Chronic kidney disease mediates cardiac dysfunction associated with increased resident cardiac macrophages.

BACKGROUND: The leading cause of death in end-stage kidney disease is related to cardiovascular disease. Macrophages are known to be involved in both chronic kidney disease (CKD) and heart failure, however their role in the development of cardiorenal syndrome is less clear. We thus sought to investigate the role of macrophages in uremic cardiac disease. METHODS: We assessed cardiac response in two experimental models of CKD and tested macrophage and chemokine implication in monocytopenic CCR2-/- and anti-CXCL10 treated mice. We quantified CXCL10 in human CKD plasma and tested the response of human iPSC-derived cardiomyocytes and primary cardiac fibroblasts to serum from CKD donors. RESULTS: We found that reduced kidney function resulted in the expansion of cardiac macrophages, in particular through local proliferation of resident populations. Influx of circulating monocytes contributed to this increase. We identified CXCL10 as a crucial factor for cardiac macrophage expansion in uremic disease. In humans, we found increased plasma CXCL10 concentrations in advanced CKD, and identified the production of CXCL10 in cardiomyocytes and cardiac fibroblasts. CONCLUSIONS: This study provides new insight into the role of the innate immune system in uremic cardiomyopathy.

Glomerular endothelial derived vesicles mediate podocyte dysfunction: A potential role for miRNA.

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

A triglyceride-rich lipoprotein environment exacerbates renal injury in the accelerated nephrotoxic nephritis model.

Hyperlipidaemia accompanies chronic renal disease either as a consequence of the renal dysfunction or as part of generalized metabolic derangements. Under both situations, the lipid profile is characterized by accumulation of triglyceride-rich lipoproteins (TGRLs). This lipid profile is recognized as a risk factor for cardiovascular complications. Whether it may pose a risk for renal injury as well remains unclear. A hyper-TGRL state was generated in C57BL/6 mice using poloxamer-407 (P-407) and immune complex-mediated renal injury was triggered using the accelerated nephrotoxic nephritis (ANTN) model. The hyper-TGRL animals were hypersensitive to ANTN demonstrated by greater haematuria and glomerular cellularity. These changes were accompanied by increased glomerular accumulation of CD68+ macrophages. The hypersensitive response to ANTN was not seen in low-density lipoprotein receptor knock-out mice fed with a high fat diet, where triglyceride levels were lower but cholesterol levels comparable to those obtained using P-407. These data indicate that a hyper-TGRL state might be more detrimental to the kidneys than low-density lipoprotein-driven hypercholesterolaemia during immune complex-mediated nephritis. We speculate that the hyper-TGRL environment primes the kidney to exacerbated renal damage following an inflammatory insult with increased accumulation of macrophages that may play a key role in mediating the injurious effects.

CD14 expression is increased on monocytes in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis and correlates with the expression of ANCA autoantigens.

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14(high) CD16(neg/low)), intermediate (CD14(high) CD16(high)) and non-classical (CD14(low) CD16(high)) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0.01). Patients with PR3-ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0.01); however, levels in patients with MPO-ANCA disease in remission were lower than active MPO-ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0.79, P < 0.0001 and r = 0.42, P < 0.005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3-ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.

Angiotensin II subtype 2 receptor blockade and deficiency attenuate the development of atherosclerosis in an apolipoprotein E-deficient mouse model of diabetes.

AIMS/HYPOTHESIS: Most of the known actions of angiotensin II have been considered primarily to be the result of angiotensin II subtype 1 receptor activation. However, recent data suggest that the angiotensin II subtype 2 receptor (AT(2)R) may modulate key processes linked to atherosclerosis. The aim of this study was to investigate the role of AT(2)R in diabetes-associated atherosclerosis using pharmacological blockade and genetic deficiency. METHODS: Aortic plaque deposition was assessed in streptozotocin-induced diabetic apolipoprotein E (Apoe) knockout (KO) and At ( 2 ) r (also known as Agtr2)/Apoe double-KO (DKO) mice. Control and diabetic Apoe-KO mice received an AT(2)R antagonist PD123319 (5 mg kg(-1) day(-1)) via osmotic minipump for 20 weeks (n = 7-8 per group). RESULTS: Diabetes was associated with a sixfold increase in plaque area (diabetic Apoe-KO: 12.7 +/- 1.4% vs control Apoe-KO: 2.3 +/- 0.4%, p < 0.001) as well as a significant increase in aortic expression of the gene At ( 2 ) r (also known as Agtr2). The increase in plaque area with diabetes was attenuated in AT(2)R antagonist-treated diabetic Apoe-KO mice (7.1 +/- 0.5%, p < 0.05) and in diabetic At ( 2 ) r/Apoe DKO mice (9.2 +/- 1.3%, p < 0.05). These benefits occurred independently of glycaemic control or BP, and were associated with downregulation of a range of pro-inflammatory cytokines, adhesion molecules, chemokines and various extracellular matrix proteins. CONCLUSIONS/INTERPRETATION: This study provides evidence for AT(2)R playing a role in the development of diabetes-associated atherosclerosis. These findings suggest a potential utility of AT(2)R blockers in the prevention and treatment of diabetic macrovascular complications.

The anti inflammatory effects of high density lipoproteins.

