The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate.

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules  CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.

Evaluation of Sydnone-Based Analogues of Combretastatin A-4 Phosphate (CA4P) as Vascular Disrupting Agents for Use in Cancer Therapy.

The combretastatins have attracted significant interest as small-molecule therapies for cancer due to their ability to function as vascular disrupting agents. We have successfully prepared a range of combretastatin analogues that are based on a novel sydnone heterocycle core, and their potential as tubulin binders has been assessed in vitro and in vivo. The most potent candidate was found to disrupt microtubules and affect cellular morphology at sub-micromolar levels. Moreover, it was found to bind reversibly to tubulin and significantly increase endothelial cell monolayer permeability, in a similar manner to combretastatin A4. Surprisingly, the compound did not exhibit efficacy in vivo, possibly due to rapid metabolism.

Early Subretinal Allograft Rejection Is Characterized by Innate Immune Activity.

Successful subretinal transplantation is limited by considerable early graft loss despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a nonimmunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation, and the neutrophil chemoattractant KC/GRO/CINC was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, nonimmunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7, and 28 days postoperatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b and F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ɛ) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using the Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p < 0.001) reduced between postoperative day (POD) 3 (90 ± 4%) and POD 7 (20 ± 7%). CD11b+, F4/80+, and Gr1 Ly-6G+ cells increased significantly (p < 0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Colabeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7, and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ɛ was low and did not differ significantly between time points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal, for the first time, a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response.

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model.

PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001). CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability.

Long-term vision rescue by human neural progenitors in a rat model of photoreceptor degeneration.

PURPOSE: As a follow-up to previous studies showing that human cortical neural progenitor cells (hNPC(ctx)) can sustain vision for at least 70 days after injection into the subretinal space of Royal College of Surgeons (RCS) rats, the authors examined how functional rescue is preserved over long periods and how this relates to retinal integrity and donor cell survival. METHODS: Pigmented dystrophic RCS rats (n = 15) received unilateral subretinal injections of hNPC(ctx) at postnatal day (P) 21; control rats (n = 10) received medium alone and were untreated. All animals were maintained on oral cyclosporine A. Function was monitored serially by measuring acuity (using an optomotor test) and luminance thresholds (recording from the superior colliculus) at approximately P90, P150, and P280. Eyes were processed for histologic study after functional tests. RESULTS: Acuity and luminance thresholds were significantly better in hNPC(ctx)-treated animals than in controls (P < 0.001) at all time points studied. Acuity was greater than 90%, 82%, and 37% of normal at P90, P150, and P270, whereas luminance thresholds in the area of best rescue remained similar the whole time. Histologic studies revealed substantial photoreceptor rescue, even up to P280, despite progressive deterioration in rod and cone morphology. Donor cells were still present at P280, and no sign of donor cell overgrowth was seen. CONCLUSIONS: Long-term rescue of function and associated morphologic substrates was seen, together with donor cell survival even in the xenograft paradigm. This is encouraging when exploring further the potential for the application of hNPC(ctx) in treating retinal disease.

Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration.

PURPOSE: It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. METHODS: Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. RESULTS: Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. CONCLUSIONS: RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Protection of visual functions by human neural progenitors in a rat model of retinal disease.

BACKGROUND: A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat. METHODOLOGY/PRINCIPAL FINDINGS: Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90-100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed. CONCLUSIONS/SIGNIFICANCE: Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo.

Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease.

Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.