97 research outputs found
Stem Cells and Cardiac Disease: Where are We Going?
During the last 10 years we have witnessed the development of a new field in research termed Stem Cell Therapy.
Classically, it was considered that cells had a limited division and differentiation ability; however, this dogma was
challenged when new exciting results about cell multi/pluripotency were presented to the scientific community. It was
found that cells from one adult tissue source were able to originate cells of a very different type. The possibility of transplanting
these cells into damaged organs with the aim of substituting sick or dead tissue, triggered many studies to understand
the plasticity of the stem cells and their potential in pathological situations. Nowadays, much more is understood
about stem cells, although of course, many questions, especially about their mechanism of action, still need to be answered.
Their benefit after transplantation has been shown experimentally and even clinically in some cases; however, the
degree of stem cell contribution through their own differentiation into the transplanted tissue, has turned out to be generally
low, and increasing evidence indicates that a trophic effect must play an important role in such a benefit. A better understanding
of the paracrine mechanisms involved could be of great relevance in order to develop new therapies focused
on stimulating endogenous cells. On the other hand, more sophisticated methods for cell transplantation combined with
bio-engineering techniques have been devised in cardiac disease models. In this review we will try to provide a critical
overview of the stem cell studies performed until now and to discuss some of the questions raised about the mechanisms
that are involved in their putative reparative effect in cardiovascular diseases, and their origin
An imbalance in Akt/mTOR is involved in the apoptotic and acantholytic processes in a mouse model of pemphigus vulgaris
Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by the presence of IgG autoantibodies against Dsg3. Our aim was to investigate the molecular events implicated in the development and localization of apoptosis and acantholysis in PV. We used a passive transfer mouse model together with immunohistochemical (IHC) techniques and the TUNEL assay, with quantification analysis in the basal layer of the epidermis. The activated signalling molecules analysed and apoptotic cells detected showed an identical localization. Herein, we found for the first time in vivo an increased expression of activated HER receptor isoforms in the basal layer in PV lesions. Besides, we observed the almost total lack of activated Akt compared with a higher level of activated mTOR within the basal cells of the epidermis. Our observations strongly support that the restriction of acantholysis to the basal layer may be due, at least in part, to the selective and increased presence of activated HER receptor isoforms in these cells. After phosphorylation of HER receptor isoforms, intracellular signalling pathways are activated in the basal layer. In addition, the imbalance in Akt/mTOR that takes place in the basal cells may provide intracellular signals necessary for the development of apoptosis and acantholysi
Cutaneous Biology: In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades
Pemphigus vulgaris (PV) is an autoimmune disease characterized by
mucocutaneous intraepithelial blisters and pathogenic autoantibodies against
desmoglein 3. The mechanism of blister formation in pemphigus has not been
defined; however, in vitro data suggest a role for activation of intracellular
signalling cascades. OBJECTIVES: To investigate the contribution of these
signalling pathways to the mechanism of PV IgG-induced acantholysis in vivo.
METHODS: We used the passive transfer mouse model. Mice were injected with IgG
fractions of sera from a patient with PV, with or without pretreatment with
inhibitors of proteins that mediate intracellular signalling cascades. RESULTS:
Inhibitors of tyrosine kinases, phospholipase C, calmodulin and the
serine/threonine kinase protein kinase C prevented PV IgG-induced acantholysis in
vivo. CONCLUSIONS: These observations strongly support the role of intracellular
signalling cascades in the molecular mechanism of PV IgG-induced acantholysi
Plasticity and cardiovascular applications of multipotent adult progenitor cells
Cardiovascular disease is the leading cause of death worldwide, which
has encouraged the search for new therapies that enable the treatment of
patients in palliative and curative ways. In the past decade, the potential
benefit of transplantation of cells that are able to substitute for the injured
tissue has been studied with several cell populations, such as stem cells.
Some of these cell populations, such as myoblasts and bone marrow cells,
are already being used in clinical trials. The laboratory of CM Verfaillie has
studied primitive progenitors, termed multipotent adult progenitor cells,
which can be isolated from adult bone marrow. These cells can differentiate
in vitro at the single-cell level into functional cells that belong to the three
germ layers and contribute to most, if not all, somatic cell types after
blastocyst injection. This remarkably broad differentiation potential makes
this particular cell population a candidate for transplantation in tissues
in need of regeneration. Here, we focus on the regenerative capacity of
multipotent adult progenitor cells in several ischemic mouse models, such
as acute and chronic myocardial infarction and limb ischemia
Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium
The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in
infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms.
Methods and results: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56+), myotubes (SM
cultured until confluence) and fibroblasts-like cells (CD56−). Gene expression revealed up-regulation of pro-angiogenic (PGF), antiapoptotics
(BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In
line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in
angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2,
MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein
endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and
cardiomyocytes (pb0.05) and a decrease in apoptosis of cardiomyocytes (pb0.05). Analysis of nude rats transplanted with human SM
demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-β, and IGF-1 1 month after transplant.
Conclusions: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of
myogenic cell transplantation in infarcted myocardium.
© 2008 European Society of Cardiology. Published by Elsevie
PEGylated-PLGA microparticles containing VEGF for long term drug delivery
The potential of poly(lactic-co-glycolic) acid (PLGA) microparticles as carriers for vascular endothelial growth factor (VEGF) has been demonstrated in a previous study by our group, where we found improved angiogenesis and heart remodeling in a rat myocardial infarction model (Formiga et al., 2010). However, the observed accumulation of macrophages around the injection site suggested that the efficacy of treatment could be reduced due to particle phagocytosis.
The aim of the present study was to decrease particle phagocytosis and consequently improve protein delivery using stealth technology. PEGylated microparticles were prepared by the double emulsion solvent evaporation method using TROMS (Total Recirculation One Machine System). Before the uptake studies in monocyte-macrophage cells lines (J774 and Raw 264.7), the characterization of the microparticles developed was carried out in terms of particle size, encapsulation efficiency, protein stability, residual poly(vinyl alcohol) (PVA) and in vitro release. Microparticles of suitable size for intramyocardial injection (5 mu m) were obtained by TROMS by varying the composition of the formulation and TROMS conditions with high encapsulation efficiency (70-90%) and minimal residual PVA content (0.5%). Importantly, the bioactivity of the protein was fully preserved. Moreover, PEGylated microparticles released in phosphate buffer 50% of the entrapped protein within 4 h, reaching a plateau within the first day of the in vitro study. Finally, the use of PLGA microparticles coated with PEG resulted in significantly decreased uptake of the carriers by macrophages, compared with non PEGylated microparticles, as shown by flow cytometry and fluorescence microscopy.
On the basis of these results, we concluded that PEGylated microparticles loaded with VEGF could be used for delivering growth factors in the myocardium
Transplantation of mesenchymal stem cells exerts a greater long-term effect than bone marrow mononuclear cells in a chronic myocardial infarction model in rat
The aim of this study is to assess the long-term effect of mesenchymal stem cells (MSC) transplantation in
a rat model of chronic myocardial infarction (MI) in comparison with the effect of bone marrow mononuclear
cells (BM-MNC) transplant. Five weeks after induction of MI, rats were allocated to receive intramyocardial
injection of 106 GFP-expressing cells (BM-MNC or MSC) or medium as control. Heart function
(echocardiography and 18F-FDG-microPET) and histological studies were performed 3 months after transplantation
and cell fate was analyzed along the experiment (1 and 2 weeks and 1 and 3 months). The main
findings of this study were that both BM-derived populations, BM-MNC and MSC, induced a long-lasting
(3 months) improvement in LVEF (BM-MNC: 26.61 ± 2.01% to 46.61 ± 3.7%, p < 0.05; MSC: 27.5 ±
1.28% to 38.8 ± 3.2%, p < 0.05) but remarkably, only MSC improved tissue metabolism quantified by 18FFDG
uptake (71.15 ± 1.27 to 76.31 ± 1.11, p < 0.01), which was thereby associated with a smaller infarct size
and scar collagen content and also with a higher revascularization degree. Altogether, results show that MSC
provides a long-term superior benefit than whole BM-MNC transplantation in a rat model of chronic MI
Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia–reperfusion model
The use of pro-angiogenic growth factors in ischemia models has been associated with limited success in the
clinical setting, in part owing to the short lived effect of the injected cytokine. The use of a microparticle
system could allow localized and sustained cytokine release and consequently a prolonged biological effect
with induction of tissue revascularization. To assess the potential of VEGF165 administered as continuous
release in ischemic disease, we compared the effect of delivery of poly(lactic–co-glycolic acid) (PLGA)
microparticles (MP) loaded with VEGF165 with free-VEGF or control empty microparticles in a rat model of
ischemia–reperfusion. VEGF165 loaded microparticles could be detected in the myocardium of the infarcted
animals for more than a month after transplant and provided sustained delivery of active protein in vitro and
in vivo. One month after treatment, an increase in angiogenesis (small caliber caveolin-1 positive vessels)
and arteriogenesis (α-SMA-positive vessels) was observed in animals treated with VEGF microparticles
(pb0.05), but not in the empty microparticles or free-VEGF groups. Correlating with this data, a positive
remodeling of the heart was also detected in the VEGF-microparticle group with a significantly greater LV
wall thickness (pb0.01). In conclusion, PLGA microparticle is a feasible and promising cytokine delivery
system for treatment of myocardial ischemia. This strategy could be scaled up and explored in pre-clinical
and clinical studies
Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes
Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein-3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell-cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase-3 activation, Bcl-2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes
Catheter-based intramyocardial injection of FGF1 or NRG1-loaded MPs improves cardiac function in a preclinical model of ischemia-reperfusion
Cardiovascular protein therapeutics such as neuregulin (NRG1) and acidic-fibroblast growth factor
(FGF1) requires new formulation strategies that allow for sustained bioavailability of the drug in the
infarcted myocardium. However, there is no FDA-approved injectable protein delivery platform due
to translational concerns about biomaterial administration through cardiac catheters. We therefore
sought to evaluate the efficacy of percutaneous intramyocardial injection of poly(lactic-co-glycolic
acid) microparticles (MPs) loaded with NRG1 and FGF1 using the NOGA MYOSTAR injection catheter
in a porcine model of ischemia-reperfusion. NRG1- and FGF1-loaded MPs were prepared using a
multiple emulsion solvent-evaporation technique. Infarcted pigs were treated one week after ischemiareperfusion
with MPs containing NRG1, FGF1 or non-loaded MPs delivered via clinically-translatable
percutaneous transendocardial-injection. Three months post-treatment, echocardiography indicated
a significant improvement in systolic and diastolic cardiac function. Moreover, improvement in bipolar
voltage and decrease in transmural infarct progression was demonstrated by electromechanical
NOGA-mapping. Functional benefit was associated with an increase in myocardial vascularization
and remodeling. These findings in a large animal model of ischemia-reperfusion demonstrate the
feasibility and efficacy of using MPs as a delivery system for growth factors and provide strong evidence
to move forward with clinical studies using therapeutic proteins combined with catheter-compatible
biomaterials
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