CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of an osteoblast-like, procalcifying cell phenotype. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Endothelial Overexpression of TGF-ß-Induced Protein Impairs Venous Thrombus Resolution: Possible Role in CTEPH.

Endothelial cells play a critical role during venous thrombus remodeling, and unresolved, fibrotic thrombi with irregular vessels obstruct the pulmonary artery in patients with chronic thromboembolic pulmonary hypertension (CTEPH). This study sought to identify endothelial mediators of impaired venous thrombus resolution and to determine their role in the pathogenesis of the vascular obstructions in patients with CTEPH. Endothelial cells outgrown from pulmonary endarterectomy specimens (PEA) were processed for mRNA profiling, and nCounter gene expression and immunohistochemistry analysis of PEA tissue microarrays and immunoassays of plasma were used to validate the expression in CTEPH. Lentiviral overexpression in human pulmonary artery endothelial cells (HPAECs) and exogenous administration of the recombinant protein into C57BL/6J mice after inferior Vena cava ligation were employed to assess their role for venous thrombus resolution. RT2 PCR profiler analysis demonstrated the significant overexpression of factors downstream of transforming growth factor beta (TGFß), that is TGFß-Induced Protein (TGFBI or BIGH3) and transgelin (TAGLN), or involved in TGFß signaling, that is follistatin-like 3 (FSTL3) and stanniocalcin-2 (STC2). Gene expression and immunohistochemistry analysis of tissue microarrays localized potential disease candidates to vessel-rich regions. Lentiviral overexpression of TGFBI in HPAECs increased fibrotic remodeling of human blood clots in vitro, and exogenous administration of recombinant TGFBI in mice delayed venous thrombus resolution. Significantly elevated plasma TGFBI levels were observed in patients with CTEPH and decreased after PEA. Our findings suggest that overexpression of TGFBI in endothelial promotes venous thrombus non-resolution and fibrosis and is causally involved in the pathophysiology of CTEPH.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFß1 and IL-27.

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Circulating Soluble EPCR Levels Are Reduced in Patients with Ischemic Peripheral Artery Disease and Associated with Markers of Endothelial and Vascular Function.

(1) Background: Endothelial dysfunction initiates cardiovascular pathologies, including peripheral artery disease (PAD). The pathophysiology of impaired new vessel formation in the presence of angiogenic stimuli, such as ischemia and inflammation, is unknown. We have recently shown in mice that reduced endothelial protein C receptor (EPCR) expression results in defective angiogenesis following experimental hindlimb ischemia. (2) Purpose: To determine soluble (s)EPCR levels in the plasma of patients with PAD and to compare them with the protein C activity and biomarkers of endothelial function, inflammation, and angiogenesis. (3) Methods and Results: Clinical tests of vascular function and immunoassays of plasma from patients with PAD stage II were compared to age- and sex-matched individuals with and without cardiovascular risk factors or PAD stage III/IV patients. sEPCR levels were significantly lower in PAD stage II patients compared to subjects with risk factors, but no PAD, and further decreased in PAD stage III/IV patients. Plasma protein C activity or levels of ADAM17, a mediator of EPCR shedding, did not differ. Significant associations between sEPCR and the ankle-brachial index (p = 0.0359), age (p = 0.0488), body mass index (p = 0.0110), and plasma sE-selectin levels (p = 0.0327) were observed. High-sensitive CRP levels and white blood cell counts were significantly elevated in PAD patients and associated with serum glucose levels, but not sEPCR. In contrast, plasma TNFα or IL1ß levels did not differ. Circulating levels of VEGF were significantly elevated in PAD stage II patients (p = 0.0198), but not associated with molecular (sE-selectin) or functional (ankle-brachial index) markers of vascular health. (4) Conclusions: Our findings suggest that circulating sEPCR levels may be useful as biomarkers of endothelial dysfunction, including angiogenesis, in persons older than 35 years and that progressive loss of endothelial protein C receptors might be involved in the development and progression of PAD.

Deletion of endothelial leptin receptors in mice promotes diet-induced obesity.

Obesity promotes endothelial dysfunction. Endothelial cells not only respond, but possibly actively promote the development of obesity and metabolic dysfunction. Our aim was to characterize the role of endothelial leptin receptors (LepR) for endothelial and whole body metabolism and diet-induced obesity. Mice with tamoxifen-inducible, Tie2.Cre-ERT2-mediated deletion of LepR in endothelial cells (End.LepR knockout, KO) were fed high-fat diet (HFD) for 16 weeks. Body weight gain, serum leptin levels, visceral adiposity and adipose tissue inflammation were more pronounced in obese End.LepR-KO mice, whereas fasting serum glucose and insulin levels or the extent of hepatic steatosis did not differ. Reduced brain endothelial transcytosis of exogenous leptin, increased food intake and total energy balance were observed in End.LepR-KO mice and accompanied by brain perivascular macrophage accumulation, whereas physical activity, energy expenditure and respiratory exchange rates did not differ. Metabolic flux analysis revealed no changes in the bioenergetic profile of endothelial cells from brain or visceral adipose tissue, but higher glycolysis and mitochondrial respiration rates in those isolated from lungs. Our findings support a role for endothelial LepRs in the transport of leptin into the brain and neuronal control of food intake, and also suggest organ-specific changes in endothelial cell, but not whole-body metabolism.

Not All Quiet on the Atherosclerosis Front.

In recent decades, research has identified the key cellular processes that take place during atherosclerotic plaque development and progression, including endothelial dysfunction, inflammation and lipoprotein oxidation, which result in macrophage and mural cell activation, death and necrotic core formation [...].

Endothelial-dependent S-Sulfhydration of tissue factor pathway inhibitor regulates blood coagulation.

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. Here we investigated whether endothelial cell-driven oxidative post-translational modifications could have an impact on TFPI activity. We focused on S-sulfhydration, which is a hydrogen sulfide-dependent post-translational modification that, in endothelial cells, is regulated by the enzyme cystathionine γ-lyase (CSE). The study made use of human primary endothelial cells and blood from healthy individuals or subjects with atherosclerosis as well as from mice lacking endothelial CSE. TFPI was S-sulfhydrated in endothelial cells from healthy individuals and mice, while the loss of endothelial CSE expression/activity reduced its modification. Non-S-sulfhydrated TFPI was no longer able to interact with factor Xa, which facilitated the activation of tissue factor. Similarly, non-S-sulfhydratable TFPI mutants bound less protein S, while supplementation with hydrogen sulfide donors, preserved TFPI activity. Phenotypically, loss of TFPI S-sulfhydration increased clot retraction, suggesting that this post-translational modification is a new endothelial cell-dependent mechanism that contributes to the regulation of blood coagulation.

Arginase-1 Deletion in Erythrocytes Promotes Vascular Calcification via Enhanced GSNOR (S-Nitrosoglutathione Reductase) Expression and NO Signaling in Smooth Muscle Cells.

BACKGROUND: Erythrocytes (red blood cells) participate in the control of vascular NO bioavailability. The purpose of this study was to determine whether and how genetic deletion of ARG1 (arginase-1) affects vascular smooth muscle cell NO signaling, osteoblastic differentiation, and atherosclerotic lesion calcification. METHODS: Atherosclerosis-prone mice with conditional, erythrocyte-restricted deletion of ARG1 (apoE-/- red blood cell.ARG1 knockout) were generated and vascular calcification studied using molecular imaging of the osteogenic activity agent OsteoSense, Alizarin staining or immunohistochemistry, qPCR of osteogenic markers and ex vivo assays. RESULTS: Atherosclerotic lesion size at the aortic root did not differ, but calcification was significantly more pronounced in apoE-/- mice lacking erythrocyte ARG1. Incubation of murine and human VSMCs with lysed erythrocyte membranes from apoE-/- red blood cell. ARG1-knockout mice accelerated their osteogenic differentiation, and mRNA transcripts of osteogenic markers decreased following NO scavenging. In addition to NO signaling via sGC (soluble guanylyl cyclase), overexpression of GSNOR (S-nitrosoglutathione reductase) enhanced degradation of S-nitrosoglutathione to glutathione and reduced protein S-nitrosation of HSP (heat shock protein)-70 were identified as potential mechanisms of vascular smooth muscle cell calcification in mice lacking ARG1 in erythrocytes, and calcium phosphate deposition was enhanced by heat shock and prevented by GSNOR inhibition. Messenger RNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline also were elevated in VSMCs, paralleled by increased proliferation, myofibroblast marker and collagen type 1 expression. CONCLUSIONS: Our findings support an important role of erythrocyte ARG1 for NO bioavailability and L-arginine metabolism in VSMCs, which controls atherosclerotic lesion composition and calcification.

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury.

BACKGROUND: Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. METHODS: Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ERT2 recombinase under the Myh11 promoter with PTP1Bflox/flox mice and subjected to FeCl3 carotid artery injury. RESULTS: Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA+ and PDGFRß+ myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11-Cre reporter cells in the remodeling adventitia, and SCA1+ CD45- vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor ß (TGFß) signaling components or enzymes involved in extracellular matrix remodeling and TGFß liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRß and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. CONCLUSION: Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription.

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia.

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.

Tubulin-folding cofactor E deficiency promotes vascular dysfunction by increased endoplasmic reticulum stress.

