F; arrows and S1 Table), suggesting that the two isoforms can assemble together to form matrix fibers. Similarly, each of the two FN isoforms supported the formation of FBN-1 fibers (Fig 7G; arrowheads) as well as the deposition of FBLN-4 (Fig 7H; arrowheads). cFN contributed more than pFN in supporting FBN-1 fibers but equally in FBLN-4 deposition, corresponding with the in vivo data (Fig 6A, 6B and 6E).
p-value of 0.01 and ÃÃÃ a p-value of 0.001 in Fig 6E ( Fibronectin in postnatal vascular stability The results for LTBP-4 (Fig 7I; arrowheads) also correlated with the in vivo role of cFN in promoting LTBP-4 assembly and showed very little contribution of pFN. However, it is important to note that because of the selected sources of FN isoforms (mouse plasma for pFN and SMCs for cFN) to closely mimic the in vivo situation, the actual amounts of individual isoforms could be different. This could contribute to the differences observed between the cFNcontaining condition (cFN+/pFN−) and the condition with pFN only (cFN−/pFN+). Tropoelastin and elastic fibers could not be detected in this cell system. For all proteins tested, the presence of both pFN and cFN mediated longer total fiber lengths and more junctions compared to the presence of only one FN isoform, suggesting the two isoforms of FN can contribute together in self-assembly and in guiding the assembly/deposition of other matrix proteins (Fig 7, right panel; S1 Table).

Distinct roles of FN isoforms in ECM assembly
To analyze if there are actually differences between pFN and cFN in fiber formation and to guide the assembly of other ECM proteins, another cell model was developed that could assess the effects of equal amounts of purified cFN and pFN, unlike the SMC cell culture model, in which cFN was produced by the cells and pFN was present in the mouse plasma added. Mouse embryonic fibroblasts from Fn null embryos are well known to assemble exogenously provided FN [16]. In a culture medium devoid of pFN, these cells grow under FN-free conditions. The absence of any FN fibers in this cell system was validated by indirect immunofluorescence (Fig 8A; Tris-buffered saline [TBS] control). An equal amount (25 μg/mL) of soluble purified pFN or cFN was added into the medium and the quality and quantity of fibers were analyzed. The fibers formed were clearly different between the two FN isoforms (Fig 8A). cFN formed more mature and elongated fibers (Fig 8A; arrows), whereas pFN formed fewer fibers and the fibers were shorter in length (Fig 8A; arrowheads). Similar consequences were observed for both FN isoforms in guiding FBN-1 ( Fig 8B) and FBLN-4 (Fig 8C). For LTBP-4, cFN supported assembly/deposition, but pFN could barely support any fiber formation, corresponding well with the in vivo data (Fig 8D). For all proteins tested, cFN mediated longer total fiber lengths and more fiber junctions (Fig 8, right panel; S2 Table). These results demonstrate that pFN and cFN assemble differently and translate these differences onto dependent downstream ECM formation and deposition. Taken together, the data from the two cell culture models and the in vivo results suggest that there are some similar functions but other different roles that cFN and pFN play in self-assembling and in supporting the assembly of other ECM proteins.
precipitates from the conditioned medium. (C-E) 4-OH Tamox and EtOH treated SMC were stained after 7 days in culture by indirect immunofluorescence against (C) FN, (D) FBN-1, and (E) FBLN-4. Note the absence of FBN-1 and FBLN-4 networks when FN is deleted. Scale bar for C-E is 100 μm (n = 3). Underlying data are provided in S1 Data. (F-I) To test the role of pFN and cFN in facilitating self-assembly and that of FBN-1, FBLN-4, and LTBP-4 in conditions replicating the mouse models, four cell culture conditions were generated (see Materials and methods for details): (1) cFN+/pFN+; (2) cFN+/pFN−; (3) cFN−/ pFN+; and (4) cFN−/pFN−. Indirect immunofluorescence analyses were performed after 5 days of cell growth with antibodies against (F) FN, (G) FBN-1, (H) FBLN-4, and (I) LTBP-4. Note that in the presence of pFN and cFN individually, short immature fibers develop (arrowheads), whereas the presence of both FN isoforms support enhanced FN self-assembly as well as the assembly and/or deposition of FBN-1, FBLN-4, and LTBP-4 (arrows). The graphs show a histogram distribution of branch lengths for the cFN+/pFN+, cFN+/pFN−, and cFN−/pFN+ samples. No measureable fibers were present in the cFN−/ pFN− samples. Underlying data are provided in S1 Data. Total fiber lengths and junction numbers are listed in S1 Table. The  scale bar

