Analyses of cellular multimerin 1 receptors: in vitro evidence of binding mediated by αΙΙbβ3 and αvβ3

2005 ◽  
Vol 94 (11) ◽  
pp. 1004-1011 ◽  
Author(s):  
Frédéric Adam ◽  
Shilun Zheng ◽  
Nilesh Joshi ◽  
David Kelton ◽  
Amin Sandhu ◽  
...  

SummaryMultimerin 1 (MMRN1) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family.In vivo, MMRN1 is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRN1 binding to activated platelets and endothelial cells, we investigated the identity of the major MMRN1 receptors on these cells using wild-type and RGE-forms of recombinant MMRN1. Ligand capture, cell adhesion, ELISA and flow cytometry analyses of platelet-MMRN1 binding, indicated that MMRN1 binds to integrins αIIbβ3 and αvβ3. Endothelial cell binding to MMRN1 was predominantly mediated by αvβ3 and did not require the MMRN1 RGD site or cellular activation. Like many other αvβ3 ligands, MMRN1 had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRN1 processing, indicated that MMRN1 adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRN1 attachment to cells and indicate that MMRN1 is a ligand for αIIbβ3 and αvβ3.

2018 ◽  
Vol 2 (S1) ◽  
pp. 11-12
Author(s):  
Mark H. Murdock ◽  
Jordan T. Chang ◽  
George S. Hussey ◽  
Nduka M. Amankulor ◽  
Johnathan A. Engh ◽  
...  

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and common primary tumor type in the central nervous system across all age groups; affected adults have a life expectancy of just 14 months. As glioma cells invade the surrounding normal parenchyma they remodel the composition and ultrastructure of the surrounding extracellular matrix (ECM), suggesting that the native (i.e., “normal”) microenvironment is not ideal for their survival and proliferation. Recent reports describe suppressive and/or lethal effects of mammalian ECM hydrogels derived from normal (nonneoplastic) sources upon various cancer types. ECM-based bioscaffolds placed at sites of neoplastic tissue resection in humans have never been reported to facilitate cancer recurrence. The objective of the present research is to evaluate mammalian ECM as a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECM hydrogels from porcine dermis, small intestine, and urinary bladder were produced as described previously. Primary glioma cells were graciously supplied by Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered through ATCC. Cells were plated onto tissue culture plastic at ~60% confluence and allowed to attach for 24 hours before treatment. The saline-soluble fraction (SSF) of ECM was obtained by mixing lyophilized, comminuted ECM with 0.9% saline for 24 hours then filtering the resulting mixture through a 10 kDa molecular weight cutoff column. All assays and kits were followed according to the manufacturer’s instructions. Cell viability was measured via MTT assay (Vybrant® MTT Cell Proliferation Assay, Invitrogen) and by live/dead staining (LIVE/DEAD® Cell Imaging Kit, Invitrogen). Time lapse videos were created by taking images every 20 minutes for 18 hours (phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagent was ordered from Biotium. Temozolomide was ordered through Abmole. All in vivo work was conducted according to protocols approved by the University of Pittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS: ECM hydrogels derived from porcine dermis, small intestine, or urinary bladder all decreased the viability of primary glioma cells in vitro, with urinary bladder extracellular matrix (UBM) having the most dramatic effects. The SSF of UBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, was sufficient to recapitulate this detrimental effect upon neoplastic cells in vitro and was used for the remainder of the experiments described herein. In a cell viability assay normalized to the media treatment, non-neoplastic CHME5 and N1E-115 cells scored 103% and 114% after 48 hours when treated with UBM-SSF and 2 primary high-grade glioma cell types scored 17% and 30.5% with UBM-SSF (n=2). Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence of UBM-SSF for 18 hours while most primary glioma cells shriveled and died within this time. Darkfield time-lapse video of wells containing Nucview dye, fluorescent upon cleavage by active caspase-3, confirmed that within 12 hours most primary glioma cells underwent apoptosis while CHME5 and HFF did not. In culture with primary astrocytes, high grade primary glioma cells, and U-87 MG glioma cells for 24 hours, UBM-SSF was found to significantly increase the population of primary astrocytes compared with media (p<0.05) while decreasing the 2 glioma cell types to approximately one-third as many cells as the media control (p<0.0001). A dose-response of temozolomide from 0 to 10,000 μM showed that when treating 2 non-neoplastic cell types (CHME5 and HFF) and 2 types of primary glioma cell there was no difference in survivability at any concentration. Contrasted to this, a dose-response of UBM-SSF from 350 to 7000 μg/mL showed that the non-neoplastic cells survived significantly better than the glioma cells at concentrations of 875 μg/mL and upward (p<0.05). In preliminary animal experiments, large primary glioma tumors in the flanks of athymic nude mice were resected and replaced with either UBM SSF or Matrigel (an ECM product of neoplastic cell origin). After 7 days the resection sites with UBM-SSF had little tumor regrowth if any compared with the dramatic recurrence seen in the Matrigel injection sites (n=2). In a separate survival study comparing PBS to UBM-SSF injections in the flank-resection model, all animals given PBS had to be sacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSF were sacrificed at 15, 24, and 39 days (n=3), indicating a moderate increase in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OF IMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZ in 2005, the standard of care for patients with glioblastoma multiforme has not improved. These findings indicate that non-neoplastic ECM contains potent bioactive regulators capable of abrogating malignancy. Our in vitro data suggest these molecules appear to have no deleterious effect on non-neoplastic cells while specifically inducing apoptosis in glioma cells. Our in vivo data suggest that these molecules may be useful in delaying glioma recurrence, thus resulting in extended lifespan. Delivering soluble fractions of ECM to a tumor site may represent a novel approach to glioma therapy, sidestepping traditional cytotoxic therapies in favor of utilizing putative endogenous anti-tumor pathways.


