Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization

Abstract

In this study, we evaluate the potential involvement of collagenase--3 (MMP13), a matrix metalloproteinase (MMP) family member, in the exudative form of age--related macular degeneration (AMD) characterized by a neovascularisation into the choroid. RT--PCR analysis revealed that human neovascular membranes issued from patients with DMLA expressed high levels of Mmp13. The contribution of MMP13 in choroidal neovascularization (CNV) formation was explored by using a murine model of laser--induced CNV and applying it to wild type mice (WT) and Mmp13--deficient mice (Mmp13 --/--mice). Angiogenic and inflammatory reactions were explored by immunohistochemistry. The implication of bone marrow (BM)--derived cells was determined by BM engraftment into irradiated mice and by injecting mesenchymal stem cells (MSC) isolated from WT BM. The deficiency of Mmp13 impaired CNV formation which was fully restored by WT BM engraftment and partially rescued by several injections of WT MSC. The present study sheds light on a novel function of MMP13 during BM--dependent choroidal vascularization and provides evidence for a role for MSC in the pathogenesis of CNV. Keywords: CNV, MMP13, angiogenesis, bone marrow, mesenchymal stem cells. Non--standard abbreviations used: AMD: age--related macular degeneration; BM: bone marrow ; CAM: choroiallantoic membrane ; CNV: choroidal neovascularisation ; MSC: mesenchymal stem cells ; RPE: retinal pigmented epithelium ; TIMP: tissue inhibitor of metalloprotease. 3 INTRODUCTIONAge--related macular degeneration (AMD) is one of the most common irreversible causes of blindness among people over 50 years [1]. Ninety percents of all vision loss due to AMD occurs in the exudative form which is characterized by choroidal neovascularization (CNV). The newly formed blood vessels arising from choriocapillaries are directed to the subretinal macular region with subsequent bleeding and/or fluid leakage into the subretinal space, local retinal detachment and retinal photoreceptor damage [2]. The pathophysiology of AMD is complex and age--related changes that induce pathologic neovascularization are incompletely understood. In combination with the rapidly growing knowledge on basic mechanisms in angiogenesis, new evidence in pathogenesis of macular disease have led to novel developments in therapeutic strategies. Indeed, angiogenic factors such as VEGF play an important role in choroidal neovascular formation [3--5] and anti--VEGF molecules represent a substantial tool against AMD [6]. In the process of CNV, the vascular overgrowth is coupled with a localized proteolysis, extracellular remodelling and cell migration involving different proteolytic systems among which the matrix metalloproteinases (MMPs) are key players [7--9].An involvement of MMPs in the progression of retinal and choroidal neovascularization is supported by both experimental and clinical data. A mutation of Timp--3 gene (tissue inhibitor of metalloproteinase--3) is the cause of a rare familial form of macular dystrophy associated with ...

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

“…Both neutrophil and macrophage depletion reduce CNV formation [14][15][16] . The recruitment of BM-derived cells (endothelial progenitor cells, pericytes, mesenchymal stem cells) in CNV lesions has been reported 5,[17][18][19] and mimics the vasculogenic process observed in human patients 17 . Vascular endothelial growth factor (VEGF-A) rapidly emerged as a potent angiogenic factor involved in CNV formation, diverting much of the focus of research.…”

“…Transgenic mouse technologies have since allowed rapid expansion in the exploration of key regulators of CNV, leading to the identification of other important VEGF family members, such as placental growth factor (PlGF) 23,24 and proteases 19,[25][26][27][28] . More recently, the interplay between complement factor H, the complement membrane attack complex, a chemokine (CCL2) and VEGF during CNV development has been established 29,30 .…”

“…When evaluating pre-miR or siRNA, appropriate scramble sequences are recommended as controls. In cases of BM engraftment, an experimental group consisting of irradiated and BM-engrafted animals is mandatory because we have observed that BM engraftment can impact CNV formation 5,19 . In general, the use of one eye for drug testing and the second eye as a control is useful because it controls for variation between mice.…”

In vivo Bioluminescence Imaging Monitoring of Stem Cells' Participation in Choroidal Neovascularization

“…In previous studies, stem cell research has been mainly based on in vitro studies, postmortem histological analysis or molecular biology experiments [2,15,16]. In the present study, molecular imaging using the Fluc reporter gene and D -luciferin reporter probe can be used as a dynamic and reproducible in vivo imaging method of stem cell survival in CNV lesions in a mouse model.…”

Mesenchymal Stem Cell Transplantation for Retinal Degenerations and Dystrophies: Present and Future

“…It's the leading cause of irreversible blindness in the western countries and the rate is continuously going up because of the rapid increasing of averaged age (Kolar, 2010). Generally, AMD is classified as dry or nonexudative AMD and wet or neovascular AMD, according to the presence or the absence of Neovascularization (NV Lecomte et al, 2011). Some studies showed that nearly 50% of cells in the new vessels were GFP positive, indicating they arise from bone-marrow.…”