IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.

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.

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.

Transient atrial inflammation in a murine model of Coxsackievirus B3-induced myocarditis.

Atrial dysfunction is a relatively common complication of acute myocarditis, although its pathophysiology is unclear. There is limited information on myocarditis-associated histological changes in the atria and how they develop in time. The aim of this study therefore was to investigate inflammation, fibrosis and viral genome in the atria in time after mild CVB3-induced viral myocarditis (VM) in mice. C3H mice (n = 68) were infected with 105 PFU of Coxsackievirus B3 (CVB3) and were compared with uninfected mice (n = 10). Atrial tissue was obtained at days 4, 7, 10, 21, 35 or 49 post-infection. Cellular infiltration of CD45+ lymphocytes, MAC3+ macrophages, Ly6G+ neutrophils and mast cells was quantified by (immuno)histochemical staining. The CVB3 RNA was determined by in situ hybridization, and fibrosis was evaluated by elastic van Gieson (EvG) staining. In the atria of VM mice, the numbers of lymphocytes on days 4 and 7 (p < .05) and days 10 (p < .01); macrophages on days 7 (p < .01) and 10 (p < .05); neutrophils on days 4 (p < .05); and mast cells on days 4 and 7 (p < .05) increased significantly compared with control mice and decreased thereafter to basal levels. No cardiomyocyte death was observed, and the CVB3 genome was detected in only one infected mouse on Day 4 post-infection. No significant changes in the amount of atrial fibrosis were found between VM and control mice. A temporary increase in inflammation is induced in the atria in the acute phase of CVB3-induced mild VM, which may facilitate the development of atrial arrhythmia and contractile dysfunction.

Liraglutide treatment attenuates inflammation markers in the cardiac, cerebral and renal microvasculature in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes mellitus (DM) induces cardiac and cerebral microvascular dysfunction via increased glycation, oxidative stress and endothelial activation. Liraglutide, a glucagon-like peptide-1 analogue, inhibited NOX2 and adhesion molecules in isolated endothelial cells. Here, we have studied how Liraglutide affects advanced glycation, NOX expression and inflammation of the cardiac, cerebral and renal microvasculature in diabetic rats. METHODS: DM was induced in Sprague-Dawley rats (n = 15) via intraperitoneal streptozotocin (STZ) injection (60 mg/kg bodyweight). Ten control rats remained nondiabetic. From day 9 post-STZ injection, Liraglutide (200 µg/kg bodyweight; n = 7) or vehicle (n = 8) was injected subcutaneously daily until termination on day 29. The advanced glycation endproduct N-ε-(carboxymethyl)lysine (CML), NOX2, NOX4, ICAM-1 and VCAM-1 were subsequently immunohistochemically analysed and quantified to compare Liraglutide treatment with placebo. RESULTS: In the heart, Liraglutide treatment significantly reduced the DM-increased scores/cm2 for CML in both ventricles (from 253 ± 53 to 72 ± 12; p = .003) and atria (343 ± 29 to 122 ± 8; p = .0001) and for NOX2, ICAM-1 and VCAM-1, but not for NOX4. Also in the cerebrum and cerebellum of the brain, Liraglutide significantly reduced the scores/cm2 for CML (to 60 ± 7 (p = .0005) and 47 ± 13 (p = .02), respectively), and for NOX2 and NOX4. In the kidney, the DM-induced expression of ICAM-1 and VCAM-1 was decreased in the blood vessels and glomeruli by Liraglutide treatment. Liraglutide did not affect blood glucose levels or bodyweight. CONCLUSIONS: Our study implies that Liraglutide protects the cardiac, cerebral and renal microvasculature against diabetes-induced dysfunction, independent of lowering blood glucose in a type 1 diabetes rat model.

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

Short-term LPS induces aortic valve thickening in ApoE*3Leiden mice.

BACKGROUND: Recently, it was shown that 12 weeks of lipopolysaccharide (LPS) administration to nonatherosclerotic mice induced thickening of the aortic heart valve (AV). Whether such effects may also occur even earlier is unknown. As most patients with AV stenosis also have atherosclerosis, we studied the short-term effect of LPS on the AVs in an atherosclerotic mouse model. METHODS: ApoE*3Leiden mice, on an atherogenic diet, were injected intraperitoneally with either LPS or phosphate buffered saline (PBS), and sacrificed 2 or 15 days later. AVs were assessed for size, fibrosis, glycosaminoglycans (GAGs), lipids, calcium deposits, iron deposits and inflammatory cells. RESULTS: LPS injection caused an increase in maximal leaflet thickness at 2 days (128.4 µm) compared to PBS-injected mice (67.8 µm; P = 0.007), whereas at 15 days this was not significantly different. LPS injection did not significantly affect average AV thickness on day 2 (37.8 µm), but did significantly increase average AV thickness at day 15 (41.6 µm; P = 0.038) compared to PBS-injected mice (31.7 and 32.3 µm respectively). LPS injection did not affect AV fibrosis, GAGs and lipid content. Furthermore, no calcium deposits were found. Iron deposits, indicative for valve haemorrhage, were observed in one AV of the PBS-injected group (a day 2 mouse; 9.1%) and in five AVs of the LPS-injected group (both day 2- and 15 mice; 29.4%). No significant differences in inflammatory cell infiltration were observed upon LPS injection. CONCLUSION: Short-term LPS apparently has the potential to increase AV thickening and haemorrhage. These results suggest that systemic inflammation can acutely compromise AV structure.

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.

Infectious myocarditis: the role of the cardiac vasculature.

