Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes.

BACKGROUND: We examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes (T1D). METHODS: T1D individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and peptidoglycan), and Ang II (angiotensin II); Akita mice were orally administered a Lactobacillus paracasei (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. Akita mice with genetic overexpression of humanAce2 by small intestine epithelial cells (Vil-Cre.hAce2KI-Akita) were similarly examined. After 9 months of T1D, circulatory, enteral, and ocular end points were assessed. RESULTS: T1D subjects exhibit elevations in gut-derived circulating immune cells (ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3ß (glycogen synthase kinase-3 beta)/c-Myc (cellular myelocytomatosis oncogene)-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis. CONCLUSIONS: Dysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In Akita mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.

Glial, Neuronal, Vascular, Retinal Pigment Epithelium, and Inflammatory Cell Damage in a New Western Diet-Induced Primate Model of Diabetic Retinopathy.

This study investigated retinal changes in a Western diet (WD)-induced nonhuman primate model of type 2 diabetes. Rhesus nonhuman primates, aged 15 to 17 years, were fed a high-fat diet (n = 7) for >5 years reflective of the traditional WD. Age-matched controls (n = 6) were fed a standard laboratory primate diet. Retinal fundus photography, optical coherence tomography, autofluorescence imaging, and fluorescein angiography were performed before euthanasia. To assess diabetic retinopathy (DR), eyes were examined using trypsin digests, lipofuscin autofluorescence, and multimarker immunofluorescence on cross-sections and whole mounts. Retinal imaging showed venous engorgement and tortuosity, aneurysms, macular exudates, dot and blot hemorrhages, and a marked increase in fundus autofluorescence. Post-mortem changes included the following: decreased CD31 blood vessel density (P < 0.05); increased acellular capillaries (P < 0.05); increased density of ionized calcium-binding adaptor molecule expressing amoeboid microglia/macrophage; loss of regular distribution in stratum and spacing typical of ramified microglia; and increased immunoreactivity of aquaporin 4 and glial fibrillary acidic protein (P < 0.05). However, rhodopsin immunoreactivity (P < 0.05) in rods and neuronal nuclei antibody-positive neuronal density of 50% (P < 0.05) were decreased. This is the first report of a primate model of DR solely induced by a WD that replicates key features of human DR.

ß-secretase 1 overexpression by AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration.

We reported previously that ß-site amyloid precursor protein cleaving enzyme (BACE1) is strongly expressed in the normal retina and that BACE1-/- mice develop pathological phenotypes associated with age-related macular degeneration (AMD). BACE1 expression is increased within the neural retina and retinal pigment epithelium (RPE) in AMD donor eyes suggesting that increased BACE1 is compensatory. We observed that AAV-mediated BACE1 overexpression in the RPE was maintained up to 6 months after AAV1-BACE1 administration. No significant changes in normal mouse visual function or retinal morphology were observed with low-dose vector while the high-dose vector demonstrated some early pathology which regressed with time. No increase in ß-amyloid was observed. BACE1 overexpression in the RPE of the superoxide dismutase 2 knockdown (SOD2 KD) mouse, which exhibits an AMD-like phenotype, prevented loss of retinal function and retinal pathology, and this was sustained out to 6 months. Furthermore, BACE1 overexpression was able to inhibit oxidative stress, microglial changes, and loss of RPE tight junction integrity (all features of AMD) in SOD2 KD mice. In conclusion, BACE1 plays a key role in retina/RPE homeostasis, and BACE1 overexpression offers a novel therapeutic target in the treatment of AMD.

Sulforaphane recovers cone function in an Nrf2-dependent manner in middle-aged mice undergoing RPE oxidative stress.

Purpose: Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has therapeutic efficacy in numerous animal models of human disease, including mouse models of retinal degeneration. However, despite dozens of clinical trials, the compound remains to be tested as a clinical treatment for ocular disease. Numerous cellular activities of SFN have been identified, including the activation of Nrf2, a transcription factor that induces a battery of target gene products to neutralize oxidative and xenobiotic stresses. As Nrf2 expression and function reportedly decrease with aging, we tested whether the loss of the transcription factor limits the therapeutic efficacy of SFN against retinal degeneration. Methods: Six- to 8-month-old wild-type and Nrf2 knockout mice were treated with SFN beginning 1 month after ribozyme-mediated knockdown of superoxide dismutase 2 (SOD2) mRNA in the RPE. The impacts of MnSOD (the protein product of SOD2) knockdown and the efficacy of SFN were evaluated using a combination of electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT), and postmortem histology. Results: SFN restored the ERG photopic b-wave suppressed by MnSOD loss in wild-type mice, but not in the Nrf2 knockout mice. In contrast, ERG scotopic a- and b-wave loss was not restored for either genotype. SFN significantly improved retinal thickness in the Nrf2 knockout mice with MnSOD knockdown, but this was not observed in the wild-type mice. In both genotypes, SFN treatment reduced morphological markers of RPE atrophy and degeneration, although these improvements did not correlate proportionally with functional recovery. Conclusions: These findings highlight the capacity of SFN to preserve cone function, as well as the potential challenges of using the compound as a standalone treatment for age-related retinal degeneration under conditions associated with reduced Nrf2 function.

Decreased Expression of Soluble Epoxide Hydrolase Suppresses Murine Choroidal Neovascularization.

Neovascular or "wet" age-related macular degeneration (nAMD) is a leading cause of blindness among older adults. Choroidal neovascularization (CNV) is a major pathological feature of nAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible vision loss. There is a critical need to develop novel therapeutic strategies to address limitations of the current anti-vascular endothelial growth factor biologics. Previously, we identified soluble epoxide hydrolase (sEH) as a possible therapeutic target for CNV through a forward chemical genetic approach. The purpose of this study was to validate sEH as a target by examining retinal expression of sEH protein and mRNA by immunohistochemistry and RNAscope in situ hybridization, respectively, and to assess the efficacy of an adeno-associated virus (AAV) vector designed to knock down the sEH gene, Ephx2, in the murine laser-induced (L-) CNV model. nAMD patient postmortem eye tissue and murine L-CNV showed overexpression of sEH in photoreceptors and retinal pigment epithelial cells. Ephx2 knockdown significantly reduced CNV and normalized mRNA expression levels of CNV-related inflammatory markers. Thus, this study further establishes sEH as a promising therapeutic target against CNV associated with nAMD.

Supplemental nitrite increases choroidal neovascularization in mice.

Low doses of nitrite, close to physiological levels, increase blood flow in normal and ischemic tissues through a nitric oxide (NO) dependent mechanism. Given that nitrite therapy and dietary supplementation with vegetables high in nitrate (e.g. beets) are gaining popularity we decided to determine if low doses of nitrite impact the development of choroidal neovascularization (CNV), a key feature of wet age related macular degeneration (AMD). Sodium nitrite (at 50 mg/L, 150 mg/L, and 300 mg/L), nitrate (1 g/L) or water alone were provided in the drinking water of C57BL/6 J mice aged 2 or 12 months. Mice were allowed to drink ad libitum for 1 week at which time laser-induced choroidal neovascularization (L-CNV) was induced. The mice continued to drink the supplemented water ad libitum for a further 14 days at which point optical coherence tomography (OCT) was performed to determine the volume of the CNV lesion. Blood was drawn to determine nitrite and nitrate levels and eyes taken for histology. CNV volume was 2.86 × 107 µm3 (±0.4 × 107) in young mice on water alone but CNV volume more than doubled to >6.9 × 107 µm3 (±0.8 × 107) in mice receiving 300 mg/L nitrite or 7.34 × 107 µm3 (±1.4 × 107) in 1 g/L nitrate (p < 0.01). A similar trend was observed in older mice. CNV volume was 5.3 × 107 µm3 (±0.5 × 107) in older mice on water alone but CNV volume almost doubled to approximately 9.3 × 107 µm3 (±1.1 × 107) in mice receiving 300 mg/L nitrite or 8.7 × 107 µm3 (±0.9 × 107) 1 g/L nitrate (p < 0.01). Plasma nitrite levels were highest in young mice receiving 150 mg/L in the drinking water with no changes in plasma nitrate observed. In older mice, drinking water nitrite did not significantly change plasma nitrite, but plasma nitrate was increased. Plasma nitrate was elevated in both young and old mice provided with nitrate supplemented drinking water. Our data demonstrate that the CNV lesion is larger in older mice compared to young and that therapeutic levels of oral nitrite increase the volume of CNV lesions in both young and older mice. Therapeutic nitrite or nitrate supplementation should be used with caution in the elderly population prone to CNV.

Potential role for age as a modulator of oral nitrate reductase activity.

Reduction of salivary nitrate to nitrite by oral nitrate reductase (NR) expressing bacteria has emerged as an integral pathway in regulating nitric oxide (NO) homeostasis and signaling. The oral microbiome is critical for this pathway. Variations in this pathway may underlie variable responses in the magnitude by which dietary or therapeutic nitrate modulates NO-signaling. The relationships between oral microbes and NR activity, and the factors that affect this relationship remain unclear however. Using a cross-sectional study design, the objective of this study was to determine the relationships between oral microbes and oral NR activity using a protocol that directly measures initial NR activity. Tongue swabs were collected from 28 subjects ranging in age from 21 to 73y. Initial NR activity showed a bell-shaped dependence with age, with activity peaking at ~40-50y and being lower but similar between younger (20-30y) and older (51-73) individuals. Microbiome relative abundance and diversity analyses, using 16s sequencing, demonstrated differences across age and identified both NR expressing and non-expressing bacteria in modulating initial NR activity. Finally, initial NR activity was measured in 3mo and 13mo old C57BL/6J mice. No differences in bacterial number were observed. However initial NR activity was significantly (80%) lower in 13mo old mice. Collectively, these data suggest that age is a variable in NR activity and may modulate responsiveness to dietary nitrate.

Characterizing temporal and spatial recruitment of systemically administered RPE65-programmed bone marrow-derived cells to the retina in a mouse model of age-related macular degeneration.

PURPOSE: Previously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer. METHODS: C57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP+ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP+ RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP+ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections. RESULTS: GFP+ RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP+ BMDC into the RPE layer and that these GFP+ human RPE65 expressing cells co-localized with murine RPE65. No GFP+ cells were observed in the neural retina or RPE layer of normal uninjured control eyes. CONCLUSIONS: Our study shows that systemically administered GFP+ RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

Systemic Injection of RPE65-Programmed Bone Marrow-Derived Cells Prevents Progression of Chronic Retinal Degeneration.

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.

A Non-Canonical Role for ß-Secretase in the Retina.

It has long been established that ß-Secretase (BACE) plays a critical role in the formation of amyloid plaques in Alzheimer's Disease patients, but it is only recently that the importance of ß-secretases in retinal pathophysiology has been recognized. BACE expression is elevated in response to stress, and downregulation results in lysosomal abnormalities and mitochondrial changes. Inhibition of BACE can lead to reduced retinal function, retinal thinning, lipofuscin accumulation and vascular dysfunction in mice. Furthermore, BACE inhibition accelerates choroidal neovascularization (CNV) in mice. We propose that BACE plays an important role in retinal homeostasis and that BACE upregulation in response to stress is a protective measure.

Pathogenic role of diabetes-induced PPAR-α down-regulation in microvascular dysfunction.

Two independent clinical studies have reported that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on microvascular complications of diabetes, including diabetic retinopathy (DR) in type 2 diabetic patients. However, the expression and function of PPARα in the retina are unclear. Here, we demonstrated that PPARα is expressed in multiple cell types in the retina. In both type 1 and type 2 diabetes models, expression of PPARα, but not PPARß/δ or PPARγ, was significantly down-regulated in the retina. Furthermore, high-glucose medium was sufficient to down-regulate PPARα expression in cultured retinal cells. To further investigate the role of PPARα in DR, diabetes was induced in PPARα knockout (KO) mice and wild-type (WT) mice. Diabetic PPARα KO mice developed more severe DR, as shown by retinal vascular leakage, leukostasis, pericyte loss, capillary degeneration, and over-expression of inflammatory factors, compared with diabetic WT mice. In addition, overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation. Furthermore, PPARα overexpression inhibited endothelial cell migration and proliferation. These findings revealed that diabetes-induced down-regulation of PPARα plays an important role in DR. Up-regulation or activation of PPARα may represent a novel therapeutic strategy for DR.

Aryl hydrocarbon receptor deficiency causes dysregulated cellular matrix metabolism and age-related macular degeneration-like pathology.

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that regulates xenobiotic metabolism and detoxification. Herein, we report a previously undescribed role for the AhR signaling pathway as an essential defense mechanism in the pathogenesis of early dry age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We found that AhR activity and protein levels in human retinal pigment epithelial (RPE) cells, cells vulnerable in AMD, decrease with age. This finding is significant given that age is the most established risk factor for development of AMD. Moreover, AhR(-/-) mice exhibit decreased visual function and develop dry AMD-like pathology, including disrupted RPE cell tight junctions, accumulation of RPE cell lipofuscin, basal laminar and linear-like deposit material, Bruch's membrane thickening, and progressive RPE and choroidal atrophy. High-serum low-density lipoprotein levels were also observed in AhR(-/-) mice. In its oxidized form, this lipoprotein can stimulate increased secretion of extracellular matrix molecules commonly found in deposits from RPE cells, in an AhR-dependent manner. This study demonstrates the importance of cellular clearance via the AhR signaling pathway in dry AMD pathogenesis, implicating AhR as a potential target, and the mouse model as a useful platform for validating future therapies.

CD82 restrains pathological angiogenesis by altering lipid raft clustering and CD44 trafficking in endothelial cells.

BACKGROUND: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. METHODS AND RESULTS: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. CONCLUSIONS: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes.

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0-200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.

Targeting the IRE1α/XBP1 and ATF6 arms of the unfolded protein response enhances VEGF blockade to prevent retinal and choroidal neovascularization.

Although anti-vascular endothelial growth factor (VEGF) treatments reduce pathological neovascularization in the eye and in tumors, the regression is often not sustainable or is incomplete. We investigated whether vascular endothelial cells circumvent anti-VEGF therapies by activating the unfolded protein response (UPR) to override the classic extracellular VEGF pathway. Exposure of endothelial cells to VEGF, high glucose, or H2O2 up-regulated the X-box binding protein-1/inositol-requiring protein-1 (IRE1) α and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in α-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1α, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1α, ATF6, or CRYAB resulted in an approximately 40% reduction of in vitro angiogenesis, which was further reduced in combination with a neutralizing antibody against extracellular VEGF. Blockade of IRE1α or ATF6 in the oxygen-induced retinopathy or choroidal neovascularization mouse models caused an approximately 35% reduction in angiogenesis. However, combination therapy of VEGF neutralizing antibody with UPR inhibitors or siRNAs reduced retinal/choroidal neovascularization by a further 25% to 40%, and this inhibition was significantly greater than either treatment alone. In conclusion, activation of the UPR sustains angiogenesis by preventing degradation of intracellular VEGF. The IRE1α/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis.

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.

Studying melanin and lipofuscin in RPE cell culture models.

The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols.