Publications by authors named "Luis R-Borlado"

13 Publications

  • Page 1 of 1

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

J Trauma Acute Care Surg 2021 08;91(2S Suppl 2):S81-S88

From the Coagulation and Blood Research (Blood) (T.C.C., X.W., J.D.K., J.G.-M., C.L.S., B.L., A.P.C., J.A.B.), United States Army Institute of Surgical Research, San Antonio, Texas; Capricor Therapeutics Institute (J.J.M., K.A.P., L.R.-B., N.A.A., L.S.M.), Beverly Hills, California; Department of Biological Chemistry (S.J.G.), Johns Hopkins, Baltimore, Maryland; and Department of Biomedical Engineering (C.R.R.), The University of Texas at San Antonio, San Antonio, Texas.

Background: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined.

Methods: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock.

Results: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy.

Conclusion: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.
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http://dx.doi.org/10.1097/TA.0000000000003218DOI Listing
August 2021

Mechanistic and therapeutic distinctions between cardiosphere-derived cell and mesenchymal stem cell extracellular vesicle non-coding RNA.

Sci Rep 2021 04 21;11(1):8666. Epub 2021 Apr 21.

Capricor Therapeutics, Inc., 8840 Wilshire Blvd., Beverly Hills, CA, 90211, USA.

Cell therapy limits ischemic injury following myocardial infarction (MI) by preventing cell death, modulating the immune response, and promoting tissue regeneration. The therapeutic efficacy of cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs) is associated with extracellular vesicle (EV) release. Prior head-to-head comparisons have shown CDCs to be more effective than MSCs in MI models. Despite differences in cell origin, it is unclear why EVs from different adult stem cell populations elicit differences in therapeutic efficacy. Here, we compare EVs derived from multiple human MSC and CDC donors using diverse in vitro and in vivo assays. EV membrane protein and non-coding RNA composition are highly specific to the parent cell type; for example, miR-10b is enriched in MSC-EVs relative to CDC-EVs, while Y RNA fragments follow the opposite pattern. CDC-EVs enhance the Arg1/Nos2 ratio in macrophages in vitro and reduce MI size more than MSC-EVs and suppress inflammation during acute peritonitis in vivo. Thus, CDC-EVs are distinct from MSC-EVs, confer immunomodulation, and protect the host against ischemic myocardial injury and acute inflammation.
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http://dx.doi.org/10.1038/s41598-021-87939-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060398PMC
April 2021

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization.

Sci Rep 2019 03 15;9(1):4647. Epub 2019 Mar 15.

Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin),  OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.
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http://dx.doi.org/10.1038/s41598-019-39571-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420620PMC
March 2019

CXCL6 is an important paracrine factor in the pro-angiogenic human cardiac progenitor-like cell secretome.

Sci Rep 2017 10 2;7(1):12490. Epub 2017 Oct 2.

Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.
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http://dx.doi.org/10.1038/s41598-017-11976-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624898PMC
October 2017

Rationale and Design of a Clinical Trial to Evaluate the Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With Acute Myocardial Infarction and Left Ventricular Dysfunction: The Randomized Multicenter Double-Blind Controlled CAREMI Trial (Cardiac Stem Cells in Patients With Acute Myocardial Infarction).

Circ Res 2017 Jun 22;121(1):71-80. Epub 2017 May 22.

From the Department of Cardiology, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañon, Facultad de Medicina, Universidad Complutense, Centro de Investigación Biomédica en Red-Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain (R.S.-R., A.C.P., M.E.F.-S., R.Y., J. Bermejo, F.F.-A.); Coretherapix S.L.U./Tigenix Group, Madrid, Spain (L.R.B., I.P., M.M., I.G.); HLA et Medicine (HLA-MED), Hôpital Saint-Louis, Paris, France (R.A.-D., D.C.); Department of Cardiovascular Medicine, University Hospitals and KU Leuven, Belgium (P.C., J. Bogaert, S.J.); Department of Cardiac Surgery, Complejo Hospitalario de Navarra, Pamplona, Spain (R.S.); and Department of Development and Cardiovascular Repair, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (A.B.).

Rationale: Stem cell therapy has increased the therapeutic armamentarium in the fight against ischemic heart disease and heart failure. The administration of exogenous stem cells has been investigated in patients suffering an acute myocardial infarction, with the final aim of salvaging jeopardized myocardium and preventing left ventricular adverse remodeling and functional deterioration. However, phase I and II clinical trials with autologous and first-generation stem cells have yielded inconsistent benefits and mixed results.