2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Julie Williams ◽  
Sanlin Robinson ◽  
Babak Alaei ◽  
Kimberly Homan ◽  
Maryam Clausen ◽  
...  

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3917-3917
Author(s):  
Frederic Adam ◽  
Shilun Zheng ◽  
Nilesh Joshi ◽  
Youko Suehiro ◽  
David S. Kelton ◽  
...  

Abstract Multimerin is a large soluble protein, with an uncertain function, found in platelets, megakaryocytes, endothelium and extracellular matrix fibers but not in plasma. The observation that multimerin contains structural features of an adhesive protein, including an Arg-Gly-Asp (RGD) sequence, led us to investigate its ability to support adhesion of platelets, megakaryocytes, endothelial cells and other cell types. Multimerin had the ability to support the adhesion of both platelets and megakaryocytes and this required cellular activation and the multimerin RGD site. Studies of normal and Glanzmann platelets indicated that multimerin interacted with the major platelet integrin receptor, αIIbβ3 and radioimmunoprecipitation analyses confirmed that multimerin bound to αIIbβ3. Multimerin also supported adhesion of endothelial cells, neutrophils and other cells including smooth muscle cells, fibroblast cells, human embryonic kidney (HEK293) and epithelial cells. Unlike platelets, these cells do not express αIIbβ3; this indicated that other integrin or non-integrin receptors could be involved in cellular adhesion to multimerin. Comparisons of cell adhesion to wild-type and RGE-multimerin indicated that unlike platelets and megakaryocytes, some other cell types (e.g. endothelial cells, smooth muscle cells and neutrophils) were capable of adhering to RGE-multimerin. This suggested that cellular adhesion to multimerin occurs by both RGD and non-RGD dependent mechanisms. Finally, unlike platelets, megakaryocytes and neutrophils, adhesion of other cell types to multimerin did not require cellular activation. In conclusion, our data indicate multimerin has fairly broad proadhesive properties, involving RGD and non-RGD dependent mechanisms, and that cellular receptors including αIIbβ3 interact with multimerin to mediate its binding to activated platelets, endothelial cells and potentially other cell types.


