LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.

Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.

Targeting cell-type-specific, choroid-peripheral immune signaling to treat age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness featuring pathogenic neovascularization of the choroidal vasculature (CNV). Although systemic immunity plays a role in AMD, the ocular signals that recruit and activate immune cells remain poorly defined. Using single-cell RNA sequencing, we prospectively profile peripheral blood mononuclear cells from 65 individuals including AMD and controls, which we integrate with existing choroid data. We generate a network of choroid-peripheral immune interactions dysregulated in AMD, including known AMD-relevant gene vascular endothelial growth factor (VEGF) receptor 2. Additionally, we find CYR61 is upregulated in choroidal veins and may signal to circulating monocytes. In mice, we validate that CYR61 is abundant in endothelial cells within CNV lesions neighboring monocyte-derived macrophages. Mechanistically, CYR61 activates macrophage anti-angiogenic gene expression, and ocular Cyr61 knockdown increases murine CNV size, indicating CYR61 inhibits CNV. This study highlights the potential of multi-tissue human datasets to identify disease-relevant and potentially therapeutically modifiable targets.

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function.

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, ß, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor-knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Loss of stearoyl-CoA desaturase 2 disrupts inflammatory response in macrophages.

Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed "inflammaging." Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.

The ZFP36 family of RNA binding proteins regulates homeostatic and autoreactive T cell responses.

RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell-specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell-specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell-specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion.

Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

Longitudinal Growth Differentiation Factor 15 (GDF15) and Long-term Intraocular Pressure Fluctuation in Glaucoma: A Pilot Study.

PURPOSE: Growth Differentiation Factor 15 (GDF15) was previously identified as a molecular marker of retinal ganglion cell stress in rodent models of glaucoma and was elevated in the aqueous humor (AH) of patients with primary open-angle glaucoma as a possible risk factor for glaucoma progression. The purpose of this study was to determine whether changes in the AH GDF15 levels were associated with intraocular pressure (IOP) changes in eyes undergoing glaucoma surgery. METHODS: Here, we performed a prospective, longitudinal pilot study in nine patients to determine whether changes in AH GDF15 levels from surgery to post-surgery follow-up were associated with IOP fluctuation. An initial AH sample was taken from the peripheral corneal paracentesis during planned glaucoma surgery, and a second sample was taken during an outpatient follow-up visit, approximately six months later. RESULTS: There was a statistically significant correlation between GDF15 fold change and IOP standard deviation (r = 0.87, P = 0.003), IOP range (r = 0.87, P = 0.003), and maximum IOP (r = 0.86, P = 0.003). There was no correlation between the GDF15 fold change and baseline IOP (r = 0.50, P = 0.17), final IOP (r = 0.038, P = 0.92), or mean IOP (r = 0.40, P = 0.28). CONCLUSION: Our findings in this pilot study suggest that longitudinal changes in AH GDF15 may be associated with IOP fluctuation during the postoperative period. Further studies are necessary to corroborate these findings in a larger patient population and to explore the possibility that AH GDF15 may be used not only to improve treatment algorithms but also as a surrogate endpoint in clinical trials.

Increased Aqueous Humor GDF15 Is Associated with Worse Visual Field Loss in Pseudoexfoliative Glaucoma Patients.

Purpose: To determine whether increased growth differentiation factor 15 (GDF15) in aqueous humor (AH) is associated with worse visual field loss in patients with pseudoexfoliative glaucoma (PXG). Methods: We recruited 12 patients (6 males, 6 females) with primary open-angle glaucoma (POAG) or PXG who were scheduled to undergo glaucoma surgery. AH was obtained from the initial peripheral paracentesis for the planned glaucoma surgery, and GDF15 levels were quantified with enzyme-linked immunosorbent assay by an investigator masked to clinical information. Humphrey visual field testing was performed as a part of routine care; results were obtained by reviewing the medical record. Results: AH GDF15 was detectable in patients with POAG and PXG. Increased AH GDF15 was significantly associated with worse mean deviation in patients with POAG (r = -0.94; 95% confidence interval [CI], -0.99 to -0.33; P = 0.02) and PXG (r = -0.92; 95% CI, -0.99 to -0.41; P = 0.01). Conclusions: AH GDF15 is detectable in patients with PXG and POAG. Elevated AH GDF15 is strongly associated with worse mean deviation in both subgroups. These findings suggest that GDF15 may be a molecular marker of glaucoma severity that is generalizable to multiple types of glaucoma regardless of the underlying etiology. Translational Relevance: This study provides proof of concept that GDF15, a molecular marker of retinal ganglion stress that was initially identified in rodent models, may have clinical utility as a measure of glaucoma severity not only in POAG but also in PXG.

