Hepatic Fat and Macrophages Are Increased in Livers of Diabetic Patients without Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: Diabetes mellitus (DM), especially type 2, is strongly associated with non-alcoholic fatty liver disease (NAFLD). Recent studies indicate that particularly in DM patients, "simple" liver steatosis can progress into more severe disease. However, little is known about putative hepatic histopathological changes in DM patients without NAFLD. In this study, we therefore analysed fat content and inflammatory cell infiltration in the livers of deceased DM and non-DM patients without NAFLD, and analysed age/sex effects hereon. METHODS: Hepatic fat and inflammatory cells were studied through (immuno)histochemical analysis in liver tissue from 24 DM patients and 66 non-diabetic controls, without histopathological characteristics of NAFLD. RESULTS: We observed a 2-fold increase in fat percentage/mm2 and a near 5-fold increase in the number of fat-containing cells/mm2 in DM patients compared to non-diabetic controls. Fat content was significantly higher in patients with type 2 DM, but not type 1 DM, compared to non-diabetic controls, while the number of CD68+ cells/mm2 was significantly elevated in both DM groups. CONCLUSION: Hepatic fat and number of macrophages are increased in patients with DM without NAFLD, which may reflect a higher risk on development of steatosis and steatohepatitis.

NOX2 and NOX5 are increased in cardiac microvascular endothelium of deceased COVID-19 patients.

BACKGROUND: Cardiac injury and inflammation are common findings in COVID-19 patients. Autopsy studies have revealed cardiac microvascular endothelial damage and thrombosis in COVID-19 patients, indicative of microvascular dysfunction in which reactive oxygen species (ROS) may play a role. We explored whether the ROS producing proteins NOX2, NOX4 and NOX5 are involved in COVID-19-induced cardio-microvascular endothelial dysfunction. METHODS: Heart tissue were taken from the left (LV) and right (RV) ventricle of COVID-19 patients (n = 15) and the LV of controls (n = 14) at autopsy. The NOX2-, NOX4-, NOX5- and Nitrotyrosine (NT)-positive intramyocardial blood vessels fractions were quantitatively analyzed using immunohistochemistry. RESULTS: The LV NOX2+, NOX5+ and NT+ blood vessels fractions in COVID-19 patients were significantly higher than in controls. The fraction of NOX4+ blood vessels in COVID-19 patients was comparable with controls. In COVID-19 patients, the fractions of NOX2+, NOX5+ and NT+ vessels did not differ significantly between the LV and RV, and correlated positively between LV and RV in case of NOX5 (r = 0.710; p = 0.006). A negative correlation between NOX5 and NOX2 (r = -0.591; p = 0.029) and between NOX5 and disease time (r = -0.576; p = 0.034) was noted in the LV of COVID-19 patients. CONCLUSION: We show the induction of NOX2 and NOX5 in the cardiac microvascular endothelium in COVID-19 patients, which may contribute to the previously observed cardio-microvascular dysfunction in COVID-19 patients. The exact roles of these NOXes in pathogenesis of COVID-19 however remain to be elucidated.

Myocardial infarction coincides with increased NOX2 and Nε-(carboxymethyl) lysine expression in the cerebral microvasculature.

BACKGROUND: Myocardial infarction (MI) is associated with mental health disorders, in which neuroinflammation and cerebral microvascular dysfunction may play a role. Previously, we have shown that the proinflammatory factors Nε-(carboxymethyl)lysine (CML) and NADPH oxidase 2 (NOX2) are increased in the human infarcted heart microvasculature. The aim of this study was to analyse the presence of CML and NOX2 in the cerebral microvasculature of patients with MI. METHODS: Brain tissue was obtained at autopsy from 24 patients with MI and nine control patients. According to their infarct age, patients with MI were divided into three groups: 3-6 hours old (phase I), 6 hours-5 days old (phase II) and 5-14 days old (phase III). CML and NOX2 in the microvasculature were studied through immunohistochemical analysis. RESULTS: We observed a 2.5-fold increase in cerebral microvascular CML in patients with phase II and phase III MI (phase II: 21.39±7.91, p=0.004; phase III: 24.21±10.37, p=0.0007) compared with non-MI controls (8.55±2.98). NOX2 was increased in microvessels in patients with phase II MI (p=0.002) and phase III MI (p=0.04) compared with controls. No correlation was found between CML and NOX2 (r=0.58, p=0.13). CONCLUSIONS: MI coincides with an increased presence of CML and NOX2 in the brain microvasculature. These data point to proinflammatory alterations in the brain microvasculature that may underlie MI-associated mental health disorders.

