Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5-fold. Finally, adenovirus-mediated VEGF 165 gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia.

Abstract-During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit ϩ and CD34 ϩ cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit ϩ and CD34 ϩ cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit ϩ cells 3 days after coronary ligation; at this time point, epicardial c-kit ϩ cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit ϩ , together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage. Key Words: epicardium Ⅲ infarction Ⅲ stem cells Ⅲ cardiovascular differentiation M yocardial infarction (MI) in the mammalian heart is associated with an acute inflammatory response, leading to the replacement of injured cardiomyocytes with granulation tissue and scar. 1 Recently it has been shown that the heart is a dynamic organ in which spontaneous myocyte regeneration together with myocyte death are major determinants of cardiac homeostasis in physiologic and pathologic conditions. 2 Myocardial regeneration appears to be mediated by multipotent cardiac stem cells (CSCs), resident in the heart, that give rise to new myocytes and vascular structures. A variety of studies document the presence of CSCs in the mouse, 3-10 rat, 11 dog, 12 and human adult heart. 7,13 The adult myocardium is enveloped by a layer of epithelial cells called epicardium that during embryogenesis, plays an important role in the formation of the coronary vasculature. The epicardium has an extracardiac origin: at approximately stage 18 in the avian heart and 10.5 days post coitum in the mouse, a cluster of cells derived from septum transversum in mammals and located close to the liver primordium in other vertebrates, populates the myocardial external surface of the heart. 14 Epicardial cells synthesize a dense layer of extracellular matrix that resides between them and the myocardium in the subepicardial space. A subset of these cells delaminate from the epicardium a...

FAS is a reliable prognostic marker to predict DFS and OS in patients with early breast cancer.

High-mobility group box 1 (HMGB1) protein is a multifunctional cytokine involved in inflammatory responses and tissue repair. In this study, it was examined whether HMGB1 plays a role in skin wound repair both in normoglycemic and diabetic mice. HMGB1 was detected in the nucleus of skin cells, and accumulated in the cytoplasm of epidermal cells in the wounded skin. Diabetic human and mouse skin showed more reduced HMGB1 levels than their normoglycemic counterparts. Topical application of HMGB1 to the wounds of diabetic mice enhanced arteriole density, granulation tissue deposition, and accelerated wound healing. In contrast, HMGB1 had no effect in normoglycemic mouse skin wounds, where endogenous HMGB1 levels may be adequate for optimal wound closure. Accordingly, inhibition of endogenous HMGB1 impaired wound healing in normal mice but had no effect in diabetic mice. Finally, HMGB1 had a chemotactic effect on skin fibroblasts and keratinoyctes in vitro. In conclusion, lower HMGB1 levels in diabetic skin may play an important role in impaired wound healing and this defect may be overcome by the topical application of HMGB1.

No abstract

AimsHMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts.Methods and ResultsAdult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit+ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20–25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206.ConclusionsHMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.

MicroRNA-155 (miR-155) is an important regulator of B cells in mice. B cells have a critical role in the pathogenesis of rheumatoid arthritis (RA). Here we show that miR-155 is highly expressed in peripheral blood B cells from RA patients compared with healthy individuals, particularly in the IgD-CD27- memory B-cell population in ACPA+ RA. MiR-155 is highly expressed in RA B cells from patients with synovial tissue containing ectopic germinal centres compared with diffuse synovial tissue. MiR-155 expression is associated reciprocally with lower expression of PU.1 at B-cell level in the synovial compartment. Stimulation of healthy donor B cells with CD40L, anti-IgM, IL-21, CpG, IFN-α, IL-6 or BAFF induces miR-155 and decreases PU.1 expression. Finally, inhibition of endogenous miR-155 in B cells of RA patients restores PU.1 and reduces production of antibodies. Our data suggest that miR-155 is an important regulator of B-cell activation in RA.

Objective-High mobility group box 1 protein (HMGB1) is a cytokine released by necrotic and inflammatory cells in response to injury. We examined the role of HMGB1 in skeletal muscle regeneration after hindlimb ischemia. Methods and Results-Unilateral hindlimb ischemia was induced in mice by femoral artery dissection. HMGB1 levels increased in regenerating skeletal muscle and the blockade of endogenous HMGB1 by the administration of its truncated form, the BoxA, resulted in the reduction of vessel density. In contrast, intramuscular administration of HMGB1 enhanced perfusion and increased the number of regenerating fibers. To separately study the myogenic and the angiogenic effects of HMGB1, in vitro experiments were performed with isolated myoblasts and endothelial cells.Myoblasts were found to express the HMGB1 receptor RAGE and TLR4 which were downregulated during in vitro myogenic differentiation. HMGB1 was extracellularly released by differentiated myoblasts and exerted a chemotactic activity on myogenic cells. This effect was partially dependent on RAGE and was inhibited by BoxA treatment. Finally, HMGB1 stimulated tubular-like structure formation by endothelial cells through the activation of extracellular signal-regulated kinase (ERK) and JNK signal transduction pathways. Key Words: hindlimb ischemia Ⅲ HMGB1 Ⅲ cytokines Ⅲ regeneration S keletal muscle regeneration, which occurs after tissue damage, is a complex multistep process leading to the formation of myotubes and to the restoration of the vascular network. 1 Muscle injury triggers signals important for neutrophil and macrophage chemoattraction in the damaged tissue. Further, inflammatory cells remove cellular debris and produce growth factors and cytokines which orchestrate the regenerative process activating muscle satellite cells to proliferate and differentiate into myotubes. 2 High Mobility group box 1 protein (HMGB1) is a multifunctional ubiquitous protein of 215 aminoacids mostly located in the nucleus where it bends DNA regulating physical interaction between transcription factors and chromatin. 3 In addition to its nuclear role, HMGB1 is an extracellular mediator of the immune response to infection and injury. HMGB1 reaches the external milieu either by passive release from necrotic cells or active secretion by immune cells stimulated with cytokines and bacterial endotoxins (eg, Lipopolysaccharide [LPS]). 4 -7 The extracellular protein signals tissue injury and evokes inflammatory response inducing the release of a variety of proinflammatory cytokines by monocytes, neutrophils, and dentritic cells. 8 Moreover, HMGB1 exhibits a chemotactic effect on smooth muscle cells, 9 stem cells of vascular origin 10,11 and endothelial precursor cells [12][13][14] . Further, it induces endothelial cell sprouting in vitro 12-14 and myogenic differentiation of rat L6 myoblasts. 15 We recently demonstrated that HMGB1 is a potent regenerative cytokine: in fact, its delivery in infarcted mouse hearts induces cardiac stem cell activation and differentiati...