Ross G. Gerrity

Prediction of the Localization of High-Risk Coronary Atherosclerotic Plaques on the Basis of Low Endothelial Shear Stress An Intravascular Ultrasound and Histopathology Natural History Study

Background— Low endothelial shear stress (ESS) promotes the development of atherosclerosis; however, its role in the progression of atherosclerotic plaques and evolution to inflamed high-risk plaques has not been studied. Our hypothesis was that the lowest values of ESS are responsible for the development of high-risk coronary atherosclerotic plaques associated with excessive expansive remodeling. Methods and Results— Twenty-four swine, treated with streptozotocin to induce diabetes and fed a high-fat diet, were allocated into early (n=12) and late (n=12) atherosclerosis groups. Intima-media thickness was assessed by intravascular ultrasound in the coronary arteries at weeks 4 and 8 in the early group and weeks 23 and 30 in the late group. Plaques started to develop after week 8, leading to marked heterogeneity in plaque severity at week 30. ESS was calculated in plaque-free subsegments of interest (n=142) in the late group at week 23. Coronary arteries (n=31) of this group were harvested at week 30, and the subsegments of interest were identified and analyzed histopathologically. Low ESS was an independent predictor of the development of high-risk plaques, characterized by intense lipid accumulation, inflammation, thin fibrous cap, severe internal elastic lamina degradation, and excessive expansive remodeling. The severity of high-risk plaque characteristics at week 30 was significantly correlated with the magnitude of low ESS at week 23. Conclusions— The magnitude of low ESS determines the complexity and heterogeneity of atherosclerotic lesions and predicts the development of high-risk plaque.

Regulation of macrophage foam cell formation by αvβ3 integrin: Potential role in human atherosclerosis

The accumulation of macrophage foam cells in atherosclerotic lesions is associated with both initiation and progression of this disease. Scavenger receptors CD36 and SRA are the primary receptors responsible for conversion of macrophages into foam cells. Integrin αVβ3 plays a role in the differentiation of several cell types, but its involvement in the transition of macrophages into foam cells and the potential role of this receptor in atherosclerosis have not been examined. Using an in vitro model of single surface receptor activation by binding with an immobilized monoclonal antibody specific to αVβ3 integrin we show that ligation of αVβ3 integrin prevents differentiation of blood monocytes and macrophages into the foam cell phenotype via coordinate down-regulation of CD36 and SRA. This effect of αVβ3 integrin ligation can be reproduced by contact with endothelial cells, whereas the inhibition of αVβ3 receptor ligation restores the uptake of oxidized low-density lipoprotein. Moreover, we found that αVβ3 integrin is readily detected in situ on macrophages in early and advanced atherosclerotic lesions and that in vitro exposure to oxidized low-density lipoprotein up-regulates αVβ3 integrin expression. We hypothesize that αVβ3 integrin regulates macrophage functional maturation into foam cells in a persistent manner, and therefore, by targeting αVβ3 receptor it could potentially be possible to regulate progression of atherosclerosis in humans.

Cysteine-674 oxidation and degradation of sarcoplasmic reticulum Ca(2+) ATPase in diabetic pig aorta.

The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.

Diabetes and Arterial Extracellular Matrix Changes in a Porcine Model of Atherosclerosis

Patients with diabetes are at substantially increased risk for atherosclerosis and clinical cardiovascular events. Because arterial extracellular matrix contains several molecules, including biglycan, versican, hyaluronan, and elastin, that may affect plaque lipid retention and stability, we determined whether diabetes affects plaque content of these molecules in a porcine model of hyperlipidemia and diabetes. Coronary artery sections were studied from non-diabetic normolipidemic (n=11, N-NL), diabetic normolipidemic (n=10, DM-NL), non-diabetic hyperlipidemic (n=16, N-HL), and diabetic hyperlipidemic (n=15, DM-HL) animals. Hyaluronan, biglycan, versican, and apolipoprotein B (apoB) were detected with mono-specific peptides or antisera, and elastin with Movats pentachrome stain, and contents of each were quantified by computer-assisted morphometry. In the hyperlipidemic groups, diabetes was associated with a 4-fold increase in intimal area, with strong correlations between intimal area and immunostained areas for hyaluronan (R 2 = 0.83, p<0.0001), biglycan (R 2 = 0.72, p<0.0001), and apoB (R 2 = 0.23, p=0.0069). In contrast, median (interquartile range) intimal elastin content was significantly lower with diabetes [N-HL: 5.2% (2.4–8.2%) vs DM-HL: 1.5% (0.5–4.2%), p=0.01], and there was a strong negative correlation between intimal total and elastin areas (Spearman r = −0.62, p=0.001). In this porcine model, diabetes was associated with multiple extracellular matrix changes that have been associated with increased lesion instability, greater atherogenic lipoprotein retention, and accelerated atherogenesis.

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells.

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38MAPK activities and their protein levels by 2–4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38MAPK activities and protein expression by 65–75% in HVSMC transiently transfected with NPRA, as compared with only 18–22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90–95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38MAPK, blocked the Erk2 and p38MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38MAPK) activities and protein levels in a 2-fold manner: by antagonizing the upstream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.

Monocyte-Induced Downregulation of Nitric Oxide Synthase in Cultured Aortic Endothelial Cells

Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell-induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by NG-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200 x 10(3) per well)-dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin-and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1 beta neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress-induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-alpha. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.

Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

Attenuation of inflammation and expansive remodeling by Valsartan alone or in combination with Simvastatin in high-risk coronary atherosclerotic plaques

AIMS We investigated the role of Valsartan (V) alone or in combination with Simvastatin (S) on coronary atherosclerosis and vascular remodeling, and tested the hypothesis that V or V/S attenuate the pro-inflammatory effect of low endothelial shear stress (ESS). METHODS Twenty-four diabetic, hyperlipidemic swine were allocated into Early (n=12) and Late (n=12) groups. In each group animals were treated with Placebo (n=4), V (n=4) and V/S (n=4) and followed for 8 weeks in the Early group and 30 weeks in the Late group. Blood pressure, serum cholesterol and glucose were similar across the treatment subgroups. ESS was calculated in plaque-free subsegments of interest (n=109) in the Late group at week 23. Coronary arteries of this group were harvested at week 30, and the subsegments of interest were identified, and analyzed histopathologically. RESULTS V alone or with S reduced the severity of inflammation in high-risk plaques. Both regimens attenuated the severity of enzymatic degradation of the arterial wall, reducing the severity of expansive remodeling. V alone or with S attenuated the pro-inflammatory effect of low ESS. CONCLUSIONS V alone or with S exerts a beneficial effect of reducing and stabilizing high-risk plaque characteristics independent of a blood pressure- and lipid-lowering effect.

Proteoglycan distribution in areas of differing permeability to Evans blue dye in the aortas of young pigs. An ultrastructural study.

We studied proteoglycan distribution In areas of spontaneously occurring high and low permeability by TEM examination of ruthenium red-stained sections of the aortic arch of normollpemlc and hyperllpemlc pigs. We noted granules of two sizes: those smaller than 20 nm contained heparan sulphate, and those from 20 to 50 nm In size contained chondroltln or dermatan sulphate. In the aortas of pigs fed a normal diet, there were significantly more granules of both types In low permeability areas than in areas permeable to Evans blue dye. This is consistent with the theory that glycosamlnoglycan provides a component for the control of aortic permeability. In the aortas of pigs fed cholesterol, there was an accumulation of llpid-fllled monocytes In areas of high permeability and an Increase In proteoglycan granule concentration, suggesting an Increase In glycosamlnoglycan concentration, which may be the precursor to extracellular lipld deposition.

Hyperlipemia and oxidation of LDL induce vascular smooth muscle cell growth: an effect mediated by the HLH factor Id3.

Hyperlipemia and oxidized LDL (ox-LDL) are important independent cardiovascular risk factors. Ox-LDL has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. However, the effects of hyperlipemia and the molecular mechanisms mediating hyperlipemia and ox-LDL effects on VSMC growth are poorly understood. The helix-loop-helix (HLH) transcription factor, Id3, is a redox-sensitive gene expressed in VSMC in response to mitogen stimulation and vascular injury. Accordingly, we hypothesize that Id3 is an important mediator of ox-LDL and hyperlipemia-induced VSMC growth. Aortas harvested from hyperlipemic pigs demonstrated significantly more Id3 than normolipemic controls. Primary VSMC were stimulated with ox-LDL, native LDL, sera from hyperlipemic pigs, or normolipemic pigs. VSMC exposed to hyperlipemic sera demonstrated increased Id3 expression, VSMC growth and S-phase entry and decreased p21cip1 expression and transcription. Cells stimulated with ox-LDL demonstrated similar findings of increased growth and Id3 expression and decreased p21cip1 expression. Moreover, the effects of ox-LDL on growth were abolished in cells devoid of the Id3 gene. Results provide evidence that the HLH factor Id3 mediates the mitogenic effect of hyperlipemic sera and ox-LDL in VSMC via inhibition of p21cip1 expression, subsequently increasing DNA synthesis and proliferation.