NOX2 Expression Is Increased in Keratinocytes After Burn Injury.

Reepithelialization is crucial for effective wound repair in burn wounds. Reactive oxygen species (ROS) have shown to be important in this. Recent studies suggest that NOX proteins produce ROS in keratinocytes. In the present study, we have studied NOX proteins in burn wounds, including the effect of C1-esterase inhibitor (C1inh) hereon, which is the endogenous inhibitor of complement activity whereof we have shown previously that it also increased the rate of reepithelialization in burn wounds. Skin tissue derived from healthy control Wistar rats (n = 6) were compared with burn-injured rats, with (n = 7) or without C1inh treatment (n = 7). After 14 days, rats were terminated. From the burn-injured rats, the entire wound and nonburned skin from the hind leg, that is, internal control was excised. From the control rats, dorsal skin was excised. In these skin samples, NOX2 and NOX4 were analyzed immunohistochemically. In nonburned rats, NOX2 was found in keratinocytes in both the basal layer and suprabasal layer of the epidermis; and the number of NOX2-positive keratinocytes was 367/mm2 (254-378). In burned rats, the number of NOX2-positive keratinocytes was significantly increased in the newly forming epidermis in the burned area to 1019/mm2 (649-1172), especially in the suprabasal layer, but significantly decreased in remote nonburned skin to 22/mm2 (6-89). C1inh treatment counteracted these changes in epidermal NOX2 expression in burned rats, both in the burned area as in remote nonburned skin. No NOX4 expression was found in the epidermis in none of the groups. NOX2 expression was increased in keratinocytes in newly forming epidermis after burn injury. C1inh, a drug that increases the rate of reepithelialization, counteracted this effect. These results suggest a role for NOX2 in the reepithelialization of burn wounds.

Elevated monocyte-specific type I interferon signalling correlates positively with cardiac healing in myocardial infarct patients but interferon alpha application deteriorates myocardial healing in rats.

Monocytes are involved in adverse left ventricular (LV) remodelling following myocardial infarction (MI). To provide therapeutic opportunities we aimed to identify gene transcripts in monocytes that relate to post-MI healing and evaluated intervention with the observed gene activity in a rat MI model. In 51 MI patients treated by primary percutaneous coronary intervention (PCI), the change in LV end-diastolic volume index (EDVi) from baseline to 4-month follow-up was assessed using cardiovascular magnetic resonance imaging (CMR). Circulating monocytes were collected at day 5 (Arterioscler Thromb Vasc Biol 35:1066-1070, 2015; Cell Stem Cell 16:477-487, 2015; Curr Med Chem 13:1877-1893, 2006) after primary PCI for transcriptome analysis. Transcriptional profiling and pathway analysis revealed that patients with a decreased LV EDVi showed an induction of type I interferon (IFN) signalling (type I IFN pathway: P value < 0.001; false discovery rate < 0.001). We subsequently administered 15,000 Units of IFN-α subcutaneously in a rat MI model for three consecutive days following MI. Cardiac function was measured using echocardiography and infarct size/cardiac inflammation using (immuno)-histochemical analysis. We found that IFN-α application deteriorated ventricular dilatation and increased infarct size at day 28 post-MI. Moreover, IFN-α changed the peripheral monocyte subset distribution towards the pro-inflammatory monocyte subset whereas in the myocardium, the presence of the alternative macrophage subset was increased at day 3 post-MI. Our findings suggest that induction of type I IFN signalling in human monocytes coincides with adverse LV remodelling. In rats, however, IFN-α administration deteriorated post-MI healing. These findings underscore important but also contradictory roles for the type I IFN response during cardiac healing following MI.

StemBell therapy stabilizes atherosclerotic plaques after myocardial infarction.

BACKGROUND AIMS: After a myocardial infarction (MI) atherosclerosis is accelerated leading to destabilization of the atherosclerotic plaque. mesenchymal stromal cells are a promising therapeutic option for atherosclerosis. Previously, we demonstrated a novel stem cell delivery technique, with adipose stem cells coupled to microbubbles (i.e., StemBells) as therapy after MI. In this study, we aim to investigate the effect of StemBell therapy on atherosclerotic plaques in an atherosclerotic mouse model after MI. METHODS: MI was induced in atherosclerotic Apolipoprotein E-deficient mice that were fed a high-fat Western diet. Six days post-MI, the mice received either 5 × 105/100 µL StemBells or vehicle intravenously. The effects of StemBell treatment on the size and stability of aortic root atherosclerotic plaques and the infarcted heart were determined 28 days post-MI via (immuno)histological analyses. Moreover, monocyte subtypes and lipids in the blood were studied. RESULTS: StemBell treatment resulted in significantly increased cap thickness, decreased intra-plaque macrophage density and increased percentage of intra-plaque anti-inflammatory macrophages and chemokines, without affecting plaque size and serum cholesterol/triglycerides. Furthermore, StemBell treatment significantly increased the percentage of anti-inflammatory macrophages within the infarcted myocardium but did not affect cardiac function nor infarct size. Finally, also the average percentage of anti-inflammatory monocytes in the circulation was increased after StemBell therapy. DISCUSSION: StemBell therapy increased cap thickness and decreased intra-plaque inflammation after MI, indicative of stabilized atherosclerotic plaque. It also induced a shift of circulating monocytes and intra-plaque and intra-cardiac macrophages towards anti-inflammatory phenotypes. Hence, StemBell therapy may be a therapeutic option to prevent atherosclerosis acceleration after MI.

PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure.

BACKGROUND: Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A2 (sPLA2-IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA2-IIA herein. METHODS: The effects of PX-18, an inhibitor of both sPLA2-IIA and apoptosis, on residual endothelium and the presence of sPLA2-IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. RESULTS: The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA2-IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA2-IIA. CONCLUSIONS: In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA2-IIA inhibition.

Arterial Blood Pressure Induces Transient C4b-Binding Protein in Human Saphenous Vein Grafts.

BACKGROUND: Complement is an important mediator in arterial blood pressure-induced vein graft failure. Previously, we noted activation of cell protective mechanisms in human saphenous veins too. Here we have analyzed whether C4b-binding protein (C4bp), an endogenous complement inhibitor, is present in the vein wall. METHODS: Human saphenous vein segments obtained from patients undergoing coronary artery bypass grafting (n = 55) were perfused in vitro at arterial blood pressure with either autologous blood for 1, 2, 4, or 6 hr or with autologous blood supplemented with reactive oxygen species scavenger N-acetylcysteine. The segments were subsequently analyzed quantitatively for presence of C4bp and complement activation product C3d using immunohistochemistry. RESULTS: Perfusion induced deposition of C3d and C4bp within the media of the vessel wall, which increased reproducibly and significantly over a period of 4 hr up to 3.8% for C3d and 81% for C4bp of the total vessel area. Remarkably after 6 hr of perfusion, the C3d-positive area decreased significantly to 1.3% and the C4bp-positive area to 19% of the total area of the vein. The areas positive for both C4bp and C3d were increased in the presence of N-acetylcysteine. CONCLUSIONS: Exposure to arterial blood pressure leads to a transient presence of C4bp in the vein wall. This may be part of a cell-protective mechanism to counteract arterial blood pressure-induced cellular stress and inflammation in grafted veins.

CD45 is a more sensitive marker than CD3 to diagnose lymphocytic myocarditis in the endomyocardium.

To diagnose lymphocytic myocarditis (LM), immunohistopathological examination of endomyocardial biopsies (EMBs) is used with a cutoff value of at least 14 leukocytes per mm2, composed of CD3- and CD68-positive cells. We hypothesized that a more common leukocyte marker, CD45, instead of CD3 could increase the diagnostic sensitivity. Hearts of mice with acute viral myocarditis (n = 9) and of controls (n = 7) and the EMB sampling area of the left ventricular posterior wall (LVPW) obtained from autopsied hearts of patients diagnosed with LM (n = 18) and controls (n = 6) were stained with anti-CD68, anti-CD3, and anti-CD45. When applying the threshold of at least 14 leukocytes per mm2, 33% of the mice would be diagnosed with LM with the use of CD3+CD68 and 89% with the use of CD45+CD68. In the EMB sampling area of autopsied hearts, using the cutoff value of at least 14 leukocytes per mm2, CD3+CD68 could only confirm 17% of the diagnosis of LM, whereas CD45+CD68 could confirm 50% of the LM cases. Moreover, we compared inflammation in the EMB sampling area of the LVPW to the remaining myocardium of the LVPW and observed a significant increase of CD45+CD68 cells per mm2 in patients with LM. In conclusion, the use of the common leukocyte marker CD45 increases the sensitivity of the diagnosis of LM. Furthermore, the inflammatory infiltrate in the EMB sampling area is significantly increased compared with the remaining LVPW, indicating that the sampling area constitutes the highest chance for histological diagnosis of LM.

