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Published ahead of print on November 15, 2007, doi:10.1165/rcmb.2007-0281OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 435-445, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0281OC

Arg-Gly-Asp–Containing Domains of Fibrillins-1 and -2 Distinctly Regulate Lung Fibroblast Migration

Stephen E. McGowan1, Amey J. Holmes1, Robert P. Mecham2 and Timothy M. Ritty3

1 Department of Veterans Affairs Research Service and University of Iowa Carver College of Medicine, Iowa City, Iowa; 2 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri; and 3 Department of Orthopaedics and Rehabilitation, Pennsylvania State University School of Medicine, Hershey, Pennsylvania

Correspondence and requests for reprints should be addressed to Stephen E. McGowan, M.D., Division of Pulmonary, Critical Care, and Occupational Medicine, Department of Internal Medicine, C33B GH, University of Iowa Hospital, 200 Hawkins Dr., Iowa City, IA 52242. E-mail: Stephen-mcgowan{at}uiowa.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Development of the extracellular matrix is a critical feature of alveolar formation and actively involves pulmonary interstitial fibroblasts. The elastic fiber network is an interconnected system of load-bearing fibers that also influences the behavior of adjacent cells, particularly the interstitial lung fibroblasts (LF). We hypothesized that discrete domains of fibrillins-1 and -2 interact with LF integrins and direct their migration in the presence of platelet-derived growth factor (PDGF)-A. Surfaces coated with recombinant peptides lacking or including an arginine-glycine-aspartic acid (RGD) motif were used to study LF migration across porous filters and on protein-coated glass. Exon 24 of fibrillin-2 (Fib2 24), which encodes for an RGD-containing transforming growth factor-β–binding (TB) domain, stimulated migration with greater directional persistence and more effectively stimulated trans-filter migration at low concentrations. Exons 36–44 of fibrillin-1 (Fib1 36–44), which include epidermal growth factor–like domains and an RGD-containing TB domain, induce more lamlellipodia and more widespread remodeling of the leading edge, resulting in greater migration velocity than did Fib2 24. Distinct structural features in regions that surround the RGD motifs may differentially regulate how the PDGF receptor-{alpha} promotes integrin distribution and actin filament remodeling at the cell's leading edge. Understanding how fibrillins regulate LF migration may help elucidate how the elastic fiber system could be restored as an interconnected unit, which fails to occur in emphysematous lungs.

Key Words: alveolus • fibrillin • elastin • integrin • cell migration



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This research defines interactions between proteins in the elastic fiber and lung fibroblasts that may be involved in the alveolar septation process. The findings foster the development of new treatments for emphysema.

 
In mammalian species whose young are not required to walk immediately after birth (humans and rats as opposed to sheep, for example), most of the pulmonary alveoli develop after birth (1). Mammalian alveolar formation occurs in a stereotypic fashion whereby simplified air sacs, which reside at the ends of the small conducting airways at birth, gain complexity by progressively dividing into smaller segments (alveoli) (2). The growing alveolar septa contain a central structural core of fibroblasts and extracellular matrix, which is covered by the more superficial gas-exchange surface. Fibers in the central core are primarily produced by alveolar fibroblasts and maintain the cellular integrity of the septum during the distortion that accompanies phasic respiration (3). The factors that regulate the initiation, localization, and termination of the synthesis of these fibrillar proteins remain incompletely understood.

Mice that are platelet-derived growth factor (PDGF)-A–null lack fibroblasts, which normally contain {alpha}-smooth muscle actin ({alpha}SMA) and synthesize elastin, and exhibit deficient secondary septation (4, 5). Septation may fail because fibroblasts, which are present in primary septa, do not migrate during secondary septal elongation. Cell migration is augmented by interactions between PDGFs and integrins. Binding of PDGF-AA or -BB to their plasma membrane tyrosine kinase receptors initiates an intracellular cascade, causing phosphorylation of focal adhesion kinase, and an increase in the migration of fibroblasts (6). PDGFs promote actin remodeling and dorsal wave formation, where new lamellipodia form at the leading edge (7). More direct effects of PDGFs on integrins include (1) direct association of PDGF-receptor-β with {alpha}Vβ3, but not β1 integrins; (2) localization of integrins at the leading edge through recruitment and activation of the small GTPase, Rac; and (3) promotion of integrin recycling via the early endosomes to the leading edge through another GTPase, Rab4A (8–10). Because PDGF-A is required for alveolar formation, it is important to understand how PDGF modifies interactions between cellular integrins and extracellular proteins during cell migration. For example, type I collagen is bound by the β1 portion of {alpha}1β1, {alpha}2β1, {alpha}10β1, and {alpha}11β1 integrins and promotes the migration of smooth muscle cells and fibroblasts (11).

Elastic fibers are very resilient, have a long biological half-life, and are composed of at least seven different insoluble polymers (12). As one of the earliest proteins that are observed in nascent elastic fibers, fibrillins may serve as scaffolding on which other components such as tropoelastin are deposited, and thereby influence the location of elastic fibers in the alveolar septum (3, 12–14). Fibrillins interact with proteins on the cell surface, including integrins, and influence the adhesion to, spreading, and migration of ligamentum nuchae fibroblasts and mesangial cells in vitro (14–17). Cellular contacts with fibrillin may also help assemble a cadre of molecules (including fibulin-5, microfibril-associated glycoproteins, and lysyl oxidase) at the cell surface that are required for elastic fiber formation (18–20).

Three fibrillin genes have been identified in mammals—fibrillins-1, -2, and -3—and the fibrillin-1 gene product is the most abundant in adults and is synthesized throughout life (21). Fibrillin-2 is maximal during early gestation and its synthesis is largely complete by birth. Fibrillin-3 is expressed in fetal human but not mouse or rat lungs (22). Fibrillin-1 is the most abundant fibrillin in pulmonary elastic fibers after birth, is synthesized as a monomer that polymerizes extracellularly, and undergoes a series of post-translational modifications (23).

The coding regions of the fibrillin genes are primarily composed of epidermal growth factor (EGF)-like repeats, which encode for calcium-binding motifs and transforming growth factor-β–binding protein-like (TB) repeats that contain 8-cysteine domains and are involved in disulfide bonding (24). Exons 37 of fibrillin-1 and fibrillin-2, both TB regions, each contain an arginine-glycine-aspartic acid (RGD) motif and N-glycosylation sites (25). Exon 24 in fibrillin-2 contains an additional RGD, although the adjacent residues differ from those in exon 37 (24, 26). Cellular attachment has been shown to involve interactions between {alpha}Vβ3 or β1integrins and RGD motifs in fibrillins-1 and -2 (14, 25). An alternate heparin-binding cell-binding domain in the carboxy terminal region of fibrillin-1 also mediates attachment (27). This heparin-binding domain mediates interactions between cells and fibrillin polymers that are required for cellular migration and normal gastrulation in Xenopus embryos (28). We have examined how peptides representing particular exons in the fibrillin-1 and fibrillin-2 proteins influence neonatal lung fibroblast attachment and migration.

