Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy.

Purpose: Myo/Nog cells are the source of myofibroblasts in the lens and synthesize muscle proteins in human epiretinal membranes (ERMs). In the current study, we examined the response of Myo/Nog cells during ERM formation in a mouse model of proliferative vitreoretinopathy (PVR). Methods: PVR was induced by intravitreal injections of gas and ARPE-19 cells. PVR grade was scored by fundus imaging, optical coherence tomography, and histology. Double label immunofluorescence localization was performed to quantify Myo/Nog cells, myofibroblasts, and leukocytes. Results: Myo/Nog cells, identified by co-labeling with antibodies to brain-specific angiogenesis inhibitor 1 (BAI1) and Noggin, increased throughout the eye with induction of PVR and disease progression. They were present on the inner surface of the retina in grades 1/2 PVR and were the largest subpopulation of cells in grades 3 to 6 ERMs. All α-SMA-positive (+) cells and all but one striated myosin+ cell expressed BAI1 in grades 1 to 6 PVR. Folds and areas of retinal detachment were overlain by Myo/Nog cells containing muscle proteins. Low numbers of CD18, CD68, and CD45+ leukocytes were detected throughout the eye. Small subpopulations of BAI1+ cells expressed leukocyte markers. ARPE-19 cells were found in the vitreous but were rare in ERMs. Pigmented cells lacking Myo/Nog and muscle cell markers were present in ERMs and abundant within the retina by grade 5/6. Conclusions: Myo/Nog cells differentiate into myofibroblasts that appear to contract and produce retinal folds and detachment. Targeting BAI1 for Myo/Nog cell depletion may be a pharmacological approach to preventing and treating PVR.

Acute Response and Neuroprotective Role of Myo/Nog Cells Assessed in a Rat Model of Focal Brain Injury.

Focal brain injury in the form of a needlestick (NS) results in cell death and induces a self-protective response flanking the lesion. Myo/Nog cells are identified by their expression of bone morphogenetic protein inhibitor Noggin, brain-specific angiogenesis inhibitor 1 (BAI1) and the skeletal muscle specific transcription factor MyoD. Myo/Nog cells limit cell death in two forms of retinopathy. In this study, we examined the acute response of Myo/Nog cells to a NS lesion that extended from the rat posterior parietal cortex to the hippocampus. Myo/Nog cells were identified with antibodies to Noggin and BAI1. These cells were the primary source of both molecules in the uninjured and injured brain. One day after the NS, the normally small population of Myo/Nog cells expanded approximately eightfold within a 1 mm area surrounding the lesion. Myo/Nog cells were reduced by approximately 50% along the lesion with an injection of the BAI1 monoclonal antibody and complement. The number of dying cells, identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), was unchanged at this early time point in response to the decrease in Myo/Nog cells. However, increasing the number of Myo/Nog cells within the lesion by injecting BAI1-positive (+) cells isolated from the brains of other animals, significantly reduced cell death and increased the number of NeuN+ neurons compared to brains injected with phosphate buffered saline or exogenous BAI1-negative cells. These findings demonstrate that Myo/Nog cells rapidly react to injury within the brain and increasing their number within the lesion is neuroprotective.

Myo/Nog cells are nonprofessional phagocytes.

Myo/Nog cells were discovered in the chick embryo epiblast. Their expression of MyoD reflects a commitment to the skeletal muscle lineage and capacity to differentiate into myofibroblasts. Release of Noggin by Myo/Nog cells is essential for normal morphogenesis. Myo/Nog cells rapidly respond to wounding in the skin and eyes. In this report, we present evidence suggesting that Myo/Nog cells phagocytose tattoo ink in tissue sections of human skin and engulf cell corpses in cultures of anterior human lens tissue and magnetic beads injected into the anterior chamber of mice in vivo. Myo/Nog cells are distinct from macrophages in the skin and eyes indicated by the absence of labeling with an antibody to ionized calcium binding adaptor molecule 1. In addition to their primary roles as regulators of BMP signaling and progenitors of myofibroblasts, Myo/Nog cells behave as nonprofessional phagocytes defined as cells whose primary functions are unrelated to phagocytosis but are capable of engulfment.

Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage.

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.

Myo/Nog cells expressing muscle proteins are present in preretinal membranes from patients with proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a complication of rhegmatogenous retinal detachment and ocular trauma. The disease is characterized by development of membranes that may apply traction to the retina and cause redetachment. Membrane contractions are attributed to myofibroblasts arising from retinal pigment epithelial cells, glia and fibroblasts. The progenitors of myofibrobasts in the lens are Myo/Nog cells that express the skeletal muscle transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. The retina and choroid also contain Myo/Nog cells that respond to stress. We examined preretinal PVR membranes from three ocular trauma patients with retinal detachment for Myo/Nog cells and their expression of muscle proteins. Myo/Nog cells were identified by co-localization of antibodies to the G8 antigen and Noggin. Greater than 80% of all cells in sections from two of three patients expressed both G8 and Noggin. Myo/Nog cells lacked pigment. Alpha smooth muscle actin (α-SMA) and striated myosin II heavy chain were present in the majority of Myo/Nog cells in these two patients. Differentiation of Myo/Nog cells was paralleled by low levels of MyoD. Membrane sections from the third patient consisted mostly of connective tissue with very few cells. A small subpopulation in these sections expressed both G8 and Noggin, and muscle proteins were detected in only a minority of G8-positive (+) cells. In all three patients, greater than 99% of cells with MyoD, α-SMA and striated muscle myosin co-expressed G8. These findings suggest that contractile myofibroblasts in PVR membranes may be derived from differentiating Myo/Nog cells.

Depletion of Myo/Nog Cells in the Lens Mitigates Posterior Capsule Opacification in Rabbits.

Purpose: Posterior capsule opacification (PCO) is a vision-impairing disease that occurs in some adults and most children after cataract surgery. Contractile myofibroblasts contribute to PCO by producing wrinkles in the lens capsule that scatter light. Myofibroblasts in the lens originate from Myo/Nog cells named for their expression of the MyoD transcription factor and bone morphogenetic protein inhibitor noggin. In this study we tested the effects of depleting Myo/Nog cells on development of PCO. Methods: Myo/Nog cells were eliminated by injecting the G8 antibody conjugated to 3DNA nanocarriers for the cytotoxin doxorubicin (G8:3DNA:Dox) during cataract surgery in rabbits. The severity of PCO was scored by slit lamp analysis, gross and histologic observation, and immunofluorescence localization of α-smooth muscle actin. Results: G8:3DNA:Dox specifically induced cell death in Myo/Nog cells in the lens. None of the lenses administered G8:3DNA containing 9 to 36 µM doxorubicin developed greater than trace levels of central PCO and few myofibroblasts were present on the capsule. Less than 9% of these lenses exhibited greater than mild levels of peripheral PCO. Doxorubucin itself reduced PCO; however, myofibroblasts and wrinkles were abundant in the lens, and off-target effects were observed in the ciliary processes and cornea. Conclusions: Myo/Nog cells are the primary source of myofibroblasts in the lens after cataract surgery. Targeted depletion of Myo/Nog cells has potential for preventing PCO and preserving vision.

Preclinical study of the long-range safety and anti-inflammatory effects of high-dose oral meglumine.

Meglumine is a methylamino derivative of sorbitol that is an approved drug excipient. Recent preclinical studies suggest that administration of high-dose oral meglumine can exert beneficial medicinal effects to treat diabetes, obesity, and fatty liver disease (NAFLD/nonalcoholic steatohepatitis [NASH]). Here we address gaps in knowledge about the pharmacology and toxicology of this substance administered at high concentrations to explore its medicinal potential. We observed that high-dose meglumine limited secretion of proinflammatory cytokines and cell adhesion molecules from activated human THP-1 or murine RAW264.7 monocytes. Preclinical pharmacokinetic analysis in Swiss mice confirmed that meglumine was orally available. Informed by this data, oral doses of 18 to 75 mM meglumine were administered ad libitum in the drinking water of Sprague-Dawley rats and two cohorts of C57BL/6 mice housed in different vivariums. In a 32-week study, urinary isoprostane levels trended lower in subjects consistent with the possibility of anti-inflammatory effects. In full lifespan studies, there was no detrimental effect on longevity. Heart function evaluated in C57BL/6 mice using an established noninvasive cardiac imaging system showed no detrimental effects on ejection fraction, fractional shortening, left ventricle function or volume, and cardiac output in mice up to 15-month old, with a potential positive trend in heart function noted in elderly mice consistent with earlier reported benefits on muscle stamina. Finally, in a transgenic model of inflammation-associated skin carcinogenesis, the incidence, number, and growth of skin tumors trended lower in subjects receiving meglumine. Overall, the evidence obtained illustrating the long-range safety of high-dose oral meglumine support the rationale for its evaluation as a low-cost modality to limit diabetes, hypertriglyceridemia, and NAFLD/NASH.

RhoB antibody alters retinal vascularization in models of murine retinopathy.

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy. Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies.

Deletion of GLUT1 in mouse lens epithelium leads to cataract formation.

The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

Myo/Nog cells are present in the ciliary processes, on the zonule of Zinn and posterior capsule of the lens following cataract surgery.

Myo/Nog cells, named for their expression of MyoD and noggin, enter the eye during early stages of embryonic development. Their release of noggin is critical for normal morphogenesis of the lens and retina. Myo/Nog cells are also present in adult eyes. Single nucleated skeletal muscle cells designated as myofibroblasts arise from Myo/Nog cells in cultures of lens tissue. In this report we document the presence of Myo/Nog cells in the lens, ciliary body and on the zonule of Zinn in mice, rabbits and humans. Myo/Nog cells were rare in all three structures. Their prevalence increased in the lens and ciliary body of rabbits 24 h following cataract surgery. Rabbits developed posterior capsule opacification (PCO) within one month of surgery. The number of Myo/Nog cells continued to be elevated in the lens and ciliary body. Myo/Nog cells containing alpha smooth muscle actin and striated muscle myosin were present on the posterior capsule and overlaid deformations in the capsule. Myo/Nog cells also were present on the zonule fibers and external surface of the posterior capsule. These findings suggest that Myo/Nog contribute to PCO and may use the zonule fibers to migrate between the ciliary processes and lens.

Role of Myo/Nog Cells in Neuroprotection: Evidence from the Light Damaged Retina.

PURPOSE: To identify Myo/Nog cells in the adult retina and test their role in protecting retinal photoreceptors from light damage. METHODS: Light damage was induced by exposing albino rats raised in dim cyclic light to 1000 lux light for 24 hours. In one group of rats, Myo/Nog cells were purified from rat brain tissue by magnetic cell sorting following binding of the G8 monoclonal antibody (mAb). These cells were injected into the vitreous humour of the eye within 2 hours following bright light exposure. Retinal function was assessed using full-field, flash electroretinogram (ERG) before and after treatment. The numbers of Myo/Nog cells, apoptotic photoreceptors, and the expression of glial fibrillary acidic protein (GFAP) in Muller cells were assessed by immunohistochemistry. RESULTS: Myo/Nog cells were present in the undamaged retina in low numbers. Light induced damage increased their numbers, particularly in the choroid, ganglion cell layer and outer plexiform layer. Intravitreal injection of G8-positive (G8+) cells harvested from brain mitigated all the effects of light damage examined, i.e. loss of retinal function (ERG), death of photoreceptors and the stress-induced expression of GFAP in Muller cells. Some of the transplanted G8+ cells were integrated into the retina from the vitreous. CONCLUSIONS: Myo/Nog cells are a subpopulation of cells that are present in the adult retina. They increase in number in response to light induced stress. Intravitreal injection of Myo/Nog cells was protective to the retina, in part, by reducing retinal stress as measured by the Muller cell response. These results suggest that Myo/Nog cells, or the factors they produce, are neuroprotective and may be therapeutic in neurodegenerative retinal diseases.

IDO1 is an Integral Mediator of Inflammatory Neovascularization.

The immune tolerogenic effects of IDO1 (indoleamine 2,3-dioxygenase 1) have been well documented and genetic studies in mice have clearly established the significance of IDO1 in tumor promotion. Dichotomously, the primary inducer of IDO1, the inflammatory cytokine IFNγ (interferon-γ), is a key mediator of immune-based tumor suppression. One means by which IFNγ can exert an anti-cancer effect is by decreasing tumor neovascularization. We speculated that IDO1 might contribute to cancer promotion by countering this anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of the pro-tumorigenic inflammatory cytokine IL6 (interleukin-6). In this study, we investigated how genetic loss of IDO1 affects neovascularization in mouse models of oxygen-induced retinopathy and lung metastasis. Neovascularization in both models was significantly reduced in mice lacking IDO1, was similarly reduced with loss of IL6, and was restored in both cases by concomitant loss of IFNγ. Likewise, the lack of IDO1 or IL6 resulted in reduced metastatic tumor burden and increased survival, which the concomitant loss of IFNγ abrogated. This insight into IDO1's involvement in pro-tumorigenic inflammatory neovascularization may have important ramifications for IDO1 inhibitor development, not only in cancer where clinical trials are currently ongoing, but in other disease indications associated with neovascularization as well.

Neuroprotective effect of Myo/Nog cells in the stressed retina.

Myo/Nog cells are essential for eye development in the chick embryo and respond to injury in adult tissues. These cells express mRNA for the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein (BMP) inhibitor Noggin and the cell surface protein recognized by the G8 monoclonal antibody (mAb). In this study, we determined that Myo/Nog cells are present in low numbers in the retina of the mouse eye. G8-positive Myo/Nog cells were distinguished from neuronal, Müller and microglial cells that were identified with antibodies to calretinin, Chx10, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1, respectively. In the neonatal retina, the number of Myo/Nog cells increased in parallel with cell death induced by transient exposure to hyperoxia. In this model of retinopathy of prematurity, depletion of Myo/Nog cells by intravitreal injection of the G8 mAb and complement increased cell death. These findings demonstrate that Myo/Nog cells are a distinct population of cells, not previously described in the retina, which increases in response to retinal damage and mitigate hypoxia-induced cell death.

Myo/Nog cells: targets for preventing the accumulation of skeletal muscle-like cells in the human lens.

Posterior capsule opacification (PCO) is a vision impairing condition that arises in some patients following cataract surgery. The fibrotic form of PCO is caused by myofibroblasts that may emerge in the lens years after surgery. In the chick embryo lens, myofibroblasts are derived from Myo/Nog cells that are identified by their expression of the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and the epitope recognized by the G8 monoclonal antibody. The goal of this study was to test the hypothesis that depletion of Myo/Nog cells will prevent the accumulation of myofibroblasts in human lens tissue. Myo/Nog cells were present in anterior, equatorial and bow regions of the human lens, cornea and ciliary processes. In anterior lens tissue removed by capsulorhexis, Myo/Nog cells had synthesized myofibroblast and skeletal muscle proteins, including vimentin, MyoD and sarcomeric myosin. Alpha smooth muscle actin (α-SMA) was detected in a subpopulation of Myo/Nog cells. Areas of the capsule denuded of epithelial cells were surrounded by Myo/Nog cells. Some of these cell free areas contained a wrinkle in the capsule. Depletion of Myo/Nog cells eliminated cells expressing skeletal muscle proteins in 5-day cultures but did not affect cells immunoreactive for beaded filament proteins that accumulate in differentiating lens epithelial cells. Transforming growth factor-betas 1 and 2 that mediate an epithelial-mesenchymal transition, did not induce the expression of skeletal muscle proteins in lens cells following Myo/Nog cell depletion. This study demonstrates that Myo/Nog cells in anterior lens tissue removed from cataract patients have undergone a partial differentiation to skeletal muscle. Myo/Nog cells appear to be the source of skeletal muscle-like cells in explants of human lens tissue. Targeting Myo/Nog cells with the G8 antibody during cataract surgery may reduce the incidence of PCO.

Meglumine exerts protective effects against features of metabolic syndrome and type II diabetes.

Metabolic syndrome, diabetes and diabetes complications pose a growing medical challenge worldwide, accentuating the need of safe and effective strategies for their clinical management. Here we present preclinical evidence that the sorbitol derivative meglumine (N-methyl-D-glucamine) can safely protect against several features of metabolic syndrome and diabetes, as well as elicit enhancement in muscle stamina. Meglumine is a compound routinely used as an approved excipient to improve drug absorption that has not been ascribed any direct biological effects in vivo. Normal mice (SV129) administered 18 mM meglumine orally for six weeks did not display any gastrointestinal or other observable adverse effects, but had a marked effect on enhancing muscle stamina and at longer times in limiting weight gain. In the established KK.Cg-Ay/J model of non-insulin dependent diabetes, oral administration of meglumine significantly improved glycemic control and significantly lowered levels of plasma and liver triglycerides. Compared to untreated control animals, meglumine reduced apparent diabetic nephropathy. Sorbitol can improve blood glucose uptake by liver and muscle in a manner associated with upregulation of the AMPK-related enzyme SNARK, but with undesirable gastrointestinal side effects not seen with meglumine. In murine myoblasts, we found that meglumine increased steady-state SNARK levels in a dose-dependent manner more potently than sorbitol. Taken together, these findings provide support for the clinical evaluation of meglumine as a low-cost, safe supplement offering the potential to improve muscle function, limit metabolic syndrome and reduce diabetic complications.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

RhoB deficiency in thymic medullary epithelium leads to early thymic atrophy.

RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-ß receptor type II (TGFßRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-ß signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-ß signaling in thymic medullary epithelium.

