Single-Cell Analyses Offer Insights into the Different Remodeling Programs of Arteries and Veins.

Arteries and veins develop different types of occlusive diseases and respond differently to injury. The biological reasons for this discrepancy are not well understood, which is a limiting factor for the development of vein-targeted therapies. This study contrasts human peripheral arteries and veins at the single-cell level, with a focus on cell populations with remodeling potential. Upper arm arteries (brachial) and veins (basilic/cephalic) from 30 organ donors were compared using a combination of bulk and single-cell RNA sequencing, proteomics, flow cytometry, and histology. The cellular atlases of six arteries and veins demonstrated a 7.8× higher proportion of contractile smooth muscle cells (SMCs) in arteries and a trend toward more modulated SMCs. In contrast, veins showed a higher abundance of endothelial cells, pericytes, and macrophages, as well as an increasing trend in fibroblasts. Activated fibroblasts had similar proportions in both types of vessels but with significant differences in gene expression. Modulated SMCs and activated fibroblasts were characterized by the upregulation of MYH10, FN1, COL8A1, and ITGA10. Activated fibroblasts also expressed F2R, POSTN, and COMP and were confirmed by F2R/CD90 flow cytometry. Activated fibroblasts from veins were the top producers of collagens among all fibroblast populations from both types of vessels. Venous fibroblasts were also highly angiogenic, proinflammatory, and hyper-responders to reactive oxygen species. Differences in wall structure further explain the significant contribution of fibroblast populations to remodeling in veins. Fibroblasts are almost exclusively located outside the external elastic lamina in arteries, while widely distributed throughout the venous wall. In line with the above, ECM-targeted proteomics confirmed a higher abundance of fibrillar collagens in veins vs. more basement ECM components in arteries. The distinct cellular compositions and transcriptional programs of reparative populations in arteries and veins may explain differences in acute and chronic wall remodeling between vessels. This information may be relevant for the development of antistenotic therapies.

The intricate cellular ecosystem of human peripheral veins as revealed by single-cell transcriptomic analysis.

The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11+DESlo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11+DEShi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.

The Transcriptomics of the Human Vein Transformation After Arteriovenous Fistula Anastomosis Uncovers Layer-Specific Remodeling and Hallmarks of Maturation Failure.

Introduction: The molecular transformation of the human preaccess vein after arteriovenous fistula (AVF) creation is poorly understood. This limits our ability to design efficacious therapies to improve maturation outcomes. Methods: Bulk RNA sequencing (RNA-seq) followed by paired bioinformatic analyses and validation assays were performed in 76 longitudinal vascular biopsies (veins and AVFs) from 38 patients with stage 5 chronic kidney disease or end-stage kidney disease undergoing surgeries for 2-stage AVF creation (19 matured, 19 failed). Results: A total of 3637 transcripts were differentially expressed between veins and AVFs independent of maturation outcomes, with 80% upregulated in fistulas. The postoperative transcriptome demonstrated transcriptional activation of basement membrane and interstitial extracellular matrix (ECM) components, including preexisting and novel collagens, proteoglycans, hemostasis factors, and angiogenesis regulators. A postoperative intramural cytokine storm involved >80 chemokines, interleukins, and growth factors. Postoperative changes in ECM expression were differentially distributed in the AVF wall, with proteoglycans and fibrillar collagens predominantly found in the intima and media, respectively. Interestingly, upregulated matrisome genes were enough to make a crude separation of AVFs that failed from those with successful maturation. We identified 102 differentially expressed genes (DEGs) in association with AVF maturation failure, including upregulation of network collagen VIII in medial smooth muscle cells (SMCs) and downregulation of endothelial-predominant transcripts and ECM regulators. Conclusion: This work delineates the molecular changes that characterize venous remodeling after AVF creation and those relevant to maturation failure. We provide an essential framework to streamline translational models and our search for antistenotic therapies.

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).

Systemic Profile of Cytokines in Arteriovenous Fistula Patients and Their Associations with Maturation Failure.

Background: Systemic cytokines are elevated in patients with chronic kidney disease (CKD) and on hemodialysis compared with the general population. However, whether cytokine levels interfere with vascular remodeling, increasing the risk of arteriovenous fistula (AVF) failure, remains unknown. Methods: This is a case-control study of 64 patients who underwent surgery for AVF creation (32 with AVF maturation failure and 32 matching controls with successful maturation). A total of 74 cytokines, including chemokines, interferons, interleukins, and growth factors, were measured in preoperative plasma samples using multiplex assays. Sixty-two patients were included in the statistical analyses. Associations with AVF failure were assessed using paired comparisons and conditional logistic regressions accounting for paired strata. Results: Seven cytokines were significantly higher in patients with AVF maturation failure than in matching controls (G-CSF, IL-6, MDC, RANTES, SDF-1α/ß, TGFα, and TPO). Of these, G-CSF (odds ratio [OR]=1.71; 95% confidence interval [95% CI], 1.05 to 2.79 per 10 pg/ml), MDC (OR=1.60, 95% CI, 1.08 to 2.38 per 100 pg/ml), RANTES (OR=1.55, 95% CI, 1.10 to 2.17 per 100 pg/ml), SDF-1α/ß (OR=1.18, 95% CI, 1.04 to 1.33 per 1000 pg/ml), and TGFα (OR=1.39, 95% CI 1.003, 1.92 per 1 pg/ml) showed an incremental association by logistic regression. Conclusions: This study identified a profile of plasma cytokines associated with adverse maturation outcomes in AVFs. These findings may open the doors for future therapeutics and markers for risk stratification.

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.

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.

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.

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 17ß-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 TGFß 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 TGFß production and by regulation of the estrogen receptor.

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.

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 17ß-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 17ß-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.

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

We recently showed that 17ß-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.

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.

The silk moth Bombyx mori U1 and U2 snRNA variants are differentially expressed.

Five U1 and eight U2 isoforms of the silk moth Bombyx mori exhibiting internal nucleotide differences have been previously identified and characterized in various tissues and developmental stages. In this investigation, it is demonstrated that the levels of some snRNA variants differ in egg and silk gland tissue and change during development. Qualitative and quantitative differences in the U1 and U2 variant populations were observed at three developmental points (early, middle and late) of the silk gland (SG) during the fifth instar larval stage of the silk moth. Statistical analyses of the various isoform populations across the fifth instar larval and egg stages show significant differences for some of the U1 and U2 variants. The representation of variant sequences in expressed U1 and U2 sequences (RT-PCR libraries) and in a whole-genome shotgun (WGS) assembly database was confirmed. In addition, conserved elements in the promoter 5'-flanking region of the U1 and U2 variants were identified in the WGS.

Superimposing polymorphism: the case of a point mutation within a polymorphic Alu insertion.

The COL3A1 Alu insertion is a member of the AluY subfamily. It has been found to be absent in non-human primates and polymorphic in worldwide human populations. The integration of the element into the human genome seems to have preceded the initial migration(s) of anatomically modern humans out of the African continent. Although the insertion has been detected in populations from all the continents, its highest frequency values are located within sub-Saharan Africa. The sequence alignment of the COL3A1 insertion from several African individuals revealed a bi-allelic single nucleotide polymorphism (SNP) at the downstream terminus of the element's poly-A tract. Once discovered, a selective PCR procedure was designed to determine the frequency of both alleles in 19 worldwide populations. The A-allele in this binary SNP experiences a clinal increase in the eastward direction from Africa to Southeast Asia and Mongolia, reaching fixation in the two latter regions. The T variant, on the other hand, exhibits a westward clinal increase outside of Africa, with its lowest frequency in Asia and achieving fixation in northern Europe. The presence of this internal SNP extends the usefulness provided by the polymorphic Alu insertion (PAI). It is possible that superimposing polymorphisms like this one found in the COL3A1 locus may accentuate signals from genetic drift events allowing for visualization of recent dispersal patterns.

Gender and estrogen supplementation increases severity of experimental choroidal neovascularization.

Observational clinical studies suggest that post-menopausal women may be at risk for more severe age-related macular degeneration, and that estrogen loss due to menopause may contribute. We sought to determine the effect of gender and estrogen status on the severity of choroidal neovascularization (CNV) in a mouse model for experimental choroidal neovascularization. Laser-induced CNV was performed in mice with or without estrogen supplementation. At various times, eyes were removed for analysis of severity of CNV lesions or for extraction of choroidal mRNA to evaluate iNOS, TNF-alpha, MMP-9, and ER-alpha expression, which are molecules relevant to angiogenic processes. Also, splenic macrophages were analysed for iNOS to determine the effect of estrogen treatment in vitro. Finally, laser-induced CNV was performed in iNOS -/- mice. Our result showed that aged female mice had significantly larger CNV than age-matched males. Ovariectomy in adult mice did not increase severity, but paradoxically estrogen supplementation after ovariectomy did increase CNV severity. More severe CNV were associated with a significant decrease in choroidal iNOS mRNA. Splenic macrophages from estrogen supplemented mice showed a significant increased in TNF-alpha mRNA expression (eight fold difference compared to the control) but only a mild change in iNOS mRNA levels (2-3 fold difference). In vitro data further showed that nitric oxide production in splenic macrophages at different estrogen levels was not different from controls. Finally, CNV severity was significantly more severe in iNOS -/- mice, compared to iNOS +/+ mice after laser treatment. In conclusion, aged female mice developed more severe CNV than do males. Estrogen replacement seems to increase severity, possibly by suppressing the upregulation of choroidal iNOS and activating macrophages. The putative beneficial or detrimental role of estrogen biology in age-related macular degeneration must be more carefully evaluated and may vary with the stage of age-related macular degeneration (atrophic or neovascular) as well as with the specific target cell type (monocytes vs. endothelial cell or vascular smooth muscle cell).

Nicotine increases size and severity of experimental choroidal neovascularization.

PURPOSE: Cigarette smoking is the strongest environmental risk factor for all forms of age-related macular degeneration (AMD). In the present study, the influence of nicotine on the severity of choroidal neovascularization (CNV) in a mouse model of neovascular AMD and its effects on vascular smooth muscle cells derived from mouse choroid were investigated. METHODS: A mouse model for CNV was used to study the effects of nicotine in young and middle-aged mice. Nicotine was administered orally in the drinking water to achieve serum levels consistent with those of chronic smokers. Hexamethonium, a nonspecific nicotinic receptor antagonist, was injected subconjunctivally to counteract the effects of nicotine. A mouse choroidal vascular smooth muscle cell line was exposed to nicotine, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), or a combination of one of the factors and nicotine. Cell growth was determined by cell counts, and the activity of matrix metalloproteinase (MMP)-2 and -9 was quantified by gel zymography. RESULTS: Nicotine administration resulted in increased size and vascularity of CNV, and older mice developed a greater relative increase than younger mice. This effect was blocked by subconjunctival hexamethonium. Choroidal vascular smooth muscle cells demonstrated a statistically significant increase in growth after exposure to a combination of PDGF and nicotine. Nicotine also reversed VEGF-induced suppression of MMP-2 activity. CONCLUSIONS: Nicotine increases size and severity of experimental CNV in the present mouse model, possibly by potentiating PDGF-mediated upregulation of proliferation of choroidal smooth muscle cells or by other mechanisms. These results suggest that non-neuronal nicotinic receptor activation probably mediates some of the harmful effects of cigarette smoking in wet AMD.