We hypothesized that microfibrils may influence where alveolar fibroblasts synthesize and deposit elastin and thereby ensure that the elastic fiber network is interconnected and most abundant at locations of maximal strain during respiration. We also formulated a more specific sub-hypothesis: the RGD moieties in fibrillins-1 and -2 enhance the attachment and migration of lung fibroblasts (LF) in vitro, in the presence of PDGF. This enhancement is associated with specific cellular processes that participate in the remodeling of integrin attachments at the leading edge of the cells. We have used recombinant fibrillin peptides that either contain or lack the RGD motif and adjacent EGF-like domains to study the adhesion and migration of neonatal rat LF. Migration was studied using two different approaches: transmigration across porous filters with the lower surface coated with fibrillin peptides or on a planar surface of fibrillin peptides on a glass coverslip. These two approaches are complementary and allowed us to study different properties of the LF.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Isolation of Neonatal LF
LF were isolated from rats at Postnatal Day 8 by digesting the lungs with a mixture or trypsin and collagenase and separating the less dense, lipid-laden fibroblasts using Percoll (29, 30). The cells were grown to confluence and were then removed from the tissue culture plates using 0.2% trypsin, frozen in fetal bovine serum (FBS) containing 10% DMSO, and maintained in the vapors of liquid nitrogen. Aliquots of cells were thawed, cultured in Ham's F-12 medium containing 5% FBS and used without further sub-cultivation. Migration and adhesion assays were performed in MCDB-201 medium on surfaces that were coated with various concentrations of fibrillin peptides.

Preparation of Fibrillin Peptides
pQE bacterial plasmids containing various domains of the human fibrillin 1 (Fib1)- and fibrillin 2 (Fib2)-coding regions have been described (13, 27). Fib1 36–44 contains exons 36–44 of fibrillin 1, which includes an RGD motif in exon 37; Fib 1–30 contains only exon 30 of fibrillin 1, which encodes an EGF-like domain and lacks an RGD motif; Fib2 24 contains exon 24 of fibrillin 2, which contains an RGD. To exclude the effect of an adjacent EGF-like domain, we studied Fib2 24 rather than Fib2 37–38, which contains an EGF-like domain in exon 38. The presence of exon 38 renders Fib2 37–38 more structurally similar to Fib1 36–44 than is Fib 2 24. The plasmids were propagated in an Escherichia coli strain that also contains the Rep4 plasmid, allowing inducible expression of the recombinant peptides in the presence of isopropyl β D 1-thiogalactopyranoside (IPTG). The peptides were purified from bacterial cell lysates in the presence of 8 M urea using Ni-NTA (nickel bound to nitriloacetic acid) agarose. Purity and Mr were confirmed using SDS-PAGE and silver staining. Fib 23–44 (LEEC) was contained in the pEE14 mammalian expression vector, which had been stably transfected into CHO-K1 cells, and its expression was driven by the CMV immediate early gene promoter (13, 31). Transfected CHO-K1 cells were propagated in Glasgow modified Eagle Medium without glutamine and containing 100 mM of L-methionine sulfoximine. Before collection of the conditioned medium, the growth medium was changed to Hy-QCCM5 serum-free medium (InVitrogen Life Technologies, Carlsbad, CA). The recombinant peptide was purified from the conditioned medium, after concentration by ultrafiltration on a YM-100 membrane (Amicon, Danvers, MA) followed by elution from a Superose 6HR column (10 mm x 300 mm; Pharmacia Biotech, Piscataway, NJ) in 100 mM ammonium bicarbonate, pH 7.5 at a flow rate of 30 ml/hour (31). Protein purity was assessed using SDS-PAGE and Western immunoblotting.

Type I collagen was included in the migration assays for several reasons: (1) relatively few LF adhered to polycarbonate filters and glass in the absence of collagen, limiting the accuracy of assays that required enumeration of cells; (2) LF retracted during haptotaxis on the glass surface of the perfused migration chamber, preventing an accurate assessment of migration; and (3) PDGF does not increase fibroblast migration on collagen, which enabled an assessment of how PDGF promotes the migration on fibrillin (8).

Adhesion Assay
The adhesion of LF to fibrillin peptides was assessed using Costar 3590, 96-well microplates (Corning Life Science, Lowell, MA). Quadruplicate wells were coated overnight at 4°C with 0, 0.1, 0.4 1, or 4 µg/ml of the various peptides in 0.14 M NaCl, 50 mM Tris-HCl, pH 7.4 (TBS), 0.02% sodium azide. After removing the unbound peptides, the wells were washed and the surface was incubated for 1 hour in 1 mg of bovine serum albumin (BSA) in TBS without azide. Fifty thousand LF were added to each well and incubated at 37° for 2 hours in 5% CO2 to allow attachment. The nonadherent LF were removed by washing the wells with warm TBS without azide. The adherent cells were quantified by assaying their DNA contents using Yo-Pro-1 dye (InVitrogen, Molecular Probes, San Diego, CA) (32). The adherent LF were first lysed in 50 µl of 0.1% Triton X-100 in H2O for 30 minutes at room temperature. An equal volume of 4 µM Yo-Pro-1 in 10 mM Tris-HCl pH 7.4, 2 M NaCl, 1 mM EDTA was added and the fluorescence intensity was assessed using a FluoStar Microplate reader (BMG-Labtech, Durham, NC) at excitation and emission wavelengths of 485 and 530 nm, respectively. A standard curve was generated using calf-thymus DNA. The DNA contained in known quantities of LF was quantified using calf-thymus DNA to determine the ng of DNA per 1,000 LF. In all cases, the quantity of DNA that was contained in LF that adhered to the microwells that only received BSA was subtracted from the quantity of DNA in wells that were coated with the fibrillin proteins.

To assess whether {alpha}V-integrin is involved in LF adhesion, wells were coated with Fib1 36–44. Lung fibroblasts were pre-incubated for 30 minutes with various concentrations of anti-{alpha}V (monoclonal rat anti-CD51, clone RMV-7; eBioscience, San Diego, CA) at 4°C and the cell suspensions in the antibody solutions were added to quadruplicate wells. After incubating for 2 hours at 37°C, the DNA in adherent LF was quantified. Three separate experiments were performed.

