Anti-inflammatory immune skewing is atheroprotective: Apoe−/−FcγRIIb−/− mice develop fibrous carotid plaques.

BACKGROUND: Stroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals. METHODS AND RESULTS: Plaques were induced by implantation of a shear stress­modifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foam­cell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg­1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)­10, immune complexes, and regulatory T cells (Tregs) and lower IL­12, IL­1ß, and tumor necrosis factor alpha (TNF­α) in DKO mice. Similarly, in vitro stimulation produced higher IL­10 and Arg­1 and lower iNOS, IL­1ß, and TNF­α in DKO versus Apoe−/− macrophages. These results define a systemic anti­inflammatory phenotype. CONCLUSIONS: We hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti­inflammatory Treg/M2 polarization that is atheroprotective.

Orai3 is an estrogen receptor α-regulated Ca²âº channel that promotes tumorigenesis.

Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration. We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1. ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%). Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells. Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume). These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers.

Ultrasound biomicroscopy for longitudinal studies of carotid plaque development in mice: validation with histological endpoints.

Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo E⁻/⁻ mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (v(max)) and duration of the systolic velocity peak (t(s)/t(t)). Pre-plaque (2 week post-surgery) PW-Doppler parameters (v(max) and t(s)/t(t)) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (v(max) and t(s)/t(t)) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.

Transforming growth factor-ß1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein.

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-ß1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-ß-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-ß-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Ligation of macrophage Fcγ receptors recapitulates the gene expression pattern of vulnerable human carotid plaques.

Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated.

Hic-5 is required for fetal gene expression and cytoskeletal organization of neonatal cardiac myocytes.

Cardiac costameres link the extracellular matrix to the sarcomere at the z-disc and contain proteins such as integrins and other signaling molecules implicated in the regulation of pathological hypertrophy. Paxillin family members, hic-5 and paxillin, are scaffolding proteins associated with the integrin complex that have been shown to mediate numerous protein interactions in other cell types. While paxillin has been described in postnatal heart, hic-5 has not been identified. Our results provide evidence of hic-5 in neonatal cardiac myocytes co-localized with paxillin and alpha-actinin at the z-discs and the ends of actin filaments. Treatment with the hypertrophic agonist phenylephrine resulted in increased hic-5 expression while having no effect on paxillin levels. To see if increased hic-5 expression was sufficient to induce changes in cytoskeletal organization, hic-5 was overexpressed in myocytes by adenoviral infection. Hic-5 overexpression significantly increased the number of cells with organized cytoskeleton. Using siRNA mediated knockdown, we examined the requirement of hic-5 and paxillin in regulation of phenylephrine induced gene expression and cytoskeletal organization. Our results indicate that hic-5, not paxillin is required for upregulation of ANF and alpha-skeletal actin genes as well as in cytoskeletal reorganization. Finally, we demonstrated that hic-5 upregulation occurs downstream of MEK1/2-ERK1/2 signaling as inhibition of MEK1/2 using U0126 inhibitor completely inhibited hic-5 upregulation by PE. In a complimentary study, we showed that hic-5 knockdown had no effect on PE induced ERK1/2 phosphorylation. These findings demonstrate a novel role for hic-5 in the regulation of actin cytoskeleton and fetal gene expression.

Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway.

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its incidence of cardiotoxicity compromises its therapeutic index. DOX-induced heart failure is thought to be caused by reduction/oxidation cycling of DOX to generate oxidative stress and cardiomyocyte cell death. Resveratrol (RV), a stilbene found in red wine, has been reported to play a cardioprotective role in diseases associated with oxidative stress. The objective of this study was to test the ability of RV to protect against DOX-induced cardiomyocyte death. We hypothesized that RV protects cardiomyocytes from DOX-induced oxidative stress and subsequent cell death through changes in mitochondrial function. DOX induced a rapid increase in reactive oxygen species (ROS) production in cardiac cell mitochondria, which was inhibited by pretreatment with RV, most likely owing to an increase in MnSOD activity. This effect of RV caused additional polarization of the mitochondria in the absence and presence of DOX to increase mitochondrial function. RV pretreatment also prevented DOX-induced cardiomyocyte death. The protective ability of RV against DOX was abolished when Sirt1 was inhibited by nicotinamide. Our data suggest that RV protects against DOX-induced oxidative stress through changes in mitochondrial function, specifically the Sirt1 pathway leading to cardiac cell survival.

Use of resveratrol to improve the effectiveness of cisplatin and doxorubicin: study in human gynecologic cancer cell lines and in rodent heart.

OBJECTIVE: The purpose of this study was to investigate whether resveratrol adds to the growth inhibitory effects of cisplatin and doxorubicin on ovarian and uterine cancer cells and to evaluate whether resveratrol diminishes the cardiac toxicity of doxorubicin in rodent heart. STUDY DESIGN: Human ovarian (OVCAR-3) and uterine (Ishikawa) cancer cells in culture were treated with cisplatin and doxorubicin, respectively, with and without resveratrol; and cell growth and viability were evaluated. Neonatal rat ventricular myocytes received doxorubicin in the presence and absence of resveratrol, and cell viability was evaluated. Mice received doxorubicin +/- resveratrol, and electrocardiograms were evaluated. Data were analyzed with analysis of variance and Scheffe's test. RESULTS: Resveratrol combined with cisplatin or with doxorubicin demonstrated an additive growth-inhibitory anticancer effect with a left shift of the cisplatin and doxorubicin dose/response curves. Resveratrol increased the viability of neonatal rat ventricular myocytes that were treated with doxorubicin and reduced doxorubicin-induced bradycardia and QTc interval prolongation in mice. CONCLUSION: Resveratrol adds to the growth inhibitory/anticancer activity of cisplatin and doxorubicin in vitro and protects against doxorubicin-induced cardiac toxicity both in vitro and in mice.

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability.

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.

Histamine H1 and H2 receptor-mediated vasoreactivity of human internal thoracic and radial arteries.

BACKGROUND: Although internal thoracic arteries (ITAs) and radial arteries (RAs) have been shown to have similar patency, RAs tend to be more vasospastic postoperatively compared with ITAs. Therefore, the purpose of this study was to examine the effect of histamine subclass 1 (H1) receptors and histamine subclass 2 (H2) receptors on vasoreactivity in human ITAs and RAs. METHODS: Vessels were obtained from coronary artery bypass grafting patients. Human arterial rings (2 mm) were mounted in tissue baths, and baseline contractility was determined. Histamine concentration response curves (10(-9)-10(-3) mol/L) were performed in the absence or presence of diphenhydramine (H1 antagonist, 10(-4) mol/L) or famotidine (H2 antagonist, 10(-4) mol/L). Comparison of curves was performed by 2-way analysis of variance with repeated measures and a Bonferroni post-t test. RESULTS: Maximal contraction to histamine was significantly greater in RA (8.3 +/- 0.8 g, n = 6) than in ITA (2.9 +/- 0.3, n = 6), (P < .05). However, there was no difference in sensitivity. Histamine-mediated responses of both RA and ITA were blocked by pre-exposure to H1 antagonist, whereas an H2 antagonist only partially inhibited RA responses while blocking most of the ITA response to histamine. CONCLUSION: These studies suggest that H1 receptors alone cause contraction in RA but not in ITA, which may have potential linkage to patency and vasospasm. Further studies are necessary to identify the exact role of H2 receptors in ITA.

Interaction of ACE2 and integrin beta1 in failing human heart.

ACE2 purified from failing human heart was found to form a complex with integrin beta1 by immunoprecipitation, Western blotting, activity assay, and ESI tandem mass spectroscopy. The ACE2/integrin complex showed a Km of 6.8 microM and a Vmax of 2.13 pmol/min/microl purified enzyme. Activity was optimal at pH 7.5 with Ang II substrate.

Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase II-delta 2.

Previous studies demonstrated a requirement for multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (delta(2) or delta(C)) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII delta(2) were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr(17), a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII delta(2) inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII delta(2) autophosphorylation on Thr(287) after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII delta(2) phosphorylated substrate in vitro without added Ca(2+)/calmodulin and in the intact cell without added Ca(2+)-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII delta(2) on Thr(287). Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII delta(2), an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII delta(2) mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII delta(2) isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII delta(2) autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.

Increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme Homologue ACE2.

BACKGROUND: The formation of angiotensin-(1-7) from either angiotensin (Ang) I or Ang II in failing human hearts is not well understood. METHODS AND RESULTS: Angiotensinase activity in left and right ventricular membranes from 14 idiopathic dilated cardiomyopathy (IDC), 8 primary pulmonary hypertension (PPH), and 13 nonfailing human hearts was measured with either 125I-Ang I or 125I-Ang II as substrate. Ang-(1-7)-forming activity from 125I-Ang I was inhibited by thiorphan. With 125I-Ang II as substrate, Ang-(1-7) formation was inhibited by the ACE2-specific inhibitor C16. Western blotting with an anti-ACE2 antibody confirmed the presence of ACE2. Angiotensinase activity with 125I-Ang I as substrate was increased in failing IDC left ventricles (LVs) compared with nonfailing LVs (P<0.001). Ang-(1-7)-forming activity with 125I-Ang II as substrate was increased in both failing LVs and right ventricles (RVs) of IDC hearts and only in failing RVs of PPH hearts (PPH LV, 51.12+/-5.25; PPH RV, 89.97+/-11.21; IDC LV, 139.7+/-21.96; and IDC RV, 192.7+/-5.43; NF LV, 32.89+/-5.38; NF RV 40.49+/-10.66 fmol/min per milligram (P<0.05 PPH RV versus PPH LV; P<0.05 PPH RV versus NF RV; P<0.001 IDC LV versus NF LV; P<0.001 IDC RV versus NF RV). CONCLUSIONS: Ang-(1-7)-forming activity from both Ang I and Ang II was increased in failing human heart ventricles but was mediated by at least two different angiotensinases. The first, which demonstrated substrate preference for Ang I, was neutral endopeptidase (NEP)-like. The second was ACE2, as demonstrated by Western blotting and inhibition of activity with C16.

A lethal perinatal cardiac phenotype resulting from altered integrin function in cardiomyocytes.

BACKGROUND: Integrins are heterodimeric receptors that couple the extracellular matrix to intracellular signaling pathways and the cyoskeleton. Integrins are strain transducers and candidates for modulators or effectors of cardiac hypertrophy. METHODS: To begin to probe this function, we have transgenically expressed a chimeric protein that alters integrin function in cardiomyocytes. The transgene (Tac-beta(1D)) consists of the biologically inert extracellular and transmembrane domain of the interleukin-2 receptor alpha subunit (Tac) fused to the cytoplasmic tail of the human beta(1D) integrin driven by the cardiac alpha-myosin heavy chain promoter. Transgene expression results in a severe, usually fatal, perinatal cardiac phenotype, characterized by initial electrocardiographic abnormalities followed by extensive myocyte loss, macrophage infiltration, and replacement fibrosis. RESULTS: Expression of Tac-beta(1D) resulted in displacement of endogenous beta(1D) integrin from Z-lines and T-tubules, decreased expression of endogenous beta(1D), and disrupted the fibronectin pericellular matrix. These results are consistent with an essential role for beta(1) integrins in maintenance of cardiomyocyte viability and interaction with extracellular matrix. CONCLUSION: The appearance of conduction abnormalities before morphologic changes suggests that integrins are important in the development or maintenance of the conducting system of the heart.