Bum-Yong Kang

Molecular Imaging of Atherosclerotic Plaques Targeted to Oxidized LDL Receptor LOX-1 by SPECT/CT and Magnetic Resonance

Background—The oxidized low-density lipoprotein receptor (LDLR) LOX-1 plays a crucial role in atherosclerosis. We sought to detect and assess atherosclerotic plaque in vivo by using single-photon emission computed tomography/computed tomography and magnetic resonance imaging and a molecular probe targeted at LOX-1. Methods and Results—Apolipoprotein E−/− mice fed a Western diet and LDLR−/− and LDLR−/−/LOX-1−/− mice fed an atherogenic diet were used. Imaging probes consisted of liposomes decorated with anti–LOX-1 antibodies or nonspecific immunoglobulin G, 111indium or gadolinium, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine fluorescence markers. In vivo imaging was performed 24 hours after intravenous injection (150 &mgr;L) of LOX-1 or nonspecific immunoglobulin G probes labeled with either 111indium (600 &mgr;Ci) or gadolinium (0.075 mmol/kg), followed by aortic excision for phosphor imaging and Sudan IV staining, or fluorescence imaging and hematoxylin/eosin staining. The LOX-1 probe also colocalized with specific cell types, apoptosis, and matrix metalloproteinase-9 expression in frozen aortic sections. Single-photon emission computed tomography/computed tomography imaging of the LOX-1 probe showed aortic arch “hot spots” in apolipoprotein E−/− mice (n=8), confirmed by phosphor imaging. Magnetic resonance imaging showed significant Gd enhancement in atherosclerotic plaques in LDLR−/− mice with the LOX-1 (n=7) but not with the nonspecific immunoglobulin G (n=5) probe. No signal enhancement was observed in LDLR−/−/LOX-1−/− mice injected with the LOX-1 probe (n=5). These results were confirmed by ex vivo fluorescence imaging. The LOX-1 probe bound preferentially to the plaque shoulder, a region with vulnerable plaque features, including extensive LOX-1 expression, macrophage accumulation, apoptosis, and matrix metalloproteinase-9 expression. Conclusions—LOX-1 can be used as a target for molecular imaging of atherosclerotic plaque in vivo. Furthermore, the LOX-1 imaging signal is associated with markers of rupture-prone atherosclerotic plaque.

The Nox4 Inhibitor GKT137831 Attenuates Hypoxia-Induced Pulmonary Vascular Cell Proliferation

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.

Oxidized LDL Receptor 1 (OLR1) as a Possible Link between Obesity, Dyslipidemia and Cancer

Recent studies have linked expression of lectin-like ox-LDL receptor 1 (OLR1) to tumorigenesis. We analyzed microarray data from Olr1 knockout (KO) and wild type (WT) mice for genes involved in cellular transformation and evaluated effects of OLR1 over-expression in normal mammary epithelial cells (MCF10A) and breast cancer cells (HCC1143) in terms of gene expression, migration, adhesion and transendothelial migration. Twenty-six out of 238 genes were inhibited in tissues of OLR1 KO mice; the vast majority of OLR1 sensitive genes contained NF-κB binding sites in their promoters. Further studies revealed broad inhibition of NF-kB target genes outside of the transformation-associated gene pool, with enrichment themes of defense response, immune response, apoptosis, proliferation, and wound healing. Transcriptome of Olr1 KO mice also revealed inhibition of de novo lipogenesis, rate-limiting enzymes fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd1) and ELOVL family member 6 (Elovl6), as well as lipolytic phospholipase A2 group IVB (Pla2g4b). In studies comparing MCF10A and HCC1143, the latter displayed 60% higher OLR1 expression. Forced over-expression of OLR1 resulted in upregulation of NF-κB (p65) and its target pro-oncogenes involved in inhibition of apoptosis (BCL2, BCL2A1, TNFAIP3) and regulation of cell cycle (CCND2) in both cell lines. Basal expression of FASN, SCD1 and PLA2G4B, as well as lipogenesis transcription factors PPARA, SREBF2 and CREM, was higher in HCC1143 cells. Over-expression of OLR1 in HCC1143 cells also enhanced cell migration, without affecting their adherence to TNFα-activated endothelium or transendothelial migration. On the other hand, OLR1 neutralizing antibody inhibited both adhesion and transmigration of untreated HCC1143 cells. We conclude that OLR1 may act as an oncogene by activation of NF-kB target genes responsible for proliferation, migration and inhibition of apoptosis and de novo lipogenesis genes.

