Capillary Cell Apoptosis Research Articles

One potential mechanism of cardiac toxicity induced by arsenic trioxide: Apoptosis in coronary arterial and blood capillary endothelial cells

One potential mechanism of cardiac toxicity induced by arsenic trioxide: Apoptosis in coronary arterial and blood capillary endothelial cells

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-kappaB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-kappaB and caspase-3 by about 30-40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-kappaB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.

Role of Mitochondrial Superoxide Dismutase in the Development of Diabetic Retinopathy

Apoptosis of retinal capillary cells is an early event in the pathogenesis of retinopathy in diabetes, and oxidative stress has been linked to accelerated apoptosis of retinal capillary cells. Mitochondria are the major endogenous source of superoxide, and superoxide is considered to be a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes. The purpose of the present study is to determine the role of mitochondrial superoxide dismutase (MnSOD) in the development of diabetic retinopathy. The effect of overexpression of MnSOD on glucose-induced endothelial cell oxidative stress, nitrosative stress, and apoptosis was determined by using bovine retinal endothelial cells. Furthermore, the effect of diabetes in rats (11 months' duration) on the activity and the mRNA expression of retinal MnSOD were also determined. MnSOD activity in the nontransfected control retinal endothelial cells was 20% compared with the total SOD activity and was increased to 60% in the MnSOD-transfected cells. MnSOD overexpression prevented a glucose-induced increase in oxidative stress (8-hydroxy guanosine levels), nitrosative stress (nitrotyrosine formation), and apoptosis of retinal endothelial cells. MnSOD enzyme activity and its mRNA were decreased significantly in the retina obtained from the diabetic rats, and these abnormalities were prevented by long-term lipoic acid therapy. The results of this study suggest a protective role for MnSOD in retinal capillary cell death and, ultimately, in the pathogenesis of retinopathy in diabetes. Understanding the role of MnSOD to modify the course of retinopathy could elucidate important molecular targets for future pharmacological interventions.

ER stress and apoptosis‐inducing mutations in carbonic anhydrase IV associated with retinitis pigmentosa (RP17) respond to chemical chaperones

We examined two human mutations in the carbonic anhydrase IV (CA IV) gene, which are associated with RP17, an autosomal dominant form of retinitis pigmentosa. CA IV is highly expressed in the choriocapillaris, but not expressed in the retina. The R14W CA IV mutation (Rebello et al., Proc. Natl. Acad. Sci. USA 101:6617–6622, 2004) is located in the signal sequence. The subsequently discovered R219S CA IV mutation is located in the sequence of the mature protein. The biological effects of these mutations were studied in transfected COS cells. Although the signal sequence mutation had only a modest effect on total enzyme production (70% of normal), the R219S mutant protein was completely inactive. Both mutations induced upregulation of BiP, PERK, and CHOP, markers of ER stress. Both suppressed levels of production of other secretory proteins. Both dramatically induced apoptosis as measured by Annexin V binding and TUNEL staining. The markers of ER stress and the number of cells undergoing apoptosis were both greatly reduced by growth in 4-phenylbutyric acid or CA inhibitors, which appeared to act as nonspecific and specific chemical chaperones, respectively. We propose that autosomal dominant RP17 associated with both CA IV mutations is a protein folding disease produced by apoptosis in capillary endothelial cells that nourish the overlying retina. These results suggest that patients with RP17 may respond clinically to treatment with chemical chaperones. This research is supported by NIH grant DK40163 to W.S.S.

Overexpression of mitochondrial superoxide dismutase in mice protects the retina from diabetes-induced oxidative stress

The retina experiences mitochondrial dysfunction in diabetes, superoxide levels are elevated, and mitochondrial superoxide dismutase (MnSOD) activity is decreased. Inhibition of superoxide accumulation in diabetes prevents mitochondrial dysfunction, apoptosis of retinal capillary cells, and the development of retinal histopathology. The purpose of this study is to examine the effect of overexpression of MnSOD on oxidative stress, DNA damage, and nitrative stress in the retina of diabetic mice. After 7 weeks of diabetes in MnSOD overexpressing (hemizygous) mice (MnSOD-Tg) and in their age-matched nontransgenic mice, parameters of oxidative stress and nitrative stress were measured in the retina. Overexpression of MnSOD prevented diabetes-induced decreases in retinal GSH levels and the total antioxidant capacity. In the same retina, MnSOD overexpression also inhibited diabetes-induced increases in the levels of 8-OHdG and nitrotyrosine. This suggests that MnSOD could be implicated in the pathogenesis of retinopathy by protecting the retina from increased oxidative damage experienced in diabetic conditions. Thus, understanding how changes in mitochondrial function result in the development of diabetic retinopathy could help identify SOD mimics to inhibit its development.

Aspirin at Low-Intermediate Concentrations Protects Retinal Vessels in Experimental Diabetic Retinopathy Through Non–Platelet-Mediated Effects

The prevention of diabetic retinopathy requires drugs that leverage the benefits of glycemic control without adding the burden of side effects. Aspirin at dosages of 1-1.5 g/day has prevented manifestations of diabetic retinal microangiopathy in a clinical trial as well as in studies with dogs. Because lower and safer doses of aspirin could be used if its beneficial effects on retinopathy were due to antithrombotic effects, we compared the effects of a selective antiplatelet drug (clopidogrel) to those of aspirin in streptozotocin-induced diabetic rats. Clopidogrel did not prevent neuronal apoptosis, glial reactivity, capillary cell apoptosis, or acellular capillaries in the retina of diabetic rats. Aspirin, at doses yielding serum levels (<0.6 mmol/l) well below the anti-inflammatory range for humans, prevented apoptosis of capillary cells and the development of acellular capillaries but did not prevent neuroglial abnormalities. The aldose reductase inhibitor sorbinil, used as the benchmark for the effect of the other drugs, prevented all abnormalities. The diabetic rat retina showed increased expression of the transcription factor CCAAT/enhancer-binding protein-beta, one of the known targets of low-intermediate concentrations of aspirin. Thus we found a spectrum of drug efficacy on the prevention of experimental diabetic retinopathy, ranging from the absent effect of a selective antiplatelet drug to the prevention of all abnormalities by an aldose reductase inhibitor. Aspirin at low-intermediate concentrations selectively prevented microangiopathy. The minimal effective dose of aspirin should now be sought.

Diabetic Retinopathy: Mitochondrial Dysfunction and Retinal Capillary Cell Death

Oxidative stress is increased in the retina in diabetes; the levels of oxidatively modified DNA and nitrosylated proteins are elevated, and antioxidant defense enzymes are impaired. The levels of superoxides are elevated in the retina, and the mitochondria become dysfunctional with proapoptotic protein, Bax, translocating from the cytosol into the mitochondria, and cytochrome c leaking out from the mitochondria. This is accompanied by increased retinal capillary cell apoptosis, and the formation of acellular capillaries and pericyte ghosts, the early signs of retinopathy in animal models of diabetic retinopathy. Inhibition of superoxides inhibits glucose -induced mitochondrial dysfunction, activation of caspase-3, and cell death in retinal capillary cells. In animal models, long-term administration of lipoic acid or other antioxidants inhibits the development of diabetic retinopathy via inhibition of accumulation of oxidatively modified DNA and nitrotyrosine and capillary cell apoptosis in the retina. Understanding the role of mitochondria in the development of retinopathy in diabetes should help identify therapies that can neutralize superoxides and inhibit their dysfunction and, ultimately, the development of retinopathy.

Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), an adhesion molecule expressed on hematopoietic and endothelial cells, mediates apoptosis, cell proliferation, and migration and maintains endothelial integrity in addition to its roles as a modulator of lymphocyte and platelet signaling and facilitator of neutrophil transmigration. Recent data suggest that CD31 functions as a scaffolding protein to regulate phosphorylation of the signal transducers and activators of transcription (STAT) family of signaling molecules, particularly STAT3 and STAT5. STAT3 regulates the acute phase response to innate immune stimuli such as lipopolysaccharide (LPS) and promotes recovery from LPS-induced septic shock. Here we demonstrate that CD31-deficient mice have reduced survival during endotoxic LPS-induced shock. As compared to wild-type controls, CD31-deficient mice showed enhanced vascular permeability; increased apoptotic cell death in liver, kidney, and spleen; and elevated levels of serum tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFNgamma), MCP-1, MCP-5, sTNRF, and IL-6. In response to LPS in vivo and in vitro, splenocytes and endothelial cells from knockout mice had reduced levels of phosphorylated STAT3. These results suggest that CD31 is necessary for maintenance of endothelial integrity and prevention of apoptosis during septic shock and for STAT3-mediated acute phase responses that promote survival during septic shock.

Effect of advanced glycation end products on accelerated apoptosis of retinal capillary cells under in vitro conditions

Advanced glycation end-products (AGEs) are considered to play an important role in the development of retinopathy in diabetes, and are shown to induce retinal vascular changes resembling that of diabetic retinopathy. We have shown that apoptosis of retinal capillary cells is accelerated in diabetes. The aim of this study is to investigate the role of AGEs in accelerated retinal capillary cell death in in vitro conditions, and to identify the possible mechanism involved. Bovine retinal endothelial cells and pericytes were incubated in the presence of 5 μM AGE-bovine serum albumin (AGE-BSA) or untreated control BSA (BSA) for up to five days. The cell death was determined by performing ELISA for cytoplasmic histone-associated DNA fragments and by measuring the activity of caspase-3. Incubation of endothelial cells or pericytes with AGE-BSA increased oxidative stress and NO by 60%, and in the same cells nuclear transcriptional factor (NF- kB) was also activated by over 60%. AGE-BSA induced their apoptosis by 55%, and activated caspase-3 by about 50% compared to the cells incubated with unmodified BSA. Co-addition of AGE-BSA and antioxidants (N-acetyl cysteine or α-lipoic acid) inhibited oxidative stress, nitrotyrosine formation, NF- kB activation and capillary cell apoptosis. These data strongly suggest that increased AGE in diabetes could play an important role in retinal capillary apoptosis and that oxidative stress is involved in this process. Inhibition of AGEs in the retinal capillary cells could prevent their apoptosis, and ultimately, the development of retinopathy in diabetes.

Effect of Long-Term Administration of α-Lipoic Acid on Retinal Capillary Cell Death and the Development of Retinopathy in Diabetic Rats

Oxidative stress is increased in the retina in diabetes, and it is considered to play an important role in the development of retinopathy. alpha-Lipoic acid, a thiol antioxidant, has been shown to have beneficial effects on polyneuropathy and on the parameters of oxidative stress in various tissues, including nerve, kidney, and retina. The purpose of this study was to examine the effect of alpha-lipoic acid on retinal capillary cell apoptosis and the development of pathology in diabetes. Retina was used from streptozotocin-induced diabetic rats receiving diets supplemented with or without alpha-lipoic acid (400 mg/kg) for 11 months of diabetes. Capillary cell apoptosis (by terminal transferase-mediated dUTP nick-end labeling) and formation of acellular capillaries were investigated in the trypsin-digested retinal microvessels. The effect of alpha-lipoic acid administration on retinal 8-hydroxy-2'deoxyguanosine (8-OHdG) and nitrotyrosine levels was determined by enzyme-linked immunosorbent assay. alpha-Lipoic acid administration for the entire duration of diabetes inhibited capillary cell apoptosis and the number of acellular capillaries in the retina, despite similar severity of hyperglycemia in the two diabetic groups (with and without alpha-lipoic acid). Retinal 8-OHdG and nitrotyrosine levels were increased by over twofold and 70%, respectively, in diabetes, and alpha-lipoic acid administration inhibited these increases. Our results demonstrate that the long-term administration of alpha-lipoic acid has beneficial effects on the development of diabetic retinopathy via inhibition of accumulation of oxidatively modified DNA and nitrotyrosine in the retina. alpha-Lipoic acid supplementation represents an achievable adjunct therapy to help prevent vision loss in diabetic patients.

Role of Interleukin-1β in the Development of Retinopathy in Rats: Effect of Antioxidants

Diabetic retinopathy is shown to share many similarities with chronic inflammatory disease, and in diabetes, accelerated apoptosis of retinal capillary cells is evident before histopathology can be seen. The purpose of this study was to examine the effect of interleukin (IL)-1beta on capillary cell apoptosis in rat retina and to determine the effect of antioxidants on diabetes-induced changes in retinal IL-1beta. The effect of injection of IL-1beta into the vitreous (5 ng/5 microL) of normal rats on capillary cell apoptosis (detected by terminal transferase dUTP nick-end labeling [TUNEL]) and formation of acellular capillaries was investigated in the trypsin digested retinal microvessels. The levels of IL-1beta were quantified (by ELISA and Western blot) in the retina of rats diabetic for 2 months, and the effect of administration of antioxidants on diabetes-induced changes in retinal IL-1beta was determined. The number of TUNEL-positive capillary cells in the retinal microvessels obtained from normal rats that received intravitreal injection of IL-1beta was increased by more than threefold and that of acellular capillaries by more than twofold, compared with the microvessels obtained from rats that received an intravitreal injection of PBS (5 microL) or BSA (5 ng/5 microL). IL-1beta also increased the levels of 8-hydroxy-2'-deoxyguanosine (an indicator of oxidative stress) and nitric oxide by more than 40% and activated NF-kappaB by 35% to 55%. Two months of diabetes in rats increased retinal IL-1beta levels by more than twofold, and antioxidants inhibited such increases. IL-1beta, by activation of NF-kappaB and an increase in oxidative stress, plays an important role in the retinal microvascular disease that is characteristic of diabetic retinopathy. Antioxidants inhibit diabetes-induced increases in retinal IL-1beta. These studies offer a possible rationale to test IL-1beta-targeted therapies to inhibit the development of retinopathy in diabetes.

