Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Disulfide-HMGB1 signals through TLR4 and TLR9 to induce inflammatory macrophages capable of innate-adaptive crosstalk in human liver transplantation.

Ischemia-reperfusion injury (IRI) during orthotopic liver transplantation (OLT) contributes to graft rejection and poor clinical outcomes. The disulfide form of high mobility group box 1 (diS-HMGB1), an intracellular protein released during OLT-IRI, induces pro-inflammatory macrophages. How diS-HMGB1 differentiates human monocytes into macrophages capable of activating adaptive immunity remains unknown. We investigated if diS-HMGB1 binds toll-like receptor (TLR) 4 and TLR9 to differentiate monocytes into pro-inflammatory macrophages that activate adaptive immunity and promote graft injury and dysfunction. Assessment of 106 clinical liver tissue and longitudinal blood samples revealed that OLT recipients were more likely to experience IRI and graft dysfunction with increased diS-HMGB1 released during reperfusion. Increased diS-HMGB1 concentration also correlated with TLR4/TLR9 activation, polarization of monocytes into pro-inflammatory macrophages, and production of anti-donor antibodies. In vitro, healthy volunteer monocytes stimulated with purified diS-HMGB1 had increased inflammatory cytokine secretion, antigen presentation machinery, and reactive oxygen species production. TLR4 inhibition primarily impeded cytokine/chemokine and costimulatory molecule programs, whereas TLR9 inhibition decreased HLA-DR and reactive oxygen species production. diS-HMGB1-polarized macrophages also showed increased capacity to present antigens and activate T memory cells. In murine OLT, diS-HMGB1 treatment potentiated ischemia-reperfusion-mediated hepatocellular injury, accompanied by increased serum alanine transaminase levels. This translational study identifies the diS-HMGB1/TLR4/TLR9 axis as potential therapeutic targets in OLT-IRI recipients.

Disulfide High-Mobility Group Box 1 Drives Ischemia-Reperfusion Injury in Human Liver Transplantation.

BACKGROUND AND AIMS: Sterile inflammation is a major clinical concern during ischemia-reperfusion injury (IRI) triggered by traumatic events, including stroke, myocardial infarction, and solid organ transplantation. Despite high-mobility group box 1 (HMGB1) clearly being involved in sterile inflammation, its role is controversial because of a paucity of patient-focused research. APPROACH AND RESULTS: Here, we examined the role of HMGB1 oxidation states in human IRI following liver transplantation. Portal blood immediately following allograft reperfusion (liver flush; LF) had increased total HMGB1, but only LF from patients with histopathological IRI had increased disulfide-HMGB1 and induced Toll-like receptor 4-dependent tumor necrosis factor alpha production by macrophages. Disulfide HMGB1 levels increased concomitantly with IRI severity. IRI+ prereperfusion biopsies contained macrophages with hyperacetylated, lysosomal disulfide-HMGB1 that increased postreperfusion at sites of injury, paralleling increased histone acetyltransferase general transcription factor IIIC subunit 4 and decreased histone deacetylase 5 expression. Purified disulfide-HMGB1 or IRI+ blood stimulated further production of disulfide-HMGB1 and increased proinflammatory molecule and cytokine expression in macrophages through a positive feedback loop. CONCLUSIONS: These data identify disulfide-HMGB1 as a mechanistic biomarker of, and therapeutic target for, minimizing sterile inflammation during human liver IRI.

Ligation of HLA Class I Molecules Induces YAP Activation through Src in Human Endothelial Cells.