FGF2-induced STAT3 activation regulates pathologic neovascularization.

Cell-autonomous endothelial cell (EC) fibroblast growth factor receptor (FGFR) signaling through FGFR1/2 is essential for injury-induced wound vascularization and pathologic neovascularization as in blinding eye diseases such as age-related macular degeneration. Which FGF ligand(s) is critical in regulating angiogenesis is unknown. Utilizing ex vivo models of choroidal endothelial sprouting and in vivo models of choroidal neovascularization (CNV), we demonstrate here that only FGF2 is the essential ligand. Though FGF-FGFR signaling can activate multiple intracellular signaling pathways, we show that FGF2 regulates pathogenic angiogenesis via STAT3 activation. The identification of FGF2 as a critical mediator in aberrant neovascularization provides a new opportunity for developing multi-target therapies in blinding eye diseases especially given the limitations of anti-VEGF monotherapy.

Oxysterol Signatures Distinguish Age-Related Macular Degeneration from Physiologic Aging.

Macrophage aging is pathogenic in numerous diseases, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. Although prior studies have explored the functional consequences of macrophage aging, less is known about its cellular basis or what defines the transition from physiologic aging to disease. Here, we show that despite their frequent self-renewal, macrophages from old mice exhibited numerous signs of aging, such as impaired oxidative respiration. Transcriptomic profiling of aged murine macrophages revealed dysregulation of diverse cellular pathways, especially in cholesterol homeostasis, that manifested in altered oxysterol signatures. Although the levels of numerous oxysterols in human peripheral blood mononuclear cells and plasma exhibited age-associated changes, plasma 24-hydroxycholesterol levels were specifically associated with AMD. These novel findings demonstrate that oxysterol levels can discriminate disease from physiologic aging. Furthermore, modulation of cholesterol homeostasis may be a novel strategy for treating age-associated diseases in which macrophage aging is pathogenic.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye.

Impaired autophagy in macrophages promotes inflammatory eye disease.

Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.

IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis.

Macrophage dysfunction plays a pivotal role during neovascular proliferation in diseases of ageing including cancers, atherosclerosis and blinding eye disease. In the eye, choroidal neovascularization (CNV) causes blindness in patients with age-related macular degeneration (AMD). Here we report that increased IL10, not IL4 or IL13, in senescent eyes activates STAT3 signalling that induces the alternative activation of macrophages and vascular proliferation. Targeted inhibition of both IL10 receptor-mediated signalling and STAT3 activation in macrophages reverses the ageing phenotype. In addition, adoptive transfer of STAT3-deficient macrophages into eyes of old mice significantly reduces the amount of CNV. Systemic and CD163(+) eye macrophages obtained from AMD patients also demonstrate STAT3 activation. Our studies demonstrate that impaired SOCS3 feedback leads to permissive IL10/STAT3 signalling that promotes alternative macrophage activation and pathological neovascularization. These findings have significant implications for our understanding of the pathobiology of age-associated diseases and may guide targeted immunotherapy.

Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis.

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2(Flk1-Cre) or Fgfr1/2(Tie2-Cre) mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2(Flk1-Cre) mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2(Flk1-Cre) and Fgfr1/2(Tie2-Cre) mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity.

Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.

Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer, and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.

VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting.

Although our understanding of the molecular regulation of adult neovascularization has advanced tremendously, vascular-targeted therapies for tissue ischemia remain suboptimal. The master regulatory transcription factors of the hypoxia-inducible factor (HIF) family are attractive therapeutic targets because they coordinately up-regulate multiple genes controlling neovascularization. Here, we used an inducible model of epithelial HIF-1 activation, the TetON-HIF-1 mouse, to test the requirement for VEGF in HIF-1 mediated neovascularization. TetON-HIF-1, K14-Cre, and VEGF(flox/flox) alleles were combined to create TetON-HIF-1:VEGF(Δ) mice to activate HIF-1 and its target genes in adult basal keratinocytes in the absence of concomitant VEGF. HIF-1 induction failed to produce neovascularization in TetON-HIF-1:VEGF(Δ) mice despite robust up-regulation of multiple proangiogenic HIF targets, including PlGF, adrenomedullin, angiogenin, and PAI-1. In contrast, endothelial sprouting was preserved, enhanced, and more persistent, consistent with marked reduction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic microscopy, which provides noninvasive, label-free, high resolution, and wide-field vascular imaging, revealed the absence of both capillary expansion and arteriovenous remodeling in serially imaged individual TetON-HIF-1:VEGF(Δ) mice. Impaired TetON-HIF-1:VEGF(Δ) neovascularization could be partially rescued by 12-O-tetradecanoylphorbol-13-acetate skin treatment. These data suggest that therapeutic angiogenesis for ischemic cardiovascular disease may require treatment with both HIF-1 and VEGF.

Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.

Neovascularization is a crucial component of tumor growth and ischemia. Although prior work primarily used disease models, delineation of neovascularization in the absence of disease can reveal intrinsic mechanisms of microvessel regulation amenable to manipulation in illness. We created a conditional model of epithelial HIF-1 induction in adult mice (TetON-HIF-1 mice). Longitudinal photoacoustic microscopy (L-PAM) was coincidentally developed for noninvasive, label-free serial imaging of red blood cell-perfused vasculature in the same mouse for weeks to months. TetON-HIF-1 mice evidenced 3 stages of neovascularization: development, maintenance, and transgene-dependent regression. Regression occurred despite extensive and tight pericyte coverage. L-PAM mapped microvascular architecture and quantified volumetric changes in neocapillary morphogenesis, arteriovenous remodeling, and microvessel regression. Developmental stage endothelial proliferation down-regulation was associated with a DNA damage checkpoint consisting of p53, p21, and endothelial γ-H2AX induction. The neovasculature was temporally responsive to VEGFR2 immuno-blockade, with the developmental stage sensitive, and the maintenance stage resistant, to DC101 treatment. L-PAM analysis also pinpointed microvessels ablated or resistant to VEGFR2 immuno-blockade. HIF-1-recruited myeloid cells did not mediate VEGFR2 inhibitor resistance. Thus, HIF-1 neovascularization in the absence of disease is self-regulated via cell autonomous endothelial checkpoints, and resistant to angiogenesis inhibitors independent of myeloid cells.

Magnetic resonance imaging defines cervicovaginal anatomy, cancer, and VEGF trap antiangiogenic efficacy in estrogen-treated K14-HPV16 transgenic mice.

Noninvasive detection of dysplasia provides a potential platform for monitoring the efficacy of chemopreventive therapy of premalignancy, imaging the tissue compartments comprising dysplasia: epithelium, microvasculature, and stromal inflammatory cells. Here, using respiratory-gated magnetic resonance imaging (MRI), the anatomy of premalignant and malignant stages of cervical carcinogenesis in estrogen-treated K14-HPV16 transgenic mice was noninvasively defined. Dynamic contrast enhanced (DCE)-MRI was used to quantify leakage across premalignant dysplastic microvasculature. Vascular permeability as measured by DCE-MRI, K(trans), was similar in transgenic (0.053 +/- 0.020 min(-1); n = 32 mice) and nontransgenic (0.056 +/- 0.029 min(-1); n = 17 mice) animals despite a 2-fold increase in microvascular area in the former compared with the latter. DCE-MRI did detect a significant decrease in vascular permeability accompanying diminution of dysplastic microvasculature by the antiangiogenic agent, vascular endothelial growth factor Trap (K(trans) = 0.052 +/- 0.013 min(-1) pretreatment; n = 6 mice versus K(trans) = 0.019 +/- 0.008 min(-1) post-treatment; n = 5 mice). Thus, we determined that the threshold of microvessel leakage associated with cervical dysplasia was <17 kDa and highlighted the potential of DCE-MRI to noninvasively monitor the efficacy of antiangiogenic drugs or chemoprevention regimens targeting the vasculature in premalignant cervical dysplasia.