Antonio C. Campos-de-Carvalho

Bone marrow mononuclear cell therapy for patients with cirrhosis: a Phase 1 study

Background: Bone marrow‐derived cell therapy has been investigated in patients with severe liver disease.

Adipose-Derived Stem-Cell Treatment of Skeletal Muscle Injury

BACKGROUND The aim of the present study was to investigate whether adipose-derived stem cells could contribute to skeletal muscle-healing. METHODS Adipose-derived stem cells of male rats were cultured and injected into the soleus muscles of female rats. Two and four weeks after injections, muscles were tested for tetanic force (50 Hz). Histological analysis was performed to evaluate muscle collagen deposition and the number of centronucleated muscle fibers. In order to track donor cells, chimerism was detected with use of real-time polymerase chain reaction targeting the male sex-determining region Y (SRY) gene. RESULTS Two weeks after cell injection, tetanus strength and the number of centronucleated regenerating myofibers, as well as the number of centronucleated regenerating myofibers, were higher in the treated group than they were in the control group (mean and standard error of the mean, 79.2 ± 5.0% versus 58.3 ± 8.1%, respectively [p < 0.05]; and 145 ± 36 versus 273 ± 18 per 10³ myofibers, respectively [p < 0.05]). However, there were no significant differences at four weeks. Treatment did not decrease collagen deposition. Male gene was not detected in female host tissue at two and four weeks after engraftment by polymerase chain reaction analysis. CONCLUSIONS Adipose-derived stem-cell therapy increased muscle repair and force at two weeks, but not four weeks, after injection, suggesting that adipose-derived stem-cell administration may accelerate muscle repair; however, the rapid disappearance of injected cells suggests a paracrine mechanism of action.

Gap junction modulation by extracellular signaling molecules: the thymus model

Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

Time course of echocardiographic and electrocardiographic parameters in myocardial infarct in rats.

In animal models the evaluation of myocardial infarct size in vivo and its relation to the actual lesion found post mortem is still a challenge. The purpose of the current study was to address if the conventional electrocardiogram (ECG) and/or echocardiogram (ECHO) could be used to adequately predict the size of the infarct in rats. Wistar rats were infarcted by left coronary ligation and then ECG, ECHO and histopathology were performed at 1, 7 and 28 days after surgery. Correlation between infarct size by histology and Q wave amplitude in lead L1 was only found when ECGs were performed one day post-surgery. Left ventricular diastolic and systolic dimensions correlated with infarct size by ECHO on day 7 post-infarction. On days 7 and 28 post-infarction, ejection indexes estimated by M-mode also correlated with infarct size. In summary we show that conventional ECG and ECHO methods can be used to estimate infarct size in rats. Our data suggest that the 7-day interval is actually the most accurate for estimation of infarct size by ECHO.

Connexin40 messenger ribonucleic acid is positively regulated by thyroid hormone (TH) acting in cardiac atria via the TH receptor.

Thyroid hormone (TH) regulates many cardiac genes via nuclear thyroid receptors, and hyperthyroidism is frequently associated with atrial fibrillation. Electrical activity propagation in myocardium depends on the transfer of current at gap junctions, and connexins (Cxs) 40 and 43 are the predominant junction proteins. In mice, Cx40, the main Cx involved in atrial conduction, is restricted to the atria and fibers of the conduction system, which also express Cx43. We studied cardiac expression of Cx40 and Cx43 in conjunction with electrocardiogram studies in mice overexpressing the dominant negative mutant thyroid hormone receptor-beta Delta337T exclusively in cardiomyocytes [myosin heavy chain (MHC-mutant)]. These mice develop the cardiac hypothyroid phenotype in the presence of normal serum TH. Expression was also examined in wild-type mice rendered hypothyroid or hyperthyroid by pharmacological treatment. Atrial Cx40 mRNA and protein levels were decreased (85 and 55%, respectively; P < 0.001) in MHC-mt mice. Atrial and ventricular Cx43 mRNA levels were not significantly changed. Hypothyroid and hyperthyroid animals showed a 25% decrease and 40% increase, respectively, in Cx40 mRNA abundance. However, MHC-mt mice presented very low Cx40 mRNA expression regardless of whether they were made hypothyroid or hyperthyroid. Atrial depolarization velocity, as represented by P wave duration in electrocardiograms of unanesthetized mice, was extremely reduced in MHC-mt mice, and to a lesser extent also in hypothyroid mice (90 and 30% increase in P wave duration). In contrast, this measure was increased in hyperthyroid mice (19% decrease in P wave duration). Therefore, this study reveals for the first time that Cx40 mRNA is up-regulated by TH acting in cardiac atria via the TH receptor and that this may be one of the mechanisms contributing to atrial conduction alterations in thyroid dysfunctions.

Sustained IGF-1 secretion by adipose-derived stem cell improves infarcted heart function.

The mechanism by which stem cell-based therapy improves heart function is still unknown, but paracrine mechanisms seem to be involved. Adipose-derived stem cells (ADSCs) secrete several factors, including insulin-like growth factor-1 (IGF-1), which may contribute to myocardial regeneration. Our aim was to investigate whether the overexpression of IGF-1 in ADSCs (IGF-1-ADSCs) improves treatment of chronically infarcted rat hearts. ADSCs were transduced with a lentiviral vector to induce IGF-1 overexpression. IGF-1-ADSCs transcribe100- to 200-fold more IGF-1 mRNA levels compared to nontransduced ADSCs. IGF-1 transduction did not alter ADSC immunophenotypic characteristics even under hypoxic conditions. However, IGF-1-ADSCs proliferate at higher rates and release greater amounts of growth factors such as IGF-1, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) under normoxic and hypoxic conditions. Importantly, IGF-1 secreted by IGF-1-ADSCs is functional given that Akt-1 phosphorylation was remarkably induced in neonatal cardiomyocytes cocultured with IGF-1-ADSCs, and this increase was prevented with phosphatidylinositol 3-kinase (PI3K) inhibitor treatment. Next, we tested IGF-1-ADSCs in a rat myocardial infarction (MI) model. MI was performed by coronary ligation, and 4 weeks after MI, animals received intramyocardial injections of either ADSCs (n = 7), IGF-1-ADSCs (n = 7), or vehicle (n = 7) into the infarcted border zone. Left ventricular function was evaluated by echocardiography before and after 6 weeks of treatment, and left ventricular hemodynamics were assessed 7 weeks after cell injection. Notably, IGF-1-ADSCs improved left ventricular ejection fraction and cardiac contractility index, but did not reduce scar size when compared to the ADSC-treated group. In summary, transplantation of ADSCs transduced with IGF-1 is a superior therapeutic approach to treat MI compared to nontransduced ADSCs, suggesting that gene and cell therapy may bring additional benefits to the treatment of MI.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats.

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 +/- 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 +/- 0.02 cm, P < 0.01) vs control (0.16 +/- 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 +/- 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 +/- 1.36%, P < 0.05) vs control (2.2 +/- 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 +/- 1.3%, P < 0.01; collagen III: 1.3 +/- 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 +/- 0.06%, P < 0.05; collagen III: 1.5 +/- 0.8%, P < 0.01) vs control (collagen I: 0.38 +/- 0.11%; collagen III: 0.25 +/- 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 +/- 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 +/- 21%, P < 0.01) vs control (7.9 +/- 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Neonatal lupus syndrome: the heart as a target of the immune system.

Neonatal lupus erythematosus (NLE) is an auto-immune disease related to systemic lupus erythematosus (SLE). Unlike SLE it is not a spontaneous syndrome but rather an acquired one. In NLE the most common disease manifestations are a transient cutaneous lesion and cardiac conduction disturbances. The cutaneous lesions and other non-cardiac manifestations of NLE are transient and disappear about six months after birth, at the time when maternal antibodies disappear from the neonatal circulation. This fact suggests that maternal antibodies may cross the placenta leading to an inflammatory reaction in the fetal tissues. NLE is the principal cause of atria-ventricular block, when it is not associated with congenital birth defects. All the clinical studies to date correlate the heart block in NLE with the presence of certain types of circulating maternal antibodies, against the Ro/SSA nuclear proteins, in the serum of the newborn. In this paper we discuss animal models that have been developed by our and others groups to study the participation of the anti-Ro/SSA antibodies in the pathogenesis of the cardiac conduction blockades that occur in NLE.

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.

Generation of human iPS cell line ihFib3.2 from dermal fibroblasts.

The human ihFib3.2 iPS cell line was generated from dermal fibroblasts obtained from a healthy donor. Lentiviral particles were produced with the polycistronic hSTEMCCA vector with Oct4, Sox2, cMyc and Klf4 as reprogramming factors.