Transmigration of LF across Polycarbonate Filters
Lung fibroblasts were grown to confluence and then starved overnight by reducing serum concentration to 0.5% in MCDB 201. The AP48 chemotaxis chamber and the 8-µm polycarbonate filters were obtained from Neuro Probe (Gaithersburg, MD). Various concentrations, ranging from 0.05 to 2 µg/ml, of the fibrillin peptides were placed in the lower chambers. After assembling the apparatus, 2.5 µg of rat tail tendon collagen (Type 1; Sigma-Aldrich Chemical Co., St. Louis, MO) in 0.02 M acetic acid was added to the upper wells and the apparatus was kept at 4°C overnight. The next morning, the solution was removed from the upper chamber, and the wells were washed three times with 0.145 M NaCl, 0.0015 M KH2PO4, 0.0027 M KCl, 0.0086 M Na2HPO4, pH 7.4 (PBS). The top plate of the chemotaxis apparatus was removed with the filter still attached. The bottom wells were evacuated, washed with PBS, and refilled with MCDB medium containing 1 mg/ml BSA and 50 ng of PDGF-AA per ml (R&D Systems, Minneapolis, MN). The apparatus was reassembled with the top plate and filter and a suspension containing 2.5 x 104 LF was added to the top chamber (33). The apparatus was incubated at 37°C in a humidified atmosphere containing 5% CO2 for 4 hours. The apparatus was disassembled and the filter was carefully inverted and floated in PBS. A weight was applied to one edge of the filter and a clamp to the opposite edge. The top side of the filter was scraped over a rubber squeegee, as directed in the Neuro Probe instruction manual. The filter was fixed in methanol at room temperature for 20 minutes. After washing the filter it was stained for 5 minutes with Diff Quik stain (IMEB Corp., San Marcos, CA). After washing and dehydration by gradually increasing the ethanol concentration in the bath from 70% to 100%, the filters were allowed to air dry on a glass slide. Immersion oil was applied to adhere the filter to glass slide. The slides were evaluated using an Olympus BX40 microscope (Olympus, Center Valley, PA) using a x100 oil objective. The regions corresponding to the wells were located and the focus was adjusted to identify the top and bottom sides of the filter. The plane of focus was adjusted to the bottom of the filter, and all of the cells in the region corresponding to the well were counted. The focus was adjusted up and down to ensure that only cells on the bottom of the filter were counted. The data were expressed as cells migrated per 20 oil fields.

Preparation of Coverslips to Study Haptotaxis of LF on Fibrillin
To prepare the coverslips to receive the fibrillin coating, the coverslip was first coated with a solution containing nickel, which was accomplished as follows (34, 35). The coverslips were first cleaned by successive exposures to acetone, ethanol, a solution of 5% ammonium hydroxide and 3% H2O2 in deionized H2O, and then air dried. Next, the coverslips were exposed to 20% 3-aminopropyl triethoxysilane in 100% ethanol (vol/vol) overnight, rinsed twice with 95% ethanol, and then autoclaved at 120°C for 40 minutes. The coverslips were then coated with a 0.1% solution of poly-octadecene-maleic anhydride (POMA) in tetrahydrofuran using a spin coater (Specialty Coating Systems, Indianapolis, IN). The coverslips were autoclaved at 120°C for 20 min and stored at 4°C individually in wells of a 12-well plate in a sterile environment. Next they were incubated overnight at room temperature in 10 mM of N{alpha}-N{alpha}-bis carboxymethyl-lysine in 0.1 M sodium phosphate (pH 8.0). The coverslips were washed with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20 and then blocked for 2 hours with PBS containing 2% BSA in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20. At room temperature, the coverslips were then successively washed with (1) 50 mM Tris HCl, pH 7.5; (2) sterile deionized H2O; (3) 100 mM EDTA, and once again in sterile deionized H2O. The coverslips were incubated for 20 minutes with 20 mM NiSO4, washed with deionized water, followed by 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and finally highly purified water. The surface-bound nickel was allowed to bind the 6-histidine–tagged Fib1 36–44 or Fib2 24 peptides for 6 hours at room temperature and then the coverslips were incubated overnight with PBS containing either 10 or 12.5 µg of collagen per ml. Equivalent molar quantities of RGD were presented when Fib1 36–44 and Fib2 24 were present at 10 and 4 µg per ml, respectively. The coating concentration of Fib1 30 was 10 µg per ml.

Migration and Time-Lapse Photography of LF Haptotaxis
Migration was evaluated using a POCmini chamber (HemoGenex, Colorado Springs, CO) in the closed chamber perfusion format. The top, 22-mm cover glass had been coated with the fibrillin peptides, whereas the bottom coverslip provided a transparent floor for the chamber. Before the migration assay, LF were maintained in Ham's F-12 medium containing 5% FBS. The medium was changed to MCDB-201 containing 5% by volume of Serum-Plus (SAFC Biosciences, Lenexa, KS) to yield an equivalent concentration of 1% serum. Twenty-five thousand (6.6 x 103/cm2) LF were allowed to adhere to the coated top coverslip for 6 hours and the POCmini apparatus was assembled. The chamber was perfused with MCDB-201 medium containing 5% Serum-Plus at 0.5 ml per hour while the chamber was maintained at 35°C using a TC-324 temperature controller and heater (Warner Instruments, Hamden, CT). We used MCDB-201 medium containing 2 mg/ml human serum albumin during the adherence and migration, but consistently observed disruption of focal contacts and contraction of many of the LF. We could not reliably assess migration under these conditions. The POCmini chamber was fixed to a motorized mechanical stage (Ludl Electronics, Hawthorne, NY) on an Olympus BX40 microscope and viewed using a phase-contrast condenser and an Olympus LUC PLAN FL x20 objective (N.A. 0.45) with a long working distance. Images were acquired every 15 minutes from five different fields, captured by an Optronix MicroFire charged-coupled device (CCD) camera, and saved in a grayscale TIF format. The Optronix Picture Frame software was used to prepare image sequences for each field, which were then analyzed using IP-Lab for Windows (BD-Scanalytics, Rockville, MD). An obvious nuclear landmark (usually a nucleolus) was chosen and its path was marked at hourly intervals over the 18-hour course of the experiment. The x- and y-coordinates of this structure were used to calculate the root mean squared distance moved at each hourly interval (36). These data were used to determine the instantaneous velocity, total distance moved, and to plot the path of the cells. The accumulated distances were assessed using the generalized least-square method of Levenberg-Marquardt to calculate root mean squared speed (S) and persistence (P) for each cell that moved and stayed within the microscopic field during the entire 18 hours. The data for D2 versus time in hours were fit to the equation: D2 = S2P2(T/P-1 + e–T/P). D2 is the square of the distance moved in the preceding 1 hour (squared displacement in µm2); P is the persistence time in hours, T is time in hours, and S is speed or velocity in µm per hour. Directional persistence is a measure of the persistence of motion at a similar velocity and direction, and is based on a diffusion model that predicts the probability that either speed or direction will change with time (37). This calculation was performed using nonlinear regression and Prism4 (Graph Pad, San Diego, CA). The data for individual cells were combined and the mean and SEM were calculated.

