Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

MicroRNA Regulation of Adipose Derived Stem Cells in Aging Rats

Background

Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells.

Methodology and Principal Findings

Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction.

Conclusions and Significance

Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.     

Alterations in the self-renewal and differentiation ability of bone marrow mesenchymal stem cells in a mouse model of rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease primarily involving the synovium. Evidence in recent years has suggested that the bone marrow (BM) may be involved, and may even be the initiating site of the disease. Abnormalities in haemopoietic stem cells'' (HSC) survival, proliferation and aging have been described in patients affected by RA and ascribed to abnormal support by the BM microenvironment. Mesenchymal stem cells (MSC) and their progeny constitute important components of the BM niche. In this study we test the hypothesis that the onset of inflammatory arthritis is associated with altered self-renewal and differentiation of bone marrow MSC, which alters the composition of the BM microenvironment.

Methods

We have used Balb/C Interleukin-1 receptor antagonist knock-out mice, which spontaneously develop RA-like disease in 100% of mice by 20 weeks of age to determine the number of mesenchymal progenitors and their differentiated progeny before, at the start and with progression of the disease.

Results

We showed a decrease in the number of mesenchymal progenitors with adipogenic potential and decreased bone marrow adipogenesis before disease onset. This is associated with a decrease in osteoclastogenesis. Moreover, at the onset of disease a significant increase in all mesenchymal progenitors is observed together with a block in their differentiation to osteoblasts. This is associated with accelerated bone loss.

Conclusions

Significant changes occur in the BM niche with the establishment and progression of RA-like disease. Those changes may be responsible for aspects of the disease, including the advance of osteoporosis. An understanding of the molecular mechanisms leading to those changes may lead to new strategies for therapeutic intervention.     

PlGF repairs myocardial ischemia through mechanisms of angiogenesis, cardioprotection and recruitment of myo-angiogenic competent marrow progenitors

Rationale

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.

Objective

We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.

Methods and Results

Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1⁺/Lin⁻ (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.

Conclusions

Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.     

Suppression of Cytochrome P450 Reductase Enhances Long-Term Hematopoietic Stem Cell Repopulation Efficiency in Mice

Background

Bone marrow microenvironment (niche) plays essential roles in the fate of hematopoietic stem cells (HSCs). Intracellular and extracellular redox metabolic microenvironment is one of the critical factors for the maintenance of the niche. Cytochrome P450 reductase (CPR) is an obligate electron donor to all microsomal cytochrome P450 enzymes (P450 or CYP), and contributes to the redox metabolic process. However, its role in maintaining HSCs is unknown.

Objective

To examine the effects of low CPR expression on HSCs function using a mouse model of globally suppressed Cpr gene expression (Cpr Low, CL mice).

Methods

Hematopoietic cell subpopulations in bone marrow (BM) and peripheral blood (PB) from WT and CL mice were examined for their repopulation and differentiation ability upon BM competitive transplantation and enriched HSC (LKS⁺) transplantation. Effects of low CPR expression on hematopoiesis were examined by transplanting normal BM cells into CL recipients. Reactive oxygen species (ROS), cell cycle, and apoptosis in CL mice were analyzed by flow cytometry for DCF-DA fluorescence intensity, Ki67 protein, and Annexin-V, respectively.

Results

The levels of ROS in BM cells, HPCs and HSCs were comparable between CL and WT mice. In comparison to WT mice, the number of LT-HSCs or ST-HSCs was lower in CL mice while CMPs, GMPs and MEPs in CL mice were higher than that in WT control. Competitive transplantation assay revealed enhanced repopulation capacity of HSCs with low CPR expression, but no difference in differentiation potential upon in vitro experiments. Furthermore, lymphoid differentiation of donor cells decreased while their myeloid differentiation increased under CL microenvironment although the overall level of donor hematopoietic repopulation was not significantly altered.

Conclusions

Our studies demonstrate that suppressing CPR expression enhances the repopulation efficiency of HSCs and a low CPR expression microenvironment favors the differentiation of myeloid over lymphoid lineage cells.     

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) Regulates Hematopoiesis and Bone Formation In Vivo

Background

Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown.

Methodology/Principal Findings

We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1⁺KIT⁺ cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis.

Conclusions/Significance

Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.     

Comparison of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes with Human Mesenchymal Stem Cells following Acute Myocardial Infarction

Introduction

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently been shown to express key cardiac proteins and improve in vivo cardiac function when administered following myocardial infarction. However, the efficacy of hiPSC-derived cell therapies, in direct comparison to current, well-established stem cell-based therapies, is yet to be elucidated. The goal of the current study was to compare the therapeutic efficacy of human mesenchymal stem cells (hMSCs) with hiPSC-CMs in mitigating myocardial infarction (MI).

