Xiang-An Li

HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages

Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor-dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor-induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor-induced atherosclerosis. Finally, ritonavir increased PPAR-gamma and CD36 mRNA levels in a PKC- and PPAR-gamma-dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.

High Density Lipoprotein Binding to Scavenger Receptor, Class B, Type I Activates Endothelial Nitric-oxide Synthase in a Ceramide-dependent Manner

Recently it has been demonstrated that high density lipoprotein (HDL) binding to scavenger receptors, class B, type I (SR-BI) stimulates endothelial nitric-oxide synthase (eNOS) activity. In the present studies we used a Chinese hamster ovary cell system and a human microvascular endothelial cell line to confirm that HDL stimulates eNOS activity in a SR-BI-dependent manner. Importantly, we have extended these studies to examine the mechanism whereby HDL binding to SR-BI stimulates eNOS. eNOS can be stimulated by an increase in intracellular calcium, by phosphorylation by Akt kinase, or by an increase in intracellular ceramide. Calcium imagining studies and experiments with the calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester demonstrated that HDL binding to SR-BI does not induce an increase in intracellular calcium. Antibodies specific for activated Akt kinase demonstrated that HDL binding to SR-BI does not induce Akt kinase activation. However, HDL binding to SR-BI caused a reversible increase in intracellular ceramide levels from 97 ± 14 pmol/mg of protein to 501 ± 21 pmol/mg of protein. In addition, C2-ceramide stimulated eNOS to the same extent as HDL, whereas C2-dihydroceramide did not stimulate eNOS. We conclude that HDL binding to SR-BI stimulates eNOS by increasing intracellular ceramide levels and is independent of an increase in intracellular calcium or Akt kinase phosphorylation.

HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI–dependent manner

Cardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sex - specifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.

The role of caveolae and caveolin in vesicle-dependent and vesicle-independent trafficking

Caveolae can mediate endocytosis, transcytosis, and potocytosis. Our understanding of these processes as well as the elucidation of the molecular machinery involved has greatly expanded. In addition, caveolin, a 22 kDa protein often associated with caveolae, can promote the trafficking of sterol through the cytoplasm independent of vesicles. Caveolin also influences the formation, morphology, and function of caveolae. The ability of caveolae and caveolin to mediate macromolecular transport directly impacts a variety of physiological and pathophysiological processes.

Immunohistochemical localization of C-reactive protein-binding sites in human atherosclerotic aortic lesions by a modified streptavidin-biotin-staining method.

One‐step fluorescein‐conjugated polyclonal antibody technique has shown that C‐reactive protein (CRP) was located only extracellularly in human atherosclerotic lesions. In this report a more sensitive streptavidin‐biotin technique was applied to detect the localization of CRP in human athere sclerotic lesions. lmmunohistochemical staining with polyclonal and monoclonal anti‐human CRP antibodies both produced a brown color extracellularly in the necrotic lesions, and intracelluarly in CD68+ foam cells. The latter suggests an uptake of CRP‐lipid complexes by macro‐phages. The staining is human CRP‐specific because it was eliminated by preabsorption of the monoclonal antibody with pure human CRP, or by substitution of the primary antibody with non‐immune rabbit serum. By overlaid CRP‐binding study, a positive stain was observed on intimal smooth muscle cells and foam cells, suggesting that they have CRP‐binding sites unless the CRP‐binding activity was generated de novo through the fixation procedure. Accordingly, it is hypothesized that CRP may facilitate the uptake of lipids by macrophages accumulating in atherosclerotic lesions. Further, CRP might participate in cytolysis, which enlarges the necrotic area, and/or in phagocytosis that scavenges the necrotic tissue.

Salt Inactivates Endothelial Nitric Oxide Synthase in Endothelial Cells

There is a 1-4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (from 137 to 142 mmol/L) caused a 25% decrease in NOS activity. Importantly, the decrease in NOS activity was in a salt concentration-dependent manner. The NOS activity was decreased by 25, 45, and 70%, with the increase of 5, 10, and 20 mmol/L of NaCl, respectively. Using Chinese hamster ovary cells stably expressing eNOS, we confirmed the inhibitory effects of salt on eNOS activity. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excludes an osmotic effect. Using an ex vivo aortic angiogenesis assay, we demonstrated that salt attenuated the nitric oxide (NO)-dependent proliferation of endothelial cells. By directly monitoring blood pressure changes in response to salt infusion, we found that in vivo infusion of salt induced an acute increase in blood pressure in a salt concentration-dependent manner. In conclusion, our findings demonstrated that eNOS is sensitive to changes in salt concentration. A 5-mmol/L rise in salt concentration, within the range observed in essential hypertension patients or in individuals with high salt intake, could significantly suppress eNOS activity. This salt-induced reduction in NO generation in endothelial cells may contribute to the development of hypertension.

Platelets Protect from Septic Shock by Inhibiting Macrophage-Dependent Inflammation via the Cyclooxygenase 1 Signalling Pathway

Although it has long been known that patients with sepsis often have thrombocytopenia and that septic patients with severe thrombocytopenia have a poor prognosis and higher mortality, the role of platelets in the pathogenesis of sepsis is poorly understood. Here we report a protective role of platelets in septic shock. We show that experimental thrombocytopenia induced by intraperitoneal injection of an anti-glycoprotein Ibα monoclonal antibody increases mortality and aggravates organ failure, whereas transfusion of platelets reduces mortality in lipopolysaccharide-induced endotoxemia and a bacterial infusion mouse sepsis model. Plasma concentrations of proinflammatory cytokines TNF-α and IL-6 are elevated by thrombocytopenia and decreased by platelet transfusion in septic mice. Furthermore, we identify that platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the COX1/PGE₂/EP4-dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating severely septic patients.

ABANDON THE MOUSE RESEARCH SHIP? NOT JUST YET!

ABSTRACT Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiologic alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently, the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without overinterpretation. Hopefully, a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.

Scavenger Receptor BI Protects against Septic Death through Its Role in Modulating Inflammatory Response

Sepsis is a leading cause of death that is characterized by uncontrolled inflammatory response. In this study, we report that scavenger receptor BI (SR-BI), a high density lipoprotein receptor, is a critical survival factor of sepsis. We induced sepsis using an established septic animal model, cecal ligation and puncture (CLP). CLP induced 100% fatality in SR-BI-null mice but only 21% fatality in wild type littermates. SR-BI-null mice exhibited aberrant inflammatory responses with delayed inflammatory cytokine generation at the early stage of sepsis and highly elevated inflammatory cytokine production 20 h after CLP treatment. To understand the mechanisms underlying SR-BI protection, we elucidated the effect of mac ro phage SR-BI on inflammatory cytokine generation. Macrophages from SR-BI-null mice produced significantly higher levels of inflammatory cytokines than those of wild type controls in response to LPS. Importantly, transgenic mice overexpressing SR-BI were more resistant to CLP-induced septic death. Using an HEK-BlueTM cell system, we demonstrated that expression of SR-BI suppressed TLR4-mediated NF-κB activation. To understand why SR-BI-null mice had a delayed inflammatory response, we elucidated the effect of SR-BI on LPS clearance during sepsis. Compared with wild type controls, SR-BI-null mice had lower plasma LPS levels in the early stage of sepsis and elevated plasma LPS levels 20 h following CLP treatment. In conclusion, our findings demonstrate that SR-BI is a critical protective modulator of sepsis in mice. SR-BI exerts its protective function through its role in modulating inflammatory response in mac ro phages and facilitating LPS recruitment and clearance.

A Novel Ligand-independent Apoptotic Pathway Induced by Scavenger Receptor Class B, Type I and Suppressed by Endothelial Nitric-oxide Synthase and High Density Lipoprotein

Scavenger receptor class B, type I (SR-BI)/ApoE double null mice develop severe atherosclerosis within 4 weeks, whereas ApoE null mice take several months to develop the disease, indicating that SR-BI plays a pivotal role in atherosclerosis. Importantly, SR-BI/ApoE double null mice have lower plasma cholesterol levels than ApoE null mice, suggesting involvement of a non-lipids mechanism. In the present study, we revealed a novel ligand-independent apoptotic pathway induced by SR-BI, and regulated by endothelial nitric-oxide synthase (eNOS) and high density lipoprotein (HDL). SR-BI significantly induces apoptosis in three independent cell systems. In contrast to known ligand-dependent apoptotic pathways, SR-BI-induced apoptosis is ligand-independent. We further showed that SR-BI-induced apoptosis is suppressed by eNOS and HDL. By using a single site mutation, we demonstrated that SR-BI induces apoptosis through a highly conserved CXXS redox motif. We finally demonstrated that SR-BI-induced apoptosis is via the caspase-8 pathway. We hypothesize that in healthy cells, the SR-BI apoptotic pathway is turned off by eNOS and HDL which prevents inappropriate apoptotic damage to the vascular wall. When HDL levels are low, oxidative stress causes the relocation of eNOS away from caveolae, which turns on SR-BI-induced apoptosis and rapidly clears damaged cells to prevent further inflammatory damage to neighboring cells. The current studies offer a new paradigm in which to study the non-cholesterol effects of SR-BI, HDL, and eNOS on the development of atherosclerosis and potentially other cardiovascular diseases.