Potential contributory role of H-Ras, a small G-protein, in the development of retinopathy in diabetic rats.

Hyperglycemia is thought to be the underlying factor in the development of diabetic retinopathy, but the mechanisms involved remain partially understood. Diabetes increases oxidative stress, and reactive oxygen species affect the interactions between a small-molecular- weight G-protein, H-Ras, and several of its effector proteins. The purpose of this study was to examine the regulatory role of H-Ras in glucose-induced apoptosis of retinal endothelial cells. The expressions of H-Ras and its effector protein (Raf-1) were compared in the retina obtained from diabetic rats (2-8 months' duration) and age-matched normal rats and in retinal endothelial cells exposed to high-glucose medium. The effect of inhibition of H-Ras function on glucose-induced capillary cell death and the potential involvement of oxidative stress in diabetes-induced activation of H-Ras were also determined. The expressions of H-Ras and Raf-1 were increased in the retina in diabetes, and antioxidant therapy prevented diabetes-induced increased protein and mRNA expressions. The inhibitors of Ras farnesylation inhibited glucose-induced nitric oxides and apoptosis in isolated retinal endothelial cells. Thus, the results suggest that functional activation of H-Ras might be one of the signaling steps involved in glucose-induced capillary cell apoptosis and supports the role of H-Ras in the development of retinopathy in diabetes.

Do the signalling proteins for angiogenesis exist as a modular complex? The case for the angosome

The vasculature remains quiescent during much of adult life, but new blood vessels can be rapidly produced when required by a process referred to as angiogenesis. Angiogenesis involves a complex series of events including the proliferation, migration, differentiation and apoptosis of capillary endothelial cells, as well as changes in vascular permeability. This hypothesis argues that in the quiescent vasculature the many factors that regulate angiogenesis are normally held together as part of an inactive modular unit, and that when angiogenesis is stimulated the modular unit dissociates thus enabling angiogenic regulators to become active. I have termed this modular unit the “angosome”. It is proposed that the angosome is present in the caveolae of capillary endothelial cells. Caveolae are flask-shaped invaginations in the plasmalemma that compartmentalise signalling molecules. Endothelial cells are particularly rich in caveolae. Many of the structural and functional aspects of caveolae are controlled by the protein caveolin, one form of which, caveolin-1, interacts directly or indirectly with most of the regulatory molecules involved in angiogenesis. Caveolin-1 forms oligomers of 14–16 sub-units and I propose that oligomers of caveolin-1 form the scaffold that holds together the angosome. There is evidence that caveolin-1 is up-regulated in the differentiated, quiescent vasculature and down-regulated in proliferating endothelial cells. Since the presence of caveolin-1 can inhibit pro-angiogenic factors, it may act as a “master-switch” co-ordinating events during angiogenesis. Thus when the vasculature is quiescent the angosome may hold angiogenic factors in an inactive state and when angiogenesis is required, the angosome must disassociate to enable angiogenic factors to become active.

Diabetes-Induced Mitochondrial Dysfunction in the Retina

Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells. Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay. Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis. Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Bone morphogenetic protein 4 mediates apoptosis of capillary endothelial cells during rat pupillary membrane regression.

Programmed capillary regression is essential for development, but little is known about the mechanism behind this phenomenon. In this study, we characterized the molecular determinants of capillary regression utilizing the pupillary membrane (PM) in the newborn rat's eye. We observed in the 1-day-culture system that apoptotic endothelial cells decrease in number with the addition of a natural antagonist, Noggin, strongly suggesting the involvement of the bone morphogenetic protein (BMP) family in PM regression. In addition, the lens-conditioned medium (Lens-CM) induced apoptosis of HUVE cells and inhibited endothelial tubulogenesis, which were completely blocked by both Noggin and the BMP4-specific neutralizing antibody. Activation of BMP4 pathway in endothelial cells was confirmed by both the up-regulation of Msx genes correlated with apoptosis and the translocation of Smad1 into the nucleus. We showed a transient expression of BMP4 in Lens-CM by immunoprecipitation assay. Furthermore, the transcorneal injection of BMP4 in rats enhanced the apoptosis of PMs, while that of Noggin attenuated it. These results indicate that BMP4 pathways play pivotal roles in capillary regression in a paracrine manner between lens and PMs.

Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite.

Hyperoxia exposure induces capillary endothelial cell apoptosis in the developing retina, leading to vaso-obliteration followed by proliferative retinopathy. Previous in vivo studies have shown that endothelial nitric oxide synthase (NOS3) and peroxynitrite are important mediators of the vaso-obliteration. Now we have investigated the relationship between hyperoxia, NOS3, peroxynitrite, and endothelial cell apoptosis by in vitro experiments using bovine retinal endothelial cells (BREC). We found that BREC exposed to 40% oxygen (hyperoxia) for 48 h underwent apoptosis associated with activation of caspase-3 and cleavage of the caspase substrate poly(ADP-ribose) polymerase. Hyperoxia-induced apoptosis was associated with increased formation of nitric oxide, peroxynitrite, and superoxide anion and was blocked by treatment with uric acid, nitro-L-arginine methyl ester, or superoxide dismutase. Analyses of the phosphatidylinositol 3-kinase/Akt kinase survival pathway in cells directly treated with peroxynitrite revealed inhibition of VEGF- and basic FGF-induced activation of Akt kinase. These results suggest that hyperoxia-induced formation of peroxynitrite induces BREC apoptosis by crippling key survival pathways and that blocking peroxynitrite formation prevents apoptosis. These data may have important clinical implications for infants at risk of retinopathy of prematurity.

Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage.

Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin). It specifically binds to tissue kallikrein and inhibits kallikrein activity. Our study was designed to test its effects on retinal neovascularization and vascular permeability. Endothelial cell proliferation was determined by [(3)H] thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry. Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model. Vascular permeability was assayed by the Evans blue method. Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA. Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells. Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model. Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR. Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production. KBP competed with (125)I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model. Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system. Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP.

Caldesmon regulates apoptosis and cell cycle progression in capillary endothelial cells and inhibits angiogenesis in vitro

Caldesmon regulates apoptosis and cell cycle progression in capillary endothelial cells and inhibits angiogenesis in vitro

Caldesmon-dependent switching between capillary endothelial cell growth and apoptosis through modulation of cell shape and contractility.

Caldesmon (CaD), a protein component of the actomyosin filament apparatus, modulates cell shape and cytoskeletal structure when overexpressed. When capillary endothelial cells were infected with an adenoviral vector encoding GFP-CaD under Tet-Off control, progressive inhibition of contractility, loss of actin stress fibers, disassembly of focal adhesions, and cell retraction resulted. This was accompanied by a cell shape (rounding)-dependent increase in apoptosis and concomitant inhibition of cell cycle progression. Cell growth also was inhibited in low expressor cells in which cell tension was suppressed independently of significant changes in cell shape, cytoskeletal structure, or focal adhesions. Thus, changes in both cytoskeletal structure and contractility appear to be central to the mechanism by which extracellular matrix-dependent changes in capillary cell shape influence growth and apoptosis during angiogenesis, and hence the cytoskeleton may represent a potential target for anti-angiogenesis therapy.

Insulin up-regulates a Glc3Man9GlcNAc2-PP-Dol pool in capillary endothelial cells not essential for angiogenesis.

Endothelial cells line blood vessels, and their proliferation during neovascularization ( i.e., angiogenesis) is essential for a normal growth and development as well as for tumor progression and metastasis. Mechanistic details indicated that down-regulation of Glc(3)Man(9)GlcNAc(2)-PP-Dol level reduced angiogenesis and induced apoptosis in capillary endothelial cells (Martínez JA, Torres-Negrón I, Amigó LA, Banerjee DK, Cellular and Molec Biochem 45, 137-152 (1999)). Unlike in any other insulin-responsive cells, insulin reduced capillary endothelial cell proliferation by increasing the cell doubling time. But, when analyzed, the rate of lipid-linked oligosaccharide-PP-Dol (LLO) synthesis as well as its turnover ( i.e., t(1/2)) were increased in insulin treated cells. No major differences in their molecular size were observed. This corroborated with an enhanced glycosylation of Factor VIIIC, an N-linked glycoprotein (essential cofactor of the blood coagulation cascade) and a marker for the capillary endothelial cell. Increased LLO synthesis was independent of elevating either Dol-P level or Man-P-Dol synthase gene (dpm) transcription. Insulin however, enhanced 2-deoxy-glucose transport across the endothelial cell plasma membrane and caused increased secretion of Factor VIIIC, thus, supporting the existence of additional LLO pool(s), and arguing favorably that growth retardation of capillary endothelial cells by insulin turned a highly proliferative cell into a highly secretory cell.