Objective: In the search for new and more efficient cellular regenerative products, interesting cardioprotective, immunoregulatory, and cardioregenerative properties have been demonstrated for human cardiac stem cells. On the other hand, allogeneic cells show several advantages over autologous sources: they can be produced in large quantities, easily administered off-the-shelf early after an acute myocardial infarction, comply with stringent criteria for product homogeneity, potency, and quality control, and may exhibit a distinctive immunologic behavior.

Methods And Results: With a promising preclinical background, CAREMI (Cardiac Stem Cells in Patients With Acute Myocardial Infarction) has been designed as a double-blind, 2:1 randomized, controlled, and multicenter clinical trial that will evaluate the safety, feasibility, and efficacy of intracoronary delivery of allogeneic human cardiac stem cell in 55 patients with large acute myocardial infarction, left ventricular dysfunction, and at high risk of developing heart failure.

Conclusions: This phase I/II clinical trial represents a novel experience in humans with allogeneic cardiac stem cell in a rigorously imaging-based selected group of acute myocardial infarction patients, with detailed safety immunologic assessments and magnetic resonance imaging-based efficacy end points.

Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439398.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.310651DOI Listing
June 2017

Minimizing the risk of allo-sensitization to optimize the benefit of allogeneic cardiac-derived stem/progenitor cells.

Sci Rep 2017 01 24;7:41125. Epub 2017 Jan 24.

Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS-976, Université Paris Diderot, Hôpital Saint-Louis, Paris, France.

Allogeneic human cardiac-derived stem/progenitor cells (hCPC) are currently under clinical investigation for cardiac repair. While cellular immune response against allogeneic hCPC could be part of their beneficial-paracrine effects, their humoral immune response remains largely unexplored. Donor-specific HLA antibodies (DSA-HLA-I/DSA-HLA-II), primary elements of antibody-mediated allograft injury, might present an unidentified risk to allogeneic hCPC therapy. Here we established that the binding strength of anti-HLA monoclonal antibodies delineates hCPC proneness to antibody-mediated injury. In vitro modeling of clinical setting demonstrated that specific DSA-HLA-I of high/intermediate binding strength are harmful for hCPC whereas DSA-HLA-II are benign. Furthermore, the Luminex-based solid-phase assays are suitable to predict the DSA-HLA risk to therapeutic hCPC. Our data indicate that screening patient sera for the presence of HLA antibodies is important to provide an immune-educated choice of allogeneic therapeutic cells, minimize the risk of precipitous elimination and promote the allogeneic reparative effects.
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http://dx.doi.org/10.1038/srep41125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259698PMC
January 2017

Deregulated expression of Cdc6 in the skin facilitates papilloma formation and affects the hair growth cycle.

Cell Cycle 2015 ;14(24):3897-907

a DNA Replication Group; Molecular Oncology Program; Spanish National Cancer Reserch Center (CNIO) ; Madrid , Spain.

Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice.
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http://dx.doi.org/10.1080/15384101.2015.1120919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825757PMC
September 2016

Exploring analytical proteomics platforms toward the definition of human cardiac stem cells receptome.

Proteomics 2015 Apr 5;15(7):1332-7. Epub 2015 Feb 5.

iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

Human cardiac stem cells (hCSC) express a portfolio of plasma membrane receptors that are involved in the regulatory auto/paracrine feedback loop mechanism of activation of these cells, and consequently contribute to myocardial regeneration. In order to attain a comprehensive description of hCSC receptome and overcoming the inability demonstrated by other technologies applied in receptor identification, mainly due to the transmembrane nature, high hydrophobic character and relative low concentration of these proteins, we have exploited and improved a proteomics workflow. This approach was based on the enrichment of hCSC plasma membrane fraction and addition of prefractionation steps prior to MS analysis. More than 100 plasma membrane receptors were identified. The data reported herein constitute a valuable source of information to further understand cardiac stem cells activation mechanisms and the subsequent cardiac repair process. All MS data have been deposited in the ProteomeXchange with identifier PXD001117 (http://proteomecentral.proteomexchange.org/dataset/PXD001117).
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http://dx.doi.org/10.1002/pmic.201400318DOI Listing
April 2015

Natural killer cell crosstalk with allogeneic human cardiac-derived stem/progenitor cells controls persistence.

Cardiovasc Res 2014 Nov 11;104(2):290-302. Epub 2014 Sep 11.

Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS940, Institut Universitaire d'Hématologie, Université Paris-Diderot and Laboratoire d'Immunologie et d'Histocompatibilité, Transplantex, Hôpital Saint Louis, CIB-HOG, AP-HP, Batiment Bazin, 1 Avenue Claude Vellefaux, Paris 75010, France

Aims: Allogeneic human cardiac-derived stem/progenitor cells (hCPC) are promising candidates for cardiac repair. They interact with T cells, major effectors of the adaptive immune response, inducing 'paracrine' anti-inflammatory effects that could sustain tissue repair/regeneration. Natural killer (NK) cells are major effectors of the innate immune system that might influence the persistence of therapeutic stem/progenitor cells. Therefore, to get through successful clinical translation and anticipate allogeneic hCPC persistence, we defined their crosstalk with NK cells under steady state and inflammatory conditions.

Methods And Results: By using an experimental model of allogeneic hCPC/NK cell interaction, we demonstrate that hCPC moderately trigger cytokine-activated, but not resting, NK cell killing that occurs through formation of lytic immunological synapse and NK cell natural cytotoxicity. Yet, inflammatory context substantially decreases their capacity to set cytokine-activated NK cell functions towards NK cell-cytotoxicity and protects hCPC from NK cell killing. Allogeneic hCPC also restrain NK cell-cytotoxicity against conventional targets and inflammatory cytokine secretion biasing the latter towards anti-inflammatory cytokines. Thus, hCPC are unprivileged targets for allogeneic NK cells and can restrain NK cell functions in allogeneic setting.

Conclusion: Collectively, our data suggest that allogeneic hCPC/innate NK cells crosstalk within injured inflamed myocardium would permit their retention and might contribute to attenuating inflammation and to preventing adverse cardiac remodelling.
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http://dx.doi.org/10.1093/cvr/cvu208DOI Listing
November 2014

Allogenicity of human cardiac stem/progenitor cells orchestrated by programmed death ligand 1.

Circ Res 2013 Feb 12;112(3):451-64. Epub 2012 Dec 12.

Institut National de la Santé et de la Recherche Médicale UMRS940, Institut Universitaire d’Hématologie, Université Paris-Diderot and Laboratoire d’Immunologie et d’Histocompatibilité, Hôpital Saint Louis, Paris, France.

Rationale: Transplantation of allogeneic cardiac stem/progenitor cells (CPC) in experimental myocardial infarction promoted cardiac regeneration and improved heart function. Although this has enhanced prospects of using allogeneic CPC for cardiac repair, the mechanisms regulating the behavior of these allogeneic cells, which are central to clinical applications, remain poorly understood.

Objective: T cells orchestrate the allogeneic adaptive immune response. Therefore, to provide insight into the mechanisms regulating the immunologic behavior of human CPC (hCPC), we investigated the allogeneic T-cell response elicited by cryopreserved c-kit-selected hCPC.

Methods And Results: By using an experimental model of allogeneic stimulation, we demonstrate that, whether under inflammatory conditions or not, hCPC do not trigger conventional allogeneic Th1 or Th2 type responses but instead induce proliferation and selective expansion of suppressive CD25(high)CD127(low)human leukocyte antigen-DR(+)FoxP3(high) effector regulatory T cells. The regulatory T-cell proliferation and amplification were dependent on the interaction with the B7 family member programmed death ligand 1 (PD-L1), which is substantially expressed on hCPC and increased under inflammatory conditions. Thus, hCPC in allogeneic settings acquire the capacity to downregulate an ongoing immune response, which was dependent on PD-L1.

Conclusions: Collectively, these data reveal that hCPC in allogeneic settings have a tolerogenic immune behavior, promoting a contact PD-L1-dependent regulatory response and a PD-L1-dependent allogeneic-driven immunomodulation. Our study attributes an important role for PD-L1 in the immune behavior of allogeneic hCPC and raises the possibility of using PD-L1 expression as a marker to identify and select low-risk high-benefit allogeneic cardiac repair cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.112.276501DOI Listing
February 2013

CDC6: from DNA replication to cell cycle checkpoints and oncogenesis.

Carcinogenesis 2008 Feb 28;29(2):237-43. Epub 2007 Nov 28.

DNA replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre, Melchor Fernández Almagro 3, E-28029 Madrid, Spain.

Cell division cycle 6 (CDC6) is an essential regulator of DNA replication in eukaryotic cells. Its best-characterized function is the assembly of prereplicative complexes at origins of replication during the G(1) phase of the cell division cycle. However, CDC6 also plays important roles in the activation and maintenance of the checkpoint mechanisms that coordinate S phase and mitosis, and recent studies have unveiled its proto-oncogenic activity. CDC6 overexpression interferes with the expression of INK4/ARF tumor suppressor genes through a mechanism involving the epigenetic modification of chromatin at the INK4/ARF locus. In addition, CDC6 overexpression in primary cells may promote DNA hyperreplication and induce a senescence response similar to that caused by oncogene activation. These findings indicate that deregulation of CDC6 expression in human cells poses a serious risk of carcinogenesis.
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http://dx.doi.org/10.1093/carcin/bgm268DOI Listing
February 2008
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