Quantitative enumeration of vascular smooth muscle cells and endothelial cells derived from bone marrow precursors in experimental choroidal neovascularization
Introduction
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population (Evans, 2001). AMD is a multifactorial disease related to age, systemic health, genetic and environmental risk factors (Evans, 2001). Inflammatory and immune mechanisms have recently been hypothesized to play a role in the pathogenesis of the disease (Penfold et al., 1985, Penfold et al., 2001). Choroidal neovascularization (CNV) is the major complication associated with vision loss in AMD (Young, 1987, Starr et al., 1998). The morphology and cellular composition of CNV is much more complex than the traditional model of CNV that conceptualizes this lesion as endothelial lined capillary tubes. Histopathology has demonstrated a wide degree of variable cellularity and fibrosis among different lesions (Grossniklaus et al., 2002). Thus, CNV are composed of many different cell types in addition to endothelial cells (Lopez et al., 1991, Seregard et al., 1994). These include RPE cells, myofibroblasts, vascular smooth muscle cells, CD 34 progenitor cells as well as inflammatory cells such as macrophages (Lopez et al., 1991, Lopez et al., 1996, Grossniklaus et al., 1992, Thomas et al., 1993, Csaky et al., 2004a, Csaky et al., 2004b). However, no systematic detailed analysis has been conducted on the origin of the different cell populations and their contribution to human or experimental CNV.
Inflammatory cells, specifically blood-derived macrophages, are the most obvious nonocular cell type that might be recruited to CNV. Macrophages can either be tissue-resident macrophages (which appear to be recruited early in ocular development and are long lived within the choroid) or blood-derived macrophages, which form part of the leukocyte population that is responsible for the innate immune response (Espinosa-Heidmann et al., 2003a, Espinosa-Heidmann et al., 2003b, McMenamin, 1997, McMenamin, 1999). Their importance has been recently confirmed in both human and experimental CNV (Espinosa-Heidmann et al., 2003a, Espinosa-Heidmann et al., 2003b, Sakurai et al., 2003, Grossniklaus et al., 2000, Grossniklaus et al., 2002).
The traditional paradigm of angiogenesis assumes that vascular cells in CNV are derived only from preexisting resident cells from the adjacent normal vascular bed. However, recruitment of vascular cells from a circulating population of cells has become another potential important source of cells forming pathological or physiological neovascularization, including CNV (Asahara et al., 1999a). Our group as well as other laboratories has demonstrated that circulating bone marrow-derived vascular precursor cells also contribute significantly to CNV composition (Espinosa-Heidmann et al., 2003a, Espinosa-Heidmann et al., 2003b, Sengupta et al., 2003).
Circulating precursor cells have been noted to exist for a wide range of populations, including epithelial cells (hepatocytes, pneumocytes), myocardiocytes, endothelial cells, VSMC, glial cells and even neurons (Asahara et al., 1997, Asahara et al., 1999b, Isner and Asahara, 1999, Cornacchia et al., 2001, Religa et al., 2002, LaBarge and Blau, 2002, Bailey and Fleming, 2003, Otto, 2002, Kajstura et al., 2004, Black et al., 2004, Picard-Riera et al., 2004). Recent studies have shown that vascular cells within CNV are derived from circulating precursors, (Espinosa-Heidmann et al., 2003a, Sengupta et al., 2003) but their relative contribution to the different cell types has not been studied. Surprisingly, the published studies have focused mostly on the recruitment of endothelial progenitor cells to sites of neovascularization, and ignored the contribution of marrow precursors to other cell types within neovascular tissues, including CNV (Csaky et al., 2004a, Csaky et al., 2004b).
In this study, we examined the contribution of different cell types to CNV using immunohistochemistry of mouse eyes with experimental CNV. We used markers for macrophages, endothelial cells, vascular smooth muscle cells, RPE cells, lymphocytes, and neutrophils. We further sought to determine the relative contribution of circulating precursor cells and monocytes to each particular cell population within developing CNV at different time points. Not surprisingly, we found that blood-derived macrophages were the predominant form of monocyte at all time points during CNV formation. We also observed that a significant frequency of endothelial and vascular smooth muscle cells were bone marrow-derived, whereas all RPE cells appeared to be entirely derived from preexisting tissue resident cells.
Section snippets
Mice
Mice used in this study were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A total of 20 female C57BL/6 mice 9–11 months old were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as recipient mice for bone marrow transplantation. Bone marrow was obtained from seven adult C57BL/6 female mice transgenic for the chicken β-actin promoter-GFP and cytomegalovirus enhancer (The Jackson Laboratory).
Bone marrow transplant
Briefly, donor mice were
Results
We used immunohistochemistry to identify different cell populations within CNV. CNV lesions were examined at 2 weeks after laser, when they have reached their maximal size (Espinosa-Heidmann et al., 2002). Endothelial cells were visualized with PECAM immunostaining and BS-1 lectin histochemistry; vascular smooth muscle cells with antibodies against αSMA and desmin; macrophages with antibodies against F4/80 and Iba1; and RPE cells with antibodies against RPE 65 and cytokeratin 18. At 2 weeks
Discussion
Histologic analysis of human CNV demonstrate surprising variability of cellularity and fibrosis that is not well-appreciated by many clinicians and investigators (Grossniklaus et al., 2002). CNV are often described as vascular channels lined with endothelial cells surrounded by pericytes and a stroma composed mainly of RPE cells and fibrocytes (Lopez et al., 1991). Most studies have focused on the role of endothelial cells. However, Grossniklaus has demonstrated some CNV to be very cellular
Acknowledgements
Supported by NIH grants EY/AI 13318 and RO3 EY 015292, and an unrestricted grant from Research to Prevent Blindness, Inc.
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These authors contributed equally to this work and therefore should be considered equivalent senior authors.