Right Atrial Adaptation to Precapillary Pulmonary Hypertension: Pressure-Volume, Cardiomyocyte, and Histological Analysis.

BACKGROUND: Precapillary pulmonary hypertension (precPH) patients have altered right atrial (RA) function and right ventricular (RV) diastolic stiffness. OBJECTIVES: This study aimed to investigate RA function using pressure-volume (PV) loops, isolated cardiomyocyte, and histological analyses. METHODS: RA PV loops were constructed in control subjects (n = 9) and precPH patients (n = 27) using magnetic resonance and catheterization data. RA stiffness (pressure rise during atrial filling) and right atrioventricular coupling index (RA minimal volume / RV end-diastolic volume) were compared in a larger cohort of patients with moderate (n = 39) or severe (n = 41) RV diastolic stiffness. Cardiomyocytes were isolated from RA tissue collected from control subjects (n = 6) and precPH patients (n = 9) undergoing surgery. Autopsy material was collected from control subjects (n = 6) and precPH patients (n = 4) to study RA hypertrophy, capillarization, and fibrosis. RESULTS: RA PV loops showed 3 RA cardiac phases (reservoir, passive emptying, and contraction) with dilatation and elevated pressure in precPH. PrecPH patients with severe RV diastolic stiffness had increased RA stiffness and worse right atrioventricular coupling index. Cardiomyocyte cross-sectional area was increased 2- to 3-fold in precPH, but active tension generated by the sarcomeres was unaltered. There was no increase in passive tension of the cardiomyocytes, but end-stage precPH showed reduced number of capillaries per mm2 accompanied by interstitial and perivascular fibrosis. CONCLUSIONS: RA PV loops show increased RA stiffness and suggest atrioventricular uncoupling in patients with severe RV diastolic stiffness. Isolated RA cardiomyocytes of precPH patients are hypertrophied, without intrinsic sarcomeric changes. In end-stage precPH, reduced capillary density is accompanied by interstitial and perivascular fibrosis.

Atrial epicardial adipose tissue abundantly secretes myeloperoxidase and activates atrial fibroblasts in patients with atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) secretome induces fibrosis. Fibrosis, primarily extracellular matrix (ECM) produced by fibroblasts, creates a substrate for atrial fibrillation (AF). Whether the EAT secretome from patients with AF activates human atrial fibroblasts and through which components, remains unexplored. RESEARCH AIMS: (a) To investigate if the EAT secretome from patients with versus without AF increases ECM production in atrial fibroblasts. (b) To identify profibrotic proteins and processes in the EAT secretome and EAT from patients with, who will develop (future onset), and without AF. METHODS: Atrial EAT was obtainded during thoracoscopic ablation (AF, n = 20), or open-heart surgery (future onset and non-AF, n = 35). ECM gene expression of human atrial fibroblasts exposed to the EAT secretome and the proteomes of EAT secretome and EAT were assessed in patients with and without AF. Myeloperoxidase and neutrophil extracellular traps (NETs) were assessed immunohistochemically in patients with paroxysmal, persistent, future onset, and those who remain free of AF (non-AF). RESULTS: The expression of COL1A1 and FN1 in fibroblasts exposed to secretome from patients with AF was 3.7 and 4.7 times higher than in patients without AF (p < 0.05). Myeloperoxidase was the most increased protein in the EAT secretome and EAT from patients with versus without AF (FC 18.07 and 21.57, p < 0.005), as was the gene-set neutrophil degranulation. Immunohistochemically, myeloperoxidase was highest in persistent (FC 13.3, p < 0.0001) and increased in future onset AF (FC 2.4, p = 0.02) versus non-AF. Myeloperoxidase aggregated subepicardially and around fibrofatty infiltrates. NETs were increased in patients with persistent versus non-AF (p = 0.03). CONCLUSION: In AF, the EAT secretome induces ECM gene expression in atrial fibroblasts and contains abundant myeloperoxidase. EAT myeloperoxidase was increased prior to AF onset, and both myeloperoxidase and NETs were highest in persistent AF, highlighting the role of EAT neutrophils in the pathophysiology of AF.

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.

Systematic study of tissue factor expression in solid tumors.

BACKGROUND: Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. AIMS: To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. METHODS AND RESULTS: TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. CONCLUSION: This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.

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.

Bone marrow adipocytes fuel emergency hematopoiesis after myocardial infarction.

After myocardial infarction (MI), emergency hematopoiesis produces inflammatory myeloid cells that accelerate atherosclerosis and promote heart failure. Since the balance between glycolysis and mitochondrial metabolism regulates hematopoietic stem cell homeostasis, metabolic cues may influence emergency myelopoiesis. Here, we show in humans and female mice that hematopoietic progenitor cells increase fatty acid metabolism after MI. Blockade of fatty acid oxidation by deleting carnitine palmitoyltransferase (Cpt1A) in hematopoietic cells of Vav1Cre/+Cpt1Afl/fl mice limited hematopoietic progenitor proliferation and myeloid cell expansion after MI. We also observed reduced bone marrow adiposity in humans, pigs and mice following MI. Inhibiting lipolysis in adipocytes using AdipoqCreERT2Atglfl/fl mice or local depletion of bone marrow adipocytes in AdipoqCreERT2iDTR mice also curbed emergency hematopoiesis. Furthermore, systemic and regional sympathectomy prevented bone marrow adipocyte shrinkage after MI. These data establish a critical role for fatty acid metabolism in post-MI emergency hematopoiesis.

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.

Hypoxia-inducible factors individually facilitate inflammatory myeloid metabolism and inefficient cardiac repair.

Hypoxia-inducible factors (HIFs) are activated in parenchymal cells in response to low oxygen and as such have been proposed as therapeutic targets during hypoxic insult, including myocardial infarction (MI). HIFs are also activated within macrophages, which orchestrate the tissue repair response. Although isoform-specific therapeutics are in development for cardiac ischemic injury, surprisingly, the unique role of myeloid HIFs, and particularly HIF-2α, is unknown. Using a murine model of myocardial infarction and mice with conditional genetic loss and gain of function, we uncovered unique proinflammatory roles for myeloid cell expression of HIF-1α and HIF-2α during MI. We found that HIF-2α suppressed anti-inflammatory macrophage mitochondrial metabolism, while HIF-1α promoted cleavage of cardioprotective MerTK through glycolytic reprogramming of macrophages. Unexpectedly, combinatorial loss of both myeloid HIF-1α and HIF-2α was catastrophic and led to macrophage necroptosis, impaired fibrogenesis, and cardiac rupture. These findings support a strategy for selective inhibition of macrophage HIF isoforms and promotion of anti-inflammatory mitochondrial metabolism during ischemic tissue repair.

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.

Depletion of activated macrophages with a folate receptor-beta-specific antibody improves symptoms in mouse models of rheumatoid arthritis.

OBJECTIVES: Most therapies for autoimmune and inflammatory diseases either neutralize or suppress production of inflammatory cytokines produced by activated macrophages (e.g., TNFα, IL-1, IL-6, IL-17, GM-CSF). However, no approved therapies directly target this activated subset of macrophages. METHODS: First, we undertook to examine whether the folate receptor beta (FR-ß) positive subpopulation of macrophages, which marks the inflammatory subset in animal models of rheumatoid arthritis, might constitute the prominent population of macrophages in inflamed lesions in humans. Next, we utilized anti-FR-ß monoclonal antibodies capable of mediating antibody-dependent cell cytotoxicity (ADCC) to treat animal models of rheumatoid arthritis and peritonitis. RESULTS: Human tissue samples of rheumatoid arthritis, Crohn's disease, ulcerative colitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, chronic obstructive pulmonary disease, systemic lupus erythematosus, psoriasis, and scleroderma are all characterized by dramatic accumulation of macrophages that express FR-ß, a protein not expressed on resting macrophages or any other healthy tissues. A monoclonal antibody to FR-ß accumulates specifically in inflamed lesions of murine inflammatory disease models and successfully treats such models of rheumatoid arthritis and peritonitis. More importantly, elimination of FR-ß-positive macrophages upon treatment with an anti-FR-ß monoclonal antibody promotes the departure of other immune cells, including T cells, B cells, neutrophils, and dendritic cells from the inflamed lesions. CONCLUSIONS: These data suggest that specific elimination of FR-ß-expressing macrophages may constitute a highly specific therapy for multiple autoimmune and inflammatory diseases and that a recently developed human anti-human FR-ß monoclonal antibody (m909) might contribute to suppression of this subpopulation of macrophages.

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.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction.

BACKGROUND AND AIMS: Neovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. METHODS: Using cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. RESULTS: NIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). CONCLUSIONS: NIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

Cancer drug addiction is relayed by an ERK2-dependent phenotype switch.

Observations from cultured cells, animal models and patients raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR-Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for 'addiction genes'. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial-mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

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.