Cellular shape was compared after LF had been allowed to migrate for 6 hours on collagen, Fib1 36–44, or Fib2 24. Only cells that did not divide during a 12-hour period surrounding this time point were analyzed. The lamellipodia at the leading edge were counted, the perimeters of the cells were traced using IP-Lab software, and the perimeter and area were calculated in µm and µm2, respectively. The ratio of the perimeter to the area (P/A) was calculated as an index of the complexity of the cell shape (P/A should be greater in cells with more protrusions).

The areas occupied by the leading edges of LF were assessed during migration on only collagen or on collagen plus either Fib1 36–44 or Fib2 24. The perimeters of the cells were traced at hourly intervals during a 6-hour period of the time-lapse recording when the LF were the most motile. The areas circumscribed by the perimeters, the lengths of the major axes of the cells, and the centroids were calculated using IP Lab. By tracing the cells at hourly intervals, we were able to determine the area of protrusion of the leading edge (% positive flow) = (area at time n – area at time n – 1 / area at time n) x 100 (38). The % negative flow was calculated using the areal difference between the retractions of the trailing edges of same tracings. By comparing the distance between the centroids of the protrusion and retraction at a particular 1-hour interval to the major axis of the cell at time n, we calculated the polarity (distance between centroids / length along the major axis of the cell) (39). Twelve cells were analyzed for each condition, and the mean % positive flow and polarity over the entire 6-hour period were calculated for each cell. Selection bias was minimized by progressing through the sequences of the fields that were photographed in a systematic way and identifying all of the cells in field 1 that could be analyzed, performing the analyses, and then moving onto field 2. Four cells were analyzed in each of three separate experiments for each coating condition.

Immunohistochemistry
The association of {alpha}Vβ3 integrins with the RGD motifs in the fibrillin peptides was examined by immunolocalization of the 6-His portion of the peptides and the {alpha}V chain (CD51) of the integrin. Paxillin was also visualized to localize focal contacts. The effects of the various fibrillin peptides and PDGF-A on the intracellular distribution of the intracellular GTPase, Rac, were also studied.

Circular glass coverslips were coated with Fib1 30, Fib1 36–44, or Fib2 24 in the presence of collagen, or with only collagen following the procedure that was used for the studies of LF haptotaxis. The LF adhered to the coverslips for 6 hours at 37°C in MCDB-201 medium containing 5% Serum-Plus, and some coverslips were exposed to 20 ng of PDGF-A per ml for 4 hours at 37°C. The coverslips were then washed with PBS and fixed for 30 minutes in 1% paraformaldehyde at 25°C. The LF were permeabilized for 20 minutes with 0.1% (vol/vol) Triton X-100 in PBS that also contained 2 mg of bovine serum albumin (BSA) per ml. In some instances permeabilization was conducted before and in other cases after exposure to the anti-CD51 antibody. After rinsing with PBS containing BSA, the coverslips were incubated with the primary antibody for 2 hours at room temperature and rinsed with PBS containing BSA. To localize the fibrillins, CD51, and paxillin, the following dilutions of primary antibodies were used: 1:200 for mouse monoclonal anti-6His-biotin (Qiagen, Valencia, CA), 1:200 for goat anti-CD51 (eBioscience, San Diego, CA), and 1:1000 for anti-paxillin-TRITC (mouse monoclonal; BD Pharmingen, San Diego, CA). The LF were exposed to the anti-6His and anti-CD51 primary antibodies for 1.5 hours, and the coverslips were rinsed and then incubated for 1 hour with 1:1,000 dilution of Alexafluor 488 swine anti-goat IgG (InVitrogen, Molecular Probes, Carlsbad, CA). After rinsing, the coverslips were incubated with a solution containing anti-Paxillin and AlexaFluor 633–streptavidin (1:1,000 dilution to visualize the anti-6His; InVitrogen, Molecular Probes). After additional rinses, coverslips were mounted using mounting medium (0.05 M Tris-HCl pH 8.0, 90% glycerol, and 10 mg/ml 1,4,-Diazobicyclo[2,2,2] octane).

To localize collections of Rac, LF were adhered and spread on a separate set of coverlips that were prepared in the same fashion; however, autofluorescence was quenched by incubating in 200 mM glycine, 50 mM NaCl for 15 minutes. The fixed coverslips were incubated for 2 hours at 25°C with a 1:200 dilution mouse-monoclonal anti-Rac1 (BD Pharmingen) in PBS containing 0.1% Triton X-100 and 2 mg of BSA per ml. After rinsing with PBS containing Triton X-100 and BSA, the coverslips were incubated with a 1:500 dilution of goat anti-mouse IgG-biotin for 1.5 hours. After rinsing again, the coverslips were incubated for 30 minutes with a mixture of phalloidin-FITC (1:800 dilution; Sigma-Aldrich) and AlexaFluor 633–streptavidin (InVitrogen, Molecular Probes), at a 1:1,000 dilution in PBS containing Triton X-100 and BSA. A final rinse was performed using PBS and Triton X-100 without BSA and the coverslips were mounted. Three experiments were performed to examine fibrillin and integrin co-localization, and three experiments were performed to examine Rac and phalloidin. In an additional experiment, coverslips were prepared and LF were adhered in the presence of collagen plus either Fib1 30, Fib1 36–44, or Fib2 24. After incubating for 4 hours in the presence of absence of PDGF-A, the cells were fixed and actin was visualized with phalloidin. Using epifluorescence microscopy, 200 cells were evaluated for each condition and LF that exhibited either a filamentous or subcortical distribution of actin were enumerated.

Confocal Microscopy
A Zeiss LSM 510 scanning confocal microscope was used at a 512 x 512 pixel resolution, scan speed of 4, and 4 scans were averaged. A x40 objective was used, the optical slice was 6.6 µm, and the pinhole was 6.14 airy units for the examination of fibrillin and integrin co-localization. A x63 objective was used, the optical slice was 3.1 µm, and the pinhole was 2.8 and 3.5 airy units, respectively, for the examination of Rac and phalloidin. Representative images for each treatment condition were examined using ImageJ software and saved in the tagged image format (TIF). Composites were prepared using either Adobe Photoshop 6 or Adobe Illustrator CS2.

Quantification of Discrete Collections of Rac
Confocal microscopic images showing the fluorescence from anti–Rac-Alexafluor 633 were extracted from the Zeiss MDB files using Image J, converted to an RGB format, and saved as TIF files. These files were opened in IP Lab (BD-Bioscience, Rockville, MD), and a histogram of the number of pixels versus intensity was displayed. Because the intensity varied among images, a uniform increment, relative to the maximal intensity for each image, was used to ascertain the lower limit of intensity for segmentation. The pixels within this normalized range of intensity were identified by segmentation and gave a good representation of the perceived (by eye) boundaries of the discrete collections of Rac. The segmented images were uniformly subjected to erosion using a mask, which eliminated small artifactual collections of pixels. The number, centroid, and pixel area of Rac collections was systematically ascertained for each LF that was completely positioned within the image boundaries. This procedure provided a uniform set of criteria for excluding the large perinuclear collections and reduced the subjectivity associated with counting visually ascertained collections. All of the cells which fell completely within the boundaries of the images were analyzed.

Statistical Analyses
The data are expressed as mean ± 1 SEM. Only LF that moved more than 100 µm during the course of the time-lapse experiment were considered in the analysis of cellular haptokinesis. At least three separate time-lapse experiments were performed for each of the coating conditions and the data for individual cells were pooled across all of the experiments for a particular condition. This enabled us to analyzed at least 56 cells for each condition. The variance was expressed as one SEM rather than SD, as others have done (40). The data from the analyses of cellular adhesion and migration across the porous filters were normally distributed and were analyzed using a two-way ANOVA for adhesion and a one-way ANOVA for migration. The haptokinesis was not normally distributed and the nonparametric ANOVA on ranks was used for all of the comparisons from the time-lapse experiments except for the polarity, which was normally distributed. The Student-Newman-Keuls and the Dunn's post hoc tests were used for the parametric and nonparametric analyses, respectively. The Kruskal-Wallis post hoc test was used for the analysis of polarity. Differences were considered significant when P was less than 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Adhesion of LF to Plastic Coated with Fibrillin Peptides
The wells of microtiter plates were coated with Fib1 30, which lacks an RGD site, or either Fib1 36–44 or Fib 2 24, which both contain an RGD site. Figure 1 shows a representative SDS-PAGE gel after silver staining of Fib 1 30, and demonstrates that the isolated peptide migrated as one predominant band. Fib 1 36–44 and Fib2 24 exhibited similar levels of purity (not shown). Figure 2A shows that adhesion in the presence of peptides that contained an RGD was significantly higher than in the presence of Fib1 30, when peptide concentrations were added at concentrations at or greater than 0.4 µg/ml for Fib1 36–44 and at 4 µg/ml for Fib2 24. Pre-incubating LF with 20 µg of an anti-{alpha}V antibody per ml significantly reduced adhesion to Fib1 36–44 (Figure 2B). Because the RGD motif promoted adhesion, we also evaluated the effect of the RGD motif on the migration of LF.


Figure 1
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  		Figure 1. Purity of fibrillin 1 30 peptide. The recombinant bacterial product was subjected to SDS-PAGE and silver staining. Protein standards are shown on the left (std) and the lanes labeled as 1 and 5 contain 1 and 5 µg, respectively, of recombinant peptide.

 

Figure 2
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  		Figure 2. Arginine-glycine-aspartic acid (RGD)-containing fibrillin polypeptides increase lung fibroblast (LF) adhesion to plastic. (A) Naked polystyrene wells were coated with increasing concentrations of either Fib1 36–44, Fib2–24 (contain RGD), or Fib1 30 (lacks RGD). Adherent LF were enumerated and data were pooled from four separate experiments in which the means from quadruplicate wells were determined. Bars represent mean ± 1 SEM, *P < 0.05 comparing Fib1 36–44 to Fib1 30, #comparing Fib2 24 to Fib1 30. (B) Inhibition of the adhesion of LF to Fib1 36–44 by anti–{alpha}V-integrin was assessed as in A and the mean ± 1 SEM number of adherent LF are shown (n = 3, P < 0.05 compared with an 20 µg/ml of nonimmune IgG).

 
Fibrillin Peptides Increase LF Migration across Polycarbonate Filters
Because fibrillin peptides were added to only the lower chambers of the chemotaxis apparatus, whereas collagen was added to the top of the filter, LF adhesion to the top of the filter was primarily to collagen. Localization of the fibrillin to the bottom surface of the filter provided a step-increase in fibrillin concentration that could promote directional migration of the LF. Increasing concentrations of Fib1 36–44, Fib1 23–44, or Fib2 24 progressively promoted LF migration, whereas Fib1 30, which lacked the RGD motif, did not increase migration above that which was observed with only PDGF (Figure 3). Only Fib2 24 augmented migration when the fibrillin peptide concentration was 0.05 µg/ml. At concentrations equal to or exceeding 0.5 µg/ml, all three RGD-containing fibrillin peptides promoted migration except at 2 µg/ml, at which the effect of Fib1 23–44 was no longer significant.


Figure 3
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  		Figure 3. Fibrillin polypeptides containing RGD increase LF transmigration. The regions of the lower surface of a polycarbonate filter that were contained within the wells were coated with various concentrations of fibrillin peptides. LF were placed in the upper compartments and PDGF-AA in the lower compartments of a modified Boyden chamber. After fixation, the LF that migrated to the bottom of the filter were enumerated. The quantities of LF (mean ± 1 SEM, n = 4 separate analyses, for each condition) that migrated are shown, *P < 0.05, one-way ANOVA comparing an RGD-containing peptide with Fib1 30, at each concentration. PDGF was included in all wells except those shown for medium only. In some cases two asterisks are shown because two points overlap.

 
Fibrillin Peptides Increase LF Haptotaxis
To more precisely determine why the RGD-containing exons in Fib1 and Fib2 increased LF migration, we studied the movement of LF on surfaces that were coated with fibrillin peptides. When combined with time-lapse photography, this allows one to assess the movement of individual cells and to evaluate the velocity and persistence, two parameters that have been linked to biochemical events within the cell. During the migration period, the LF were exposed to a unidirectional flow of medium at 0.5 ml/hour. Coating with collagen alone promoted adhesion and allowed the cells to spread. The LF did not migrate in the absence of PDGF (data not shown), so all of the data that are shown were obtained in the presence of 50 ng of PDGF per ml. Representative cellular paths for LF on only collagen or collagen with either Fib1 36–44 or Fib2 24 are shown in Figure 4. When LF were exposed to only collagen or collagen plus Fib1 30 (lacks RGD), they migrated with a slower velocity than in the presence of Fib1 36–44 (Figure 5A). Addition of Fib1 36–44 to collagen significantly increased the velocity of migration by approximately 3-fold, but there was also a significant (~ 2-fold) decrease in persistence. The velocity of migration on coverslips, which had been coated with 4 µg of Fib2 24 per ml, was significantly higher than on coverslips coated with Fib1 30 but not those that were only coated with collagen. The persistence was significantly higher for LF that were migrating on coverslips coated with Fib2 24 compared with coverslips coated with either collagen alone or with collagen plus Fib1 36–44. The velocity of migration on Fib 1 30, which lacks an RGD motif, was similar to that observed in the presence of only collagen, and significantly less than in the presence of Fib1 36–44 or Fib2 24. The effects of different concentrations of Fib2 24 on LF migration are shown in Figure 5B. Coverslips coated with 4 µg of Fib2 24 per ml supported more rapid migration than did coverslips coated with 10 µg per ml.


Figure 4
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  		Figure 4. Haptokinesis of LF on glass coverslips that were coated with collagen in the absence or presence of fibrillin polypeptides (10 µg per ml of Fib1 36–44 or 4 µg per ml of Fib2 24). The LF resided at the origins of the cellular paths shown in the left column at the beginning of the time-lapse exposure. The x and y coordinates (·) of the cells are shown at hourly intervals. The right column shows the instantaneous velocities (the velocity of the cell during the preceding hour), which varied during the course of the experiment. Representative results are shown for one cell for each coating condition.

 

Figure 5
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  		Figure 5. Fibrillin 1 36–44 increases the velocity but diminished the persistence of LF. (A) Comparison of velocity and directional persistence of LF migrating on a glass surface that was treated with only collagen, or collagen plus either 10 µg of Fib1 30 per ml, 10 µg of Fib1–36–44, or 4 µg of Fib2 24 per ml. Velocity (open bars) and persistence (hatched bars) are shown as means and 1 SEM of 56, 91, 62, and 61 cells for only collagen, or with Fib1 30, Fib1 36–44, or Fib2 24, respectively. **Velocity for Fib1 36–44 compared with only collagen, Fib2 24, and Fib1 30; #velocity for Fib2 24, compared with Fib1 30: P < 0.05 ANOVA on ranks, Dunn's post hoc test; opersistence for Fib1 30 compared with only collagen; *persistence of Fib1 36–44 compared with only collagen, Fib1 30, or Fib2 24; {tau}Fib2 24 compared with collagen: P < 0.05, ANOVA on ranks, Dunn's post hoc test. (B) Effects of different concentrations of Fib2 24 [Fib2 24] along with a constant concentration of collagen on velocity (open bars) and persistence (hatched bars). Mean ± 1 SEM are shown using 56, 61, and 77 cells for 10, 4, and 2 µg of Fib2 24 per ml, respectively. *P < 0.05 for 4 compared with 10 mg of Fib2 24 per ml. ANOVA on ranks, Dunn's post hoc test.

 
Fibrillin Peptides Modify How LF Spread during Migration
The shape of cells that had been present on the coverslips for 6 hours in the presence of PDGF-AA were compared after coating with only collagen or collagen and equimolar RGD concentrations of Fib1 30, Fib1 36–44, or Fib2 24 per ml. The shapes of representative LF that are spread on collagen, Fib1 36–44, or Fib2 24 are shown in Figure 6. Lamellipodia that occupied the leading edge of the cells were also enumerated. Four parameters were assessed: area, perimeter, the ratio of perimeter to area (P/A) and number of lamellipodia. The results shown in Figure 7 indicate that LF that spread on Fib1 36–44 had a more convoluted (larger) perimeter, a larger P/A, and more lamellipodia at the leading edge.


Figure 6
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  		Figure 6. The shapes of three representative LF that are spread on collagen only or collagen plus either 10 µg of Fib1 36–44 or 4 µg of Fib2 24 per ml. The leading edges are highlighted in white, and on Fib1 36–44 more than one lamellipodium occupies the leading edge.

 

Figure 7
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  		Figure 7. Fib1 36–44 alters the shape of LF during haptotaxis. LF migrating on only collagen (open bars), Fib1 30 (vertical striped bars), Fib1 36–44 (hatched bars), or on Fib2 24 (cross-hatched bars). (A) Area in µm2 has been divided by 10 to permit use of the same ordinate scale as perimeter in µm. (B) Perimeter (P) divided by area (A, P/A, µm–1) has been multiplied by 10 to permit use of the same ordinate scale as the number of lamellipodia. Bars are mean ± 1 SEM for 40 LF migrating on only collagen (open bars), 52 LF migrating on 10 µg of Fib1 30 per ml + collagen (vertical striped bars), 33 LF on 10 µg of Fib1 36–44 + collagen (hatched bars), and 33 LF on 4 µg of Fib2 24 per ml + collagen (cross-hatched bars), respectively. In A, P < 0.05 perimeter for Fib1 36–44 compared with *collagen or with #Fib2 24. In B, *P/A for Fib1 36–44 compared with collagen, Fib1 30, or Fib2 24; **number of lamellipodia for Fib1 36–44 compared with collagen, Fib1 30, or Fib2 24. ANOVA on ranks, Dunn's post hoc test.

 
The Area of Protrusions in the Leading Edge Is Increased by Fibrillins
Cellular movement is accompanied by advancement of the leading edge and retraction at the trailing edge. Because the velocity increased after exposure to Fib1 36–44, we quantified advancement of the leading edge at 1-hour intervals during a period when a cell was migrating. The area change of the leading edge was expressed relative to the area of the entire cell to normalize for differences in cell size (percent positive flow). Fibrillin 1 36–44 and Fib2 24 each significantly increased the % positive flow relative to that observed when the surface was coated with only collagen or collagen along with Fib1 30 (Figure 8). Lung fibroblasts that migrated on Fib1 36–44 exhibited a lower polarity than LF that migrated on Fib1 30, Fib2 24, or only collagen. The percent negative flow (hourly areal change in the trailing edge relative to total cell area) was not altered by any of the fibrillin peptides (data not shown).