Discussion
FN is a critical player in vascular biology, with essential roles in embryonic development, in various cardiovascular diseases, and in pathologies in which vascular development is a key process, such as in tumor progression. However, virtually nothing is known about the role of FN and its isoforms during physiological postnatal vascular development. Here, we report that deficiency of both hepatocyte-derived pFN and SMC-derived cFN arrests early postnatal aortic development, whereas either one FN isoform is sufficient to support vascular maturation at this developmental stage. We have identified a novel function of pFN in which it represents a safeguard in maintaining blood vessel wall integrity by translocating into the vessel wall. This is also the first report to show, in vivo, the role of FN as a "master organizer" for ECM assembly and to establish that both pFN and cFN have critical functions in vascular wall protein assembly/deposition.
From the initial global KO, it became clear that FN has a critical role in the development and homeostasis of the major blood vessels, as the mice die during embryogenesis around E9.5 with a variety of phenotypes, including defects in blood vessel development [30]. Deletion of both alternatively spliced EDA and EDB exons (present only in cFN) from the Fn gene led to embryonic lethality by E10.5, displaying also multiple cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, and defective placental angiogenesis [31]. While cFN is essential for cardiovascular development during embryogenesis, we established from our study that postnatal conditional deletion of cFN in SMCs induced at P1-P3 only results in minor aortic phenotypes not affecting the life span of the mice, as pFN is able to rescue the functions. Similarly, postnatal deletion of pFN around P3 in our study did not result in any obvious aortic phenotype, because SMC-derived cFN is sufficient to maintain the vascular integrity. Therefore, both SMC-derived cFN and pFN are individually dispensable for the postnatal development and homeostasis of the thoracic aorta. However, the deletion of both FNs around P3 resulted in the death of most (>80%) dKO mice until P30. These data demonstrate that at least one of the FNs is required for the continued postnatal development and stability of the thoracic aorta and that pFN can compensate for the loss of SMC-derived cFN. However, Murphy and colleagues reported that when FN was deleted globally (absence of both cFN and pFN) in adult mice, no vascular phenotype was observed, except when the mice were challenged by disturbed blood flow in the carotid arteries, resulting in enlargement of the aorta and hemorrhage [52]. The dependency on at least one-SMC-derived cFN or pFN-during early postnatal aortic development is likely due to the requirement of deposition of new ECM as the aorta grows and matures within the first 4 postnatal weeks [53]. It is known that new elastin deposition during this phase occurs at the fenestrations in the elastic lamellae [54]. As the dKO mice die during this expansion phase, it is possible that FNs support a role in the deposition of new elastin at these fenestrations. This assumption is further supported by (i) ultrastructural tissue analysis, which showed irregular elastic lamellae, and (ii) biochemical data that showed reduced newly deposited elastin at P8 in the dKO mice. Taken together, these ECM deficiencies in the aortic media of the dKO mice are likely the cause for the postnatal lethality of these mice.
cFN iKO mice showed similar matrix defects in the tunica media compared to dKO mice at P8 when analyzed by immunofluorescence (Fig 6). However, at later time points (8 months) no deficiencies in matrix components were observed in the cFN iKO, as compared to control mice (S1 Fig). This indicates that it takes likely somewhat longer than P8 (5 days after tamoxifen injection) for pFN to fully rescue the functions of the deleted SMC-derived cFN, so that the cFN iKO mice develop and survive normally. Our cell culture data also support this hypothesis, as pFN can take over the functions of cFN, but only partially, when analyzed at the same time point (Fig 8).
It was shown in cell culture studies that pFN and cFN have similar but not identical cellular and biochemical functions. For example, the FN isoforms were equally effective in promoting cell spreading and mediating cell attachment to collagen and gelatin substrates [55,56]. On the other hand, EDA/EDB containing cFN incorporated more efficiently into existing fibers compared to pFN [57]. We have identified qualitative and quantitative differences in the FN and FN-dependent fiber systems between the two FN isoforms. cFN formed and supported extended fibers, while pFN formed and guided shorter fibers. This is potentially the result of differential cross-linking of the two isoforms by transglutaminases, where cFN forms a very high molecular weight complex but does not form the intermediate multimers observed in pFN cross-linking [58]. In addition, different transglutaminases act on the two FN isoforms [59].
Our in vivo and cell culture data also provide new insights into the functional relationship of pFN and cFN, in which the two isoforms interact to self-assemble and support the deposition of elastogenic ECM proteins. We and others have previously shown in cell culture that the assembly of fibrillins are strictly dependent on FN fibers [20,21]. It is thought that small units of fibrillin-containing microfibrils are transferred to FN fibers for further elongation and maturation [60]. Here, we demonstrate in cell culture and in vivo that both pFN and cFN support the assembly of FBN-1 and/or the deposition of microfibrils onto the elastic laminae, with greater contribution from cFN. We also identified a FN dependency for FBLN-4 assembly/ deposition in cell culture and in vivo, which extends the list of FN-depending ECM fiber systems. It is currently not known, however, whether FBLN-4 interacts directly with FN or through other mediators, potentially including FBN-1. Two other critical elastogenic proteins, LTBP-4 and tropoelastin, showed a differential pattern, both requiring cFN for their assembly/ deposition in vivo (and in cell culture for LTBP-4), whereas pFN was not required. The common aspect for both proteins is that their assembly is hierarchically dependent on the presence of FBN-1 and microfibrils [26,61] and thus only indirectly on the presence of FN fibers. One interpretation of these data is that cFN has additional functional or stabilizing consequences (not shared by pFN) on microfibrils that enable them to promote LTBP-4 and tropoelastin assembly. The data for FBN-1 and LTBP-4 are in contrast to what was found in tumors in which nearly complete FN deletion did not affect the assembly/deposition of these proteins [62]. However, in that study, FN deletion was induced much later (5-6 weeks) than in our study, followed by tumor analysis at 12-13 weeks. It is possible that the dependency of FBN-1 and LTBP-4 on FN is primarily required in the early postnatal phase when blood vessels still undergo intensive remodeling and growth, or, alternatively, it is possible that this dependency is tissue specific (aorta versus tumor).
The concept of translocation of pFN from blood into tissues is known for several organs, including liver, spleen, brain, and bone [48, [63][64][65]. Here, we provide novel data showing the function of the translocated pFN in the vascular wall of the aorta. Translocation and accumulation of pFN in the tunica media are not required for normal postnatal development and homeostasis of the aorta, evidenced by the absence of a phenotype in the pFN KO. However, in the absence of SMC-derived cFN in the tunica media, the translocated pFN is able to completely rescue a fetal phenotype (obvious in the dKO), leading to the concept of a critical safeguard function of pFN in maintaining vascular stability and health. This may help explain vascular manifestations in a variety of disorders associated with defective cFN expression, including atherosclerosis For the translocation of pFN to the vascular wall, we have excluded a passive diffusion through a potentially leaky endothelial cell layer, as both 250 kDa dextran and the 2 kDa fluorophore do not enter the tunica media. Transcytosis, the transport of molecules across cells through coupled endocytosis and exocytosis transport pathways, is a hallmark of endothelial cells that is frequently mediated through caveolae [66]. Active transcytosis of albumin through endothelial cells, for example, has been shown to be dependent on caveolae [67,68]. While it is not known whether FN uses this pathway, FN can be turned over through integrin β1-mediated (e.g., α5β1) endocytosis regulated by caveolin-1 [69,70]. Thus, it is possible that pFN transcytosis follows a similar transport mechanism.
In summary, this study illuminates the critical role of FN isoforms in postnatal vascular development. It reveals a novel function of pFN, in which it transfers to the vessel walls to safeguard the role of cFN. The two isoforms play important differential and similar functions in maintaining the integrity and matrix stability of the vasculature.

