Barbara M. Schreiber

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

The A2b adenosine receptor protects against vascular injury

The A2b adenosine receptor (A2bAR) is highly abundant in bone marrow macrophages and vascular smooth muscle cells (VSMC). To examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis after angioplasty. While considering related mechanisms, we noted higher levels of TNF-α, an up-regulator of CXCR4, and of VSMC proliferation in the injured KO mice. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is up-regulated in lesions of the KO mice. In addition, aortic smooth muscle cells derived from A2bAR KO mice display greater proliferation in comparison with controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study highlights the significance of the A2bAR in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation.

Apolipoprotein E Is Synthesized in the Retina by Müller Glial Cells, Secreted into the Vitreous, and Rapidly Transported into the Optic Nerve by Retinal Ganglion Cells

We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [S]methionine/cysteine into the vitreous, 44% of [S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [S]Met/Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Müller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Müller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics.

Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles

We have shown that apolipoprotein E (ApoE) is synthesized by Muller cells, the major glial cell of the rabbit retina, and secreted into the vitreous after which it is taken up by retinal ganglion cells and rapidly transported into the optic nerve [Amaratunga et al., J. Biol. Chem. 271 (1996) 5628-5632]. In this report we demonstrate that the ApoE secreted by Muller cells in vivo and in culture is efficiently assembled into lipoprotein particles. Apolipoprotein J (ApoJ) is also synthesized by these cells and assembled into lipoprotein particles. The lipoproteins are triglyceride-rich and contain cholesterol esters and free cholesterol. They are heterogeneous, with densities between 1.006 and 1.18 and diameters between 14 and 45 nm. We discuss the possible role of these lipoproteins in supplying the needs of neurons for lipids, especially long axonal projection neurons such as retinal ganglion cells, which are vulnerable to age-related neurodegenerative diseases including Alzheimers disease.

Absence of adipocyte fatty acid binding protein prevents the development of accelerated atherosclerosis in hypercholesterolemic mice

Lipid deposition in arterial walls due to elevated levels of plasma cholesterol is central to the development of atherosclerosis and involves the uptake of oxidized low‐density lipoprotein (oxLDL) by macrophages. Fatty acid binding proteins (FABPs) belong to a family of low molecular weight cytoplasmic proteins that are involved with intracellular transport and metabolism of fatty acids, and adipocyte FABP (aP2) has recently been shown to be expressed in macrophages. Here, we investigate the role of aP2 in the development of atherosclerosis in mice. We show that atherosclerotic lesions from hypercholesterolemic, apolipoprotein E deficient (ApoE−/−) mice (but not arterial walls from normal mice) contain high levels of aP2 mRNA. We also identified aP2 in inflammatory cells that localized in these lesions, as confirmed by its presence in isolated mouse and human macrophages, and demonstrated that aP2 is induced by oxLDL. To determine the importance of aP2 in atherosclerosis, we generated mice lacking both ApoE and aP2 (ApoE−/−aP2−/−). In comparison with ApoE−/− mice, ApoE−/−aP2−/− mice developed trivial lesions that were markedly smaller, less complex, and less macrophage‐rich even though the ApoE−/−aP2−/− mice remained hypercholesterolemic. Absence of aP2 did not prevent lesion formation and macrophage accumulation in transplant‐associated arteriosclerosis that does not depend on elevated levels of cholesterol. These results indicate a critical role for aP2 in the development of hypercholesterolemia‐induced atherosclerosis.

Role of prostaglandin E2 EP receptors and cAMP in the expression of connective tissue growth factor

Abstract Wild-type (WT) Rat-1 fibroblasts express undetectable quantities of the prostaglandin E2 (PGE2) EP1, EP2, and EP3 receptor types and detectable amounts of the EP4 receptor. In the WT Rat-1, PGE2 enhances connective tissue growth factor (CTGF) mRNA. PGE2 does not stimulate cAMP production in these cells. However, forskolin induces cAMP production and ablates TGFβ-stimulated increases in CTGF mRNA. A similar pattern of CTGF expression in response to PGE2 and forskolin is observed in neonatal rat primary smooth muscle cell cultures. When WT Rat-1 cells are stably transfected with the EP2 receptor, PGE2 causes a sizable elevation in intracellular cAMP and ablates the TGFβ-stimulated increase in CTGF mRNA. PGE2 does not have this effect on cells expressing the EP1, EP3, or EP4 receptor subtypes. These results demonstrate the importance of the EP2 receptor and cAMP in the inhibition of TGFβ-stimulated CTGF production and suggest a role for PGE2 in increasing CTGF mRNA levels in the absence of the EP2 receptor. Involvement of inositol phosphate in this upregulation of CTGF expression by PGE2 is doubtful. None of the cell lines containing the four EP transfectants nor the WT Rat-1 cells responded to PGE2 with inositol phosphate production.

Serum amyloid A in Alzheimer's disease brain is predominantly localized to myelin sheaths and axonal membrane*

Immunohistochemical localization of the injury specific apolipoprotein, acute phase serum amyloid A (A-apoSAA), was compared in brains of patients with neuropathologically confirmed Alzheimers disease (AD), multiple sclerosis (MS), Parkinsons disease (PD), Picks disease (Picks), dementia with Lewy bodies (DLB), coronary artery disease (CAD), and schizophrenia. Affected regions of both AD and MS brains showed intense staining for A-apoSAA in comparison to an unaffected region and non-AD/MS brains. The major site of A-apoSAA staining in both diseases was the myelin sheaths of axons in layers V and VI of affected cortex. A-apoSAA contains a cholesterol binding site near its amino terminus and is likely to have a high affinity for cholesterol-rich myelin. These findings, along with our recent evidence that A-apoSAA can inhibit lipid synthesis in vascular smooth muscle cells suggest that A-apoSAA plays a role in the neuronal loss and white matter damage occurring in AD and MS.

A3 adenosine receptor deficiency does not influence atherogenesis

Atherosclerosis is a multifactorial disease, the progression of which is modulated by several factors, including inflammation and hypercholesterolemia. The A3 adenosine receptor (A3AR) has been reported to affect mast cell degranulation leading to inflammation, as well as to influence cardiovascular homeostasis. Here, we show that its deletion can also impact vascular smooth muscle cell (VSMC) proliferation in vitro. Based on these observations, we hypothesized that A3AR deficiency would affect atheromatous lesion development in vivo. Our results indicate that the expression of the matrix enzyme lysyl oxidase (LO) is increased while the proliferation potential of VSMC is decreased in A3AR‐null aortas. This is in accordance with the previously reported inverse correlation between LO level and proliferation. Nevertheless, we found that A3‐deficiency does not protect vessels against atherogenesis. This was demonstrated in mouse models of high fat diet‐induced atherosclerosis and guidewire‐induced femoral artery injury. We conclude that the contributions of the A3AR to inflammation and to modulating LO levels are not significant enough to control vascular response to injury.

The role of the carboxy terminus of tropoelastin in its assembly into the elastic fiber.

Tropoelastin, the soluble precursor protein of insoluble amorphous elastin, contains repeating segments that are important for the characteristic elasticity and crosslinking sites of mature elastin. In addition, there is a unique carboxy terminal domain that is encoded by exon 36 of the elastin gene, and it has been suggested that this region may play a role in the process of insolubilization. The contribution of exon 36 to the maturation of tropoelastin into insoluble elastin was probed in these studies. Neonatal rat aortic smooth muscle cells were cultured and the fate of [3H] Lys labeled human recombinant tropoelastin (hrTE) molecules added to the cultures was monitored. In comparison to the hrTE containing the region encoded by exon 36, hrTE molecules lacking this domain were less efficiently incorporated into elastin, as evidenced by a decrease in NaOH insoluble radioactivity. Specific residues within the domain encoded by exon 36 were targeted for further study in experiments in which the two Cys residues were reduced and alkylated, and/or the four basic Arg-Lys-Arg-Lys residues at the carboxy terminus were removed. Both of these modifications resulted in decreased incorporation into elastin equivalent to the complete removal of the carboxy terminus. Prior treatment of the cell layer with elastase reduced the efficiency of insolubilization of hrTE containing the domain encoded by exon 36, but had no effect on the processing of molecules lacking this region. These data suggest that exon 36 of the elastin gene contributes to normal efficient incorporation of tropoelastin into the elastin fiber.

Apolipoprotein serum amyloid A down-regulates smooth-muscle cell lipid biosynthesis

The addition of acute-phase apolipoprotein serum amyloid A (SAA) to cultured aortic smooth-muscle cells caused a decrease in the incorporation of [(14)C]acetate into lipids. Optimal inhibition of lipid biosynthesis was achieved with 2 microM SAA, and the effect was maintained for up to 1 week when SAA was included in the culture medium. Lipid extracts were subjected to TLC and it was determined that the SAA-induced decrease in [(14)C]acetate incorporation into lipids was attributable to decreases in cholesterol, phospholipid and triglyceride levels. The accumulated mass of cholesterol and phospholipid in SAA-treated cultures was significantly less than that of controls, with no change in the accumulated protein. Moreover, SAA had no effect on either protein synthesis or DNA synthesis, suggesting that SAA specifically alters lipid synthesis. By using a peptide corresponding to the cholesterol-binding domain of acute-phase SAA (amino acids 1-18), it was shown that this region of the molecule was as effective as the full-length protein in decreasing lipid synthesis and the accumulation of cholesterol and phospholipid. The implications of these findings for atherosclerosis and Alzheimers disease are discussed.