Safety of assisted reproductive techniques in gynecological cancer patients.

PURPOSE OF REVIEW: Gynecological cancer is a very important cause of comorbidity and mortality in women. The current delay in motherhood is increasing the incidence of women under 40 years of age that have not yet achieved their maternity goals when they are diagnosed and standard treatment negatively impacts the reproductive potential of cancer survivors. In this review, we update the information available about the safety of fertility-sparing treatments in young gynecological cancer patients, as well as the safety and efficacy of assisted reproductive techniques (ART) in such group. We also evaluate the long-term gynecological cancer risk in women requiring ART. RECENT FINDINGS: Although eligibility criteria continue to be very strict, there are more and more reports of fertility-sparing approaches outside of what traditionally has been considered safe. Molecular assessment is starting to be used in the selection of appropriate candidates. Data increasingly shows the long term safety and the efficacy of ART and pregnancy in these patients. SUMMARY: Appropriate selection is key to safely preconize fertility-sparing alternatives. Because subfertility may be a result of these procedures, ART could be indicated in this setting. Neither ART nor pregnancy appear to increase recurrences or affect survival rates.

Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation.

The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins.

Deciphering the therapeutical potentials of rosmarinic acid.

Lemon balm is herbal tea used for soothing stomach cramps, indigestion, and nausea. Rosmarinic acid (RA) is one of its chemical constituents known for its therapeutic potentials against cancer, inflammatory and neuronal diseases such as the treatment of neurofibromatosis or prevention from Alzheimer's diseases (AD). Despite efforts, recovery and purification of RA in high yields has not been entirely successful. Here, we report its aqueous extraction with optimal conditions and decipher the structure by nuclear magnetic resonance (NMR) spectroscopy. Using various physical-chemical and biological assays, we highlight its anti-aggregation inhibition potentials against the formation of Tau filaments, one of the hallmarks of AD. We then examine its anti-cancer potentials through reduction of the mitochondrial reductase activity in tumor cells and investigate its electrochemical properties by cyclic voltammetry. Our data demonstrates that RA is a prominent biologically active natural product with therapeutic potentials for drug discovery in AD, cancer therapy and inflammatory diseases.

Peptide Conjugates Derived from flg15, Pep13, and PIP1 That Are Active against Plant-Pathogenic Bacteria and Trigger Plant Defense Responses.

Thirty peptide conjugates were designed by combining an antimicrobial peptide (BP16, BP100, BP143, KSL-W, BP387, or BP475) at the N- or C-terminus of a plant defense elicitor peptide (flg15, BP13, Pep13, or PIP1). These conjugates were highly active in vitro against six plant-pathogenic bacteria, especially against Xanthomonas arboricola pv. pruni, Xanthomonas fragariae and Xanthomonas axonopodis pv. vesicatoria. The most active peptides were those incorporating Pep13. The order of the conjugation influenced the antibacterial activity and the hemolysis. Regarding the former, peptide conjugates incorporating the elicitor peptide flg15 or Pep13 at the C-terminus were, in general, more active against Pseudomonas syringae pv. actinidiae and P. syringae pv. syringae, whereas those bearing these elicitor peptides at the N-terminus displayed higher activity against Erwinia. amylovora and the Xanthomonas species. The best peptide conjugates displayed MIC values between 0.8 and 12.5 µM against all the bacteria tested and also had low levels of hemolysis and low phytotoxicity. Analysis of the structural and physicochemical parameters revealed that a positive charge ranging from +5 to +7 and a moderate hydrophobic moment/amphipathic character is required for an optimal biological profile. Interestingly, flg15-BP475 exhibited a dual activity, causing the upregulation of the same genes as flg15 and reducing the severity of bacterial spot in tomato plants with a similar or even higher efficacy than copper oxychloride. Characterization by nuclear magnetic resonance (NMR) of the secondary structure of flg15-BP475 showed that residues 10 to 25 fold into an α-helix. This study establishes trends to design new bifunctional peptides useful against plant diseases caused by plant-pathogenic bacteria. IMPORTANCE The consequences of plant pathogens on crop production together with the lack of effective and environmentally friendly pesticides evidence the need of new agents to control plant diseases. Antimicrobial and plant defense elicitor peptides have emerged as good candidates to tackle this problem. This study focused on combining these two types of peptides into a single conjugate with the aim to potentiate the activity of the individual fragments. Differences in the biological activity of the resulting peptide conjugates were obtained depending on their charge, amphipathicity, and hydrophobicity, as well as on the order of the conjugation of the monomers. This work provided bifunctional peptide conjugates able to inhibit several plant-pathogenic bacteria, to stimulate plant defense responses, and to reduce the severity of bacterial spot in tomato plants. Thus, this study could serve as the basis for the development of new antibacterial/plant defense elicitor peptides to control bacterial plant pathogens.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

Purpose: We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)-derived cells to the neurosensory retina (NSR) and RPE layer. Methods: GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 µm) with 30 applications were placed 50 to 100 µm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results: Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle-dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions: SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle-dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Hematopoietic stem/progenitor involvement in retinal microvascular repair during diabetes: Implications for bone marrow rejuvenation.