Ethics statement
All animal studies strictly followed the guidelines imposed by the Canadian Council on Animal Care and were approved by the McGill University Animal Care Committee (protocol 2010-5893). Mice were humanely killed by an anesthetic overdose using a ketamine/xylazine/ acepromazine cocktail followed by cardiac perfusion.

Mouse models
Fn(fl/fl) mice were generated as previously described [32] and were used as controls throughout the study, unless otherwise mentioned. Sma-Cre-ER T2 /+ mice were a generous gift from Drs. Pierre Chambon and Daniel Metzger and generated as described

Tamoxifen injection
To delete Fn (deleted region encompassing start codon, sequence for signal peptide, and exon/ intron border of exon 1) in SMCs of cFN iKO and dKO mice, and to induce β-galactosidase expression in the Reporter-Cre mice, 0.1 mg of tamoxifen (Sigma) was injected intragastrically daily from P1 to P3 using a 2 mg/mL tamoxifen solution in sunflower oil [71]. The same tamoxifen treatment regimen was followed for all the mouse strains analyzed with cFN iKO, dKO, or Reporter-Cre (including control Fn(fl/fl), pFN KO (Fn(fl/fl);Alb-Cre/+) and Reporter control (Rosa26(fl/fl))).
For histological and immunohistological staining procedures, mice were humanely killed by an anesthetic overdose using a ketamine/xylazine/acepromazine cocktail. Mice were then cardiac-perfused with 10% neutral-buffered formalin, and aortae were harvested. After an overnight incubation in 10% neutral buffered formalin, the tissues were dehydrated through an increasing EtOH gradient and embedded in paraffin. Four micrometer sections were deparaffinized in CitriSolv (Decon Labs) and rehydrated with decreasing EtOH concentrations in H 2 O. The sections were stained with Hematoxylin and Eosin (general histology and cell orientation), Masson's trichrome stain (collagen), picrosirius red (collagen), or Hart's elastin stain using standard procedures. For immunostaining, the sections were treated for antigen retrieval with 10 mM citric acid, 0.05% Tween 20 (pH 6) at 98˚C, followed by treatment with bacterial type XXIV proteinase (10 μg/mL; Sigma #P8038). The sections were then incubated with primary antibody (monoclonal anti-human SMA Clone 1A4 [Dako #M0851] at a 1:100 dilution and polyclonal anti-mouse fibrillin-1 Cterminal half [generated in the lab] at 1:3,000). The immunostaining procedure was performed using the Dako Envision+ System-HRP (AEC) staining system (Dako #K4004 and #K4008).

pFN and dextran translocation experiments
pFN and dextran translocation experiments were performed with litters injected with tamoxifen (P1-P3) to generate cFN iKOs and controls (Fn(fl/fl)). Human pFN (Millipore #FC010) or 250 kDa amino dextran (Fina Biosolutions #AD 250 × 100) were covalently coupled to the water-soluble activated fluorophore Oyster-680 N-hydroxysuccinimide ester (Boca Scientific) in 100 mM NaHCO 3 , 500 mM NaCl, pH 8.4. Oyster-680 emits light at 693 nm, which does not overlap with the known autofluorescence of elastic lamellae in the aorta. Unbound fluorophore was removed by dialysis against the coupling buffer, and the degree of labeling was calculated to be 3-4 molecules Oyster-680 per molecule of pFN or dextran. The labeled pFN or dextran was injected in mice intraperitoneally from P8 to P10 (0.13 mg per mouse per day) using an established protocol [48,74]. TBS-deactivated Oyster-680 alone used at the same molar concentration served as control. Eighteen hours after the last injection, the mice were dissected and tissues harvested after being humanely killed following anesthetic overdose with a ketamine/xylazine/acepromazine cocktail. Eight micrometer OCT-embedded cryosections were analyzed with a Cy5 filter set. Elastin autofluorescence was analyzed using GFP filter set to visualize the elastic lamellae label-free.

Analysis of pFN in blood
Blood was collected by cardiac puncture from pFN KO and control (Fn(fl/fl)) mice after they were humanely killed. Plasma preparation was performed using EDTA-coated tubes (Sarstedt). Three microliter aliquots of plasma were analyzed by standard western blotting under reducing conditions. The polyclonal rabbit anti-mouse FN antiserum (Millipore #AB2033) was used 1:500 diluted as primary antibody, with the 1:200 diluted Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Laboratories) as secondary antibody.

Necropsy
To investigate the cause of lethality in dKO mice, litters injected with tamoxifen (P1-P3) were monitored twice every day. Dead pups were found often for dKOs but occasionally we found dead pups from other tamoxifen-injected strains, which were used as controls for the necropsy. Dead pups were fixed in 10% neutral buffered formalin (overnight) and washed twice in phosphate buffered saline to prevent postmortem changes. Necropsy was performed to visualize gross anatomy of the dead pups, with a focus on the aorta and thoracic cavity. Images were recorded using an AmScope dissection microscope (ZM-1TZ2-FOR-GT-10M) equipped with a digital camera. All other analyses throughout this study were performed on mice dissected immediately after cardiac perfusion to optimally preserve the tissue structure and avoid any postmortem changes.

Transmission electron microscopy
For transmission electron microscopic analysis, mice were humanely killed by an anesthetic overdose with a ketamine/xylazine/acepromazine cocktail, followed by cardiac perfusion with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) (fixative). The aortae were then harvested and incubated in the fixative overnight. This was followed by a postfixation step with 1% osmium tetraoxide + 1.5% aqueous potassium ferrocyanide. Aortae were then dehydrated with an increasing concentration of acetone, followed by embedding in Epon. Ninety nanometer sections were transferred onto 200 mesh copper TEM grids and counterstained stained using 4% aqueous uranyl acetate and Reynold's lead before imaging with an FEI Tecnai 12 BioTwin 120 kV transmission electron microscope.

Biochemical protein analysis
Thoracic aortae from all four mouse strains at P8 (post-tamoxifen injection) were extracted by 0.1 N NaOH at 95˚C for 45 minutes. After centrifugation, the pellets containing insoluble elastin were subjected to hydrolysis and amino acid analysis using a three buffer gradient system and post-column ninhydrin derivatization. The content of elastin cross-links (Isodes and Des) were analyzed in an aliquot of the NaOH-insoluble fraction containing elastin after CF-11 preclearance by amino acid analysis (leucine equivalence factors IDES: 3.4; DES: 3.4). The amino acid composition of the NaOH-insoluble protein fraction was typical for pure elastin [75].

Quantitative PCR
Descending aortae were harvested from tamoxifen-injected control, dKO, cFN iKO, and pFN KO (littermates) at P8. Total RNA was isolated using the Trizol method (Life Technologies). RNA was reverse transcribed using ProtoScript II First Strand cDNA synthesis kit (New England Biolabs). A QuantStudio 5 Real Time PCR System (Applied Biosystems) was used to perform the quantitative PCR using SYBR Select Master Mix (Applied Biosystems). Specific primers were used for all the genes analyzed. Gapdh was used as the control gene. Relative quantification of mRNA levels (fold change) was calculated using the 2 −ΔΔCt method. Significance was determined using the two-tailed t test.

