729-2 Macrophage-Monocyte Invasion is Associated with Greater Cardiac Hypertrophy in Hypertensive Rats

Cardiac hypertrophy is an adaptive response to increased wall stress in hypertension. Although the mechanisms underlying this adaptive response are not clearly understood, a genetic predisposition appears to playa role. We sought to examine the hypothesis that an inflammatory response occurs in the heart early in the course of development of hypertension. We measured blood pressure, heart weight and cardiac macrophage-monocyte invasion in 6 week old SHR and WKY rats with renal hypertension (1 Kidney-1 Clip, 1K1C) or sham-operated controls (SHAM). Macrocyte-monophage invasion was measured as the extent of immunohistochemical staining with ED1, a cytoplasmic antigen in this cell type. Measurements were made at 7 and 21 days post procedure. Renal hypertensive rats, both SHR and WKY, had higher blood pressures at both time points than SHR sham, which in turn had higher blood pressures that WKY sham rats. Heart weight/body weight ratios were highest in SHR-1 K1 C rats, followed by SHR SHAM and WKY 1K1C, and least in WKY sham rats. ED1 staining was also highest in SHR 1K-lC rats, followed by SHR sham rats, and was considerably lower in WKY rats, 1K-1C or sham. Data atthe 21 day time point are shown below:BP (mmHg)Heart wt/body wt (mg/g)ED1 (counts/HPF)SHR 1K1C2054.320.0SHR SHAM1703.415.0WKY 1K1C2033.712.5WKY SHAM1432.812.5Thus, significant monocyte-macrophage invasion is a hitherto unrecognized feature of cardiac hypertrophy in SHR rats. We speculate that growth factors released by such cells may contribute to hypertensive cardiac hypertrophy

Vascular types I and II transforming growth factor-beta receptor expression: differential dependency on tyrosine kinases during induction by TGF-β

AbstractRecent evidence indicates that the type II transforming growth factor-β (TGF-β) receptor (TβRII) is a serine-threonine-tyrosine kinase. However, the significance of its tyrosine kinase is unclear. We investigated in vascular smooth muscle cells the effects of tyrosine kinase inhibition on the expression of TGF-β receptor types I (ALK-5) and II (TβRII) mRNA, induced by TGF-β1. TGF-β1 elevated ALK-5 mRNA levels 5-fold; essentially similar TGF-β1-dependent elevations were observed with growth factors, PDGF-BB and FGF-2. The tyrosine kinase inhibitor genistein abolished these TGF-β1 and growth factor responses. TGF-β1 also elevated TβRII mRNA levels which were not inhibited by genistein. We conclude that tyrosine kinases participate in defining how cells respond to TGF-β

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension

IntroductionDepletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension.MethodsMale C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 μg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220−), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay.ResultsBortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10–11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9–11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9–11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9–11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9–11).ConclusionReductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension

Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein: evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the Gα(i2), Gα(i3), Gα(s), Gβ, and Gβ protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca mobilization, we investigated the effect of PT on Ca responses using fluorescence spectroscopy and the Ca indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 μg/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca response was inhibited by approximately 72%, suggesting that the major fraction of the Ca response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the β-subunit of G proteins because quantitative immunoblots showed that levels of the β-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca pool because Ca release induced by agents that release Ca directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [H]inositol trisphosphate (IP) formation because the level of inhibition of IP formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT- sensitive species is the major G protein pathway that couples thrombin- receptor activation to Ca mobilization. This G protein appears to be involved not in the mechanisms that generate IP but rather possibly in coupling at the level of the intracellular Ca store

Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP: evidence for the involvement of calmodulin-dependent kinases

The role of Ca2+/calmodulin-dependent processes in the activation of the Na+/H+ antiport of primary cultures of rat aortic smooth muscle was studied using 22Na+ uptake and measurement of intracellular pH (pHi) with the fluorescent pH dye 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein. Antiport activation following exposure to serum and by the induction of an intracellular acidosis could be markedly attenuated by calmodulin antagonists. Ionomycin also transiently elevated pHi and 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ influx, effects consistent with activation of the antiport; these effects were abolished in cells exposed to calmodulin antagonists or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Activation of the antiport following intracellular acidosis was markedly affected by cellular ATP depletion. A comparison of the abilities of control and 2-deoxy-D-glucose-treated cells to increase 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ influx in response to graded acidifications indicated that attenuation of Na+/H+ antiport activity was due to both a shift of its pHi dependence and to a reduction in maximal activity. The results suggest that the Na+/H+ antiport of rat aortic smooth muscle is dependent on Ca2+/calmodulin-dependent processes, presumably phosphorylation, which influences its activity by modulating (i) an intracellular proton dependent regulatory mechanism (allosteric site) and (ii) the maximum activity of the antiport

Hematopoietic Cell–Restricted Deletion of CD36 Reduces High-Fat Diet–Induced Macrophage Infiltration and Improves Insulin Signaling in Adipose Tissue

OBJECTIVE: The fatty acid translocase and scavenger receptor CD36 is important in the recognition and uptake of lipids. Accordingly, we hypothesized that it plays a role in saturated fatty acid-induced macrophage lipid accumulation and proinflammatory activation. RESEARCH DESIGN AND METHODS: In vitro, the effect of CD36 inhibition and deletion in lipid-induced macrophage inflammation was assessed using the putative CD36 inhibitor, sulfosuccinimidyl oleate (SSO), and bone marrow-derived macrophages from mice with (CD36KO) or without (wild-type) global deletion of CD36. To investigate whether deletion of macrophage CD36 would improve insulin sensitivity in vivo, wild-type mice were transplanted with bone marrow from CD36KO or wild-type mice and then fed a standard or high-fat diet (HFD) for 20 weeks. RESULTS: SSO treatment markedly reduced saturated fatty acid-induced lipid accumulation and inflammation in RAW264.7 macrophages. Mice harboring CD36-specific deletion in hematopoietic-derived cells (HSC CD36KO) fed an HFD displayed improved insulin signaling and reduced macrophage infiltration in adipose tissue compared with wild-type mice, but this did not translate into protection against HFD-induced whole-body insulin resistance. Contrary to our hypothesis and our results using SSO in RAW264.7 macrophages, neither saturated fatty acid-induced lipid accumulation nor inflammation was reduced when comparing CD36KO with wild-type bone marrow-derived macrophages. CONCLUSIONS: Although CD36 does not appear important in saturated fatty acid-induced macrophage lipid accumulation, our study uncovers a novel role for CD36 in the migration of proinflammatory phagocytes to adipose tissue in obesity, with a concomitant improvement in insulin action

Depletion of B2 but Not B1a B Cells in BAFF Receptor-Deficient ApoE−/− Mice Attenuates Atherosclerosis by Potently Ameliorating Arterial Inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation