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Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-[beta]1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy., Introduction Proper wound healing depends on effective hemostasis, which allows regeneration and reconstitution of defective tissue function (1). Coronary thrombosis causing myocardial infarction (MI) (2) leads to irreversible tissue injury, [...]

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1[alpha] and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-[gamma] signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells., Introduction Myeloid-derived suppressor cells (MDSCs) with potent immune-suppressive activity are widely implicated in negative regulation of immune responses in many pathological conditions including cancer, chronic inflammation, infections, autoimmune diseases, and [...]

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea- induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage- dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder., Introduction Platelets are small, anuclear blood cells that are essential for blood clotting and the maintenance of vascular integrity (1, 2). They are produced by megakaryocytes, large polyploid precursor cells [...]

Thrombocytopenia, an abnormally low number of circulating platelets, results from inadequate platelet production, splenic platelet sequestration, or accelerated platelet clearance. Platelet transfusions are now the cornerstone for treating thrombocytopenia. With an ever-expanding demand for platelets, and with many patients having an inadequate response to platelet transfusions, new strategies are needed to treat thrombocytopenia. In this issue of the JCI, Fuentes et al. present provocative data regarding the use of direct megakaryocyte infusions as a novel approach to manage this vexing clinical problem., It was only 100 years ago that James Wright reported that his modification of the Romanowsky stain could unequivocally identify platelets on a peripheral blood smear (1). Applying his new [...]

Magnetic resonance imaging (MRI) can track progenitor cells following direct intramyocardial injection. However, in the vast majority of post-myocardial infarction (MI) clinical trials, cells are delivered by the intracoronary (IC) route, which results in far greater dispersion within the myocardium. Therefore, we assessed whether the more diffuse distribution of cells following IC delivery could be imaged longitudinally with MRI. In 11 pigs (7 active, 4 controls), MI was induced by 90-min balloon occlusion of the left anterior descending coronary artery. Seven (0) days [median (interquartile range)] following Ml, bone marrow progenitor cells (BMCs) were colabeled with an iron-fluorophore and a cell viability marker and delivered to the left anterior descending coronary artery distal to an inflated over-the-wire percutaneous transluminal coronary angioplasty balloon. [T2.sup.*]-weighted images were used to assess the location of the magnetically labeled cells over a 6-wk period post-MI. Immediately following cell delivery, hypointensity characteristic of the magnetic label was observed in the infarct border rather than within the infarct itself. At 6 wk, the cell signal hypointensity persisted, albeit with significantly decreased intensity. BMC delivery resulted in significant improvement in infarct volume and ejection fraction (EF): infarct volume in cell-treated animals decreased from 7.1 [+ or -] 1.5 to 4.9 [+ or -] 1.0 ml (P < 0.01); infarct volume in controls was virtually unchanged at 4.64 [+ or -] 2.1 to 4.39 [+ or -] 2.1 ml (P = 0.7). EF in cell-treated animals went from 30.4 [+ or -] 5.2% preinjection to 34.5 [+ or -] 2.5% 6 wk postinjection (P = 0.013); EF in control animals went from 34.3 [+ or -] 4.7 to 31.9 [+ or -] 6.8% (P = 0.5). Immunohistochemical analysis revealed intracellular colocalization of the iron fluorophore and cell viability dye with the labeled cells continuing to express the same surface markers as at baseline. MRI can track the persistence and distribution of magnetically labeled BMCs over a 6-wk period following IC delivery. Signal hypointensity declines with time, particularly in the first week following delivery. These cells maintain their original phenotype during this time course. Delivery of these cells appears safe and results in improvement in infarct size and left ventricular ejection fraction. magnetic resonance imaging; myocardial infarction doi: 10.1152/ajpheart.01260.2008.

The bone marrow compartment is enriched in stem and progenitor cells, and an unidentified subpopulation of these cells can contribute to lung epithelial repair. Here we identify this subpopulation and quantitate its relative contribution to injured airway epithelium. A subpopulation of adherent human and murine bone marrow cells that expresses Clara cell secretory protein (CCSP) was identified using flow cytometry. When cultured at the air-liquid interface in ex vivo cultures, [Ccsp.sup.+] cells expressed type I and type II alveolar markers as well as basal cell markers and active epithelial sodium channels. [Ccsp.sup.+] cells preferentially homed to naphthalene-damaged airways when delivered transtracheally or intravenously, with the former being more efficient than the latter. Interestingly, naphthalene-induced lung damage transiently increased Ccsp expression in bone marrow and peripheral circulation. Furthermore, lethally irradiated Ccsp-null mice that received tagged wild-type bone marrow contained donor-derived epithelium in both normal and naphthalene-damaged airways. This study therefore identifies what we believe to be a newly discovered cell in the bone marrow that might have airway reconstitution potential in the context of cell-based therapies for lung disease. Additionally, these data could reconcile previous controversies regarding the contribution of bone marrow to lung regeneration., Introduction Adult bone marrow contains a variety of stem cell populations that can be recruited to injured tissues. Although controversial, studies have suggested the possible involvement of bone marrow--derived hematopoietic, [...]

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF. hsa-mir199a | hsa-mir346 | stem cells | plasticity

Recently, parathyroid hormone (PTH) was shown to support survival of progenitor cells in bone marrow. The release of progenitor cells occurs in physiological and pathological conditions and was shown to contribute to neovascularization in tumors and ischemic tissues. In the present study we sought to investigate prospectively the effect of primary hyperparathyroidism (PHPT) on mobilization of bone marrow-derived progenitor cells. In 22 patients with PHPT and 10 controls, defined subpopulations of circulating bone marrow-derived progenitor cells (BMCs) were analyzed by flow cytometry ([CD45.sup.+]/ [CD34.sup.+]/[CD31.sup.+] cells indicating endothelial progenitor cells, [CD45.sup.+]/[CD34.sup.+]/c-[kit.sup.+] cells indicating hematopoietic stem cells, and [CD45.sup.+]/[CD34.sup.+]/[CXCR4.sup.+] cells indicating progenitor cells with the homing receptor CXCR4). Cytokine serum levels (SCF, SDF-1, VEGF, EPO, and G-CSF) were assessed using ELISA. Levels of PTH and thyroid hormone as well as serum electrolytes, renal and liver parameters, and blood count were analyzed. Our data show for the first time a significant increase of circulating BMCs and an upregulation of SDF-1 and VEGF serum levels in patients with PHPT. The number of circulating BMCs returned to control levels measured 16.7 [+ or -] 2.3 mo after surgery. There was a positive correlation of PTH levels with the number of [CD45.sup.+]/[CD34.sup.+]/[CD31.sup.+], [CD45.sup.+]/[CD34.sup.+]/c-[kit.sup.+], and CD[45.sup.+]/CD[34.sup.+]/CXCR[4.sup.+] cells. However, there was no correlation between cytokine serum concentrations (SDF-1, VEGF) and circulating BMCs. Serum levels of G-CSF, EPO, and SCF known to mobilize BMCs were even decreased or remained unchanged, suggesting a direct effect of PTH on stem cell mobilization. Our data suggest a new function of PTH mobilizing BMCS into peripheral blood. parathyroid hormone; mobilization; cytokines

Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). Hydrogen peroxide ([H.sub.2][O.sub.2]) has been shown to increase VEGF expression and to be involved in angiogenesis. We tested the hypothesis that pretreatment with [H.sub.2][O.sub.2] enhances the efficacy of BMCs for neovascularization. [H.sub.2][O.sub.2] pretreatment was done by incubating mouse BMCs in 5 [micro]M [H.sub.2][O.sub.2] for 30 rain, followed by washing twice with PBS. The [H.sub.2][O.sub.2]-pretreated and untreated BMCs were then studied in vitro and in vivo. RT-PCR analysis showed that expression of VEGF and Flk-1 mRNA was significantly higher in [H.sub.2][O.sub.2]-pretreated BMCs than in untreated BMCs after 12 and 24 h of culture (P < 0.01). Pretreatment with [H.sub.2][O.sub.2] also effectively enhanced the VEGF production and endothelial differentiation from BMCs after 1 and 7 days of culture (P < 0.05). To estimate the angiogenic potency in vivo, [H.sub.2][O.sub.2]-pretreated or untreated BMCs were intramuscularly implanted into the ischemic hindlimbs of mice. After 14 days of treatment, many of the [H.sub.2][O.sub.2]-pretreated BMCs were viable, showed endothelial differentiation, and were incorporated in microvessels. Conversely, the survival and incorporation of the untreated BMCs were relatively poor. Microvessel density and blood flow in the ischemic hindlimbs were significantly greater in the mice implanted with [H.sub.2][O.sub.2]-pretreated BMCs than in those implanted with untreated BMCs (P < 0.05). These results show that the short-term pretreatment of BMCs with low-dose [H.sub.2][O.sub.2] is a novel, simple, and feasible method of enhancing their angiogenic potency. cell therapy; reactive oxygen species; differentiation doi:10.1152/ajpheart.00786.2006

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6-8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ~90 rain of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H. shock; bone marrow cells

The Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial was conducted to determine whether intracoronary infusion of enriched bone marrow (BMC) is associated with improved global left ventricular function in patients with myocardial infarction treated with state-of-the-art methods. Results show that after acute myocardial infarction, the intracoronary administration of BMC enhances recovery of left ventricular contractile function.

A randomized, controlled trial, known as the Autologous Stem-Cell Transplantation in Acute Myocardial Infarction (ASTAMI) study, was conducted to investigate whether intracoronary injection of autologous mononuclear bone marrow (BMC) results in a clinically important improvement in left ventricular function as measured by the left ventricular ejection factor (LVEF). Results showed no effects of intracoronary injection of autologous mononuclear BMC on global left ventricular function.

Byline: Cora K Ogle (1,2), John F Valente (1,2), Xialing Guo (2), Bing-Guo Li (2), James D. Ogle (2), J. Wesley Alexander (1,2) Abstract: The normal course of hematopoiesis is controlled by growth factors and cytokines and, therefore, should be susceptible to alterations induced by systemic mediator release such as that seen following thermal injury. We hypothesized that a brief exposure of developing macrophages to the postthermal injury state would result in functionally altered progeny. We measured the production of inflammatory mediators by rat, bone-marrow macrophage precursors harvested 24 h following a 30% TBSA burn after subsequent maturation in a controlled, in vitro environment. Interleukin (IL)-6, tumor necrosis factor (TNF), and prostaglandin (PG) E.sub.2 levels in response to 24 h stimulation with lipopolysaccharide (LPS) were measured following 4 or 8 days of incubation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or both. Flow cytometric analysis showed that bone marrow cells harvested from burn and sham animals cultured in GM-CSF developed principally into macrophages (His48.sup.-, R21A6A.sup.+, CD11b.sup.+. Unstimulated cells produced negligent levels of cytokines and PGE.sub.2. Stimulated burn-derived cells released greater amounts of IL-6 and TNF at 4 or 8 days of culture depending on the conditions. Elevated PGE.sub.2 release was noted in all GM-CSF containing cultures, with burn-derived cells showing a trend towards reduced prostaglandin release. Detection of mRNA for cytokines after LPS stimulation showed no change in IL-6 or TNF transcripts. A short exposure to the systemic effects of thermal injury preprogramed macrophage progenitor cells with the propensity to develop into inflammatory macrophages, secreting higher levels of TNF and IL-6. This shift towards proinflammatory functions in these cells suggests they could be a source of enhanced inflammatory mediator release at 4 or more days post thermal injury. Author Affiliation: (1) Department of Surgery, University of Cincinnati, Cincinnati, Ohio (2) Shriners Burns Institute, 3229 Burnet Avenue, Cincinnati, Ohio, 45229 Article History: Registration Date: 10/09/2004

Byline: S. Joshi, P. Sunita, C. Deshmukh, S. Gujral, P. Amre, C. Nair Background: Imatinib mesylate has shown promising results in chronic myeloid leukemia (CML) in all phases. This drug [...]

Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells. Shinoka T, Matsumura G, Hibino N, Naito Y, Watanabe M, Konuma T, Sakamoto T, Nagatsu M, Kurosawa [...]