Oxidative Stress Modulates PPARγ in Vascular Endothelial Cells

The peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in vascular regulation. However, the impact of oxidative stress on PPAR gamma expression and activity has not been clearly defined. Human umbilical vein endothelial cells (HUVECs) were exposed to graded concentrations of H(2)O(2) for 0.5-72h, or bovine aortic endothelial cells (BAECs) were exposed to alterations in extracellular thiol/disulfide redox potential (E(h)) of the cysteine/cystine couple. Within 2h, H(2)O(2) reduced HUVEC PPAR gamma mRNA and activity and reduced the expression of two PPAR gamma-regulated genes without altering PPAR gamma protein levels. After 4h H(2)O(2) exposure, mRNA levels remained reduced, whereas PPAR gamma activity returned to control levels. PPAR gamma mRNA levels remained depressed for up to 72 h after exposure to H(2)O(2), without any change in PPAR gamma activity. Catalase prevented H(2)O(2)-induced reductions in PPAR gamma mRNA and activity. H(2)O(2) (1) reduced luciferase expression in HUVECs transiently transfected with a human PPAR gamma promoter reporter, (2) failed to alter PPAR gamma mRNA half-life, and (3) transiently increased expression and activity of c-Fos and phospho-c-Jun. Treatment with the AP1 inhibitor curcumin prevented H(2)O(2)-mediated reductions in PPAR gamma expression. In addition, medium having an oxidized E(h) reduced BAEC PPAR gamma mRNA and activity. These findings demonstrate that oxidative stress, potentially through activation of inhibitory redox-regulated transcription factors, attenuates PPAR gamma expression and activity in vascular endothelial cells through suppression of PPAR gamma transcription.

Curcumin reduces angiotensin II-mediated cardiomyocyte growth via LOX-1 inhibition.

BACKGROUND Curcumin, a natural polyphenolic compound, has been shown to reduce cardiomyocyte growth. Angiotensin II type 1 receptor (AT1R) and lectin-like oxidized low density lipoprotein (ox-LDL) receptor-1 (LOX-1) are major stimuli for cardiomyocyte growth via activation of oxidant signals. We postulated that curcumin may reduce Ang II-mediated cardiomyocyte growth via AT1R and LOX-1 inhibition. METHODS Adult mouse cardiomyocytes (HL-1) were incubated overnight in serum-free medium, and then treated with solvents or curcumin, the AT1R inhibitor losartan or anti-LOX-1 antibody for 3 hours, and the cells were then stimulated with Ang II. We measured cardiomyocyte growth, and associated intracellular redox signals using reverse transcriptase-polymerase chain reaction and quantitative real-time RT-PCR. We also examined the effect of curcumin on cardiomyocyte biology with forced overexpression of LOX-1 gene. RESULTS Curcumin (5-10 microM), losartan, and anti-LOX-1 antibody markedly attenuated Ang II-mediated oxidant stress, and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor-kappaB (NF-kappaB). Attenuation of redox state by curcumin resulted in abrogation of Ang II-mediated cardiomyocyte growth and atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes. Curcumin also reduced Ang II-mediated upregulation of AT1R and LOX-1. The forced upregulation of LOX-1 enhanced the expression of genes for AT1R, ANP, and BNP, and curcumin pretreatment reduced LOX-1 and AT1R expression and LOX-1-mediated increase in hypertrophy markers. CONCLUSIONS Curcumin attenuates Ang II-mediated cardiomyocyte growth by inhibiting LOX-1 and AT1R expression and suppressing the heightened intracellular redox state.Background: Curcumin, a natural polyphenolic compound, has been shown to reduce cardiomyocyte growth. Angiotensin II type 1 receptor (AT1R) and lectin-like oxidized low density lipoprotein (ox-LDL) receptor-1 (LOX-1) are major stimuli for cardiomyocyte growth via activation of oxidant signals. We postulated that curcumin may reduce Ang II-mediated cardiomyocyte growth via AT1R and LOX-1 inhibition. Methods: Adult mouse cardiomyocytes (HL-1) were incubated overnight in serum-free medium, and then treated with solvents or curcumin, the AT1R inhibitor losartan or anti-LOX-1 antibody for 3 hours, and the cells were then stimulated with Ang II. We measured cardiomyocyte growth, and associated intracellular redox signals using reverse transcriptase-polymerase chain reaction and quantitative real-time RT-PCR. We also examined the effect of curcumin on cardiomyocyte biology with forced overexpression of LOX-1 gene. Results: Curcumin (5-10 μM), losartan, and anti-LOX-1 antibody markedly attenuated Ang II-mediated oxidant stress, and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor-κB (NF-κB). Attenuation of redox state by curcumin resulted in abrogation of Ang II-mediated cardiomyocyte growth and atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes. Curcumin also reduced Ang II-mediated upregulation of AT1R and LOX-1. The forced upregulation of LOX-1 enhanced the expression of genes for AT1R, ANP, and BNP, and curcumin pretreatment reduced LOX-1 and AT1R expression and LOX-1-mediated increase in hypertrophy markers. Conclusions: Curcumin attenuates Ang II-mediated cardiomyocyte growth by inhibiting LOX-1 and AT1R expression and suppressing the heightened intracellular redox state.

The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O(2)) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for 3 wk with or without gavage with RSG (10 mg·kg(-1)·day(-1)) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O(2)(•-) (mtO(2)(•-)) and H(2)O(2) (mtH(2)O(2)) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72 h increased mtO(2)(•-) and mtH(2)O(2). To assess the contribution of mtO(2)(•-) and mtH(2)O(2) to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (Tg(hSOD2)) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H(2)O(2) (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, Tg(hSOD2) mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO(2)(•-) and mtH(2)O(2). Targeting mtH(2)O(2) attenuates PH pathogenesis, whereas targeting mtO(2)(•-) exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H(2)O(2) release. These studies suggest that targeted reductions in mtH(2)O(2) generation may be particularly effective in preventing hypoxia-induced PH.

Hypoxia mediates mutual repression between microRNA-27a and PPARγ in the pulmonary vasculature.

Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models. In contrast, activating PPARγ attenuates hypoxia-induced PH and endothelin 1 (ET-1) expression. To further explore mechanisms of hypoxia-induced PH and reductions in PPARγ, we examined the effects of hypoxia on selected microRNA (miRNA or miR) levels that might reduce PPARγ expression leading to increased ET-1 expression and PH. Our results demonstrate that exposure to hypoxia (10% O2) for 3-weeks increased levels of miR-27a and ET-1 in the lungs of C57BL/6 mice and reduced PPARγ levels. Hypoxia-induced increases in miR-27a were attenuated in mice treated with the PPARγ ligand, rosiglitazone (RSG, 10 mg/kg/d) by gavage for the final 10 d of exposure. In parallel studies, human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O2) or hypoxic (1% O2) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR-27a and ET-1 expression, and reduced PPARγ expression. These alterations were attenuated by treatment with RSG (10 µM) during the last 24 h of hypoxia exposure. Overexpression of miR-27a or PPARγ knockdown increased HPAEC proliferation and ET-1 expression and decreased PPARγ levels, whereas these effects were reversed by miR-27a inhibition. Further, compared to lungs from littermate control mice, miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. Knockdown of either SP1 or EGR1 was sufficient to significantly attenuate miR-27a expression in HPAECs. Collectively, these studies provide novel evidence that miR-27a and PPARγ mediate mutually repressive actions in hypoxic pulmonary vasculature and that targeting PPARγ may represent a novel therapeutic approach in PH to attenuate proliferative mediators that stimulate proliferation of pulmonary vascular cells.

Rosuvastatin Attenuates Ang II–Mediated Cardiomyocyte Hypertrophy via Inhibition of LOX-1

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, also known as statins, have been shown to reduce cardiac remodeling. Angiotensin II (Ang II) type 1 receptor (AT1R) and oxidized low-density lipoprotein (ox-LDL) via its lectin-like ox-LDL receptor (LOX-1) are major stimuli for cardiomyocyte growth. We postulated that rosuvastatin, a potent HMG-CoA reductase inhibitor, may reduce Ang II—mediated cardiomyocyte growth via AT1R and LOX-1 inhibition. HL-1 adult mouse cardiomyocytes were incubated overnight in serum-free medium, and then treated with rosuvastatin, the AT1R inhibitor losartan or anti-LOX-1 antibody for 3 hours. The cells were then stimulated with Ang II. We measured cardiomyocyte growth, and associated intracellular redox signals using reverse transcription— polymerase chain reaction (RT-PCR) and real-time quantitative PCR. Losartan and anti-LOX-1 antibody markedly attenuated Ang II—mediated oxidant stress, and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p40pho x and gp91 phox subunits) and nuclear factor-κB (NF-κB). Rosuvastatin attenuated the Ang II—mediated upregulation of both subunits of NAPDH oxidase as well as NF-κB. Rosuvastatin also reduced Ang II—mediated upregulation of AT1R and LOX-1. In other experiments, LOX-1 was upregulated in cardiomyocytes by transfection with pCI-neo/LOX-1, which also enhanced the expression AT1R messenger RNA (mRNA), and rosuvastatin pretreatment reduced the expression of both LOX-1 and AT1R in this system. Thus, rosuvastatin attenuates Ang II—mediated cardiomyocyte growth by inhibiting LOX-1 and AT1R expression and suppressing the heightened intracellular redox state.

Statins and angiogenesis: is it about connections?

Statins, inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, have been shown to induce both angiogenic and angiostatic responses. We attempted to resolve this controversy by studying the effects of two different statins, rosuvastatin and simvastatin, in two different assay systems. In the matrigel angiogenesis assay, both statins enhanced tube formation by human umbilical vein endothelial cells (HUVECs, p<0.01 vs. control). In the ex vivo mouse aortic ring sprouting assay, both statins virtually abolished new vessel formation (p<0.01). As a basic difference between the two models of angiogenesis is dispersed state of endothelial cells vs. compact monolayer, we analyzed influence of statins on endothelial junction proteins. RT-PCR analysis and cytoimmunostaining of HUVECs treated with simvastatin revealed increased expression of VE-cadherin (p<0.05). The blockade of VE-cadherin with a specific antibody reversed simvastatin-induced tube formation (p<0.002). These data suggest that statins through VE-cadherin stimulation modulate cell-cell adhesion and diminish the ability of cells to proliferate and migrate. The observations of reduced angiogenesis in the intact vessel may relate to anti-atherosclerotic and anti-cancer effects of statins, and provide a feasible explanation for conflicting data under different experimental conditions.