Analyses of cell cultures
SMCs were isolated from the tunica media of aortae harvested from Fn(fl/fl); Sma-Cre-ER T2 / + mice using an established explant-based method [76]. Cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum and 100 μg/ mL penicillin/streptomycin and 2 mM glutamine. Cells were treated with 1 μM of activated 4-OH Tamox for 3 days to induce the deletion of Fn (deleted region as described above). EtOH, the solvent for 4-OH Tamox, was used as a control at a final concentration of 0.02% (same concentration as used for the 4-OH Tamox treatment). For experiments, cells were grown in medium containing fetal bovine serum depleted of pFN by gelatin sepharose chromatography [21]. Deletion of cFN was analyzed by RT-PCR from mRNA isolated from cells using RNeasy mRNA extraction kit (Qiagen). RT-PCR was performed as described above. Deletion was also analyzed at protein level by western blotting with 15 μL of conditioned medium (obtained after 24 hours) using 1:500 diluted anti FN antibody (Millipore #AB2033). Indirect immunofluorescence was performed after 7 days with 70% methanol/ 30% acetone fixed and permeabilized cells. Goat serum diluted in phosphate buffered saline (1:10) was used for blocking. Primary antibodies were prepared in blocking buffer and included polyclonal rabbit anti-mouse FN (Millipore #AB2033-1:500 diluted), anti-mouse FBN-1 (1:500 diluted), and anti-human FBLN-4 (1:500 diluted). Alexa Fluor 488 goat antirabbit IgG (H+L) (1:200 diluted) were used as secondary antibodies. DOC extraction was performed using standard procedures as described in [77]. DOC-soluble and -insoluble fractions were analyzed by western blotting using 1:500 diluted anti-FN antibody (Millipore #AB2033).
Experiments were designed to mimic the three KO mouse models and the control in cell culture as follows: 4-OH Tamox and EtOH-treated cells were cultured in the presence of 10% mouse plasma prepared from either wild-type or pFN KO mice. The combinations used were: (1) cFN+/pFN+, EtOH-treated cells (cFN present) were grown in medium containing mouse plasma from control mice (pFN present); (2) cFN+/pFN−, EtOH-treated cells (cFN present) were grown in medium containing mouse plasma from pFN KO mice (pFN absent); (3) cFN−/pFN+, 4-OH Tamox-treated cells (cFN absent) were grown in medium containing mouse plasma from control mice (pFN present); and (4) cFN−/pFN−, 4-OH Tamox treated cells (cFN absent) were grown in medium containing mouse plasma from pFN KO mice (pFN absent). After 5 days, cells were fixed with 4% paraformaldehyde and immunofluorescence was performed as described above, additionally with primary LTBP-4 antibody (1:500 diluted) and secondary Cy5 AffiniPure Goat anti-Rabbit IgG (H+L) antibody (Jackson Laboratories, 1:200 diluted). No LTBP-4 fibers were observed in this setup. However, when, instead of using mouse plasma, 40 μg/mL of purified soluble pFN was added to the system, LTBP-4 fibers formed. This could be due to factors present in the mouse plasma, which might affect LTBP-4 assembly.
Mouse embryonic fibroblasts differentiated from Fn null mouse embryonic stem cells were prepared as described [78]. As for the SMCs, the Fn −/− embryonic fibroblasts (which do not produce cFN) were grown in pFN-depleted cell culture medium, providing an experimental fibroblast system without pFN and cFN. Affinity chromatography on immobilized gelatin was used to purify human pFN from plasma, as described in detail [79], and cFN from human fibroblast-conditioned medium, as previously described [80]. Twelve and a half micrograms of purified pFN or cFN was added to 500 μL of culture medium at the time of cell seeding. Cells were fixed with 4% paraformaldehyde after an 8 day culture period and analyzed by indirect immunofluorescence, as outlined above.