1986 ◽  
Vol 163 (5) ◽  
pp. 1292-1307 ◽  
Author(s):  
D M Klinman ◽  
J F Mushinski ◽  
M Honda ◽  
Y Ishigatsubo ◽  
J D Mountz ◽  
...  

PBMC from patients with autoimmune diseases and from normal controls were studied for the expression of several cellular oncogenes. Gene expression was assessed by Northern blot analysis of poly(A)+ RNA obtained from leukapheresis samples. Patients with SLE expressed significantly more c-myc protooncogene RNA than did normal controls. Increased expression of the N-ras protooncogene was found in that subset of patients whose autoimmune disease was very active. Cells from individuals with SLE, but not from those with other autoimmune illnesses, showed significantly decreased levels of the c-myb and c-fos protooncogenes. To examine the implications of these findings, B and T cells were purified from apheresis samples donated by normal volunteers. When mitogen was used to activate the B cells in vitro, their pattern of protooncogene expression changed to resemble that found in freshly isolated cells from lupus patients. These results suggest that the differences detected in the expression of protooncogenes by patients with SLE may be due to the abnormal activation of their B cells in vivo. The pattern of protooncogene expression found in patients with other autoimmune illnesses is consistent with the activation of additional cell types in those diseases.


2020 ◽  
Author(s):  
Andrew Tae-Jun Kwon ◽  
Kohta Mohri ◽  
Satoshi Takizawa ◽  
Takahiro Arakawa ◽  
Maiko Takahashi ◽  
...  

AbstractAntibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Myung-Chul Kim ◽  
Nicholas Borcherding ◽  
Kawther K. Ahmed ◽  
Andrew P. Voigt ◽  
Ajaykumar Vishwakarma ◽  
...  

AbstractRegulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.


Author(s):  
Maria Veronica Lipreri ◽  
Nicola Baldini ◽  
Gabriela Graziani ◽  
Sofia Avnet

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.


1997 ◽  
Vol 138 (2) ◽  
pp. 471-480 ◽  
Author(s):  
Martín I. García-Castro ◽  
Robert Anderson ◽  
Janet Heasman ◽  
Christopher Wylie

Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.


2010 ◽  
Vol 16 (3) ◽  
pp. 781-793 ◽  
Author(s):  
Claudio E. Pedraza ◽  
Benedetto Marelli ◽  
Florencia Chicatun ◽  
Marc D. McKee ◽  
Showan N. Nazhat

1977 ◽  
Vol 145 (1) ◽  
pp. 136-150 ◽  
Author(s):  
A E Butterworth ◽  
J R David ◽  
D Franks ◽  
A A Mahmoud ◽  
P H David ◽  
...  

After earlier observations that antibody-dependent, cell-mediated damage to 51Cr-labeled schistosomula can be ablated by pretreatment of a mixed preparation of human peripheral blood leukocytes with an anti-eosinophil serum and complement, we investigated the cytotoxic effects of eosinophil-enriched cell preparations. Preparations containing up to 98.5% eosinophils and devoid of neutrophils were effective in mediating antibody-dependent damage to schistosomula. Preparations enriched in mononuclear cells or in neutrophils, and devoid of eosinophils, were inactive. Eosinophils from some patients with eosinophilia induced by schistosomiasis were less active on a cell-to-cell basis than cells from normal individuals. The possibility that such cells were initially blocked by immune complexes was considered, and it was found that reasonable cytotoxicity by purified eosinophils from patients with eosinophilia could be generated by overnight cultures. A possible requirement for cooperation between eosinophils and other cell types was also studied. Lymphocytes, neutrophils and monocytes failed to enhance eosinophil-mediated cytotoxicity. These results provide further evidence that the eosinophil is the only cell in man responsible for antibody-dependent, complement-independent damage to schistosomula in vitro. Eosinophils from individuals, however, differ in their cytotoxic potential by a mechanism yet to be elucidated. The possible relationship of these findings to immunity in vivo is discussed.


Sign in / Sign up

Export Citation Format

Share Document