The widespread nature of diabetes affects all organ systems of an individual including the bone marrow. Long-term damage to the cellular and extracellular components of the bone marrow leads to a rapid decline in the bone marrow-hematopoietic stem/progenitor cells (HS/PCs) compartment. This review will highlight the importance of bone marrow microenvironment in maintaining bone marrow HS/PC populations and the contribution of these key populations in microvascular repair during the natural history of diabetes. The autonomic nervous system can initiate and propagate bone marrow dysfunction in diabetes. Systemic pharmacological strategies designed to protect the bone marrow-HS/PC population from diabetes induced-oxidative stress and advanced glycation end product accumulation represent a new approach to target diabetic retinopathy progression. Protecting HS/PCs ensures their participation in vascular repair and reduces the risk of vasogdegeneration occurring in the retina.

Fragment-Based NMR Study of the Conformational Dynamics in the bHLH Transcription Factor Ascl1.

The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.

Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury.

The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.

CX3CR1 deficiency accelerates the development of retinopathy in a rodent model of type 1 diabetes.

In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild-type (WT), CX3CR1 null (CX3CR1gfp/gfp, KO), and heterozygous (CX3CR1+/gfp, Het) mice were compared in the presence and absence of streptozotocin (STZ)-induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. The induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose, they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10. KEY MESSAGES: • CX3CR1 deletion in STZ-diabetic mice accelerated the onset of diabetic retinopathy (DR). • The early onset of DR was associated with increased retinal cell apoptosis. • The early onset of DR was associated with increased recruitment of bone marrow-derived macrophages to the retina. • Bone marrow-derived macrophages from CX3CR1 KO diabetic mice expressed more TNF-α and less IL-10. • The role of IL-10 in protection from progression of DR is highlighted.

Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice.

Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system.

Ataxia Telangiectasia Mutated Dysregulation Results in Diabetic Retinopathy.

Ataxia telangiectasia mutated (ATM) acts as a defense against a variety of bone marrow (BM) stressors. We hypothesized that ATM loss in BM-hematopoietic stem cells (HSCs) would be detrimental to both HSC function and microvascular repair while sustained ATM would be beneficial in disease models of diabetes. Chronic diabetes represents a condition associated with HSC depletion and inadequate vascular repair. Gender mismatched chimeras of ATM(-/-) on wild type background were generated and a cohort were made diabetic using streptozotocin (STZ). HSCs from the STZ-ATM(-/-) chimeras showed (a) reduced self-renewal; (b) decreased long-term repopulation; (c) depletion from the primitive endosteal niche; (d) myeloid bias; and (e) accelerated diabetic retinopathy (DR). To further test the significance of ATM in hematopoiesis and diabetes, we performed microarrays on circulating angiogenic cells, CD34(+) cells, obtained from a unique cohort of human subjects with long-standing (>40 years duration) poorly controlled diabetes that were free of DR. Pathway analysis of microarrays in these individuals revealed DNA repair and cell-cycle regulation as the top networks with marked upregulation of ATM mRNA compared with CD34(+) cells from diabetics with DR. In conclusion, our study highlights using rodent models and human subjects, the critical role of ATM in microvascular repair in DR.

Loss of survival factors and activation of inflammatory cascades in brain sympathetic centers in type 1 diabetic mice.

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter (P<0.05) and reduced neuronal expression of IGFBP-3 (-32%, P<0.05) and IGF-I (-15%, P<0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P<0.01), and IDO⁺ cell density rose by (62%, P<0.05). CD39 expression dropped by 30% (P<0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized (P<0.05). Our results suggest that increased IDO and early loss of CD39⁺ protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.

CNS inflammation and bone marrow neuropathy in type 1 diabetes.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

Circulating mononuclear progenitor cells: differential roles for subpopulations in repair of retinal vascular injury.

PURPOSE: We examined effect on retinal vascular homing of exogenous CD34(+) and CD14(+) progenitor cells using mouse models of chronic (streptozotocin [STZ]-induced diabetes) and acute (ischemia-reperfusion [I/R]) ocular vascular injury. METHODS: STZ-treated mice of short or long duration (≤4, ≥11 months) diabetes, along with age- and sex-matched controls, were given intravitreous injections of human CD34(+) and CD14(+) cells isolated from healthy or diabetic donors alone or in combination. I/R injured mice were given diabetic or nondiabetic CD34(+) cells with mesenchymal stem cells (MSCs) or diabetic CD34(+) cells manipulated by ex vivo fucosylation with ASC-101. Injected cells were localized by fluorescent immunocytochemistry, and the degree of retinal vascular colocalization quantified morphometrically. Permeability was assessed by fluorescent albumin leakage. RESULTS: Diabetic CD14(+) cells associated with vessels to a greater degree than diabetic CD34(+) cells. Vascular permeability was reduced only by nondiabetic cells and only at the highest number of cells tested. Diabetic CD34(+) cells consistently demonstrated reduced migration. There was a 2-fold or 4-fold increase over control in the specific localization of diabetic CD34(+) cells within the vasculature when these cells were co-administered with MSCs or ex vivo fucosylated prior to injection, respectively. CONCLUSIONS: Diabetic CD14(+) cells, unlike diabetic CD34(+) cells, retain robust homing characteristics. CD34(+) or CD14(+) subsets rather than whole bone marrow or peripheral blood cells may prove more beneficial in autologous cell therapy for diabetics. Co-administration with MSCs or ex vivo fucosylation may enhance utility of CD34(+) cells in cell therapy for diabetic ocular conditions like macular ischemia and retinal nonperfusion.

Activation of the ACE2/angiotensin-(1-7)/Mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors.

We tested the hypothesis that activation of the protective arm of the renin angiotensin system, the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(1-7) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(1-7) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(1-7) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(1-7)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Bone marrow-CNS connections: implications in the pathogenesis of diabetic retinopathy.

Diabetic retinopathy is the fourth most common cause of blindness in adults. Current therapies, including anti-VEGF therapy, have partial efficacy in arresting the progression of proliferative diabetic retinopathy and diabetic macular edema. This review provides an overview of a novel, innovative approach to viewing diabetic retinopathy as the result of an inflammatory cycle that affects the bone marrow (BM) and the central and sympathetic nervous systems. Diabetes associated inflammation may be the result of BM neuropathy which skews haematopoiesis towards generation of increased inflammatory cells but also reduces production of endothelial progenitor cells responsible for maintaining healthy endothelial function and renewal. The resulting systemic inflammation further impacts the hypothalamus, promoting insulin resistance and diabetes, and initiates an inflammatory cascade that adversely impacts both macrovascular and microvascular complications, including diabetic retinopathy (DR). This review examines the idea of using anti-inflammatory agents that cross not only the blood-retinal barrier to enter the retina but also have the capability to target the central nervous system and cross the blood-brain barrier to reduce neuroinflammation. This neuroinflammation in key sympathetic centers serves to not only perpetuate BM pathology but promote insulin resistance which is characteristic of type 2 diabetic patients (T2D) but is also seen in T1D. A case series of morbidly obese T2D patients with retinopathy and neuropathy treated with minocycline, a well-tolerated antibiotic that crosses both the blood-retina and blood-brain barrier is presented. Our results indicates that minocycine shows promise for improving visual acuity, reducing pain from peripheral neuropathy, promoting weight loss and improving blood pressure control and we postulate that these observed beneficial effects are due to a reduction of chronic inflammation.

Long-term effects of intravitreal injection of GMP-grade bone-marrow-derived CD34+ cells in NOD-SCID mice with acute ischemia-reperfusion injury.

PURPOSE: To determine long-term safety of intravitreal administration of good manufacturing practice (GMP)-grade human bone-marrow-derived CD34(+) cells in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mice with acute retinal ischemia-reperfusion injury, a model for retinal vasculopathy. METHOD: Acute ischemia-reperfusion injury was induced in the right eye of adult NOD-SCID mice (n = 23) by transient elevation of intraocular pressure. Seven days later, 12 injured eyes and 5 normal contralateral eyes were injected each intravitreally with 5 × 10(4) CD34(+) cells isolated under GMP conditions from a healthy human donor bone marrow using an immunomagnetic cell isolation system. The remaining 11 injured eyes were not treated and served as controls. Mice were euthanized 1 day, 4 months, and 8 months later. Both eyes were enucleated and examined by immunohistochemical analysis and hematoxylin and eosin staining. Among mice followed for 8 months, electroretinography (ERG) was performed on both eyes before euthanization. All major organs were examined grossly and histologically after serial sectioning. RESULTS: Immunohistochemical staining 4 months after injection showed detectable CD34(+) cells in the retinal vasculature. ERG at 8 months after CD34(+) cell injection showed signals that were similar in untreated eyes. Histology of the enucleated eyes injected with CD34(+) cells showed no intraocular tumor or abnormal tissue growth after 8 months. Histologic analysis of all major organs showed no abnormal proliferation of human cells. CONCLUSIONS: Intravitreal administration of GMP-grade human bone-marrow-derived CD34(+) cells appears to be well tolerated long-term in eyes with acute retinal ischemic injury. A clinical trial will start to further explore this therapy.

γ-Secretase inhibition of murine choroidal neovascularization is associated with reduction of superoxide and proinflammatory cytokines.

PURPOSE: This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines. METHODS: γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA. RESULTS: γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis. CONCLUSIONS: γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV.

PEDF regulates vascular permeability by a γ-secretase-mediated pathway.

Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and ß-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and ß-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability.

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.