NOX5 expression is increased in intramyocardial blood vessels and cardiomyocytes after acute myocardial infarction in humans.

Reactive oxygen species producing NADPH oxidases play important roles under different (patho)physiological conditions. NOX1, NOX2, and NOX4 are important sources of reactive oxygen species in the heart, but knowledge of the calcium-dependent NOX5 in the heart is lacking. The presence of NOX5 was studied via RT-PCR in heart tissue from patients with end-stage heart failure; the tissue was obtained during cardiac transplantation surgery. NOX5 positivity and cellular localization were studied via IHC and digital-imaging microscopy in heart tissues of patients who did not have heart disease and in infarction areas of patients who died of myocardial infarctions of different durations. Furthermore, NOX5 expression was analyzed in vitro by using Western blot analysis. NOX5 RNA was found in the hearts of controls and patients with ischemic cardiomyopathy. In controls, NOX5 localized to the endothelium of a limited number of intramyocardial blood vessels and to a limited number of scattered cardiomyocytes. In infarcted hearts, NOX5 expression increased, especially in infarctions >12 hours, which manifested as an increase in NOX5-positive intramyocardial blood vessels, as well as in endothelium, smooth muscle, and cardiomyocytes. NOX5 was found in cardiomyocyte cytoplasm, plasma membrane, intercalated disks, and cross striations. Western blot analysis confirmed NOX5 expression in isolated human cardiomyocytes. For the first time to our knowledge, we demonstrate NOX5 expression in human intramyocardial blood vessels and cardiomyocytes, with significant increases in the affected myocardium after acute myocardial infarction.

Loss of DPP4 activity is related to a prothrombogenic status of endothelial cells: implications for the coronary microvasculature of myocardial infarction patients.

Pro-coagulant and pro-inflammatory intramyocardial (micro)vasculature plays an important role in acute myocardial infarction (AMI). Currently, inhibition of serine protease dipeptidyl peptidase 4 (DPP4) receives a lot of interest as an anti-hyperglycemic therapy in type 2 diabetes patients. However, DPP4 also possesses anti-thrombotic properties and may behave as an immobilized anti-coagulant on endothelial cells. Here, we studied the expression and activity of endothelial DPP4 in human myocardial infarction in relation to a prothrombogenic endothelial phenotype. Using (immuno)histochemistry, DPP4 expression and activity were found on the endothelium of intramyocardial blood vessels in autopsied control hearts (n = 9). Within the infarction area of AMI patients (n = 73), this DPP4 expression and activity were significantly decreased, coinciding with an increase in Tissue Factor expression. In primary human umbilical vein endothelial cells (HUVECs), Western blot analysis and digital imaging fluorescence microscopy revealed that DPP4 expression was strongly decreased after metabolic inhibition, also coinciding with Tissue Factor upregulation. Interestingly, inhibition of DPP4 activity with diprotin A also enhanced the amount of Tissue Factor encountered and induced the adherence of platelets under flow conditions. Ischemia induces loss of coronary microvascular endothelial DPP4 expression and increased Tissue Factor expression in AMI as well as in vitro in HUVECs. Our data suggest that the loss of DPP4 activity affects the anti-thrombogenic nature of the endothelium.

NOX2, p22phox and p47phox are targeted to the nuclear pore complex in ischemic cardiomyocytes colocalizing with local reactive oxygen species.

BACKGROUND: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. METHODS: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. RESULTS: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. CONCLUSION: We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes.

Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity.

AIMS: Increased levels of homocysteine (Hcy) form an independent risk factor for cardiovascular disease. In a previous study we have shown that Hcy induced phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane in cardiomyocytes, inducing a pro-inflammatory phenotype. In the present study the mechanism(s) involved in Hcy-induced PS exposure were analyzed. METHODS: H9c2 rat cardiomyoblasts were subjected to 2.5 mM D,L-Hcy and analyzed for RhoA translocation and activity, Rho Kinase (ROCK) activity and expression and flippase activity. In addition, the effect of ROCK inhibition with Y27632 on Hcy-induced PS exposure and flippase activity was analyzed. Furthermore, GTP and ATP levels were determined. RESULTS: Incubation of H9c2 cells with 2.5 mM D,L-Hcy did not inhibit RhoA translocation to the plasma membrane. Neither did it inhibit activation of RhoA, even though GTP levels were significantly decreased. Hcy did significantly inhibit ROCK activation, but not its expression, and did inhibit flippase activity, in advance of a significant decrease in ATP levels. ROCK inhibition via Y27632 did not have significant added effects on this. CONCLUSION: Hcy induced PS exposure in the outer leaflet of the plasma membrane in cardiomyocytes via inhibition of ROCK and flippase activity. As such Hcy may induce cardiomyocytes vulnerable to inflammation in vivo in hyperhomocysteinaemia patients.

