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Supplementary Figure 1 and Table 1 from CXCL13 Is Highly Produced by Sézary Cells and Enhances Their Migratory Ability via a Synergistic Mechanism Involving CCL19 and CCL21 Chemokines

Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the sFLT-1 receptor in a breast cancer animal model. MCF-7 cells, which produce VEGF, were used to establish solid tumors in the mammary fat pads of female nude mice. After six weeks tumors were injected either with Adsflt, or a negative control virus (AdCMV.βgal). After six months, average tumor volume in the Adsflt-infected group (33 ± 22 mm3) was decreased by 91% relative to that of the negative control group (388 ± 94 mm3; Psflt-infected tumors exhibited complete regression. The vascular density in Adsflt-infected tumors was reduced by 50% relative to that of negative controls (Psflt–infected tumors were preceded by apoptosis of tumor vascular endothelial cells. Mice treated with Adsflt intratumorally showed no delay in the healing of cutaneous wounds, providing preliminary evidence that Ad-mediated sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects.

Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Ad

Background Inflammatory bowel disease (IBD) predisposes to colorectal cancer (CRC) with some specific features that distinguish it from sporadic CRC. Magnesium (Mg) homeostasis is severely compromised in IBD patients, which may affect both inflammation and tumor development. Efficient transcellular Mg transport in intestinal cells depends on the transient receptor potential melastatin (TRPM) channels type 6 and 7, but their expression has never been investigated in the context of IBD-related CRC. Aims We sought to study the expression pattern of TRPM6 and TRPM7 in CRC, and to compare IBD-related cases to sporadic cases. Methods TRPM6 and TRPM7 protein expression was evaluated by immunohistochemistry in surgical specimens from 16 IBD and 13 NON-IBD CRC patients. Results TRPM7 expression was higher in tumor tissue than in the adjacent non-neoplastic tissue in both IBD and NON-IBD patients. Overall, adenocarcinomas showed a higher TRPM7 expression than adenomas. TRPM7 expression also positively correlated with tumor grade. Conversely, TRPM6 expression was higher in tumor tissues in both IBD and NON-IBD CRC, but it did not correlate with tumor stage or grade. Conclusions We report a possible participation of TRPM6 and 7 in both IBD-related and sporadic CRC and suggest that TRPM7 might serve as a marker of malignant transformation and lack of differentiation.

The regenerative effects of cardiac ckit+ stem cells (ckit+CSCs) in acute myocardial infarction (MI) have been studied extensively, but how these cells exert a protective effect on cardiomyocytes is not well known. Growing evidences suggest that in adult stem cells injury triggers inflammatory signaling pathways which control tissue repair and regeneration. Aim of the present study was to determine the mechanisms underlying the cardioprotective effects of ckit+CSCs following transplantation in a murine model of MI. Following isolation and in vitro expansion, cardiac ckit+CSCs were subjected to normoxic and hypoxic conditions and assessed at different time points. These cells adapted to hypoxia as showed by the activation of HIF-1α and the expression of a number of genes, such as VEGF, GLUT1, EPO, HKII and, importantly, of alarmin receptors, such as RAGE, P2X7R, TLR2 and TLR4. Activation of these receptors determined an NFkB-dependent inflammatory and reparative gene response (IRR). Importantly, hypoxic ckit+CSCs increased the secretion of the survival growth factors IGF-1 and HGF. To verify whether activation of the IRR in a hypoxic microenvironment could exert a beneficial effect in vivo, autologous ckit+CSCs were transplanted into mouse heart following MI. Interestingly, transplantation of ckit+CSCs lowered apoptotic rates and induced autophagy in the peri-infarct area; further, it reduced hypertrophy and fibrosis and, most importantly, improved cardiac function. ckit+CSCs are able to adapt to a hypoxic environment and activate an inflammatory and reparative response that could account, at least in part, for a protective effect on stressed cardiomyocytes following transplantation in the infarcted heart.

The regenerative effects of cardiac ckit+ stem cells (ckit+CSCs) in acute myocardial infarction (MI) have been studied extensively, but how these cells exert a protective effect on cardiomyocytes is not well known. Growing evidences suggest that in adult stem cells injury triggers inflammatory signaling pathways which control tissue repair and regeneration. Aim of the present study was to determine the mechanisms underlying the cardioprotective effects of ckit+CSCs following transplantation in a murine model of MI. Following isolation and in vitro expansion, cardiac ckit+CSCs were subjected to normoxic and hypoxic conditions and assessed at different time points. These cells adapted to hypoxia as showed by the activation of HIF-1α and the expression of a number of genes, such as VEGF, GLUT1, EPO, HKII and, importantly, of alarmin receptors, such as RAGE, P2X7R, TLR2 and TLR4. Activation of these receptors determined an NFkB-dependent inflammatory and reparative gene response (IRR). Importantly, hypoxic ckit+CSCs increased the secretion of the survival growth factors IGF-1 and HGF. To verify whether activation of the IRR in a hypoxic microenvironment could exert a beneficial effect in vivo, autologous ckit+CSCs were transplanted into mouse heart following MI. Interestingly, transplantation of ckit+CSCs lowered apoptotic rates and induced autophagy in the peri-infarct area; further, it reduced hypertrophy and fibrosis and, most importantly, improved cardiac function. ckit+CSCs are able to adapt to a hypoxic environment and activate an inflammatory and reparative response that could account, at least in part, for a protective effect on stressed cardiomyocytes following transplantation in the infarcted heart.

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., MiR-155 is thought to inhibit PU.1 and thereby drive antigen-induced B-cell maturation. Here the authors show that patients with rheumatoid arthritis have high B-cell miR-155 expression and that an antagomir can rescue PU.1 expression, suggesting potential therapeutic avenues to treat rheumatoid arthritis.

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 + and CD34 + 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 + and CD34 + 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 + cells 3 days after coronary ligation; at this time point, epicardial c-kit + cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit + , 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.

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. Conclusions— HMGB1 plays a role in skeletal muscle regeneration modulating, in an autocrine-paracrine manner, myoblast and endothelial cell functions.

Laminar shear stress (LSS) represents a major athero-protective stimulus. However, the mechanisms for this effect are poorly characterized. As chemokine receptors modulate endothelial cell functions, we hypothesized that at least some LSS effects on endothelial cells (ECs) may be due to LSS-dependent changes in chemokine receptor expression and function. Exposure of Human umbilical vein endothelial cells (HUVECs) to 15 dynes/cm2/sec(-1) LSS strongly inhibited CXC chemokine receptor 4 (CXCR4) expression at the transcriptional level and impaired stromal-derived factor (SDF)-1/CXCL12-driven chemotaxis. On the contrary, low shear stress (SS; 4 dynes/cm2/sec(-1)) only marginally affected CXCR4 expression when compared with static control cells. Differently from CXCR4, the expression of SDF-1 mRNA was not affected by LSS treatment. CXCR4 overexpression induced a dose-dependent endothelial cell apoptosis that was enhanced by SDF-1 treatment and was caspase-dependent. CXCR4 overexpression inhibited the LSS-mediated antiapoptotic effect on ECs and was associated to impairment of LSS-induced ERK1/2 phosphorylation. These findings suggest that LSS-induced CXCR4 down-regulation may contribute to endothelial cell survival. Interestingly, the expression of the proatherogenic chemokines MCP-1 and IL-8 was induced by SDF-1 treatment and by CXCR4 overexpression in HUVECs. Further, the known LSS-induced inhibition of MCP-1 expression was impaired in CXCR4 overexpressing ECs. Finally, CXCR4 was abundantly expressed by human atherosclerotic plaque endothelium that is exposed to low/absent shear stress, while it was poorly expressed by minimally diseased carotid artery endothelium. In conclusion, LSS-dependent CXCR4 down-regulation may contribute to atheroprotection by favoring the integrity of the endothelial barrier and by inhibiting MCP-1 and IL-8 expression.

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.

Several chemokines have been shown to regulate cellular apoptosis following discrete stimuli. It was previously demonstrated that the CC chemokine CCL1 (I-309) rescues thymic lymphoma cells from apoptois by unknown mechanisms. The aim of our study was to characterize the role of the CC chemokine receptor 8 (CCR8), the only described receptor for CCL1, in the rescue of murine thymic lymphoma cells and murine thymocytes from dexamethasone (dex)-induced apoptosis. We show here that the CCR8-restricted agonist Kaposi sarcoma-associated herpesvirus-encoded chemokine viral macrophage-inflammatory protein-1 (vMIP-1) rescues thymic lymphoma cells from dex-induced apoptosis, similar to CCL1, and that such rescue is extracellular-regulated kinase-dependent. Although it has been hypothesized that the rescuing effect of CCL1 from apoptosis could be CCR8-mediated, here, we formally demonstrate the role of such receptor as its selective antagonist encoded by the MC148 gene of molluscum contagiosum virus MC148/vMCC-I inhibits v-MIP-1- and CCL1-induced rescue activity. In addition, CCR8 ligands inhibit dex-induced apoptosis of murine thymocytes with potential implications for thymic selection.

Aims and background Many normal and human cancer tissues express fatty acid synthase (FAS), the major enzyme required for endogenous fatty acid biosynthesis. Strong expression of FAS seems to be associated with a poor prognosis. This study examines the strength of FAS and other common markers of relapse in poorly differentiated breast carcinoma. Materials and methods Fifty-one patients with poorly differentiated ductal infiltrating breast carcinomas were followed up for more than 10 years. Immunohistochemical detection of FAS was associated with morphological features of the tumors, with immunohistochemical expression of c-erbB-2, cathepsin D, estrogen and progesterone receptor status and with DNA ploidy in order to detect a statistical correlation. Results The chi-square test revealed a correlation between FAS and peritumoral lymphatic vessel invasion (PLVI) (P = 0.001). Univariate analysis showed that FAS was correlated with disease-free survival (DFS) (P = 0.0001). Other prognosticators associated with DFS were PLVI (P = 0.002), estrogen (P = 0.008) and progesterone receptor status (P = 0.007). Bivariate analysis showed that FAS was a further prognostic discriminant of DFS within the ER, PgR and PLVI subsets. Discussion FAS is a reliable prognosticator of recurrence in poorly differentiated early breast carcinomas. Association of FAS with PLVI may be useful to plan a correct follow-up in patients with breast neoplasms.

AIMS: HMGB1 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 RESULTS: Adult 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. CONCLUSIONS: HMGB1 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.

AimsAcidification is associated with a variety of pathological and physiological conditions. In the present study, we aimed at investigating whether acidic pH may regulate endothelial cell (EC) functions via the chemokine receptor CXCR4, a key modulator of EC biological activities.Methods and resultsExposure of ECs to acidic pH reversibly inhibited mRNA and protein CXCR4 expression, CXCL12/stromal cell-derived factor (SDF)-1-driven EC chemotaxis in vitro, and CXCR4 expression and activation in vivo in a mouse model. Further, CXCR4 signalling impaired acidosis-induced rescue from apoptosis in ECs. The inhibition of CXCR4 expression occurred transcriptionally and was hypoxia-inducible factor (HIF)-1-dependent as demonstrated by both HIF-1 and HIF-1 dominant negative overexpression, by HIF-1 silencing, and by targeted mutation of the-29 to-25 hypoxia response element (HRE) in the-357/-59 CXCR4 promoter fragment. Moreover, chromatin immunoprecipitation (ChIP) analysis showed endogenous HIF-1 binding to the CXCR4 promoter that was enhanced by acidification.ConclusionThe results of the present study identify CXCR4 as a key player in the EC response to acidic pH and show, for the first time, that HRE may function not only as an effector of hypoxia, but also as an acidosis response element, and raise the possibility that this may constitute a more general mechanism of transcriptional regulation at acidic pH. © The Author 2009. For permissions please.

Stem cells expressing c-kit have been identified in the adult epicardium. In mice, after myocardial infarction, these cells proliferate, migrate to the injury site and differentiate toward myocardial and vascular phenotype. We hypothesized that, acutely after myocardial infarction, pericardial sac integrity and pericardial fluid (PF) may play a role on epicardial cell gene expression, proliferation and differentiation. Microarray analysis indicated that, in the presence of an intact pericardial sac, myocardial infarction modulated 246 genes in epicardial cells most of which were related to cell proliferation, cytoskeletal organization, wound repair and signal transduction. Interestingly, WT1, Tbx18 and RALDH2, notably involved in epicardial embryonic development, were markedly up-regulated. Importantly, coexpression of stem cell antigen c-kit and WT1 and/or Tbx18 was detected by immunohistochemistry in the mouse epicardium during embryogenesis as well as in adult mouse infarcted heart. Injection of human pericardial fluid from patients with acute myocardial ischemia (PFMI) in the pericardial cavity of non-infarcted mouse hearts, enhanced, epicardial cell proliferation and WT1 expression. Further, PFMI supplementation to hypoxic cultured human epicardial c-kit(+) cells increased WT1 and Tbx18 mRNA expression. Finally, insulin-like growth factor 1, hepatocyte growth factor and high mobility group box 1 protein, previously involved in cardiac c-kit(+) cell proliferation and differentiation, were increased in PFMI compared to the pericardial fluid of non ischemic patients. In conclusion, myocardial infarction reactivates an embryonic program in epicardial c-kit(+) cells; soluble factors released in the pericardial fluids following myocardial necrosis may play a role in this process.

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance. [Cancer Res 2008;68(17):7137–46]

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1+progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1+compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

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. © 2008 The Society for Investigative Dermatology.

Objective: Rat α adducin point mutation (F316Y) has been associated with primary systemic arterial hypertension. As microcirculatory abnormalities are present in most forms of hypertension, the aim of the present study was to investigate whether rat α adducin may regulate endothelial cell (EC) functions in vitro and in vivo. Methods and results: The overexpression of rat wild type α adducin (WT-Add1) in ECs induced capillary-like structure development in Matrigel in vitro and enhanced capillary formation in Matrigel implants in vivo in CD1 mice. In contrast, the overexpression of the mutated form (MUT-Add1) of rat α adducin had a Null effect in vitro and lacked any significant activity in vivo. Further, adenovirus-mediated rat WT-Add1 but not MUT-Add1 gene transfer to murine ischemic hindlimb enhanced capillary formation in skeletal muscles. Gene profiling of human umbilical vein endothelial cells overexpressing α adducin was performed in order to identify putative effector molecules of α adducin-mediated activities on ECs. Interestingly, among a number of genes involved in angiogenesis regulation, retinoic acid-induced protein (RAI17) was found to be upregulated in WT-Add1 vs MUT-Add1 overexpressing cells, possibly representing a key molecule/axis for the functional Add1-induced effect. Conclusions: Rat WT α adducin enhanced EC functions both in vitro and in vivo. The expression of the F316Y variant, associated with the hypertensive phenotype, had a Null effect and might contribute to endothelial rarefaction/dysfunction in hypertension. RAI17 was found to be a putative effector molecule differentially regulated by the overexpression of the two forms of Add1 in endothelial cells. © 2007 European Society of Cardiology.

Background MicroRNA-155 (miR-155) has been shown to be a key regulator of B cell biology. The regulation of the transcription factor PU.1 by miR-155 is required for high-affinity IgG1 production and B cell maturation. However, the role of miR-155 in the activation of B in Rheumatoid Arthritis (RA) has not been explored. Objectives To investigate miR-155 expression in RA B cells and its association with B cell activation and synovial inflammation. Methods 53 RA patients underwent synovial (ST) biopsy. ST samples were categorized on a basis of immunostaining for CD68, CD21, CD3 and CD20 cells as diffuse or aggregate pattern. MiR-155 expression in RA ST was evaluated by in situ hybridization (ISH)(n=15). B cells from peripheral blood (PB) and matched synovial fluid (SF) of RA (n=19) and PB of healthy controls (HC)(n=10) were isolated by CD19+ microbeads. B-cell subsets were determined through Flow-Cytometry. IL-6 and BAFF levels in PB and SF were measured by ELISA. MiR-155 and PU.1 expression was determined by qPCR in B cells from PB and SF of RA (n=10) and on ST samples of osteoarthritis (OA)(n=3), RA diffuse (n=8) and RA aggregate (n=8) patients. Finally, HC PB B cells (n=5) were cultured with or without IL-6 (30ng/ml) or BAFF (20ng/ml) and miR-155 and PU.1 expression was assessed by qPCR. Results 23 (43,4%) RA patients had an aggregate pattern of ST biopsy. RA with an aggregate pattern were more likely anti-CCP+ compared to RA with diffuse pattern (p=0.04).Moreover, anti-CCP plasma levels directly correlated with the synovial aggregate grade (r=0.39; p=0.01). IL-6 and BAFF levels were higher in SF than in PB of RA regardless of the synovial infiltrate pattern (p=0.001 for both). ISH showed that miR-155 was expressed at significantly higher levels in ST of aggregate RA compared to diffuse RA (p=0.03). qPCR further confirmed that miR-155 was significantly increased in ST of aggregate RA compared to diffuse RA (p=0.03) and OA (p=0.03) respectively. Consistently, PU.1 staining was found to be lower within synovial lymphoid aggregates in RA patients. On the single cell level, miR-155 was expressed significantly higher in RA PB B-cells compared to HC (p=0.0002). In addition, miR-155 was over-expressed in SF B-cells compared to matched PB B-cells (p=0.05) in RA. Consistently, PU.1 expression was lower in SF B-cells compared to matched PB B-cells (p=0.001). Moreover, anti-CCP+ RA showed higher miR-155 expression in PB B-cells compared to anti-CCP- RA (p=0.02) and HC (p=0.001). CD19+/IgD-CD27- cells were significantly over-represented in PB of aggregate compared to diffuse RA (p=0.04). Finally, IL-6 and BAFF in vitro stimulation of HC B-cells induced miR-155 overexpression (p=0.04 and p=0.03) whereas PU.1 was significantly down-regulated (p=0.01 and p=0.03). Conclusions MiR-155 is over-expressed in B-cells of RA and is associated to anti-CCP positivity, an aggregate synovial pattern and a PU.1 lower expression. IL-6 and BAFF are significantly increased in the SF environment and induce in vitro miR-155 expression in B-cells. Thus, miR-155 may represent a key regulator of B-cells in RA patients with a memory phenotype. Disclosure of Interest None declared

Background and objectives MicroRNAs (miRs) are post-transcriptional regulators implicated in Systemic sclerosis (SSc). MiR-34a and miR-155 are related to endothelial senescence and inflammation. The aim of the study is to investigate the expression of miR-34a and miR-155 in peripheral blood (PB) CD14 cells and in clinically involved and uninvolved skin paired samples in SSc. Materials and methods Twenty-seven patients with Raynaud phenomenon (RP) were enrolled, divided into 3 groups: long standing SSc (lsSSc) fulfilling the 1987 ACR criteria (n = 10), early SSc (eSSc) reaching 9 points using the Very Early Diagnosis of Systemic Sclerosis criteria (n = 9) and primary RP (n = 8) respectively. Matched healthy controls (HC) (n = 7) were enrolled. Immunostaining for CD68 was performed on paired skin tissues from SSc patients. miR-34a and miR-155 expression was evaluated by qPCR on CD14 cells isolated by CD14 specific microbeads from PB and on clinically involved and uninvolved skin paired samples. Using HumanTargetScan cross-referenced methodology, IL-6 receptor (IL6-R) was selected as target of miR-34a and miR-155 and experimentally confirmed by qPCR. IL-6 and IL-6R plasma levels were determined by ELISA. CD14 cells from PB of HC (n = 4) were cultured in RPMI, stimulated with IL-6 (30ng/ml) or LPS (100ng/ml) and collected after 48 h to assess miR-34a and miR-155 expression by qPCR. Results MiR-155 is over-expressed either in eSSc (p = 0.0002) and lsSSc (p = 0.04) compared to HC. MiR-34a expression is increased only in lsSSc compared to HC (p = 0.01). Primary RP patients did not differ for miR-155 and miR-34a expression from HC (p = 0.71). CD68 cells are over-represented in clinically involved skin compared to uninvolved skin paired samples (p = 0.02). MiR-34a is over-expressed in clinically involved skin samples compared to uninvolved skin (p = 0.04). IL-6R expression is significantly lower in lsSSc and eSSc patients compared to primary RP (p = 0.03 and p = 0.03). IL-6 plasma levels were higher in lsSSc (p = 0.01) as well as in eSSc (p = 0.003) compared to HC whereas no significant difference was found in IL-6R plasma levels. MiR-34a expression directly correlates with the skin score value (R = 0.52, p = 0.03) in SSc patients and with IL-6 plasma levels (R = 0.42; p = 0.01). SSc patients with digital ulcers have higher miR-34a expression than SSc patients without ulcers (p = 0.01). Finally, miR-34a and miR-155 are induced in CD14 cells by IL-6 and LPS in vitro stimulation. Conclusions MiR-34a and miR-155 expression is unbalanced in CD14 cells and paired skin samples of SSc patients linked to the IL-6/IL-6R pathway. MiRNA expression profile could help to differentiate patients with primary and SSc associated RP.