Orthopedic surgery increases atherosclerotic lesions and necrotic core area in ApoE-/- mice.

BACKGROUND AND AIMS: Observational studies show a peak incidence of cardiovascular events after major surgery. For example, the risk of myocardial infarction increases 25-fold early after hip replacement. The acuteness of this increased risk suggests abrupt enhancement in plaque vulnerability, which may be related to intra-plaque inflammation, thinner fibrous cap and/or necrotic core expansion. We hypothesized that acute systemic inflammation following major orthopedic surgery induces such changes. METHODS: ApoE-/- mice were fed a western diet for 10 weeks. Thereafter, half the mice underwent mid-shaft femur osteotomy followed by realignment with an intramedullary K-wire, to mimic major orthopedic surgery. Mice were sacrificed 5 or 15 days post-surgery (n = 22) or post-saline injection (n = 13). Serum amyloid A (SAA) was measured as a marker of systemic inflammation. Paraffin embedded slides of the aortic root were stained to measure total plaque area and to quantify fibrosis, calcification, necrotic core, and inflammatory cells. RESULTS: Surgery mice showed a pronounced elevation of serum amyloid A (SAA) and developed increased plaque and necrotic core area already at 5 days, which reached significance at 15 days (p = 0.019; p = 0.004 for plaque and necrotic core, respectively). Macrophage and lymphocyte density significantly decreased in the surgery group compared to the control group at 15 days (p = 0.037; p = 0.024, respectively). The density of neutrophils and mast cells remained unchanged. CONCLUSIONS: Major orthopedic surgery in ApoE-/- mice triggers a systemic inflammatory response. Atherosclerotic plaque area is enlarged after surgery mainly due to an increase of the necrotic core. The role of intra-plaque inflammation in this response to surgical injury remains to be fully elucidated.

Dopamine induces lipid accumulation, NADPH oxidase-related oxidative stress, and a proinflammatory status of the plasma membrane in H9c2 cells.

Excess catecholamine levels are suggested to be cardiotoxic and to underlie stress-induced heart failure. The cardiotoxic effects of norepinephrine and epinephrine are well recognized. However, although cardiac and circulating dopamine levels are also increased in stress cardiomyopathy patients, knowledge regarding putative toxic effects of excess dopamine levels on cardiomyocytes is scarce. We now studied the effects of elevated dopamine levels in H9c2 cardiomyoblasts. H9c2 cells were cultured and treated with dopamine (200 µM) for 6, 24, and 48 h. Subsequently, the effects on lipid accumulation, cell viability, flippase activity, reactive oxygen species (ROS) production, subcellular NADPH oxidase (NOX) protein expression, and ATP/ADP and GTP/GDP levels were analyzed. Dopamine did not result in cytotoxic effects after 6 h. However, after 24 and 48 h dopamine treatment induced a significant increase in lipid accumulation, nitrotyrosine levels, indicative of ROS production, and cell death. In addition, dopamine significantly reduced flippase activity and ATP/GTP levels, coinciding with phosphatidylserine exposure on the outer plasma membrane. Furthermore, dopamine induced a transient increase in cytoplasmic and (peri)nucleus NOX1 and NOX4 expression after 24 h that subsided after 48 h. Moreover, while dopamine induced a similar transient increase in cytoplasmic NOX2 and p47phox expression, in the (peri)nucleus this increased expression persisted for 48 h where it colocalized with ROS. Exposure of H9c2 cells to elevated dopamine levels induced lipid accumulation, oxidative stress, and a proinflammatory status of the plasma membrane. This can, in part, explain the inflammatory response in patients with stress-induced heart failure.

Modulators of Macrophage Polarization Influence Healing of the Infarcted Myocardium.