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).

We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 µM extracellular L -Hcy), (2) incubation with 50 µM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 µM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 µM extracellular L -Hcy) with or without 10 µM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 µM, and 100 µM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.

Intravenous clusterin administration reduces myocardial infarct size in rats.

BACKGROUND: Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. METHODS: Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n=15) or vehicle (n=13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. RESULTS: Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. CONCLUSIONS: Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.

p47phox-Dependent Reactive Oxygen Species Stimulate Nuclear Translocation of the FoxO1 Transcription Factor During Metabolic Inhibition in Cardiomyoblasts.

Reactive oxygen species (ROS) control forkhead box O (FOXO) transcription factor activity by influencing their nuclear translocation. However, knowledge of the ROS cellular source(s) involved herein remains scarce. Recently, we have shown p47phox-dependent activation of ROS-producing NADPH oxidase (NOX) at the nuclear pore in H9c2 rat cardiomyoblasts in response to ischemia. This localizes NOX perfectly to affect protein nuclear translocation, including that of transcription factors. In the current study, involvement of p47phox-dependent production of ROS in the nuclear translocation of FOXO1 was analyzed in H9c2 cells following 4 h of metabolic inhibition (MI), which mimics the effects of ischemia. Nuclear translocation of FOXO1 was determined by quantitative digital-imaging fluorescence and western blot analysis. Subsequently, the effect of inhibiting p47phox-dependent ROS production by short hairpin RNA (shRNA) transfection on FOXO1 translocation was analyzed by digital-imaging microscopy. MI induced a significant translocation of FOXO1 into the nucleus. Transfection with p47phox-shRNA successfully knocked-down p47phox expression, reduced nuclear nitrotyrosine production, an indirect marker for ROS production, and inhibited the nuclear translocation of FOXO1 following MI. With these results, we show for the first time that nuclear import of FOXO1 induced by MI in H9c2 depends critically on p47phox-mediated ROS production.

Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.

Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 µM PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells.

S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine.

OBJECTIVE: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 µM extracellular l-Hcy); and incubation with (2) 50 µM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 µM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 µM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3). CONCLUSION: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

Prolonged C1 inhibitor administration improves local healing of burn wounds and reduces myocardial inflammation in a rat burn wound model.

In a previous study, the authors found persistent presence of acute inflammation markers such as C-reactive protein and complement factors locally in burn wounds. This persistence of acute inflammation may not only delay local burn wound healing but also have a systemic effect, for instance on the heart. Here, the effects of C1 esterase inhibitor (C1inh), an inhibitor of complement activation, on burn wound progression and the heart were analyzed in rats. Dorsal full-thickness burn wounds (2 × 4 cm) were induced on female Wistar rats (n = 14). The rats were divided into two groups (n = 7): a control group (just burns) and a C1inh group. C1inh was administered daily intravenously for 14 days. The burn wound, healthy skin from the hind leg (internal control), and the heart were then fixed in formalin. Tissues were analyzed for granulation tissue formation, reepithelialization, amount and type of infiltrating inflammatory cells (granulocytes and macrophages), and inflammatory markers (complement factors C3 and C4). C1inh treatment significantly reduced the amount of granulation tissue and significantly increased reepithelialization. C1inh also significantly reduced macrophage infiltration. Burns induced infiltration of macrophages into the ventricles of the heart and remarkably also into the atria of the heart. This effect could be counteracted by C1inh. These data show that systemic treatment with C1inh acts at different levels resulting in improved healing locally in burn wounds and systemically reduced inflammation in the heart. Therefore, C1inh might be a possible therapeutic intervention for burn wound patients.