Dan Jane-wit

An autoimmune-mediated strategy for prophylactic breast cancer vaccination

Although vaccination is most effective when used to prevent disease, cancer vaccine development has focused predominantly on providing therapy against established growing tumors. The difficulty in developing prophylactic cancer vaccines is primarily due to the fact that tumor antigens are variations of self proteins and would probably mediate profound autoimmune complications if used in a preventive vaccine setting. Here we use several mouse breast cancer models to define a prototypic strategy for prophylactic cancer vaccination. We selected α-lactalbumin as our target vaccine autoantigen because it is a breast-specific differentiation protein expressed in high amounts in the majority of human breast carcinomas and in mammary epithelial cells only during lactation. We found that immunoreactivity against α-lactalbumin provides substantial protection and therapy against growth of autochthonous tumors in transgenic mouse models of breast cancer and against 4T1 transplantable breast tumors in BALB/c mice. Because α-lactalbumin is conditionally expressed only during lactation, vaccination-induced prophylaxis occurs without any detectable inflammation in normal nonlactating breast tissue. Thus, α-lactalbumin vaccination may provide safe and effective protection against the development of breast cancer for women in their post–child-bearing, premenopausal years, when lactation is readily avoidable and risk for developing breast cancer is high.

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-&ggr;, 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-&ggr;––producing CD4+ T cells in a manner dependent on the activation of noncanonical nuclear factor-&kgr;B signaling. Noncanonical nuclear factor-&kgr;B 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-&ggr;––producing T cells and enhanced cardiac allograft vasculopathy lesion formation. Conclusions— Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-&kgr;B signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-&kgr;B 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.

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 αvβ3 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.—Devalliere, J., Chang, W. G., Andrejecsk, J. W., Abrahimi, P., Cheng, C. J., Jane‐wit, D., Saltzman, W. M., Pober, J. S. Sustained delivery of proangiogenic microRNA‐132 by nanoparticle transfection improves endothelial cell transplantation. FASEB J. 28, 908–922 (2014). www.fasebj.org

Interacting Mechanisms in the Pathogenesis of Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.

Mechanisms of Dysfunction in Senescent Pulmonary Endothelium

Age-dependent changes in pulmonary endothelium contribute to worsened clinical outcomes in elderly individuals. Due to altered pulmonary endothelial responses, older participants have increased vulnerability to infection-related sequelae, higher prevalence of pulmonary hypertension, mitigated DNA repair mechanisms, and attenuated parenchymal healing. Aberrant signaling in pulmonary endothelium undergird these clinical processes. In this review, we provide an overview of the work that has elucidated age-related molecular derangements in pulmonary endothelial cells. In particular, we summarize studies describing mishandling of intracellular reactive oxygen species, pathological nitric oxide signaling, and deficient recruitment of endothelial stem cell precursors. We conclude with a summary of potential future avenues of investigation. The signaling pathways associated with pulmonary endothelial senescence reviewed herein suggest a number of putative therapeutic drug targets. Further elucidation of the cellular processes associated with aging in the pulmonary endothelium may provide critical insights into the rational design of therapies that may subvert or even reverse the effects of aging on a molecular level.

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

Significance Complement activation contributes to host defense and immunopathology. We recently discovered that membrane attack complexes (MAC), the terminal effector mechanisms of complement, activate proinflammatory functions in human endothelial cells (ECs) via noncanonical NF-ΚB signaling. Here we elucidate the initial steps of how MACs activate this pathway. MACs formed on the surface of human ECs are rapidly internalized via clathrin-mediated endocytosis into Rab5+ endosomes, which subsequently recruit activated Akt in a Rab5-dependent manner. Akt recruitment results in NIK protein stabilization on the surface of the endosome within 30 min, initiating noncanonical NF-ΚB signaling. MAC internalization in ECs lining human coronary arteries in vivo similarly activates noncanonical NF-ΚB signaling. Our findings suggest new therapeutic targets for controlling complement-mediated inflammation. Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB–inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5+endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC+ endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt+NIK+ signalosome on Rab5+ endosomes.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell–cell interactions of circulating leukocytes with IL-1– or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17–induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17–activated PCs, but not ECs, induce pertussis toxin–sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17–activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17–treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17–activated PCs can modulate neutrophil functions within the perivascular tissue space.

