Leigh Brian

Graft-Extrinsic Cells Predominate in Vein Graft Arterialization

Objective—Vein graft disease involves neointimal smooth muscle cells, the origins of which are unclear. This study sought to characterize and quantitate vein graft infiltration by cells extrinsic to the graft in a mouse model of vein graft disease. Methods and Results—Inferior vena cava-to-carotid artery interposition grafting between C57Bl/6 and congenic &bgr;-galactosidase–expressing ROSA26 mice was performed. Vein grafts were harvested 6 weeks postoperatively and stained with X-gal. More than 60% of neointimal cells derived from the recipient, and 50% of these cells expressed smooth muscle &agr;-actin. The distribution of donor and recipient-derived cells within this vein graft wall layer was distinctly focal, consistent with focal infiltration and expansion of progenitor cells. When bone marrow transplantation with congenic green fluorescent protein (GFP)-expressing cells was used in vein graft recipients 1 month before surgery, abundant GFP-expressing cells appeared in the media, but not the neointima, of mature grafts. Endothelial cells in mature grafts derived from graft-intrinsic and graft-extrinsic sources and were, in part, of bone marrow origin. Conclusions—Cells extrinsic to the graft, including bone marrow-derived cells, predominate during vein graft remodeling.

Expression of Tumor Necrosis Factor Receptor-1 in Arterial Wall Cells Promotes Atherosclerosis

Objective—Mechanisms by which tumor necrosis factor-α (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis. Methods and Results—Carotid artery-to-carotid artery interposition grafting was performed with tnfr1−/− and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1−/− grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1−/− grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe−/−/tnfr1−/− mice, as compared with cognate apoe−/− mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity. Conclusions—TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

Phosphorylation of the Platelet-derived Growth Factor Receptor-β and Epidermal Growth Factor Receptor by G Protein-coupled Receptor Kinase-2 MECHANISMS FOR SELECTIVITY OF DESENSITIZATION

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-β (PDGFRβ) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRβ, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRβ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRβ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRβ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRβ mutant, GRK2-mediated PDGFRβ phosphorylation required receptor tyrosine kinase activity, as does PDGFRβ ubiquitination. Agonist-induced ubiquitination of the PDGFRβ, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRβ down-regulation. Like the vast majority of GRK2 substrates, the PDGFRβ, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRβ, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.

Vein Graft Neointimal Hyperplasia Is Exacerbated by Tumor Necrosis Factor Receptor-1 Signaling in Graft-Intrinsic Cells

Objective—Vein graft remodeling and neointimal hyperplasia involve inflammation, graft-intrinsic cells, and recruitment of vascular progenitor cells. We sought to examine if the inflammatory cytokine tumor necrosis factor (TNF) affects vein graft remodeling via its p55 TNF receptor-1 (p55). Methods and Results—Inferior vena cava-to-carotid artery interposition grafting was performed between p55−/− and congenic (C57Bl/6) wild-type (WT) mice. Immunofluorescence revealed TNF in early (2-week) vein grafts. Six weeks postoperatively, luminal and medial areas were indistinguishable among all vein graft groups. However, neointimal area was reduced in p55−/− grafts: by 40% in p55−/− grafts placed in p55−/− recipients, and by 21% in p55−/− grafts placed in WT recipients, compared with WT grafts in WT recipients (P<0.05). In 2-week-old vein grafts, p55 deficiency reduced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 expression by 50% to 60%, and increased the extent of graft endothelialization. In vitro, TNF promoted chemokine expression and [3H]thymidine incorporation in vascular smooth muscle cells (SMCs) from WT, but not from p55−/− mice. However, responses of WT and p55−/− SMCs to other growth factors were equivalent. Conclusions—Signaling via p55, in vein graft-intrinsic cells, contributes to the pathogenesis of vein graft neointimal hyperplasia.

Kruppel-like factor 15 is critical for vascular inflammation

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.

