Thrombin and protease-activated receptors (PARs) in atherothrombosis

SummaryThrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes,T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extra-cellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation inVSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overallTRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.

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A review of bioeffects induced by focused ultrasound combined with microbubbles on the neurovascular unit

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x211046129 ◽

2021 ◽

pp. 0271678X2110461

Author(s):

Si Chen ◽

Arash Nazeri ◽

Hongchae Baek ◽

Dezhuang Ye ◽

Yaoheng Yang ◽

...

Keyword(s):

Endothelial Cells ◽

Focused Ultrasound ◽

Neurovascular Unit ◽

Cell Types ◽

Current Treatment ◽

Clinical Applications ◽

Brain Diseases ◽

Great Promise ◽

Cellular Effects ◽

Cell Type Specific

Focused ultrasound combined with circulating microbubbles (FUS+MB) can transiently enhance blood-brain barrier (BBB) permeability at targeted brain locations. Its great promise in improving drug delivery to the brain is reflected by a rapidly growing number of clinical trials using FUS+MB to treat various brain diseases. As the clinical applications of FUS+MB continue to expand, it is critical to have a better understanding of the molecular and cellular effects induced by FUS+MB to enhance the efficacy of current treatment and enable the discovery of new therapeutic strategies. Existing studies primarily focus on FUS+MB-induced effects on brain endothelial cells, the major cellular component of BBB. However, bioeffects induced by FUS+MB expand beyond the BBB to cells surrounding blood vessels, including astrocytes, microglia, and neurons. Together these cell types comprise the neurovascular unit (NVU). In this review, we examine cell-type-specific bioeffects of FUS+MB on different NVU components, including enhanced permeability in endothelial cells, activation of astrocytes and microglia, as well as increased intraneuron protein metabolism and neuronal activity. Finally, we discuss knowledge gaps that must be addressed to further advance clinical applications of FUS+MB.

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TFAM-deficient mouse skin fibroblasts: an ex vivo model of mitochondrial dysfunction

Disease Models & Mechanisms ◽

10.1242/dmm.048995 ◽

2021 ◽

Author(s):

Manuel J. Del Rey ◽

Carolina Meroño ◽

Cristina Municio ◽

Alicia Usategui ◽

María Mittelbrunn ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Ex Vivo ◽

Stress Test ◽

Critical Role ◽

Substantial Reduction ◽

Mouse Skin ◽

Cell Types ◽

Ex Vivo Model ◽

Primary Mouse ◽

Inflammatory Phenotype

Mitochondrial dysfunction in different cell types is associated to several pathological processes and potentially contributes to chronic inflammatory and ageing-related diseases. Mitochondrial Transcription Factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of the Tfamfl/fl UBC-Cre/ERT2+/+ mice, we sought to develop a cellular in vitro system to investigate the role of mitochondrial dysfunction in the stromal cell component. We describe an inducible model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblast (SK-FB) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction of Tfam and mtDNA-encoded mRNA expression in Cre(+) cultured for low (LP) and high passages (HP). Ultimately, Tfam knockout translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction of the mitochondrial respiratory chain (MRC) complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT Cre(+) SK-FB. Functionally, we determined the mitochondrial function and the glycolytic activity by mito-stress and glycolysis-stress test respectively. These analysis showed that mitochondrial dysfunction was developed after long-term 4-OHT treatment in HP Cre(+) SK-FB and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FB showed a senescent and pro-inflammatory phenotype. In conclusion, we have generated and validated the first ex vivo model of fibroblast mitochondrial dysfunction that results in a pro-inflammatory phenotype applicable to explore this process in other cell types in a variety of pathological conditions.

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Nonhemostatic Activities of Factor Xa: Are There Pleiotropic Effects of Anti-FXa Direct Oral Anticoagulants?

