Catalase, a therapeutic target in the reversal of estrogen-mediated aging.

Despite increasing interest in the reversal of age-related processes, there is a paucity of data regarding the effects of post-menopausal-associated estrogen loss on cellular function. We studied human adipose-derived mesenchymal stem cells (hASCs) isolated from women younger than 45 years old (pre-menopause, pre-hASC) or older than 55 years old (post-menopause, post-hASC). In this study, we provide proof of concept that the age-related ineffective functionality of ASCs can be reversed to improve their ability in promoting tissue repair. We found reduced estrogen receptor expression, decreased estrogen receptor activation, and reduced sensitivity to 17ß-estradiol in post-hASCs. This correlated with decreased antioxidants (catalase and superoxide dismutase [SOD] expression) and increased oxidative stress compared with pre-hASCs. Increasing catalase expression in post-hASCs restored estrogen receptor (ER) expression and their functional capacity to promote tissue repair as shown in human skin ex vivo wound healing and in vivo mouse model of lung injury. Our results suggest that the consequences of 17ß-estradiol decline on the function of hASCs may be reversible by changing the oxidative stress/antioxidant composition.

Anti-fibrotic effects of different sources of MSC in bleomycin-induced lung fibrosis in C57BL6 male mice.

BACKGROUND AND OBJECTIVE: IPF is a fatal and debilitating lung disorder increasing in incidence worldwide. To date, two approved treatments only slow disease progression, have multiple side effects and do not provide a cure. MSC have promising therapeutic potential as a cell-based therapy for many lung disorders based on the anti-fibrotic properties of the MSC. METHODS: Critical questions remain surrounding the optimal source, timing and efficacy of cell-based therapies. The present study examines the most effective sources of MSC. Human MSC were derived from adipose, WJ, chorionic membrane (CSC) and chorionic villi (CVC). MSC were injected into the ageing mouse model of BLM-induced lung fibrosis. RESULTS: All sources decreased Aschroft and hydroxyproline levels when injected into BLM-treated mice at day 10 with the exception of CSC cells that did not change hydroxyproline levels. There were also decreases in mRNA expression of αv -integrin and TNFα in all sources except CSC. Only ASC- and WJ-derived cells reduced AKT and MMP-2 activation, while Cav-1 was increased by ASC treatment as previously reported. BLM-induced miR dysregulation of miR-29 and miR-199 was restored only by ASC treatment. CONCLUSION: Our data suggest that sources of MSC may differ in the pathway(s) involved in repair.

MicroRNA let-7 Downregulates Ligand-Independent Estrogen Receptor-mediated Male-Predominant Pulmonary Fibrosis.

Rationale: The relevance of hormones in idiopathic pulmonary fibrosis (IPF), a predominantly male lung disease, is unknown.Objectives: To determine whether the ER (estrogen receptor) facilitates the development of pulmonary fibrosis and is mediated in part through microRNA regulation of ERα and ERα-activated profibrotic pathways.Methods: ER expression in male lung tissue and myofibroblasts from control subjects (n = 6) and patients with IPF (n = 6), aging bleomycin (BLM)-treated mice (n = 7), and BLM-treated AF2ERKI mice (n = 7) was determined. MicroRNAs that regulate ER and fibrotic pathways were assessed. Transfections with a reporter plasmid containing the 3' untranslated region of the gene encoding ERα (ESR1) with and without miRNA let-7 mimics or inhibitors or an estrogen response element-driven reporter construct (ERE) construct were conducted.Measurements and Main Results: ERα expression increased in IPF lung tissue, myofibroblasts, or BLM mice. In vitro treatment with let-7 mimic transfections in human myofibroblasts reduced ERα expression and associated fibrotic pathways. AF2ERKI mice developed BLM-induced lung fibrosis, suggesting a role for growth factors in stimulating ER and fibrosis. IGF-1 (insulin-like growth factor 1) expression was increased and induced a fourfold increase of an ERE construct.Conclusions: Our data show 1) a critical role for ER and let-7 in lung fibrosis, and 2) that IGF may stimulate ER in an E2-independent manner. These results underscore the role of sex steroid hormones and their receptors in diseases that demonstrate a sex prevalence, such as IPF.

An Optimized and Advanced Algorithm for the Quantification of Immunohistochemical Biomarkers in Keratinocytes.

Advancements in pathology have given rise to software applications intended to minimize human error and improve efficacy of image analysis. Still, the subjectivity of image quantification performed manually and the limitations of the most ubiquitous tissue stain analysis software requiring parameters tuned by the observer, reveal the need for a highly accurate, automated nuclear quantification software specific to immunohistochemistry, with improved precision and efficiency compared with the methods currently in use. We present a method for the quantification of immunohistochemical biomarkers in keratinocyte nuclei proposed to overcome these limitations, contributing sensitive shape-focused segmentation, accurate nuclear detection, and automated device-independent color assessment, without observer-dependent analysis parameters.

Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly.

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific SC neuronal populations and circuits.

Single-nucleus RNA sequencing of developing superior colliculus identifies neuronal diversity and candidate mediators of circuit assembly.

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment of neural connectivity. Here we perform single-nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Using these data, we find that Pax7 expression is restricted to a subset of GABAergic neurons. Our data provide a valuable resource for interrogating the mechanisms of circuit development and identifying markers for manipulating specific SC neuronal populations and circuits.

Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo.

Background: MicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum, and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis. Methods: Exosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models. Results: U-IPFexo or MF-IPFexo expressed miR-let-7d, miR-29a-5p, miR-181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 min after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models. Conclusions: Our results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models. Funding: This work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).

Testosterone and 17ß-estradiol have opposite effects on podocyte apoptosis that precedes glomerulosclerosis in female estrogen receptor knockout mice.

Podocyte damage and apoptosis are thought to be important if not essential in the development of glomerulosclerosis. Female estrogen receptor knockout mice develop glomerulosclerosis at 9 months of age due to excessive ovarian testosterone production and secretion. Here, we studied the pathogenesis of glomerulosclerosis in this mouse model to determine whether testosterone and/or 17ß-estradiol directly affect the function and survival of podocytes. Glomerulosclerosis in these mice was associated with the expression of desmin and the loss of nephrin, markers of podocyte damage and apoptosis. Ovariectomy preserved the function and survival of podocytes by eliminating the source of endogenous testosterone production. In contrast, testosterone supplementation induced podocyte apoptosis in ovariectomized wild-type mice. Importantly, podocytes express functional androgen and estrogen receptors, which, upon stimulation by their respective ligands, have opposing effects. Testosterone induced podocyte apoptosis in vitro by androgen receptor activation, but independent of the TGF-ß1 signaling pathway. Pretreatment with 17ß-estradiol prevented testosterone-induced podocyte apoptosis, an estrogen receptor-dependent effect mediated by activation of the ERK signaling pathway, and protected podocytes from TGF-ß1- or TNF-α-induced apoptosis. Thus, podocytes are target cells for testosterone and 17ß-estradiol. These hormones modulate podocyte damage and apoptosis.

Estrogen receptor beta protects against in vivo injury in RPE cells.

Epidemiological data suggest that estrogen deficiency in postmenopausal women may contribute to the severity of AMD. We discovered that 17beta-estradiol (E2) was a crucial regulator of the severity of extracellular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)beta regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of pro-MMP-2, MMP-14 and TIMP-2 correlated with increased Bruch's membrane thickening or sub-retinal deposit formation (basal laminar deposits) in ERKObeta mice. In addition RPE isolated from ERKObeta mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKObeta mice. These data suggest that mice which lack ERbeta are more susceptible to in vivo injury associated with environmental light and high fat diet.

17 beta-estradiol and tamoxifen upregulate estrogen receptor beta expression and control podocyte signaling pathways in a model of type 2 diabetes.

Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

Inhibition of C-jun N-terminal kinase improves insulin sensitivity but worsens albuminuria in experimental diabetes.

C-jun N-terminal kinase (JNK) regulates both the development of insulin resistance and inflammation. Podocytes of the widely used db/db mouse model of diabetic nephropathy lose their ability to respond to insulin as albuminuria develops, in comparison to control db/+ mice. Here we tested whether JNK inhibition or its gene deletion would prevent albuminuria in experimental diabetes. Phosphorylated/total JNK was significantly increased in vivo in glomeruli of db/db compared to db/+ mice. Treatment of podocytes isolated from these two strains of mice with tumor necrosis factor-alpha caused greater phosphorylation of JNK in those obtained from diabetic animals. When db/db mice were treated with a cell-permeable TAT-JNK inhibitor peptide, their insulin sensitivity and glycemia significantly improved compared to controls. We induced diabetes in JNK1 knockout mice with streptozotocin and found that they had significantly better insulin sensitivity compared to diabetic wild-type or JNK2 knockout mice. Albuminuria was, however, worse in all mice treated with the JNK inhibitor and in diabetic JNK2 knockout mice compared to controls. Nephrin expression was also reduced in JNK inhibitor-treated mice compared to controls. A similar degree of mesangial expansion was found in all diabetic mice. Our study shows that targeting JNK to improve systemic insulin sensitivity does not necessarily prevent diabetic nephropathy.

