Monobody adapter for functional antibody display on nanoparticles for adaptable targeted delivery applications.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

Eculizumab Therapy for Chronic Antibody-Mediated Injury in Kidney Transplant Recipients: A Pilot Randomized Controlled Trial.

We hypothesized that de novo donor-specific antibody (DSA) causes complement-dependent endothelial cell injury in kidney transplants, as assessed by expression of endothelial cell-associated transcripts (ENDATs), that may be attenuated through complement inhibition. In total, 15 participants (five control, 10 treatment) with DSA and deteriorating renal function were enrolled. The treatment group received 6 mo of eculizumab followed by 6 mo of observation, whereas controls were observed. The primary end point was percentage change in estimated GFR (eGFR) trajectory over the treatment period. The treatment group had an improved eGFR trajectory versus control, based on our predetermined two-sided 0.10 significance level (p = 0.09). Within-subject analysis of treated participants at 6-mo intervals did not show significant change (p = 0.60). Modeling C1q status showed that C1q-positive patients had significantly higher mean eGFR than patients with negative C1q (p = 0.04). Biopsies revealed elevated renal ENDATs in most participants, but ENDATs were not reduced with complement inhibition. Our data suggest that eculizumab treatment may stabilize kidney function in patients with chronic persistent DSA based on our pilot a priori significance threshold. ENDAT expression predicative of acute humoral injury is not reduced with complement inhibition in this chronic setting. Further studies will be necessary to determine which patients may benefit from eculizumab.

Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis.

We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKL(Ser358) and Drp1(Ser616) phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKL(Ser358), and coincidence of phospho-MLKL(ser358) and phospho-Drp1(Ser616) at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.

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.

Blood Vessels in Allotransplantation.

Human vascularized allografts are perfused through blood vessels composed of cells (endothelium, pericytes, and smooth muscle cells) that remain largely of graft origin and are thus subject to host alloimmune responses. Graft vessels must be healthy to maintain homeostatic functions including control of perfusion, maintenance of permselectivity, prevention of thrombosis, and participation in immune surveillance. Vascular cell injury can cause dysfunction that interferes with these processes. Graft vascular cells can be activated by mediators of innate and adaptive immunity to participate in graft inflammation contributing to both ischemia/reperfusion injury and allograft rejection. Different forms of rejection may affect graft vessels in different ways, ranging from thrombosis and neutrophilic inflammation in hyperacute rejection, to endothelialitis/intimal arteritis and fibrinoid necrosis in acute cell-mediated or antibody-mediated rejection, respectively, and to diffuse luminal stenosis in chronic rejection. While some current therapies targeting the host immune system do affect graft vascular cells, direct targeting of the graft vasculature may create new opportunities for preventing allograft injury and loss.

In vitro self-assembly of human pericyte-supported endothelial microvessels in three-dimensional coculture: a simple model for interrogating endothelial-pericyte interactions.

We describe a method for coculture of macro- or microvascular human endothelial cells (ECs) and pericytes (PCs) within a 3-dimensional (3-D) protein matrix resulting in lumenized EC cords invested by PCs. To prevent apoptotic cell death of ECs in 3-D culture, human umbilical vein or dermal microvascular ECs were transduced to express the antiapoptotic protein Bcl-2. To prevent PC-mediated gel contraction, the collagen-fibronectin gel was polymerized within a polyglycolic acid nonwoven matrix. Over the first 24-48 h, EC-only gels spontaneously formed cords that developed lumens via vacuolization; such vascular networks were maintained for up to 7 days. In EC-PC cocultures, PCs were recruited to the EC networks. PC investment of EC cords both limited the lumen diameter and increased the degree of vascular network arborization. Peg and socket junctions formed between ECs and PCs in this system, but dye transfer, indicative of gap junction formation, was not observed. This simple system can be used to analyze bidirectional signals between ECs and PCs in a 3-D geometry.

Transforming growth factor beta expression by human vascular cells inhibits interferon gamma production and arterial media injury by alloreactive memory T cells.

Arteriosclerosis is characterized by the local activation of effector T cells leading to production of proinflammatory cytokines, such as IFN (interferon)-γ and IL-17, within the vessel wall. Conversely, the production of antiinflammatory cytokines, for example, TGF-ß, by regulatory lymphocytes is known to inhibit both the differentiation of naïve T cells into effector T cells and the development of arteriosclerosis in murine models. We investigated the role of TGF-ß on the alloreactivity of human effector memory T cells (Tem). Quiescent vascular cells, but not Tem, expressed TGF-ß. Blockade of TGF-ß activity in cocultures of CD4(+) Tem with allogeneic endothelial cells significantly increased IFN-γ, but not IL-17, secretion. Additionally, serologic neutralization of TGF-ß in immunodeficient mouse hosts of human coronary artery grafts into which allogeneic human T cells were adoptively transferred resulted in heavier medial infiltration by Tem, greater loss of medial smooth muscle cells and increased IFN-γ production within the grafts without significantly reducing either intimal injury or IL-17 production. Protective effects of TGF-ß may be limited by fewer TGF-ß-expressing vascular cells within the intimal compartment, by a reduction in the expression of TGF-ß by vascular cells in rejecting grafts, or possibly to less effective suppression of Tem than naïve T cells.

TNF receptors differentially signal and are differentially expressed and regulated in the human heart.

Tumor necrosis factor (TNF) utilizes two receptors, TNFR1 and 2, to initiate target cell responses. We assessed expression of TNF, TNFRs and downstream kinases in cardiac allografts, and compared TNF responses in heart organ cultures from wild-type ((WT)C57BL/6), TNFR1-knockout ((KO)), TNFR2(KO), TNFR1/2(KO) mice. In nonrejecting human heart TNFR1 was strongly expressed coincidentally with inactive apoptosis signal-regulating kinase-1 (ASK1) in cardiomyocytes (CM) and vascular endothelial cells (VEC). TNFR2 was expressed only in VEC. Low levels of TNF localized to microvessels. Rejecting cardiac allografts showed increased TNF in microvessels, diminished TNFR1, activation of ASK1, upregulated TNFR2 co-expressed with activated endothelial/epithelial tyrosine kinase (Etk), increased apoptosis and cell cycle entry in CM. Neither TNFR was expressed significantly by cardiac fibroblasts. In (WT)C57BL/6 myocardium, TNF activated both ASK1 and Etk, and increased both apoptosis and cell cycle entry. TNF-treated TNFR1(KO) myocardium showed little ASK1 activation and apoptosis but increased Etk activation and cell cycle entry, while TNFR2(KO) myocardium showed little Etk activation and cell cycle entry but increased ASK1 activation and apoptosis. These observations demonstrate independent regulation and differential functions of TNFRs in myocardium, consistent with TNFR1-mediated cell death and TNFR2-mediated repair.

Generation of NO by bystander human CD8 T cells augments allogeneic responses by inhibiting cytokine deprivation-induced cell death.

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in bystander human CD8 T cells, augments the accumulation of allogeneically activated human CD8 T cells in vitro and in vivo. Here, we report that iNOS-derived NO does not affect T-cell proliferation but rather inhibits cell death of activated human CD8 T cells after activation by allogeneic endothelial cells in culture. Exogenous NO did not affect activation-induced cell death of human CD8 T cells but specifically reduced death of activated T cells due to cytokine deprivation. NO-mediated inhibition of T-cell death did not involve cGMP signaling, and NO did not affect the expression of Bcl-2-related proteins known to regulate cytokine deprivation-induced cell death. However, NO inhibited the activity of caspases activated as a consequence of cytokine deprivation in activated T cells. This protective effect correlated with S-nitrosylation of caspases and was phenocopied by z-VAD.fmk and z-LEHD.fmk, pharmacological inhibitors of caspases. In summary, our findings indicate that NO augments the accumulation of activated human T cells principally by inhibiting cytokine deprivation-induced cell death through S-nitrosylation of caspases.

Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo.

This pilot study examines noninvasive MR monitoring of tissue-engineered vascular grafts (TEVGs) in vivo using cells labeled with iron oxide nanoparticles. Human aortic smooth muscle cells (hASMCs) were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The labeled hASMCs, along with human aortic endothelial cells, were incorporated into eight TEVGs and were then surgically implanted as aortic interposition grafts in a C.B-17 SCID/bg mouse host. USPIO-labeled hASMCs persisted in the grafts throughout a 3 wk observation period and allowed noninvasive MR imaging of the human TEVGs for real-time, serial monitoring of hASMC retention. This study demonstrates the feasibility of applying noninvasive imaging techniques for evaluation of in vivo TEVG performance.

Endothelial cell dysfunction, injury and death.

Vascular endothelial cells (ECs) perform a number of functions required to maintain homeostasis. Inflammation can cause EC injury and death which disrupt these processes and result in endothelial dysfunction. Three common mediators of EC injury in inflammation are macrophage-derived cytokines, such as tumour necrosis factor (TNF); neutrophil-generated reactive oxygen species (ROS) and cytolytic T lymphocytes (CTL). Here we describe the distinct but overlapping biochemical pathways of injury elicited by these different agents.

Combining altered levels of effector transcripts in circulating T cells with a marker of endothelial injury is specific for active graft-versus-host disease.

Cytotoxic T lymphocytes (CTLs) are important effector cells of graft-versus-host disease (GVHD) and vascular endothelial cells are target cells of allospecific CTL. A combined assessment of T-cell activation and endothelial injury should result in a specific and sensitive test for GVHD. We examined circulating T lymphocytes for effector molecules involved in CTL-mediated endothelial injury. We analyzed CD4 and CD8 T lymphocytes of 24 long-term survivors of allogeneic stem cell transplantation with or without GVHD, and nine healthy, age-matched controls for signs of CTL activation and endothelial injury. IFN-gamma transcript levels in CD8 T cells were significantly elevated in SCT recipients with GVHD compared to patients without GVHD (767 CD3epsilon units/T cell (376-2050) vs 211 CD3epsilon units/T cell (159-274), P=0.01). Fas ligand transcript levels in CD4 T cells were significantly elevated in SCT recipients without GVHD compared to patients with GVHD (20 CD3epsilon units/T cell (0-78) vs 0 CD3epsilon units/T cell (0-0), P=0.01). Von Willebrand factor plasma levels were high in patients with GVHD, but normal in patients without GVHD (209 (186-254) vs 120 (100-141), P=0.0005). This assessment of T-cell activation and endothelial injury results in a sensitive and specific test to identify patients with active chronic GVHD.

Obstacles facing translational research in academic medical centers.

Over the last quarter of the 20th century, there has been a boom in biomedical research discoveries that, for the most part, has not been successfully exploited for improving medical therapy or diagnosis. This lack of success is surprising because there is a broad consensus within academic medical centers (AMCs) that a primary mission is to move scientific discoveries into meaningful clinical outcomes, and there are numerous opportunities for doing so. We illustrate the latter point with 10 clinical opportunities for translating scientific discoveries from our field of vascular biology and transplantation. We attribute the limited success of translation to various factors, chief of which is that translation is rarely straightforward and requires continuing research in both the clinic and the laboratory. Translational research is hindered by insufficient targeted resources, a shortage of qualified investigators, an academic culture that hinders collaboration between clinical and laboratory-based investigators, a traditional structure of the AMC that favors departmental efforts over interdisciplinary programs, an increasing regulatory burden, and a lack of specific mechanisms within the AMC for facilitating solutions to these problems. We offer several suggestions to reduce these impediments.

Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.

Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with either of two cell surface receptors, TNFR-1 and TNFR-2. The levels and regulation of expression of these two receptors has been extensively analyzed in cultured cells, but little is known of TNFR expression in situ. We analyzed the expression of TNFR-1 and -2 in normal human renal kidney and in renal transplants undergoing acute cellular rejection. Immunohistochemistry and immunogold electron microscopy indicated a strong expression of TNFR-1 on the endothelium of glomeruli of normal kidney. Immunogold colocalization for TNFR-1 and a marker of the trans-Golgi network (TGN-46) demonstrated TNFR-1 within the Golgi complex in endothelial cells in normal kidney, confirming our previous studies with cultured cells. TNFR-1 expression was lost in glomeruli from acutely rejecting kidney, but TNFR-1 was detected in abundance on infiltrating leukocytes in the interstitium of allografts with acute rejection. In contrast, TNFR-2 was demonstrated predominantly in epithelial cells of distal convoluted tubule (DCT) in acute rejection kidney near TNF-expressing leukocytes. TNF was absent in normal kidney, but present in rejecting allograft. TNF was found in infiltrating leukocytes and in adjacent tubular epithelial cells. In situ hybridization showed TNFR-1 mRNA within the endothelium of the glomeruli and of a few arterioles in normal kidney, whereas TNFR-2 mRNA was seen in tubular epithelial cells of the DCT in acute transplant rejection. These data reveal that there is both differential expression and regulation of the two TNF receptors in human kidney.

Human endothelial cell presentation of antigen and the homing of memory/effector T cells to skin.

Dermal microvascular endothelial cells (ECs) form a continuous lining that normally bars blood-borne T lymphocytes from entering the skin, but as part of the response to foreign antigen, dermal ECs undergo alterations in their surface proteins so as to provide signals to circulating T cells that lead to their activation and recruitment. Several observations suggest that human dermal microvascular ECs may help initiate cutaneous immune reactions by presentation of cognate antigens to circulating T memory cells: (1) antigen-specific inflammatory responses in the skin, as in other organs, involve accumulation of memory and effector T cell populations that are enriched in cells specific for the eliciting antigen; (2) recall responses to intradermal protein antigens in the skin start very rapidly within two hours of challenge; (3) dermal microvascular ECs in humans and other large mammals basally display high levels of class I and class II MHC molecules, the only known purpose of which is to present antigenic peptides to lymphocytes; (4) the lumen of dermal capillaries are narrower than the diameter of circulating T cells, ensuring surface contact; and (5) cultured human ECs effectively present antigens to resting memory T cells isolated from the circulation. Upon contact with activated T cells or their secreted products (cytokines), dermal ECs themselves become activated, increasing their capacity to recruit memory and effector T cell populations in an antigen-independent manner. Specifically, activated ECs express inducible leukocyte adhesion molecules such as E-selectin, ICAM-1, and VCAM-1; and several lines of evidence, including neutralizing antibody experiments and gene knockouts, have supported a role of these molecules in T cell recruitment. Dermal ECs have unique expression patterns of adhesion molecules that can determine the subsets of memory T cells that are recruited into the skin. For example, slow internalization of E-selectin allows more persistent expression of this protein on the surface of dermal ECs, favoring interactions with CLA-1+ T cells. VCAM-1 expression, normally confined to venular EC may extend to capillaries within the dermal papillae and contribute to epidermal inflammation, recruiting alpha4beta7 integrin-expressing T cells that also express the cadherin-binding integrin alphaEbeta7. New models involving transplantation of normal and genetically modified human dermal ECs into immunodeficient mice may be used to further explore these properties.

Tumor necrosis factor receptor-associated factors (TRAFs).

Tumor necrosis factor receptor-associated factors (TRAFS) were initially discovered as adaptor proteins that couple the tumor necrosis factor receptor family to signaling pathways. More recently they have also been shown to be signal transducers of Toll/interleukin-1 family members. Six members of the TRAF family have been identified. All TRAF proteins share a C-terminal homology region termed the TRAF domain that is capable of binding to the cytoplasmic domain of receptors, and to other TRAF proteins. In addition, TRAFs 2-6 have RING and zinc finger motifs that are important for signaling downstream events. TRAF proteins are thought to be important regulators of cell death and cellular responses to stress, and TRAF2, TRAF5 and TRAF6 have been demonstrated to mediate activation of NF-kappaB and JNK. TRAF proteins are expressed in normal and diseased tissue in a regulated fashion, suggesting that they play an important role in physiological and pathological processes.

Human T cells infiltrate and injure pig coronary artery grafts with activated but not quiescent endothelium in immunodeficient mouse hosts.

BACKGROUND: We have previously demonstrated that human artery grafts transplanted to immunodeficient mice are infiltrated and injured by unsensitized allogeneic human T cells. We extended our investigations to human anti-porcine xenoresponses in this model. METHODS: Pig coronary artery segments were interposed into the infrarenal aorta of severe combined immunodeficiency/beige mice. After 7 days, certain recipients were reconstituted with human leukocytes and/or treated with proinflammatory cytokines. The grafts were harvested after 1-70 days and examined by histology, immunohistochemistry, and morphometry. RESULTS: Pig artery grafts from untreated mice had no evidence of injury, leukocytic infiltrate, or endothelial cell activation up to 70 days postoperatively, despite deposition of murine complement. Host reconstitution with human peripheral blood mononuclear cells resulted in a discrete population of circulating T cells that did not infiltrate or injure the grafts up to 28 days after adoptive transfer. Administration of porcine interferon-gamma for up to 28 days sustained the expression of graft vascular cell adhesion molecule-1 and major histocompatibility complex antigens, but did not initiate recruitment of human leukocytes. In contrast, treatment with human tumor necrosis factor for 7 days induced the de novo expression of porcine E-selectin by graft endothelial cells and elicited human T cell infiltration and human peripheral blood mononuclear cell-dependent vascular injury. CONCLUSIONS: The human peripheral blood mononuclear cell-severe combined immunodeficiency/beige mouse model identifies a significant difference between human T cell allogeneic and xenogeneic responses in vivo. Xenografts with quiescent endothelium are not infiltrated or injured by T cells under the same conditions in which allografts are rejected. Activation of pig coronary artery endothelial cells by human tumor necrosis factor, but not porcine interferon-gamma, elicits cellular xenoresponses.

TNF signaling in vascular endothelial cells.

Vascular endothelium is a major target of actions of the proinflammatory cytokine tumor necrosis factor (TNF). Increasingly, the intracellular pathways that are activated in response to TNF have been elucidated. Many of these pathways have proven to be cell type-specific, requiring that observations made in other cell types be confirmed or ruled out in endothelial cells (EC). In this review the authors will summarize the state of the field, emphasizing studies in cultured human EC.

Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.