Fibroblast Growth Factor-9 Enhances M2 Macrophage Differentiation and Attenuates Adverse Cardiac Remodeling in the Infarcted Diabetic Heart

Inflammation has been implicated as a perpetrator of diabetes and its associated complications. Monocytes, key mediators of inflammation, differentiate into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages upon infiltration of damaged tissue. However, the inflammatory cell types, which propagate diabetes progression and consequential adverse disorders, remain unclear. The current study was undertaken to assess monocyte infiltration and the role of fibroblast growth factor-9 (FGF-9) on monocyte to macrophage differentiation and cardioprotection in the diabetic infarcted heart. Db/db diabetic mice were assigned to sham, myocardial infarction (MI), and MI+FGF-9 groups. MI was induced by permanent coronary artery ligation and animals were subjected to 2D transthoracic echocardiography two weeks post-surgery. Immunohistochemical and immunoassay results from heart samples collected suggest significantly increased infiltration of monocytes (Mean +/- SEM; MI: 2.02% +/- 0.23% vs. Sham 0.75% +/- 0.07%; p \u3c 0.05) and associated pro-inflammatory cytokines (TNF-alpha, MCP-1, and IL-6), adverse cardiac remodeling (Mean +/- SEM; MI: 33% +/- 3.04% vs. Sham 2.2% +/- 0.33%; p \u3c 0.05), and left ventricular dysfunction (Mean +/- SEM; MI: 35.4% +/- 1.25% vs. Sham 49.19% +/- 1.07%; p \u3c 0.05) in the MI group. Importantly, treatment of diabetic infarcted myocardium with FGF-9 resulted in significantly decreased monocyte infiltration (Mean +/- SEM; MI+FGF-9: 1.39% +/- 0.1% vs. MI: 2.02% +/- 0.23%; p \u3c 0.05), increased M2 macrophage differentiation (Mean +/- SEM; MI+FGF-9: 4.82% +/- 0.86% vs. MI: 0.85% +/- 0.3%; p \u3c 0.05) and associated anti-inflammatory cytokines (IL-10 and IL-1RA), reduced adverse remodeling (Mean +/- SEM; MI+FGF-9: 11.59% +/- 1.2% vs. MI: 33% +/- 3.04%; p \u3c 0.05), and improved cardiac function (Fractional shortening, Mean +/- SEM; MI+FGF-9: 41.51% +/- 1.68% vs. MI: 35.4% +/- 1.25%; p \u3c 0.05). In conclusion, our data suggest FGF-9 possesses novel therapeutic potential in its ability to mediate monocyte to M2 differentiation and confer cardiac protection in the post-MI diabetic heart

Regulation of PTEN/Akt Pathway Enhances Cardiomyogenesis and Attenuates Adverse Left Ventricular Remodeling following Thymosin beta 4 Overexpressing Embryonic Stem Cell Transplantation in the Infarcted Heart

Thymosin beta 4 (T beta 4), a small G-actin sequestering peptide, mediates cell proliferation, migration, and angiogenesis. Whether embryonic stem (ES) cells, overexpressing T beta 4, readily differentiate into cardiac myocytes in vitro and in vivo and enhance cardioprotection following transplantation post myocardial infarction (MI) remains unknown. Accordingly, we established stable mouse ES cell lines, RFP-ESCs and T beta 4-ESCs, expressing RFP and an RFP-T beta 4 fusion protein, respectively. In vitro, the number of spontaneously beating embryoid bodies (EBs) was significantly increased in T beta 4-ESCs at day 9, 12 and 15, compared with RFP-ESCs. Enhanced expression of cardiac transcriptional factors GATA-4, Mef2c and Txb6 in T beta 4-EBs, as confirmed with real time-PCR analysis, was accompanied by the increased number of EB areas stained positive for sarcomeric alpha-actin in T beta 4-EBs, compared with the RFP control, suggesting a significant increase in functional cardiac myocytes. Furthermore, we transplanted T beta 4-ESCs into the infarcted mouse heart and performed morphological and functional analysis 2 weeks after MI. There was a significant increase in newly formed cardiac myocytes associated with the Notch pathway, a decrease in apoptotic nuclei mediated by an increase in Akt and a decrease in levels of PTEN. Cardiac fibrosis was significantly reduced, and left ventricular function was significantly augmented in the T beta 4-ESC transplanted group, compared with controls. It is concluded that genetically modified T beta 4-ESCs, potentiates their ability to turn into cardiac myocytes in vitro as well as in vivo. Moreover, we also demonstrate that there was a significant decrease in both cardiac apoptosis and fibrosis, thus improving cardiac function in the infarcted heart

