CTGF-D4 Amplifies LRP6 Signaling to Promote Grafts of Adult Epicardial-derived Cells That Improve Cardiac Function After Myocardial Infarction.

Transplantation of stem/progenitor cells holds promise for cardiac regeneration in patients with myocardial infarction (MI). Currently, however, low cell survival and engraftment after transplantation present a major barrier to many forms of cell therapy. One issue is that ligands, receptors, and signaling pathways that promote graft success remain poorly understood. Here, we prospectively isolate uncommitted epicardial cells from the adult heart surface by CD104 (ß-4 integrin) and demonstrate that C-terminal peptide from connective tissue growth factor (CTGF-D4), when combined with insulin, effectively primes epicardial-derived cells (EPDC) for cardiac engraftment after MI. Similar to native epicardial derivatives that arise from epicardial EMT at the heart surface, the grafted cells migrated into injured myocardial tissue in a rat model of MI with reperfusion. By echocardiography, at 1 month after MI, we observed significant improvement in cardiac function for animals that received epicardial cells primed with CTGF-D4/insulin compared with those that received vehicle-primed (control) cells. In the presence of insulin, CTGF-D4 treatment significantly increased the phosphorylation of Wnt co-receptor LRP6 on EPDC. Competitive engraftment assays and neutralizing/blocking studies showed that LRP6 was required for EPDC engraftment after transplantation. Our results identify LRP6 as a key target for increasing EPDC engraftment after MI and suggest amplification of LRP6 signaling with CTGF-D4/insulin, or by other means, may provide an effective approach for achieving successful cellular grafts in regenerative medicine.

Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.

Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP(+)) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETB(R)) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETB(R) expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETB(R)-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1-STAT3-ETB(R) axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury.

Priming with ligands secreted by human stromal progenitor cells promotes grafts of cardiac stem/progenitor cells after myocardial infarction.

Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells including fibroblasts and stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/progenitor cells in culture. We hypothesized that secreted factors in conditioned medium (CdM) from adult bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/CPCs) and improve graft success after myocardial infarction (MI). Incubation of adult rat CPCs in CdM from human MSCs isolated by plastic adherence or by magnetic sorting against CD271 (a.k.a., p75 low-affinity nerve growth factor receptor; p75MSCs) induced phosphorylation of STAT3 and Akt in CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30× p75MSC CdM for 30 minutes prior to transplantation into subepicardial tissue 1 day after MI markedly increased engraftment compared with vehicle priming. Screening CdM with neutralizing/blocking antibodies identified connective tissue growth factor (CTGF) and Insulin as key factors in p75MSC CdM that protected CPCs. Human CTGF peptide (CTGF-D4) and Insulin synergistically promoted CPC survival during hypoxia in culture. Similar to CdM priming, priming of CSCs with CTGF-D4 and Insulin for 30 minutes prior to transplantation promoted robust engraftment, survival, and migration of CSC derivatives at 1 week and 1 month after MI. Our results indicate that short-term priming of human CSCs with CTGF-D4 and Insulin may improve graft success and cardiac regeneration in patients with MI.

Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma.

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

The Neural Progenitor Cell-Associated Transcription Factor FoxG1 Regulates Cardiac Epicardial Cell Proliferation.

The epicardium is a layer of mesothelial cells that covers the surface of the heart. During development, epicardial cells undergo epithelial-to-mesenchymal transition (EMT) to form multipotent precursors that migrate into the heart and contribute to the coronary vasculature by differentiating into adventitial fibroblasts, smooth muscle cells, and endothelial cells. Epicardial cells also provide paracrine signals to cardiac myocytes that are required for appropriate heart growth. In adult hearts, a similar process of epicardial cell EMT, migration, and differentiation occurs after myocardial infarction (MI, heart attack). Pathological cardiac hypertrophy is associated with fibrosis, negative remodeling, and reduced cardiac function. In contrast, aerobic exercises such as swimming and running promote physiological (i.e., beneficial) hypertrophy, which is associated with angiogenesis and improved cardiac function. As epicardial cell function(s) during physiological hypertrophy are poorly understood, we analyzed and compared the native epicardial cells isolated directly from the hearts of running-exercised mice and age-matched, nonrunning littermates. To obtain epicardial cells, we enzymatically digested the surfaces of whole hearts and performed magnetic-activated cell sorting (MACS) with antibodies against CD104 (integrin ß4). By cDNA microarray assays, we identified genes with increased transcription in epicardial cells after running exercise; these included FoxG1, a transcription factor that controls neural progenitor cell proliferation during brain development and Snord116, a small noncoding RNA that coordinates expression of genes with epigenetic, circadian, and metabolic functions. In cultured epicardial cells, shRNA-mediated FoxG1 knockdown significantly decreased cell proliferation, as well as Snord116 expression. Our results demonstrate that FoxG1 regulates epicardial proliferation, and suggest it may affect cardiac remodeling.

