The binding of HDL to scavenger receptor–BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-β-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-β-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-β-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.
Chatchawin Assanasen, Chieko Mineo, Divya Seetharam, Ivan S. Yuhanna, Yves L. Marcel, Margery A. Connelly, David L. Williams, Margarita de la Llera-Moya, Philip W. Shaul, David L. Silver
Platelet activation is a hallmark of severe preeclampsia, and platelet PGH synthase 1–derived (PGHS1-derived) thromboxane A2 (TxA2) has been implicated in its pathogenesis. However, genetic disruption of PGHS1 delays parturition. We created hypomorphic PGHS1 (PGHS1Neo/Neo) mice, in which the substantial but tissue-dependent variability in the inhibition of PGHS1-derived eicosanoids achieved by low-dose aspirin treatment is mimicked, to assess the relative impact of this strategy on hemostatic and reproductive function. Depression of platelet TxA2 by 98% in PGHS1Neo/Neo mice decreased platelet aggregation and prevented thrombosis. Similarly, depression of macrophage PGE2 by 75% was associated with selectively impaired inflammatory responses. PGF2α at 8% WT levels was sufficient to induce coordinated temporal oxytocin receptor (OTR) expression in uterus and normal ovarian luteolysis in PGHS1Neo/Neo mice at late gestation, while absence of PGHS1 expression in null mice delayed OTR induction and the programmed decrease of serum progesterone during parturition. Thus, extensive but tissue-dependent variability in PG suppression, as occurs with low-dose aspirin treatment, prevents thrombosis and impairs the inflammatory response but sustains parturition. PGHS1Neo/Neo mice provide a model of low-dose aspirin therapy that elucidates how prevention or delay of preeclampsia might be achieved without compromising reproductive function.
Ying Yu, Yan Cheng, Jinjin Fan, Xin-Sheng Chen, Andres Klein-Szanto, Garret A. FitzGerald, Colin D. Funk
MMPs are implicated in LV remodeling after acute myocardial infarction (MI). To analyze the role of MMP-2, we generated MI by ligating the left coronary artery of MMP-2–KO and WT mice, the latter of which were administered orally an MMP-2–selective inhibitor or vehicle (TISAM). The survival rate was significantly higher in MMP-2–KO and TISAM-treated mice than in control WT mice. The main cause of mortality in control WT mice was cardiac rupture, which was not observed in MMP-2–KO or TISAM-treated mice. Control WT mice, but not MMP-2–KO or TISAM-treated mice, showed activation of the zymogen of MMP-2, strong gelatinolytic activity, and degradation of ECM components, including laminin and fibronectin, in the infarcted myocardium. Although infarcted cardiomyocytes in control WT mice were rapidly removed by macrophages, the removal was suppressed in MMP-2–KO and TISAM-treated mice. Macrophage migration was induced by the infarcted myocardial tissue from control WT mice and was inhibited by treatment of macrophages with laminin or fibronectin peptides prior to migration assay. These data suggest that inhibition of MMP-2 activity improves the survival rate after acute MI by preventing cardiac rupture and delays post-MI remodeling through a reduction in macrophage infiltration.
Shin-ichiro Matsumura, Shiro Iwanaga, Satsuki Mochizuki, Hiroyuki Okamoto, Satoshi Ogawa, Yasunori Okada
Regions in the vasculature that are exposed to steady laminar blood flow are protected from atherosclerosis as compared with regions where flow is disturbed. We found that flow decreased TNF-mediated VCAM1 expression by inhibiting JNK and p38. JNK inhibition correlated with inhibition of apoptosis signal–regulating kinase 1 (ASK1), a JNK and p38 activator. Thioredoxin-interacting protein (TXNIP) is a stress-responsive protein that inhibits thioredoxin (TRX) activity. Since thioredoxin inhibits ASK1, we hypothesized that changes in TXNIP-TRX-ASK1 interactions mediate the antiinflammatory effects of flow. To explore this, we used perfused vessels and cultured ECs. Exposure of rabbit aortae or ECs to normal flow (12 dyn/cm2, 24 hours) was associated with decreased TXNIP expression and increased TRX activity compared with exposure to low flow (0.4 dyn/cm2). Normal flow inhibited TNF activation of JNK/p38 and VCAM1 expression. In cultured ECs, reduction of TXNIP expression by small interfering RNA increased TRX binding to ASK1 and inhibited TNF activation of JNK/p38 and VCAM1 expression. Conversely, overexpression of TXNIP stimulated JNK and p38. In aortae from TXNIP-deficient mice, TNF-induced VCAM1 expression was inhibited. The data suggest that TXNIP and TRX are key components of biomechanical signal transduction and establish them as potentially novel regulators of TNF signaling and inflammation in ECs.
Hideyuki Yamawaki, Shi Pan, Richard T. Lee, Bradford C. Berk
Tissue kallikrein (TK), the major kinin-forming enzyme, is synthesized in several organs, including the kidney and arteries. A loss-of-function polymorphism of the human TK gene (R53H) induces a substantial decrease in enzyme activity. As inactivation of the TK gene in the mouse induces endothelial dysfunction, we investigated the vascular, hormonal, and renal phenotypes of carriers of the 53H allele. In a crossover study, 30 R53R-homozygous and 10 R53H-heterozygous young normotensive white males were randomly assigned to receive both a low sodium–high potassium diet to stimulate TK synthesis and a high sodium–low potassium diet to suppress TK synthesis, each for 1 week. Urinary kallikrein activity was 50–60% lower in R53H subjects than in R53R subjects. Acute flow-dependent vasodilatation and endothelium-independent vasodilatation of the brachial artery were both unaffected in R53H subjects. In contrast, R53H subjects consistently exhibited an increase in wall shear stress and a paradoxical reduction in artery diameter and lumen compared with R53R subjects. Renal and hormonal adaptation to diets was unaffected in R53H subjects. The partial genetic deficiency in TK activity is associated with an inward remodeling of the brachial artery, which is not adapted to a chronic increase in wall shear stress, indicating a new form of arterial dysfunction affecting 5–7% of white people.
Michel Azizi, Pierre Boutouyrie, Alvine Bissery, Mohsen Agharazii, Francis Verbeke, Nora Stern, Alessandra Bura-Rivière, Stéphane Laurent, François Alhenc-Gelas, Xavier Jeunemaitre
We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.
Young-sup Yoon, Andrea Wecker, Lindsay Heyd, Jong-Seon Park, Tengiz Tkebuchava, Kengo Kusano, Allison Hanley, Heather Scadova, Gangjian Qin, Dong-Hyun Cha, Kirby L. Johnson, Ryuichi Aikawa, Takayuki Asahara, Douglas W. Losordo
Advanced congestive heart failure is associated with activation of the renin-angiotensin system and skeletal muscle wasting. We previously showed that angiotensin II infusion in rats produces cachexia secondarily to increased muscle proteolysis and also decreases levels of circulating and skeletal muscle IGF-1. Here we show that angiotensin II markedly downregulates phospho-Akt and activates caspase-3 in skeletal muscle, leading to actin cleavage, an important component of muscle proteolysis, and to increased apoptosis. These changes are blocked by muscle-specific expression of IGF-1, likely via the Akt/mTOR/p70S6K signaling pathway. We also demonstrate that mRNA levels of the ubiquitin ligases atrogin-1 and muscle ring finger–1 are upregulated in angiotensin II–infused WT, but not in IGF-1–transgenic, mice. These findings strongly suggest that angiotensin II downregulation of IGF-1 in skeletal muscle is causally related to angiotensin II–induced wasting. Because the renin-angiotensin system is activated in many catabolic conditions, our findings have broad implications for understanding mechanisms of skeletal muscle wasting and provide a rationale for new therapeutic approaches.
Yao-Hua Song, Yangxin Li, Jie Du, William E. Mitch, Nadia Rosenthal, Patrick Delafontaine
The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.
Xuri Li, Marc Tjwa, Lieve Moons, Pierre Fons, Agnes Noel, Annelii Ny, Jian Min Zhou, Johan Lennartsson, Hong Li, Aernout Luttun, Annica Pontén, Laetitia Devy, Ann Bouché, Hideyasu Oh, Ann Manderveld, Silvia Blacher, David Communi, Pierre Savi, Françoise Bono, Mieke Dewerchin, Jean-Michel Foidart, Monica Autiero, Jean-Marc Herbert, Désiré Collen, Carl-Henrik Heldin, Ulf Eriksson, Peter Carmeliet
Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.
Patrick Most, Sven T. Pleger, Mirko Völkers, Beatrix Heidt, Melanie Boerries, Dieter Weichenhan, Eva Löffler, Paul M.L. Janssen, Andrea D. Eckhart, Jeffrey Martini, Matthew L. Williams, Hugo A. Katus, Andrew Remppis, Walter J. Koch
PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.
Andrew C. Li, Christoph J. Binder, Alejandra Gutierrez, Kathleen K. Brown, Christine R. Plotkin, Jennifer W. Pattison, Annabel F. Valledor, Roger A. Davis, Timothy M. Willson, Joseph L. Witztum, Wulf Palinski, Christopher K. Glass