Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix Nancy Boudreau,* Carolyn J. Sympson, Zena Werb, Mina J. Bissell N. Boudreau and M. J. Bissell Life Sciences Division, Lawrence Berkeley Laboratory 1 Cyclotron Road, Building 83, Berkeley, CA 94720, USA. C. J. Sympson Life Sciences Division, Lawrence Berkeley Laboratory 1 Cyclotron Road, Building 83, Berkeley, CA 94720, USA Laboratory of Radiobiology and Environmental Health University of California, San Francisco, CA 94143, USA. Z. Werb Laboratory of Radiobiology and Environmental Health University of California, San Francisco, CA 94143, USA. *To whom correspondence should be addressed. LBNL/DOE funding & contract number: DE-AC02-05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

Background In patients with type 1 diabetes mellitus who do not have uremia and have not received a kidney transplant, pancreas transplantation does not ameliorate established lesions of diabetic nephropathy within five years after transplantation, but the effects of longer periods of normoglycemia are unknown. Methods We studied kidney function and performed renal biopsies before pancreas transplantation and 5 and 10 years thereafter in eight patients with type 1 diabetes but without uremia who had mild to advanced lesions of diabetic nephropathy at the time of transplantation. The biopsy samples were analyzed morphometrically. Results All patients had persistently normal glycosylated hemoglobin values after transplantation. The median urinary albumin excretion rate was 103 mg per day before transplantation, 30 mg per day 5 years after transplantation, and 20 mg per day 10 years after transplantation (P=0.07 for the comparison of values at base line and at 5 years; P=0.11 for the comparison between base ...

Reversal of Lesions of Diabetic Nephropathy after Pancreas Transplantation

Inflammation in diabetic retinopathy.

Pericytes are uniquely positioned within the neurovascular unit to serve as vital integrators, coordinators and effectors of many neurovascular functions, including angiogenesis, blood-brain barrier (BBB) formation and maintenance, vascular stability and angioarchitecture, regulation of capillary blood flow and clearance of toxic cellular byproducts necessary for proper CNS homeostasis and neuronal function. New studies have revealed that pericyte deficiency in the CNS leads to BBB breakdown and brain hypoperfusion resulting in secondary neurodegenerative changes. Here we review recent progress in understanding the biology of CNS pericytes and their role in health and disease.

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Central nervous system pericytes in health and disease

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.

Very long term persistence of plasmid dna and foreign gene expression in mouse muscle

To identify events and mechanisms that might contribute to the poor reversibility of diabetic complications, we examined whether diabetes or high glucose induces changes in gene expression and whether such changes outlast the presence of the metabolic abnormalities. The study focused on fibronectin because the increased amounts of this glycoprotein found in diabetic tissues and thickened basement membranes are as yet unexplained. In streptozotocin-induced diabetic rats, fibronectin mRNA levels were increased to 304 +/- 295% of control (mean +/- SD) in the kidney cortex (P less than 0.02), and to 271 +/- 273% of control in the heart (P less than 0.02), while actin mRNA levels remained unchanged. Elevation of fibronectin mRNA persisted for weeks after restoration of near-normoglycemia. In cultured human endothelial cells, high glucose-induced overexpression of fibronectin and collagen IV remained detectable after replating and multiple cell divisions in the absence of high glucose. Cells shifted to normal-glucose medium after prolonged exposure to high glucose also exhibited a proliferative advantage over cells chronically maintained in normal glucose. Thus, diabetes increases fibronectin expression in tissues that are known targets of the complications, and the effect is not readily reversible. The in vitro studies suggest that hyperglycemia may be responsible for these events through induction of self-perpetuating changes in gene expression.

Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory.

Although the degree of hyperglycemia is a powerful and independent risk factor for diabetic microvascular disease, it has not been established if and how high glucose per se can induce the typical lesions of microangiopathy. We have investigated in human vascular endothelial cells the expression of messenger RNA (mRNA) for collagen type IV and fibronectin, the two glycoproteins characteristically increased in diabetic basement membranes. In 12 confluent primary cultures exposed for 11 +/- 1 d (mean +/- SD) to 30 mM glucose and exhibiting cell number and thymidine incorporation similar to control cultures, the levels of collagen IV and fibronectin mRNA were, respectively, 238 +/- 140 and 221 +/- 231 percent of control (P less than 0.01). The effects of high glucose were selective (the levels of collagen I and c-myc mRNA remained unchanged), independent of the proliferative activity of the cultures and of the plating substratum, and maintained throughout multiple passages. However, several days of exposure to high glucose were required before their appearance. These observations establish that high glucose is a perturbation sufficient to mimic the effects of diabetes on the regulation of basement membrane components and propose that modifications in gene expression may pertain to the chain of events leading to diabetic angiopathy.

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Increased expression of basement membrane components in human endothelial cells cultured in high glucose.

Retinal microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy. To examine this further, type 1 streptozotocin-induced diabetic rats and type 2 Zucker diabetic fatty rats were treated by intravitreal injection of the tumor necrosis factor-specific inhibitor pegsunercept, and the impact was measured by analysis of retinal trypsin digests. For type 2 diabetic rats, the number of endothelial cells and pericytes positive for diabetes-enhanced activated caspase-3 decreased by 81% and 86%, respectively, when treated with pegsunercept (P < 0.05). Similarly, the number of diabetes-enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive endothelial cells and pericytes decreased by 81% and 67% respectively when treated with pegsunercept (P < 0.05). Diabetes-increased activated caspase-3- and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive microvascular cell numbers were both reduced by 81% and 80%, respectively, in pegsunercept-treated type 1 diabetic rats (P < 0.05). Inhibition of tumor necrosis factor reduced type 1 diabetes-enhanced pericyte ghost formation by 87% and the number of type 2 diabetes-enhanced pericyte ghosts by 62% (P < 0.05). Similarly, increased acellular capillary formation caused by type 1 and type 2 diabetes was reduced by 68% and 67%, respectively, when treated with pegsunercept (P < 0.05). These results demonstrate a previously unrecognized role of tumor necrosis factor-α in promoting the early pathogenesis of diabetic retinopathy leading to loss of retinal microvascular cells and demonstrate the potential therapeutic benefit of modulating its activity.

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Diabetes-Enhanced Tumor Necrosis Factor-α Production Promotes Apoptosis and the Loss of Retinal Microvascular Cells in Type 1 and Type 2 Models of Diabetic Retinopathy

Growing evidence that high glucose may be a causative agent of the thickened vascular basement membranes that characterize diabetic microangiopathy prompted this investigation of the underlying mechanisms. When exposed to 30 mM glucose, 70% of 52 primary cultures of human endothelial cells, each derived from a single umbilical vein, showed increased levels of fibronectin (median 181% of control, range 104-549%) and collagen IV mRNA (175% of control, range 101-807%). The response of the two transcripts to high glucose was concordant in 77% of the 52 cultures studied (P = 0.01), required 5 days of exposure, and was accompanied by proportionally increased synthesis of the respective protein. Laminin B1 expression was also upregulated by high glucose, concordantly with that of fibronectin and collagen IV. Increased fibronectin and collagen IV mRNA levels resulted from increased gene transcription (median 183 and 236% of control, respectively) without evidence of translational regulation, were not triggered by hypertonicity or signals originating from the matrix, and were also induced by hexoses with limited (D-galactose) or no (L-glucose) access to metabolic pathways but capable of inducing nonenzymatic glycosylation. There was no amplification of the overexpressed genes. Thus, high glucose upregulates in a coordinated fashion the transcription of genes coding for basement membrane components through effects exerted intracellularly or at the cell-matrix boundary and modulated by individual characteristics of the target cells.

Characteristics and Mechanisms of High-Glucose–Induced Overexpression of Basement Membrane Components in Cultured Human Endothelial Cells

Purpose. It is unknown why human corneal endothelium exhibits limited capacity to divide while the endothelia of other species, such as rabbit, divide in vivo at wounding and in culture. A potentially valuable source of information concerning why human endothelium has such a limited proliferative capacity lies in elucidating any differences in the molecular events governing the cell cycle of these two species. A recent study of the relative expression of cell cycle-associated proteins in donor corneas suggests that human corneal endothelial cells in vivo have not exited the cell cycle but are arrested in G1-phase. The purpose of the current study was to identify differences in cell cycle protein expression in human and rabbit endothelium that would explain the difference in their relative proliferative capacities. Specifically, the authors first ascertained the relative proliferative status of rabbit corneal endothelial cells in vivo. The expression and intracellular distribution of G1-phase regulatory proteins was then determined in both species, and the results were compared. Methods. Corneas from New Zealand white rabbits (weight range, 2 to 3 kg) and from human donors (age range, 6 months to 67 years) were fresh frozen, cryostat sectioned, and prepared for indirect immunofluorescence microscopy using an established protocol. The following monoclonal antibodies were localized in rabbit corneal endothelium only : cyclins D, E, A, and B1 ; protein kinase p34 cdc2 ; and Ki67, a marker of actively cycling cells. Localization patterns for the following G1-phase regulatory proteins were compared in both human and rabbit corneal endothelia : the tumor suppressors, pRb, p53, and p16 INK4 , and the transcription factor, E2F. Reverse transcription-polymerase chain reaction studies were conducted to detect mRNA for Ki67 in human and rabbit corneal cells. Results. Cyclins D, E, and A were localized in the cytoplasm of rabbit corneal endothelium, whereas cyclins B1 and p34 were detected in the nucleus. No Ki67 protein or mRNA expression was detected in the endothelium of either species. In human and rabbit endothelia, p53 and p16 INK4 were localized to the cytoplasm, whereas pRb was detected in the nucleus. E2F exhibited a nuclear and a cytoplasmic localization in each species. Conclusions. The corneal endothelium of rabbits stained positively for cyclins D, E, and A and did not stain for Ki67, suggesting that, as in humans, rabbit corneal endothelium in vivo is arrested in Gl-phase upstream from Ki67 synthesis. Cyclin E was located in the cytoplasm of rabbit cells, whereas it was found in the nucleus in human endothelium. The apparent difference in cellular distribution of cyclin E in these two species may be significant because this cyclin is active during the G1-/S-phase transition. It is possible that in situ human and rabbit corneal endothelial cells are arrested at different points within Gl-phase and/or that the difference in relative proliferative capacity exhibited by the corneal endothelium in these two species may be caused by differences in their relative ability to overcome G1-phase arrest.

Sayon Roy

Papers

Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory.

Increased expression of basement membrane components in human endothelial cells cultured in high glucose.

Diabetes-Enhanced Tumor Necrosis Factor-α Production Promotes Apoptosis and the Loss of Retinal Microvascular Cells in Type 1 and Type 2 Models of Diabetic Retinopathy

Characteristics and Mechanisms of High-Glucose–Induced Overexpression of Basement Membrane Components in Cultured Human Endothelial Cells

Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ.