Mian Chen

A major role for VCAM-1, but not ICAM-1, in early atherosclerosis

VCAM-1 and ICAM-1 are endothelial adhesion molecules of the Ig gene superfamily that may participate in atherogenesis by promoting monocyte accumulation in the arterial intima. Both are expressed in regions predisposed to atherosclerosis and at the periphery of established lesions, while ICAM-1 is also expressed more broadly. To evaluate functions of VCAM-1 in chronic disease, we disrupted its fourth Ig domain, producing the murine Vcam1(D4D) allele. VCAM-1(D4D) mRNA and protein were reduced to 2-8% of wild-type allele (Vcam1(+)) levels but were sufficient to partially rescue the lethal phenotype of VCAM-1-null embryos. After crossing into the LDL receptor-null background, Vcam1(+/+) and Vcam1(D4D/D4D) paired littermates were generated from heterozygous intercrosses and fed a cholesterol-enriched diet for 8 weeks. The area of early atherosclerotic lesions in the aorta, quantified by en face oil red O staining, was reduced significantly in Vcam1(D4D/D4D) mice, although cholesterol levels, lipoprotein profiles, and numbers of circulating leukocytes were comparable to wild-type. In contrast, deficiency of ICAM-1 either alone or in combination with VCAM-1 deficiency did not alter nascent lesion formation. Therefore, although expression of both VCAM-1 and ICAM-1 is upregulated in atherosclerotic lesions, our data indicate that VCAM-1 plays a dominant role in the initiation of atherosclerosis.

Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis

Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule–1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow–derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1–deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.

Resident Intimal Dendritic Cells Accumulate Lipid and Contribute to the Initiation of Atherosclerosis

Rationale: Atherosclerosis is an inflammatory disease in which leukocytes and oxidatively modified lipids accumulate in the arterial intima. Previously, we showed that dendritic cells (DCs) accumulate preferentially in regions predisposed to atherosclerosis in the normal murine aortic intima. The function of these cells in atherogenesis is unknown. Objective: Our goal was to determine the role of resident intimal DCs in the initiation of atherosclerosis. Methods and Results: En face immunostaining of nascent atherosclerotic lesions in low-density lipoprotein receptor–deficient (Ldlr−/−) mice fed a cholesterol-rich diet for 5 or 10 days demonstrated that foam cells expressed DC markers CD11c, 33D1, and major histocompatibility complex class II. Transmission electron microscopy revealed that the majority of intimal lipid was intracellular. The role of resident intimal DCs in lesion formation was verified by their conditional depletion using transgenic mice expressing the simian diphtheria toxin receptor in CD11c+ cells. A single injection of diphtheria toxin depleted intimal CD11c+ DCs by >98% within 24 hours, with 25% and 75% recovery at 1 and 3 weeks, respectively. When bred onto the Ldlr−/− background, intimal DC depletion with diphtheria toxin during 5 days of lesion formation reduced the intimal lipid area by 55% relative to undepleted controls. Transmission electron microscopy revealed few foam cells in DC-depleted mice and abundant accumulation of subendothelial extracellular lipid. Conclusions: Induction of hypercholesterolemia in mice triggers rapid ingestion of lipid by resident intimal DCs, which initiate nascent foam cell lesion formation.

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2′-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6Chigh monocyte recruitment in low density lipoprotein receptor–null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti–GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.

Mechanisms of Thrombin Receptor Agonist Specificity CHIMERIC RECEPTORS AND COMPLEMENTARY MUTATIONS IDENTIFY AN AGONIST RECOGNITION SITE

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn in the Xenopus receptors amino-terminal exodomain and Glu for Leu in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg in the human agonist peptide might normally interact with Glu in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu was converted to Arg. An “arginine scan” of the receptors extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu region and to a lesser degree Phe as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the “correct” agonist and inhibit responsiveness to an “incorrect” agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.

Thrombin Receptor Activating Mutations ALTERATION OF AN EXTRACELLULAR AGONIST RECOGNITION DOMAIN CAUSES CONSTITUTIVE SIGNALING

Constitutively active thrombin receptors were generated while constructing chimeric receptors to identify the structural basis for thrombin receptor agonist specificity. Substitution of eight amino acids from the Xenopus receptors second extracellular loop (XECL2B) for the cognate sequence in the human thrombin receptor was sufficient to confer robust constitutive activity. Smaller substitutions within the XECL2B site yielded less constitutive activation, and substitution of several unrelated sequences at this site caused no activation. Expression of the XECL2B receptor caused high basal Ca efflux in Xenopus oocytes and high basal phosphoinositide hydrolysis and reporter gene induction in COS cells. Of note, a mutant receptor in which all four of the Xenopus thrombin receptors extracellular segments replaced the cognate human sequences showed much less constitutive activity than XECL2B and preserved responsiveness to agonist. This partial complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures suggests that the XECL2B mutation causes constitutive activation by altering interactions among the human receptors extracellular domains. Thus, a change in an extracellular loop of a G protein-coupled receptor can transmit information across the cell membrane to cause signaling, perhaps via a conformational change similar to that caused by agonist binding. Indeed, the site of the activating mutation in XECL2B coincides with a putative agonist-docking site, supporting the hypothesis that agonist interactions with the thrombin receptors extracellular loops contribute to receptor activation.

D-series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury

Recent evidence suggests that specialized lipid mediators derived from polyunsaturated fatty acids control resolution of inflammation, but little is known about resolution pathways in vascular injury. We sought to determine the actions of D‐series resolvin (RvD) on vascular smooth muscle cell (VSMC) phenotype and vascular injury. Human VSMCs were treated with RvD1 and RvD2, and phenotype was assessed by proliferation, migration, monocyte adhesion, superoxide production, and gene expression assays. A rabbit model of arterial angioplasty with local delivery of RvD2 (10 nM vs. vehicle control) was employed to examine effects on vascular injury in vivo. Local generation of proresolving lipid mediators (LC‐MS/MS) and expression of RvD receptors in the vessel wall were assessed. RvD1 and RvD2 produced dose‐dependent inhibition of VSMC proliferation, migration, monocyte adhesion, superoxide production, and proinflammatory gene expression (IC50≈0.1–1 nM). In balloon‐injured rabbit arteries, cell proliferation (51%) and leukocyte recruitment (41%) were reduced at 3 d, and neointimal hyperplasia was attenuated (29%) at 28 d by RvD2. We demonstrate endogenous biosynthesis of proresolving lipid mediators and expression of receptors for RvD1 in the artery wall. RvDs broadly reduce VSMC responses and modulate vascular injury, suggesting that local activation of resolution mechanisms expedites vascular homeostasis.—Miyahara, T., Runge, S., Chatterjee, A., Chen, M., Mottola, G., Fitzgerald, J. M., Serhan, C. N., Conte, M. S. D‐series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury. FASEB J. 27, 2220–2232 (2013). www.fasebj.org

The Thrombin Receptor Second Cytoplasmic Loop Confers Coupling to Gq-like G Proteins in Chimeric Receptors ADDITIONAL EVIDENCE FOR A COMMON TRANSMEMBRANE SIGNALING AND G PROTEIN COUPLING MECHANISM IN G PROTEIN-COUPLED RECEPTORS

Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptors amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled β2-adrenergic receptor (β2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric β2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a β2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and β2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and β2AR despite the thrombin receptors strikingly different liganding mechanism.

Tethered Ligand Library for Discovery of Peptide Agonists

We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptors amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a “tethered ligand library,” which displayed the thrombin receptors amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors.

Discoidin Domain Receptor 1 on Bone Marrow–Derived Cells Promotes Macrophage Accumulation During Atherogenesis

Rationale: We described a critical role for the discoidin domain receptor (DDR)1 collagen receptor tyrosine kinase during atherosclerotic plaque development. Systemic deletion of Ddr1 in Ldlr−/− mice accelerated matrix accumulation and reduced plaque size and macrophage content. However, whether these effects reflected an independent role for macrophage DDR1 during atherogenesis remained unresolved. Methods: In the present study, we performed sex-mismatched bone marrow transplantation using Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice to investigate the role of macrophage DDR1 during atherogenesis. Chimeric mice with deficiency of DDR1 in bone marrow–derived cells (Ddr1−/−→+/+) or control chimeric mice that received Ddr1+/+;Ldlr−/− marrow (Ddr1+/+→+/+) were fed an atherogenic diet for 12 weeks. Results: We observed a 66% reduction in atherosclerosis in the descending aorta and a 44% reduction in plaque area in the aortic sinus in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice. Furthermore, we observed a specific reduction in the number of donor-derived macrophages in Ddr1−/−→+/+ plaques, suggesting that bone marrow deficiency of DDR1 attenuated atherogenesis by limiting macrophage accumulation in the plaque. We have also demonstrated that the effects of DDR1 on macrophage infiltration and accumulation can occur at the earliest stage of atherogenesis, the formation of the fatty streak. Deficiency of DDR1 limited the appearance of 5-bromodeoxyuridine–labeled monocytes/macrophages in the fatty streak and resulted in reduced lesion size in Ldlr−/− mice fed a high fat diet for 2 weeks. In vitro studies to investigate the mechanisms involved revealed that macrophages from Ddr1−/− mice had decreased adhesion to type IV collagen and decreased chemotactic invasion of type IV collagen in response to monocyte chemoattractant protein-1. Conclusions: Taken together, our data support an independent and critical role for DDR1 in macrophage accumulation at early and late stages of atherogenesis.