Ab cross-linking of HLA class I (HLA I) molecules on the surface of endothelial cells (EC) triggers proliferative and prosurvival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. Despite the importance of Ab-mediated rejection in transplantation, the mechanisms involved remain incompletely understood. In this study, we examined the regulation of yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in human ECs challenged with Abs that bind HLA I. In unstimulated ECs, YAP localized mainly in the cytoplasm. Stimulation of these cells with Ab W6/32 induced marked translocation of YAP to the nucleus. The nuclear import of YAP was associated with a rapid decrease in YAP phosphorylation at Ser127 and Ser397, sites targeted by LATS1/2 and with the expression of YAP-regulated genes, including connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Transfection of small interfering RNAs targeting YAP/TAZ blocked the migration of ECs stimulated by ligation of HLA I, indicating that YAP mediates the increase in EC migration induced by HLA I ligation. Treatment of intact ECs with Src family inhibitors induced cytoplasmic localization of YAP in unstimulated ECs and, strikingly, blocked the nuclear import of YAP induced by Ab-induced HLA I activation in these cells and the increase in the expression of the YAP-regulated genes CTGF and CYR61 induced by HLA I stimulation. Our results identify the Src/YAP axis as a key player in promoting the proliferation and migration of ECs that are critical in the pathogenesis of transplant vasculopathy.

HLA Class II-Triggered Signaling Cascades Cause Endothelial Cell Proliferation and Migration: Relevance to Antibody-Mediated Transplant Rejection.

Transplant recipients developing donor-specific HLA class II (HLA-II) Abs are at higher risk for Ab-mediated rejection (AMR) and transplant vasculopathy. To understand how HLA-II Abs cause AMR and transplant vasculopathy, we determined the signaling events triggered in vascular endothelial cells (EC) following Ab ligation of HLA-II molecules. HLA-II expression in EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF-α/IFN-γ. Ab ligation of class II stimulated EC proliferation and migration. Class II Ab also induced activation of key signaling nodes Src, focal adhesion kinase, PI3K, and ERK that regulated downstream targets of the mammalian target of rapamycin (mTOR) pathway Akt, p70 ribosomal S6 kinase, and S6 ribosomal protein. Pharmacological inhibitors and small interfering RNA showed the protein kinases Src, focal adhesion kinase, PI3K/Akt, and MEK/ERK regulate class II Ab-stimulated cell proliferation and migration. Treatment with rapalogs for 2 h did not affect HLA-II Ab-induced phosphorylation of ERK; instead, mTOR complex (mTORC)1 targets were dependent on activation of ERK. Importantly, suppression of mTORC2 for 24 h with rapamycin or everolimus or treatment with mTOR active-site inhibitors enhanced HLA-II Ab-stimulated phosphorylation of ERK. Furthermore, knockdown of Rictor with small interfering RNA caused overactivation of ERK while abolishing phosphorylation of Akt Ser473 induced by class II Ab. These data are different from HLA class I Ab-induced activation of ERK, which is mTORC2-dependent. Our results identify a complex signaling network triggered by HLA-II Ab in EC and indicate that combined ERK and mTORC2 inhibitors may be required to achieve optimal efficacy in controlling HLA-II Ab-mediated AMR.

Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial cell-monocyte interactions via mTOR in transplant antibody-mediated rejection.

Antibody-mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long-term survival of solid organ transplants. AMR is caused by donor-specific antibodies to HLA, which contribute to TAV by initiating outside-in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab-mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab-activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM-1) clustering, and monocyte firm adhesion to HLA I Ab-activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1-/- recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM-1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell-monocyte interactions during AMR.

HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+ in human aortic endothelial cells.

Following transplantation, HLA class I antibodies targeting donor endothelium stimulate cell proliferation and migration, which contribute to the development of transplant vasculopathy and chronic allograft rejection. Dynamic remodeling of the actin cytoskeleton regulates cell proliferation and migration in endothelial cells (ECs), but the mechanism(s) involved remain incompletely understood. We explored anti-HLA class I antibody-mediated alterations of the cytoskeleton in human aortic ECs (HAECs) and contrasted these findings to thrombin-induced cytoskeleton remodeling. Our results identify two different signaling pathways leading to myosin light chain (MLC) phosphorylation in HAECs. Stimulation of HAECs with thrombin at 1 U/ml induced a robust elevation of intracellular Ca(2+) concentration, increased MLC phosphorylation, and promoted stress fiber formation via MLC kinase (MLCK) and Rho kinase (ROK) in an ERK-independent manner. In contrast, HAECs stimulated with HLA class I antibodies did not promote any detectable change in intracellular Ca(2+) concentration but instead induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. Stimulation of HAECs with low-dose thrombin (1 mU/ml) induced signaling cascades that were similar to stimulation with HLA class I antibodies. HLA class I antibodies also stimulated the translocation of mammalian target of rapamycin complex 2 (mTORC2) and ERK1/2 from the cytoplasm to the plasma membrane independently of stress fiber assembly. These findings identify novel roles for HLA class I signaling in ECs and provide new insights into the role of ERK1/2 and mTORC2 in cytoskeleton regulation, which may be important in promoting transplant vasculopathy, tumor angiogenesis, and atherosclerosis.

