Death Of Human Endothelial Cells Research Articles

Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration.

Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy.

Coumaric Acid Induces Mitochondrial Damage and Oxidative-Mediated Cell Death of Human Endothelial Cells

Evidence that higher natural antioxidants (NA) intake provides cardiovascular protection is contradictory. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from the interaction of NA with endothelial cells (ECs) are actively investigated. Here, we show that moderately high doses of coumaric acid (CA) induced intracellular reactive oxygen species (ROS) production, mitochondrial membrane depolarization and ECs death. Treatment of ECs with cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented the oxidative-mediated cell damage indicating mitochondrial involvement in CA-induced ECs impairment. CA-induced intracellular ROS generation was counteracted by the specific cytochrome P450 (CYP) 2C9 inhibitor sulfaphenazole (SPZ). SPZ also prevented CA-induced mitochondrial membrane depolarization and ECs death, implicating CYP2C9 in mediating the cellular response upon CA treatment. Our results indicate that moderately high doses of CA can promote CYP2C9-mediated oxidative stress eliciting mitochondrial-dependent ECs death and may pave the way toward mechanistic insight into NA effects on cardiovascular cells.

Oxidative stress with an activation of the renin–angiotensin system in human vascular endothelial cells as a novel mechanism of uric acid-induced endothelial dysfunction

Oxidative stress is known to be a major mechanism of endothelial dysfunction, which plays a key role in the development of cardiovascular disease. Although uric acid is one of the most important antioxidants, recent studies have suggested that uric acid may have a causal role in endothelial dysfunction. In order to understand the paradoxical association of uric acid with oxidative stress and vascular disease, we investigated whether uric acid induced oxidative stress in human vascular endothelial cells. We also examined whether uric acid-induced changes in redox status were related to aging and death of endothelial cells or an activation of local renin-angiotensin system, another mediator of endothelial dysfunction. Endothelial senescence and apoptosis were evaluated by senescence-associated beta-galactosidase staining and annexin V-propidium iodide staining in primary isolated human umbilical vein endothelial cells (HUVECs). Production of reactive oxygen species was assessed by dichlorofluorescein diacetate staining. mRNA expression of angiotensinogen, angiotensin-converting enzyme and the receptors of angiotensin II was evaluated by real-time PCR, and angiotensin II levels were measured in uric acid-stimulated HUVECs. Uric acid-induced senescence and apoptosis in HUVECs at concentrations more than 6 and 9 mg/dl, respectively. Uric acid-induced alterations in cell proliferation, senescence and apoptosis were blocked by probenecid, enalaprilat or telmisartan. Uric acid significantly increased production of reactive oxygen species beginning at 5 min, and uric acid-induced senescence and apoptosis of HUVECs were ameliorated by N-acetylcysteine or tempol. Uric acid also upregulated the expression of angiotensinogen, angiotensin-converting enzyme and angiotensin II receptors and increased angiotensin II levels, which was ameliorated with tempol. Uric acid-induced aging and death of human endothelial cells are medicated by local activation of oxidative stress and the renin-angiotensin system, which provides a novel mechanism of uric acid-induced endothelial dysfunction. Therapies targeting uric acid maybe beneficial in cardiovascular disease.

Role of endonuclease G in senescence-associated cell death of human endothelial cells

Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells.

Tanshinone IIA isolated from Salvia miltiorrhiza elicits the cell death of human endothelial cells

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is known to exhibit potent cytotoxicity against various human carcinoma cells in vitro. However, the mechanism by which tanshinone IIA produces this anti-tumor effect remains unknown. Since anti-neovascularization has generally been regarded as an effective strategy for anti-cancer therapy, we decided to investigate the mechanism underlying tanshinone IIA-mediated death of human endothelial cells. In this study, we demonstrate that tanshinone IIA elicits human endothelial cell death independent of oxidative stress. These events are partially calcium-dependent and actually dependent upon NAD(P)H: quinone oxidoreductase (NQO1) activity. Tanshinone IIA induces an increase in intracellular calcium, which triggers the release of cytochrome c, thus causing loss of the mitochondrial membrane potential (MMP), resulting in the subsequent activation of caspases. Blocking the induction of Ca2+ perturbation with BAPTA-AM partially rescued cells from tanshinone IIA-induced cytotoxicity. Additionally, blocking NQO1 activity with dicoumoral or inhibiting caspase activities with the general caspase inhibitor, z-VAD-fmk, prevented cell death induced by tanshinone IIA. Therefore, our results imply that tanshinone IIA-mediated cytotoxicity against human endothelial cells may occur through activation of NQO1, which induces a calcium imbalance and mitochondrial dysfunction, thus stimulating caspase activity.

Senescence-associated cell death of human endothelial cells: the role of oxidative stress

Replicative senescence of human endothelial cells was analyzed, using primary endothelial cells from the human umbilical vein endothelial cells (HUVEC) as an experimental model system. We had shown before that senescent HUVEC arrest in the G1 phase of the cell cycle and that a subpopulation of the senescent cells undergoes cell death. We now demonstrate that cell death occurs by apoptosis, characterized by activation of caspase 3. Using the redox-sensitive dye dihydrorhodamine 123, a significant accumulation of reactive oxygen species is detected in senescent but not young endothelial cells. To determine if increased oxidative stress may contribute to the senescent phenotype, cells were treated with tert-butyl hydroperoxide (tBHP), which is known to increase oxidative stress by decreasing the intracellular glutathione levels. We show here that mild tBHP stress induces a phenotype of premature senescence in a subpopulation of the treated cells, which closely resembles the phenotype of naturally senescent HUVEC, including growth arrest, senescence-associated β-gal activity, and apoptotic cell death. These results establish a model of premature senescence for human endothelial cells, which will be suitable to analyze mechanisms of age-associated cell death.

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

Effects of First Generation E1E3-Deleted and Second Generation E1E3E4-Deleted/Modified Adenovirus Vectors on Human Endothelial Cell Death

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying β-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFTR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.