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.

Multipotent stromal cell therapy for cavernous nerve injury-induced erectile dysfunction.

INTRODUCTION: Erectile dysfunction (ED) following radical prostatectomy (RP) is a result of inadvertent damage to the cavernous nerves that run close to the prostate capsula. The mechanisms behind the development of post-RP ED are increasingly recognized and include cavernosal fibrosis and cavernosal smooth muscle apoptosis, resulting from cavernous nerve degeneration due to neuropraxia. In recent years, cell-based therapies have received increasing attention regarding their potential for recovery of erectile function following cavernous nerve injury (CNI). Multipotent stromal cells (MSCs) are an attractive cell source for this application based on their regenerative potential and their clinical applicability. AIM: To review available evidence on the efficacy and mechanisms of action of MSC application for the treatment of ED, with an emphasis on ED following CNI. METHODS: A nonsystematic review was conducted on the available English literature between 1966 and 2011 on the search engines SciVerse-sciencedirect, SciVerse-scopus, Google Scholar, and PubMed. RESULTS: MSCs from both bone marrow and adipose tissue have shown beneficial effects in a variety of animal models for ED. While MSC application in chronic disease models such as diabetes, aging, and hyperlipidemia may result in cell engraftment and possibly MSC differentiation, this observation has not been made in the acute CNI rat model. In the latter setting, MSC effects seem to be established by cell recruitment toward the major pelvic ganglion and local paracrine interaction with the host neural tissue. CONCLUSIONS: While the type of model may influence the mechanisms of action of this MSC-based therapy, MSCs generally display efficacy in various animal models for ED. Before translation to the clinic is established, various hurdles need to be overcome.

Human Growth Factor/Immunoglobulin Complexes for Treatment of Myocardial Ischemia-Reperfusion Injury.

Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor 2 (FGF2) are receptor tyrosine kinase agonists that promote cell survival after tissue injury and angiogenesis, cell proliferation and migration during tissue repair and regeneration. Both ligands have potential as systemic treatments for ischemia-reperfusion injury, however clinical use of HGF and FGF2 has been limited by poor pharmacokinetic profiles, i.e., their susceptibility to serum proteases, rapid clearance and short half-lives. Previously, we reported vaso- and cardioprotective protein complexes formed between HGF and polyclonal, non-specific immunoglobulin (IgG) with therapeutic efficacy in a rat model of myocardial ischemia with reperfusion (MI/R). Here, using a pre-clinical porcine MI/R model, we demonstrate human HGF/IgG complexes provide significant myocardial salvage, reduce infarct size, and are detectable in myocardial tissue 24 h after intracoronary injection. Furthermore, we show that multiple daily infusions of HGF/IgG complexes after MI do not lead to production of HGF-specific auto-antibodies, an important concern for administered biologic drugs. In experiments to identify other growth factors that non-covalently interact with IgG, we found that human FGF2 associates with IgG. Similar to human HGF/IgG complexes, FGF2/IgG complexes protected primary human cardiac endothelial cells under simulated ischemia (1% oxygen and nutrient deprivation) for 48-72 h. Molecular modeling studies suggested that FGF2 and HGF both interact with the Fc domain of IgG. Also, we tested whether an Fc-fusion protein would bind FGF2 to form complexes. By native gel electrophoretic assays and biochemical pulldowns, we found that Jagged1, a Notch1 ligand that controls stem cell self-renewal and tissue regeneration, bound FGF2 when presented as a Jagged1- Fc fusion protein. Our results suggest that human growth factor/IgG and FGF2/Fc- fusion complexes have potential to provide a biologics platform to treat myocardial ischemia-reperfusion and other forms of tissue injury.

Proliferating reactive astrocytes are regulated by Notch-1 in the peri-infarct area after stroke.

BACKGROUND AND PURPOSE: The formation of reactive astrocytes is common after central nervous system injuries such as stroke. However, the signaling pathway(s) that control astrocyte formation and functions are poorly defined. We assess the effects of Notch 1 signaling in peri-infarct-reactive astrocytes after stroke. METHODS: We examined reactive astrocyte formation in the peri-infarct area 3 days after distal middle cerebral artery occlusion with or without γ-secretase inhibitor treatment. To directly study the effects of inhibiting a γ-secretase cleavage target in reactive astrocytes, we generated glial fibrillary acidic protein-CreER™:Notch 1 conditional knockout mice. RESULTS: Gamma-secretase inhibitor treatment after stroke decreased the number of proliferative glial fibrillary acidic protein-positive reactive astrocytes and RC2-positive reactive astrocytes directly adjacent to the infarct core. The decrease in reactive astrocytes correlated with an increased number of CD45-positive cells that invaded into the peri-infarct area. To study the influence of reactive astrocytes on immune cell invasion, ex vivo immune cell invasion assays were performed. We found that a γ-secretase-mediated pathway in astrocytes affected Jurkat cell invasion. After tamoxifen treatment, glial fibrillary acidic protein-CreER™:Notch 1 conditional knockout mice had a significantly decreased number of proliferating reactive astrocytes and RC2-positive reactive astrocytes. Tamoxifen treatment also led to an increased number of CD45-positive cells that invaded the peri-infarct area. CONCLUSIONS: Our results demonstrate that proliferating and RC2-positive reactive astrocytes are regulated by Notch 1 signal transduction and control immune cell invasion after stroke.

Stem and progenitor cells for neurological repair: minor issues, major hurdles, and exciting opportunities for paracrine-based therapeutics.

The transplantation of cultured stem and progenitor cells is a key element in the rapidly growing field of regenerative medicine. Based on their ability to rescue and/or repair injured tissue and partially restore organ function, multiple types of stem/progenitor cells have already entered into clinical trials. However, despite several decades of intense research, the goal to apply culture-expanded stem/progenitor cells in a manner that can effectively replace cells after injury has yet to be realized. Many sources of potentially useful cells are available, but something is clearly missing. In addition, recent studies suggest that paracrine effects of secreted or released factors are responsible for most of the benefits observed after cell transplantation, rather than direct cell replacement. These data call into question the need for cell transplantation for many types of therapy, in particular for acute injuries such as myocardial infarction and stroke. In this review, we examine current progress in the area of cell transplantation and minor issues and major hurdles regarding the clinical application of different cell types. We discuss the "paracrine hypothesis" for the action of transplanted stem/progenitor cells as an opportunity to identify defined combinations of biomolecules to rescue and/or repair tissues after injury. Although many of the concepts in this review will apply to multiple injury/repair systems, we will focus primarily on stem/progenitor cell-based treatments for neurological disorders and stroke.

Incomplete reprogramming after fusion of human multipotent stromal cells and bronchial epithelial cells.

Bone marrow-derived progenitor cells can fuse with cells of several different tissues, including lung, especially following injury. Despite many reports of cell fusion, few studies have examined the function of the resulting hybrid cells. We cocultured human multipotent stromal cells (hMSCs) and normal human bronchial epithelial cells (NHBEs) and observed the formation of hMSC/NHBE heterokaryons. The heterokaryons expressed several proteins characteristic of epithelial cells, such as keratin and occludin. Hybrid cells also expressed the mRNAs and proteins for 2 important ion channels that maintain bronchial and alveolar fluid balance: the cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial Na(+) channel (ENaC). By immunocytochemistry, CFTR was expressed in many hybrid cells but was absent or low in others. Whole-cell patch-clamp recordings demonstrated a glibenclamide-sensitive current in the presence of barium chloride, consistent with functional CFTR channels, in control NHBEs and hMSC/NHBE heterokaryons. Total cell capacitance measurements showed that the membrane surface area of heterokaryons was similar to that of NHBEs. Heterokaryons expressed the α- and γ-ENaC subunits but did not express the ß-ENaC subunit, indicating the inability to form a complete ENaC channel. In addition, hybrid cells formed by the fusion of hMSCs with immortalized bronchial cells that expressed CFTR ΔF508 did not lead to reprogramming of the hMSC nucleus and expression of wild-type CFTR mRNA. Our data show that reprogramming can be incomplete following fusion of adult progenitor cells and somatic cells and may lead to altered cell function.

Isolation of locally derived stem/progenitor cells from the peri-infarct area that do not migrate from the lateral ventricle after cortical stroke.

BACKGROUND AND PURPOSE: Neurogenesis can arise from neural stem/progenitor cells of the subventricular zone after strokes involving both the cortex and striatum. However, it is controversial whether all types of stroke and strokes of different sizes activate neurogenesis from the subventricular zone niche. In contrast with cortical/striatal strokes, repair and remodeling after mild cortical strokes may involve to a greater extent local cortical stem/progenitor cells and cells from nonneurogenic niches. METHODS: We compared stem/progenitor cell responses after focal cortical strokes produced by distal middle cerebral artery occlusion and cortical/striatal strokes produced by the intraluminal suture model. To label migrating neuroblasts from the subventricular zone, we injected DiI to the lateral ventricle after distal middle cerebral artery occlusion. By immunohistochemistry, we characterized cells expressing stem/progenitor cell markers in the peri-infarct area. We isolated cortical stem/progenitor cells from the peri-infarct area after distal middle cerebral artery occlusion and assayed their self-renewal and differentiation capacity. RESULTS: In contrast with cortical/striatal strokes, focal cortical strokes did not induce neuroblast migration from the subventricular zone to the infarct zone after distal middle cerebral artery occlusion. By immunohistochemistry, we observed subpopulations of reactive astrocytes in the peri-infarct area that coexpressed radial glial cell markers such as Sox2, Nestin, and RC2. Clonal neural spheres isolated from the peri-infarct area after distal middle cerebral artery occlusion differentiated into neurons, astrocytes, oligodendrocytes, and smooth muscle cells. Notably, neural spheres isolated from the peri-infarct area also expressed RC2 before differentiation. CONCLUSIONS: Mild cortical strokes that do not penetrate the striatum activate local cortical stem/progenitor cells but do not induce neuroblast migration from the subventricular zone niche.

Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice.

Cultured adherent bone marrow stromal cells (BMSCs) are capable of forming ectopic hematopoietic microenvironments (HMEs) in immunodeficient mice. However, the cell surface phenotype of the native bone marrow stem/progenitor cell that gives rise to BMSCs that support hematopoiesis remains poorly defined. We recently reported the derivation of human BMSC-like cells (CD133BMSCs) by magnetic cell sorting against Prominin-1 (CD133), an epitope expressed by embryonic, fetal, and adult stem cells. Here we demonstrate that CD133BMSCs are capable of forming ectopic HMEs. Cultured adherent CD133BMSCs derived from sorted CD133-positive cells lacked CD133 expression, but were uniformly positive for CD146, an epitope recently described to identify self-renewing osteoprogenitor cells that could transfer the HME. CD133BMSCs were genetically-tagged by lentivirus, expanded, and seeded into HA/TCP/fibrin constructs that were implanted subcutaneously. After 60days, CD133BMSCs produced human osteocytes, osteoblasts, adipocytes, and reticular cells that supported murine hematopoiesis. CD133BMSCs that were not transduced with lentivirus also formed HMEs. Control constructs seeded with human dermal fibroblasts formed connective tissue, but failed to form HMEs. Our data indicate that CD133 expression identifies a native human bone marrow stem/progenitor cell that gives rise to BMSCs capable of forming the HME.

Transplantation of nonhematopoietic adult bone marrow stem/progenitor cells isolated by p75 nerve growth factor receptor into the penis rescues erectile function in a rat model of cavernous nerve injury.

PURPOSE: Radical prostatectomy for prostate cancer frequently results in erectile dysfunction and decreased quality of life. We investigated the effects of transplanting nonhematopoietic adult bone marrow stem/progenitor cells (multipotent stromal cells) into the corpus cavernosum in a rat model of bilateral cavernous nerve crush injury. MATERIALS AND METHODS: Multipotent stromal cells were isolated from the bone marrow of transgenic green fluorescent protein rats by plastic adherence (rat multipotent stromal cells) or magnetic activated cell sorting using antibodies against p75 low affinity nerve growth factor receptor (p75 derived multipotent stromal cells). Bilateral cavernous nerve crush injury was induced in adult male Sprague-Dawley rats. Immediately after injury 8 rats each were injected intracavernously with phosphate buffered saline (vehicle control), fibroblasts (cell control), rat multipotent stromal cells (cell treatment) or p75 derived multipotent stromal cells (cell treatment). Another 8 rats underwent sham operation (phosphate buffered saline injection). Four weeks after the procedures we assessed erectile function by measuring the intracavernous-to-mean arterial pressure ratio and total intracavernous pressure during cavernous nerve stimulation. RESULTS: Intracavernous injection of p75 derived multipotent stromal cells after bilateral cavernous nerve crush injury resulted in a significantly higher mean intracavernous-to-mean arterial pressure ratio and total intracavernous pressure compared with all other groups except the sham operated group (p <0.05). Rats injected with typical multipotent stromal cells had partial erectile function rescue compared with animals that received p75 derived multipotent stromal cells. Fibroblast (cell control) and phosphate buffered saline (vehicle control) injection did not improve erectile function. Enzyme-linked immunosorbent assay suggested that basic fibroblast growth factor secreted by p75 derived multipotent stromal cells protected the cavernous nerve after bilateral cavernous nerve crush injury. CONCLUSIONS: Transplantation of adult stem/progenitor cells may provide an effective treatment for erectile dysfunction after radical prostatectomy.

Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform.

BACKGROUND AIMS: Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest. METHODS: We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo. RESULTS: Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC. CONCLUSIONS: These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.

The magnitude and temporal dependence of apoptosis early after myocardial ischemia with or without reperfusion.

In view of the conventional wisdom in the cardiology literature that apoptosis is extensive early after myocardial ischemia, predicated largely from results with the TUNEL assay known to be nonspecific, this study was performed to delineate its extent with multiple assays and at multiple intervals. Coronary occlusion with and without subsequent revascularization was induced in 10-wk-old C57BL6 mice subjected to 1 or 4 h of transient ligation followed by 24 h of reperfusion, or 24 h persistent ligation. Apoptosis was quantified throughout the left ventricle immunohistochemically by assay of TUNEL, single-stranded DNA (ssDNA), and cleaved caspase 3; electron microscopy (EM); and activity assays of caspase 3 and 8. TUNEL staining was marked, but ssDNA and cleaved caspase 3 staining were significantly less (P<0.001 compared with TUNEL), and apoptosis defined by EM was virtually absent in all groups. Caspase 3 and caspase 8 activities per milligram protein were not significantly different from those in normal hearts. Only rare, potentially apoptotic cells were seen by EM in hearts from any group. Thus, the results with TUNEL were not specific, and the extent of apoptosis was markedly less than that predicated on the results with the TUNEL procedure. Apoptosis is de minimus early after transitory or persistent ischemia, though it is overestimated by TUNEL assays. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of myocardium early after ischemic insults.

CD133 identifies a human bone marrow stem/progenitor cell sub-population with a repertoire of secreted factors that protect against stroke.

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

Internalized FGF-2-Loaded Nanoparticles Increase Nuclear ERK1/2 Content and Result in Lung Cancer Cell Death.

: Innovative cancer treatments, which improve adjuvant therapy and reduce adverse events, are desperately needed. Nanoparticles provide controlled intracellular biomolecule delivery in the absence of activating external cell surface receptors. Prior reports suggest that intracrine signaling, following overexpression of basic fibroblast growth factor (FGF-2) after viral transduction, has a toxic effect on diseased cells. Herein, the research goals were to 1) encapsulate recombinant FGF-2 within stable, alginate-based nanoparticles (ABNs) for non-specific cellular uptake, and 2) determine the effects of ABN-mediated intracellular delivery of FGF-2 on cancer cell proliferation/survival. In culture, human alveolar adenocarcinoma basal epithelial cell line (A549s) and immortalized human bronchial epithelial cell line (HBE1s) internalized ABNs through non-selective endocytosis. Compared to A549s exposed to empty (i.e., blank) ABNs, the intracellular delivery of FGF-2 via ABNs significantly increased the levels of lactate dehydrogenase, indicating that FGF-2-ABN treatment decreased the transformed cell integrity. Noticeably, the nontransformed cells were not significantly affected by FGF-2-loaded ABN treatment. Furthermore, FGF-2-loaded ABNs significantly increased nuclear levels of activated-extracellular signal-regulated kinase ½ (ERK1/2) in A549s but had no significant effect on HBE1 nuclear ERK1/2 expression. Our novel intracellular delivery method of FGF-2 via nanoparticles resulted in increased cancer cell death via increased nuclear ERK1/2 activation.

Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension.

Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow-derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)-transgenic male rats was transplanted into GFP-negative female rats. The chimeric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP-positive BMD cells engrafted in the lungs of MCT-treated rats. Microarray analyses and double-immunohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT-treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion.