Contribution of Social Isolation, Restraint, and Hindlimb Unloading to Changes in Hemodynamic Parameters and Motion Activity in Rats

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient

Glutaredoxin-1 Overexpression Enhances Neovascularization and Diminishes Ventricular Remodeling in Chronic Myocardial Infarction

Oxidative stress plays a critical role in the pathophysiology of cardiac failure, including the modulation of neovascularization following myocardial infarction (MI). Redox molecules thioredoxin (Trx) and glutaredoxin (Grx) superfamilies actively maintain intracellular thiol-redox homeostasis by scavenging reactive oxygen species. Among these two superfamilies, the pro-angiogenic function of Trx-1 has been reported in chronic MI model whereas similar role of Grx-1 remains uncertain. The present study attempts to establish the role of Grx-1 in neovascularization and ventricular remodeling following MI. Wild-type (WT) and Grx-1 transgenic (Grx-1Tg/+) mice were randomized into wild-type sham (WTS), Grx-1Tg/+ Sham (Grx-1Tg/+S), WTMI, Grx-1Tg/+MI. MI was induced by permanent occlusion of the LAD coronary artery. Sham groups underwent identical time-matched surgical procedures without LAD ligation. Significant increase in arteriolar density was observed 7 days (d) after surgical intervention in the Grx-1Tg/+MI group as compared to the WTMI animals. Further, improvement in myocardial functional parameters 30 d after MI was observed including decreased LVIDs, LVIDd, increased ejection fraction and, fractional shortening was also observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Moreover, attenuation of oxidative stress and apoptotic cardiomyocytes was observed in the Grx-1Tg/+MI group as compared to the WTMI animals. Increased expression of p-Akt, VEGF, Ang-1, Bcl-2, survivin and DNA binding activity of NF-κB were observed in the Grx-1Tg/+MI group when compared to WTMI animals as revealed by Western blot analysis and Gel-shift analysis, respectively. These results are the first to demonstrate that Grx-1 induces angiogenesis and diminishes ventricular remodeling apparently through neovascularization mediated by Akt, VEGF, Ang-1 and NF-κB as well as Bcl-2 and survivin-mediated anti-apoptotic pathway in the infarcted myocardium

Increased Expression of Fatty-Acid and Calcium Metabolism Genes in Failing Human Heart

Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca(2+))-handling in the human heart.RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6).Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca(2+)-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca(2+)-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM.DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca(2+)-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca(2+)-handling genes

A Combined Synthetic-Fibrin Scaffold Supports Growth and Cardiomyogenic Commitment of Human Placental Derived Stem Cells

Aims: A potential therapy for myocardial infarction is to deliver isolated stem cells to the infarcted site. A key issue with this therapy is to have at one\u27s disposal a suitable cell delivery system which, besides being able to support cell proliferation and differentiation, may also provide handling and elastic properties which do not affect cardiac contractile function. In this study an elastic scaffold, obtained combining a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (s-IPN) with fibrin, was used as a substrate for in vitro studies of human amniotic mesenchymal stromal cells (hAMSC) growth and differentiation. Methodology/Principal Findings: After hAMSC seeding on the fibrin side of the scaffold, cell metabolic activity and proliferation were evaluated by WST-1 and bromodeoxyuridine assays. Morphological changes and mRNAs expression for cardiac differentiation markers in the hAMSCs were examined using immunofluorescence and RT-PCR analysis. The beginning of cardiomyogenic commitment of hAMSCs grown on the scaffold was induced, for the first time in this cell population, by a nitric oxide (NO) treatment. Following NO treatment hAMSCs show morphological changes, an increase of the messenger cardiac differentiation markers [troponin I (TnI) and NK2 transcription factor related locus 5 (Nkx2.5)] and a modulation of the endothelial markers [vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR)]. Conclusions/Significance: The results of this study suggest that the s-IPN PEtU-PDMS/fibrin combined scaffold allows a better proliferation and metabolic activity of hAMSCs cultured up to 14 days, compared to the ones grown on plastic dishes. In addition, the combined scaffold sustains the beginning of hAMSCs differentiation process towards a cardiomyogenic lineage

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy

Migration towards SDF-1 selects angiogenin-expressing bone marrow monocytes endowed with cardiac reparative activity in patients with previous myocardial infarction

INTRODUCTION: Chemokine-directed migration is crucial for homing of regenerative cells to the infarcted heart and correlates with outcomes of cell therapy trials. Hence, transplantation of chemokine-responsive bone marrow cells may be ideal for treatment of myocardial ischemia. To verify the therapeutic activity of bone marrow mononuclear cells (BM-MNCs) selected by in vitro migration towards the chemokine stromal cell-derived factor-1 (SDF-1) in a mouse model of myocardial infarction (MI), we used BM-MNCs from patients with previous large MI recruited in the TransACT-1&2 cell therapy trials. METHODS: Unfractioned BM-MNCs, SDF-1-responsive, and SDF-1-nonresponsive BM-MNCs isolated by patients recruited in the TransACT-1&2 cell therapy trials were tested in Matrigel assay to evaluate angiogenic potential. Secretome and antigenic profile were characterized by flow cytometry. Angiogenin expression was measured by RT-PCR. Cells groups were also intramyocardially injected in an in vivo model of MI (8-week-old immune deficient CD1-FOXN1(nu/nu) mice). Echocardiography and hemodynamic measurements were performed before and at 14 days post-MI. Arterioles and capillaries density, infiltration of inflammatory cells, interstitial fibrosis, and cardiomyocyte proliferation and apoptosis were assessed by immunohistochemistry. RESULTS: In vitro migration enriched for monocytes, while CD34(+) and CD133(+) cells and T lymphocytes remained mainly confined in the non-migrated fraction. Unfractioned total BM-MNCs promoted angiogenesis on Matrigel more efficiently than migrated or non-migrated cells. In mice with induced MI, intramyocardial injection of unfractionated or migrated BM-MNCs was more effective in preserving cardiac contractility and pressure indexes than vehicle or non-migrated BM-MNCs. Moreover, unfractioned BM-MNCs enhanced neovascularization, whereas the migrated fraction was unique in reducing the infarct size and interstitial fibrosis. In vitro studies on isolated cardiomyocytes suggest participation of angiogenin, a secreted ribonuclease that inhibits protein translation under stress conditions, in promotion of cardiomyocyte survival by migrated BM-MNCs. CONCLUSIONS: Transplantation of bone marrow cells helps post-MI healing through distinct actions on vascular cells and cardiomyocytes. In addition, the SDF-1-responsive fraction is enriched with angiogenin-expressing monocytes, which may improve cardiac recovery through activation of cardiomyocyte response to stress. Identification of factors linking migratory and therapeutic outcomes could help refine regenerative approaches. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-015-0028-y) contains supplementary material, which is available to authorized users

Fermitins, the orthologs of mammalian Kindlins, regulate the development of a functional cardiac syncytium in Drosophila melanogaster.

The vertebrate Kindlins are an evolutionarily conserved family of proteins critical for integrin signalling and cell adhesion. Kindlin-2 (KIND2) is associated with intercalated discs in mice, suggesting a role in cardiac syncytium development; however, deficiency of Kind2 leads to embryonic lethality. Morpholino knock-down of Kind2 in zebrafish has a pleiotropic effect on development that includes the heart. It therefore remains unclear whether cardiomyocyte Kind2 expression is required for cardiomyocyte junction formation and the development of normal cardiac function. To address this question, the expression of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kind2, was silenced in Drosophila cardiomyocytes. Heart development was assessed in adult flies by immunological methods and videomicroscopy. Silencing both Fit1 and Fit2 led to a severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. A null allele of Fit1 was generated but this had no impact on the heart. Similarly, the silencing of Fit2 failed to affect heart function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1 null flies disrupted syncytium development, leading to severe cardiomyopathy. The data definitively demonstrate a role for Fermitins in the development of a functional cardiac syncytium in Drosophila. The findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. These findings support the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans