Bone marrow (BM) derived vascular reparative cells, myeloid angiogenic cells (MACs), work with vascular wall derived cells, endothelial colony forming cells (ECFCs), to orchestrate vessel repair. Both populations of cells are found in the circulation making their identification at times problematic. Importantly, the circulating levels of both ECFCs and MACs reflect vascular injury and disease. In humans, MACs are often identified by the surface marker CD34. Circulating levels of CD34+ cells are reduced following coronary angioplasty, acute coronary syndromes, unstable angina, and in individuals with diabetic complications including diabetic retinopathy (DR). The aim of this chapter is to summarize the literature pertinent to the role of MACs and ECFCs in vascular repair specifically in the eye with the goal of understanding the potential of these cells as treatment for retinal degenerative diseases.

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.

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.

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.

We recently found indicators of hypothalamic inflammation and neurodegeneration linked to the loss of neuroprotective factors including insulin-like growth factor (IGF-1) and IGF binding protein-2 (IGFBP-3) in mice made diabetic using streptozotocin (STZ). In the current work, a genetic model of type-1 diabetes (Ins2(Akita) mouse) was used to evaluate changes in neuronal activity and concomitant changes in the proinflammatory mediator high-mobility group box-1 (HMBG1). We found basal hypothalamic neuronal activity as indicated by manganese-enhanced magnetic resonance imaging (MEMRI) was significantly decreased in 8 months old, but not 2 months old Ins2(Akita) diabetic mice compared to controls. In tissue from the same animals we evaluated the expression of HMBG1 using immunohistochemistry and confocal microscopy. We found decreased HMBG1 nuclear localization in the paraventricular nucleus of the hypothalamus (PVN) in 8 months old, but not 2 months old diabetic animals indicating nuclear release of the protein consistent with an inflammatory state. Adjacent thalamic regions showed little change in HMBG1 nuclear localization and neuronal activity as a result of diabetes. This work extends our previous findings demonstrating changes consistent with hypothalamic neuroinflammation in STZ treated animals, and shows active inflammatory processes are correlated with changes in basal hypothalamic neuronal activity in Ins2(Akita) 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.

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.

We recently found indicators of hypothalamic inflammation and neurodegeneration linked to the loss of neuroprotective factors including insulin-like growth factor (IGF-1) and IGF binding protein-2 (IGFBP-3) in mice made diabetic using streptozotocin (STZ). In the current work, a genetic model of type-1 diabetes (Ins2 mouse) was used to evaluate changes in neuronal activity and concomitant changes in the proinflammatory mediator high-mobility group box-1 (HMBG1). We found basal hypothalamic neuronal activity as indicated by manganeseenhanced magnetic resonance imaging (MEMRI) was significantly decreased in 8 month old, but not 2 month old Ins2 diabetic mice compared to controls. In tissue from the same animals we evaluated the expression of HMBG1 using immunohistochemistry and confocal microscopy. We found decreased HMBG1 nuclear localization in the paraventricular nucleus of the hypothalamus (PVN) in 8 month old, but not 2 month old diabetic animals indicating nuclear release of the protein consistent with an inflammatory state. Adjacent thalamic regions showed little change in HMBG1 nuclear localization and neuronal activity as a result of diabetes. This work extends our previous findings demonstrating changes consistent with hypothalamic neuroinflammation in STZ treated animals, and shows active inflammatory processes are correlated with changes in basal hypothalamic neuronal activity in Ins2 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.

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.