Infectious myocarditis is the result of an immune response to a microbial infection of the heart. The blood vessels of the heart, both the intramyocardial microvasculature and the large epicardial coronary arteries, play an important role in the pathogenesis of infectious myocarditis. First of all, in addition to cardiomyocytes, endothelial cells of the cardiac (micro)vasculature are direct targets for infection. Moreover, through the expression of adhesion molecules and antigen presenting Major Histocompatibility Complex molecules, the blood vessels assist in shaping the cellular immune response in infectious myocarditis. In addition, damage and dysfunction of the cardiac (micro)vasculature are associated with thrombus formation as well as aberrant regulation of vascular tone including coronary vasospasm. These in turn can cause cardiac perfusion abnormalities and even myocardial infarction. In this review, we will discuss the role of the cardiac (micro)vasculature in the pathogenesis of infectious myocarditis.

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.

Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.

OBJECTIVE: Venous thromboembolism is a major contributor to global disease burden. Leukocytes and platelets initiate thrombogenesis on blood stasis and initiate the formation of a fibrin, VWF (von Willebrand factor), and neutrophil extracellular trap scaffold for erythrocytes. However, there is little knowledge on how erythrocytes become stably incorporated into this scaffold. Recently, we described the adhesion of calcium-loaded erythrocytes to endothelial-derived VWF strings. Because VWF is part of the scaffold of venous thrombi, we questioned whether reduced flow or stasis promotes the adhesion of normal erythrocytes to VWF and whether venous thrombi show evidence of erythrocyte-VWF interactions. APPROACH AND RESULTS: In the present work, we perfused, under controlled shear conditions, washed, normal erythrocytes over surface-immobilized plasma and extracellular matrix proteins and showed that normal erythrocytes specifically bind to VWF. The interaction between erythrocytes and VWF significantly increased when the wall shear stress was reduced. Next, we investigated whether erythrocyte-VWF interactions support the structure of venous thrombi. High-resolution immunofluorescence imaging of human venous thrombi showed a striking pattern between erythrocytes, VWF, and fibrin, which suggests that VWF plays a supporting role, linking erythrocytes to fibrin in the thrombus. CONCLUSIONS: Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

Myocardial infarction accelerates atherosclerosis.

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischaemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, Apoe-/- mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. The progenitors then seeded the spleen, yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.

A comparison in therapeutic efficacy of several time points of intravenous StemBell administration in a rat model of acute myocardial infarction.

BACKGROUND: Adipose-derived stromal cells (ASCs) are a promising new therapeutic option for patients with acute myocardial infarction (AMI). Previously, we found that ASCs coupled to antibody-targeted microbubbles (StemBells [StBs]) improved cardiac function when administered intravenously 7 days post-AMI in rats. In this study, we compared the efficacy of intravenous StB administration at different administration time points following AMI in rats. METHODS: AMI, followed by reperfusion, was induced in four groups of male Wistar rats, which subsequently received an intravenous 1 × 106 StB bolus 1 day post-AMI (StB1; n = 8), 7 days post-AMI (StB7; n = 9), at both time points (StB1+7; n = 7) or neither (Control; n = 7). The effect onrdiac function was determined using echocardiography prior to AMI, 7 days post-AMI and 42 days post-AMI. The effect on infarct size and macrophages in the infarct core were determined (immuno)histochemically 42 days post-AMI. RESULTS: At 42 days post-AMI, all three StB groups had a significantly improved fractional shortening compared with the control group. Between the StB-treated groups, the effects did not differ significantly at 42 days post-AMI. At 7 days post-AMI, the StB1 group had a significantly improved fractional shortening compared with the control and StB7 groups. No significant changes in infarct size or macrophage numbers were found compared with the control group for any StB group. CONCLUSIONS: StB administration resulted in long-term improvement of cardiac function, independent of the time point of administration. When administered at 1 day post-AMI, this improvement was already evident at 7 days post-AMI.

Neutrophil extracellular traps coincide with a pro-coagulant status of microcirculatory endothelium in burn wounds.

Burn-induced tissue loss is partly related to secondary expansion of necrosis into vital dermis neighboring the initial burn injury. An important factor herein is the severe loss of perfusion of the burn wound, probably caused by microvascular damage induced by the intense local inflammatory responses as well as burn-induced hypercoagulation. We hypothesize that the formation of neutrophilic extracellular traps (NETs) play an important role in this. The purpose of this study was to investigate postburn intravascular thrombosis, NETs formation and the coagulant state in the microvasculature of burns in both animal models and patients. We used two in vivo burn wound models: rats and pigs. In rats, the entire wound was excised at day 14 postburn and in pigs burn wound biopsies were collected at different time points up to 60 days postburn. To confirm the data in patients, eschar from the burn wound was obtained from burn wound patients at different time points after wounding. The number of intravascular thrombi, the presence of intravascular NETs and the number of tissue factor (TF) positive blood vessels in the burn wound was determined. In rats, a significant increase in intravascular thrombi and TF expression was observed 14 days postburn, that in majority coincided with NETs. In pigs, a significant increase in intravascular thrombi and TF expression was found over time up to 60 days postburn, that in majority coincided with NETs too. Also in eschar of burn wound patients, a significant increase in intravascular thrombi was noted, that in majority coincided with NETs, already 0.5 days postburn and remained elevated up to 46 days postburn. This study shows the presence of NETosis in microcirculatory thrombosis of burn wounds and a switch in the microcirculatory endothelium toward a procoagulant phenotype.

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.