It is well recognised that increased levels of high density lipoprotein (HDL) protect against atherosclerosis and correlate with improved prognosis for vascular disease associated events. While many of the atheroprotective effects of HDL are ascribed to the ability to remove cholesterol from the vasculature through the reverse cholesterol transport system, recent work has shown that HDL may be atheroprotective through its other functions, such as regulation of endothelial adhesion molecule expression, stimulation of endothelial nitric oxide synthase and inhibition of the damaging effects of oxidised low density lipoproteins. Recently, HDL has also been described to interact with circulating cells inhibiting both leukocyte and platelet activation, therefore having further systemic anti-inflammatory functions. This review summarises the studies and models used to examine the anti-inflammatory effects of HDL and details data describing the ability to inhibit leukocyte activation, contributing to the hypothesis that raised HDL is beneficial in the context of inflammation in atherosclerosis. Further, HDL modification in disease and current therapeutic strategies such as reconstituted HDL particles and apoA I mimetic peptides is discussed to provide insights to the potential applicability of raising HDL to regress cardiovascular disease.

Reduced plaque formation induced by rosiglitazone in an STZ-diabetes mouse model of atherosclerosis is associated with downregulation of adhesion molecules.

Adhesion molecules have been implicated in the development and progression of cardiovascular disease, which is highly prevalent in people with diabetes. Adhesion molecules can mediate adhesion of leukocytes to the endothelium. Furthermore, P-selectin expressed on platelets is able to mediate the adhesion of leukocytes to platelets. In this study, we examine the in-vivo and in-vitro effects of rosiglitazone with particular emphasis on three important adhesion molecules (VCAM-1, ICAM-1 and P-selectin). In the aorta of STZ-diabetic apolipoprotein E-deficient (apoE KO) mice, rosiglitazone significantly reduced both total and arch plaque area. The mechanism for this appeared to be reduced macrophage infiltration into the atherosclerotic plaque which was also associated with reduced mRNA levels for VCAM-1, ICAM-1, MCP-1 and P-selectin in the aorta. In-vitro studies revealed reduced cell adhesion of monocytic cells (THP-1) to fibrinogen and endothelial cells (HUVEC) after incubation with rosiglitazone. Furthermore, the reduction in leukocyte adhesion also correlated with significant reductions in mRNA levels for VCAM-1, ICAM-1 and P-selectin indicating that reduced macrophage infiltration in atherosclerotic plaques may occur as a result of a direct effect of rosiglitazone on adhesion molecules in both monocytes and endothelial cells. Thus, we have shown that rosiglitazone appears to have direct anti-atherosclerotic effects in an animal model of diabetes-associated atherosclerosis which are at least partly due to effects on VCAM-1, ICAM-1, MCP-1 and P-selectin expression which leads to decreased leukocyte adhesion and macrophage infiltration.

Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion.

Raised levels of soluble P-selectin (sP-selectin) have been reported in the plasma of patients with vascular diseases; however, the functional importance of this ligand remains unclear. In this study we have examined a potential role for plasma sP-selectin in regulating neutrophil adhesion in patients with peripheral arterial occlusive disease (PAOD). Patients with PAOD had significantly higher levels of sP-selectin (mean+/-SD: 73.3+/-13.0 versus 16.7+/-6.4 ng/mL) and enhanced whole blood leukocyte adhesion to platelets under shear. To examine whether the raised sP-selectin levels can directly influence leukocyte adhesion, isolated neutrophils were incubated with plasma from PAOD patients before and after immunodepletion of sP-selectin. Neutrophil adhesion to fibrinogen increased 2-fold following incubation with PAOD plasma, which was abrogated on sP-selectin immunodepletion. We subsequently demonstrated that recombinant sP-selectin dose-dependently (75 to 250 ng/mL) increased leukocyte adhesion to fibrinogen and platelet monolayers. This increase was PSGL-1 and Src kinase-dependent and correlated with an increase in sP-selectin-mediated Mac-1 activation. sP-selectin-stimulated neutrophil adhesion to platelet monolayers was inversely correlated with shear, such that at low shear (50 s(-1)) a 92.7%+/-15.7 increase in adhesion was observed decreasing to 38.5%+/-11.9 at 150 s(-1) and 10.1%+/-7.4 at 300 s(-1). These studies suggest a potentially important role for sP-selectin in modulating neutrophil adhesion in patients with PAOD, particularly at sites of low shear, where it raises the possibility that raised plasma sP-selectin levels may enhance leukocyte recruitment to vascular injury and promote disease progression.

C-reactive protein mediates CD11b expression in monocytes through the non-receptor tyrosine kinase, Syk, and calcium mobilization but not through cytosolic peroxides.

OBJECTIVE: C-Reactive protein (CRP) can modulate integrin surface expression on monocytes following Fcgamma receptor engagement. We have investigated the signal transduction events causing this phenotypic alteration. METHODS: CRP-induced signalling events were examined in THP-1 and primary monocytes, measuring Syk phosphorylation by Western blotting, intracellular Ca(2+) ([Ca(2+)](i)) by Indo-1 fluorescence and surface expression of CD11b by flow cytometry. Cytosolic peroxides were determined by DCF fluorescence. RESULTS: CRP induced phosphorylation of Syk and an increase in [Ca(2+)](i) both of which were inhibitable by the Syk specific antagonist, piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pre-treatment of primary monoytes with the Ca(2+) mobiliser, thapsigargin, increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca(2+)](i) chelator, BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, alpha-tocopherol), expression of surface CD11b not being inhibited by antioxidants alone. CONCLUSION: CRP induces CD11b expression in monocytes through a peroxide independent pathway involving both Syk phosphorylation and [Ca(2+)](i) release.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells.

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 micro g/ml for 0-60 min at 37 degrees C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 micro g/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.