Regulation of the Platelet-derived Growth Factor Receptor-β by G Protein-coupled Receptor Kinase-5 in Vascular Smooth Muscle Cells Involves the Phosphatase Shp2

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-β (PDGFRβ), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRβ activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRβ were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRβ seryl phosphorylation by 3-fold and reduced PDGFRβ-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRβ tyrosyl phosphorylation by ∼35%. Physiologic SMC GRK5 activity also increased PDGFRβ association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRβ Tyr1009 (the docking site for Shp2), and reduced phosphorylation of PDGFRβ Tyr1021. Consistent with having increased PDGFRβ-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRβ inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRβ/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRβ/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRβs. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRβ in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRβ inositol phosphate signaling and enhances PDGFRβ-triggered Src activation.

Human Umbilical Cord Blood–Derived Endothelial Cells Reendothelialize Vein Grafts and Prevent Thrombosis

Objective—To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. Methods and Results—Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface &agr;5&bgr;1 integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdcscid/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. Conclusion—The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.

Tumor Necrosis Factor Receptor-2 Signaling Attenuates Vein Graft Neointima Formation by Promoting Endothelial Recovery

Objective—Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization. Methods and Results—Inferior vena cava-to-carotid artery interposition grafting was performed between p75−/− and congenic (C57B1/6J) wild-type (WT) mice. Six weeks postoperatively, neointimal and medial dimensions were greater in p75−/− grafts placed into p75−/− recipients (by 42% or 60%, respectively; P<0.05), when compared with WT veins grafted into WT recipients. Relative to WT vein grafts, p75 deficiency augmented early (2-week-old) graft vascular cell adhesion molecule (VCAM)-1 expression (by 2.4-fold, P<0.05), increased endothelial cell apoptosis (2-fold), and delayed graft re-endothelialization. Both cellular proliferation in early, and collagen I content of mature (6-week-old) vein grafts were increased (by 70% and 50%, respectively) in p75−/− grafts. P75 deficiency augmented TNF-induced apoptosis of cultured endothelial cells, but did not affect TNF-stimulated SMC proliferation or migration induced by co-cultured macrophages. Conclusions—TNF signaling via p75 reduces vein graft neointimal hyperplasia through mechanisms involving reduction of adhesion molecule expression and endothelial cell apoptosis.

G Protein–Coupled Receptor Kinase-5 Attenuates Atherosclerosis by Regulating Receptor Tyrosine Kinases and 7-Transmembrane Receptors

Objective—G protein–coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-&kgr;B (NF-&kgr;B). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. Methods and Results—Grk5−/−/Apoe−/− mice developed 50% greater aortic atherosclerosis than Apoe−/− mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe−/− mice, carotid interposition grafts from Grk5−/− mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5−/− with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-&bgr; in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-&kgr;B-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-&kgr;B activity in macrophages. Conclusion—GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-&kgr;B activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-&bgr;.

Kalirin Promotes Neointimal Hyperplasia by Activating Rac in Smooth Muscle Cells

Objective—Kalirin is a multifunctional protein that contains 2 guanine nucleotide exchange factor domains for the GTPases Rac1 and RhoA. Variants of KALRN have been associated with atherosclerosis in humans, but Kalirin’s activity has been characterized almost exclusively in the central nervous system. We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs). Approach and Results—Kalirin-9 protein is expressed abundantly in aorta and bone marrow, as well as in cultured SMCs, endothelial cells, and macrophages. Moreover, arterial Kalirin was upregulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in 3 models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin Rho-guanine nucleotide exchange factor -1 inhibitor 1-(3-nitrophenyl)-1H-pyrrole-2,5-dione. With reduced Kalirin function, SMCs showed normal RhoA activation but diminished Rac1 activation, assessed as reduced Rac-GTP levels, p21-activated kinase autophosphorylation, and SMC migration. Kalrn–/+ SMCs proliferated 30% less rapidly than wild-type SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ≈60% less in Kalrn–/+ and SMC-specific Kalrn–/+ mice than in control mice. Conclusion—Kalirin functions as a guanine nucleotide exchange factor for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.