Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

RATIONALE: The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. OBJECTIVE: To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. METHODS AND RESULTS: To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CONCLUSIONS: CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.

Three tapasin docking sites in TAP cooperate to facilitate transporter stabilization and heterodimerization.

The TAP translocates peptide Ags into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex, an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains of TAP1 and TAP2. The third binding site is present in the core transmembrane (TM) domain of TAP1 and is used only by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular, the role of tapasin binding to the core TM domain of TAP1 single chains is mysterious because this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic TM helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants that lack tapasin binding to either N-terminal domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization.

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.

Augmenting CAR T Cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both LTßR on cancer cells and HVEM on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with LTßR on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

A national mapping assessment of blood collection and transfusion service facilities in Afghanistan.

BACKGROUND: The purpose of this study was to assess functionality and resources of facilities providing blood collection and transfusion services in Afghanistan. STUDY DESIGN AND METHODS: This national cross-sectional assessment included facilities collecting or transfusing blood identified through official data sources and private key informants. At each facility, study representatives completed a standardized instrument assessing presence of records logbook, electricity, refrigeration, and required transfusion-transmitted infection (TTI; human immunodeficiency virus, syphilis, and hepatitis B and C) test kits. Descriptive statistics were generated, with differences analyzed using chi-square or Fisher's exact tests. RESULTS: Between August and November 2010, a total of 243 facilities were surveyed with public (52.3%, n = 127) and private (43.2%, n = 105) sector comprising the majority. Most (63%) facilities were urban, with 23.5% located in Kabul province. Of 92,682 units collected nationally in the 12 months before evaluation, 7.5% (n = 6952) had no disposition record. Many (62%, n = 151) facilities had an established recordkeeping system; the remainder provided estimates. Half of surveyed facilities had regular power supply (57.8%), refrigerators for storing blood (52.3%), or all necessary TTI test kits (62.1%). Military (83.3%) and public (74.8%) facilities were more likely to have all TTI test kits present compared to private (46.7%, p < 0.01) but not nongovernmental organization (40.0%, p = 0.37) facilities. CONCLUSION: In Afghanistan, blood donation and transfusion occur with substantial differences in data recording and TTI test availability, with private facilities less likely to have these resources. Efforts are needed to improve available resources and ensure that facilities are in compliance with national standards for donor screening.

Blood supply safety in Afghanistan: a national assessment of high-volume facilities.

BACKGROUND: Little information is available regarding blood supply safety in Afghanistan. The purpose of this study was to assess blood safety through serologic and observational measures in Afghanistan. STUDY DESIGN AND METHODS: This cross-sectional assessment included the 40 highest-volume facilities collecting and transfusing blood nationally identified in a previous survey. At each facility, study representatives completed a standardized instrument assessing staff performance of transfusion-related activities and performed rapid testing for human immunodeficiency virus, syphilis, and hepatitis B and C with rapid diagnostic tests on clinically discarded specimens. Reactive samples received confirmatory testing. Descriptive statistics were generated, with differences analyzed using chi-square or Fisher's exact tests. RESULTS: Between November 2010 and May 2011, a total of 332 blood donor collection procedures were observed. Only 52.4% of observed encounters correctly screened and deferred donors by international criteria. Public and private facilities demonstrated glove use, proper sharps disposal, and patient counseling and relayed screening test results in less than 75% of observed events, significantly less likely than military facilities (p < 0.01). Of 1612 specimens assessed, confirmed cases of hepatitis B (n = 6), hepatitis C (n = 1), and syphilis (n = 3) were detected among units already prescreened and accepted for transfusion. CONCLUSION: Lapses in proper donor screening contributed to the presence of confirmed-positive units available for transfusion, as detected in this study. Steps must be taken to ensure standardization of testing kits requirements, documentation, and mandatory training and continuing education for blood bank staff with regard to counseling, drawing, processing, and transfusion of blood products.

Emerging Intraoperative Imaging Technologies in Urologic Oncology.

As surgical cases become more complex, intraoperative imaging is increasingly being used. This article discusses emerging imaging technologies used in prostate, kidney, and bladder cancer surgery, including ultrasound, fluorescence-based, and enhanced endoscopy techniques including their strengths and limitations.

A comparative population-based analysis of peritoneal carcinomatosis in patients undergoing robotic-assisted and open radical cystectomy.