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.

Bot1p is required for mitochondrial translation, respiratory function, and normal cell morphology in the fission yeast Schizosaccharomyces pombe.

Maintenance of cell morphology is essential for normal cell function. For eukaryotic cells, a growing body of recent evidence highlights a close interdependence between mitochondrial function, the cytoskeleton, and cell cycle control mechanisms; however, the molecular details of this interconnection are still not completely understood. We have identified a novel protein, Bot1p, in the fission yeast Schizosaccharomyces pombe. The bot1 gene is essential for cell viability. bot1Delta mutant cells expressing lower levels of Bot1p display altered cell size and cell morphology and a disrupted actin cytoskeleton. Bot1p localizes to the mitochondria in live cells and cofractionates with purified mitochondrial ribosomes. Reduced levels of Bot1p lead to mitochondrial fragmentation, decreased mitochondrial protein translation, and a corresponding decrease in cell respiration. Overexpression of Bot1p results in cell cycle delay, with increased cell size and cell length and enhanced cell respiration rate. Our results show that Bot1p has a novel function in the control of cell respiration by acting on the mitochondrial protein synthesis machinery. Our observations also indicate that in fission yeast, alterations of mitochondrial function are linked to changes in cell cycle and cell morphology control mechanisms.

Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness.

CONTEXT: The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood. OBJECTIVE: The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM. DESIGN: In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness. SETTING: The study was conducted at an academic medical center. PATIENTS: Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007). INTERVENTION: LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline. MAIN OUTCOME MEASURES: Activity of MMP-2 and invasiveness of LAMD-SM cells were measured. RESULTS: LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs. CONCLUSION: The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

Estrogen receptor subtype expression and regulation is altered in papillary thyroid cancer after menopause.

BACKGROUND: Estrogen receptors can regulate growth in papillary thyroid cancer and may affect prognosis after menopause. This study examines changes of estrogen receptor subtype ratio expression in papillary thyroid cancer cell lines derived from pre- and postmenopausal women. METHODS: Cells were harvested from papillary thyroid cancer and non-papillary thyroid cancer thyroid tissue (control) from pre- (n = 9) and postmenopausal women (n = 11). Protein expression of estrogen receptor α, estrogen receptor ß, and phosphorylated extracellular signal-regulated kinase and protein kinase B were analyzed. Matrix metalloproteinase-2 activity was determined as a measure of tumor invasiveness. Mitochondrial retrograde signaling was altered with ethidium bromide to determine its effect on estrogen receptor α protein expression. RESULTS: Estrogen receptor α expression was increased in postmenopausal papillary thyroid cancer cells compared with controls but was unchanged in premenopausal papillary thyroid cancer. Estrogen receptor ß expression did not change in either group. Increased matrix metalloproteinase-2 activity was observed only in postmenopausal papillary thyroid cancer. Premenopausal papillary thyroid cancer cells demonstrated increased extracellular signal-regulated kinase and unchanged protein kinase B activation. Conversely, postmenopausal papillary thyroid cancer cells had decreased extracellular signal-regulated kinase and increased protein kinase B activation. Ethidium bromide treatment resulted in increased estrogen receptor α protein expression only in premenopausal papillary thyroid cancer cells. CONCLUSION: Increased estrogen receptor α expression may be involved in papillary thyroid cancer aggressiveness after menopause. This process may be regulated by differential activation of intracellular pathways and differing sensitivities to mitochondrial signaling regulation.

Estrogen receptor subtype ratio change protects against podocyte damage.

Women are relatively protected against the development and progression of glomerulosclerosis (GS) prior to menopause. However, the "female advantage" is lost in women who are either diabetic, post-menopausal or both. We showed that 17ß-estradiol (E2) was effective in prevention of diabetic GS development in part through the stabilization of podocyte cytoskeleton and a change in estrogen receptor (ER) subtype ratio. The objective of this study was to examine whether resveratrol (RSV), reported to have estrogen-like action and renoprotective activity against diabetic GS, would affect similar pathways. After in vitro treatment with RSV we found a change in the ERα and ERß expression ratio in favor of ERß, suppression of heat shock protein 25 (Hsp25) expression and increase in ß1-integrin expression, important for maintaining podocyte cytoskeleton. We noted a reduction of insulin-like growth factor 1 receptor (IGFR1) expression, decrease in extracellular signal-regulated kinase (ERK) activation, decrease in reactive oxygen species (ROS), and decrease in cleaved-caspase 3 expression. We found an increase in [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and an increase in matrix metalloproteinases (MMP-2 and MMP-9) activity. Using cre-loxP strategy we developed podocyte-specific ERα knockout mice to show the importance of ERß. In isolated podocytes, we confirmed reduction of ERα expression in conjunction with a decrease in IGFR1 expression, ERK and increase of MMP-2 similar to that of our in vitro treatment with RSV. Taken together these data suggest an important role for ERß and ER subtype ratio in podocyte stabilization. Therefore RSV or other regulators of ER pathways could offer protection against diabetic and age-related podocyte changes.

Retinal pigment epithelium protection from oxidant-mediated loss of MMP-2 activation requires both MMP-14 and TIMP-2.

PURPOSE: Eyes with age-related macular degeneration (AMD) demonstrate accumulation of specific deposits and extracellular matrix (ECM) molecules under the retinal pigment epithelium (RPE). Metalloproteinases (MMP) are crucial regulators of basement membrane and ECM turnover. Accordingly, loss of RPE MMP activity most likely leads to excessive accumulation of collagen and other ECM, a potential mechanism for formation of deposits. A prior study showed that MMP-2 activity, but not pro-MMP-2 protein, decreases after RPE oxidative injury, indicating that oxidant injury disrupts the enzymatic cleavage of pro-MMP-2. Activation of MMP-2 requires the formation of a tri-molecular complex of pro-MMP-2, MMP-14, and tissue inhibitor of metalloproteinases (TIMP)-2. Therefore, a study was conducted to investigate the impact of oxidant injury on the interaction between these three molecules. METHODS: Human GFP-RPE cells were oxidant injured by transient exposure to H2O2 and myeloperoxidase, and the time course of recovery determined. Supernatants and cell lysates were collected for analysis of MMP-2, MMP-14, and TIMP-2 activity, mRNA and protein expression. In some studies, overexpression with either MMP-14 or TIMP-2 was performed to revert the cells to a preinjury phenotype. RESULTS: Transient injury resulted in a decrease of both MMP-14 and TIMP-2 activity and protein. Overexpression of each single molecule failed to prevent the injury-induced decrease of MMP-2 activity. In contrast, overexpression of MMP-14 together with the addition of exogenous TIMP-2 prevented the reduction of MMP-2 activation. CONCLUSIONS: Loss of MMP-2 activity after oxidant injury is caused by the downregulation of MMP-14 and TIMP-2. Overexpression of either MMP-14 or TIMP-2 alone before oxidant injury is not enough to prevent loss of MMP-2 activity. All three components of the tri-molecular complex must be present to preserve normal MMP-2 activity after oxidant injury.

Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced sub-RPE deposits in vivo.

PURPOSE: To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS: An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 microM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS: In vitro, high doses of HQ (400-250 microM) killed a significant fraction of RPE cells ( approximately 60% of control). Low doses (50-100 microM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS: In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.

Cigarette smoke-related oxidants and the development of sub-RPE deposits in an experimental animal model of dry AMD.

PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model. METHODS: Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy. RESULTS: As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch's membrane. The choriocapillaris endothelium was variably hypertrophic. CONCLUSIONS: Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch's membrane, and accumulation of deposits within Bruch's membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.

Estrogen deficiency promotes cigarette smoke-induced changes in the extracellular matrix in the lungs of aging female mice.

Female smokers have a faster decline in lung function with increasing age and overall develop a greater loss of lung function than male smokers. This raises the question of whether estrogen status in women affects susceptibility to cigarette smoke (CS)-induced lung disease. Mouse models suggest that female mice are more susceptible than males to CS-induced lung disease. Moreover, young CS-exposed female mice develop emphysema earlier than male mice. The purpose of this study was to characterize the relationship of estrogen status on the pattern and severity of CS-induced lung disease. In this study, 15-month-old female C57BL/6J mice were ovariectomized and administered either placebo (pla) or 17ß-estradiol (E2, 0.025 mg) 2 weeks after ovariectomy. They were further divided into those that were exposed to CS and no-smoke controls (NSC). Mice were exposed to CS in stainless steel inhalation chambers 3 hours a day, 5 days a week for 6 months, and sacrificed after 24 weeks of CS exposure. Blood and urine were collected at sacrifice to measure estrogen and cotinine levels, a metabolite of nicotine. Uterine weight was recorded as an indicator of estrogen status. Results showed that CS in the absence of E2 induced a decrease in hydroxyproline content, macrophage number, and respiratory chain complex-1 protein. CS without E2 also resulted in an increase in matrix metalloproteinase-2 activity and apoptosis and a change in the ratio of estrogen receptor subtype. These findings were abrogated with administration of E2, suggesting that estrogen deficiency increases susceptibility to CS-induced lung disease.