Ramprasad Srinivasan

Myeloid-Specific Krüppel-Like Factor 2 Inactivation Increases Macrophage and Neutrophil Adhesion and Promotes Atherosclerosis

Rationale: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2−/−) mice were similar to myeKlf2+/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2+/+ macrophages, myeKlf2−/− macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2−/− mice also adhered more robustly to endothelial cells, and fewer myeKlf2−/− neutrophils survived in culture over a 24-hour period in comparison with myeKlf2+/+ neutrophils. When myeKlf2−/− mice were mated to Ldlr−/− mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2−/−Ldlr−/− mice compared with myeKlf2+/+Ldlr−/− littermates. The increased atherosclerosis in myeKlf2−/−Ldlr−/− mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2+/+Ldlr−/− mice. Conclusions: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Splice variants of Tissue Factor promote monocyte-endothelial interactions by triggering the expression of cell adhesion molecules via integrin-mediated signaling

Summary.  Background: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective: To examine the role of integrin signaling in TF‐dependent recruitment of monocytes by endothelial cells. Methods: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ‐TF) and recombinant asTF were assessed. Results: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid‐rich plaques. Tumor lesions, as well as stromal CD68+ monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ‐TF, and this was completely blocked by anti‐β1 integrin antibody. asTF‐ and LZ‐TF‐treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF‐elicited adhesion was more pronounced than that elicited by LZ‐TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E‐selectin, ICAM‐1 and VCAM‐1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3‐fold under MCP‐1 gradient. Conclusions: TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non‐proteolytic, integrin‐mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Transplanted Perivascular Adipose Tissue Accelerates Injury-Induced Neointimal Hyperplasia

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF‐overexpressing high‐grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell‐derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell‐derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis

Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. # CLINICAL PERSPECTIVE {#article-title-42}Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Nonproteolytic Properties of Murine Alternatively Spliced Tissue Factor: Implications for Integrin-Mediated Signaling in Murine Models

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr−/− mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

Splice variants of Tissue Factor and integrin-mediated signaling

Full-length Tissue Factor (flTF) - the obligatory co-factor for the serine protease (factor) VII/VIIa - serves as the initiator of blood coagulation. The flTF/VIIa complex triggers a sequence of proteolytic events that lead to the formation of a hemostatic plug. Aside from hemostatic maintenance, flTF can contribute to thrombogenesis in some settings. The proteolytic properties of the flTF/VIIa complex (as well as the flTF/VIIa/Xa complex) account for non-hemostatic functions of flTF, largely exerted through activation of intracellular signaling via Protease Activated Receptors (PARs). The flTF-PAR nexus impacts several kinases highly significant in the pathobiology of cancer and cardiovascular disease. Over the past decade, many advances have been made in the understanding of PAR-mediated functions of flTF, an important highlight of which was the finding that a sub-set of integrins - a diverse family of integral membrane proteins - cross-regulate flTF-elicited signaling. Concomitantly, an alternatively spliced TF form (asTF) was discovered in human and mouse. Initial studies characterizing asTF revealed that it is differentially expressed during development, continuously present in circulating blood and solid tissues, and possesses very low pro-coagulant activity. Hypomorphic nature of asTFs cofactor activity is the source of an ongoing controversy over whether asTF is pro-coagulant, and how it may contribute to hemostatic maintenance and/or its aberrations. Very recently, a novel concept emerged in asTF biology: asTF can evidently trigger intracellular signaling that promotes the formation of new vessels from the existing ones (angiogenesis) and monocyte-endothelial interactions, via interaction with integrins. We provide a brief overview of the fl/asTF-integrin nexus with an emphasis on asTFs non-proteolytic, integrin-mediated biological activity.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of MCP-1

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Red Blood Cell Dysfunction Induced by High-Fat DietCLINICAL PERSPECTIVE: Potential Implications for Obesity-Related Atherosclerosis

Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. # CLINICAL PERSPECTIVE {#article-title-42}Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.