Telomere dysfunction contributes to reduced cell viability, altered differentiation, and impaired regenerative/proliferative responses. Recent advances indicate that telomerase activity confers a pro-angiogenic phenotype to endothelial cells and their precursors. We have investigated whether telomerase contributes to tissue regeneration following hind limb ischemia and vascular endothelial growth factor 165 (VEGF(165)) treatment. VEGF delivery induced angiogenesis and increased expression of the telomerase reverse transcriptase (TERT) and telomerase activity in skeletal muscles and satellite and endothelial cells. Adenovirus-mediated transfer of wild type TERT but not of a dominant negative mutant, TERTdn, significantly induced capillary but not arteriole formation. However, when co-delivered with VEGF, TERTdn abrogated VEGF-dependent angiogenesis, arteriogenesis, and blood flow increase. This effect was paralleled by in vitro evidence that telomerase inhibition by 3'-azido-3'-deoxythymidine in VEGF-treated endothelial cells strongly reduced capillary density and promoted apoptosis in the absence of serum. Similar results were obtained with adenovirus-mediated expression of TERTdn and AKTdn, both reducing endogenous TERT activity and angiogenesis on Matrigel. Mechanistically, neo-angiogenesis in our system involved: (i) VEGF-dependent activation of telomerase through the nitric oxide pathway and (ii) telomerase-dependent activation of endothelial cell differentiation and protection from apoptosis. Furthermore, detection of TERT in activated satellite cells identified them as VEGF targets during muscle regeneration. Because TERT behaves as an angiogenic factor and a downstream effector of VEGF signaling, telomerase activity appears required for VEGF-dependent remodeling of ischemic tissue at the capillaries and arterioles level.

Background— The absence of functional dystrophin in Duchenne muscular dystrophy (DMD) patients and in mdx mice results in progressive muscle degeneration associated with necrosis, fibrosis, and inflammation. Because vascular supply plays a key role in tissue repair, we examined whether new blood vessel development was altered in mdx mice. Methods and Results— In a model of hindlimb ischemia on femoral artery dissection, hindlimb perfusion, measured by laser Doppler imaging, was higher in mdx mice (0.67±0.26) than in wild-type (WT) mice (0.33±0.18, P mdx mice (13.6±8.4 mm/mm 3 ) compared with WT mice (7.8±4.6 mm/mm 3 , P mdx mice showed an accelerated wound closure rate compared with WT mice. Moreover, a vascularization assay in Matrigel plugs containing basic fibroblast growth factor injected subcutaneously revealed an increased length density of arterioles in mdx (46.9±14.7 mm/mm 3 ) versus WT mice (19.5±5.8 mm/mm 3 , P mdx mice sustained formation of endothelium-derived tubular structures in vitro more efficiently than WT serum. Conclusions— These results demonstrate that arteriogenesis is enhanced in mdx mice both after ischemia and skin wounding and in response to growth factors.

Objective: The antiapoptotic effect of p21Waf1/Cip1/Sdi1 (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia. Methods: In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment. Results: At T0, HUVEC were either exposed to SS (15 dyn/cm2/s−1), treated with SNP or kept in static condition (ST) for 1–12 h; after additional 12 h in ST, 30–35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia. Conclusions: The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow.

We evaluated the kinetics of transgene expression and humoral and cellular immune responses against viral antigens and the product of the reporter gene LacZ in young (4 months) and old (20 months) Wistar rats. Animals received the intramuscular injection of a recombinant E1-deleted human type 5 adenovirus encoding beta-gal (Ad-LacZ) on days 0 and 30. The transgene expression evaluated on day 2 after infection revealed a significantly higher beta-gal activity in young than in old animals (1.9-fold increase, p

Abstract —The effect of retinoic acid (RA) on endothelial cells is still controversial and was examined in the present study. In bovine aortic endothelial cells (BAECs), all-trans RA (ATRA) and 9- cis RA (9CRA), but not 13- cis RA (13CRA), induced fibroblast growth factor-2 (FGF-2) production and exhibited a biphasic dose-dependent effect to enhance BAEC proliferation and differentiation into tubular structures on reconstituted basement membrane proteins (Matrigel); both processes were inhibited by FGF-2–neutralizing antibody. The pan RA receptor (RAR)-selective ligand (E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl)-1-propenyl] benzoic acid and the RARα-selective ligand 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid stimulated the production of FGF-2, whereas the addition of the RARα-antagonist RO 41-5253 inhibited this effect. In BAECs, the forced expression of RARα, but not RARβ or RARγ, enhanced FGF-2 production, whereas the RARα-dominant negative, Δ403, blocked this effect. Furthermore, RARα overexpression directly stimulated BAEC differentiation on Matrigel and potentiated the effects of ATRA in this assay. Finally, ATRA-treated BAECs coinjected with Matrigel subcutaneously in mice induced neovascularization within the Matrigel plug, and ATRA also enhanced angiogenesis in the chicken chorioallantoic membrane assay. In conclusion, RA can stimulate endothelial cell proliferation and differentiation in vitro via enhanced RARα-dependent FGF-2 production, and it can also induce angiogenesis in vivo. The full text of this article is available at http://www.circresaha.org.