Methods

Male athymic nude hyrats were subjected to permanent ligation of the left-anterior-descending (LAD) coronary artery to induce acute MI. Four experimental groups were studied: 1) control (non-MI), 2) MI, 3) hMSCs (MI+MSC), and 4) hiPSC-CMs (MI+hiPSC-derived cardiomyocytes). The hiPSC-CMs and hMSCs were labeled with superparamagnetic iron oxide (SPIO) in vitro to track the transplanted cells in the ischemic heart by high-field cardiac MRI. These cells were injected into the ischemic heart 30-min after LAD ligation. Four-weeks after MI, cardiac MRI was performed to track the transplanted cells in the infarct heart. Additionally, echocardiography (M-mode) was performed to evaluate the cardiac function. Immunohistological and western blot studies were performed to assess the cell tracking, engraftment and cardiac fibrosis in the infarct heart tissues.

Results

Echocardiography data showed a significantly improved cardiac function in the hiPSC-CMs and hMSCs groups, when compared to MI. Immunohistological studies showed expression of connexin-43, α-actinin and myosin heavy chain in engrafted hiPSC-CMs. Cardiac fibrosis was significantly decreased in hiPSC-CMs group when compared to hMSCs or MI groups. Overall, this study demonstrated improved cardiac function with decreased fibrosis with both hiPSC-CMs and hMSCs groups when compared with MI group.     

Fractalkine Depresses Cardiomyocyte Contractility

Background

Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E₂ EP4 receptor sub-type (EP4 KO) exhibit reduced cardiac function. Gene array on left ventricles (LV) showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE₂ and contributes to depressed contractility via alterations in intracellular calcium.

Methods

Fractalkine was measured in LV of 28–32 week old male EP4 KO and wild type controls (WT) by ELISA and the effect of PGE₂ on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM) from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso) stimulation.

Results

LV fractalkine was increased in EP4 KO mice but surprisingly, PGE₂ regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca²⁺ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.

Conclusions

Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.     

Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims

Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods

We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results

Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion

Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.     

Dietary supplementation with probiotics improves hematopoiesis in malnourished mice

Background

Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.

Objective

This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.

Methods

Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat''s milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.

Results

Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.

Conclusions

Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.     

The Role of Lipotoxicity in Smoke Cardiomyopathy

Background/Aims

Experimental and clinical studies have shown the direct toxic effects of cigarette smoke (CS) on the myocardium, independent of vascular effects. However, the underlying mechanisms are not well known.

Methods

Wistar rats were allocated to control (C) and cigarette smoke (CS) groups. CS rats were exposed to cigarette smoke for 2 months.

Results

After that morphometric, functional and biochemical parameters were measured. The echocardiographic study showed enlargement of the left atria, increase in the left ventricular systolic volume and reduced systolic function. Within the cardiac metabolism, exposure to CS decreased beta hydroxy acyl coenzyme A dehydrogenases and citrate synthases and increased lactate dehydrogenases. Peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were expressed similarly in both groups. CS increased serum lipids and myocardial triacylglycerols (TGs). These data suggest that impairment in fatty acid oxidation and the accumulation of cardiac lipids characterize lipotoxicity. CS group exhibited increased oxidative stress and decreased antioxidant defense. Finally, the myocyte cross-sectional area and active Caspase 3 were increased in the CS group.

Conclusion

The cardiac remodeling that was observed in the CS exposure model may be explained by abnormalities in energy metabolism, including lipotoxicity and oxidative stress.     

Bone Marrow Alterations and Lower Endothelial Progenitor Cell Numbers in Critical Limb Ischemia Patients

Background

Critical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI.

Methods

Levels of primitive (CD34⁺ and CD133⁺) progenitors and CD34⁺KDR⁺ EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial ({"type":"clinical-trial","attrs":{"text":"NCT00371371","term_id":"NCT00371371"}}NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed.

Results

Endothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1α, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34⁺ cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34⁺ cell levels (P = 0.007). CAC from CLI patients had reduced paracrine function (P<0.0001).

Conclusion

CLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34⁺-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1α/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.     

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background

An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart.

Methodology/Principal Findings

Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ).

Conclusions/Significance

The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Lineage tracing of cardiac explant derived cells

Aims

Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs.

Methods

Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies.

Results

Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone.

Conclusions

This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.     

Aging Impairs Recipient T Cell Intrinsic and Extrinsic Factors in Response to Transplantation

Background

As increasing numbers of older people are listed for solid organ transplantation, there is an urgent need to better understand how aging modifies alloimmune responses. Here, we investigated whether aging impairs the ability of donor dendritic cells or recipient immunity to prime alloimmune responses to organ transplantation.

Principal Findings

Using murine experimental models, we found that aging impaired the host environment to expand and activate antigen specific CD8⁺ T cells. Additionally, aging impaired the ability of polyclonal T cells to induce acute allograft rejection. However, the alloimmune priming capability of donor dendritic cells was preserved with aging.

Conclusion

Aging impairs recipient responses, both T cell intrinsic and extrinsic, in response to organ transplantation.     

Granulocyte Colony-Stimulating Factor Reduces Fibrosis in a Mouse Model of Chronic Pancreatitis

Background

Chronic pancreatitis (CP) is a necroinflammatory process resulting in extensive pancreatic fibrosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic stem cell mobilizer, has been shown to exert an anti-fibrotic effect partly through the enrichment of bone marrow (BM) cells in fibrotic organ. We aimed to test the effect of G-CSF on fibrosis in a mouse model of CP.

