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The Journal of Immunology, 2007, 178, 7911 -7922
Copyright © 2007 by The American Association of Immunologists, Inc.
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RNA Interference Elucidates the Role of Focal Adhesion Kinase in HLA Class I-Mediated Focal Adhesion Complex Formation and Proliferation in Human Endothelial Cells1

Yi-Ping Jin*, Yael Korin*, Xiaohai Zhang*, Peter T. Jindra*, Enrique Rozengurt{dagger} and Elaine F. Reed2,*

* Department of Pathology and {dagger} Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr418, p85 PI3K, and Akt at both Thr308 and Ser473 sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try118 and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chronic rejection is the leading cause of late heart, lung, and renal allograft loss, and is estimated to affect >40% of recipients within 5 years following transplantation. The hallmark of chronic rejection is transplant-associated vasculopathy (TAV),3 which is characterized by intimal thickening, occlusion of the vessels of the graft, and deterioration of organ function. Recent studies have shown that angiogenesis, which, mediated by endothelial cell (EC) proliferation and migration, is a common feature of allografts diagnosed with TAV (1, 2, 3).

Anti-HLA Ab have long been implicated in the process of chronic allograft rejection because numerous studies have shown that patients developing posttransplant anti-donor Ab to the mismatched HLA class I and class II molecules of the allograft are at increased risk of developing TAV and graft loss (4, 5). Although the development of posttransplant anti-HLA Ab is linked to TAV, the physiologic and pathologic effect of their binding to the endothelium of the transplanted organ has only recently been explored. Studies by our group and others have shown that ligation of HLA class I molecules on the surface of EC by murine and human mAbs and polyclonal Abs promotes diverse biological functions, including cellular proliferation and survival in a model relevant to the development of TAV (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Engagement of class I molecules by anti-HLA Ab induces Rho activation; stimulates tyrosine phosphorylation of intracellular proteins, including Src, focal adhesion kinase (FAK), and paxillin; and enhances proliferative responses to basic fibroblast growth factor (bFGF) (6, 7, 8, 9, 10, 12, 13, 14, 15). A key step in the HLA class I signaling pathway is phosphorylation of FAK, a central element of the focal adhesion signaling complex (9). FAK is a cytoplasmic protein tyrosine kinase that discretely localizes to regions of the cell that attach to the extracellular matrix, called focal adhesions. FAK is an important mediator in regulating growth-factor and integrin signaling, cell survival, cell proliferation, and cell migration (18), and therefore plays a critical role in embryonic development, wound repair, atherosclerosis, and cancer (19). The ability of FAK to transmit survival and proliferation signals to downstream targets is dependent on its interactions with several intracellular signaling molecules, including Src, paxillin, and PI3K (19). In accordance with these findings, ligation of class I molecules triggers a prosurvival signaling cascade, resulting in phosphorylation of PI3K and Akt and up-regulation of the antiapoptotic proteins Bcl-2 and Bcl-xL in EC (10, 16, 17). Biopsies from heart allograft recipients with evidence of Ab-mediated rejection also displayed increased phosphorylation of S6 ribosomal protein and Bcl-2 expression on the vascular endothelium of the graft (10, 15).

Rapid increases in phosphorylation of Src, FAK, PI3K, and Akt are prominent events in response to HLA class I ligation on EC. However, the precise cause-effect connections in the signal transduction pathways induced by class I ligation remain to be investigated. In earlier studies, we observed that class I-induced activation of the PI3K/Akt cell survival pathway and fibroblast growth factor (FGF) receptor (FGFR) expression was inhibited by cytochalasin D and latrunculin A, suggesting that FAK regulates both class I-mediated activation of the PI3K/Akt survival pathway and cell proliferation (10). However, much of what we conjecture about FAK interactions with effectors in the class I signal transduction pathway is based upon the pharmacological inhibitors cytochalasin D and latrunculin A that prevent actin polymerization (20). This approach, although informative, is limited by the challenges inherent in establishing the specificity of these agents. Small interfering RNA (siRNA) technology has emerged as a powerful tool to examine the function of specific gene products and their relationship to other proteins in a well-characterized, cultured primary cell system (21).

The studies described in this work were aimed at elucidating the role of FAK in MHC class I-induced cell proliferation and cell survival. Using siRNA to knock down FAK in EC, we show that FAK plays a critical role in anti-HLA Ab-induced assembly of focal adhesions, stress fiber formation, and activation of the PI3K/Akt pathway. Contrary to our previous data using pharmacological inhibitors, FAK expression blockade by siRNA did not interfere with class I-induced FGFR plasma membrane expression. However, FAK knockdown inhibited class I-induced proliferative responses, demonstrating a role for FAK in regulating class I-mediated cell cycle progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and chemicals

Cell culture reagents were from Invitrogen Life Technologies. Mirus TransIT-TKO transfection reagent was purchased from Mirus. The hybridoma (HB-95) W6/32, recognizing a monomorphic epitope on HLA class I, was purchased from the American Type Culture Collection and purified by protein A-agarose affinity chromatography. The mouse IgG mAb isotype control, mAb against vinculin (V9131), and protein A-agarose were purchased from Sigma-Aldrich. Anti-FAK, 2A7, mAb, and PI3K p85 antiserum were from Upstate Biotechnology. The rabbit polyclonal Ab against FAK (C-20), c-Src (SRC2), beta-tubulin (H-235), and protein A/G plus-agarose were obtained from Santa Cruz Biotechnology. Mouse mAb against proline-rich tyrosine kinase 2 (PYK2)/cell adhesion kinase beta (catalog no. 610548) and paxillin (catalog no. 610052) were from BD Transduction Laboratories. Rabbit polyclonal Ab against phospho-FAK (Tyr925), phospho-paxillin (Tyr118), phospho-(Tyr) p85 PI3K, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt Ab were from Cell Signaling Technology. Polyclonal Ab against phospho-FAK (Tyr397), phosphor-FAK (Tyr576), and phospho-FAK (Tyr577) were obtained from BioSource International. Alexa Fluor 488 rabbit anti-mouse IgG and goat anti-rabbit IgG (catalog no. A11054), Texas Red-X phalloidin (catalog no. T7471), and Vybrant CFDA SE Cell Tracer Kit (V-12883) were purchased from Molecular Probes. FITC-conjugated donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories.