To diminish heart failure development after acute myocardial infarction (AMI), several preclinical studies have focused on influencing the inflammatory processes in the healing response post-AMI. The initial purpose of this healing response is to clear cell debris of the injured cardiac tissue and to eventually resolve inflammation and support scar tissue formation. This is a well-balanced reaction. However, excess inflammation can lead to infarct expansion, adverse ventricular remodeling and thereby propagate heart failure development. Different macrophage subtypes are centrally involved in both the promotion and resolution phase of inflammation. Modulation of macrophage subset polarization has been described to greatly affect the quality and outcome of healing after AMI. Therefore, it is of great interest to reveal the process of macrophage polarization to support the development of therapeutic targets. The current review summarizes (pre)clinical studies that demonstrate essential molecules involved in macrophage polarization that can be modulated and influence cardiac healing after AMI.

Atrial fibrillation coincides with the advanced glycation end product N(ε)-(carboxymethyl)lysine in the atrium.

Presence of advanced glycation end products (AGEs) in the heart induces a proinflammatory phenotype. However, the presence of AGEs within atrial tissue of atrial fibrillation (AF) patients is unknown and was analyzed here. Left atrial appendage tissue from 33 AF patients and 9 controls was analyzed for the presence of the major AGEs N(ε)-(carboxymethyl)lysine (CML), VCAM-1, neutrophilic granulocytes, lymphocytes, and macrophages in both the fat tissue and myocardium separately. The total amount of fibrosis was also analyzed. Presence of CML was significantly higher in blood vessels of the left atrial appendage in AF patients as compared to controls, independent of diabetes mellitus. In AF patients, VCAM-1 expression in blood vessels and the numbers of infiltrated neutrophilic granulocytes, lymphocytes, and macrophages significantly increased compared to controls, and were highest in the fat tissue; there was no significant difference in fibrosis compared to controls. Interestingly, total amount of CML and fibrosis in AF and control patients correlated positively. Finally, there was no difference between AF patients based on AF type or surgical indication in the presence of CML, VCAM-1 expression, inflammatory cells, and fibrosis. Our results indicate that in AF the intramyocardial blood vessels of the left atrial appendage have an increased CML presence and proinflammatory status coinciding with a local increase in the number of inflammatory cells.

Analysis of inflammatory cells and mediators in skin wound biopsies to determine wound age in living subjects in forensic medicine.

OBJECTIVE: In forensic medicine it is important to determine the age of skin wounds in living subjects. The aim of this study was to assess whether analysis of inflammatory cells and inflammatory mediators in skin biopsies of wounds from living subjects could improve wound age determination. METHODS: Biopsies (n=101), representing the superficial border area of a skin wound, were taken from skin injuries of known wound age (range: 4.5 hours to 25 days) of living subjects. All biopsies were analyzed for 3 inflammatory cell markers (MPO, CD45 and CD68) and 4 inflammatory mediators (MIP-1, IL-8, CML and vitronectin). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. RESULTS: MPO, CD45, MIP-1, IL-8 (inflammatory cell markers) and N(epsilon)-(carboxymethyl)lysine (CML) positivity were maximal in wounds of 0.2-2 days old and then decreased in time. Remarkably, CD45, CD68 and CML showed a minor but non-significant increase again in 10-25 days old wounds. MPO and CD68 positivity was significantly lower in 4-25 days old wounds compared to 0.2-4 days old wounds. MPO positivity was also significantly lower in 10-25 days old wounds compared to 0.2-10 days old wounds. For CD45, MIP-1, IL-8 and CML no significant differences between the age groups were found. In case of vitronectin positivity in the extravasate or when the number of MIP-1 or IL-8-positive cells was more than 10 cells/mm(2) the probability that a wound was more than 10 days old was 0%. A probability scoring system of all analyzed markers can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. CONCLUSIONS: We have developed a probability scoring system of inflammatory cells and mediators that can be used to determine wound age in skin biopsies of living subjects.

Analysis of morphological characteristics and expression levels of extracellular matrix proteins in skin wounds to determine wound age in living subjects in forensic medicine.

