Monica Napolitano

Analysis of the role of chemokines in angiogenesis.

Chemokines, a large family of inflammatory cytokines, have been shown to play a critical role in the regulation of angiogenesis during several pathophysiologic processes, such as tumor growth, wound healing and ischemia. Semiquantitative or quantitative angiogenesis assays are commonly utilized to screen the angiogenic or angiostatic activity of chemokines. These include in vitro endothelial cell activation assays and ex vivo or in vivo models of neovascularization. Chemokines may exert their regulatory activity on angiogenesis directly or as a consequence of leukocyte infiltration and/or the induction of growth factor expression. The effect of chemokines on endothelium can be assessed by performing in vitro assays on purified endothelial cell populations or by in vivo assays. Nevertheless, each model used to evaluate the angiogenic or angiostatic activity of a discrete factor has advantages and limitations. Thus, in order to avoid under- or overestimating the regulatory effect of chemokines on angiogenesis and to evaluate all aspects of the angiogenic process, multiple assays are usually performed. This review summarizes past and recent studies on chemokines as modulators of angiogenesis with particular emphasis on the methods currently used for the assessment of chemokine-mediated angiogenic or angiostatic responses.

Identification of the CC chemokines TARC and macrophage inflammatory protein-1β as novel functional ligands for the CCR8 receptor

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I‐309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation‐regulated cytokine (TARC) and macrophage inflammatory protein‐1β (MIP‐1β) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.

Adenovirus-mediated VEGF 165 gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice

It has been previously shown that vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis during wound repair and that healing-impaired diabetic mice show decreased VEGF expression levels. In order to investigate the potential benefits of gene therapy with growth factors on wound repair, a replication-deficient recombinant adenovirus vector carrying the human VEGF165 gene (AdCMV.VEGF165) was topically applied on excisional wounds of streptozotocin-induced diabetic mice. Treatment with AdCMV.VEGF165 significantly accelerated wound closure when compared with AdCMV.LacZ-treated, as well as saline-treated control mice, by promoting angiogenesis at the site of injury. Our findings suggest that AdCMV.VEGF165 may be regarded as a therapeutic tool for the treatment of diabetic ulcers.

MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients

MicroRNAs (miRNAs) are noncoding RNAs that act as negative regulators of gene expression. Interestingly, specific alterations of miRNA expression have been found in failing hearts of different etiologies. The aim of this study was to identify the miRNA expression pattern of peripheral blood mononuclear cells (PBMCs) derived from chronic heart failure (CHF) patients affected by ischemic (ICM) and nonischemic dilated (NIDCM) cardiomyopathy. The expression profile of 257 miRNAs was assessed in 7 NIDCM patients, 8 ICM patients, and 9 control subjects by quantitative real-time PCR. Significantly modulated miRNAs were validated by using an independent set of 34 CHF patients (NIDCM = 19, ICM = 15) and 19 control subjects. Three miRNAs (miR-107, -139, and -142-5p) were downmodulated in both NIDCM and ICM patients versus control subjects. Other miRNAs were deregulated in only one of the CHF classes analyzed compared with control subjects: miR-142-3p and -29b were increased in NIDCM patients, while miR-125b and -497 were decreased in ICM patients. Bioinformatic analysis of miRNA predicted targets and of gene expression modifications associated with CHF in PBMCs indicated a significant impact of the miRNA signature on the transcriptome. Furthermore, miRNAs of both the NIDCM and the ICM signature shared predicted targets among CHF-modulated genes, suggesting potential additive or synergistic effects. The present study identified miRNAs specifically modulated in the PBMCs of NIDCM and ICM patients. Intriguingly, most of these miRNAs were previously reported as deregulated in human and/or mouse failing hearts. The identified miRNAs might have a potential diagnostic and/or prognostic use in CHF.

The SDF-1/CXCR4 axis in stem cell preconditioning

We review the pivotal role of the stromal derived factor (SDF)-1 chemokine in tissue ischaemia and how it orchestrates the rapid revascularization of injured, ischaemic, and regenerating tissues via the CXC chemokine receptors CXCR4 and CXCR7. Furthermore, we discuss the effects of preconditioning (PC), which is a well-known protective phenomenon for tissue ischaemia. The positive effect of both hypoxic and acidic PC on progenitor cell therapeutic potential is reviewed, while stressing the role of the SDF-1/CXCR4 axis in this process.

Enhanced Arteriogenesis and Wound Repair in Dystrophin-Deficient mdx Mice

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<0.03). In keeping with these data, a significant increase in arteriole length density was found in mdx mice (13.6±8.4 mm/mm3) compared with WT mice (7.8±4.6 mm/mm3, P<0.03). Conversely, no difference was observed in capillary density between mice of the 2 genotypes. The enhanced regenerative response was not limited to ischemic skeletal muscle, because in a wound-healing assay, 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/mm3) versus WT mice (19.5±5.8 mm/mm3, P<0.001). Finally, serum derived from 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.

Laminar shear stress inhibits CXCR4 expression on endothelial cells: functional consequences for atherogenesis

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−1LSS 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.

High mobility group box 1 is a novel substrate of dipeptidyl peptidase-IV.

Aims/hypothesisHigh mobility group box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. Previous studies have shown that topical application of HMGB1 to skin wounds of mouse models of diabetes enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. Dipeptidyl peptidase IV (DPP-IV/CD26) is a protease whose activity is increased in diabetes and whose inhibition improves glucose tolerance. Since HMGB1 contains potential DPP-IV cleavage sites, we determined whether HMGB1 may be a substrate for DPP-IV and whether DPP-IV-mediated cleavage may alter the biological activity of HMGB1.MethodsReversed phase HPLC, mass spectrometry and western blot analyses were performed to analyse and identify HMGB1 peptides generated following DPP-IV digestion. HMGB1 angiogenic functions in the presence of DPP-IV were evaluated in vitro and in vivo. HMGB1 protein was detected in the serum of type 2 diabetic patients before and after treatment with DPP-IV inhibitors.ResultsDPP-IV cleaved HMGB1 at its N-terminal region and affected its angiogenic functions. Specifically, DPP-IV inhibited HMGB1-induced endothelial cell migration and capillary-like structure formation, as well as HMGB1-mediated vascular network formation in Matrigel implants in mice. We had previously found that HMGB1 promoted endothelial cell migration through activation of extracellular regulated kinase signalling pathway. Here we showed that such an effect was abolished in the presence of DPP-IV. Finally, the N-terminal truncated form of HMGB1 was detected in the serum of type 2 diabetic patients, in whom DPP-IV inhibitors enhanced the levels of full-length HMGB1.Conclusions/interpretationDPP-IV cleaves HMGB1 and, via this mechanism, inhibits HMGB1 angiogenic activity. Treatment with DPP-IV inhibitors may enhance HMGB1 activity in diabetic patients, thereby improving angiogenesis in this condition.

Gene expression profiles in peripheral blood mononuclear cells of chronic heart failure patients

The present study was aimed at identifying chronic heart failure (CHF) biomarkers from peripheral blood mononuclear cells (PBMCs) in patients with ischemic (ICM) and nonischemic dilated (NIDCM) cardiomyopathy. PBMC gene expression profiling was performed by Affymetrix in two patient groups, 1) ICM (n = 12) and 2) NIDCM (n = 12) New York Heart Association (NYHA) III/IV CHF patients, vs. 3) age- and sex-matched control subjects (n = 12). Extracted RNAs were then pooled and hybridized to a total of 11 microarrays. Gene ontology (GO) analysis separated gene profiling into functional classes. Prediction analysis of microarrays (PAM) and significance analysis of microarrays (SAM) were utilized in order to identify a molecular signature. Candidate markers were validated by quantitative real-time polymerase chain reaction. We identified a gene expression profiling that distinguished between CHF patients and control subjects. Interestingly, among the set of genes constituting the signature, chemokine receptor (CCR2, CX(3)CR1) and early growth response (EGR1, 2, 3) family members were found to be upregulated in CHF patients vs. control subjects and to be part of a gene network. Such findings were strengthened by the analysis of an additional 26 CHF patients (n = 14 ICM and n = 12 NIDCM), which yielded similar results. The present study represents the first large-scale gene expression analysis of CHF patient PBMCs that identified a molecular signature of CHF and putative biomarkers of CHF, i.e., chemokine receptor and EGR family members. Furthermore, EGR1 expression levels can discriminate between ICM and NIDCM CHF patients.

Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor‐1 (SDF‐1) plays a key role in bone marrow (BM) c‐kit+ stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM‐derived c‐kit+ cells and on their response to SDF‐1. Acute hindlimb ischemia was induced in streptozotocin‐treated DM and control mice; circulating c‐kit+ cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c‐kit+ cells as well as SDF‐1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM‐derived c‐kit+ cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF‐1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF‐1 ability to induce differentiation of c‐kit+ cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c‐kit+ cells from normoglycaemic mice failed to differentiate in response to SDF‐1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c‐kit+ cell number following hindlimb ischemia and inhibits SDF‐1‐mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM‐derived c‐kit+ cells.