PURPOSE: To compare the population-based incidence of peritoneal carcinomatosis following open (ORC) vs. robotic-assisted radical cystectomy (RARC). METHODS: Using the Surveillance, Epidemiology and End Results Program (SEER)-Medicare linked data, we identified 1,621 patients who underwent radical cystectomy for bladder cancer during 2009 and 2014; 18.1% (n = 294) and 81.9% (n = 1327) underwent RARC and ORC, respectively. We subsequently evaluated the rates of peritoneal carcinomatosis at 6, 12, and 24 months following surgery. Multivariable proportional hazards regression was performed to determine factors associated with development of peritoneal carcinomatosis. RESULTS: Patients who underwent RARC vs. ORC were more likely to be male (p = 0.04); however, age at diagnosis, race, comorbidities, education, and marital status (all p > 0.05) did not differ by surgical approaches. Our findings showed that there were no significant differences in the rates of peritoneal carcinomatosis between ORC and RARC at 6, 12, and 24 months. In adjusted analyses, factors associated with peritoneal carcinomatosis were advanced N stage (N0 versus N2/3: HR 0.30, 95% CI 0.16-0.55, p < 0.01), preoperative hydronephrosis (HR 1.70, 95% CI 1.09-2.65, p = 0.04), higher T stage (T1 versus T4: HR 0.34, 95% CI 0.15-0.79, p < 0.01; T2 versus T4: HR 0.39, 95% CI 0.20-0.76, p < 0.01), and use of neoadjuvant chemotherapy (HR 1.78, 95% CI 1.11-2.84, p < 0.01). However, RARC was not associated with peritoneal carcinomatosis (HR 1.36, 95% CI 0.78-2.35). CONCLUSION: In this population-based analysis, we found no difference in peritoneal carcinomatosis between robotic or open approaches to radical cystectomy. These data should be reassuring to those utilizing robotic cystectomy.

Calmodulin-dependent gating of Ca(v)1.2 calcium channels in the absence of Ca(v)beta subunits.

It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.

Blocking MHC class II on human endothelium mitigates acute rejection.

Acute allograft rejection is mediated by host CD8+ cytotoxic T lymphocytes (CTL) targeting graft class I major histocompatibility complex (MHC) molecules. In experimental rodent models, rejection requires differentiation of naive CD8+ T cells into alloreactive CTL within secondary lymphoid organs, whereas in humans, CTL may alternatively develop within the graft from circulating CD8+ effector memory T cells (TEM) that recognize class I MHC molecules on graft endothelial cells (EC). This latter pathway is poorly understood. Here, we show that host CD4+ TEM, activated by EC class II MHC molecules, provide critical help for this process. First, blocking HLA-DR on EC lining human artery grafts in immunodeficient mice reduces CD8+ CTL development within and acute rejection of the artery by adoptively transferred allogeneic human lymphocytes. Second, siRNA knockdown or CRISPR/Cas9 ablation of class II MHC molecules on EC prevents CD4+ TEM from helping CD8+ TEM to develop into CTL in vitro. Finally, implanted synthetic microvessels, formed from CRISPR/Cas9-modified EC lacking class II MHC molecules, are significantly protected from CD8+ T cell-mediated destruction in vivo. We conclude that human CD8+ TEM-mediated rejection targeting graft EC class I MHC molecules requires help from CD4+ TEM cells activated by recognition of class II MHC molecules.

Effect of Ca(v)beta subunits on structural organization of Ca(v)1.2 calcium channels.

BACKGROUND: Voltage-gated Ca(v)1.2 calcium channels play a crucial role in Ca(2+) signaling. The pore-forming alpha(1C) subunit is regulated by accessory Ca(v)beta subunits, cytoplasmic proteins of various size encoded by four different genes (Ca(v)beta(1)-beta(4)) and expressed in a tissue-specific manner. METHODS AND RESULTS: Here we investigated the effect of three major Ca(v)beta types, beta(1b), beta(2d) and beta(3), on the structure of Ca(v)1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Ca(v)1.2 to form clusters depends on the type of the Ca(v)beta subunit present. The highest density of Ca(v)1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac beta(1b) present. Ca(v)1.2 channels containing beta(3), the predominant Ca(v)beta subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between alpha(1C) and Ca(v)beta in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Ca(v)1.2 channels in the plasma membrane depends on the Ca(v)beta type. The presence of different Ca(v)beta subunits does not result in significant differences in the intramolecular distance between the termini of alpha(1C), but significantly affects the distance between the termini of neighbor alpha(1C) subunits, which varies from 67 A with beta(1b) to 79 A with beta(3). CONCLUSIONS: Thus, our results show that the structural organization of Ca(v)1.2 channels in the plasma membrane depends on the type of Ca(v)beta subunits present.

A perfusion bioreactor for intestinal tissue engineering.

BACKGROUND: Short gut syndrome is a devastating clinical problem with limited long-term treatment options. A unique characteristic of the normal intestinal epithelium is its capacity for regeneration and adaptation. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial differentiation and proliferation has been the difficulty in maintaining primary cultures of normal gut epithelium in vitro. A perfusion bioreactor system has been shown to be beneficial in long-term culture and bioengineering of a variety of tissues. The purpose of this study is to design and fabricate a perfusion bioreactor for intestinal tissue engineering. MATERIALS AND METHODS: A perfusion bioreactor is fabricated using specific parameters. Intestinal epithelial organoid units harvested from neonatal rats are seeded onto biodegradable polymer scaffolds and cultured for 2 d in the bioreactor. Cell attachment, viability, and survival are assessed using MTT assay, scanning electron micrograph, and histology. RESULTS: A functional perfusion bioreactor was successfully designed and manufactured. MTT assay and scanning electron micrograph demonstrated successful attachment of viable cells onto the polymer scaffolds. Histology confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the perfusion bioreactor for 2 days. CONCLUSIONS: A functional perfusion bioreactor can be successfully fabricated for the in-vitro cultivation of intestinal epithelial cells. With further optimization, the perfusion bioreactor may be a useful in in-vitro system for engineering new intestinal tissue.