Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of micro-RNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of non-cardiac myocytes to cardiac myocytes. However, the biologic and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether non-cardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected FSP1-Cre mice/tdTomato mice to cardiac injury by permanent ligation of the left anterior descending coronary artery (LAD) and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five-to-six weeks following injury, morphological and physiological properties of tdTomato(-) and tdTomato(+) cardiac myocyte-like cells were analyzed ex vivo. tdTomato(+) cells expressed cardiac myocyte markers, sarcomeric organization, excitation-contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato(-) cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential utility of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration following myocardial injury.
Rationale: Cardiac progenitor cells (CPCs) are believed to differentiate into the major cell types of the heart; cardiomyocytes, smooth muscle cells, and endothelial cells. We have recently identified Abi3bp as a protein important for mesenchymal stem cell (MSC) biology. Since CPCs share several characteristics with MSCs we hypothesized that Abi3bp would similarly affect CPC differentiation and proliferation. Objective: To determine whether Abi3bp regulates CPC proliferation and differentiation. Methods and Results: In vivo, genetic ablation of the Abi3bp gene inhibited CPC differentiation whereas CPC number and proliferative capacity was increased. This correlated with adverse recovery following myocardial infarction. In vitro, CPCs, either isolated from Abi3bp knockout mice or expressing an Abi3bp shRNA construct, displayed a higher proliferative capacity and, under differentiating conditions, reduced expression of both early and late cardiomyocyte markers. Abi3bp controlled CPC differentiation via integrin-?1, PKC?, and Akt. Conclusions: We have identified Abi3bp as a protein important for CPC differentiation and proliferation.
We present a precision, 1-A, digitally interfaced current controller for quantum cascade lasers, with demonstrated temperature coefficients for continuous and 40-kHz full-depth square-wave modulated operation, of 1-2 ppm/?°C and 15 ppm/?°C, respectively. High precision digital to analog converters (DACs) together with an ultra-precision voltage reference produce highly stable, precision voltages, which are selected by a multiplexer (MUX) chip to set output currents via a linear current regulator. The controller is operated in conjunction with a power multiplexing unit, allowing one of three lasers to be driven by the controller, while ensuring protection of controller and all lasers during operation, standby, and switching. Simple ASCII commands sent over a USB connection to a microprocessor located in the current controller operate both the controller (via the DACs and MUX chip) and the power multiplexer.
Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; TUNEL positive nuclei were fewer in number, and caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ? (PKC?). Inhibition of PKC? blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKC?. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKC?.
The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.
Mesenchymal stem cells (MSCs) transplanted into injured myocardium promote repair through paracrine mechanisms. We have previously shown that MSCs over-expressing AKT1 (Akt-MSCs) exhibit enhanced properties for cardiac repair. In this study, we investigated the relevance of Abi3bp toward MSC biology. Abi3bp formed extracellular deposits with expression controlled by Akt1 and ubiquitin-mediated degradation. Abi3bp knockdown/knockout stabilized focal adhesions and promoted stress-fiber formation. Furthermore, MSCs from Abi3bp knockout mice displayed severe deficiencies in osteogenic and adipogenic differentiation. Knockout or stable knockdown of Abi3bp increased MSC and Akt-MSC proliferation, promoting S-phase entry via cyclin-d1, ERK1/2, and Src. Upon Abi3bp binding to integrin-?1 Src associated with paxillin which inhibited proliferation. In vivo, Abi3bp knockout increased MSC number and proliferation in bone marrow, lung, and liver. In summary, we have identified a novel extracellular matrix protein necessary for the switch from proliferation to differentiation in MSCs.
Secreted frizzled related protein 2 (Sfrp2) is known as an inhibitor for the Wnt signaling. In recent studies, Sfrp2 has been reported to inhibit the activity of Xenopus homolog of mammalian Tolloid-like 1 metalloproteinase. Bone morphogenic protein 1 (Bmp1)/Tolloid-like metalloproteinase plays a key role in the regulation of collagen biosynthesis and maturation after tissue injury. Here, we showed both endogenous Sfrp2 and Bmp1 protein expressions were up-regulated in rat heart after myocardial infarction (MI). We hypothesize that Sfrp2 could inhibit mammalian Bmp1 activity and, hence, the exogenous administration of Sfrp2 after MI would inhibit the deposition of mature collagen and improve heart function. Using recombinant proteins, we demonstrated that Sfrp2, but not Sfrp1 or Sfrp3, inhibited Bmp1 activity in vitro as measured by a fluorogenic peptide based procollagen C-proteinase activity assay. We also demonstrated that Sfrp2 at high concentration inhibited human and rat type I procollagen processing by Bmp1 in vitro. We further showed that exogenously added Sfrp2 inhibited type I procollagen maturation in primary cardiac fibroblasts. Two days after direct injection into the rat infarcted myocardium, Sfrp2 inhibited MI-induced type I collagen deposition. As early as 2 wk after injection, Sfrp2 significantly reduced left ventricular (LV) fibrosis as shown by trichrome staining. Four weeks after injection, Sfrp2 prevented the anterior wall thinning and significantly improved cardiac function as revealed by histological analysis and echocardiographic measurement. Our study demonstrates Sfrp2 at therapeutic doses can inhibit fibrosis and improve LV function at a later stage after MI.
Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly because of poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (eg, CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival.
Renin is a key enzyme for cardiovascular and renal homeostasis and is produced by highly specialized endocrine cells in the kidney, known as juxtaglomerular (JG) cells. The nature and origin of these cells remain as mysteries. Previously, we have shown that the nuclear hormone receptor liver X receptor-alpha (LXRalpha) is a major transcriptional regulator of the expression of renin, c-myc, and other genes involved with growth/differentiation. In this study we test the hypothesis that LXRalpha plays an important role not only in renin expression but also in renin-containing cell differentiation, specifically from the mesenchymal stem cell (MSC), which may be the origin of the JG cell. Indeed, our data demonstrated that LXRalpha activation by its ligands or cAMP stimulated renin gene expression in both murine and human MSCs. Furthermore, sustained cAMP stimulation of murine MSCs overexpressing LXRalpha led to their differentiation into JG-like cells expressing renin and alpha-smooth muscle actin. These MSC-derived JG-like cells contained renin in secretory granules and released active renin in response to cAMP. In conclusion, the activation of LXRalpha stimulates renin expression and induces MSCs differentiation into renin-secreting, JG-like cells. Our results suggest that the MSC may be the origin of the juxtaglomerular cell and provide insight into novel understanding of pathophysiology of the renin-angiotensin system.
Variation in maize for response to photoperiod is related to geographical adaptation in the species. Maize possesses homologs of many genes identified as regulators of flowering time in other species, but their relation to the natural variation for photoperiod response in maize is unknown. Candidate gene sequences were mapped in four populations created by crossing two temperate inbred lines to two photoperiod-sensitive tropical inbreds. Whole-genome scans were conducted by high-density genotyping of the populations, which were phenotyped over 3 years in both short- and long-day environments. Joint multiple population analysis identified genomic regions controlling photoperiod responses in flowering time, plant height, and total leaf number. Four key genome regions controlling photoperiod response across populations were identified, referred to as ZmPR1-4. Functional allelic differences within these regions among phenotypically similar founders suggest distinct evolutionary trajectories for photoperiod adaptation in maize. These regions encompass candidate genes CCA/LHY, CONZ1, CRY2, ELF4, GHD7, VGT1, HY1/SE5, TOC1/PRR7/PPD-1, PIF3, ZCN8, and ZCN19.
We have demonstrated that mesenchymal stem cells overexpressing the survival gene Akt can confer paracrine protection to ischemic myocytes both in vivo and in vitro through the release of secreted frizzled related protein 2 (Sfrp2). However, the mechanisms mediating these effects of Sfrp2 have not been fully elucidated. In this study, we studied rat cardiomyoblasts subjected to hypoxia reoxygenation (HR) injury to test the hypothesis that Sfrp2 exerts anti-apoptotic effect by antagonizing pro-apoptotic properties of specific Wnt ligands. We examined the effect of Wnt3a and Sfrp2 on HR-induced apoptosis. Wnt3a significantly increased cellular caspase activities and TUNEL staining in response to HR. Sfrp2 attenuated significantly Wnt3a-induced caspase activities in a concentration dependent fashion. Using a solid phase binding assay, our data demonstrates that Sfrp2 physically binds to Wnt3a. In addition, we observed that Sfrp2 dramatically inhibits the beta-catenin/TCF transcriptional activities induced by Wnt3a. Impressively, Dickkopf-1, a protein that binds to the Wnt coreceptor LRP, significantly inhibited the Wnt3a-activated caspase and transcriptional activities. Similarly, siRNA against beta-catenin markedly inhibited the Wnt3a-activated caspase activities. Consistent with this, significantly fewer TUNEL positive cells were observed in siRNA transfected cells than in control cells. Together, our data provide strong evidence to support the notion that Wnt3a is a canonical Wnt with pro-apoptotic action whose cellular activity is prevented by Sfrp2 through, at least in part, the direct binding of these molecules. These results can explain the in vivo protective effect of Sfrp2 and highlight its therapeutic potential for the ischemic heart.
A thorough understanding of quantitative disease resistance (QDR) would contribute to the design and deployment of durably resistant crop cultivars. However, the molecular mechanisms that control QDR remain poorly understood, largely due to the incomplete and inconsistent nature of the resistance phenotype, which is usually conditioned by many loci of small effect. Here, we discuss recent advances in research on QDR. Based on inferences from analyses of the defense response and from the few isolated QDR genes, we suggest several plausible hypotheses for a range of mechanisms underlying QDR. We propose that a new generation of genetic resources, complemented by careful phenotypic analysis, will produce a deeper understanding of plant defense and more effective utilization of natural resistance alleles.
Administration of mesenchymal stem cells (MSCs) is an effective therapy to repair cardiac damage after myocardial infarction (MI) in experimental models. However, the mechanisms of action still need to be elucidated. Our group has recently suggested that MSCs mediate their therapeutic effects primarily via paracrine cytoprotective action. Furthermore, we have shown that MSCs overexpressing Akt1 (Akt-MSCs) exert even greater cytoprotection than unmodified MSCs. So far, little has been reported on the metabolic characteristics of infarcted hearts treated with stem cells. Here, we hypothesize that Akt-MSC administration may influence the metabolic processes involved in cardiac adaptation and repair after MI. MI was performed in rats randomized in four groups: sham group and animals treated with control MSCs, Akt-MSCs, or phosphate-buffered saline (PBS). High energy metabolism and basal 2-deoxy-glucose (2-DG) uptake were evaluated on isolated hearts using phosphorus-31 nuclear magnetic resonance spectroscopy at 72 hours and 2 weeks after MI. Treatment with Akt-MSCs spared phosphocreatine stores and significantly limited the increase in 2-DG uptake in the residual intact myocardium compared with the PBS- or the MSC-treated animals. Furthermore, Akt-MSC-treated hearts had normal pH, whereas low pH was measured in the PBS and MSC groups. Correlative analysis indicated that functional recovery after MI was inversely related to the rate of 2-DG uptake. We conclude that administration of MSCs overexpressing Akt at the time of infarction results in preservation of normal metabolism and pH in the surviving myocardium.
Maize production in sub-Saharan Africa incurs serious losses to epiphytotics of foliar diseases. Quantitative trait loci conditioning partial resistance (rQTL) to infection by causal agents of gray leaf spot (GLS), northern corn leaf blight (NCLB), and maize streak have been reported. Our objectives were to identify simple-sequence repeat (SSR) molecular markers linked to consensus rQTL and one recently identified rQTL associated with GLS, and to determine their suitability as tools for selection of improved host resistance. We conducted evaluations of disease severity phenotypes in separate field nurseries, each containing 410 F2:3 families derived from a cross between maize inbred CML202 (NCLB and maize streak resistant) and VP31 (a GLS-resistant breeding line) that possess complimentary rQTL. F2:3 families were selected for resistance based on genotypic (SSR marker), phenotypic, or combined data and the selected F3:4 families were reevaluated. Phenotypic values associated with SSR markers for consensus rQTL in bins 4.08 for GLS, 5.04 for NCLB, and 1.04 for maize streak significantly reduced disease severity in both generations based on single-factor analysis of variance and marker-interval analysis. These results were consistent with the presence of homozygous resistant parent alleles, except in bin 8.06, where markers were contributed by the NCLB-susceptible parent. Only one marker associated with resistance could be confirmed in bins 2.09 (GLS) and 3.06 (NCLB), illustrating the need for more robust rQTL discovery, fine-mapping, and validation prior to undertaking marker-based selection.
Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewarts bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetles ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.
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