Figure 8
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  		Figure 8. Fib1 36–44 enhances cellular movement at the leading edge. The areal changes at the leading and trailing edges of LF were measured at six 1-hour intervals when the cells were continually moving. The areal change at the leading edge was divided by the area of the cell (% positive flow). The distance between the centroids of the protruding and retracting zones was divided by the length of the cell along the major axis (polarity). Bars are mean and 1 SEM for 12 cells randomly selected from four separate experiments for each treatment group, except Fib1 30, 14 cells. Positive flow: *,#P < 0.05, comparing % positive flow for LF on Fib1 36–44 with LF on Fib 1 30 or only collagen, respectively; {tau},+P < 0.05, comparing % positive flow for LF on Fib2 24 with LF on Fib 1 30 or only collagen, respectively; ANOVA on ranks, Kruskal-Wallis post hoc test. Polarity: ·P < 0.05, comparing LF on Fib1 36–44 and LF on only collagen; #P < 0.05, comparing polarity for LF on Fib1 36–44 and LF on Fib1 30; one-way ANOVA, Student-Newman-Keuls post hoc test.

 
Fibrillin Peptides Co-Localize with {alpha}V-Integrins but Not with Paxillin
Figure 9 (top panel) demonstrates co-localization of 6-His-labeled Fib1 36–44 and anti-CD5–labeled {alpha}V-integrin in a perinuclear distribution, when LF were permeabilized before adding the anti-{alpha}V antibody. The {alpha}V chain has a very similar distribution, indicating that intermolecular interactions could occur between the RGD domain of Fib2 24 and {alpha}V-integrins. When the anti-CD51 antibody was added before permeabilization (Figure 9, bottom two panels), the {alpha}V-integrin was only visualized at the focal contacts and not in a perinuclear distribution. Paxillin-containing focal contacts were only located at the periphery of the cells, and were not associated with the perinuclear collections of the {alpha}V-integrin. Paxillin and the {alpha}V-chains had similar distributions in the presence of 6-His–labeled Fib1 30.


Figure 9
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  		Figure 9. Fibrillins and {alpha}V-integrins co-localize in perinuclear region and at focal contacts of LF. After coating coverslips with Fib1 36–44 or Fib2 24, LF were allowed to adhere and spread in the presence of PDGF-A. After fixation, LF were permeabilized either before or after adding anti-CD51. Fib1 36–44 of Fib2 24 were identified by the 6-His tag, {alpha}V-chains were identified using anti-CD51, and focal contacts were identified by anti-paxillin. These results are representative of three other experiments using Fib1 36–44 or Fib2 24, and the distribution of antigens was similar for LF that adhered to either Fib1 30 (not shown).

 
Figure 10 shows that Rac is abundant in the region of the nucleus, but is also located at the periphery of the cell, particularly where phalloidin-stained subcortical actin is abundant. PDGF-A promoted the formation of peripheral collections of Rac, which were particularly dense in lamellipodia. These dense lamellipodial collections of Rac were significantly more abundant in LF on Fib1 36–44, than on either Fib2 24, Fib1 30 (Figure 10B). Because the antibody that we used recognizes an epitope that is not modified by interaction with GTP, we were unable to determine whether Rac was in the active, GTP-bound state. Although it appears from Figure 10A that the distribution of actin varies with the type of fibrillin peptide that the LF were exposed to, this was not evident when the configuration of actin filaments was analyzed in a larger number of cells. The large majority of fibroblasts contained filamentous rather than subcortical actin. In the absence of PDGF-A, filamentous actin predominated in 98.4, 95.1, and 90.4 percent of fibroblasts on Fib1 20, Fib1 36–44, and Fib2 24, respectively. In the presence of PDGF-A, filamentous actin predominated in 97.2, 96.6, and 98.6 percent of fibroblasts on Fib1 20, Fib1 36–44, and Fib2 24, respectively.


Figure 10
Figure 10
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  		Figure 10. Rac is differentially distributed in the presence of Fib 1–36–44. Coverslips were coated with either Fib1 30, Fib1 36–44, or Fib2 24 and LF were allowed to adhere and spread in either the presence of the absence of PDGF-A. After fixation, Rac1 was immunostained and actin filaments were identified using phalloidin. (A) In the absence of PDGF-A the distribution of Rac was primarily perinuclear but became more diffuse in the presence of PDGF-A. Discrete collections of Rac (arrows) were observed in the protrusions of LF that had spread on Fib1 36–44; collections of Rac were less frequently observed in LF that had spread on Fib2 24. (B) Discrete collections of Rac were enumerated (see MATERIALS AND METHODS). The mean ± SEM number of Rac collections per cell from analyzing 20, 26, and 24 for LF on Fib1 30, Fib1 36–44, and Fib2 24, respectively, are shown. *P < 0.01 Fib1 36–44 compared with Fib1 30 and Fib2 24, one-way ANOVA, Student-Newman-Keuls post hoc test.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Whereas the RGD motifs in both Fib1 36–44 and Fib2 24 polypeptides had similar effects on LF adhesion and migration across porous filters, distinct differences in haptotaxis were observed when LF migrated on a coated glass surface. In the presence of Fib1 36–44, LF migration was characterized by greater velocity, more dispersed lamellipodia, and less persistence than in the presence of Fib2 24. Because the molar coating-concentration of RGD was the same for both Fib1 36–44 or Fib2 24, portions of the peptides outside the immediate RGD motif may influence the cellular response. These influences could involve differences in peptide folding that alter how the RGD motifs are presented to the LF, or differences in cellular association with or response to other portions of the polypeptides. There may also be differences in the way Fib1 36–44 and Fib2 24 promote interactions between cellular integrins and Rac GTPase. The precise mechanisms that mediate the varied haptotactic responses to Fib1 36–44 and Fib2 24 remain undefined, but our observations indicate that the context of the RGD moiety may be important.

Recent studies by Bax and associates showed that cellular adhesion is influenced by the context in which the RGD motif is presented (26). Their studies revealed that adhesion is greatly enhanced when exon 37 of fibrillin 1 is presented in association with one or more upstream EGF-like domains (26). The adhesion of human dermal fibroblasts was primarily mediated by {alpha}1β5–integrin, although {alpha}Vβ3-integrins played a lesser role. They also identified a heparin-binding site in exon 41 that was not required for adhesion but promoted the formation of focal contacts. The EGF-like Exon 36, which Bax and associates (EGF-22 by their terminology) showed enhances adhesion, is also present on our construct Fib1 36–44. Fib1 36–44 also contains the heparin-binding motif in exon 41 that was characterized by Bax and coworkers. Although Fib2 24 lacks both the flanking EGF-like domain and the heparin-binding region, we observed significant adhesion of LF to Fib2 24 at 4 µg per ml, although it was lower than Fib1 36–44 at 0.4 µg per ml.