AIMS: Assessment of endothelial function in humans by measuring flow-mediated dilation (FMD) risk-stratifies individuals with established cardiovascular disease, whereas its predictive value is limited in primary prevention. We therefore aimed to establish and evaluate novel markers of FMD at the population level. METHODS AND RESULTS: In order to identify novel targets that were negatively correlated with FMD and investigate their contribution to vascular function, we performed a genome-wide association study (GWAS) of 4175 participants of the population based Gutenberg Health Study. Subsequently, conditional knockout mouse models deleting the gene of interest were generated and characterized. GWAS analysis revealed that single-nucleotide polymorphisms (SNPs) in the tubulin-folding cofactor E (TBCE) gene were negatively correlated with endothelial function and TBCE expression. Vascular smooth muscle cell (VSMC)-targeted TBCE deficiency was associated with endothelial dysfunction, aortic wall hypertrophy, and endoplasmic reticulum (ER) stress-mediated VSMC hyperproliferation in mice, paralleled by calnexin up-regulation and exacerbated by the blood pressure hormone angiotensin II. Treating SMMHC-ERT2-Cre+/-TBCEfl/fl mice with the ER stress modulator tauroursodeoxycholic acid amplified Raptor/Beclin-1-dependent autophagy and reversed vascular dysfunction. CONCLUSION: TBCE and tubulin homeostasis seem to be novel predictors of vascular function and offer a new drug target to ameliorate ER stress-dependent vascular dysfunction.

The gut microbiota instructs the hepatic endothelial cell transcriptome.

The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence-activated cell sorting, was used to isolate hepatic sinusoidal ECs from germ-free (GF) and conventionally raised (CONV-R) mice for transcriptome analysis by RNA sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were affected. The absence of microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine metabolism and the sphingosine-1-phosphate pathway showed drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

Extracellular Vesicles and Thrombosis: Update on the Clinical and Experimental Evidence.

Extracellular vesicles (EVs) compose a heterogenous group of membrane-derived particles, including exosomes, microvesicles and apoptotic bodies, which are released into the extracellular environment in response to proinflammatory or proapoptotic stimuli. From earlier studies suggesting that EV shedding constitutes a cellular clearance mechanism, it has become evident that EV formation, secretion and uptake represent important mechanisms of intercellular communication and exchange of a wide variety of molecules, with relevance in both physiological and pathological situations. The putative role of EVs in hemostasis and thrombosis is supported by clinical and experimental studies unraveling how these cell-derived structures affect clot formation (and resolution). From those studies, it has become clear that the prothrombotic effects of EVs are not restricted to the exposure of tissue factor (TF) and phosphatidylserines (PS), but also involve multiplication of procoagulant surfaces, cross-linking of different cellular players at the site of injury and transfer of activation signals to other cell types. Here, we summarize the existing and novel clinical and experimental evidence on the role and function of EVs during arterial and venous thrombus formation and how they may be used as biomarkers as well as therapeutic vectors.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

BACKGROUND: Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. METHODS: ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. RESULTS: Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-ß1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. CONCLUSION: Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis.

Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied. Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis ( n = 46), atherosclerotic carotid artery plaques ( n = 9), and abdominal aortic aneurysms ( n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson-Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals. Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.

Brain-Derived Neurotrophic Factor Expression and Signaling in Different Perivascular Adipose Tissue Depots of Patients With Coronary Artery Disease.

Background Brain-derived neurotrophic factor (BDNF) is expressed in neuronal and nonneuronal cells and may affect vascular functions via its receptor, tropomyosin-related kinase B (TrkB). In this study, we determined the expression of BDNF in different perivascular adipose tissue (PVAT) depots of patients with established coronary atherosclerosis. Methods and Results Serum, vascular tissue, and PVAT surrounding the proximal aorta (C-PVAT) or internal mammary artery (IMA-PVAT) was obtained from 24 patients (79% men; mean age, 71.7±9.7 years; median body mass index, 27.4±4.8 kg/m2) with coronary atherosclerosis undergoing elective coronary artery bypass surgery. BDNF protein levels were significantly higher in C-PVAT compared with IMA-PVAT, independent of obesity, metabolic syndrome, or systemic biomarkers of inflammation. mRNA transcripts of TrkB, the BDNF receptor, were significantly reduced in aorta compared with IMA. Vessel wall TrkB immunosignals colocalized with cells expressing smooth muscle cell markers, and confocal microscopy and flow cytometry confirmed BDNF receptor expression in human aortic smooth muscle cells. Significantly elevated levels of protein tyrosine phosphatase 1B, a negative regulator of TrkB signaling in the brain, were also observed in C-PVAT. In vitro, inhibition of protein tyrosine phosphatase 1B blunted the effects of BDNF on smooth muscle cell proliferation, migration, differentiation, and collagen production, possibly by upregulation of low-affinity p75 neurotrophin receptors. Expression of nerve growth factor or its receptor tropomyosin-related kinase A did not differ between C-PVAT and IMA-PVAT. Conclusions Elevated expression of BDNF in parallel with local upregulation of negative regulators of neurotrophin signaling in perivascular fat and lower TrkB expression suggest that vascular BDNF signaling is reduced or lost in patients with coronary atherosclerosis.

Specialized regulatory T cells control venous blood clot resolution through SPARC.

The cells and mechanisms involved in blood clot resorption are only partially known. We show that regulatory T cells (Tregs) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. We describe a clot Treg population that forms the matricellular acid- and cysteine-rich protein SPARC (secreted protein acidic and rich in cysteine) and show that SPARC enhances monocyte MMP activity and that SPARC+ Tregs are crucial for blood clot resorption. By comparing different treatment times, we define a therapeutic window of Treg expansion that accelerates clot resorption.