MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells.

in PLoS ONE (2011), 6(2), 16979

BACKGROUND: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the ... [more ▼]

BACKGROUND: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated. METHODOLOGY/PRINCIPAL FINDINGS: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization. CONCLUSIONS/SIGNIFICANCE: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB. [less ▲]

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity.

in Cellular & Molecular Life Sciences (2010)

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ... [more ▼]

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2. [less ▲]

1H NMR metabolomic approach of the laser-induced choroidal neovascularization in mice

Poster (2008, August)

Anti-angiogenic therapy of exudative age-related macular degeneration: current progress and emerging concepts

in Trends in Molecular Medicine (2007), 13(8), 345-352

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients. The more aggressive exudative form is characterized by abnormal blood-vessel development that occurs beneath ... [more ▼]

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients. The more aggressive exudative form is characterized by abnormal blood-vessel development that occurs beneath the retina as a result of choroidal neovascularization (CNV). Vascular endothelial growth factor [VEGF) has emerged as the key mediator of CNV formation; this has led to intensive research on VEGF and the recent approval of anti-VEGF compounds by the US Food and Drug Administration. Despite this successful introduction of anti-angiogenic therapies into the clinical setting, there is still a lack of treatments that definitively reverse damaged vision. Here, we consider the importance of putative molecular targets other than VEGF that might have been underestimated. Emerging cellular mechanisms offer additional opportunities for innovative therapeutic approaches. [less ▲]

Tumoral and choroidal vascularization: differential cellular mechanisms involving plasminogen activator inhibitor type I.

in American Journal of Pathology (2007), 171(4), 1369-80

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal ... [more ▼]

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1(-/-) mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1(-/-) BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis. [less ▲]

ADAMTS-2, a metalloproteinase containing a disintegrin domain and thrombospondin type I repeats, a new regulator of angiogenesis

in Journal of Vascular Research (2005), 42(Suppl. 2), 76

Enzymes of the ADAMTS family are closely related to MMPs and ADAMs. They further contain specific domains, such as the “ThromboSpondin type I” (TSP1) repeats able to strongly repress angiogenesis. The ... [more ▼]

Enzymes of the ADAMTS family are closely related to MMPs and ADAMs. They further contain specific domains, such as the “ThromboSpondin type I” (TSP1) repeats able to strongly repress angiogenesis. The primary function of ADAMTS-2 is to process procollagen type I, II, III and V into mature molecules by excising the amino-propeptide. We further hypothesized that it could modulate angiogenesis through its TSP1 repeats. Recombinant ADAMTS-2 induced morphological changes in HUVEC and HMEC cultured on gelatin, collagen and fibronectin. It also significantly reduced their proliferation, attachment and spreading. Similar effects were observed when using inactive ADAMTS-2 mutated at the Zn++-binding catalytic site. ADAMTS-2 did not alter the initial steps of formation of capillary-like structures by HUVEC in vitro. However, these structures appeared more rapidly disrupted in presence of ADAMTS-2 than in control conditions. Immunostaining with monoclonal antibodies against ADAMTS-2 indicate that it is tightly immobilized at the endothelial cell surface by an heparin-sensitive binding. With the aim to identify mechanism(s)leading to the modulation of angiogenesis by ADAMTS-2, we investigated various signalling pathways critical for EC. Phosphorylation status of FAK was not altered by ADAMTS-2 while a downregulation of phosphorylation of p42/44 MAPK was observed. Our data suggest that ADAMTS-2 reduces angiogenesis by regulating endothelial cell adhesion and proliferation, and by alteration of the stability of the capillary-like structures. These effects do not seem to be mediated by an integrin-dependent signaling pathway. Choroidal neovascularization induced in TS2+/+ or TS2-/- mice by LASER burns was used as in vivo model. Several genes involved in the healing and angiogenesis processes (fibrillar collagens, VEGF, TGF, CTGF, …) were not differently regulated in TS2+/+ and TS2-/- mice 5 days after the LASER impact. Wound capillaries visualized by confocal microscopy after FITC-conjugated dextran injection, were significantly increased (p<0,05) in TS2-/- mice suggesting an increased angiogenic response in the KO animals. The results obtained in in vivo and in vitro models indicate that ADAMTS-2 is involved in the control of angiogenesis. Additional investigations are being performed to determine which domain(s) of the molecule is (are) antiangiogenic and to identify the mechanism(s) underlying this regulatory function. [less ▲]

Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization

in Investigative Ophthalmology & Visual Science (2003), 44(7), 3186-3193

PURPOSE. VEGF has been shown to be necessary, but not sufficient alone, for the development of subretinal pathologic angiogenesis. In the current study, the influence of placental growth factor (PIGF), a ... [more ▼]

PURPOSE. VEGF has been shown to be necessary, but not sufficient alone, for the development of subretinal pathologic angiogenesis. In the current study, the influence of placental growth factor (PIGF), a member of the VEGF family, in human and experimental choroidal neovascularization (CNV) was investigated. METHODS. The presence of VEGF family member mRNA was evaluated by RT-PCR in neovascular membranes extracted during surgery. The spatial and temporal pattern of VEGF isoforms and PIGF mRNA expression were explored by using the laser capture catapulting technique and RT-PCR in a murine laser-induced model and in vitro. PIGF expression was also studied in human donor eyes. The influence of endogenous PIGF was evaluated in deficient mice (PlGF(-/-)) and by antibody-mediated neutralization of the PIGF receptor. RESULTS. Human neovascular membranes consistently expressed VEGF-A, -B, and -C; PlGF; and VEGFR-1 and -2. The VEGF(120) isoform mRNA was primarily induced in early stages of angiogenesis in vivo and in vitro. PIGF mRNA expression was present in the intact choroid and significantly upregulated during the course of experimental CNV. Both deficient PIGF expression in PIGF(-/-) mice and PIGF receptor neutralization in wild-type mice prevented the development of choroidal neovascularization induced by laser. CONCLUSIONS. These observations demonstrate the participation of PIGF in experimental CNV. They identify therefore PIGF as an additional promising target for ocular antiangiogenic strategies. [less ▲]

Severe inhibition of choroidal neovascularization in mice with a combined deficiency of MMP-2 and MMP-9 genes

in Investigative Ophthalmology & Visual Science (2003, May), 44(Suppl. 2), 410

Role of plasminogen activator-plasmin system in tumor angiogenesis

in Cellular and Molecular Life Sciences : CMLS (2003), 60(3), 463-473

New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated ... [more ▼]

New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and, conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand, genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications. [less ▲]

MMP-2 and MMP-9 synergize in promoting choroidal neovascularization

in FASEB Journal (2003), 17(15), 2290-2292

Matrix metalloproteinase 2 (MMP-2) and MMP-9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age-related macular degeneration (AMD), but ... [more ▼]

Matrix metalloproteinase 2 (MMP-2) and MMP-9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age-related macular degeneration (AMD), but their precise role and potential interactions remain unclear. To address the question of MMP-2 and MMP-9 functions, mice deficient in the expression of MMP-2 (MMP-2 KO), MMP-9 (MMP-9 KO), and both MMP-2 and MMP-9 (MMP-2,9 KO) with their corresponding wild-type mice (WT) underwent CNV induction by laser-induced rupture of the Bruch's membrane. Both the incidence and the severity of CNV were strongly attenuated in double deficient compared with single gene deficient mice or corresponding WT controls. The reduced neovascularization was accompanied by fibrinogen/fibrin accumulation. Furthermore, overexpression of the endogenous MMP inhibitors TIMP-1 or TIMP-2 (delivered by adenoviral vectors) in WT mice or daily injection of a synthetic and gelatinase selective MMP inhibitor (Ro 26-2853) significantly decreased the pathological reaction. These findings suggest that MMP-2 and MMP-9 may cooperate in the development of AMD and that their selective inhibition represents an alternative strategy for the treatment of choroidal neovascularization. [less ▲]