Dual effect of oxidized LDL on cell cycle in human endothelial cells through oxidative stress.

BACKGROUND: Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the exposure time. Various steps in the cell cycle and in the apoptotic signaling cascade are modulated by O2-, and OxLDL stimulates vascular O2- formation. Here we studied the role of NADPH oxidase, a potential source for O2- formation after OxLDL stimulation, in cell proliferation, and we investigated whether OxLDL influences anti-apoptotic genes in cultured human umbilical vein endothelial cells (HUVEC). Methods and Results. OxLDL dose-dependently (10 to 300 microg/mL) stimulated O2- formation in HUVEC (detected by cytochrome c assay and by chemiluminescence of lucigenin). Low OxLDL concentrations (5 to 10 microg/mL) induced proliferation (detected by 3H-thymidine incorporation), while higher concentrations (50 to 300 microg/mL) induced apoptotic cell death (detected by Annexin assay and DNA fragmentation). Proliferation was blocked by the antioxidants SOD and catalase and by diphenyleneiodonium (10 micromol/L), an inhibitor of the O2- generating NADPH oxidase. In addition, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly reduced O2- formation after stimulation with OxLDL. The OxLDL effect on apoptosis was also blocked by antioxidants. Since endothelial cells are protected against apoptosis through anti-apoptotic genes, we investigated whether OxLDL overcomes protection against apoptosis through suppression of the anti-apoptotic gene A20, a zinc-finger protein. OxLDL suppressed the expression of A20 in a dose-dependent manner. CONCLUSION: These data indicate that OxLDL has a dual effect on cell cycle in HUVEC, inducing proliferation at low and apoptosis at higher concentrations. Both effects are mediated by O2- formation, with NADPH oxidase being a major source for O2-. Thus, OxLDL contributes importantly to vascular cellular turnover through the induction of oxidative stress.

Differential role of angiotensin II receptor subtypes on endothelial superoxide formation.

The physiological role of the angiotensin II AT2 receptor subtype is not fully characterized. We studied whether AT2 receptor could antagonize AT1 mediated superoxide formation in endothelial cells. In quiescent human umbilical vein endothelial cells (HUVEC) superoxide formation was measured after long-term incubation (6 h) with angiotensin II in the presence or absence of its receptor blocker candesartan (AT1) or PD123319 (AT2) using the cytochrome c assay. In separate experiments, the effects of AT2 mediated effects on activities of cellular phosphates including the src homology 2 domain containing phosphatases (SHP-1) was studied. The basal superoxide formation (0.19+/-0.03 nmol superoxide mg protein(-1) min(-1)) in HUVEC was increased by 37.1% after exposure to angiotensin II (100 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 microM) as well as the tyrosine kinase inhibitor genistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the superoxide formation by 73.7% in intact cells. Stimulation of AT2 went along with an increased activity of tyrosine phosphatases in total cell lysates (29.8%) and, in particular, a marked stimulation of src homology 2 domain containing phosphatases (SHP-1, by 293.4%). The tyrosine phosphatase inhibitor vanadate, in turn, prevented the AT2 mediated effects on superoxide formation. The expression of both angiotensin II receptor subtypes AT1 and AT2 was confirmed by RT - PCR analysis. It is concluded that AT2 functionally antagonizes the AT1 induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.

KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells.

Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.

Oxidative stress mediates apoptosis induced by oxidized low-density lipoprotein and oxidized lipoprotein(a).

We measured oxidative stress caused by lipoproteins in human umbilical vein endothelial cells and rabbit aorta. Oxidized lipoprotein(a) was the more potent stimulus over oxidized low density lipoprotein, and we believe that both influence the pathogenesis of atherosclerosis.

Atherogenic lipoproteins, oxidative stress, and cell death.

BACKGROUND: Glomerulosclerosis and atherosclerosis are chronic inflammatory processes that may be influenced by oxidized lipoproteins, oxidized low-density lipoproteins (oxLDL), and oxidized lipoprotein(a) [oxLp(a)]. We hypothesize that these lipoproteins contribute to the development of glomerulosclerosis and atherosclerosis through the induction of oxidative stress, which influences cell viability. We therefore determined the impact of oxLDL and oxLp(a) on O2- formation and on necrotic and apoptotic cell death in vascular and glomerular cells. METHODS: The impact of human LDL and Lp(a) (oxidized with CuSO4) on O2- formation (detected with a chemiluminescence method), apoptosis, and necrosis (determined with the annexin assay) was studied in cultured human umbilical vein endothelial cells (ECs) and in cultured human mesangial cells (MCs). RESULTS: O2- formation was increased by 10 micrograms/ml oxLDL (by factor 2.5 in ECs) and by 5 micrograms/ml oxLp(a) (by factor 3.5 in ECs). OxLDL and oxLp(a) both significantly and dose-dependently increased the rate of apoptotic cell death in ECs and in MCs, with oxLp(a) being the more potent stimulus that also caused necrosis. The induction of apoptosis by oxLDL and oxLp(a) in ECs and MCs was enhanced by inhibition of the endogenous superoxide dismutase (SOD) with diethyl-dithio-carbamate and was blunted by the antioxidants N-acetylcysteine, vitamin C + E, SOD, and catalase, suggesting that oxidative stress was the stimulus for apoptosis. CONCLUSIONS: These data suggest that oxLDL and oxLp(a) contribute to inflammation by stimulating O2- formation, leading to apoptotic cell death in the vascular wall and in the glomerulus. The oxidized lipoproteins may thereby influence the pathogenesis of atherosclerosis and glomerulosclerosis.

Lp(a) and LDL induce apoptosis in human endothelial cells and in rabbit aorta: role of oxidative stress.

BACKGROUND: Atherogenic lipoproteins cause injury to the vascular wall in the early phase of atherogenesis. We assessed the effects of native (nLDL) and oxidized (oxLDL) low-density lipoprotein (LDL) and lipoprotein (a) [Lp(a)] on O2- formation and cell death in cultured human umbilical vein endothelial cells (HUVECs) and rabbit aorta (RA). METHODS AND RESULTS: O2- formation of HUVECs and RA segments was not influenced by nLDL, but was dose dependently increased by oxLDL and was moderately increased by nLp(a). oxLp(a) was the most potent stimulus for O2- formation, increasing it in HUVECs by 356% at 5 micrograms/ml and in RA by 294% at 100 micrograms/ml. Apoptosis was detected by DNA fragmentation and Annexin assay in HUVECs and by TUNEL staining in RA. Incubation of HUVECs and RA with oxLDL, but not nLDL, dose and time dependently induced apoptosis with only a minimal effect on necrosis. nLp(a) elicited a small but significant effect on apoptosis, whereas oxLp(a) induced apoptosis more potently than oxLDL in HUVECs and RA and caused necrotic cell death in HUVECs. Induction of apoptosis by oxLDL and oxLp(a) in RA was enhanced by the superoxide dismutase (SOD) inhibitor, diethyl-dithio-carbamate, and was blunted by SOD and catalase in HUVECs and RA, suggesting that O2- formation was involved. The concentration of lysophosphatidylcholine, a lipoprotein oxidation product and stimulus for O2- formation, was significantly enhanced by factor 5 in oxLDL and by factor 7 in oxLp(a) compared with native lipoproteins. CONCLUSION: Atherogenic lipoproteins stimulate O2- formation and induction of apoptosis in HUVECs and RA, and may thereby influence the pathogenesis of atherosclerosis.

Effects of extremely low frequency electromagnetic field (EMF) on collagen type I mRNA expression and extracellular matrix synthesis of human osteoblastic cells.

Human osteoblastic cells were grown in a three-dimensional (3-D) cell culture model and used to test the effects of a 20 Hz sinusoidal electromagnetic field (EMF; 6 mT and 113 mV/cm max) on collagen type I mRNA expression and extracellular matrix formation in comparison with the effects of growth factors. The cells were isolated from trabecular bone of a healthy individual (HO-197) and from a patient presenting with myositis ossificans (MO-192) and grown in a collagenous sponge-like substrate. Maximal enhancement of collagen type I expression after EMF treatment was 3.7-fold in HO-197 cells and 5.4-fold in MO-192 cells. Similar enhancement was found after transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I) treatment. Combined treatment of the cells with EMF and the two growth factors TGF-beta and IGF-I did not act synergistically. MO-192 cells produced an osteoblast-characteristic extracellular matrix containing collagen type I, alkaline phosphatase, and osteocalcin, together with collagen type III, TP-1, and TP-3, two epitopes of an osteoblastic differentiation marker. The data suggest that the effects of EMFs on osteoblastic differentiation are comparable to those of TGF-beta and IGF-I. We conclude that EMF effects in the treatment of skeletal disorders and in orthopedic adjuvant therapy are mediated via enhancement of collagen type I mRNA expression, which may lead to extensive extracellular matrix synthesis.

Bax and Bcl-xs are induced at the onset of apoptosis in involuting mammary epithelial cells.

Mammary gland involution is a physiological process in which the entire organ is remodeled through the process of apoptosis. Apoptosis of secretory alveolar cells is initiated at the time of weaning, followed by the collapse and disappearance of the entire lobuloalveolar compartment. While apoptotic figures were rare in mammary epithelium of lactating mice, their number increased after weaning and reached a maximum on day 3 of involution. Active cell death continued until day 5 after weaning and only little parenchyma remained on day 8, when remodeling of the gland was completed. Bax mRNA levels increased during the first day of involution independent of the presence or absence of p53. Bax protein was detected in an increasing number of cells after weaning, peaking at day 3 and decreasing thereafter. Low levels of bcl-x mRNA and protein were present during lactation, followed by a sharp increase during the first 2 days of involution. The bcl-xS splice variant of bcl-x can promote cell death, and bcl-xL has a protective function in cell culture. The ratio of bcl-xS versus bcl-xL remained stable in the virgin, pregnant and lactating gland. However, during the first 2 days of involution, bcl-xS expression increased six-fold more than bcl-xL. To further evaluate the role of Bcl-xS which was less abundant in the mammary cells than Bcl-xL, cotransfection studies were performed in cell culture. They confirmed that Bcl-xS protein can facilitate apoptosis even when Bcl-xL is present in excess. These findings point to a significant role for Bax and Bcl-xS in the regulation of apoptosis of secretory alveolar cells during involution.

Expression of a viral oncoprotein during mammary gland development alters cell fate and function: induction of p53-independent apoptosis is followed by impaired milk protein production in surviving cells.

The disruption of cell cycle regulation is associated with developmental abnormalities and tumorigenesis. The SV40 large T antigen (Tag) interferes with cell cycle control by interacting with the pRb family and p53. Mice carrying a transgene composed of the whey acidic protein (WAP) gene promoter and the Tag coding sequence express Tag during pregnancy and are unable to nurse their young. Tag expression induced apoptosis in mammary epithelial cells during late pregnancy. At least 5% of mammary epithelial cells were undergoing apoptosis at any one time. In contrast, less than 0.2% of mammary epithelial cells in nontransgenic littermates was undergoing apoptosis. Apoptosis in Tag mice was associated with increased steady-state RNA levels of bax and bcl-xL + S, with a relative increase in bcl-xs expression. Since p53 was sequestered by Tag, it is likely that p53-independent mechanisms precipitated apoptosis. The Tag-expressing mammary alveolar cells that did not undergo apoptosis continued to differentiate through late pregnancy, as measured by the sequential activation of milk protein gene expression. However, milk protein production, processing, and secretion was impaired, resulting in lactation failure.

Apoptosis and remodeling of mammary gland tissue during involution proceeds through p53-independent pathways.

Mammary gland involution is a physiological process that follows lactation and results in the rapid disappearance of the entire lobulo-alveolar compartment. Coincident with the onset of involution, milk protein gene expression ceases and alveolar cells undergo programmed cell death. In mammary epithelial tissue culture cells in vitro, both p53-dependent and p53-independent apoptosis pathways have been identified. We investigated whether p53 induces apoptosis during mammary gland involution in vivo and participates in tissue remodeling. Toward this end, we examined the process of involution in the presence and absence of functional p53 in mouse models: wild-type, transgenic mice that express SV40 T-antigen specifically in mammary tissue during pregnancy; and mice that carry nonfunctional p53 alleles in their germ line. Mammary gland whole-mount and histological analyses revealed that involution and remodeling, with the concomitant disappearance of the lobulo-alveolar structures, proceeded normally in the absence of functional p53. In addition, the absence of functional p53 did not alter the involution related pattern of bax (death inducer) gene expression or the ratio of RNAs encoding bcl-xs (death inducer) to bcl-xL (survival inducer).

Bone matrix deposition of T1, a homologue of interleukin 1 receptors.

The mouse T1 glycoprotein is a secreted molecule of the immunoglobulin superfamily with significant homology to interleukin 1 receptors. It is expressed during bone development, and the extracellular diffusible gene product is found associated with newly formed bone but not cartilage matrix. During osteogenic differentiation of mandibular condyles of newborn mice in vitro, T1 gene expression is induced shortly after cultivation and is observed throughout the differentiation process. The temporal expression pattern of the gene is an mandibular condyles indicates that T1 expression is an early marker of osteogenic differentiation. This view is substantiated by the analysis of T1 gene regulation in continuous osteogenic cell lines. Both in differentiating osteoblast-like KM-1K cells derived from mandibular condyles and in MC3T3 cells, T1 gene activity is preferentially associated with early differentiation stages. In mandibular condyles, the secreted extracellular T1 protein is deposited into newly formed osteoid but not into cartilage matrix. This novel bone matrix protein may locally modulate the availability of its ligand.

Adipose differentiation of cartilage in vitro.

Xiphoids of newborn mice consist of young chondrogenic cells of primary cartilage. During in vitro cultivation, xiphoids showed, morphologically, characteristics of adipose differentiation. This process progressed with time and by day 21 of the culture most of the cells in the xiphoids represented morphological mature adipocytes. During this period, the level of mRNA of lipoprotein lipase, and adipocyte-characteristic gene, increased steadily, while the level of collagen type II mRNA decreased. Continuous DNA synthesis during the cultivation period, even in mature adipocytes confirmed the viability of the cells. Mandibular condyles of newborn mice obtain chondroprogenitor cells as well as young and mature chondroblasts and represent secondary cartilage. Under identical culture conditions mandibular condyles obtained from the same mice undergo osteogenic differentiation and form mature bone within 7 to 10 days. Common to both xiphoids and mandibular condyles is the capacity to transdifferentiate, but they show distinct, divergent differentiation pathways. These findings indicate that cartilagenous tissue of xiphoids undergoes transdifferentiation into adipose tissue in vitro.

Osteosarcomagenic doses of radium (224Ra) and infectious endogenous retroviruses enhance proliferation and osteogenic differentiation of skeletal tissue differentiating in vitro.

Cartilage tissue from embryonic mice which undergoes osteogenic differentiation during in vitro cultivation was used to study the effect of osteosarcomagenic doses of alpha-irradiation and bone-tumor-inducing retroviruses on proliferation and phenotypic differentiation of skeletal cells in a defined tissue culture model. Irradiated mandibular condyles showed dose-dependent enhancement of cell proliferation at day 7 of the culture and increased osteogenic differentiation at day 14. Maximal effects were found with 7.4 Bq/ml of 224Ra-labeled medium. Doses of 740 and 7400 Bq/ml of 224Ra-labeled medium induced increasing cell death. Retrovirus infection enhanced osteogenic differentiation and extended the viability of irradiated cells. After transplantation none of the treated tissues developed tumors in syngeneic mice.

Rapid and sensitive mRNA phenotyping for interleukins (IL-1 to IL-6) and colony-stimulating factors (G-CSF, M-CSF, and GM-CSF) by reverse transcription and subsequent polymerase chain reaction.

Oligonucleotide primer pairs specific for interleukins (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, as well as for granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA/cDNA were synthesized in order to detect cytokine transcripts by reverse transcription and subsequent polymerase chain reaction (RT/PCR). Analysis of RNA preparations of the human bladder carcinoma cell line 5637 by this methodology reveals expression of mRNAs for IL-1 alpha, IL-1 beta, IL-6, G-CSF, M-CSF, and for GM-CSF, whereas mRNAs for IL-2, IL-3, IL-4, and IL-5 are not detectable. These results are in agreement with data obtained by classical methods. Thus, for the cytokines IL-2, IL-3, IL-4, and IL-5, it was not possible to detect a phenomenon described as 'illegitimate transcription,' defined as the low level transcription of any gene in any cell type. This finding is of importance for the applicability of mRNA phenotyping employing RT/PCR for the determination of mRNA expression patterns. For M-CSF mRNA detection, two oligonucleotide primer pairs had to be used to distinguish between the alpha-(pcCSF17) and beta-splicing forms and to overcome the problem of non-amplification of a larger fragment in the presence of a competing smaller one, defined here as 'incomplete positivity.' For G-CSF, IL-4, IL-2, and IL-5, RT/PCR reveals two fragments. Restriction enzyme analysis of the additional fragments suggests that they may arise from alternative splicing events. For G-CSF and IL-4, exons 3 and 2 seem to be spliced out, respectively. The additional fragments for IL-2 and IL-5 RT/PCR have not yet been further characterized, but the size of the fragments makes it seem probable that exons 2 and 3 are spliced out for IL-2 and IL-5, respectively. The biological role of these alternative mRNAs has yet to be determined.