Simone Pereira-Simon

Therapeutic benefits of young, but not old, adipose-derived mesenchymal stem cells in a chronic mouse model of bleomycin-induced pulmonary fibrosis.

The observation that pulmonary inflammatory lesions and bleomycin (BLM)-induced pulmonary fibrosis spontaneously resolve in young mice, whereas remaining irreversible in aged mice suggests that impairment of pulmonary regeneration and repair is associated with aging. Because mesenchymal stem cells (MSCs) may promote repair after injury, we postulated that differences in MSCs from aged mice may underlie postinjury fibrosis in aging. The potential for young-donor MSCs to inhibit BLM-induced pulmonary fibrosis in aged male mice (>22 months) has not been studied. Adipose-derived MSCs (ASCs) from young (4 months) and old (22 months) male mice were infused 1 day after intratracheal BLM administration. At 21-day sacrifice, aged BLM mice demonstrated lung fibrosis by Ashcroft score, collagen content, and α(v)-integrin messenger RNA (mRNA) expression. Lung tissue from aged BLM mice receiving young ASCs exhibited decreased fibrosis, matrix metalloproteinase (MMP)-2 activity, oxidative stress, and markers of apoptosis vs BLM controls. Lung mRNA expression of tumor necrosis factor-alpha was also decreased in aged BLM mice receiving young-donor ASCs vs BLM controls. In contrast, old-donor ASC treatment in aged BLM mice did not reduce fibrosis and related markers. On examination of the cells, young-donor ASCs had decreased mRNA expression of MMP-2, insulin-like growth factor (IGF) receptor, and protein kinase B (AKT) activation compared with old-donor ASCs. These results show that the BLM-induced pulmonary fibrosis in aged mice could be blocked by young-donor ASCs and that the mechanisms involve changes in collagen turnover and markers of inflammation.

Macrophage Activation Associated with Chronic Murine Cytomegalovirus Infection Results in More Severe Experimental Choroidal Neovascularization

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.

In vivo 17β-estradiol treatment contributes to podocyte actin stabilization in female db/db mice

We recently showed that 17β-estradiol (E(2)) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E(2) administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E(2)-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C(3) botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E(2) female db/db mice. We found that in vivo E(2) protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E(2) treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease.

Oxidant Stress and Mitochondrial Signaling Regulate Reversible Changes of ERα Expression and Apoptosis in Aging Mouse Glomeruli and Mesangial Cells

Estrogen actions are largely dependent on the intracellular estrogen receptor (ER) levels. During aging the decline of estrogens or ER leads to a loss in antiinflammatory protection and an increase in oxidant stress due to changes in mitochondrial function. Estrogens/ER may also coordinate signaling between the nucleus and mitochondria through ERK activation, which paradoxically decreases ER expression. The changes in ER expression and transcriptional activation that occur with aging as well as the mitochondria-to-nuclear signaling pathways have not been studied in the glomerulus. We found that ER expression and transcriptional activation decreased with age. Whereas ER levels decreased by greater than 90%, serum 17β-estradiol levels decreased by less than 30%, suggesting alternative mechanisms for ER decrease. Because we postulated that this was due in part to age-related oxidant stress, we treated mesangial cells (MCs) with ethidium bromide (EtBr) to deplete mitochondria. EtBr treatment resulted in decreased ERK activation and reactive oxygen species, which were associated with increased ERα expression and transcriptional activation in old MCs. EtBr treatment also decreased apoptosis and caspase-9 protein expression in old MCs. These data suggest that loss of several of the functions of 17β-estradiol during aging could be mainly due to decreased ERα expression, that the ER loss is reversible by reducing reactive oxygen species, and that mitochondrial retrograde signaling plays a role in this regulation.

Inhibition of Advanced Glycation End Products (AGEs) Accumulation by Pyridoxamine Modulates Glomerular and Mesangial Cell Estrogen Receptor α Expression in Aged Female Mice.

Age-related increases in oxidant stress (OS) play a role in regulation of estrogen receptor (ER) expression in the kidneys. In this study, we establish that in vivo 17β-estradiol (E2) replacement can no longer upregulate glomerular ER expression by 21 months of age in female mice (anestrous). We hypothesized that advanced glycation end product (AGE) accumulation, an important source of oxidant stress, contributes to these glomerular ER expression alterations. We treated 19-month old ovariectomized female mice with pyridoxamine (Pyr), a potent AGE inhibitor, in the presence or absence of E2 replacement. Glomerular ERα mRNA expression was upregulated in mice treated with both Pyr and E2 replacement and TGFβ mRNA expression decreased compared to controls. Histological sections of kidneys demonstrated decreased type IV collagen deposition in mice receiving Pyr and E2 compared to placebo control mice. In addition, anti-AGE defenses Sirtuin1 (SIRT1) and advanced glycation receptor 1 (AGER1) were also upregulated in glomeruli following treatment with Pyr and E2. Mesangial cells isolated from all groups of mice demonstrated similar ERα, SIRT1, and AGER1 expression changes to those of whole glomeruli. To demonstrate that AGE accumulation contributes to the observed age-related changes in the glomeruli of aged female mice, we treated mesangial cells from young female mice with AGE-BSA and found similar downregulation of ERα, SIRT1, and AGER1 expression. These results suggest that inhibition of intracellular AGE accumulation with pyridoxamine may protect glomeruli against age-related oxidant stress by preventing an increase of TGFβ production and by regulation of the estrogen receptor.

Mesenchymal stromal cells prevent bleomycin-induced lung and skin fibrosis in aged mice and restore wound healing

Fibrosis can develop in nearly any tissue leading to a wide range of chronic fibrotic diseases. However, current treatment options are limited. In this study, we utilized an established aged mouse model of bleomycin‐induced lung fibrosis (BLM) to test our hypothesis that fibrosis may develop simultaneously in multiple organs by evaluating skin fibrosis and wound healing. Fibrosis was induced in lung in aged (18–22‐month‐old) C57BL/6 male mice by intratracheal BLM administration. Allogeneic adipose‐derived mesenchymal stromal cells (ASCs) or saline were injected intravenously 24 hr after BLM administration. Full thickness 8‐mm punch wounds were performed 7 days later to study potential systemic anti‐fibrotic and wound healing effects of intravenously delivered ASCs. Mice developed lung and skin fibrosis as well as delayed wound closure. Moreover, we observed similar changes in the expression of known pro‐fibrotic factors in both lung and skin wound tissue, including miR‐199 and protein expression of its corresponding target, caveolin‐1, as well as phosphorylation of protein kinase B. Importantly, ASC‐treated mice exhibited attenuation of BLM‐induced lung and skin fibrosis and accelerated wound healing, suggesting that ASCs may prime injured tissues and prevent end‐organ fibrosis.