Delivery of cardiovascular progenitors with biomimetic microcarriers reduces adverse ventricular remodeling in a rat model of chronic myocardial infarction.

Despite tremendous progress in cell-based therapies for heart repair, many challenges still exist. To enhance the therapeutic potential of cell therapy one approach is the combination of cells with biomaterial delivery vehicles. Here, we developed a biomimetic and biodegradable micro-platform based on polymeric microparticles (MPs) capable of maximizing the therapeutic potential of cardiac progenitor cells (CPCs) and explored its efficacy in a rat model of chronic myocardial infarction. The transplantation of CPCs adhered to MPs within the infarcted myocardial microenvironment improved the long-term engraftment of transplanted cells for up to one month. Furthermore, the enhancement of cardiac cellular retention correlated with an increase in functional recovery. In consonance, better tissue remodeling and vasculogenesis were observed in the animals treated with cells attached to MPs, which presented smaller infarct size, thicker right ventricular free wall, fewer deposition of periostin and greater density of vessels than animals treated with CPCs alone. Finally, we were able to show that part of this beneficial effect was mediated by CPC-derived extracellular vesicles (EVs). Taken together, these findings indicate that the biomimetic microcarriers support stem cell survival and increase cardiac function in chronic myocardial infarction through modulation of cardiac remodeling, vasculogenesis and CPCs-EVs mediated therapeutic effects. The biomimetic microcarriers provide a solution for biomaterial-assisted CPC delivery to the heart. STATEMENT OF SIGNIFICANCE: In this study, we evaluate the possibility of using a biomimetic and biodegradable micro-platform to improve cardiovascular progenitor therapy. The strategy reported herein serves as an injectable scaffold for adherent cells due to their excellent injectability through cardiac catheters, capacity for biomimetic three-dimensional stem cell support and controllable biodegradability. In a rat model of chronic myocardial infarction, the biomimetic microcarriers improved cardiac function, reduced chronic cardiac remodeling and increased vasculogenesis through the paracrine signaling of CPCs. We have also shown that extracellular vesicles derived from CPCs cultured on biomimetic substrates display antifibrotic effects, playing an important role in the therapeutic effects of our tissue-engineered approach. Therefore, biomimetic microcarriers represent a promising and effective strategy for biomaterial-assisted CPC delivery to the heart.

The involvement of ADAM10 in acantholysis in mucocutaneous pemphigus vulgaris depends on the autoantibody profile of each patient.

BACKGROUND: Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. OBJECTIVES: Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). METHODS: We used three PV-IgG fractions from different patients containing high or low levels of anti-Dsg1 and anti-Dsg3 antibodies, and the presence or not of anti-desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. RESULTS: Although all of the PV-IgG fractions produced suprabasal acantholysis, only those containing anti-Dsg1/3, but not anti-Dsc2/3 antibodies, induced ADAM10 activation in a Src-dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti-Dsc2/3 antibodies, in addition to anti-Dsg1/3, triggered earlier and ADAM10-independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. CONCLUSIONS: All PV-IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti-Dsg antibodies or the presence of non-Dsg antibodies, such as anti-Dsc, more severe cell-cell epidermal detachment will occur at different times, and in an ADAM10-dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non-Dsg proteins, and therefore more specific therapeutic approaches in PV should be used. What's already known about this topic? Suprabasal acantholysis in pemphigus vulgaris (PV) may be triggered by both desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient is associated with a distinct intracellular signalling pattern. What does this study add? In patients with PV with anti-Dsg3 and anti-Dsg1, but not anti-desmocollin (Dsc)3 antibodies, ADAM10 activation is induced in an Src-dependent way, together with an increase in the epidermal growth factor receptor (EGFR) ligands EGF and betacellulin. The presence of anti-Dsc3 antibodies triggers an earlier and ADAM10-independent acantholysis, without increasing EGFR ligands, and is associated with more severe epidermal detachment. Lower levels of anti-Dsc3 antibodies are associated with less severe acantholysis. What is the translational message? In some patients with PV, the severity and the timing for cell-cell detachment seem to depend on the level of anti-Dsg1/3 antibodies, although other as yet uncharacterized antibodies may also participate. These patients with PV would exhibit inhibition of acantholysis by Src, ADAM10, EGF and EGFR inhibitors. In other patients, the presence of non-Dsg antibodies, such as anti-Dsc2/3, would produce an earlier and more severe ADAM10-independent suprabasal acantholysis.

Angiogenic therapy for cardiac repair based on protein delivery systems.

Cardiovascular diseases remain the first cause of morbidity and mortality in the developed countries and are a major problem not only in the western nations but also in developing countries. Current standard approaches for treating patients with ischemic heart disease include angioplasty or bypass surgery. However, a large number of patients cannot be treated using these procedures. Novel curative approaches under investigation include gene, cell, and protein therapy. This review focuses on potential growth factors for cardiac repair. The role of these growth factors in the angiogenic process and the therapeutic implications are reviewed. Issues including aspects of growth factor delivery are presented in relation to protein stability, dosage, routes, and safety matters. Finally, different approaches for controlled growth factor delivery are discussed as novel protein delivery platforms for cardiac regeneration.

Regulation of apoptosis by peptides of fibronectin in human monocytes.

Synthetic peptides with sequences present in extracellular matrix protein fibronectin have been described to stimulate human monocytes. We describe now that one of these peptides, FN6, induces apoptotic effects on monocytes and we investigate the molecular mechanisms involved in the regulation of this response. Incubation of monocytes with FN6 induces the activation of the small GTPase Rac. In turn, Rac mediates the increase of both JNK and p38 activities in a sustained fashion, as well as the phosphorylation levels of their respective substrates c-Jun and ATF-2. FN6 also stimulates caspases -9 and -3 and the delayed proteolysis of its substrates PARP and D4-GDI. In addition, initiator caspases-1 and -5 were activated by FN6 treatment of monocytes but, in contrast to that observed for caspases-9 and -3, this effect was not dependent on JNK or p38 activities. These kinases also mediated the increase of Bax levels, but only in some conditions Bcl-2 depletion caused by the peptide. Moreover, whereas initially only caspase-1 is involved in caspase-3 activation, later on caspase-9 seems also to participate. Therefore, we demonstrate that FN6 stimulation allows multiple, JNK and p38-dependent and -independent interacting signals to regulate the apoptotic response in human monocytes.

In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades.

BACKGROUND: 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 acantholysis.

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.

Correlation between profile of circulating mononuclear cells and clinical manifestations in patients with pemphigus vulgaris.

Phenotypes of 38 samples of mononuclear (PBMC) cells from 11 different patients with pemphigus vulgaris (PV) at different stages of the disease were explored looking for a possible relationship between cell immunity, mucocutaneous or mucosal lesion intensity and capacity of serum autoantibodies to elicit the disease in mice. PBMC from 5 patients with mucocutaneous lesions and sera with IgG capable of inducing the disease in neonatal mice had a high proportion of mature monocytes with CD14low DRhigh, and co-expressing CD16 and CD11b. In addition, a high proportion of CD19+CD5+ activated B cells and a very low proportion of naive CD4+CD45RA+ and CD8+CD11b+ T lymphocytes was observed. Monocytes from these patients expressed inducible nitric oxide synthase (iNOS). In contrast, PBMC from 6 patients, with lesions restricted to mucosal membranes and IgG lacking the capacity to induce the disease in mice, contained a high proportion of CD14high DRlow co-expressing CD16 circulating macrophages, CD8+CD11b+ T cells, and a low proportion of activated B lymphocytes. The results suggest a possible association between proportion of different antigen presenting cells (monocytes with high HLA-DR and low CD14 expression and activated B lymphocytes, or differentiated monocytes/macrophages), type of PV and capacity of serum autoantibodies to elicit the disease in mice.