Methods

CP was induced in C57Bl/6J mice by consecutive cerulein injection (50 µg/kg/day, 2 days a week) for 6 weeks. Mice were then treated with G-CSF (200 µg/kg/day, 5 day a week) or normal saline for 1 week, and sacrificed at week 7 or week 9 after first cerulein injection. Pancreatic histology, pancreatic matrix metallopeptidase 9 (MMP-9), MMP-13 and collagen expression were examined. Pancreatic myofibroblasts were isolated and cultured with G-CSF. Collagen, MMP-9 and MMP-13 expression by myofibroblasts was examined. The BM-mismatched mice model was used to examine the change of BM-derived myofibroblasts and non-myofibroblastic BM cells by G-CSF in the pancreas.

Results

The pancreatic collagen expression were significantly decreased in the G-CSF-treated group sacrificed at week 9. While collagen produced from myofibroblasts was not affected by G-CSF, the increase of MMP13 expression was observed in vitro. There were no effect of G-CSF in the number of myofibroblasts and BM-derived myofibroblasts. However, the number of non-myofibroblastic BM cells and macrophages were significantly increased in the pancreata of cerulein- and G-CSF-treated mice, suggesting a potential anti-fibrotic role of non-myofibroblastic BM cells and macrophages stimulated by G-CSF.

Conclusions

Our data indicated that G-CSF contributed to the regression of pancreatic fibrosis. The anti-fibrotic effects were possibly through the stimulation of MMP-13 from myofibroblasts, and the enhanced accumulation of non-myofibroblastic BM cells and macrophages in the pancreas.     

Atrophic cardiac remodeling induced by taurine deficiency in wistar rats

Introduction

Micronutrient deficiency is observed in heart failure patients. Taurine, for example, represents 50% of total free amino acids in the heart, and in vivo studies have linked taurine deficiency with cardiomyopathy.

Methods

Thirty-four male Wistar rats (body weight = 100 g) were weighed and randomly assigned to one of two groups: Control (C) or taurine-deficient (T (-)). Beta-alanine at a concentration of 3% was added to the animals’ water to induce taurine deficiency in the T (-) group. On day 30, the rats were individually submitted to echocardiography; morphometrical and histopathological evaluation and metalloproteinase activity, oxidative stress and inflammation evaluation were performed. Tissue samples were collected to determine the taurine concentration in the heart.

Results

Taurine deficiency led to decreases in: ventricular wall thickness, left ventricle dry weight, myocyte sectional area, left ventricle posterior wall thickness and ventricular geometry. With regard to heart function, the velocity of the A wave, the ratio between the E and A wave, the ejection fraction, fractional shortening and cardiac output values were decreased in T (-) rats, suggesting abnormal diastolic and systolic function. Increased fibrosis, inflammation and increased activation of metalloproteinases were not observed. Oxidative stress was increased in deficient animals.

Conclusions

These data suggest that taurine deficiency promotes structural and functional cardiac alterations with unique characteristics.     

Association between Cardiac Autonomic Function, Oxidative Stress and Inflammatory Response in Impaired Fasting Glucose Subjects: Cross-Sectional Study

Background

The worldwide burden of diabetes in 2030 is projected around 552 million. Diabetes leads to higher risk for cardiovascular diseases (CVD). Altered cardiac autonomic function (CAF) measured by heart rate variability (HRV) is observed in early stages of diabetes but the relationship between impaired fasting glucose (IFG) and HRV is still debatable. The aim of the study was to evaluate the association between CAF, oxidative stress, insulin resistance (IR), and inflammatory response in IFG subjects.

Subjects and Methods

Cross-sectional blinded study. Volunteers recruited from health awareness camps underwent CAF and biochemical tests. Based on fasting plasma glucose (FPG) participants (n = 123) were divided into two groups, normal fasting glucose (n = 76) and IFG (n = 47). The comparison of parameters between the groups was carried out using student t test and Mann-Whitney U test for parametric and non-parametric data respectively. The correlation between the parameters was analyzed by Spearman’s rank correlation using SPSS 13.0.

Results

The resting cardiovagal modulation parameters, heart rate response to forced timed breathing, and orthostatic stress were reduced in IFG subjects. Fasting plasma lipid profile, coronary atherogenic lipid risk factors, IR, thiobarbituric acid reactive substance (TBARS), high sensitive C-reactive protein, and tumor necrosis factor alpha were increased and total antioxidant capacity (TAC) was decreased significantly in IFG group but no significant alteration was observed in high-density lipoprotein (HDL-c). Cardiovagal modulation parameters were negatively correlated with triglycerides, FPG, insulin, IR, TBARS, and inflammatory markers and positively with TAC.

Conclusion

There is a continuous interplay between the altered CAF, hyperinsulinemia, IR, oxidative stress parameters, inflammatory response, and IFG in which one factor perpetuates another leading to the progression of disease.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.