The adhesion of LF was significantly enhanced by coating plastic with RGD-containing Fib 1 or Fib 2 peptides, and was increased by more than 2-fold, compared with a peptide that lacked the RGD, when the wells were coated using 4 µg of peptide per ml. Others have observed that fibrillins increase the adhesion of bovine chondroblasts and ligamentum nuchae fibroblasts or various transformed epithelial or meshenchymal cell lines (15, 16). As we have observed with rat LF, the adhesion of bovine nuchal ligament fibroblasts is also inhibited by blocking the function of {alpha}Vβ3-integrins (16).

Fib2 24 supported migration of LF across collagen coated polycarbonate filters to a greater extent than Fib1 36–44 at a fibrillin concentration of 0.5 µg per ml. However, migration on Fib2 24 and Fib1 36–44 or Fib1 23–44 were similar when the bottom surface of the filter had been coated with 2 µg of either peptide per ml. Others have suggested that the RGD in exon 24 of Fib 2 does not participate in cell attachment based on Sakamoto and associates finding that a 12–amino acid peptide containing the RGD did not disrupt cellular adhesion to purified fibrillin-1 (16). Our findings indicate that the RGD in exon 24 of Fib 2 promotes cell adhesion and migration when it is associated with the remaining 73 amino acids in this exon. We studied the direct attachment of LF to peptides containing the RGD motif, whereas Sakamoto and associates used the short peptide containing the Fib2 24-RGD as a competitor to disrupt attachment (16, 41). We observed co-localization of Fib2 24 and {alpha}V-integrin (Figure 9), which is also consistent with (although not proof of) a physical interaction between these two proteins.

Lung fibroblasts clearly migrate more slowly than some other types of cells (neutrophils and some epithelial cells, for example). Precise quantitation of velocity is more difficult in slowly moving cells, because cell spreading could be mistaken for movement, and the velocity is not constant (Figure 4). Others have observed saltatory movement of more slowly migrating cells (42). We have used the random-walk model, which others have shown accurately describes the behavior of fibroblasts from other sources on surfaces that are coated with extracellular matrix molecules (43). We used a prominent landmark in the nucleus rather than the cell centroid to plot the location of the cell at different times, because the nuclear land mark is less influenced by alterations in cell shape, in the absence of movement. Because cellular movement exhibits a stochastic response, it was necessary to analyze at least 50 cells (44). There was significant variation in velocity and persistence among the cells that migrated on a particular substrate, and this variation was not normally distributed. Therefore we used nonparametric statistical methods, which ranked the cells according to the magnitude of either velocity or persistence, and then compared the effects of the different substrates. This reduced the effects of cell-to-cell variation, and promoted a meaningful comparison of the populations of cells within the various treatment (coating substrate) groups.

Others have shown that integrins influence the velocity and persistence of migration through several potential mechanisms. Persistence is directly correlated with the coating concentration of integrins (40). However, this is unlikely to account for the differences that we observed, because our coating concentrations were adjusted to provide equal mols of RGD moieties for Fib1 36–44 and Fib2 24. Furthermore, the persistence was not significantly altered by varying the concentration of Fib2 24. However, differences in concentration may contribute to the higher persistence that was observed on Fib1 30 plus collagen, because both coating proteins were present at 10 µg per ml, compared with 10 µg per ml for the collagen-only condition. We did observe a concentration-related inverse correlation with velocity of migration on Fib2 24, because the velocity was greater on 4 µg than on 10 µg of Fib2 24 per ml (Figure 5B). Therefore, it is more likely that fibrillin-peptide structural differences account for the greater persistence on Fib2 24 than on Fib1 36–44 (Figure 5A). There are two structural differences between Fib1 36–44 and Fib2 24 that may contribute to the observed differences in LF migration. Fib1 36–44 has an EGF-like domain just upstream to exon 37, which produces a hairpin loop in the protein and promotes exposure of the RGD to integrins (45). In addition, exon 41 of Fib1 36–44 encodes for an arginine-arginine dipeptide heparin-binding motif that promotes the stabilization of focal plaques (26). Pankov and associates showed that persistence is inversely correlated with the intracellular levels of activated Rac (9). If the regions encoded by exon 36 and/or exon 41 in Fib1 36–44 promote Rac-activity, then their absence in Fib2 24 may foster greater persistence. We observed that LF migrating on Fib2 24 exhibit fewer lamellipodia at the leading edge (Figure 7) and greater polarity (Figure 8) than LF migrating on Fib1 36–44, which is consistent with the findings of Pankov and coworkers (9). Furthermore, Figure 10 demonstrates that in the presence of PDGF-A, LF have significantly more collections of Rac, as well as more lamellopodia when migrating on Fib1 36–44 compared with Fib 2 24. This suggests that Fib1 36–44 and PDGF-A may influence Rac to promote less persistent migration than on Fib2 24.

Guo and associates have recently shown that PDGF promoted the p21-activated kinase activation of Rac, resulting in greater guanalyl cyclase activity (46). This process fostered the formation of lamellipodia and a greater velocity of migration. Therefore Fib1 36–44 may enhance the effect of PDGF, resulting in greater velocity and lower persistence than for LF migrating on Fib 2 24. Our observation that Fib1 36–44 supports greater areal change (% positive flow) at the leading edge suggests that there is more turnover of integrin–fibrillin peptide contacts and actin reorganization, a known effect of PDGF in fibroblasts (7). The precise features of Fib1 36–44 that could foster this process require further elucidation.

Disruption of the elastic fiber network is one of the hallmarks of pulmonary emphysema (47, 48). Whereas the levels of elastin may be restored in emphysematous lungs, the elastic fiber network remains discontinuous and dysfunctional. Rat LF deposit tropoelastin as they migrate in vitro (49). Structural and functional restoration of the network likely involves directional migration of interstitial LF along contiguous and continuous pathways. Our studies provide new insight into how fibrillin interacts with LF and could assist in the development of new strategies for restoring a functional elastic fiber network in pulmonary emphysema.


   Footnotes
 
This research was supported by the Department of Veterans Affairs Research Service.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0281OC on November 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 23, 2007

Accepted in final form September 25, 2007


   References
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 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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