A Nanoprimer To Improve the Systemic Delivery of siRNA and mRNA.

Despite tremendous interest in gene therapies, the systemic delivery of nucleic acids still faces substantial challenges. To successfully administer nucleic acids, one approach is to encapsulate them in lipid nanoparticles (LNPs). However, LNPs administered intravenously substantially accumulate in the liver where they are taken up by the reticuloendothelial system (RES). Here, we administer prior to the LNPs a liposome designed to transiently occupy liver cells, the Nanoprimer. This study demonstrates that the pretreatment of mice with the Nanoprimer decreases the LNPs' uptake by the RES. By accumulating rapidly in the liver cells, the Nanoprimer improves the bioavailability of the LNPs encapsulating human erythropoietin (hEPO) mRNA or factor VII (FVII) siRNA, leading respectively to more hEPO production (by 32%) or FVII silencing (by 49%). The use of the Nanoprimer offers a new strategy to improve the systemic delivery of RNA-based therapeutics.

Tissue scaffolds functionalized with therapeutic elastin-like biopolymer particles.

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.

Functionalized Biopolymer Particles Enhance Performance of a Tissue-Protective Peptide under Proteolytic and Thermal Stress.

Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at subphysiological temperatures. The bifunctional submicron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites.

Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation.

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvß3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥ 6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.

Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.

BACKGROUND: Cardiac allograft vasculopathy is the major cause of late allograft loss after heart transplantation. Cardiac allograft vasculopathy lesions contain alloreactive T cells that secrete interferon-γ, a vasculopathic cytokine, and occur more frequently in patients with donor-specific antibody. Pathological interactions between these immune effectors, representing cellular and humoral immunity, respectively, remain largely unexplored. METHODS AND RESULTS: We used human panel reactive antibody to form membrane attack complexes on allogeneic endothelial cells in vitro and in vivo. Rather than inducing cytolysis, membrane attack complexes upregulated inflammatory genes, enhancing the capacity of endothelial cells to recruit and activate allogeneic interferon-γ--producing CD4(+) T cells in a manner dependent on the activation of noncanonical nuclear factor-κB signaling. Noncanonical nuclear factor-κB signaling was detected in situ within endothelial cells both in renal biopsies from transplantation patients with chronic antibody-mediated rejection and in panel-reactive antibody--treated human coronary artery xenografts in immunodeficient mice. On retransplantation into immunodeficient hosts engrafted with human T cells, panel-reactive antibody--treated grafts recruited more interferon-γ--producing T cells and enhanced cardiac allograft vasculopathy lesion formation. CONCLUSIONS: Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-κB signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-κB signaling in endothelial cells, observed in human allograft specimens and implicated in lesion pathogenesis, may represent a target for new pharmacotherapies to halt the progression of cardiac allograft vasculopathy.

LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration.

Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates ß1-integrin-mediated signaling via Akt and GSK3ß phosphorylation. Moreover, the α-parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates α-parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.

Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments.

Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases.

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.

Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft's cytoprotection.

BACKGROUND: Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor-α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC). METHODS: Endothelial cells cultures were established from WT and Gal(-/-) pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement-dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF-activated EC, and the protective functions were tested toward TNF-induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase-3/7 activity. RESULTS: First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal(-/-) pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b-9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM-1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase-3/7-dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01). CONCLUSIONS: Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.

Repopulation of intrahepatic bile ducts in engineered rat liver grafts.

Engineered liver grafts for transplantation with sufficient hepatic function have been developed both in small and large animal models using the whole liver engineering approach. However, repopulation of the bile ducts in the whole liver scaffolds has not been addressed yet. In this study, we show the feasibility of repopulating the bile ducts in decellularized rat livers. Biliary epithelial cells were introduced into the bile ducts of the decellularized liver scaffolds with or without hepatocytes in the parenchymal space. The recellularized grafts were cultured under perfusion for up to 2 days and histological analysis revealed that the biliary epithelial cells formed duct-like structures, with the viable hepatocyte mass residing in the parenchymal space, in an arrangement highly comparable to the native tissue. The grafts were viable and functional as confirmed by both albumin and urea assay results and the gene expression analysis of biliary epithelial cells in recellularized liver grafts. This study provides the proof-of-concept results for rat liver grafts co-populated with parenchymal and biliary epithelial cells.

Improving functional re-endothelialization of acellular liver scaffold using REDV cell-binding domain.

Engineering of functional vascularized liver tissues holds great promise in addressing donor organ shortage for transplantation. Whole organ decellularization is a cell removal method that retains the native vascular structures of the organ such that it can be anastomosed with the recipient circulation after recellularization with healthy cells. However, a main hurdle to successful implantation of bioengineered organ is the inability to efficiently re-endothelialize the vasculature with a functional endothelium, resulting in blood clotting which is the primary cause of failure in early transplant studies. Here, we present an efficient approach for enhancing re-endothelialization of decellularized rat liver scaffolds by conjugating the REDV cell-binding domain to improve attachment of endothelial cells (EC) on vascular wall surfaces. In order to facilitate expression and purification of the peptide, REDV was fused with elastin-like peptide (ELP) that confers thermally triggered aggregation behavior to the fusion protein. After validating the adhesive properties of the REDV-ELP peptide, we covalently coupled REDV-ELP to the blood vasculature of decellularized rat livers and seeded EC using perfusion of the portal vein. We showed that REDV-ELP increased cell attachment, spreading and proliferation of EC within the construct resulting in uniform endothelial lining of the scaffold vasculature. We further observed that REDV-ELP conjugation dramatically reduced platelet adhesion and activation. Altogether, our results demonstrate that this method allowed functional re-endothelialization of liver scaffold and show great potential toward the generation of functional bioengineered liver for long-term transplantation. STATEMENT OF SIGNIFICANCE: There is a critical need for novel organ replacement therapies as the grafts for transplantation fall short of demand. Recent advances in tissue engineering, through the use of decellularized scaffolds, have opened the possibility that engineered grafts could be used as substitutes for donor livers. However, successful implantation has been challenged by the inability to create a functional vasculature. Our research study reports a new strategy to increase efficiency of endothelialization by increasing the affinity of the vascular matrix for endothelial cells. We functionalized decellularized liver scaffold using elastin-like peptides grafted with REDV cell binding domain. We showed that REDV-ELP conjugation improve endothelial cell attachment and proliferation within the scaffold, demonstrating the feasibility of re-endothelializing a whole liver vasculature using our technique.

Co-delivery of a growth factor and a tissue-protective molecule using elastin biopolymers accelerates wound healing in diabetic mice.

Growth factor therapy is a promising approach for chronic diabetic wounds, but strategies to efficiently and cost-effectively deliver active molecules to the highly proteolytic wound environment remain as major obstacles. Here, we re-engineered keratinocyte growth factor (KGF) and the cellular protective peptide ARA290 into a protein polymer suspension with the purpose of increasing their proteolytic resistance, thus their activity in vivo. KGF and ARA290 were fused with elastin-like peptide (ELP), a protein polymer derived from tropoelastin, that confers the ability to separate into a colloidal suspension of liquid-like coacervates. ELP fusion did not diminish peptides activities as demonstrated by ability of KGF-ELP to accelerate keratinocyte proliferation and migration, and ARA290-ELP to protect cells from apoptosis. We examined the healing effect of ARA290-ELP and KGF-ELP alone or in combination, in a full-thickness diabetic wound model. In this model, ARA290-ELP was found to accelerate healing, notably by increasing angiogenesis in the wound bed. We further showed that co-delivery of ARA290 and KGF, with the 1:4 KGF-ELP to ARA290-ELP ratio, was the most effective wound treatment with the fastest healing rate, the thicker granulation tissue and regenerated epidermis after 28 days. Overall, this study shows that ARA290-ELP and KGF-ELP constitute promising new therapeutics for treatment of chronic wounds.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins.

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the ß-galactosidase (ß-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the ß-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/ß-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

The disintegrin and metalloproteinase ADAM10 mediates a canonical Notch-dependent regulation of IL-6 through Dll4 in human endothelial cells.

Although the involvement of the disintegrin and metalloproteinase ADAM10 in several areas of vascular biology is now clearly established, its role in vascular inflammation and in Notch signaling at the endothelial level remains unclear. In this study, we demonstrated that ADAM10 specifically localizes in the CD31(+) endothelial cells (ECs) in normal human cardiac tissues and in cultured primary arterial ECs. In vitro, ADAM10 drives a specific regulation of the Notch pathway in vascular ECs. Using an ADAM10 gain and loss of function approach we show an ADAM10-dependent regulation of Dll1 and Dll4 expression in association with changes in Hes1 and Hey1 expression. We also identified IL-6, IL-8, MCP-1 and sVCAM-1 as novel targets of ADAM10 upon inflammation. Although Notch pathway does not seem to be required for the production of IL-8, MCP-1 and sVCAM-1, the release of IL-6 by ECs occurred through ADAM10 and a canonical Notch signaling pathway, dependent of γ-secretase activity. Moreover, sustained expression of Dll4 mediated by ADAM10 elicits an increased release of IL-6 suggesting a strong implication of the specific Dll4 signaling in this mechanism. Modulation of IL-6 mediated by ADAM10/Notch signaling required PI3K activity. Thus, our findings suggest that ADAM10/Dll4 signaling is a major signaling pathway in ECs driving inflammatory events involved in inflammation and immune cell recruitment.

Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

Paracrine exchanges of molecular signals between alginate-encapsulated pericytes and freely suspended endothelial cells within a 3D protein gel.

Paracrine signals, essential for the proper survival and functioning of tissues, may be mimicked by delivery of therapeutic proteins within engineered tissue constructs. Conventional delivery methods are of limited duration and are unresponsive to the local environment. We developed a system for sustained and regulated delivery of paracrine signals by encapsulating living cells of one type in alginate beads and co-suspending these cell-loaded particles along with unencapsulated cells of a second type within a 3D protein gel. This system was applied to vascular tissue engineering by placing human placental microvascular pericytes (PCs) in the particulate alginate phase and human umbilical vein endothelial cells (HUVECs) in the protein gel phase. Particle characteristics were optimized to keep the encapsulated PCs viable for at least two weeks. Encapsulated PCs were bioactive in vitro, secreting hepatocyte growth factor, an angiogenic protein, and responding to externally applied HUVEC-derived signals. Encapsulated PCs influenced HUVEC behavior in the surrounding gel by enhancing the formation of vessel-like structures when compared to empty alginate bead controls. In vivo, encapsulated PCs modulated the process of vascular self-assembly by HUVECs in 3D gels following implantation into immunodeficient mice. We conclude that alginate encapsulated cells can provide functional paracrine signals within engineered tissues.

Profiling posttransplant circulating antibodies in kidney transplantation using donor endothelial cells.

BACKGROUND: Pathogenesis of antibody (Ab) responses to transplant are yet not well defined. This study aimed to detect and to analyze posttransplant circulating allo-Abs reacting toward graft endothelial cells (ECs) using primary EC cultures prospectively isolated from the transplant donor at the time of transplantation. METHODS: This study shows a retrospective analysis performed using a dedicated EC crossmatch (ECXM) assay that we developed for the experimental assessment of donor-specific EC-reactive Abs. Donor-specific ECXM was performed by flow cytometry on posttransplant sera (n=256) from an historical cohort of 22 kidney allograft recipients. RESULTS: In this study, we show that 27.3% (6/22) of recipients have a positive ECXM that strictly correlates (100%, 6/6) with the presence of anti-human leukocyte antigen (HLA) Abs posttransplantation. ECXM identifies both donor-specific Abs (DSA; 50%) and non-DSA (50%) reactive to EC. DSA and non-DSA are mostly IgG1 and exhibit peak titers ranging from 1/8 to 1/1024. ECXM indicates that DSA correspond to anti-HLA class II Abs; this immunization is late (M3-M60) but persistent (still detected at M60). In contrast, non-DSA are non-HLA-type Abs reacting with third-party EC and reflecting an early but transient immunization (ended at M3-M12). Our findings demonstrate selective regulatory pathways initiated by anti-HLA class II and non-DSA in graft EC reflected by CCR4 and interleukin 1ß up-regulation, respectively. CONCLUSIONS: We provide evidence that circulating Abs in HLA-sensitized transplant recipients include both DSA and non-HLA/non-DSA able to bind to graft EC and induce specific gene transcription.

The adaptor Lnk (SH2B3): an emerging regulator in vascular cells and a link between immune and inflammatory signaling.

A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.

Inflammation dysregulates Notch signaling in endothelial cells: implication of Notch2 and Notch4 to endothelial dysfunction.

Although the involvement of the Notch pathway in several areas of vascular biology is now clearly established, its role in vascular inflammation at the endothelial level remains to be elucidated. In this study, we demonstrated that pro-inflammatory cytokines drive a specific regulation of the Notch pathway in vascular endothelial cells (ECs). In arterial ECs, TNFα strongly modulates the pattern of Notch expression by decreasing Notch4 expression while increasing Notch2 expression. Changes in Notch expression were associated with a reduction in hes1 and hey2 expression and in CBF1 reporter gene activity, suggesting that TNFα regulates both Notch expression and activity. Notch2 and Notch4 regulations occurred independently and were found to be mostly mediated by the NFκB signaling pathways and PI3-kinase signaling pathways, respectively. Functionally, TNF-mediated Notch regulation promotes caspase-dependent EC apoptosis. Finally, our findings confirmed that dysregulated Notch signaling also occurs upon inflammation in vivo and correlates with caspase activation and apoptosis. In conclusion, inflammatory cytokines elicit a switch in Notch expression characterized by Notch2 predominance over Notch4 leading to a reduced Notch activity and promoting apoptosis. Thus, here we provide evidence for a role of soluble mediators of inflammation (i.e. cytokines) in the regulation of Notch signaling and for the implication of a dysregulated Notch pathway to endothelial and vascular dysfunction.

Notch2 signaling sensitizes endothelial cells to apoptosis by negatively regulating the key protective molecule survivin.

BACKGROUND: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively. CONCLUSIONS/SIGNIFICANCE: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.