Innate immune mechanisms in transplant allograft vasculopathy.

Purpose of reviewAllograft vasculopathy is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury and donor-specific antibody-induced complement activation confer heightened risk for allograft vasculopathy via numerous innate immune mechanisms, including MyD88, high-mobility group box 1 (HMGB1), and complement-induced noncanonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&kgr;B) signaling. Recent findingsThe role of MyD88, a signal adaptor downstream of the Toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced allograft vasculopathy. Importantly, triggering receptor on myeloid receptor 1, a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of allograft vasculopathy in mice. HMGB1, a nuclear protein that activates Toll-like receptors, also enhanced the development of allograft vasculopathy. Complement activation elicits assembly of membrane attack complexes on endothelial cells which activate noncanonical NF-&kgr;B signaling, a novel complement effector pathway that induces proinflammatory genes and potentiates endothelial cell-mediated alloimmune T-cell activation, processes which enhance allograft vasculopathy. SummaryInnate immune mediators, including HMGB1, MyD88, and noncanonical NF-&kgr;B signaling via complement activation contribute to allograft vasculopathy. These pathways represent potential therapeutic targets to reduce allograft vasculopathy after solid organ transplantation.

Recent advances in allograft vasculopathy.

Purpose of review Despite considerable advances in controlling acute rejection, the longevity of cardiac and renal allografts remains significantly limited by chronic rejection in the form of allograft vasculopathy. This review discusses recently reported mechanistic insights of allograft vasculopathy pathogenesis as well as recent clinical evaluations of new therapeutic approaches. Recent findings Although adaptive immunity is the major driver of allograft vasculopathy, natural killer cells mediate vasculopathic changes in a transplanted mouse heart following treatment with donor-specific antibody (DSA). However, natural killer cells may also dampen chronic inflammatory responses by killing donor-derived tissue-resident CD4+ T cells that provide help to host B cells, the source of DSA. DSA may directly contribute to vascular inflammation by inducing intracellular signaling cascades that upregulate leukocyte adhesion molecules, facilitating recruitment of neutrophils and monocytes. DSA-mediated complement activation additionally enhances endothelial alloimmunogenicity through activation of noncanonical NF-&kgr;B signaling. New clinical studies evaluating mammalian target of rapamycin and proteasome inhibitors to target these pathways have been reported. Summary Allograft vasculopathy is a disorder resulting from several innate and adaptive alloimmune responses. Mechanistic insights from preclinical studies have identified agents that are currently being investigated in clinical trials.

Complement C5 Inhibition Reduces T Cell–Mediated Allograft Vasculopathy Caused by Both Alloantibody and Ischemia Reperfusion Injury in Humanized Mice

Allograft vasculopathy (AV) is characterized by diffuse stenoses in the vasculature of solid organ transplants. Previously, we developed two humanized models showing that alloantibody and ischemia reperfusion injury (IRI) exacerbated T cell–mediated AV in human arterial xenografts in vivo. Herein we examined a causal role for terminal complement activation in both settings. IRI, in contrast to alloantibody, elicited widespread membrane attack complex (MAC) assembly throughout the vessel wall. Both alloantibody and IRI caused early (24 h) and robust endothelial cell (EC) activation localized to regions of intimal MAC deposition, indicated by increases in nuclear factor kappa B (NF‐κB)–inducing kinase, an MAC‐dependent activator of noncanonical NF‐kB, VCAM‐1 expression and Gr‐1+ neutrophil infiltration. Endothelial cell activation by alloantibody was inhibited by antimouse C5 mAb, but not by anti‐C5a mAb or by control mAb, implicating MAC as the primary target of anti‐C5 mAb. Antimouse C5 mAb significantly reduced alloantibody‐ and IRI‐enhanced T cell infiltration and AV‐like changes, including neointimal hyperplasia as well as intraluminal thrombosis in a subset of IRI‐treated arterial grafts. These results indicate that increased AV lesion formation in response to either alloantibody or IRI is dependent on complement C5 activation and, accordingly, inhibition of this pathway may attenuate AV.