Cell culture

Primary human aortic EC were isolated from the aortic rings of explanted donor hearts, as described previously (22), and cultured in M199 medium supplemented with 20% (v/v) FBS, penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively; both from Invitrogen Life Technologies), sodium pyruvate (1 mM), heparin (90 µg/ml; Sigma-Aldrich), and EC growth supplement (20 µg/ml; Fisher Scientific). Human aortic EC from a single donor (lot no. EC5555) was obtained from Cambrex and maintained in EC basal medium, supplemented with 5% FBS, 10 ng/ml human epidermal growth factor, 3 mg/ml bovine brain extract, 1.0 mg/ml hydrocortisone, and 5 mg/ml gentamicin (Cambrex) at 37°C in a humidified incubator (5% CO2, 95% air). Cells from passages 3–8 were used at a confluence of 70–80%. Before use in experiments, cells were grown for 16 h in medium containing 0.2% FBS.

siRNA transfection

The target sequence (GGTTCAAGCTGGATTATTT) was selected from cDNA library of FAK, which corresponds to nt 1006–1024 of the coding region of human FAK (GenBank accession no. NM 005607). siRNA duplexes (5'-GGU UCA AGC UGG AUU AUU U-3' and 5'-AAA UAA UCC AGC UUG AAC C-3') and control nontargeting siRNA duplexes (5'-UAG CGA CUA AAC ACA UCA AUU-3' and 5'-AAU UGA UGU GUU UAG UCG CUA-3') were synthesized by Dharmacon. EC were plated at a density of 70% confluency in 35-mm dishes in FBS-free medium M199 and transfected with siRNA using Mirus TransIT-TKO transfection reagents, according to the manufacturer’s protocol. For each transfection, 4 µl of the Mirus transfection reagent was mixed with 200 µl of serum-free medium OPTI-MEM I in a 5-ml tube and incubated for 5 min at room temperature. Following the incubation, 60 nM siRNA was added into the mixture and incubated for 5 min at room temperature. The cells were washed once with serum-free medium M199 and transfected with the siRNA mixture. Fresh complete medium was added to the cells 5 h after transfection, and experiments were conducted 48 h posttransfection. Dosing experiments demonstrated that optimal FAK protein inhibition was achieved using 60 nM FAK siRNA. Immunoblotting with anti-FAK Ab was performed to monitor the efficiency of FAK knockdown. Anti-vinculin mAb was used to confirm equal loading of cellular proteins in lysates.

Cell lysates, immunoprecipitation, and Western blotting

EC were treated with anti-HLA class I mAb W6/32 or control mouse IgG, and cell lysates for Western blot and immunoprecipitation were prepared, as previously described (9). The phosphorylated bands were scanned using the GS-710 Calibrated Imaging Densitometer (Bio-Rad). Data represent at least three independent experiments. Densitometry data are expressed as the percentage of maximal increase in phosphorylation above control values (mean + SE).

Flow cytometry

HLA class I expression. EC were grown in 2 ml of complete medium in 35-mm dishes coated with 0.1% gelatin until confluent. EC were detached with 0.125% trypsin/0.05% EDTA, washed with PBS containing 2.5% FBS and 0.1% sodium azide, and treated with different concentrations of anti-HLA class I mAb W6/32 for 30 min at 4°C with continuous mixing. EC were washed three times and incubated with 50 µl of 1/400 diluted FITC-conjugated anti-mouse IgG Ab (Jackson ImmunoResearch Laboratories) for 30 min at 4°C and washed three times, and cell fluorescence was analyzed on a FACSCalibur flow cytometry using CellQuest Software (BD Biosciences). Gates for forward and side scatter measurements were set on EC, and a minimum of 10,000 events was acquired.

FGFR expression

EC were stimulated with anti-HLA class I mAb W6/32 for 2 h at 37°C, and expression of FGFR was determined by indirect immunofluorescence, as previously described (7, 9).

Immunohistochemical staining

Indirect immunofluorescence analysis of FAK and paxillin was performed, as previously described (9, 23, 24). Briefly, EC were fixed in 10% buffered formalin phosphate for 20 min at room temperature. Cells were rinsed three times with PBS and permeabilized with 0.2% Triton X-100 in PBS. After extensive washing, EC were incubated for 1 h at room temperature in blocking buffer (PBS containing 5% BSA) and then stained for 60 min at room temperature with primary Ab to FAK (C-20) or paxillin. Subsequently, the cells were washed with PBS and incubated with Alexa Fluor 488 anti-rabbit IgG secondary Ab for FAK or anti-mouse IgG secondary Ab for paxillin for 30 min at room temperature. The samples were mounted with the ProLong Antifade Kit (catalog no. P7481; Molecular Probes). Fluorescence images were observed with an epifluorescence microscope (Zeiss Axioskop; Zeiss) with a Zeiss water immersion objective for FAK staining (Achoplan x40/0.75w; Zeiss) or with a Zeiss oil immersion objective for paxillin (Plan-Apochromat x63/1.4 oil; Zeiss). Images were captured as uncompressed 24-bit TIFF files with a SPOT cooled (–12°C) single charge-coupled device color digital camera (three pass method) driven by SPOT version 4.1 software (Diagnostic Instruments). Fluorescence signals were observed with HI Q filter sets for FITC (Chroma Technology). Images were processed using Adobe Photoshop CS.

F-actin staining

F-actin was visualized by direct staining with Texas Red-X phalloidin (Molecular Probes), as previously described (14). Briefly, EC grown to 80% confluence on glass coverslips coated with 0.1% gelatin were starved for 3 h in basal M199 medium containing 1% FBS, followed by treatment with mAb W6/32 or isotype control mouse IgG for different times. Treated EC were fixed in 3.7% formaldehyde solution and permeabilized with 0.1% Triton X-100/PBS. The presence of F-actin was visualized by direct staining with Texas Red-X phalloidin (Molecular Probes). Cells were analyzed with a Zeiss Axioskop epifluorescence microscope.

Cell proliferation assays

BrdU incorporation. DNA synthesis was measured by BrdU incorporation using the ABSOLUTE-S SBIP Cell Proliferation Assay Kit (A-23150) from Molecular Probes, according to the manufacturer’s protocol. Briefly, EC grown to 70% confluence in 35-mm culture dishes coated with 0.1% gelatin in medium containing 2.5% FBS were treated with W6/32 or isotype control IgG for 48 h. During the final 16 h of incubation, 6 µg/ml BrdU was added to the culture. The medium containing BrdU was removed, and 2 ml of prewarmed PBS was added and the EC were exposed to UV light for 10 min. The cells were detached with trypsin, fixed, and permeabilized in 1 ml of 70% ethanol at –20°C. After an overnight incubation, the cells were incubated for 60 min at 37°C with 10 µl of DNA-labeling solution, containing BrdUTP and TdT. The cells were washed and incubated with 20 µl (1/20 diluted) of Alexa Fluor 488 dye-labeled anti-BrdU Ab for 30 min at room temperature and then incubated with 100 µl of propidium iodide/RNase A in the dark for 30 min at room temperature. The cells were analyzed for simultaneous green (FL1) and red (FL3) fluorescence emission on a FACSCalibur flow cytometer.

CFSE labeling

EC were labeled with CFSE (Molecular Probes), according to the manufacturer’s directions, and stimulated with anti-class I mAb W6/32 for 48 h. Fluorescence was analyzed on FACSCalibur using CellQuest software. Freshly CFSE-labeled EC and CFSE-labeled EC grown in complete medium were used as negative and positive controls for staining, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
siRNA inhibits FAK expression in EC

To determine the effect of FAK siRNA on FAK expression, cells were transfected with increasing concentrations of FAK siRNA, and FAK protein expression was determined by Western blot. FAK expression was markedly inhibited in a dose-dependent manner (Fig. 1A). Densitometric analysis revealed a 62, 71, and 92% reduction in endogenous FAK protein expression following transfection with 25, 50, and 100 nM FAK siRNA, respectively (Fig. 1A, lanes 2–4). In contrast, transfection with the nontargeting siRNA had no effect on FAK expression (Fig. 1A, lanes 5–7).


Figure 1
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  		FIGURE 1. Effect of siRNA on FAK protein expression and distribution in EC. A, EC were transfected with 25–100 nM FAK siRNA or control siRNA. After 48 h, the cells were lysed and subjected to Western blot analysis using Abs to FAK, PYK2, vinculin, beta-tubulin, and beta-actin. Densitometry results of FAK and PYK2 are expressed as the percentage of maximal increase in phosphorylation above control values. B, EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h. The cells were fixed, permeabilized, and stained with anti-FAK Ab. Bar, 50 µm. Results presented in A and B are representative of three independent experiments. C, EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h. Transfected cells were detached and incubated with different concentrations of anti-class I mAb W6/32 for 30 min, followed by staining with FITC anti-mouse IgG Ab. HLA class I expression was analyzed by flow cytometry. Values shown are the mean ± SE of at least three independent experiments, and are expressed as median channel value of fluorescence.

 
To exclude the possibility that nonspecific effects of FAK siRNA contribute to the results we observed on class I-mediated activation of EC, we examined the expression of a variety of cytoskeletal proteins in the same cell lysates of EC transfected with FAK siRNA. Transfection of EC with control or FAK siRNA did not substantially change the expression of any of these cytoskeletal proteins (Fig. 1A). The PYK2, a second member of the FAK subfamily, has a conserved domain structure and significant sequence homology to FAK, and is also endogenously expressed by EC used in our experiments. Transfection of EC with FAK siRNA did not appreciably alter the expression of PYK2 (Fig. 1A), illustrating that it is feasible to knock down FAK expression without inducing a compensatory change in the level of a closely related protein, PYK2.

To further characterize the effect of FAK siRNA on FAK protein expression levels and distribution, we performed indirect immunofluorescence analysis of FAK in EC transfected with FAK siRNA and control siRNA. EC transfected with FAK siRNA showed a marked reduction in FAK expression consistent with the loss of FAK expression (Fig. 1B). In contrast, FAK was equally distributed in control cells or cells transfected with nontargeting siRNA. Immunofluorescence staining of cells following FAK siRNA treatment revealed that ~90% of the cells had reduced FAK expression.

To further assess the specificity of the FAK siRNA, we examined the expression of HLA class I proteins on EC treated with FAK siRNA. The binding capacity of the anti-class I mAb increased in a dose-dependent manner, reaching maximal binding at a concentration of 1.0 µg/ml (Fig. 1C). Cells transfected with FAK siRNA did not show a reduction in HLA class I staining, whereas FAK protein expression in lysates from these cells was dramatically inhibited. The results indicate that FAK siRNA efficiently and specifically inhibited expression of FAK in EC.

Impact of siRNA knockdown of FAK on Src protein tyrosine kinase

The Tyr397 autophosphorylation site on FAK creates a high-affinity binding site for the Src homology (SH)2 domain of Src kinases and can lead to the formation of a signaling complex between FAK and Src kinases (25, 26, 27, 28). The assembly of a complex between FAK and Src has been show to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases (29). Thus, the negative regulatory site of Src becomes hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of Src from a diffuse cellular location to focal adhesions. Recent studies have shown that a FAK mutant defective for Src binding does not effectively induce the translocation of Src to focal adhesions, indicating that a FAK-Src complex is a prerequisite for Src activation and phosphorylation of other focal adhesion proteins (29). To determine whether FAK is required for class I-mediated activation of Src, we studied the effect of siRNA knockdown of FAK on class I-mediated phosphorylation of Src at Tyr418, located in the catalytic domain of the kinase. Exposure of EC transfected with nontargeting siRNA to varying concentrations of anti-class I Ab resulted in a dose-dependent increase in phosphorylation of Src at Tyr418 (Fig. 2A). Transfection of EC with FAK siRNA prevented class I-mediated phosphorylation of Src at Tyr418. Knockdown of FAK expression by siRNA also blocked class I-induced phosphorylation of Src in a time-dependent manner. Down-regulation of FAK expression impaired class I-mediated phosphorylation of Src at Tyr418 in EC over a period from 1 to 60 min (Fig. 2B). These results support the idea that FAK-Src interactions are required for class I-mediated activation of Src.