Program Number: 3014 Presentation Time: 11:00 AM–11:15 AM Changes in retinal vessel caliber with flicker light stimulation in eyes with diabetic retinopathy Laurence S. Lim1, 2, Peng Guan Ong1, E SHyong Tai1, 2, Gemmy C. Cheung1, Wallace S. Foulds1, Tien Y. Wong1, 2. 1Ophthalmology, Singapore National Eye Center, Singapore, Singapore; 2Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Purpose: Changes in retinal vessel calibre in response to flickering light are believed to be mediated by nitric oxide release from the retinal microvascular endothelium. This study investigated the responses of retinal vessels to flickering light in diabetic patients with various grades of diabetic retinopathy(DR). Methods: This cross-sectional observational study evaluated adult subjects with diabetes mellitus. The Dynamic Vessel Analyser (DVA) was used to measure retinal vascular responses to diffuse illuminance flicker. DR was graded from retinal photography. Each eye was assigned a retinopathy severity score according to the modified Airlie House classification system, and categorized as minimal nonproliferative diabetic retinopathy (NPDR), mild NPDR, moderate NPDR, severe NPDR, or proliferative retinopathy. Eyes were also classified as having any DR (minimal NPDR or worse), moderate DR (moderate NPDR or worse), or vision-threatening DR (severe NPDR or worse, or clinically significant macular edema) according to the Eye Diseases Prevalence Research Group definitions. Results: There were 279 subjects in total, with a mean age of 59.9±9.2 years. The majority were male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7±1.4% and 13.9±10.4 years respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension and hyperlipidemia, retinal arteriolar and venular dilation responses to flicker stimulation decreased continuously with increasing severity of diabetic retinopathy.(p = 0.008 and <0.001 respectively). Subjects with reduced arteriolar dilation responses were more likely to have any DR [odds ratio (OR) 1.20 (95% confidence interval 1.01 – 1.45) per standard deviation (SD) decrease, p=0.045]. Subjects with reduced venular dilation responses were more likely to have any DR [OR 1.27(1.04 – 1.53) per SD decrease, p=0.02], moderate DR [OR 1.27 (1.06 – 1.49) per SD decrease, p = 0.007] and vision-threatening DR [OR 1.51(1.14 – 1.50) per SD decrease, p = 0.002]. Conclusions: Retinal arteriolar and venular dilation responses to flickering light are diminished in subjects with DR, and decrease progressively with more severe stages of DR. Our findings suggest that the severity of DR is correlated with measurable differences in retinal microvascular endothelial function, supporting a role for the latter in the pathogenesis of DR. Commercial Relationships: Laurence S. Lim, None; Peng Guan Ong, None; E SHyong Tai, None; Gemmy C. Cheung, None; Wallace S. Foulds, None; Tien Y. Wong, None Support: NMRC grant number R710/60/2009 Program Number: 3015 Presentation Time: 11:15 AM–11:30 AM Amacrine cell-derived VEGF is required for development and maintenance of the retinal vasculature in mice Yoshihiko Usui, Toshihide Kurihara, Peter D. Westenskow, Edith Aguilar, Liliana P. Paris, Stacey K. Moreno, Carli M. Wittgrove, Daniel Feitelberg, Martin Friedlander. Cell Biology, The Scripps Research Institute, San Diego, CA. Purpose: The retinal vasculature of many organisms including humans and mice consists of three distinct plexus layers. While it is clear that the inner retinal vascular layer develops over a pre-existing astrocytic network and that development of vascular and neuronal networks are co-dependent, it is unclear how the outer retinal vascular networks form. As retinal neurons populate the retina and mature, oxygen demands change and activation of the oxygen sensing VHL/HIF-α/VEGF pathway in maturing neurons may be a strong driving force for development and maintenance of the outer plexus layers. In this study, we examined the contribution of amacrine and horizontal cells due to their close proximity to the intermediate and outer retinal vascular layers. Methods: Transgenic mice expressing Cre recombinase specifically in amacrine and horizontal cells (Ptf1a-Cre mice) were mated with floxed VHL, HIF-1α, HIF-2α and/or VEGF mice to generate conditional knockouts. Amacrine and horizontal cells were genetically ablated using Ptf1a-Cre and forced expression of diphtheria toxin (DT) receptors. Results: We show that amacrine and horizontal cell processes tightly associate with intermediate and outer plexus retinal capillaries. Pseudo-hypoxia in Ptf1a-Cre; VHL mutants induces formation of a dense intermediate plexus compared to controls, while a dramatically attenuated intermediate plexus is observed in Ptf1a-Cre; VEGF and Ptf1a-Cre; HIF-1α mutants. Co-deletion of HIF-1α, but not HIF-2α, rescued the vascular phenotypes of Ptf1a-Cre; VHL KO mice. Amacrine and horizontal cell ablation by DT injection also suppressed the formation of the intermediate plexus and DT injection after the retinal vasculature had developed resulted in attenuation of the vasculature. In all of these genetic manipulations the deep plexus was less affected. Conclusions: Dysregulated VEGF release from amacrine and horizontal cells results in formation of a very dense intermediate vascular plexus, while elimination of VEGF (or of amacrine and horizontal cells themselves) prevents its formation. These data demonstrate a novel function of amacrine cells, directing formation of the intermediate plexus layer. Horizontal cells, on the other hand, are likely strictly dependent on the vasculature, but do not determine its formation or maintenance. Commercial Relationships: Yoshihiko Usui, None; Toshihide Kurihara, None; Peter D. Westenskow, None; Edith Aguilar, None; Liliana P. Paris, None; Stacey K. Moreno, None; Carli M. Wittgrove, None; Daniel Feitelberg, None; Martin Friedlander, None Support: The Lowy Medical Research Institute and EY11254

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 toss 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.

We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.