Jean-Marie Rakic

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

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

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.

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

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 Bruchs 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.

Role of plasminogen activator-plasmin system in tumor angiogenesis

Abstract: 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.

Presence of Oestrogen Receptor Type Beta in Human Retina

BACKGROUND/AIMS Recent studies have demonstrated the existence of two oestrogen receptor subtypes α (ORα) and β (ORβ) with significant differences of expression among organs. Since important pathologies of human eye could be linked to hormonal status, the expression of ORβ in ocular posterior segment was sought. METHODS Immunohistochemical localisation of ORβ and ORα protein and detection of OR mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) were performed in macular and extramacular regions of the retina and in the choroid on male and female donors eyes. RESULTS ORβ protein was localised in the ganglion cell layer and in the choroid. At the transcriptional level, mRNA for ORβ and for ORα were both present. Local differences in the expression level were observed, however, suggesting the possibility of variation in the ratio of ORαv ORβ. CONCLUSIONS The coexistence of two oestrogen receptor subtypes in the human ocular posterior segment raises acute questions about their potential physiological role, but offers a perspective for preferential targeting of a specific receptor subtype.

Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruchs membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7–14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature.

Matrix Metalloproteinase-9 Contributes to Choroidal Neovascularization

Age-related macular degeneration (AMD) is the primary cause of irreversible photoreceptors loss in adult patients and current therapies are limited. Increased levels of matrix metalloproteinases (MMPs) have been documented in neovascularization of severe ocular pathologies such as AMD and proliferative diabetic retinopathy. We report here that MMP-9 (gelatinase B) expression is induced and temporally regulated in the course of experimental choroidal neovascularization. We used transgenic mice expressing beta-galactosidase reporter gene under the dependence of MMP-9 promoter and RT-PCR analysis on choroidal neovascular structures microdissected from serial sections by laser pressure catapulting to show that MMP-9 expression is up-regulated concomitantly with the appearance of inflammatory cells in the subretinal lesion. In mice deficient in MMP-9 expression the development of choroidal neovascularization induced by laser photocoagulation still occurred, but at a reduced level.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

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

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.

Angiogenesis Is Not Impaired in Connective Tissue Growth Factor (CTGF) Knock-out Mice

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF−/− and heterozygous CTGF+/− mice to establish involvement of CTGF in neovascularization. CTGF−/− mice die around birth. Therefore, embryonic CTGF−/−, CTGF+/−, and CTGF+/+ bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF+/− and CTGF+/+ mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.