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Abstract Background Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn’s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease

A Cd63\u3csup\u3e+Ve\u3c/sup\u3e/C-Kit\u3csup\u3e+Ve\u3c/sup\u3e Stem Cell Population Isolated From The Mouse Heart

Cardiac cell regeneration from endogenous cardiac stem cells (CSCs) following MI is rather low. Therefore, identifying mechanisms to boost endogenous CSC activation and participation in cardiac repair appears to be the most promising strategy for MI patients. We previously engineered tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing embryonic stem (ES-TIMP-1) cells and transplanted them into the infarcted murine heart. Collected data demonstrated that TIMP-1 enhanced transplanted ES cell engraftment, survival and differentiation into cardiac myocytes post-transplantation. Therefore, we postulated that there may be a new stem cell population present in the heart that is regulated by extracellular protein TIMP-1. Furthermore, we hypothesized that this cell population has a potential for cell proliferation and differentiation into cardiac cell types. Therefore, we isolated CSCs from 4 weeks old C57BL/6 mice and cultured them in vitro in presence of ESCM, ES-TIMP-1-CM or TIMP-1. Our immunostaining data demonstrated the existence of a novel CSC subpopulation, CD63+ve/c-kit+ve. When treated with TIMP-1, these cells showed significantly (p \u3c 0.05) increased proliferation rates compared to control cells, enhanced TIMP-1 receptor (CD63), along with improved expression of phospho and total β-catenin proteins as demonstrated by Western blot analysis. Next, we demonstrate significantly (p \u3c 0.05) improved cardiac myocyte, vascular smooth muscle cell, and endothelial cell differentiation. Furthermore, our RT-PCR data shows increase in cardiac gene (GATA-4, Mef2C, and Nkx-2.5) expression when compared to ESCM and control cells. Collectively, these data, for the first time, establish the existence of a new CD63+ve/c-kit+ve CSC subpopulation that has a significant potential for proliferation and differentiation into cardiac cell types once stimulated with TIMP-1

Embryonic Stem Cells And Released Factors Stimulate C-Kit(+)/Flk-1(+) Progenitor Cells And Promote Neovascularization In Doxorubicin-Induced Cardiomyopathy

Vascular apoptosis plays a pivotal role in the development and progression of a myriad of cardiac dysfunctions, but has yet to be investigated in doxorubicin-induced cardiomyopathy (DIC). Additionally, the neovascularization potential and resulting functional consequences of embryonic stem (ES) cells and factors released from these cells in the chronic DIC myocardium remain largely unknown. To this end, we transplanted conditioned media (CM) and ES cells in the DIC-injured heart and evaluated their potential to inhibit vascular cell death, activate endogenous c-kit(+) and FLK-1(+) cells, enhance neovascularization, and augment left ventricular dysfunction. Data presented suggest transplanted CM and ES cells significantly blunt vascular cell apoptosis consequent to DIC. Quantitative immunohistochemistry data demonstrate significantly increased c-kit(+) and FLK-1(+) cells, as well as enhanced differentiated CD31(+) cells in the CM and ES cell groups relative to DIC controls. Heart function, including fractional shortening and ejection fraction, assessed by transthoracic echocardiography, was significantly improved following CM and ES cell transplantation. In conclusion, our data suggest that transplantation of CM and ES cells inhibit vascular apoptosis, activate endogenous c-kit(+) and FLK-1(+) cells and differentiate them into endothelial cells, enhance neovascularization, and improve cardiac function in the DIC-injured myocardium

Transplanted Induced Pluripotent Stem Cells Improve Cardiac Function And Induce Neovascularization In The Infarcted Hearts Of Db/Db Mice

