Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

Heme oxygenase-1 gene expression attenuates angiotensin II-mediated DNA damage in endothelial cells

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression using a retroviral vector on attenuation of angiotensin II (Ang II)-mediated oxidative stress. Comet and glutathione (GSH) levels were used as indicators of the levels of oxidative stress. Comet assay was performed to evaluate damage on DNA, whereas GSH levels were measured to determine the unbalance of redox potential. Pretreatments with inducers, such as heme 10 microM, SnCl(2) 10 microM, and inhibitors, such as tin-mesoporphyrin 10 microM was followed by treatment with Ang II 200 ng/ml. Pretreatment with heme or SnCl(2) provoked significant reductions (P < 0.01) of tail moment in the comet assay. Opposite effects were evident by pretreatment for 16 hr with tin-mesoporphyrin. A decrease in tail moment levels was found in human endothelial cells transduced with the human HO-1 gene. The addition of Ang II (200 ng/ml) to human dermal microvessel endothelial cell-1 for 16 hr resulted in a significant (P < 0.05) reduction of GSH contents control endothelial cells but not in endothelial cells transduced with HO-1 gene. The results presented indicated that stimulation or overexpression of HO-1 attenuated DNA damages caused by exposures of Ang II.     

Heme oxygenase-1 gene transfer inhibits angiotensin II-mediated rat cardiac myocyte apoptosis but not hypertrophy

Cardiac myocyte apoptosis underlies the pathophysiology of cardiomyopathy, and plays a critical role in the transition from myocardial hypertrophy to heart failure. Angiotensin II (Ang II) induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress; however, the mechanisms underlying the activation of both pathways and their interactions remain unclear. In the present study, we have investigated whether overexpression of the antioxidant protein heme oxygenase-1 (HO-1) protects against apoptosis and hypertrophy in cultured rat cardiac myocytes treated with Ang II. Our findings demonstrate that Ang II (100 nM, 24 h) alone upregulates HO-1 expression and induces both myocyte hypertrophy and apoptosis, assessed by measuring terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) staining, caspase-3 activity and mitochondrial membrane potential. Ang II elicited apoptosis was augmented in the presence of tin protoporphyrin, an inhibitor of HO activity, while HO-1 gene transfer to myocytes attenuated Ang II-mediated apoptosis but not hypertrophy. Adenoviral overexpression of HO-1 was accompanied by a significant increase in Ang II induced phosphorylation of Akt, however, Ang II-mediated p38 mitogen activated protein kinase (MAPK) phosphorylation was attenuated. Inhibition of phosphotidylinositol-3-kinase enhanced myocyte apoptosis elicited by Ang II, however, p38MAPK inhibition had no effect, suggesting that overexpression of HO-1 protects myocytes via augmented Akt activation and not through modulation of p38MAPK activation. Our findings identify the signalling pathways by which HO-1 gene transfer protects against apoptosis and suggest that overexpression of HO-1 in cardiomyopathies may delay the transition from myocyte hypertrophy to heart failure.     

Down-regulation of ether-a-go-go-related gene potassium channel protein through sustained stimulation of AT1 receptor by angiotensin II

We investigated the effects of AT₁ receptor stimulation by angiotensin II (Ang II) on human ether-a-go-go-related gene (hERG) potassium channel protein in a heterogeneous expression system with the human embryonic kidney (HEK) 293 cells which stably expressed hERG channel protein and were transiently transfected with the human AT₁ receptors (HEK293/hERG). Western-blot analysis showed that Ang II significantly decreased the expression of mature hERG channel protein (155-kDa band) in a time- and dose-dependent manner without affecting the level of immature hERG channel protein (135-kDa band). The relative intensity of 155-kDa band was 64.7 ± 6.8% of control (P < 0.01) after treatment of Ang II at 100 nM for 24 h. To investigate the effect of Ang II on the degradation of mature hERG channel protein, we blocked forward trafficking from ER to Golgi with a Golgi transit inhibitor brefeldin A (10 μM). Ang II significantly enhanced the time-dependent reduction of mature hERG channel protein. In addition, the proteasomal inhibitor lactacystin (5 μM) inhibited Ang II-mediated the reduction of mature hERG channel protein, but the lysosomal inhibitor bafilomycin A1 (1 μM) had no effect on the protein. The protein kinase C (PKC) inhibitor bisindolylmaleimide 1 (1 μM) antagonized the reduction of mature hERG channel protein induced by Ang II. The results indicate that sustained stimulation of AT₁ receptors by Ang II reduces the mature hERG channel protein via accelerating channel proteasomal degradation involving the PKC pathway.     

Heme oxygenase and the cardiovascular-renal system

Heme oxygenase (HO) has been shown to be important for attenuating the overall production of reactive oxygen species (ROS) through its ability to degrade heme and to produce carbon monoxide (CO), biliverdin/bilirubin, and the release of free iron. Excess free heme catalyzes the formation of ROS, which may lead to endothelial cell (EC) dysfunction as seen in numerous pathological conditions including hypertension and diabetes, as well as ischemia/reperfusion injury. The upregulation of HO-1 can be achieved through the use of pharmaceutical agents, such as metalloporphyrins and some HMG-CoA reductase inhibitors. Among other agents, atrial natriretic peptide and donors of nitric oxide (NO) are important modulators of the heme-HO system, either through induction of HO-1 or the biological activity of its products. Gene therapy and gene transfer, including site- and organ-specific targeted gene transfer, have become powerful tools for studying the potential role of HO-1/HO-2 in the treatment of various cardiovascular diseases as well as diabetes. HO-1 induction by pharmacological agents or gene transfer of human HO-1 into endothelial cells (ECs) in vitro increases cell-cycle progression and attenuates Ang II, TNF-, and heme-mediated DNA damage; administration in vivo acts to correct blood pressure elevation following Ang II exposure. Moreover, site-specific delivery of HO-1 to renal structures in spontaneously hypertensive rats (SHR), specifically to the medullary thick ascending limb of the loop of Henle (mTALH), has been shown to normalize blood pressure and provide protection to the mTAL against oxidative injury. In other cardiovascular situations, delivery of human HO-1 to hyperglycemic rats significantly lowers superoxide (O(2)(-)) levels and prevents EC damage and sloughing of vascular EC into the circulation. In addition, administration of human HO-1 to rats in advance of ischemia/reperfusion injury considerably reduces tissue damage. The ability to upregulate HO-1 through pharmacological means or through the use of gene therapy may offer therapeutic strategies for cardiovascular disease in the future. This review discusses the implications of HO-1 delivery during the early stages of cardiovascular system injury or in early vascular pathology and suggests that pharmacological agents that regulate HO activity or HO-1 gene delivery itself may become powerful tools for preventing the onset or progression of certain cardiovascular pathologies.     

A significant role for the heme oxygenase-1 gene in endothelial cell cycle progression

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression, using a retroviral vector, on cell cycle progression in the presence and absence of pyrrolidine dithiocarbamate (PDTC). The addition of PDTC (25 and 50 microM) to human microvessel endothelial cells over 24 h resulted in significant (P < 0.05) abnormalities in DNA distribution and cell cycle progression compared to cells overexpressing the HO-1 gene. The addition of PDTC resulted in a significantly decreased G(1) phase and an increased G(2)/M phase in the control cells, but not in cells transduced with the human HO-1 gene (P < 0.05). Further, PDTC had a potent effect on DNA distribution abnormalities in exponentially grown cells compared to subconfluent cells. Upregulation of HO activity in endothelial cells, as a result of overexpressing human HO-1, prevented PDTC-mediated abnormalities in DNA distribution. Inhibition of HO activity by tin-mesoporphyrin (SnMP) (30 microM) resulted in enhancement of PDTC-mediated abnormalities in cell cycle progression. Bilirubin or iron did not mediate DNA distribution. We conclude that an increase in endothelial cell HO-1 activity with subsequent generation of carbon monoxide, elicited by gene transfer, reversed the PDTC-mediated abnormalities in cell cycle progression and is thus a potential therapeutic means for attenuating the effects of oxidative stress-causing agents.     

Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes

Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF₂ and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF₂, P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 ± 0.8 cells/ml blood in control rats vs. 48 ± 4.8 cells/ml blood in diabetic rats (P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 ± 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 ± 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 ± 2.3 (P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 ± 2.3 to 29 ± 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes.     

Phosphatidylinositide 3-kinase regulates angiotensin II-induced cytosolic phospholipase A2 activity and growth in vascular smooth muscle cells

Angiotensin (Ang) II via the AT(1) receptor acts as a mitogen in vascular smooth muscle cells (VSMC) through stimulation of multiple signaling mechanisms, including tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, cytosolic phospholipase A(2)(cPLA(2))-dependent release of arachidonic acid (AA) is linked to VSMC growth and we have reported that Ang II stimulates cPLA(2) activity via the AT(1) receptor. The coupling of Ang II to the activation of cPLA(2) appears to involve mechanisms both upstream and downstream of MAPK such that AA stimulates MAPK activity which phosphorylates cPLA(2) to further enhance AA release. However, the upstream mechanisms responsible for activation of cPLA(2) are not well-defined. One possibility includes phosphatidylinositide 3-kinase (PI3K), since PI3K has been reported to participate in the upstream signaling events linked to activation of MAPK. However, it is not known whether PI3K is involved in the Ang II-induced activation of cPLA(2) or if this mechanism is associated with the Ang II-mediated growth of VSMC. Therefore, we used cultured rat VSMC to examine the role of PI3K in the Ang II-dependent phosphorylation of cPLA(2), release of AA, and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased [(3)H]thymidine incorporation, cell number, and the release of [(3)H]AA. Also, using Western analysis, Ang II increased the phosphorylation of MAPK and cPLA(2) which were blocked by the MAPK kinase inhibitor PD98059 (10 microM/L). Similarly, the PI3K inhibitor LY294002 (10 microM/L) abolished the Ang II-mediated increase in MAPK phosphorylation, as well as phosphoserine-PLA(2). Further, inhibition of PI3K blocked the Ang II-induced release of AA and VSMC mitogenesis. However, exogenous AA was able to restore VSMC growth in the presence of LY294002, as well as reverse the inhibition of MAPK and cPLA(2) phosphorylation by LY294002. Thus, it appears from these data that Ang II stimulates the PI3K-sensitive release of AA which stimulates MAPK to phosphorylate cPLA(2) and enhance AA release. This mechanism may play an important role in the Ang II-induced growth of VSMC.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.     

Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains

Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.     

Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II: ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES*

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.     

Inhibition of cancer cell growth by cyclin dependent kinase 4 inhibitors synthesized based on the structure of fascaplysin

Tryptamine derivatives, a new structural class of cyclin dependent kinase 4 inhibitors, have been identified during extensive biological screening of synthetic molecules. The molecules were synthesized based on the structure of fascaplysin, which is not only a specific inhibitor of the Cdk4-cyclin D1 enzyme but also a relatively toxic molecule, probably because it binds and intercalates DNA. Interestingly, the new structural analogues of fascaplysin do not interact or intercalate with double-stranded DNA, although they inhibit Cdk4-cyclin D1 specifically. We found that compound CA199 was the most potent molecule, showing at least 25-fold specificity towards Cdk4-cyclin D1 (IC50 for Cdk4-cyclin D1 = 20 microM, Cdk2 > 500 microM). CA199 inhibits the growth of different cancer cell lines at concentrations ranging from 10-40 microM. It blocks growth of asynchronous cells at G0/G1 in a retinoblastoma protein (pRb) dependent manner. Moreover, CA199 blocks growth only at early G1 in synchronised cells released from a mimosine-induced G1/S block. These observations are reminiscent of a true Cdk4 inhibitor.     

Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin–angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β₁)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β₁, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT₁) receptor antagonist losartan, an anti-TGF-β₁ antibody and the ERK 1/2 inhibitor PD98059 but not by the AT₂ receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT₁ receptor/TGF-β₁/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β₁ antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT₁ receptor/TGF-β₁/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

Adenosine protects against angiotensin II-induced apoptosis in rat cardiocyte cultures

Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A₁ and possibly A₃ receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA, 1 M), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A₁ adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A₃ adenosine receptor agonist N⁶-(3-iodobenzyl) adenosine-5-N-methyluronamide (IB-MECA, 0.1 M), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca²⁺ that lasted 40–60 sec. Pre-treatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca²⁺ elevation. In conclusion, activation of adenosine A₁ receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A₃ receptors confers partial cardioprotection.     

Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression

Arterial remodeling in response to pathological insult is a complex process that depends in part on the balance between vascular cell apoptosis and proliferation. Studies in experimental models suggest that HO-1 mediates neointimal formation while limiting lumen stenosing, indicating a differential effect on vascular endothelial (EC) and smooth muscle cells (SMC). We investigated the effect of HO-1 expression on cell cycle progression in EC and SMC. The addition of SnMP (10 microM), an inhibitor of HO activity, to EC or SMC for 24h, resulted in significant abnormalities in DNA distribution and cell cycle progression compared to cells treated with the HO-1 inducers, heme (10 microM) or SnCl(2) (10 microM). SnMP increased G(1) phase and decreased S and G(2)/M phases in EC while heme or SnCl(2) decreased G(1) phase, but increased S and G(2)/M phases (p<0.05). Opposite effects were obtained in SMC. SnMP decreased G(1) phase and increased S and G(2)/M phases while heme or SnCl(2) increased G(1) phase but decreased S and G(2)/M phases (p<0.05). Our data demonstrate that HO-1 regulates the cell cycle in a cell-specific manner; it increases EC but decreases SMC cycle progression. The mechanisms underlying the HO-1 cell-specific effect on cell cycle progression within the vascular wall are yet to be explored. Nevertheless, these findings suggest that cell-specific targeting of HO-1 expression may provide a novel therapeutic strategy for the treatment of cardiovascular diseases.     

ACE2 deficiency enhances angiotensin II-mediated aortic profilin-1 expression, inflammation and peroxynitrite production

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2(+/y)) and ACE2 knockout (ACE2KO, Ace2(-/y)) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg?1.d?1) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1-7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases.     

Angiotensin II Activates Programmed Myocyte Cell Deathin Vitro

To determine whether angiotensin II (Ang II) can induce apoptosis of neonatal ventricular myocytes, these cells were exposed to 10⁻⁹MAng II for 24 hin vitroand the effects of this intervention on programmed myocyte cell death were examined by the terminal deoxynucleotidyl transferase assay and DNA gel electrophoresis. Ang II resulted morphologically in a 2.5-fold increase in the percentage of myocytes with double strand cleavage of the DNA and biochemically in the formation of DNA fragments equal in size to mono- and oligonucleosomes. Moreover, Ang II stimulation was characterized by a 37% increase in resting level of intracellular calcium and the activation of calcium-dependent endogenous endonuclease. In contrast, pH-dependent endogenous endonuclease was not enhanced by the addition of Ang II. Ang II-induced DNA damage was inhibited by the AT₁receptor antagonist, losartan. Similarly, the calcium chelator, BAPTA-AM, prevented Ang II-mediated cell death. Conversely, the calcium ionophore, A23187, triggered programmed cell death. Finally, the selective AT₂receptor subtype blocker, PD123319, failed to reduce myocyte apoptosis. In conclusion, ligand binding of AT₁receptors may initiate programmed myocyte cell death via an elevation in cytosolic calcium and the stimulation of calcium-dependent endogenous endonuclease.