High-Frequency Fluctuation Intensity as a Predictor of Post-Stenotic Dilatation in Swine.

OBJECTIVE: To identify the turbulent components of blood flow facilitating aortic lumen dilatation in a post-stenotic dilatation (PSD) porcine model. METHODS: The porcine abdominal aorta (AA) was moderately coarctated to induce overt flow turbulence in the downstream region and to lead to dilatation in time periods between four and twelve weeks. We propose a new metric, fluctuation intensity (FI), to quantify turbulent fluctuations of pulsatile aortic flow measured within twenty minutes post-coarctation. Lumen perimeter ratio (LPR) of the distal-to-suprarenal AA was used to assess the degree of PSD. Using k-means clustering analysis, we first divided FI frequency spectrum into low- and high-frequency fluctuation intensity (LFFI and HFFI), and subsequently grouped animals with coarctation. Receiver operating characteristic (ROC) analysis was performed to evaluate the ability of the proposed metric to predict PSD. RESULTS: The frequency band of the FI spectrum in facilitating aortic lumen dilatation was identified to be 40∼200 Hz. Using sham group as the reference, pigs receiving coarctation were clustered into two groups with (group A) and without (group B) increases in HFFI values. Coarctation significantly increased LPR values in group A, but not in group B. Moreover, group A exhibited a high probability density distribution on severe elastic fiber fragmentation. ROC analysis indicated HFFI to be capable of predicting PSD with excellent sensitivity and specificity. CONCLUSION: High-intensity, high-frequency components of blood flow fluctuations induced by moderate coarctation promote elastic lamella degradation and aortic lumen dilatation. SIGNIFICANCE: HFFI application in flowmeter programs may provide a useful predictor of PSD.

Hypertension Accelerates Alzheimer's Disease-Related Pathologies in Pigs and 3xTg Mice.

Epidemiological studies suggest there is an association between midlife hypertension and increased risk of late-life Alzheimer's disease (AD). However, whether hypertension accelerates the onset of AD or is a distinct disease that becomes more prevalent with age (comorbidity) remains unclear. This study aimed to test the possible relationship between hypertension and AD pathogenesis. Two animal models were used in this study. For the first model, 7-month-old Lanyu-miniature-pigs were given the abdominal aortic constriction operation to induce hypertension and their AD-related pathologies were assessed at 1, 2, and 3 months after the operation. The results showed that hypertension was detected since 1 month after the operation in the pigs. Levels of Aß, amyloid precursor protein, RAGE, phosphorylated tau and activated GSK3ß in the hippocampi increased at 3 months after the operation. For the second model, 3xTg mice at the ages of 2, 5, and 7 months were subjected to the "two-kidney-one-clip" operation to induce hypertension. One month after the operation, blood pressure was significantly increased in the 3xTg mice in any age. Aß, amyloid plaque load, and phosphorylated tau levels increased in the operated mice. Furthermore, the operation also induced shrinkage in the dendritic arbor of hippocampal dentate gyrus granule neurons, leakage in the blood-brain barrier, activation in microglia, and impairment in the hippocampus-dependent learning and memory in the 3xTg mice. In conclusion, hypertension accelerates the onset of AD. Blood pressure control during midlife may delay the onset of AD.

The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells.

BACKGROUND: Thrombomodulin (TM), a transmembrane glycoprotein highly expressed in endothelial cells (ECs), is a potent anticoagulant maintaining circulation homeostasis. Under inflammatory states, TM expression is drastically reduced in ECs while vascular smooth muscle cells (VSMCs) show a robust expression of TM. The functional role of TM in VSMCs remains elusive. METHODS: We examined the role of TM in VSMCs activities in human aortic VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB). Using rat embryonic aorta-derived A7r5 VSMCs which do not express TM, the role of the chondroitin sulfate (CS) moiety of TM in VSMCs was delineated with cells expressing wild-type TM and the CS-devoid TM mutant. RESULTS: Expression of TM enhanced cell migration and adhesion/spreading onto type I collagen, but had no effect on cell proliferation. Knocking down TM with short hairpin RNA reduced PDGF-stimulated adhesion and migration of human aortic VSMCs. In A7r5 cells, TM-mediated cell adhesion was eradicated by pretreatment with chondroitinase ABC which degrades CS moiety. Furthermore, the TM mutant (TMS490, 492A) devoid of CS moiety failed to increase cell adhesion, spreading or migration. Wild-type TM, but not TMS490, 492A, increased focal adhesion kinase (FAK) activation during cell adhesion, and TM-enhanced cell migration was abolished by a function-blocking anti-integrin ß1 antibody. CONCLUSION: Chondroitin sulfate modification is required for TM-mediated activation of ß1-integrin and FAK, thereby enhancing adhesion and migration activity of VSMCs.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence.

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10(-7)M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated ß-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Peroxisome Proliferator-Activated Receptor γ Level Contributes to Structural Integrity and Component Production of Elastic Fibers in the Aorta.

Loss of integrity and massive disruption of elastic fibers are key features of abdominal aortic aneurysm (AAA). Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to attenuate AAA through inhibition of inflammation and proteolytic degradation. However, its involvement in elastogenesis during AAA remains unclear. PPARγ was highly expressed in human AAA within all vascular cells, including inflammatory cells and fibroblasts. In the aortas of transgenic mice expressing PPARγ at 25% normal levels (Pparg(C) (/-) mice), we observed the fragmentation of elastic fibers and reduced expression of vital elastic fiber components of elastin and fibulin-5. These were not observed in mice with 50% normal PPARγ expression (Pparg(+/-) mice). Infusion of a moderate dose of angiotensin II (500 ng/kg per minute) did not induce AAA but Pparg(+/-) aorta developed flattened elastic lamellae, whereas Pparg(C/-) aorta showed severe destruction of elastic fibers. After infusion of angiotensin II at 1000 ng/kg per minute, 73% of Pparg(C/-) mice developed atypical suprarenal aortic aneurysms: superior mesenteric arteries were dilated with extensive collagen deposition in adventitia and infiltrations of inflammatory cells. Although matrix metalloproteinase inhibition by doxycycline somewhat attenuated the dilation of aneurysm, it did not reduce the incidence nor elastic lamella deterioration in angiotensin II-infused Pparg(C/-) mice. Furthermore, PPARγ antagonism downregulated elastin and fibulin-5 in fibroblasts, but not in vascular smooth muscle cells. Chromatin immunoprecipitation assay demonstrated PPARγ binding in the genomic sequence of fibulin-5 in fibroblasts. Our results underscore the importance of PPARγ in AAA development though orchestrating proper elastogenesis and preserving elastic fiber integrity.

Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

Human cytomegalovirus UL76 elicits novel aggresome formation via interaction with S5a of the ubiquitin proteasome system.

HCMV UL76 is a member of a conserved Herpesviridae protein family (Herpes_UL24) that is involved in viral production, latency, and reactivation. UL76 presents as globular aggresomes in the nuclei of transiently transfected cells. Bioinformatic analyses predict that UL76 has a propensity for aggregation and targets cellular proteins implicated in protein folding and ubiquitin-proteasome systems (UPS). Furthermore, fluorescence recovery after photobleaching experiments suggests that UL76 reduces protein mobility in the aggresome, which indicates that UL76 elicits the aggregation of misfolded proteins. Moreover, in the absence of other viral proteins, UL76 interacts with S5a, which is a major receptor of polyubiquitinated proteins for UPS proteolysis via its conserved region and the von Willebrand factor type A (VWA) domain of S5a. We demonstrate that UL76 sequesters polyubiquitinated proteins and S5a to nuclear aggresomes in biological proximity. After knockdown of endogenous S5a by RNA interference techniques, the UL76 level was only minimally affected in transiently expressing cells. However, a significant reduction in the number of cells containing UL76 nuclear aggresomes was observed, which suggests that S5a may play a key role in aggresome formation. Moreover, we show that UL76 interacts with S5a in the late phase of viral infection and that knockdown of S5a hinders the development of both the replication compartment and the aggresome. In this study, we demonstrate that UL76 induces a novel nuclear aggresome, likely by subverting S5a of the UPS. Given that UL76 belongs to a conserved family, this underlying mechanism may be shared by all members of the Herpesviridae.

ROS-mediated downregulation of MYPT1 in smooth muscle cells: a potential mechanism for the aberrant contractility in atherosclerosis.

Reactive oxygen species (ROS) mediates the aberrant contractility in hypertension. Abnormal contractility occurs in atherosclerotic vessels but changes in proteins that regulate contractility remain poorly understood. Myosin phosphatase (MP) activity, which regulates smooth muscle relaxation, is regulated by the phosphorylation of its regulatory subunit, MP targeting subunit 1 (MYPT1). In the present study, we examined the roles of ROS in MP subunit expression both in cultured human aortic smooth muscle cells (HASMCs) and during atherosclerosis progression in apolipoprotein E-knockout (apoE-KO) mice. Furthermore, the effect of decreased MYPT1 on actin cytoskeleton and cell migration activity was assessed in HASMCs. Short hairpin RNA-mediated knockdown of MYPT1 increased stress fibers and attenuated platelet-derived growth factor-induced cell migration in HASMCs. Superoxide anion-inducing agent LY83583 downregulated MYPT1 mRNA and protein levels, but did not affect the phosphorylation of MYPT1 and catalytic subunit of MP, PP1δ. The LY83583-induced decrease in MYPT1 was abolished by co-treating with superoxide dismutase or by inhibiting NADPH oxidase with diphenyleneiodonium. Treatment of peroxynitrite, but not hydrogen peroxide (H2O2), downregulated MYPT1 protein expression and induced MYPT1 phosphorylation without affecting mRNA levels. Co-treatment with a proteasome inhibitor, MG-132, eliminated peroxynitrite-induced MYPT1 downregulation. In apoE-KO mice, MYPT1 protein, but not mRNA, levels were markedly decreased in 16-week- and 24-week-old mice. Oral estrogen treatment, which was previously shown to decrease aortic ROS levels, upregulated aortic MYPT1 expression. Moreover, reduction in MYPT1 expression correlated with increased aortic sensitivity toward vasoconstrictors. These results suggested that during atherosclerosis progression oxidative stress mediates the downregulation of MYPT1, which may inhibit smooth muscle cell migration and contribute to the aberrant contractility.

Coarctation-induced degenerative abdominal aortic aneurysm in a porcine model.

OBJECTIVE: Hemodynamic stress participates in the initiation and progression of aneurysmal degeneration. Coarctation increases flow-mediated stress on the aortic wall. We tested the hypothesis that prolonged coarctation of an infrarenal abdominal aorta (AA) segment leads to abdominal aortic aneurysm (AAA) formation in mini pigs. METHODS: An asymmetric, funnel-shaped flow path was created by constricting the infrarenal AA segment of Taiwanese Lanyu mini pigs (age, 7-10 months; male and female) wrapped with an 8-mm-wide expanded polytetrafluoroethylene Teflon strip for 4 weeks (4w), 8 weeks (8w), and 12 weeks (12w) (seven pigs per group). This mimics the tortuous aneurysm neck in human AAA, which increases downstream flow-mediated stress. Significant flow disturbance resulting from moderate coarctation was indicated by a pulsatility index reduced to one third the inherent levels. Sham control pigs received Teflon wrapping without coarctation. RESULTS: Aneurysm characterized by progressive medial degeneration occurred at the terminal AA after 12w coarctation. The outer dimension enlargement of the distal AA exceeded 50% compared with that of the proximal AA at 4w, 8w, and 12w postcoarctation (sham, 1.0; 4w, 1.7 ± 0.08; 8w, 1.5 ± 0.09; 12w, 1.7 ± 0.01). Lumen ratio of the distal-to-suprarenal AA increased time dependently, with 12w postcoarctation exhibiting significant increase (sham, 1.0 ± 0.05; 4w, 1.1 ± 0.11; 8w, 1.4 ± 0.20; 12w, 1.5 ± 0.09). In the distal AA, elastic lamellae exhibited fragmentation at 4w and more pronounced fragmentation with decreased density at 8w and 12w postcoarctation. Medial collagen density exhibited the trend to increase at 4w and 8w but was reversed at 12w postcoarctation. Smooth muscle exhibited disarray and nuclear density decrease at 8w and 12w postcoarctation (sham, 6966 ± 888/mm; 4w, 5747 ± 1340/mm; 8w, 4153 ± 323/mm; 12w, 4083 ± 465/mm). Gelatin zymography revealed that matrix metalloproteinase-9 activity markedly increased at 4w postcoarctation. CONCLUSIONS: Prolonged moderate coarctation caused regional hemodynamic stress and thereby induced degenerative AAA in the terminal AA.