Figure 2
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  		FIGURE 2. FAK knockdown prevents HLA class I-mediated phosphorylation of Src in EC. EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h and treated with the following: different concentrations of mAb W6/32 (A) or for various time points (B). EC were lysed and immunoprecipitated with anti-Src Ab, followed by immunoblotting with phospho-anti-Src Y-418 Ab. Immunoblotting with anti-Src (SRC2) Ab was performed to confirm equal loading of proteins. Immunoblotting with anti-FAK (C-20) Ab was performed to monitor the efficiency of FAK knockdown and with anti-vinculin Ab to confirm equal loading of cellular proteins in the lysates. Data represent at least three independent experiments. Densitometry results are expressed as the percentage of maximal increase in phosphorylation above control values (mean + SE).

 
FAK knockdown inhibits class I-induced phosphorylation of p85 PI3K

The capacity of FAK to transmit signals to downstream targets is dependent on its ability to interact with several intracellular signaling molecules, including PI3K. Phosphorylation of FAK at Tyr397 promotes the assembly of a signaling complex with PI3K and provides a plausible mechanism for anti-HLA class I Ab activation of PI3K/Akt signaling pathway (24). To determine whether FAK is necessary for class I-mediated PI3K activation, cultures of EC were transfected with targeting or nontargeting FAK siRNA. Treatment of EC transfected with nontargeting siRNA with varying concentrations of anti-class I Ab resulted in a dose-dependent increase in phosphorylation of p85 PI3K (Fig. 3A). Transfection of EC with FAK siRNA reduced class I-stimulated phosphorylation of PI3K by 75, 85, and 75% when cells were treated with 0.01, 0.1, and 1.0 µg/ml mAb W6/32, respectively (Fig. 3A). Down-regulation of FAK expression was sufficient to impair class I-mediated activation of PI3K in EC over a period of 1–60 min. Treatment with anti-class I Ab triggered a rapid increase in phosphorylation of p85 PI3K as early as 1 min that was sustained at high levels for at least 60 min (Fig. 3B). Transfection of EC with FAK siRNA attenuated HLA class I-induced phosphorylation of p85 PI3K compared with nontargeting siRNA-transfected cells. These results demonstrate that inhibition of FAK expression by siRNA severely impaired class I-mediated PI3K phosphorylation in EC.


Figure 3
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  		FIGURE 3. FAK knockdown prevents HLA class I-mediated phosphorylation of p85 PI3K in EC. EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h and treated with the following: different concentrations of mAb W6/32 (A) or for various time points (B). Cells were lysed and immunoprecipitated with anti-p85 PI3K Ab, followed by immunoblotting with a phospho-anti-p85 PI3K Ab. Immunoblotting with anti-PI3K Ab was performed to confirm equal loading of proteins. Immunoblotting with anti-FAK (C-20) Ab was performed to monitor the efficiency of FAK knockdown and with anti-vinculin Ab to confirm equal loading of cellular proteins in the lysates. Data represent at least three independent experiments. Densitometry results are expressed as the percentage of maximal increase in phosphorylation above control values (mean + SE).

 
FAK knockdown inhibits class I-induced phosphorylation of Akt in EC

Akt is a major substrate downstream of FAK and PI3K (30) and is activated by HLA class I ligation (10). We therefore used the approach of siRNA knockdown to explore the role of FAK in HLA class I-mediated activation of Akt. To better characterize the effects of FAK in the activation of Akt, we determined the dose response and time course for class I-induced Akt phosphorylation in control siRNA or FAK siRNA-transfected EC. Treatment of EC transfected with nontargeting siRNA with various concentrations of anti-class I mAb induced a dose-dependent increase in phosphorylation of Akt at both sites Ser473 and Thr308 (Fig. 4A), with the highest level of phosphorylation observed at the lowest concentration of anti-HLA Ab. Exposure to 0.01 µg/ml anti-class I Ab for 10 min resulted in a 5-fold increase in phosphorylation of Akt at Ser473 and a 3.7-fold increase of Akt at Thr308 (Fig. 4A). Treatment with 1 µg/ml anti-class I Ab for 10 min stimulated a 3-fold increase in phosphorylation of Akt at Ser473 and a 2.4-fold increase of Akt at Thr308. Treatment of control EC with anti-class I Ab also stimulated a time-dependent increase in phosphorylation of Akt at both Ser473 and Thr308 that peaked at 30 min (Fig. 4B). Transfection of EC with FAK siRNA resulted in an 80% inhibition of Akt phosphorylation at both Ser473 and Thr308 when cells were treated with 0.01 µg/ml mAb W6/32. Down-regulation of FAK expression was sufficient to impair class I-mediated activation of Akt in EC over a time period from 10 to 60 min. These results demonstrate that inhibition of FAK expression by siRNA blocks class I-mediated Akt phosphorylation in EC.


Figure 4
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  		FIGURE 4. Transfection of EC with FAK siRNA impairs HLA class I-mediated Akt phosphorylation. EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h and treated with the following: different concentrations of mAb W6/32 (A) or for various time points (B), and immunoblotted with phospho anti-Akt-Thr308 and Akt-Ser473 Ab. The membranes were immunoblotted with anti-Akt Ab to confirm equal loading of total Akt. Immunoblotting with anti-FAK (C-20) Ab was performed to monitor the efficiency of FAK knockdown and with anti-vinculin Ab to confirm equal loading of cellular proteins in the lysates. Data represent at least three independent experiments. Densitometry results are expressed as the percentage of maximal increase in phosphorylation above control values (mean + SE).

