Structure, Recognition, and Processing of Cisplatin-DNA Adducts.

Diabetic retinopathy (DR) is a leading cause of vision-loss globally. Of an estimated 285 million people with diabetes mellitus worldwide, approximately one third have signs of DR and of these, a further one third of DR is vision-threatening DR, including diabetic macular edema (DME). The identification of established modifiable risk factors for DR such as hyperglycemia and hypertension has provided the basis for risk factor control in preventing onset and progression of DR. Additional research investigating novel risk factors has improved our understanding of multiple biological pathways involved in the pathogenesis of DR and DME, especially those involved in inflammation and oxidative stress. Variations in DR prevalence between populations have also sparked interest in genetic studies to identify loci associated with disease susceptibility. In this review, major trends in the prevalence, incidence, progression and regression of DR and DME are explored, and gaps in literature identified. Established and novel risk factors are also extensively reviewed with a focus on landmark studies and updates from the recent literature.

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Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss

Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however, upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of the cardiac fibroblast impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis.

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Cardiac Fibrosis: The Fibroblast Awakens.

Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line.

Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia.

DNA is the accepted target for cisplatin, but recent evidence has shed doubt on DNA synthesis as the critical process. L1210/0 cells incubated for 2 hours with cisplatin progress to the G2 phase of the cell cycle and are arrested there for several days. They then either progress in the cell cycle or die. In cells that eventually die, total transcription, polyadenylated [poly(A)+] RNA synthesis, and protein synthesis were markedly inhibited only after 48 hours. Nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) levels decreased after 3 days. Cell membrane integrity was lost after 4 days. These results demonstrate that cells can be lethally damaged, yet continue to undergo apparently normal metabolic activities for several days. In a previous study, DNA double-strand breaks were detected after 1 day. We now show that by 2 days, breaks are visible as fragmentation in the nucleosome spacer regions of chromatin. This type of damage is consistent with cell death occurring by the process of apoptosis. Cell shrinkage and morphology were also consistent with this type of cell death. The slow cell death reported here appears to occur at the G2/M transition and may involve events that normally occur at this stage of the cell cycle. These results demonstrate the importance of DNA degradation as an early and possibly essential step in cell death.

Analysis of Events Associated With Cell Cycle Arrest at G2 Phase and Cell Death Induced by Cisplatin

Purpose To isolate and characterize primary retinal endothelial cells (REC) from wild type and transgenic mice to facilitate the study of their properties in vitro. Methods REC were isolated from wild type or transgenic-immortomouse by collagenase digestion of retina and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (anti-PECAM-1). The bound cells were plated on fibronectin-coated wells and expanded. The REC were characterized for expression and localization of endothelial cell markers by fluorescence-activated cell sorting (FACS) analysis and indirect immunofluorescence staining. The ability of these cells to form capillary like networks was assessed on Matrigel while the migration properties were examined in wound closure assays. Results Isolation of REC from mouse has been very difficult and has not been previously reported. Here, we describe a method for isolation of retinal endothelial cells from wild type and thrombospondin-1 deficient (TSP1-/-) immortomice. Our results indicate that nearly 100% of selected cells express the endothelial cell marker PECAM-1 and vascular endothelial-cadherin (VE-cadherin). The cells were successfully passaged and maintained in culture for several months without a significant loss in expression of endothelial cell markers. The wild type REC, like most primary endothelial cells, formed capillary-like networks on Matrigel. The ability of the REC from TSP1-/- mice to form capillary-like networks on Matrigel was severely compromised. This may be attributed, at least in part, to the enhanced migratory and less differentiated phenotype of these cells. Conclusions The retinal endothelial cells can be readily obtained from wild type and transgenic mice, which facilitate the comparison and identification of the physiologic role of specific genes in endothelial cell function.

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Isolation and characterization of murine retinal endothelial cells.

PURPOSE. Vitamin D compounds inhibit the growth of a variety of tumors in preclinical and clinical studies. Among the mechanisms suggested for this inhibition is antiangiogenesis. Retinal angiogenesis is the basis for vision loss in several major blinding diseases. The purpose of this study was to evaluate the antiangiogenic activity of calcitriol (1,25-dihydroxyvitamin D3) in vivo and its effect on retinal endothelial cell (EC) proliferation, migration, and capillary morphogenesis in vitro. METHODS. The mouse oxygen-induced ischemic retinopathy (OIR) model was used to assess the antiangiogenic activity of calcitriol. Ocular VEGF levels were determined by Western blot analysis of whole eye extracts from postnatal day (P) 15 mice during OIR. The effects of calcitriol on retinal EC proliferation, migration, and capillary morphogenesis were also assessed in vitro. RESULTS. Calcitriol-treated animals demonstrated a significant decrease in retinal neovascularization compared with control animals. This effect was dose dependent, and retinal neovascularization was significantly inhibited in calcitriol-treated mice. Although no deaths occurred, calcitriol administration was associated with increased serum calcium and a lack of increase in body weight in a dose-independent manner. The ocular level of VEGF was similar in control and calcitrioltreated animals. At a lower concentration of calcitriol, retinal EC capillary morphogenesis in solubilized basement membrane was inhibited without a significant inhibitory effect on EC proliferation and migration. The concentration of calcitriol required to inhibit retinal EC proliferation was significantly higher than that required to inhibit EC capillary morphogenesis. CONCLUSIONS. These data suggest calcitriol is a potent inhibitor of retinal neovascularization and may be of benefit in the treatment of a variety of eye diseases with a neovascular component. (Invest Ophthalmol Vis Sci. 2007;48:2327‐2334)

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Calcitriol is a potent inhibitor of retinal neovascularization.

Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis Its expression is most prominent during the late stages of vascular development and in the adult vasculature Our previous studies have shown that TSP1 expression promotes a quiescent, differentiated phenotype of vascular endothelial cells However, the physiological role TSP1 plays during vascular development and neovascularization requires further delineation Here, we investigated the role of TSP1 during development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy The retinal vascular density was increased in TSP1-deficient (TSP1-/-) mice compared with wild-type mice This finding was mainly attributed to increased number of retinal endothelial cells in TSP1-/- mice During oxygen-induced ischemic retinopathy, the developing retinal vasculature of TSP1-/- mice was less sensitive to vessel obliteration induced by hyperoxia but exhibited a similar level of neovascularization induced by normoxia compared with wild-type mice This finding is consistent with the similar pattern of VEGF expression detected in wild-type and TSP1-/- mice Furthermore, the increased expression of TSP1 during development of retinal vasculature was not affected by oxygen-induced ischemic retinopathy In addition, the regression of ocular embryonic (hyaloid) vessels, as well as the newly formed retinal vessels during oxygen-induced ischemic retinopathy, was delayed in TSP1-/- mice Therefore, TSP1 is a modulator of vascular homeostasis and its expression is essential for appropriate remodeling and maturation of retinal vasculature

https://anatomypubs.onlinelibrary.wiley.com/doi/pdf/10.1002/dvdy.10412

Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration.

Christine M. Sorenson

Papers

Analysis of Events Associated With Cell Cycle Arrest at G2 Phase and Cell Death Induced by Cisplatin

Isolation and characterization of murine retinal endothelial cells.

Calcitriol is a potent inhibitor of retinal neovascularization.

Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration.

Bcl-2 family members and disease.