Recently, we proclaimed that induced pluripotent stem (iPS) cells generated from H9c2 cells, following transplantation into infarcted nondiabetic mice, can inhibit apoptosis and differentiate into cardiac myocytes. iPS cells can be an ideal candidate to expand regenerative medicine to the clinic. Therefore, examining the wide range of their potential to differentiate into neovascular cell types remains a major interest. We hypothesized that transplanted iPS cells in the infarcted diabtetic db/db and nondiabetic mice can differentiate into vascular smooth muscle (VSM) and endothelial cells (ECs) as well as activate endogenous c-kit progenitor cells to enhance neovascularization along with improved cardiac function. We transplanted intramyocardially 50,000 iPS cells in the peri-infarct zone of infarcted db/db and C57BL/6 mice and hearts were examined at D14 post-MI. Cardiac function was examined using echocardiography. Our data implies that there was a significant (p \u3c 0.001) increase in VSM and ECs in the infarcted heart following iPS cell transplantation compared with MI and sham groups in both db/db and C57BL/6 animals. Furthermore, the MI+iPS cell transplanted group also displayed a significant (p \u3c 0.001) increase in c-kit +ve activated VSM and ECs confirmed with combined stainings of c-kit and cell specific markers, compared with respective controls. Next, our histology data in the MI+iPS cell group also establishes a significant (p \u3c 0.05) increase in coronary artery vessels compared with MI, suggesting neovascularization. Furthermore, our data demonstrates significant improved cardiac function following iPS cell transplantation compared with MI. Overall increased neovascularization in the infarcted db/db and C57BL/6 mice is associated with improved cardiac function following iPS cell transplantation. © 2011 American Chemical Society

Cell Therapy In The Heart

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development

Extra-Pulmonary Complications in SARS-CoV-2 Infection: A Comprehensive Multi Organ-System Review

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is typically presented with acute symptoms affecting upper and lower respiratory systems. As the current pandemic progresses, COVID-19 patients are experiencing a series of nonspecific or atypical extra-pulmonary complications such as systemic inflammation, hypercoagulability state, and dysregulation of the renin–angiotensin–aldosterone system (RAAS). These manifestations often delay testing, diagnosis, and the urge to seek effective treatment. Although the pathophysiology of these complications is not clearly understood, the incidence of COVID-19 increases with age and the presence of pre-existing conditions. This review article outlines the pathophysiology and clinical impact of SARS-CoV-2 infection on extra-pulmonary systems. Understanding the broad spectrum of atypical extra-pulmonary manifestations of COVID-19 should increase disease surveillance, restrict transmission, and most importantly prevent multiple organ-system complications

Fibroblast growth factor-9 enhances M2 macrophage differentiation and attenuates adverse cardiac remodeling in the infarcted diabetic heart.

Inflammation has been implicated as a perpetrator of diabetes and its associated complications. Monocytes, key mediators of inflammation, differentiate into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages upon infiltration of damaged tissue. However, the inflammatory cell types, which propagate diabetes progression and consequential adverse disorders, remain unclear. The current study was undertaken to assess monocyte infiltration and the role of fibroblast growth factor-9 (FGF-9) on monocyte to macrophage differentiation and cardioprotection in the diabetic infarcted heart. Db/db diabetic mice were assigned to sham, myocardial infarction (MI), and MI+FGF-9 groups. MI was induced by permanent coronary artery ligation and animals were subjected to 2D transthoracic echocardiography two weeks post-surgery. Immunohistochemical and immunoassay results from heart samples collected suggest significantly increased infiltration of monocytes (Mean ± SEM; MI: 2.02% ± 0.23% vs. Sham 0.75% ± 0.07%; p<0.05) and associated pro-inflammatory cytokines (TNF-α, MCP-1, and IL-6), adverse cardiac remodeling (Mean ± SEM; MI: 33% ± 3.04% vs. Sham 2.2% ± 0.33%; p<0.05), and left ventricular dysfunction (Mean ± SEM; MI: 35.4% ± 1.25% vs. Sham 49.19% ± 1.07%; p<0.05) in the MI group. Importantly, treatment of diabetic infarcted myocardium with FGF-9 resulted in significantly decreased monocyte infiltration (Mean ± SEM; MI+FGF-9: 1.39% ± 0.1% vs. MI: 2.02% ± 0.23%; p<0.05), increased M2 macrophage differentiation (Mean ± SEM; MI+FGF-9: 4.82% ± 0.86% vs. MI: 0.85% ± 0.3%; p<0.05) and associated anti-inflammatory cytokines (IL-10 and IL-1RA), reduced adverse remodeling (Mean ± SEM; MI+FGF-9: 11.59% ± 1.2% vs. MI: 33% ± 3.04%; p<0.05), and improved cardiac function (Fractional shortening, Mean ± SEM; MI+FGF-9: 41.51% ± 1.68% vs. MI: 35.4% ± 1.25%; p<0.05). In conclusion, our data suggest FGF-9 possesses novel therapeutic potential in its ability to mediate monocyte to M2 differentiation and confer cardiac protection in the post-MI diabetic heart

Development Of Multiplex Pcr And Multi-Color Fluorescent In Situ Hybridization (M-Fish) Coupled Protocol For Detection And Imaging Of Multi-Pathogens Involved In Inflammatory Bowel Disease

Background: Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn\u27s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods: The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results: Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion: We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease