Instruct: Given a scientific claim, retrieve passages that support or refute the claim.
Query: KLF4 is necessary for VSMCs to gain genetic characteristics of other cell types within atherosclerotic lesions.
Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation–sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.
Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.
Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell-cell and cell-matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor kappaB-dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell-cell and cell-matrix adhesions to elicit flow-induced inflammatory signaling.
Background: Atherosclerosis is characterized by infiltration of inflammatory cells from circulating blood. Blood cell activation could play an important role in plaque formation. Methods: We analyzed the relationship between blood cellular markers and quantitative measures of carotid wall components in 1,546 participants from the ARIC (Atherosclerosis Risk in Communities) Carotid MRI Study. Carotid imaging was performed using a gadolinium contrast-enhanced MRI and cellular phenotyping by flow cytometry. Results: Monocyte Toll-like receptor (TLR)-2 is associated with larger plaques, while CD14, myeloperoxidase, and TLR-4 associate with smaller. Platelet CD40L is associated with smaller plaques and thinner caps, while P-selectin is associated with smaller core size. Conclusions: Blood cell activation is significantly associated with atherosclerotic changes of the carotid wall.
Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.
Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.
The zinc-finger transcription factor KLF2 transduces the physical forces exerted by blood flow into molecular signals responsible for a wide range of biological responses. Following its initial recognition as a flow-responsive endothelial transcription factor, KLF2 is now known to be expressed in a range of cell types and to participate in a number of processes during development and disease such as endothelial homeostasis, vasoregulation, vascular growth/remodeling, and inflammation. In this review, we summarize the current understanding about KLF2 with a focus on its effects on vascular biology.
Objective: To investigate the regulation of CD4+CD25+ Regulatory T cells (Tregs) on pro-inflammatory adhesion molecules, Krüppel-Like Factor-2 (KLF-2) and its downstream transcriptional targets in human umbilical vein endothelial cells (HUVECs) impaired by ox-LDL and the mechanisms of it. Methods and results: HUVECs were cultured in the continuous presence of ox-LDL(0 mg/L,25 mg/L,50 mg/L,100 mg/L) for 4, 6, 12 and 24 hours to allow identification of early-and late-induced genes, respectively, whereas non-stimulated controls were taken at 0 hours. The expression of pro-inflammatory adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, KLF-2 and its target genes eNOS, PAI-1 were determined by real time RT-PCR and/or western-blot analysis. Expression of pro-inflammatory adhesion molecules, KLF-2, eNOS and PAI-1 in HUVEC cultured alone or with anti-CD3 mAbs activated Tregs, followed by addition of ox-LDL (50 mg/L) for 6 hours, are compared to expression levels in control cultures. Ox-LDL treated HUVECs increased pro-inflammatory adhesion molecules expression, as well as increased PAI-1 but decreased eNOS expression accompanied with significant downregulating of KLF-2 at a dose and time dependent manner. Furthermore, ox-LDL increased pro-inflammatory adhesion molecules but inhibited KLF2 expression was reversed by addition of Tregs. Small interfering RNA reduced endogenous KLF-2 expression and partly reversed the suppressive effect of Tregs on HUVECs activation, which strongly implicate KLF-2 as a transcriptional regulator of the Tregs-mediated effects in endothelial cells. Mechanism studies reveal that Treg-mediated KLF2 expression in HUVECs impaired by ox-LDL requires cell contact as well as soluble factors. Conclusions: Tregs could protect endothelial function that is largely dependent on KLF2 and its downstream transcriptional targets regulation involving cell-to-cell contact and soluble factors.
