Subendothelial matrix components influence endothelial cell apoptosis in vitro.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Specifically, endothelial apoptosis plays pivotal roles in atherosclerosis whereas prevention of endothelial apoptosis is a prerequisite for neovascularization in tumors and metastasis. Endothelial biology is intertwined with the composition of subendothelial basement membrane proteins. Apoptosis was induced by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells. Cells were either grown on polystyrene culture plates or on plates precoated with healthy basement membrane proteins (collagen IV, fibronectin, or laminin) or collagen I. Our results reveal that proteins of healthy basement membrane alleviate cytokine-induced apoptosis whereas precoating with collagen type I had no significant effect on apoptosis by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells compared with cells cultured on uncoated plates. Yet, treatment with transforming growth factor-ß1 significantly reduced the rate of apoptosis endothelial cells grown on collagen I. Detailed analysis reveals differences in intracellular signaling pathways for each of the basement membrane proteins studied. We provide additional insights into the importance of basement membrane proteins and the respective cytokine milieu on endothelial biology. Exploring outside-in signaling by basement membrane proteins may constitute an interesting target to restore vascular function and prevent complications in the atherosclerotic cascade.

CD34+ circulating cells display signs of immune activation in patients with acute coronary syndrome.

Bone marrow-derived endothelial progenitor cells (EPC) are released into the peripheral blood in situations of vascular repair/angiogenesis. Regulation of vascular repair and angiogenesis by EPC depends not only on the number of circulating EPC but also on their functionality. As endothelial cells can act as antigen-presenting cells in coronary artery disease (CAD), we postulated that EPC can be immune activated here as well. CD34+-EPC were isolated from peripheral blood of patients with ST-elevation myocardial infarction (STEMI, n = 12), non-STEMI/unstable angina (UA, n = 15), and stable CAD (SA, n = 18). Expression of HLA-DR, adhesion and costimulatory molecules by isolated CD34+-EPC were compared with levels in healthy controls (n = 18). There were no significant differences in VCAM-1 and CD80 expression by peripheral circulating CD34+-EPC between the four groups, yet expression of CD86 was highest in UA (p < 0.05). ICAM-1 expression was lowest in SA (p < 0.01). CD34+-EPC constitutively expressed HLA-DR across all groups. Of note, patients pretreated with HMG-CoA reductase inhibitors exhibited lower expression of VCAM-1 by CD34+-EPC throughout all patient groups; furthermore, statins significantly limited ex vivo-induced upregulation of ICAM-1 by TNF-alpha. To the best of our knowledge, this is the first study to examine the expression of immune markers in peripheral circulating CD34+-EPC ex vivo. We demonstrate that CD34+-EPC display different patterns of adhesion and costimulatory molecules in various states of CAD. Expression levels were affected by pretreatment with statins. Hence, immune activity of peripheral circulating CD34+ cells might play a pathophysiologic role in evolution of CAD.

Cell matrix contact modifies endothelial major histocompatibility complex class II expression in high-glucose environment.

Atherosclerosis is a chronic inflammatory disease. Cardiovascular risk factors such as hyperglycemia, hyperlipidemia, and arterial hypertension induce endothelial dysfunction with alterations in endothelial biosecretion and immune behavior. The aim of this study is to elucidate whether glucose-induced modifications of endothelial biosecretory and immune functions are regulated by interactions of endothelial cells (ECs) with their extracellular matrix [ECs plated on polystyrene-coated tissue culture plates (TC-EC) vs. ECs embedded within three-dimensional (3-D) collagen-based matrixes (3D-EC)]. In the absence of glucose, IFN-γ-induced phosphorylation of JAK and STAT proteins and human leukocyte antigen (HLA)-DR expression were lower in 3D-EC compared with TC-EC. Inversely, the expression of suppressor of cytokine signaling proteins (SOCS)-1 and -3 were significantly higher in naïve 3D-EC compared with naïve TC-EC. IFN-γ-induced upregulation of SOCS proteins was further amplified by the 3-D environment. Glucose significantly augmented IFN-γ-dependent signaling pathways in TC-EC. IFN-γ-induced phosphorylation of JAK and STAT proteins as well as HLA-DR expression by ECs in low- and high-glucose medium was significantly lower in 3-D than in two-dimensional environment. Glucose increased SOCS expression in TC-EC and 3D-EC to the same extent, such that expression levels in 3D-EC exceeded SOCS-1 and -3 expression in TC-EC by 1.6-2.5-fold. In conclusion, low- and high-glucose concentrations amplify IFN-γ-induced signaling pathways in TC-EC. Increased SOCS expression raises the threshold for IFN-γ to induce HLA-DR expression in a 3-D environment. This immunoprotective effect is maintained even in states of experimental hyperglycemia.

Association between admission ECG changes and long-term mortality in patients with an incidental myocardial infarction: Results from the KORA myocardial infarction registry.

BACKGROUND: The aim of this study was to examine the predictive value of specific changes in admission ECG on long-term outcome in acute myocardial infarction (AMI). METHODS: From 2000 until 2017 all AMI cases (n = 9,689) in the study area of Augsburg, Germany, were prospectively recorded. For this study, all patients with a first-time AMI, who survived the first 28 days, were considered. Median observational time was 6.7 years (IQR: 3.6-10.9). Each case was assigned to one of the following groups according to the admission ECG: 'ST-segment elevation', 'ST-segment depression', 'T-wave inversion', 'unspecific changes', 'normal ECG' and 'bundle branch block' (BBB). Multivariable adjusted COX regression models were calculated to compare long-term all-cause mortality. RESULTS: The final regression model revealed a significantly higher mortality among patients with BBB (HR: 1.52 [1.34-1.73], p-value: < 0.001) and 'ST-segment depression' (HR: 1.16 [1.03-1.29], p-value: 0.01252) compared to the STEMI group (reference group). The 'normal ECG' group (HR: 0.76 [0.66-0.87], p-value: < 0.001) on the other hand was associated with significantly lower long-term mortality. The 'T-wave inversion' group (HR: 1.08 [0.96-1.21]) and the 'unspecific changes' group (HR: 1.05 [0.94-1.17]) did not differ significantly from the STEMI group. CONCLUSION: ST-segment depressions and BBB admission ECGs go along with higher long-term mortality in AMI patients compared to STEMI cases. This should be taken into account by physicians when treating patients with NSTEMIs. Only the complete absence of AMI-related ECG changes predicts a more favorable outcome.

Function and mode of regulation of endothelial major histocompatibility complex class II.

Tissue engineering is a promising approach to implement endothelial cells as a cellular delivery therapy for vascular disease. We and others previously demonstrated that endothelial cells embedded in three-dimensional collagen-based matrices retain their full biosecretory spectrum, enabling them to serve as powerful regulators of vascular diseases. Fascinatingly, matrix embedding of endothelial cells not only allows for their implantation but also seems to provide protection from allo- and xenogeneic-triggered host immune responses. This is not an effect of simple physical shielding but a more fundamental influence of cell-matrix interconnectivity on the cellular immune phenotype. Reduced cytokine-induced levels of costimulatory and adhesion molecules associated with significantly lower expression levels of major histocompatibility class II expression on matrix-embedded human aortic endothelial cells when compared to the same cells cultured on two-dimensional polystyrene coated-tissue culture plates. Strikingly, the entire interferon-gamma-dependent signaling cascade resulting in MHC class II molecule expression is markedly suppressed in endothelial cells grown to confluence within three-dimensional scaffolds. These findings might be of pivotal importance for designing endothelial cell-based therapies in general and might enhance our understanding of the underlying pathophysiology in a broad range of cardiovascular diseases (e.g., atherosclerosis, vasculitis, chronic allograft vasculopathy).

NF-kappaB activity in endothelial cells is modulated by cell substratum interactions and influences chemokine-mediated adhesion of natural killer cells.

Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, we sought to understand if cytokine-induced NF-kappaB activity and downstream effects depend on substrate adherence of endothelial cells (EC). We compared the upstream phosphorylation cascade, activation of NF-kappaB, and expression/secretion of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC). Adhesion of natural killer (NK) cells was quantified in vitro and in vivo. NF-kappaB subunit p65 nuclear levels were significantly lower and p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS. Despite similar surface expression of TNF-alpha receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1. Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 +/- 420 vs. 1,735 +/- 135 cpm; p < 0.0002). Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix. Matrix embedding enables control of EC substratum interaction. This in turn regulates chemokine and surface molecule expression and secretion, in particular of those compounds within NF-kappaB pathways, chemoattraction of NK cells, local inflammation, and tissue repair.

Matrix adherence of endothelial cells attenuates immune reactivity: induction of hyporesponsiveness in allo- and xenogeneic models.

Endothelial integrity regulates vascular tone, luminal patency, and the immune reactivity to tissue grafts. Endothelial dysfunction is the first marker and site of disease initiation and severity. It has long been known that endothelial biochemical function is density dependent, and we have recently shown that endothelial immunobiology is anchorage dependent. Matrix-embedded endothelial cells (EC) establish a controlled anchorage state and are not only immune protected but also induce a system immune protective state. We now define this aspect of vascular and immune biology in detail. The in vitro immune response of allogeneic splenocytes (proliferation, lytic activity, and cytokine expression) on exposure to aortic EC was significantly reduced if EC were embedded within three-dimensional collagen matrices (3D-EC; P<0.005) to an even greater extent than EC that had reached confluence as monolayers on tissue culture plates (EC-TCPS). Splenocyte reactivity was enhanced with repeated exposure to EC-TCPS but minimally if preexposed to 3D-EC (P<0.002). 3D-EC induced significantly greater differentiation of splenocytes into CD4+ CD25+ Foxp3+ regulatory T cells than EC-TCPS (P<0.02). The reduced response to 3D-EC and potential protective effect to subsequent exposure were confirmed in vivo. Repeated exposure of immune-competent mice to injections of xenogeneic EC-TCPS induced vigorous host immunity. In contrast, prior implantation of 3D-EC induced hyporesponsiveness toward subsequent injection of EC-TCPS with reduced humoral response, decreased lytic activity, and lower frequency of effector splenocytes (P<0.001). EC interaction with its matrix determines phenotype, viability, and biosecretory potential. We now show that this microenvironmental interaction also influences endothelial-mediated activation of allo- and xenogeneic immune cells. 3D matrix-embedding limits the ability of EC to initiate adaptive immunity, and initial exposure to 3D-EC confers hyporesponsiveness to subsequent exposure to immunogeneic EC. These effects transcended the traditional control that confluence imposes on EC and reflects perhaps even higher order control. Our findings might offer novel insights to endothelial-mediated diseases and potential cell-based therapies.

Glucocorticoid-remediable aldosteronism in a young adult with a family history of Conn's syndrome.

Glucocorticoid-remediable aldosteronism is a hereditary form of primary hyperaldosteronism and the most common monogenic cause of hypertension. We present the case of a 24-year-old man with a family history of Conn's syndrome. Yet, in the index patient, classical characteristics of mineralocorticoid excess could be reversed by exogenous glucocorticoids.

3D matrix-embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three-dimensional growing environment significantly shields endothelial cells from cytokine-induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine-stimulated endothelial cells when cells are grown within a three-dimensional collagen-based matrix compared to cells grown on two-dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38-phosphorylation in three-dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three-dimensional matrix-embedded endothelial cells express significantly less tyrosine-phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin αv ß3 partially reversed the protective effect of three-dimensional matrix-embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue-engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.

Cell-matrix contact prevents recognition and damage of endothelial cells in states of heightened immunity.