Patients with secondary hyperparathyroidism following chronic renal disease frequently develop hyperplastic parathyroids. Hyperplastic parathyroids have an increased number of chief cells, a decreased amount of stromal fat, and a nodular or diffuse histologic pattern. Hyperplastic parathyroids may also express higher proliferative activity compared with controls. We evaluated the morphologic features and immunohistochemical expression of fatty acid synthase (FAS), Ki67, proliferating cell nuclear antigen, and p53 protein in 78 hyperplastic parathyroids from 20 patients with secondary hyperparathyroidism. Twenty normal parathyroids incidentally removed during nonneoplastic thyroid surgery were used as controls. Our results showed that hyperplastic glands overexpress FAS ( P = .06). Statistical analysis also revealed a significant association between FAS and p53 protein ( P = .006) and between FAS and hyperplastic glands with a predominant nodular pattern ( P = .02). Hyperplastic parathyroids from patients with chronic renal failure strongly express FAS. Fatty acid synthase may therefore be a potential biological indicator of highly proliferating parathyroid cells.

BACKGROUND Fatty acid synthase (FAS) is a molecule found in tumor cells from breast carcinomas of patients whose prognosis is very poor. Recently, this molecule has been identified as the key enzyme in fatty acid biosynthesis. This study was done to test the strength of FAS as a prognostic indicator for disease free survival (DFS) and overall survival (OS). METHODS Clinical records, histologic features, immunohistochemical expression of cathepsin D and c-erbB-2, and estrogen and progesterone receptor status of 110 Stage I breast carcinoma patients were all associated with FAS by a chi-square test. The patterns of DFS and OS were estimated over a ten-year follow-up period using the Kaplan-Meier method. Univariate and multivariate analysis were evaluated using a log logistic regression model. Multivariate regression analysis was based on the Cox proportional hazard model. To detect FAS, cathepsin D and c-erbB-2 expression as well as estrogen and progesterone receptor status, we used the unlabeled immunoperoxidase technique on formalin fixed, paraffin embedded tissue. RESULTS FAS was significantly associated with a higher risk of recurrence because it predicted both DFS (P = 0.0001) and OS (P = 0.003) when evaluated as a continuous variable and DFS (P = 0.0001) when evaluated with other prognostic markers. Peritumoral lymphatic vessel invasion was the other most significant independent predictor for DFS (P = 0.001) and OS (P = 0.003). CONCLUSIONS FAS is a reliable prognostic marker to predict DFS and OS in patients with early breast cancer.

Background Reported data indicate that cancer cells have increased rates of glucose metabolism, as determined by 18FDG-PET imaging in patients with malignancies. The results of many studies have demonstrated that the expression of glucose transporters, especially Glut-1, is increased in a variety of malignancies. This study was undertaken to assess the differential expression of Glut-1 and Glut-3 by benign and malignant melanocytic lesions. Methods Immunohistochemical staining for Glut-1 and Glut-3 was performed on paraffin-embedded tissue sections prepared from melanocytic nevi (12 cases), Spitz nevi (12 cases) and primary cutaneous malignant melanomas (20 cases). Results We observed immunoreactivity for Glut-1 in all melanocytic nevi, 9 of the 12 Spitz nevi and in 9 of the 20 malignant melanomas, whereas Glut-3 was expressed in all the melanocytic lesions, both benign and malignant. Conclusion These findings indicate that the glucose transporters Glut-1 and Glut-3 play a role in the glucose metabolism of melanocytic cells. Glut-1 was present in the majority of benign nevi, whereas its expression was downregulated in 55% of malignant melanomas. Our results suggest that glucose transporter Glut-1 expression can significantly discriminate between human malignant melanoma and benign melanocytic nevi, and support the idea that additional mechanisms other than Glut-1 may contribute to glucose uptake in melanomas.