Combined fascial flap and expanded skin flap for enveloping Medpor framework in microtia reconstruction.

The Medpor implant is another choice for a new auricular framework besides autogenous costal cartilage. However, its relatively frequent exposure and less-matching skin coverage discourage surgeons from using it. In this article, we present a new two-flap method, a combination of the temporoparietal fascial flap and the expanded skin flap, for wrapping the Medpor implant in microtia reconstruction. A staged surgical procedure was performed, including soft tissue expansion in the mastoid region, soft tissue expander removal, expanded skin flap and temporoparietal fascial flap formation, Medpor framework implantation, and the combined two-flap envelopment. Conventional lobule transposition and tragus reconstruction were accomplished for selected patients. In this study, a total of 22 microtias were reconstructed consecutively using this method. Eighteen patients were followed since the first surgery. The postoperative follow-up time ranged from 3 to 12 months. The draped soft tissue covering was thin enough to show the reconstructed ear with excellent, subtle contour when edema gradually vanished 3-6 months postoperatively. The new ear had a stable shape, and its skin color and texture matched the normal surrounding skin very well. No exposure or extrusion of the framework was observed in the series. The Medpor implant enveloped by both a temporoparietal fascial flap and an expanded cutaneous flap appears to be a promising alternative for the auricular framework in microtia reconstruction. Because of the wrapping tissues, auricular construction using a Medpor implant can be a safe, steady, and easily acceptable choice for both microtia patients and their physicians.

MHC class I and integrin ligation induce ERK activation via an mTORC2-dependent pathway.

The aim of this study was to characterize the interaction between mTOR and ERK in primary endothelial cells (EC) following MHC class I and integrin ligation. Ligation of MHC class I molecules or integrins on the surface of EC leads to phosphorylation of ERK at Thr202/Tyr204. We utilized small interfering RNA (siRNA) blockade of mTOR and proteins involved in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) to define a relationship between mTOR and ERK following MHC class I signaling. We found mTORC2 was responsible for MHC class I and integrin induced phosphorylation of ERK at Thr202/Tyr204. We corroborated these results demonstrating that long-term exposure to rapamycin also inhibited ERK pathway activation in response to MHC class I signaling. Our results demonstrate, for the first time, that engagement of either MHC class I or integrin on the surface of EC leads to ERK activation through an mTORC2-dependent pathway.

HLA Class I and Class II-Induced Intracellular Signaling and Molecular Associations in Primary Human Endothelial Cells.

The signaling capacity of HLA molecules in vascular cells has been well established. Intracellular signaling and association with the coreceptor integrin ß4 has been well-studied for HLA class I. However, little is known regarding HLA class II intracellular signaling in human endothelial cells. Investigation of HLA class II has been challenging due to the loss of HLA class II expression in cultured primary cells. Herein, we describe methods for inducing expression of endogenous alleles and loci of HLA class II molecules, as well as for studying intracellular signaling. This includes siRNA knockdown of proteins and coimmunoprecipitation of putative coreceptors for HLA in primary human aortic endothelial cells.

Anti-HLA antibodies can induce endothelial cell survival or proliferation depending on their concentration.

Patients exhibiting a humoral immune response to the transplanted organ are at increased risk of antibody-mediated rejection and development of transplant vasculopathy. Historically, antibodies were thought to elicit transplant rejection through complement mediated damage of the endothelium of the graft. More recently, studies from our laboratory and others have shown that antibody ligation of class I molecules on the surface of endothelial cells transduces signals resulting in functional changes including expression of cell survival proteins and cell proliferation. The intracellular events initiated by antibody ligation are dependent upon the degree of molecular aggregation and influenced by the concentration of the antibody and level of human leukocyte antigen (HLA) expression. Herein we describe our recent findings on the effect of molecular aggregation on the class I signaling pathway in human endothelial cells.

Anti-HLA class I antibodies activate endothelial cells and promote chronic rejection.

Transplant recipients exhibiting a humoral immune response to the allograft demonstrate lower graft survival and increased risk for the development of chronic rejection and transplant arteriosclerosis. Our studies suggest that anti-HLA class I antibodies (Ab) play an important role in controlling endothelial cell (EC) function by binding to class I molecules on the surface of the EC and transducing intracellular signals. Anti-HLA Ab exhibit two primary effector functions: stimulation of cell proliferation and up-regulation of cell survival genes. Importantly, the intracellular events initiated by class I ligation appear to be influenced by the concentration of the Ab. High-titered anti-HLA Ab stimulate cell proliferation whereas low-titered Ab activate the PI3K/Akt pathway and promote expression of cell survival proteins including Bcl-2 and Bcl-xL. Anti-HLA class I Ab may contribute to the process of chronic allograft rejection by promoting EC survival and proliferation.

Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells.

Anti-human leukocyte antigen (HLA) antibodies (Ab) have long been implicated in the process of acute and chronic allograft rejection, yet their mechanism(s) of action is not well understood. The aim of this study was to determine whether ligation of HLA class I molecules by anti-HLA Ab on the surface of human endothelial cells (EC) activates the PI3 Kinase (PI3K)/Akt signaling pathway and downstream target proteins of the cell death apparatus. We report that Ab ligation of major histocompatibility complex (MHC) class I molecules on the surface of EC triggers phosphorylation of Akt, PI3K, and recruitment of PI3K and Akt into a signaling unit with focal adhesion kinase. Signaling through class I also stimulated phosphorylation of Bad and upregulated expression of Bcl-2 and Bcl-xL. Pretreatment of EC with the PI3K inhibitor wortmannin blocked class I-mediated expression of Bcl-2, but not Bcl-xL, suggesting a role for the PI3K/Akt signaling pathway in regulation of class I-induced Bcl-2 expression. The intracellular events initiated by class I ligation were influenced by the concentration of the anti-HLA Ab with the lowest tested concentrations of Ab stimulating the highest level of Akt phosphorylation, Bcl-xL and Bcl-2 expression. Consistent with the in vitro experiments, analysis of biopsy samples from heart transplant recipients with evidence of Ab-mediated rejection exhibited increased Bcl-2 expression on the vascular endothelium. These results suggest that exposure of the graft endothelium to low concentrations of anti-HLA Ab may promote cell survival by transducing signals resulting in upregulation of cell survival genes.

Characterization of the endothelial cell cytoskeleton following HLA class I ligation.

BACKGROUND: Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood. METHODOLOGY AND PRINCIPAL FINDINGS: The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin. CONCLUSIONS/SIGNIFICANCE: Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes.

Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner.

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy.

HLA class I antibody-mediated endothelial cell proliferation via the mTOR pathway.

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser(473) and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells.

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 Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) 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.

[Application of both expanded cutaneous flap and temporoparietal fascia flap in ear reconstruction with Medpor framework].

OBJECTIVE: To investigate the feasibility and results of application of both expanded cutaneous flap and temporoparietal fascia flap in total ear reconstruction with Medpor framework. METHODS: The main procedure consists of two stages: Stage I-skin expansion; Stage II -auricle formation consists of orientation of Medpor implant and creation of coverage for the implant by both expanded skin flap and temporoparietal fascia flap. RESULTS: Twenty-two ears in 22 unilateral microtia patients were constructed using Medpor implants covered with both expanded cutaneous flap and temporoparietal fascia flap over the last three years, they were accepted as pleasing by the patients. CONCLUSIONS: Application of both expanded cutaneous flap and temporoparietal fascia flap can assure no extrusion of Medpor implant in ear reconstruction. Either more, the two layers of transferred tissues will not affect the profile details of the reconstructed ear. And because the skin covering the framework and fascia is derived from mastoid region, the appearance and profile of the reconstructed auricle is true to nature and close to that of the opposite one.