BACKGROUND: Autoimmunity may exacerbate vascular disease, particularly in the form of anti-endothelial cell (EC) antibodies. The increased morbidity of cardiovascular diseases in concert with diabetes mellitus, hypertension, and other systemic illnesses may reflect the increase presence and potency of these antibodies. Matrix-embedded ECs act as powerful regulators of vascular repair accompanied by significant reduction in expected systemic and local inflammation. We compared the immune response against free and matrix-embedded ECs in naïve mice and mice with heightened EC immune reactivity. METHODS AND RESULTS: Mice were presensitized to EC with repeated (days 0, 21, 35) subcutaneous injections of saline-suspended porcine EC (PAE) (5 x 10(5) cells). Controls received saline injections. On day 42, mice received 5 x 10(5) matrix-embedded or free PAEs. Circulating PAE-specific antibodies and effector T-cells were analyzed via flow cytometry, and xenoreactive lymphocytes via ELISPOT, 90 days after implantation. PAE-specific antibody-titers, frequency of CD4+-effector cells, and xenoreactive splenocytes were 2- to 4-fold lower (P<0.0001) when naïve mice were injected with matrix-embedded instead of saline-suspended PAEs. Though basal levels of circulating antibodies were significantly elevated after serial PAE injections (2210+/-341 mean fluorescence intensity, day 42) and almost doubled again 90 days after injection of a fourth set of free PAEs, antibody levels declined by half in recipients of matrix-embedded PAEs at day 42 (P<0.0001). Levels of CD4+-effector cells and xenoreactive splenocytes showed similar results. CONCLUSIONS: Implantation of free PAE elicits a significant immune response in naïve mice and even more pronounced in mice with predeveloped anti-endothelial immunity. Matrix-embedding protects xenogeneic ECs against immune reaction in naïve mice and to a similar extent in mice with heightened immune reactivity. Matrix-embedded EC might offer a promising approach for treatment of advanced cardiovascular disease.

Endothelial immunogenicity--a matter of matrix microarchitecture.

The endothelium is a highly specialized active interface between blood and the underlying tissues, maintaining vascular tone, thrombo-resistance and selective permeability to cells and proteins. It is also an important regulator of inflammatory diseases, and endothelial-leukocyte interactions often herald complex diseases with an inflammatory component. Yet, the exact mechanisms promoting immune activation of endothelial cells (EC) are incompletely understood. Knowledge is accumulating that the immediate environment defines the cellular phenotype, whereby matrix composition and spatial formation (three- versus two-dimensional) seem to act as pivotal mediators. Here we summarize current findings denoting a key role of matrix environment in regulating endothelial immunogenicity. The immune response to three-dimensional matrix-embedded EC stands in stark contrast to the response engendered by injection of these same cells in their free state. Matrix-embedding confers a quiescent endothelial state with reduced expression levels of chemokines, adhesion, costimulatory, and major histocompatibility complex II molecules. Compared to EC grown on two-dimensional tissue culture plates, cytokine-stimulation of matrix-embedded EC results in significantly reduced adhesion of natural killer cells and proliferation of co-cultivated allogeneic T cells. On the contrary, matrix-embedded EC induce an immune-inhibitory phenotype of dendritic cells and T regulatory cells to a greater extent than non-embedded EC. As vascular diseases are associated with profound changes in basement membrane composition and overall tissue architecture, our results indicate that the immediate environment of EC might play a pivotal role in initiating and regulating of different vascular diseases. Cell-matrix interconnections appear to govern endothelial immunogenicity and interactions between EC and immune cells.

Torsades de Pointes: a rare complication of an extra-adrenal pheochromocytoma.

Pheochromocytoma is an infrequent secondary cause of arterial hypertension, often associated with paroxysmal headache, sweating, weight loss, and palpitations. Cardiovascular complications of pheochromocytoma include sudden death, heart failure due to toxic cardiomyopathy, and hypertensive encephalopathy. Here we report the case of a female with an acquired long-QT-syndrome as a rare complication of an extra-adrenal pheochromocytoma. Diagnosis was made after sotalol-induced Torsades de Pointes.

Influence of polyphenol-rich diet on exercise-induced immunomodulation in male endurance athletes.

Stress is associated with increased susceptibility to infection. We investigated if the mechanism involves immunomodulation of dendritic cells and whether this can be inhibited by a polyphenol-rich diet. Blood samples were taken from a total of 100 male endurance athletes at 5 time points around a marathon run: 4 weeks before; 1 week before; and immediately, 24 h, and 72 h after. Participants were randomized into 2 double-blinded groups. One group received a polyphenol-rich beverage during a 3-week training phase before marathon while the other group received a placebo beverage. Flow cytometric analysis of dendritic cell (DC) counts and subpopulation counts (myeloid, plasmocytoid DCs) was performed. Levels of viral antigen presenting toll-like receptor (TLR) 7 messenger RNA was measured by real-time polymerase chain reaction. Marathon running induced a significant increase of circulating myeloid DCs (0.2% vs. 0.33% of whole-blood leukocytes (wbl); p < 0.01) and a significant decrease of plasmozytoid DCs (0.12% vs. 0.03% of wbl; p < 0.01) and TLR7 expression (decline of 60%; p < 0.01). Polyphenol supplementation did not significantly affect mobilization of dendritic cells but showed beneficial effects on regeneration of TLR7 expression in wbl at 3 days postmarathon (decline of 40% vs. increase of 1000%; p < 0.05). In conclusion, physical stress affects circulating DCs, with an increase of myeloid and a decrease of plasmozytoid DCs. This may partially explain the susceptibility to viral infections after strenuous exercise. These detrimental effects are not attenuated by polyphenol supplementation. However, polyphenols support regeneration of viral antigen presenting TLR7 after strenuous exercise.

Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome.

BACKGROUND: Atherosclerosis is an inflammatory disease in which monocytes and macrophages have been suggested to play an essential role. The underlying signaling mechanisms are unknown thus far. We hypothesized that the human isoform of Toll-like receptor (hTLR)-4 is involved in monocyte activation of patients with accelerated forms of atherosclerosis. METHODS AND RESULTS: Expression of hTLR4 on circulating monocytes from 30 controls, 20 patients with stable angina (SA), 40 patients with unstable angina (UA), and 28 patients with acute myocardial infarction (AMI) was compared with the use of flow-cytometry and reverse transcription-polymerase chain reaction. Regulation of interleukin (IL)-12 and B7-1 as downstream events of TLR4 activation was analyzed after lipopolysaccharide stimulation of monocytes. TLR4-transfected Chinese hamster ovary (CHO) cells were used to identify potential hTLR4 ligands in the serum of patients with UA or AMI. Circulating hTLR4+/CD14+ monocytes were approximately 2.5-fold increased above controls and patients with SA in the UA and AMI groups (P<0.0001). This was paralleled by enhanced transcript levels of TLR4 and Myd88 in patients with UA and AMI (P<0.0001) and increased expression of IL-12 (UA 35.5+/-7.8, AMI 31.8+/-7.7 versus SA 2.2+/-0.5, controls 2.1+/-0.3 pg/mL; P<0.0002) and B7-1 (UA 27.3+/-14.4, AMI 22.6+/-11.1 versus SA 3.4+/-2.5, controls 2.4+/-2.3%; P<0.0001). Compared with serum from patients with UA and AMI, challenging TLR4-transfected CHO cells with serum from SA patients yielded only a weak response (P<0.0001). Coincubation with anti-heat shock protein 60 inhibited CHO cell activation. CONCLUSIONS: UA and AMI are associated with enhanced expression and signaling events downstream of hTLR4 in circulating monocytes. These observations suggest hTLR4 activation as a signaling mechanism in immune-mediated progression of atherosclerosis.

Matrix embedding alters the immune response against endothelial cells in vitro and in vivo.

BACKGROUND: Endothelial cell (EC) dysfunction represents the first manifestation of atherosclerotic disease. Restoration of endothelium via seeding or transfection is hampered by local alterations in flow, inflammation, and metabolic activation. Perivascular EC matrix implants are shielded from these forces and still control vascular repair. The host immune response to such implants, however, remains largely unknown. We investigated the effect of embedding of ECs within 3-dimensional matrices on host immune responses in vitro and in vivo. METHODS AND RESULTS: We compared expression of major histocompatibility complex (MHC), costimulatory, and adhesion molecules by free aortic ECs or ECs embedded in Gelfoam matrices by flow-cytometry. T-cell proliferation was assessed by [3H] thymidine incorporation. Humoral immune response (ELISA and FACS analysis) and cellular (histopathology) infiltration were investigated after subcutaneous injection of free porcine aortic ECs (PAEs) or of a Gelfoam/EC block, or after concomitant injection of PAEs adjacent to Gelfoam in rats. Aortic ECs embedded in Gelfoam expressed lower levels of MHC class II, costimulatory, and adhesion molecules compared with free ECs (P<0.001), and induced 3-fold less proliferation of human CD4+ T-cells (P<0.0005). Implantation of a Gelfoam/EC block in rats nearly abrogated the immune response with 1.75- to 9.0-fold downregulation in tumor necrosis factor-alpha, interleukin-6, monocyte chemotactic protein-1, and PAE-specific immunoglobulin G (P<0.005) and 3.3- to 4.5-fold reduction in leukocytic tissue infiltration. Injecting PAEs adjacent to Gelfoam induced a significant response comparable to that of free implanted PAEs. CONCLUSIONS: Embedding ECs within 3-dimensional matrices alters the host immune response by inhibiting expression of MHC class II, costimulatory, and adhesion molecules, offering the rationale to develop novel therapies for vascular diseases.

Enhanced T-helper-1 lymphocyte activation patterns in acute coronary syndromes.

OBJECTIVES: We sought to determine whether different stages of coronary artery disease (CAD) are associated with distinct differentiation patterns of activated T cells. BACKGROUND: Atherosclerosis is an inflammatory disease. However, little is known about specific inflammatory cell activation in atherosclerosis, for example, the T-helper (Th)1/Th2-balance. METHODS: We studied 18 patients with stable angina (SA), 28 patients with acute coronary syndrome (ACS) (16 with unstable angina pectoris and 12 with ST-segment elevation myocardial infarction), 19 patients with unheralded myocardial infarction (UH), and 16 control patients. Cytokine patterns and transcription factor signaling pathways of circulating T cells were characterized using reverse transcription polymerase chain reaction and flow cytometry. RESULTS: Although interferon (IFN)-gamma(+)/CD3(+) T cells were approximately 2-fold greater in patients with SA or UH than in control subjects, there was a massive expansion of Th1 cells in patients with ACS (p < 0.001). This increase was paralleled by significantly increased mRNA transcript levels for signal-transducer-and-activator-4 (ACS 1.17 +/- 0.14 relative units [RU]; control patients 0.44 +/- 0.09 RU; SA 0.67 +/- 0.12 RU; UH 0.61 +/- 0.17 RU), interleukin-2 (ACS 1.55 +/- 0.51 RU; control patients 0.21 +/- 0.09 RU; SA 0.54 +/- 0.18 RU; UH 0.45 +/- 0.16 RU), and IFN-gamma in ACS (1.27 +/- 0.39 RU; control patients 0.35 +/- 0.09 RU; SA 0.58 +/- 0.11 RU; UH 0.53 +/- 0.24 RU; p < 0.002). Th2 and Th0 cells were not different across patient subsets. The burden of CAD was identical between SA (1.4 +/- 0.2 diseased vessels, 68 +/- 13% diameter stenosis) and ACS (1.4 +/- 0.2 diseased vessels, 64 +/- 17% diameter stenosis) but significantly greater in patients with UH (2.5 +/- 0.5 diseased vessels, 95 +/- 7% diameter stenosis; p < 0.05). CONCLUSIONS: Patients with UH have a greater burden of obstructive CAD than SA but no greater T-cell activation. Patients with ACS have the same extent of CAD than SA but significantly greater activation of Th1 cells that may contribute to the increasing instability. Differences in circulating Th1 cells might indicate different pathogenic components, leading to ACS and UH.

Statins decrease Toll-like receptor 4 expression and downstream signaling in human CD14+ monocytes.

OBJECTIVE: Anti-inflammatory effects of statins contribute to their clinical benefit. Molecular mechanisms underlying these effects have not been well explored. Because statins attenuate lipopolysaccharide (LPS) responsiveness, we hypothesized that part of the pleiotropic effects are mediated through innate immunity. METHODS AND RESULTS: Toll-like receptor (TLR) 4 expression and downstream signaling in CD14+ monocytes after incubation with simvastatin and atorvastatin were quantified via flow-cytometry, quantitative RT-PCR, kinase assay, and enzyme-linked immunosorbent assay. Incubation with intermediates/ inhibitors of the mevalonate pathway was used to identify the mode of statin action. Statin incubation resulted in a dose-dependent reduction of TLR4 expression (53+/-7.6% reduction compared with untreated monocytes; P<0.005), transcript levels (68+/-6.3%; P<0.002), decreased IRAK phosphorylation (37+/-8.3%; P<0.05), and LPS-induced IL-6, IL-12, tumor necrosis factor (TNF)-alpha, and B7-1 expression (P<0.05). Four weeks of treatment with atorvastatin significantly reduced TLR4 expression on circulating CD14+ monocytes by 36.2+/-4.2% (P<0.05). Effects of statins were reversed by mevalonate (P=0.57). Incubation with specific inhibitors of geranylgeranyltransferase (54+/-4.3%), farnesyltransferase (57+/-5.1%), or with clostridium-difficile toxin B (58+/-6.1%, P<0.01) imitated the statin effects. Whereas wortmannin and LY294002 inhibited the statin effect (P=0.27), incubation with a specific RhoA kinase inhibitor had no effect (P=0.57). CONCLUSIONS: Statins influence TLR4 expression and signaling via inhibition of protein geranylgeranylation and farnesylation. These observations imply interactions with innate immunity as one pleiotropic mechanism.