OBJECTIVE T cell immunoglobulin and mucin domain (Tim) proteins are expressed by numerous immune cells, recognize phosphatidylserine on apoptotic cells, and function as costimulators or coinhibitors. Tim-1 is expressed by activated T cells but is also found on dendritic cells and B cells. Tim-4, present on macrophages and dendritic cells, plays a critical role in apoptotic cell clearance, regulates the number of phosphatidylserine-expressing activated T cells, and is genetically associated with low low-density lipoprotein and triglyceride levels. Because these functions of Tim-1 and Tim-4 could affect atherosclerosis, their modulation has potential therapeutic value in cardiovascular disease. APPROACH AND RESULTS ldlr(-/-) mice were fed a high-fat diet for 4 weeks while being treated with control (rat immunoglobulin G1) or anti-Tim-1 (3D10) or -Tim-4 (21H12) monoclonal antibodies that block phosphatidylserine recognition and phagocytosis. Both anti-Tim-1 and anti-Tim-4 treatments enhanced atherosclerosis by 45% compared with controls by impairment of efferocytosis and increasing aortic CD4(+)T cells. Consistently, anti-Tim-4-treated mice showed increased percentages of activated T cells and late apoptotic cells in the circulation. Moreover, in vitro blockade of Tim-4 inhibited efferocytosis of oxidized low-density lipoprotein-induced apoptotic macrophages. Although anti-Tim-4 treatment increased T helper cell (Th)1 and Th2 responses, anti-Tim-1 induced Th2 responses but dramatically reduced the percentage of regulatory T cells. Finally, combined blockade of Tim-1 and Tim-4 increased atherosclerotic lesion size by 59%. CONCLUSIONS Blockade of Tim-4 aggravates atherosclerosis likely by prevention of phagocytosis of phosphatidylserine-expressing apoptotic cells and activated T cells by Tim-4-expressing cells, whereas Tim-1-associated effects on atherosclerosis are related to changes in Th1/Th2 balance and reduced circulating regulatory T cells.
Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.
RATIONALE Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.
Although the entire coronary tree is exposed to the atherogenic effect of the systemic risk factors, atherosclerotic lesions form at specific arterial regions, where low and oscillatory endothelial shear stress (ESS) occur. Low ESS modulates endothelial gene expression through complex mechanoreception and mechanotransduction processes, inducing an atherogenic endothelial phenotype and formation of an early atherosclerotic plaque. Each early plaque exhibits an individual natural history of progression, regression, or stabilization, which is dependent not only on the formation and progression of atherosclerosis but also on the vascular remodeling response. Although the pathophysiologic mechanisms involved in the remodeling of the atherosclerotic wall are incompletely understood, the dynamic interplay between local hemodynamic milieu, low ESS in particular, and the biology of the wall is likely to be important. In this review, we explore the molecular, cellular, and vascular processes supporting the role of low ESS in the natural history of coronary atherosclerosis and vascular remodeling and indicate likely mechanisms concerning the different natural history trajectories of individual coronary lesions. Atherosclerotic plaques associated with excessive expansive remodeling evolve to high-risk plaques, because low ESS conditions persist, thereby promoting continued local lipid accumulation, inflammation, oxidative stress, matrix breakdown, and eventually further plaque progression and excessive expansive remodeling. An enhanced understanding of the pathobiologic processes responsible for atherosclerosis and vascular remodeling might allow for early identification of a high-risk coronary plaque and thereby provide a rationale for innovative diagnostic and/or therapeutic strategies for the management of coronary patients and prevention of acute coronary syndromes.
In atherosclerosis, the accumulation of apolipoprotein B-lipoproteins in the matrix beneath the endothelial cell layer of blood vessels leads to the recruitment of monocytes, the cells of the immune system that give rise to macrophages and dendritic cells. Macrophages derived from these recruited monocytes participate in a maladaptive, nonresolving inflammatory response that expands the subendothelial layer due to the accumulation of cells, lipid, and matrix. Some lesions subsequently form a necrotic core, triggering acute thrombotic vascular disease, including myocardial infarction, stroke, and sudden cardiac death. This Review discusses the central roles of macrophages in each of these stages of disease pathogenesis.
AIMS Vascular endothelial dysfunction and inflammation are hallmarks of atherosclerosis. Krüppel-like factor 2 (KLF2) is a key mediator of anti-inflammatory and anti-atherosclerotic properties of the endothelium. However, little is known of the molecular mechanisms for regulating KLF2 transcriptional activation. METHODS AND RESULTS Here, we found that histone deacetylase 5 (HDAC5) associates with KLF2 and represses KLF2 transcriptional activation. HDAC5 resided with KLF2 in the nuclei of human umbilical cord vein endothelial cells (HUVECs). Steady laminar flow attenuated the association of HDAC5 with KLF2 via stimulating HDAC5 phosphorylation-dependent nuclear export in HUVEC. We also mapped the KLF2-HDAC5-interacting domains and found that the N-terminal region of HDAC5 interacts with the C-terminal domain of KLF2. Chromatin immunoprecipitation and luciferase reporter assays showed that HDAC5 through a direct association with KLF2 suppressed KLF2 transcriptional activation. HDAC5 overexpression inhibited KLF2-dependent endothelial nitric oxide synthesis (eNOS) promoter activity in COS7 cell and gene expression in both HUVECs and bovine aortic endothelial cells (BAECs). Conversely, HDAC5 silencing enhanced KLF2 transcription and hence eNOS expression in HUVEC. Moreover, we observed that the level of eNOS protein in the thoracic aorta isolated from HDAC5 knockout mice was higher, whereas expression of pro-inflammatory vascular cell adhesion molecule 1 was lower, compared with those of HDAC5 wild-type mice. CONCLUSIONS We reveal a novel role of HDAC5 in modulating the KLF2 transcriptional activation and eNOS expression. These findings suggest that HDAC5, a binding partner and modulator of KLF2, could be a new therapeutic target to prevent vascular endothelial dysfunction associated with cardiovascular diseases.
Background: Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo a phenotypic transformation into osteoblast-like cells, is one of the emergent risk factors for the accelerated atherosclerosis process characteristic of chronic kidney disease (CKD). Phosphate is an important regulator of VC. Methods: The expression of different smooth muscle cell or osteogenesis markers in response to high concentrations of phosphate or exogenous bone morphogenetic protein 2 (BMP-2) was examined by qRT-PCR and western blotting in rat VSMCs. Osteocalcin secretion was measured by radioimmunoassay. Differentiation and calcification of VSMCs were examined by alkaline phosphatase (ALP) activity assay and Alizarin staining. Short hairpin RNA-mediated silencing of β-catenin was performed to examine the involvement of Wnt/β-catenin signaling in VSMC calcification and osteoblastic differentiation induced by high phosphate or BMP-2. Apoptosis was determined by TUNEL assay and immunofluorescence imaging. Results: BMP-2 serum levels were significantly higher in CKD patients than in controls. High phosphate concentrations and BMP-2 induced VSMC apoptosis and upregulated the expression of β-catenin, Msx2, Runx2 and the phosphate cotransporter Pit1, whereas a BMP-2 neutralization antibody reversed these effects. Knockdown of β-catenin abolished the effect of high phosphate and BMP-2 on VSMC apoptosis and calcification. Conclusions: BMP-2 plays a crucial role in calcium deposition in VSMCs and VC in CKD patients via a mechanism involving the Wnt/β-catenin pathway.
Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+) , 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.
Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1(+) (Sca-1(+)/PDGFRα(+) and Sca-1(+)/PDGFRα(-)) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1(-) (Sca-1(-)/PDGFRα(+) and Sca-1(-)/PDGFRα(-)) progenitor cells. Among Sca-1(+) progenitor populations, Sca-1(+)/PDGFRα(-) cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1(+)/PDGFRα(+) cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1(+)/PDGFRα(-) cells preferentially differentiated into osteoclast-like cells. Sca-1(+) progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1(+) calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1(+) progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1(+)/PDGFRα(-) cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1(+)/PDGFRα(-) cells into Apoe(-/-) mice increased the severity of calcified atherosclerotic plaques. However, Sca-1(+)/PDGFRα(-) cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1(+)/PDGFRα(-) cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.