RhoB loss prevents streptozotocin-induced diabetes and ameliorates diabetic complications in mice.

RhoB is an early-response gene whose expression is elevated by multiple cellular stresses; this gene plays an important role in cancer, macrophage motility, and apoptosis. These factors are essential for the onset of type 1 diabetes mellitus and related complications. This study explores the role of RhoB in ß-cell depletion and hyperglycemia-associated complications and tests whether the pleiotropic effect of statins on glycemic control is RhoB dependent. We induced ß-cell depletion in RhoB(+/+), RhoB(+/-), and RhoB(-/-) mice with streptozotocin (STZ). Diabetic status was assessed by glucose tolerance and pancreatic islet loss. RhoB(-/-) mice showed a significant reduction in the severity of STZ-induced diabetes; only 13% of the STZ-treated RhoB-null animals became hyperglycemic, as opposed to 61% of the wild-type controls. Diabetes-related complications, such as wound healing rate and onset of nephropathy, were also assessed. Hyperglycemic RhoB(-/-) mice had fewer signs of nephropathy and showed faster wound healing than RhoB(+/+) animals. After assessing the diabetic status of mice treated simultaneously with STZ and simvastatin, we conclude that the effect of statins in improving glycemic control is RhoB independent. We propose that RhoB is a modifier of diabetes, important for the induction of ß-cell loss. Suppression of RhoB expression may have potential application in the treatment of diabetes and associated complications.

Relationship between mitochondrial DNA damage and photoreceptor death in developing and adult retina, assessed in normal and degenerative rat strains.

In this study, we used Real-Time PCR to study the correlation of mtDNA deletions and photoreceptor death by apoptosis in one normal (SD) and two different degenerative (RCS and P23H) rat strains. Our results show that, in the SD and RCS strains, mtDNA deletion frequency increased and fell during neonatal life, correlating with rates of photoreceptor death during the critical period of photoreceptor development, and into adulthood. Results suggest that mitochondrial damage occurs in close association with photoreceptor death, in the normal (SD) and fast degenerative (RCS) retinas. The lack of a similar association was observed in the slowly degenerative P23H-3 strain.

Photoreceptor degeneration and loss of retinal function in the C57BL/6-C2J mouse.

PURPOSE: The C57BL/6-c(2J) (c2J) mouse strain has been shown in earlier studies to be highly resistant to light damage. Subsequent studies related this resistance to an amino acid substitution (leu450met) in a pigment epithelial enzyme (RPE65), which slowed the rate of rhodopsin regeneration. The present study was conducted to examine patterns of photoreceptor death, electrophysiological function (the ERG) and trophic factor expression over the life of the C57BL/6-c(2J) retina. METHODS: Observations were made on two C57BL/6J-c(2J) substrains, one albino (Tyr/Tyr) and one pigmented (Tyr/(+)), and two nondegenerative strains, one albino (BALB/cJ) and one pigmented (C57BL/6J). Mice were raised in dim cyclic light (12 hours at 5 lux, 12 hours in the dark), and a developmental series of retinas of each strain was taken between postnatal day (P)4 and (P365(+)). Retinas were examined for cell death by using the TUNEL technique, stress-induced protein expression (FGF-2 and GFAP), and measures of retinal thickness. The dark-adapted ERG was recorded in dark-adapted conditions in early adulthood (13-15 weeks) and late adulthood (>1 year). RESULTS: In both C57BL/6-c(2J) substrains, the retina showed marked degenerative features when compared with two control strains, BALB/cJ (leucine at codon 450 in RPE65) and C57BL/6J (methionine). During development and into young adulthood, photoreceptor death rates were abnormally high, levels of two stress-inducible proteins (FGF-2 and GFAP) were abnormally high, and the ERG (electroretinogram) was significantly reduced in amplitude (<50% of values in BALB/cJ or C57BL/6J). The rate of photoreceptor death remained abnormally high into young adulthood (2-3 months) but decreased to control levels by 1 year. Accordingly, the thickness of the outer nuclear layer and the ERG were stable over the same period. CONCLUSIONS: Results suggest that a still-unidentified stress increases photoreceptor death in the C57BL/6-c(2J) retina during the critical period of photoreceptor development and into young adulthood, upregulates stress-inducible factors, and markedly limits the amplitude of the ERG. These degenerative changes do not continue after early adulthood, the retina remaining stable in structure and function into late adulthood. The degenerative changes were apparent in both albino and pigmented C57BL/6-c(2J) substrains. Their genetic cause remains unknown.