 
FAK knockdown decreases class I-mediated paxillin phosphorylation and impairs subcellular distribution

Paxillin is one of the key signaling components required for cell adhesion and migration by regulating focal adhesion formation and turnover (31). Paxillin is phosphorylated by FAK-Src on Tyr31 and Tyr118. To characterize the role of FAK in regulating activation and distribution of paxillin, we studied the capacity of anti-class I Ab to stimulate phosphorylation of paxillin at Tyr118 in EC transfected with FAK siRNA. Exposure of EC to various doses of anti-class I Ab stimulated a dose-dependent increase in phosphorylation of paxillin. Exposure to 0.01 and 1 µg/ml anti-class I Ab for 10 min resulted in a 2- and 3-fold increase in phosphorylation of paxillin at Tyr118 in EC transfected with control siRNA, respectively (Fig. 5A). Treatment with anti-class I Ab also stimulated a time-dependent increase in phosphorylation of paxillin at Tyr118 in EC transfected with control siRNA (Fig. 5B). The increased phosphorylation was detected as early as 1 min and reached a maximum level between 10 and 30 min after treatment (Fig. 5B). Class I-mediated phosphorylation of paxillin was impaired in FAK siRNA-transfected cells (Fig. 5B).


Figure 5
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  		FIGURE 5. FAK knockdown attenuates HLA class I-mediated phosphorylation of paxillin and inhibits assembly of focal adhesions in EC. A and B, EC were transfected with 60 nM FAK siRNA or control siRNA for 48 h and treated with the following: different concentrations of mAb W6/32 (A) or for various time points (B), and immunoblotted with an anti-paxillin Ab to confirm equal loading of paxillin. Immunoblotting with anti-FAK (C-20) Ab was performed to monitor the efficiency of FAK knockdown and with anti-vinculin Ab to confirm equal loading of cellular proteins in the lysates. C, EC were transfected with 60 nM FAK siRNA (d–f), control siRNA (g–i), or without siRNA (a–c); treated with 1.0 µg/ml W6/32 or isotype control IgG for 5 or 10 min; and then fixed, permeabilized, and stained with anti-paxillin mAb and Alexa Fluor 488 anti-mouse secondary Ab. Bar, 20 µm. Data represent at least three independent experiments.

 
We next explored whether down-regulation of FAK expression by siRNA would affect the subcellular distribution of paxillin following stimulation with mAb W6/32 (Fig. 5B). EC exposed to medium alone (Fig. 5Ca) or isotype control IgG (Fig. 5C, j and k) showed a diffuse pattern of paxillin staining with limited distribution to focal adhesions. Exposure of control EC to anti-class I Ab (Fig. 5C, b and c) or thrombin (Fig. 5Cl) led to a marked increase in the levels of phosphorylated paxillin and localization to focal contacts (Fig. 5C, b and c). Similarly, HLA class I ligation increased paxillin expression and led to the redistribution into well-defined focal adhesions in EC transfected with control siRNA (Fig. 5C, h and j). Treatment of EC with siRNA-targeting FAK reduced localization of phosphorylated paxillin to focal contacts (Fig. 5Cd). Furthermore, FAK knockdown completely prevented class I-induced assembly of paxillin into focal adhesions (Fig. 5C, e and f). Instead, the phosphorylated paxillin was enriched in membrane ruffles around the perimeter of the cells treated with anti-class I Ab. These results demonstrate that FAK plays an important role in regulating class I-mediated phosphorylation of paxillin and redistribution of paxillin to focal adhesion contact sites.

FAK knockdown blocks HLA class I-induced actin stress fiber formation

Paxillin localizes to focal adhesions at the ends of actin, where the actin microfilaments play an important role in cell-cell regulation through their interaction with myosin and focal adhesion proteins (31). To explore the role of FAK in regulating reorganization of the actin cytoskeleton, we determined the effect of FAK knockdown on class I-induced stress fiber formation. Long parallel actin stress fibers were distributed diffusely throughout the cytoplasm in the control EC (Fig. 6, B and C) or EC transfected with nontargeting siRNA (Fig. 6, H and I) following exposure to anti-class I Ab and was similar to results obtained with thrombin (Fig. 6L). Treatment with FAK siRNAs (Fig. 6, E and F) completely inhibited class I-mediated stress fiber formation. These results further underscore the importance of FAK in class I-mediated reorganization of the actin cytoskeleton.


Figure 6
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  		FIGURE 6. FAK knockdown blocks HLA class I-mediated actin stress fiber formation in EC. EC were transfected with 60 nM FAK siRNA (D–F), control siRNA (G–I), or without siRNA (A–C) for 48 h. Transfected EC were stimulated with 1.0 µg/ml W6/32 for 5 or 10 min at 37°C and then fixed, permeabilized, and stained with Texas Red-X phalloidin. Results are representative images of at least three independent experiments. Bar, 20 µm.

 
FAK knockdown does not affect HLA class I-mediated increase in FGFR expression

We previously reported that pretreatment of EC with cytochalasin D or latrunculin A disrupted the actin cytoskeleton and inhibited HLA class I-induced increases in FGFR expression (9). We therefore examined the role of FAK knockdown in class I-induced FGFR expression. Ligation of class I molecules on EC transfected with nontargeting siRNA led to a dose-dependent increase in FGFR expression (Fig. 7). FGFR expression levels were increased by 1.7-, 2.7-, 3-, and 3-fold in EC treated with 0.01, 0.1, 1.0, and 10 µg/ml W6/32. Transfection with FAK siRNA did not radically alter class I-induced FGFR expression in EC (Fig. 7). These results indicate that anti-class I Ab stimulates FGFR translocation and surface expression in a FAK-independent manner.


Figure 7
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  		FIGURE 7. FAK knockdown does not alter HLA class I-mediated increases in FGFR expression on EC. A–D, EC were transfected with 60 nM FAK siRNA or control siRNA and treated with the following: 0.01 µg/ml (A), 0.1 µg/ml (B), 1.0 µg/ml (C), or 10 µg/ml (D) W6/32 for 2 h at 37°C. Cells were detached and stained for FGFR1 expression using polyclonal anti-FGFR1 Ab and FITC-conjugated donkey anti-rabbit IgG secondary Ab. Cell fluorescence was analyzed on a FACSCalibur flow cytometer. E, Values shown are the mean ± SE of at least three independent experiments, and are expressed as median channel value of fluorescence.

 
Knockdown of FAK expression inhibits class I-mediated cell proliferation

Ligation of HLA class I molecules on EC triggers cell proliferation by up-regulating FGFR expression and increasing responsiveness to bFGF (8, 12). To investigate the role of FAK knockdown on class I-induced EC proliferation, EC were transfected with control or FAK siRNA and stimulated with anti-class I Ab, and DNA synthesis was analyzed by BrdU incorporation. Cells transfected with nontargeting siRNA showed a 5-fold increase in cell proliferation when stimulated with anti-class I mAb (Fig. 8A). In contrast, only a 2-fold increase in cell proliferation was observed in FAK siRNA-transfected EC following stimulation with anti-class I Ab. To determine the effect of FAK knockdown on responsiveness to bFGF, EC were transfected with targeting or nontargeting siRNA and stimulated with anti-class I Ab in the presence or absence of bFGF. The addition of bFGF to anti-class I Ab-treated control EC resulted in a proliferative response that was 4-fold greater than cultures treated with anti-class I Ab alone (Fig. 8A) and 20-fold greater than cells treated with control IgG. In contrast, transfection of EC with FAK siRNA almost completely inhibited cell proliferation triggered by anti-class I Ab and bFGF.


Figure 8
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  		FIGURE 8. FAK knockdown blocks HLA class I-mediated EC proliferation. EC were transfected with 60 nM FAK siRNA or control siRNA for 24 h and treated for 48 h with 1.0 µg/ml mAb W6/32 or isotype control mouse IgG. A, DNA synthesis was measured by BrdU incorporation on a FACSCalibur flow cytometer. B, Cell proliferation was measured by CFSE labeling. Proliferation index represents the number of proliferating cells in test cultures/number of proliferating cells in control cultures.

 
CFSE proliferation studies confirmed the results of the BrdU experiments and showed that FAK knockdown inhibits class I-induced EC proliferation. In these experiments, control or FAK siRNA-transfected EC were labeled with CFSE and then stimulated with anti-class I Ab or isotype control IgG. Forty-eight hours later, cells were harvested and analyzed by flow cytometry for decreased cell fluorescence as an indicator of cell proliferation. Similar to BrdU incorporation data, cells transfected with FAK siRNA showed a decrease in proliferation compared with nontargeted control siRNA-transfected cells (Fig. 8B). Taken together, these data show that FAK knockdown with siRNA inhibits HLA class I-mediated EC proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Angiogenesis is now appreciated to be a critical process in the development of TAV. Recent studies by Atkinson et al. (2) demonstrated that almost 90% of patients with TAV showed evidence of significant angiogenesis predominantly within the intima of the vessels of the allograft. Angiogenesis is a multistep process that involves EC activation and changes in the dynamics of actin filaments and assembly and disassembly of cell adhesions. FAK is an important tyrosine kinase that regulates the organization of the actin cytoskeleton and modulates cell adhesion and proliferation. Therefore, in the context of chronic TAV, we have postulated that FAK plays an important role in the altered growth of EC and their responses to autocrine and paracrine factors that contribute to the process of angiogenesis.

In this study, we used siRNA-mediated protein depletion of FAK to elucidate the role of this tyrosine kinase in EC proliferation and assembly of focal adhesions. We found that transfection of FAK siRNAs into primary cultures of human EC induced a dramatic decrease in the level of FAK protein expression without affecting the expression of other cytoskeletal proteins, including the focal adhesion-associated proteins vinculin, paxillin, and a second member of the FAK subfamily, PYK2. These results demonstrated that it was possible to specifically deplete FAK protein in primary cultures of human EC and provided us with a reliable experimental model to explore the relationship of FAK with other focal adhesion-associated proteins.

The autophosphorylation site Tyr397 of FAK has been shown to bind Src and PI3K, and this binding is required for FAK-mediated functions (19). Src-mediated transphosphorylation of FAK within the kinase domain at Tyr576 and Tyr577 promotes maximal activation of FAK catalytic activation. In addition to stimulating FAK phosphorylation, association of Src and FAK has been previously shown to enhance Src activity (29). In agreement with this possibility, knockdown of FAK led to a marked decrease in Src phosphorylation at Tyr418 following class I ligation. These results support the hypothesis that Src kinases are regulated by associating with FAK in response to class I stimulation.

In addition to promoting maximal FAK activation, the recruitment of Src into a FAK-Src signaling complex facilitates the phosphorylation of many FAK-associated proteins, including PI3K, Akt, and paxillin (19, 31). PI3Ks are composed of heterodimers of a regulatory subunit p85 and a catalytic subunit p110. The regulatory subunit p85 contains a SH3 domain and two SH2 domains that interact with phosphotyrosine residues on Src and FAK, providing an integration point for p110 subunit, resulting in PI3K activation (32). We found that siRNA-mediated depletion of FAK markedly impaired p85 PI3K phosphorylation induced by class I ligation. These results indicate a FAK-dependent pathway for class I-induced phosphorylation of PI3K. Upon activation, PI3Ks phosphorylate phosphatidylinositol and its derivatives, including phosphatidylinositol 4,5-bisphosphate, and produce phosphatidylinositol 3,4,5-trisphosphate. These lipid products act as second messengers for promoting cell survival via the recruitment of several kinases, including Akt (32, 33, 34). Recruitment of Akt to the membrane exposes two crucial amino acids that are phosphorylated and necessary for activation: Thr308 in the kinase domain and Ser473 in the hydrophobic C-terminal domain (19, 32). The results presented in this work demonstrate that Ab ligation of HLA class I molecules rapidly induces phosphorylation of Akt in the activation loop and C-terminal domain sites. Furthermore, treatment of EC with siRNA-targeting FAK prevented class I-mediated phosphorylation of Akt at both Thr308 and Ser473. PI3K and Akt kinase activity promote cell survival by regulating levels of the antiapoptotic proteins Bcl-2 and Bcl-xL (10). The PI3K/Akt signaling pathway also regulates EC proliferation by activating several downstream targets, including elongation factor 2 (E2F), forkhead transcription factor, S6 kinase, and S6 ribosomal protein (32). These results identify FAK as a key regulator of MHC class I-induced cell survival and cell proliferation signaling. Furthermore, our findings that phosphorylation of Akt is most prominent when EC are treated with low concentrations of anti-class I Ab are in accordance with previous studies showing exposure of EC to subsaturating concentrations of anti-HLA Ab increases the expression of Bcl-2 and Bcl-xL and renders the EC refractory to cell lysis (10, 16, 17). Therefore, similar to anti-ABO Ab, anti-HLA Ab can induce the expression of antiapoptotic proteins that may promote a state of accommodation in the allograft (35). Our studies confirm and extend these findings by identifying FAK as a key upstream tyrosine kinase controlling anti-HLA Ab-mediated prosurvival signaling.

The question of how different concentrations of anti-class I Abs mediate differential signaling events via FAK has not been addressed as yet. Six major tyrosine phosphorylation sites that contribute to the biological function of FAK have been identified at positions 397, 407, 576, 577, 861, and 925 (19). Tyrosine phosphorylation at specific residues leads to the formation of binding sites for other proteins such as Tyr397 for Src and PI3K and Tyr925 for paxillin and most likely explains differential activation of downstream signaling pathways elicited by different concentrations of Ab. This idea is in keeping with our previous studies showing a direct link between phosphorylation and FAK function (36). These studies demonstrated that increased FAK phosphorylation at Ser843 represses FAK phosphorylation of Tyr397 and inhibits cell migration. Similarly, studies by Westhoff et al. (37) showed that Src-induced phosphorylation of FAK on tyrosine residues is necessary to enhance complex formation among FAK, calpain 2, and Erk. An additional mechanism that may explain the differential effects of Ab on FAK signal transduction is the recruitment of FAK and its binding partners to different intracellular locations. Studies by Hsia et al. (38) demonstrated that the particular subcellular localization of FAK influences the recruitment of FAK to distinct intracellular pools. In their studies, FAK recruitment to the focal contacts promoted integrin-stimulated cell motility, whereas FAK accumulation at lamellipodia promoted cell invasion each through the activation of distinct signaling pathways. In addition, the direct targeting of FAK-associated signaling molecules to focal contacts has been shown to regulate cell migration vs cell cycle progression (39).

The results presented in this work also demonstrate that FAK siRNA blocks class I-mediated phosphorylation of paxillin and inhibits the assembly of focal adhesions. Paxillin is a central component of integrin signaling and cytoskeletal reorganization (31). Paxillin is phosphorylated by FAK at Tyr118 (40, 41), which provides a docking site for recruitment of the other signaling molecules to focal adhesions. Focal adhesions are sites of tight adhesion between the membrane and the extracellular matrix and the membrane and the cytoskeleton. They are assembled following the recruitment of signaling molecules such as FAK and paxillin and of structural and membrane actin-anchoring proteins such as talin, vinculin, tensin, and {alpha}-actinin, which link the microfilament network to the adhesive molecule integrins at their sites of clustering (42). In this study, we show that HLA class I ligation mediates phosphorylation of paxillin at Tyr118, increases paxillin expression, and promotes reorganization of paxillin toward cell periphery and areas of focal contacts. Transfection with FAK siRNA decreased class I-mediated phosphorylation of paxillin at Tyr118 and blocked paxillin redistribution to focal adhesions in EC, implicating FAK as a crucial regulator of this process. Because the assembly of actin into transcytoplasmic stress fibers is tightly associated with formation of focal adhesions to which actin filaments are anchored (32), we reasoned that FAK should also control HLA class I-mediated stress fiber formation. Our studies are consistent with this hypothesis and show siRNA knockdown of FAK completely disrupts HLA class I-mediated assembly of actin stress fibers. These findings are in agreement with studies that showed that overexpression of FAK-related nonkinase (FRNK), a noncatalytic COOH-terminal portion of FAK that acts as a negative regulator of FAK, can inhibit FAK-induced stress fiber formation (43). Furthermore, cross-linking of HLA class I on human fibroblasts leads to reorganization of HLA molecules in linear arrays directly superimposed over stress fibers positioned underneath the membrane (44). Collectively, our data indicate that class I-mediated FAK activation can influence cell migration by controlling stress fiber and focal adhesion complex formation. Thus, because the process of migration is a crucial component of angiogenesis, class I-mediated FAK activation may well be involved in the formation and sustenance of the vascular lesion associated with TAV.

We previously reported that ligation of HLA class I molecules with anti-HLA Ab on EC stimulates proliferation by increasing cell surface expression of FGFR and increasing responsiveness to bFGF (7, 12). FGF exert their biological effects via binding to four transmembrane tyrosine-kinase receptors (45). FGF are well known to exert their proangiogenic activity by interacting with various EC surface receptors, including tyrosine kinase receptors, heparin-sulfate proteoglycans, and integrins. Class I-mediated increases in FGFR membrane expression are most likely to be an important event for neointimal formation and vascular remodeling in organ transplants because immunolocalization of FGFR and FGF in human allografts is associated with development of transplant vasculopathies (46). Our earlier studies using latrunculin A and cytochalasin D showed that these pharmacological agents that inhibit actin polymerization and FAK phosphorylation inhibited class I-mediated FGFR surface expression on EC (9). We interpreted these findings to indicate that ligation of HLA class I molecules on the surface of EC stimulated FGFR translocation in a FAK-dependent manner. However, contrary to our previous findings, siRNA knockdown of FAK definitively showed that class I-mediated FGFR translocation is a FAK-independent process. Currently, the signaling pathway responsible for class I-mediated FGFR translocation is unknown and is under further investigation by our laboratory. Given that class I-induced FGFR translocation occurs rapidly following HLA class I cross-linking, we speculate that the signaling pathway is linked to the transport of FGFR from subcellular locations such as the Golgi apparatus (47).

Inhibition of FAK protein expression by FAK siRNA reduced class I-mediated proliferation in the presence and absence of bFGF. This suggests that although class I-mediated up-regulation of FGFR expression was not inhibited by siRNA knockdown of FAK, FAK is critically involved in class I-mediated EC proliferation downstream to bFGF ligand binding to FGFR. Whether there is a direct association between FGFR and FAK is not known. Several reports have described cross-talk between FGFR and focal adhesion complexes. Plopper et al. (48) isolated focal adhesion complexes that contained Src, FAK, and FGFR, and Klint et al. (49) reported that FGF-2 induces complex formation between Src and FAK in differentiating EC. Our data are also consistent with studies that have shown a role for FAK in regulating integrin-mediated cell cycle progression (50).

The mechanisms whereby class I molecules initiate signal transduction resulting in FAK phosphorylation are unknown. HLA molecules do not have intrinsic kinase activity, and although the H chain of class I contains serine, threonine, and tyrosine residues that can be phosphorylated, studies using constructs lacking the cytoplasmic tail have shown that signaling via class I does not require this portion of the molecule (51). Thus, class I molecules most likely associate with other molecules that have the capacity to transduce signals or generate intracellular messengers causing FAK phosphorylation. In this respect, class I molecules have previously been shown to interact with the insulin receptor and epidermal growth factor receptor (52, 53, 54), both of which have the capacity to mediate FAK phosphorylation (55, 56). In addition, accessory molecules involved in cytoskeletal reorganization and assembly of focal adhesions are most likely important for HLA class I signaling. This notion is strongly reinforced by data showing that disruption of the actin cytoskeleton and focal adhesion kinase inhibits class I-mediated signaling in EC and T cells (9, 57). Integrins have been shown to interact with FAK, linking it with proliferation and survival pathways (58). Given the remarkable similarity in the signaling pathways initiated by integrins and HLA class I molecules, we speculate that HLA class I molecules may bind and recruit integrins upon Ab ligation to transduce proliferative and survival signals. Studies are underway in our laboratory to investigate whether members of the integrin family serve as cosignaling molecules for HLA class I.

Our study was designed to elucidate the relationship between anti-HLA Ab and EC signal transduction and proliferation by using a siRNA knockdown cell culture model that allowed us to isolate the specific effect of anti-HLA Abs on FAK without immune cells and complement. Therefore, the interpretation of this data may or may not be similar in more complex tissue architecture and/or in the context of other events taking place in the transplant setting such as complement and cellular infiltrates. Hence, these studies require validation in a suitable in vivo transplant model. Furthermore, although the efficiency of the siRNA-mediated suppression of FAK was optimized with respect to the selection of the optimal siRNA sequence, dose, transfection delivery, and t1/2 of FAK protein to achieve the most favorable and specific silencing, caution must be exercised in interpreting the data because introduction of a siRNA molecule into a cell can have effects beyond the expected inhibition of target gene expression, including alterations in nontargeted genes and interference with endogenous micro RNA (59).

As an outgrowth of these investigations, several questions remain to be addressed, such as the following. 1) Can Abs alone mediate TAV? 2) Is complement required for development of TAV? 3) How do Abs mediate the changes in grafts that appear to occur over months to years? Recent studies by Uehara et al. (60) in a murine heterotopic cardiac allograft model begin to address some of these questions and confirm that alloantibody alone is sufficient to cause arterial neointimal proliferation. Their studies demonstrate that passive transfer of complement-fixing donor-specific anti-MHC class I mAb in T and B cell knockout mouse heart allograft recipients led to progressive TAV. Interestingly, a minimum threshold of a 28-day exposure to Ab was required to cause florid TAV, and even after the disappearance of anti-MHC Ab and C4d deposition, the lesions remained fully intact. These data support the concept that anti-HLA Ab can initiate the disease process. However, Abs may not be critical at later times, because the TAV lesion could progress and persist in the absence of continued Ab production or C4d deposition in this model. Using phosphorylation-specific Ab that can detect activated signaling molecules in paraffin-embedded human biopsy tissue, we recently determined the significance of the class I signaling pathway in endomyocardial biopsies from cardiac transplant recipients diagnosed with Ab-mediated rejection (15). We found increased capillary endothelial staining for the phosphorylated form of S6 ribosomal protein, a downstream target of class I signaling and FAK activation, was significantly associated with the diagnosis of Ab-mediated rejection, but not acute cellular rejection. Furthermore, we observed a link between TAV and persistent phosphorylated S6 ribosomal protein. Collectively, these in vitro and in vivo studies extend the evidence that anti-HLA Ab can participate in the process of TAV.

Together, our previous results and current data are consistent with a model whereby anti-HLA Ab-induced clustering of class I molecules stimulates FAK phosphorylation, which in turn promotes the assembly of stress fibers and assembly of FAK, Src, paxillin, PI3K, and Akt into focal adhesion complexes that act in concert as signaling units. Class I-mediated activation of FAK also regulates cell proliferation via two means, as follows: assembly and disassembly of focal adhesions and bFGF-mediated cell proliferation. Elucidation of the signaling pathway(s) elicited by ligation of HLA class I molecules by Ab should provide a better understanding of how Abs contribute to the development of TAV and may permit the development of new treatment strategies.


   Acknowledgments
 
We are grateful to James Sinnett-Smith and Xiaohua Jiang for technical assistance in developing the siRNA transfection methods. We thank Steven H. Young for help with fluorescence microscopy.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institute of Allergy and Infectious Diseases Grant R01 AI 42819 and the American Heart Association Grant-in-Aid 9750894A. Back

2 Address correspondence and reprint requests to Dr. Elaine F. Reed, Immunogenetics Center, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, 1000 Veteran Avenue, Los Angeles, CA 90095. E-mail address: ereed{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: TAV, transplant-associated vasculopathy; bFGF, basic fibroblast growth factor; EC, endothelial cell; FAK, focal adhesion kinase; FGF, fibroblast growth factor; FGFR, FGF receptor; SH, Src homology; siRNA, small interfering RNA; PYK2, proline-rich tyrosine kinase 2. Back

Received for publication November 29, 2006. Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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