BACKGROUND Although Toll-like receptor 4 (TLR-4) is involved in monocyte activation in patients with accelerated forms of atherosclerosis, the relationship between the expression of TLR-4 on circulating monocytes and coronary plaque vulnerability has not previously been evaluated. We investigated this relationship using 64-slice multidetector computed tomography (MDCT) in patients with stable angina pectoris (SAP).Methods and Results:We enrolled 65 patients with SAP who underwent MDCT. Three monocyte subsets (CD14++CD16-, CD14++CD16+, and CD14+CD16+) and expression of TLR-4 were measured by flow cytometry. Intracoronary plaques were assessed by 64-slice MDCT. We defined vulnerability of intracoronary plaques according to the presence of positive remodeling (remodeling index >1.05) and/or low CT attenuation (<35 HU). The circulating CD14++CD16+monocytes more frequently expressed TLR-4 than CD14++CD16-and CD14+CD16+monocytes (P<0.001). The relative proportion of the expression of TLR-4 on CD14++CD16+monocytes was significantly greater in patients with vulnerable plaque compared with those without (10.4 [4.1-14.5] % vs. 4.5 [2.8-7.8] %, P=0.012). In addition, the relative proportion of TLR-4 expression on CD14++CD16+monocytes positively correlated with the remodeling index (r=0.28, P=0.025) and negatively correlated with CT attenuation value (r=-0.31, P=0.013). CONCLUSIONS Upregulation of TLR-4 on CD14++CD16+monocytes might be associated with coronary plaque vulnerability in patients with SAP.
OBJECTIVES Calcific aortic valve (AV) disease is known to be an inflammation-related process. High-mobility group box-1 (HMGB1) protein and Toll-like receptor 4 (TLR4) have been reported to participate in several inflammatory diseases. The purpose of the present study was to determine whether the HMGB1-TLR4 axis is involved in calcific AV disease, and to evaluate the effect of HMGB1, and its potential mechanisms, on the pro-osteogenic phenotype change of valvular interstitial cells (VICs). METHODS Expression of HMGB1 and TLR4 in human calcific AVs was evaluated using immunohistochemical staining and immunoblotting. Cultured VICs were used as an in vitro model. The VICs were stimulated with HMGB1 for analysis, with versus without TLR4 small interfering ribonucleic acid (siRNA), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and nuclear factor kappa-B (NF-κB) inhibitors. RESULTS Enhanced accumulation of HMGB1 and TLR4 was observed in calcific valves. Moreover, we found that HMGB1 induced high levels of pro-inflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. In addition, HMGB1 induced phosphorylation of JNK MAPK and NF-κB. However, these effects were markedly suppressed by siRNA silencing of TLR4. In addition, blockade of JNK MAPK and NF-κB phosphorylation prohibited HMGB1-induced production of pro-osteogenic factors, and mineralization of VICs. CONCLUSIONS The HMGB1 protein may promote osteoblastic differentiation and calcification of VICs, through the TLR4-JNK-NF-κB signaling pathway.
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (SR-A). Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.
To generate transgenic mice that express Cre-recombinase exclusively in the megakaryocytic lineage, we modified a mouse bacterial artificial chromosome (BAC) clone by homologous recombination and replaced the first exon of the platelet factor 4 (Pf4), also called CXCL4, with a codon-improved Cre cDNA. Several strains expressing the transgene were obtained and one strain, Q3, was studied in detail. Crossing Q3 mice with the ROSA26-lacZ reporter strain showed that Cre-recombinase activity was confined to megakaryocytes. These results were further verified by crossing the Q3 mice with a strain containing loxP-flanked integrin beta1. Excision of this conditional allele in megakaryocytes was complete at the DNA level, and platelets were virtually devoid of the integrin beta1 protein. The Pf4-Cre transgenic strain will be a valuable tool to study megakaryopoiesis, platelet formation, and platelet function.
Although the accumulation of cholesterol in macrophages appears to be an initial step in atherogenesis, low-density lipoprotein (LDL), a major risk factor for atherosclerosis, does not promote cholesterol accumulation in macrophages in its native form. On the other hand, apolipoprotein (apo) A-I-containing lipoprotein removes cholesterol from cholesterol-loaded macrophages (foam cells) and prevents cholesterol from accumulating in the cells. We examined the effect of LDL on cholesterol removal by two species of apoA-I-containing lipoproteins, one containing only apoA-I (LpA-I) and the other containing apoA-I and apoA-II (LpA-I/A-II). When foam cells were incubated with LpA-I or LpA-I/A-II, cellular cholesterol mass was reduced. In contrast, when LDL was added, the cholesterol-reducing capacities of these lipoproteins were dose-dependently inhibited by LDL. In the presence of LDL, LpA-I and LpA-I/A-II removed free cholesterol preferentially from LDL rather than from the plasma membrane of foam cells. In addition, a fair amount of cellular cholesterol was directly moved to LDL rather than to LpA-I or LpA-I/A-II. The cellular cholesterol that moved to LDL was completely compensated for by the cholesterol influx from LDL to foam cells. Thus, net cholesterol efflux (a combination of influx and efflux) from foam cells was inhibited by LDL. These results, taken together, indicate that LDL may accelerate foam cell formation by inhibiting cholesterol removal from the cells and that elevated levels of plasma LDL may become a risk factor for atherosclerosis by inhibiting the function of LpA-I and LpA-I/A-II at the cellular level.
BACKGROUND Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2. METHODS AND RESULTS Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3' untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow. CONCLUSIONS Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.
Recent research has shown that adipose tissues contain abundant MSCs (mesenchymal stem cells). The origin and location of the adipose stem cells, however, remain unknown, presenting an obstacle to the further purification and study of these cells. In the present study, we aimed at investigating the origins of adipose stem cells. α-SMA (α-smooth muscle actin) is one of the markers of pericytes. We harvested ASCs (adipose stromal cells) from α-SMA-GFP (green fluorescent protein) transgenic mice and sorted them into GFP-positive and GFP-negative cells by FACS. Multilineage differentiation tests were applied to examine the pluripotent ability of the α-SMA-GFP-positive and -negative cells. Immunofluorescent staining for α-SMA and PDGF-Rβ (platelet-derived growth factor receptor β) were applied to identify the α-SMA-GFP-positive cells. Then α-SMA-GFP-positive cells were loaded on a collagen-fibronectin gel with endothelial cells to test their vascularization ability both in vitro and in vivo. Results show that, in adipose tissue, all of the α-SMA-GFP-positive cells congregate around the blood vessels. Only the α-SMA-GFP-positive cells have multilineage differentiation ability, while the α-SMA-GFP-negative cells can only differentiate in an adipogenic direction. The α-SMA-GFP-positive cells maintained expression of α-SMA during multilineage differentiation. The α-SMA-GFP-positive cells can promote the vascularization of endothelial cells in three-dimensional culture both in vitro and in vivo. We conclude that the adipose stem cells originate from perivascular cells and congregate around blood vessels.
Blood and lymphatic vessels pervade almost all body tissues and have numerous essential roles in physiology and disease. The inner lining of these networks is formed by a single layer of endothelial cells, which is specialized according to the needs of the tissue that it supplies. Whereas the general mechanisms of blood and lymphatic vessel development are being defined with increasing molecular precision, studies of the processes of endothelial specialization remain mostly descriptive. Recent insights from genetic animal models illuminate how endothelial cells interact with each other and with their tissue environment, providing paradigms for vessel type- and organ-specific endothelial differentiation. Delineating these governing principles will be crucial for understanding how tissues develop and maintain, and how their function becomes abnormal in disease.