Peripheral expansion of circulating T-helper 1 cells predicts coronary endothelial dysfunction after cardiac transplantation.

OBJECTIVE: We sought to assess the importance of Th1 cells for the development of cardiac allograft vasculopathy. BACKGROUND: Despite improvements in immunosuppressive regimens, chronic rejection still represents one of the leading causes of death beyond the first year after heart transplantation. Chronic rejection is characterized by the development of transplant vasculopathy. The exact mechanisms initiating and promoting this form of arteriosclerosis in the human setting remain unclear. METHODS: In order to assess the role of T lymphocytes we characterized differentiated T-cell subsets in 32 transplant recipients early after transplantation using RT-PCR, flow cytometry and immunhistochemistry and matched these findings with endothelial function testing as an early clinical indicator of transplant vasculopathy. RESULTS: Allograft endothelial dysfunction (ED) was defined as a compromised coronary flow reserve to acetylcholine (CFVR<2 in 8 of 32 transplant recipients). In these patients, mRNA transcript levels for the T-helper (Th)1 signature cytokines interferon (INF)-gamma (p<0.0001) and interleukin (IL)-2 (p<0.005) and STAT4 (Th1 transcription factor, p<0,05) were significantly higher than in the remaining 24 patients with normal endothelial function. This correlated with a significant increase in circulating CD3(+)/IFN-gamma(+)-T-cells (28.6 +/- 4.4% vs 8.7 +/- 5.6%; p<0.0001). In contrast, transcript levels for the Th2 signature cytokines (IL-4, IL-10) and STAT6 (Th2 transcription factor) did not differ significantly between the two groups. CONCLUSIONS: Peripheral expansion of circulating Th1 but not Th2 cells predicts coronary ED after cardiac transplantation. Therefore, quantification of circulating T cells might be a diagnostic tool to predict development of ED in patients after heart transplantation.

Evidence for a role of toll-like receptor 4 in development of chronic allograft rejection after cardiac transplantation.

BACKGROUND: Long-term success of cardiac transplantation is limited by various forms of graft rejection. The specific mechanisms initiating and controlling these highly specialized immune processes remain unclear so far. METHODS: To investigate the role of innate immunity in the development of allograft rejection, we assessed toll-like receptor (TLR)4 expression and typical downstream effects of TLR signaling (B7-1, interleukin [IL]-12, tumor necrosis factor [TNF]-alpha) in circulating CD14+ monocytes in 38 transplant recipients 1 to 3 years and in 10 transplant recipients 6 to 10 years after transplantation and compared them with 20 healthy controls using reverse transcription polymerase chain reaction, flow cytometry, and enzyme-linked immunoadsorbent assay. The results were matched with endothelial function testing as an early clinical indicator of transplant vasculopathy early after transplantation. RESULTS: Allograft endothelial dysfunction (ED) was defined as a compromised coronary flow reserve (CFVR) to acetylcholine (CFVR<2 in 13 of 38 transplant recipients). In these patients, mRNA transcript levels for TLR4 (P<0.05) and surface expression of TLR4 (P<0.005) and B7-1 (P<0.05) on circulating monocytes as well as secretion of IL-12 (P<0.02) and TNF-alpha (P<0.05) were significantly higher than in the remaining 25 patients without ED. Compared with the controls, recipients late after transplantation did not show significantly elevated levels of TLR4 or dependent mediators. These results were compared with mRNA levels in a mice model of acute and chronic rejection. Rejecting mice exhibited elevated mRNA levels for mTLR4 and mB7-1. CONCLUSIONS: Our results suggest activation of innate immunity in heart-transplant recipients through TLR4 contributes to the development of chronic rejection after cardiac transplantation.