Semaphorin 4A exerts a proangiogenic effect by enhancing vascular endothelial growth factor-A expression in macrophages.

The axon guidance cues semaphorins (Semas) and their receptors plexins have been shown to regulate both physiological and pathological angiogenesis. Sema4A plays an important role in the immune system by inducing T cell activation, but to date, the role of Sema4A in regulating the function of macrophages during the angiogenic and inflammatory processes remains unclear. In this study, we show that macrophage activation by TLR ligands LPS and polyinosinic-polycytidylic acid induced a time-dependent increase of Sema4A and its receptors PlexinB2 and PlexinD1. Moreover, in a thioglycollate-induced peritonitis mouse model, Sema4A was detected in circulating Ly6C(high) inflammatory monocytes and peritoneal macrophages. Acting via PlexinD1, exogenous Sema4A strongly increased macrophage migration. Of note, Sema4A-activated PlexinD1 enhanced the expression of vascular endothelial growth factor-A, but not of inflammatory chemokines. Sema4A-stimulated macrophages were able to activate vascular endothelial growth factor receptor-2 and the PI3K/serine/threonine kinase Akt pathway in endothelial cells and to sustain their migration and in vivo angiogenesis. Remarkably, in an in vivo cardiac ischemia/reperfusion mouse model, Sema4A was highly expressed in macrophages recruited at the injured area. We conclude that Sema4A activates a specialized and restricted genetic program in macrophages able to sustain angiogenesis and participates in their recruitment and activation in inflammatory injuries.

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies.

BACKGROUND AIMS: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS: The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS: Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS: Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.

Histone deacetylase inhibition enhances self renewal and cardioprotection by human cord blood-derived CD34 cells.

BACKGROUND: Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of "enhancement" strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit. PRINCIPAL FINDINGS: Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34(+) were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction. CONCLUSIONS: Our results show that HDAC blockade leads to phenotype changes in CD34(+) cells with enhanced self renewal and cardioprotection.

A mouse model for spatial and temporal expression of HGF in the heart.

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.

Pentraxins and atherosclerosis: the role of PTX3.

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we will concisely review the general properties of PTX3 in the context of the pentraxin superfamily and discuss recent data suggesting that PTX3 plays a cardiovascular protective effect. PTX3 may represent a new marker in vascular pathology which correlates with the risk of developing vascular events.

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models.

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

Human cardiac mesoangioblasts isolated from hypertrophic cardiomyopathies are greatly reduced in proliferation and differentiation potency.

AIMS: Our objective was to test whether progenitor cell proliferation and differentiation potential may vary depending upon the disease of the donor. METHODS AND RESULTS: Human cardiac mesoangioblasts were isolated from cardiac muscle biopsies of patients undergoing open heart surgery for correction of mitral regurgitation following an acute myocardial infarction (MR-MI) or correction of mitral and aortic regurgitation with ensuing left ventricular hypertrophy (MAR-LVH). The cells express surface markers and cardiac genes similar to mouse cardiac mesoangioblasts; they have limited self-renewing and clonogenic activity and are committed mainly to cardiogenesis. Although cardiac differentiation can be induced by 5-azacytidine or by co-culture with rat neonatal cardiomyocytes, human cells do not contract spontaneously like their mouse counterparts. When locally injected in the infarcted myocardium of immunodeficient mice, cardiac mesoangioblasts generate a chimeric heart that contains human myocytes and some capillaries; likewise, they colonize chick embryo hearts when transplanted in ovo. At variance with cells from patients with MR-MI, when isolation was performed on biopsies from MAR-LVH, cells could be isolated in much lower numbers, proliferated less extensively and failed to differentiate. CONCLUSION: Cardiac mesoangioblasts are present in the human heart but this endogenous progenitor population is progressively exhausted, possibly by continuous and inefficient regeneration attempts.

Cardioprotective function of the long pentraxin PTX3 in acute myocardial infarction.

BACKGROUND: Despite widespread clinical use as a prognostic marker in ischemic heart disorders, the actual pathogenetic role of the short pentraxin, C-reactive protein, has not undergone stringent genetic testing because of evolutionary divergence between mouse and humans. The long pentraxin PTX3 is conserved in evolution, is expressed in the heart under inflammatory conditions, and is a candidate prognostic marker in acute myocardial infarction. It was therefore important to assess whether PTX3 plays a pathogenetic role in acute myocardial infarction. METHODS AND RESULTS: In a model of acute myocardial infarction caused by coronary artery ligation and reperfusion, tissue mRNA expression and circulating levels of PTX3 increased. The interleukin-1R-MyD88 pathway plays a pivotal role in the induction of PTX3 transcript after ischemia. ptx3-deficient mice showed exacerbated heart damage (33% larger infarcts in null mice; P=0.0047). Increased myocardial damage in ptx3-deficient mice was associated with a greater no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes. In addition, ptx3-deficient mice with acute myocardial infarction showed higher circulating levels of interleukin-6 and increased C3 deposition in lesional tissue. The phenotype was reversed by exogenous PTX3. CONCLUSIONS: Thus, PTX3 plays a nonredundant, regulatory, cardioprotective role in acute myocardial infarction in mice. Our results suggest that modulation of the complement cascade contributes to the cardioprotective function of PTX3.