Angiology ◽

10.1177/0003319719840861 ◽

2019 ◽

Vol 70 (10) ◽

pp. 896-907 ◽

Cited By ~ 7

Author(s):

Styliani Papadaki ◽

Alexandros D. Tselepis

Keyword(s):

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Anticoagulation Therapy ◽

Factor Xa ◽

Cell Types ◽

Pleiotropic Effects ◽

Coagulation Cascade ◽

Protease Activated Receptors ◽

Cellular Effects ◽

Thrombotic Diseases

Factor Xa (FXa) is the key serine protease of the coagulation cascade as it is the point of convergence of the intrinsic and extrinsic pathways, leading to the formation of thrombin. Factor Xa is an established target of anticoagulation therapy, due to its central role in coagulation. Over the past years, several direct oral anticoagulants (DOACs) targeting FXa have been developed. Rivaroxaban, apixaban, and edoxaban are used in clinical practice for prevention and treatment of thrombotic diseases. Increasing evidence suggests that FXa exerts nonhemostatic cellular effects that are mediated mainly through protease-activated receptors-1 and -2 and are involved in pathophysiological conditions, such as atherosclerosis, inflammation, and fibrosis. Direct inhibition of FXa by DOACs could be beneficial in these conditions. This is a narrative review that focuses on the cellular effects of FXa in various cell types and conditions, as well as on the possible pleiotropic effects of FXa-targeting DOACs.

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Angiogenic properties of the chemokine RANTES/CCL5

Biochemical Society Transactions ◽

10.1042/bst20110651 ◽

2011 ◽

Vol 39 (6) ◽

pp. 1649-1653 ◽

Cited By ~ 40

Author(s):

Nadine Suffee ◽

Benjamin Richard ◽

Hanna Hlawaty ◽

Olivier Oudar ◽

Nathalie Charnaux ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Critical Role ◽

Cell Types ◽

Developed Countries ◽

Present Review ◽

Vasa Vasorum ◽

In Vivo Models ◽

Vitro Assay

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

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Crosstalk between Breast Cancer Cells and Bone Marrow Endothelium Require the Engagement of PI3K/AKT Signaling.

Blood ◽

10.1182/blood.v104.11.1299.1299 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1299-1299

Author(s):

Joana G. Brandao ◽

Joao T. Barata ◽

Raquel Nunes ◽

Lee M. Nadler ◽

Angelo A. Cardoso

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Endothelial Cells ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Critical Role ◽

Metastatic Breast ◽

Cell Types ◽

Akt Pathway ◽

The Impact

Abstract The presence of breast cancer cells in the patient’s bone marrow (BM) at diagnosis is associated with resistance to treatment, disease relapse and poor prognosis. Identification of the factors implicated in the homing, survival and latency of breast cancer cells in the BM should contribute to the design of more efficient therapeutic strategies for breast cancer. There is evidence that breast cancer can recruit endothelial progenitors from the BM. Also, other epithelial tumors seem to preferentially adhere to BM endothelial cells. Therefore, we hypothesized that BM endothelium may play a significant role in the biology of breast cancer cells residing in the BM. Co-cultures in Matrigel showed that breast cancer cells interact with BM endothelium to form heterotypic multicellular networks. Moreover, breast cancer cells migrate towards BM endothelium assembled as capillary-like structures, but not to structures of BM mesenchymal stem cells or BM stroma. This migration was abrogated by pertussis toxin-mediated blockade of chemokine receptor signaling, suggesting the involvement of endothelium-secreted chemokine(s). We then evaluated the impact of breast cancer cells in the survival and proliferation of BM endothelium. All breast cancer lines tested (n=4) promoted the proliferation of BM-derived endothelial cells. This effect is mediated through the engagement of the PI3K/Akt pathway (phosphorylation of Akt at Ser437 and Thr308, and activation of its downstream substrates GSK3β, PRAS-40 and FKHRL1) since its specific blockade abrogated the stimulatory effects of breast cancer on BM endothelium. We next determined whether, reciprocally, BM endothelium impacts on breast cancer cell survival. These experiments were performed in serum-free media to enhance dependency of breast cancer cells from microenvironmental stimuli. In all cases tested, BM endothelium promoted survival/proliferation of breast cancer cells. This stimulation was accompanied by the engagement of the PI3K/Akt pathway in breast cancer cells and, in three of the four lines, the phosphorylation of Erk1/2. These effects were also observed for breast cancer cells that showed constitutive activation of Akt (MCF-7 and ZR-75-1 cells). Specific blockade of PI3K/Akt abrogated the BM endothelium-promoted survival of breast cancer cells, thus demonstrating the critical role of this pathway. These studies show that crosstalk between BM endothelial cells and breast cancer cells may impact on the survival of both cell types. These findings provide new light on the mechanisms that may facilitate the development of a tumor-permissive BM microenvironment in breast cancer, and the creation of breast cancer-supporting BM niches. Importantly, this study implicates BM endothelium as a therapeutic target in breast cancer and suggest that blockade of PI3K/Akt may impact the outcome of patients with metastatic breast cancer.

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Glycolysis links reciprocal activation of myeloid cells and endothelial cells in the retinal angiogenic niche

Science Translational Medicine ◽

10.1126/scitranslmed.aay1371 ◽

2020 ◽

Vol 12 (555) ◽

pp. eaay1371 ◽

Cited By ~ 3

Author(s):

Zhiping Liu ◽

Jiean Xu ◽

Qian Ma ◽

Xiaoyu Zhang ◽

Qiuhua Yang ◽

...

Keyword(s):

Endothelial Cells ◽

Critical Role ◽

Myeloid Cells ◽

Cell Types ◽

Acetyl Coenzyme A ◽

Enhanced Production ◽

Fructose 2 ◽

Retinal Angiogenesis ◽

Cellular Components

The coordination of metabolic signals among different cellular components in pathological retinal angiogenesis is poorly understood. Here, we showed that in the pathological angiogenic vascular niche, retinal myeloid cells, particularly macrophages/microglia that are spatially adjacent to endothelial cells (ECs), are highly glycolytic. We refer to these macrophages/microglia that exhibit a unique angiogenic phenotype with increased expression of both M1 and M2 markers and enhanced production of both proinflammatory and proangiogenic cytokines as pathological retinal angiogenesis–associated glycolytic macrophages/microglia (PRAGMs). The phenotype of PRAGMs was recapitulated in bone marrow–derived macrophages or retinal microglia stimulated by lactate that was produced by hypoxic retinal ECs. Knockout of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3; Pfkfb3 for rodents), a glycolytic activator in myeloid cells, impaired the ability of macrophages/microglia to acquire an angiogenic phenotype, rendering them unable to promote EC proliferation and sprouting and pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Mechanistically, hyperglycolytic macrophages/microglia produced large amount of acetyl–coenzyme A, leading to histone acetylation and PRAGM-related gene induction, thus reprogramming macrophages/microglia into an angiogenic phenotype. These findings reveal a critical role of glycolytic metabolites as initiators of reciprocal activation of macrophages/microglia and ECs in the retinal angiogenic niche and suggest that strategies targeting the metabolic communication between these cell types may be efficacious in the treatment of pathological retinal angiogenesis.

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Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease

International Journal of Molecular Sciences ◽

10.3390/ijms222312948 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12948

Author(s):

Michael J. Duryee ◽

Dahn L. Clemens ◽

Patrick J. Opperman ◽

Geoffrey M. Thiele ◽

Logan M. Duryee ◽

...

Keyword(s):

Lipid Peroxidation ◽

Animal Model ◽

Endothelial Cells ◽

Inflammatory Response ◽

Critical Role ◽

Density Lipoprotein ◽

Cell Types ◽

Fat Accumulation ◽

Modified Proteins

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

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Glomerular Endothelial Cells Are the Coordinator in the Development of Diabetic Nephropathy

Frontiers in Medicine ◽

10.3389/fmed.2021.655639 ◽

2021 ◽

Vol 8 ◽

Author(s):

Tingting Li ◽

Kaiyuan Shen ◽

Jiawei Li ◽

Susan W. S. Leung ◽

Tongyu Zhu ◽

...

Keyword(s):

Endothelial Cells ◽

Diabetic Nephropathy ◽

Protein Interactions ◽

Immune Cells ◽

Plasma Cells ◽

Mesangial Cells ◽

Critical Role ◽

Cell Types ◽

Protein Protein Interactions ◽

Glomerular Endothelial Cells

The prevalence of diabetes is consistently rising worldwide. Diabetic nephropathy is a leading cause of chronic renal failure. The present study aimed to explore the crosstalk among the different cell types inside diabetic glomeruli, including glomerular endothelial cells, mesangial cells, podocytes, and immune cells, by analyzing an online single-cell RNA profile (GSE131882) of patients with diabetic nephropathy. Differentially expressed genes in the glomeruli were processed by gene enrichment and protein-protein interactions analysis. Glomerular endothelial cells, as well as podocytes, play a critical role in diabetic nephropathy. A subgroup of glomerular endothelial cells possesses characteristic angiogenesis genes, indicating that angiogenesis takes place in the progress of diabetic nephropathy. Immune cells such as macrophages, T lymphocytes, B lymphocytes, and plasma cells also contribute to the disease progression. By using iTALK, the present study reports complicated cellular crosstalk inside glomeruli. Dysfunction of glomerular endothelial cells and immature angiogenesis result from the activation of both paracrine and autocrine signals. The present study reinforces the importance of glomerular endothelial cells in the development of diabetic nephropathy. The exploration of the signaling pathways involved in aberrant angiogenesis reported in the present study shed light on potential therapeutic target(s) for diabetic nephropathy.

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LPS-Toll-Like Receptor-Mediated Signaling on Expression of Protein S and C4b-Binding Protein in the Liver

Gastroenterology Research and Practice ◽

10.1155/2010/189561 ◽

2010 ◽

Vol 2010 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Tatsuya Hayashi ◽

Koji Suzuki

Keyword(s):

Endothelial Cells ◽

Binding Protein ◽

Critical Role ◽

Activated Protein C ◽

Protein S ◽

Cell Types ◽

Mrna Levels ◽

Toll Like Receptor ◽

Membrane Bound

Protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role as a cofactor of anticoagulant activated protein C (APC). PS activity is regulated by C4b-binding protein (C4BP), structurally composed of sevenα-chains (C4BPα) and aβ-chain (C4BPβ). In this paper, based primarily on our previous studies, we review the lipopolysaccharide (LPS)-induced signaling which affects expression of PS and C4BP in the liver. Ourin vivostudies in rats showed that after LPS injection, plasma PS levels are significantly decreased, whereas plasma C4BP levels first are transiently decreased after 2 to 12 hours and then significantly increased after 24 hours. LPS decreases PS antigen and mRNA levels in both hepatocytes and sinusoidal endothelial cells (SECs), and decreases C4BP antigen and both C4BPαand C4BPβmRNA levels in hepatocytes. Antirat CD14 and antirat Toll-like receptor (TLR)-4 antibodies inhibited LPS-induced NFκB activation in both hepatocytes and SECs. Furthermore, inhibitors of NFκB and MEK recovered the LPS-induced decreased expression of PS in both cell types and the LPS-induced decreased expression of C4BP in hepatocytes. These data suggest that the LPS-induced decrease in PS expression in hepatocytes and SECs and LPS-induced decrease in C4BP expression in hepatocytes are mediated by MEK/ERK signaling and NFκB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism.

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Differential Serotonin Uptake Mechanisms at the Human Maternal–Fetal Interface

International Journal of Molecular Sciences ◽

10.3390/ijms22157807 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7807

Author(s):

Petra Baković ◽

Maja Kesić ◽

Maja Perić ◽

Ivona Bečeheli ◽

Marina Horvatiček ◽

...

Keyword(s):

Endothelial Cells ◽

Cord Blood ◽

Blood Platelets ◽

Organic Cation Transporter ◽

Cell Types ◽

Maternal Blood ◽

Km Value ◽

5 Ht Uptake ◽

First Time ◽

Maternal Fetal Interface

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of 5-HT uptake into human primary placental cells and cord blood platelets, all isolated immediately after birth. Trophoblasts and cord blood platelets showed 5-HT uptake with similar Michaelis constant (Km) values (~0.6 μM), typical of the high-affinity serotonin transporter (SERT). The uptake of 5-HT into trophoblasts was efficiently inhibited by various SERT-targeting drugs. In contrast, the uptake of 5-HT into feto-placental endothelial cells was not inhibited by a SERT blocker and showed a Km value (~782 μM) in the low-affinity range. Consistent with this, SERT mRNAs were abundant in term trophoblasts but sparse in feto-placental endothelial cells, whereas the opposite was found for the low-affinity plasma membrane monoamine transporter (PMAT) mRNAs. Organic cation transporter (OCT) 1, 2, and 3 mRNAs were absent or sparse in both cell types. In summary, the results demonstrate, for the first time, the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells that are in direct contact with fetal blood plasma. The data also highlight the sensitivity to various psychotropic drugs of 5-HT transport into trophoblasts facing the maternal blood. The multiple, high-, and low-affinity systems present for the cellular uptake of 5-HT underscore the importance of 5-HT homeostasis at the maternal–fetal interface.

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