OBJECTIVE: Wound age determination in living subjects is important in routine diagnostics in forensic medicine. Macroscopical description of a wound to determine wound age however is inadequate. The aim of this study was to assess whether it would be feasible to determine wound age via analysis of morphological characteristics and extracellular matrix proteins in skin biopsies of living subjects referred to a forensic outpatient clinic. METHODS: Skin biopsies (n=101), representing the border area of the wound, were taken from skin injuries of known wound age (range: 4.5h-25 days) in living subjects. All biopsies were analyzed for 3 morphological features (ulceration, parakeratosis and hemorrhage) and 3 extracellular matrix markers (collagen III, collagen IV and α-SMA). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. RESULTS: For hemorrhage, collagen III, collagen IV and α-SMA expression no relation with wound age was found. Ulceration was only found in wounds of 0.2-2, 2-4 and 4-10 days old, implying that the probability that a wound was more than 10 days old in case of ulceration is equal to 0%. Also parakeratosis was almost exclusively found in wounds of 0.2-2, 2-4 and 4-10 days old, except for one case with a wound age of 15 days old. The probability scoring system of all analyzed markers, as depicted above, however can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. CONCLUSIONS: We have developed a probability scoring system, analysing morphological characteristics and extracellular matrix proteins in superficial skin biopsies of wounds in living subjects that can be applied in forensic medicine for wound age determination.

Lymphocytes infiltrate the quadriceps muscle in lymphocytic myocarditis patients: a potential new diagnostic tool.

BACKGROUND: Diagnosing lymphocytic myocarditis (LM) is challenging because of the large variation in clinical presentation and the limitations inherent in current diagnostic tools. The objective of this study was to analyze infiltration of inflammatory cells in quadriceps skeletal muscle of LM patients and investigate the potential diagnostic value of assaying infiltrating inflammatory cells. METHODS: Quadriceps muscle tissue, obtained at autopsy from control patients (n = 9) and LM patients (n = 21), was analyzed using immunohistochemistry for infiltration of lymphocytes (CD45), macrophages (CD68), neutrophilic granulocytes (myeloperoxidase), and several lymphocyte subtypes (CD3, CD4, CD8, CD20) and using polymerase chain reaction for a panel of myocarditis-associated viruses. Additionally, quadriceps muscle from mice with acute coxsackievirus B3-induced myocarditis and control mice was analyzed for presence of lymphocytes and virus. RESULTS: In quadriceps muscle of LM patients the number of infiltrating lymphocytes were significantly increased and LM was diagnosed with specificity of 100% and sensitivity of 71%. Parvovirus B19 was the primary virus found in our patient groups, found in quadriceps tissue of 3 LM patients (although it was also found in 1 control patient). In the mice, enteroviral RNA was present in the quadriceps muscle, although enteroviral capsid proteins and lymphocyte infiltration were found primarily in the adipose tissue within and directly adjacent to the myocyte tissue, rather than in the myocyte tissue itself. CONCLUSIONS: LM is associated with lymphocyte infiltration and viral presence in quadriceps muscle. This indicates that skeletal muscle biopsy/lymphocyte quantification might be a potential diagnostic tool for LM patients.

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.

Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir.

AIMS: Monocytes are critical mediators of healing following acute myocardial infarction (AMI), making them an interesting target to improve myocardial repair. The purpose of this study was a gain of insight into the source and recruitment of monocytes following AMI in humans. METHODS AND RESULTS: Post-mortem tissue specimens of myocardium, spleen and bone marrow were collected from 28 patients who died at different time points after AMI. Twelve patients who died from other causes served as controls. The presence and localization of monocytes (CD14(+) cells), and their CD14(+)CD16(-) and CD14(+)CD16(+) subsets, were evaluated by immunohistochemical and immunofluorescence analyses. CD14(+) cells localized at distinct regions of the infarcted myocardium in different phases of healing following AMI. In the inflammatory phase after AMI, CD14(+) cells were predominantly located in the infarct border zone, adjacent to cardiomyocytes, and consisted for 85% (78-92%) of CD14(+)CD16(-) cells. In contrast, in the subsequent post-AMI proliferative phase, massive accumulation of CD14(+) cells was observed in the infarct core, containing comparable proportions of both the CD14(+)CD16(-) [60% (31-67%)] and CD14(+)CD16(+) subsets [40% (33-69%)]. Importantly, in AMI patients, of the number of CD14(+) cells was decreased by 39% in the bone marrow and by 58% in the spleen, in comparison with control patients (P = 0.02 and <0.001, respectively). CONCLUSIONS: Overall, this study showed a unique spatiotemporal pattern of monocyte accumulation in the human myocardium following AMI that coincides with a marked depletion of monocytes from the spleen, suggesting that the human spleen contains an important reservoir function for monocytes.

Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.