Light microscopy imaging
An epifluorescence microscope (AxioImager M2; Zeiss) was used to capture all bright field and immunofluorescence images with an AxioCam ICc5 color camera or an Orca Flash 4.0 sCMOS grayscale camera, respectively. Grayscale immunofluorescence images were false colored using the Zen Pro software (Zeiss).

Quantification procedures
Mean pixel intensity analysis of various fluorescent signals (EDA FN, Total FN, FBN-1, FBLN-4, LTBP-4, Tropoelastin) was performed with 8-bit TIFF images using the ImageJ software [81] on regions of the tunica media, as described by others [52]. The mean pixel intensity of the background was deducted to produce the values used for plotting the graphs. The error bars in the graphs represent standard deviation.
For collagen analysis, picrosirius red stained images of the aorta were obtained through fluorescence microscopy using a Zeiss HQ Texas Red filter set [82]. Mean pixel intensity, total intensity, and area of collagen distribution analysis in the tunica media and tunica adventitia were performed using the ImageJ software, as described above. Error bars represent standard deviation.
Quantification of total fiber lengths and junction number of ECM fibers visualized by immunofluorescence was performed using the ImageJ distribution, Fiji [83,84]. Color images were converted to 8-bit and the threshold was set to best match the fiber appearance in the original image (same threshold for all the images comparing the same protein staining). This was followed by removing the noise using the despeckle feature, followed by skeletonizing the image using "Plugins-Skeleton-Skeletonize (2D/3D)" and analyzing the processed image by clicking "Analyze-Skeleton-Analyze Skeleton (2D/3D)" [85]. This provides results about the number of junctions and branch lengths. Branches that were less than 5 μm were excluded to avoid small nonfibrous particles, which can influence the total branch length. All the other branches were added up to get total branch length, and junctions were added to get the total number of junctions per condition. A histogram was generated to depict the number of branches in each condition, binned by branch lengths.

Statistics
Two-sample t test was used to determine significance of the difference in protein staining intensities (tissue and cell immunofluorescence) and mRNA levels (qPCR). One-way ANOVA was used to analyze significance for the difference in elastin content and cross-links (biochemical analysis). Chi-squared test was used to determine the significance of the deviation of mouse strain survival from the Mendelian ratio by comparing the number of observed live mice to that of the expected Mendelian ratio. For all statistical analyses, Ã denotes p 0.05, ÃÃ denotes p 0.01, and ÃÃÃ denotes p 0.001.

S1 Fig. Analysis of matrix integrity of the aorta in cFN iKO mice at 8 months of age. (A-B)
Fibrillin-1 immunostaining of cross sections of descending aorta show no change between cFN iKO and control at 8 months (n = 3). (C-D) Hart's elastin stained cross sections of descending aorta of tamoxifen-injected control and cFN iKO at 8 months. (E-F) Quantification of breaks (E) and forks (F) in the elastic lamellae normalized to the area (10,000 μm 2 ) of the aortic section analyzed in the cFN iKO and control at 8 months (n = 8-10). There was a trend towards more elastin fiber breaks and fewer forks in the cFN iKO, but it did not reach statistical significance. Underlying data are provided in S1 Data. Scale represents 50 μm in A-D. Lumen is indicated with an asterisk in A-D. cFN iKO, cellular fibronectin inducible knockout. Quantitative PCR was performed with total RNA isolated from descending aortae of tamoxifen-injected mice, as indicated, at P8 (n = 3). mRNA levels of the proteins analyzed in immunostaining (Fig 6A-6D) were not altered except for FBN-1, validating the role of FN as a master organizer in ECM protein assembly, but not in mRNA expression. Underlying data are provided in S1 Data.  Table. (DOCX) S2 Table. (DOCX)