Transplantation of endothelial progenitor cells improves pulmonary endothelial function and gas exchange in rabbits with endotoxin-induced acute lung injury.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) have been therapeutically applied to aid vascular repair and myocardial regeneration. The number of circulating EPCs also provides invaluable outcome prediction for fatal diseases such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). However, evidence for the therapeutic potential of EPCs in subjects with ALI/ADRS is limited. METHODS: Circulating EPCs were obtained from rabbits using Ficoll centrifugation. One week after culturing EPCs in endothelial growth medium-2, ALI was induced in rabbits by intratracheal instillation of lipopolysaccharide (500 µg/kg). Autologous EPCs or saline were administered IV after induction of ALI and animals were killed 2 days later. Pulmonary artery endothelial function and gas exchange were determined. Degrees of lung injury were assessed by alveolocapillary permeability, lung hemoglobin content, and myeloperoxidase activity. RESULTS: In comparison with controls, Po(2) in arterial blood was significantly elevated and pulmonary artery endothelium-dependent relaxation response was restored in rabbits receiving EPC transplantation. Lung water, Evan's blue, and bronchoalveolar lavage protein contents were significantly reduced in the EPC transplanted group, indicating a better preservation of the alveolocapillary membrane. Transplantation of EPCs decreased the lung hemoglobin level. Furthermore, expressions of CD11b and myeloperoxidase activity were also suppressed after administration of EPCs. CONCLUSIONS: Transplantation of EPCs restored pulmonary endothelial function, preserved integrity of the alveolocapillary barrier and suppressed the lung inflammatory response, thereby improving pulmonary gas exchange in rabbits with intratracheal lipopolysaccharide-induced ALI. Transplantation of EPCs can be a novel cell-based, endothelium-targeted therapeutic strategy for prevention and treatment of ALI/ARDS.

Reactive oxygen species are involved in regulating alpha1-adrenoceptor-activated vascular smooth muscle contraction.

BACKGROUND: Reactive oxygen species (ROS) were shown to mediate aberrant contractility in hypertension, yet the physiological roles of ROS in vascular smooth muscle contraction have remained elusive. This study aimed to examine whether ROS regulate alpha1-adrenoceptor-activated contraction by altering myosin phosphatase activities. METHODS: Using endothelium-denuded rat tail artery (RTA) strips, effects of anti-oxidants on isometric force, ROS production, phosphorylation of the 20-kDa myosin light chain (MLC20), and myosin phosphatase stimulated by alpha1-adrenoceptor agonist phenylephrine were examined. RESULTS: An antioxidant, N-acetyl-L-cysteine (NAC), and two NADPH oxidase inhibitors, apocynin and VAS2870, dose-dependently inhibited contraction activated by phenylephrine. Phenylephrine stimulated superoxide anion production that was diminished by the pretreatment of apocynin, VAS2870, superoxide scavenger tiron or mitochondria inhibitor rotenone, but not by xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. Concurrently, NADPH oxidase activity in RTA homogenates increased within 1 min upon phenylephrine stimulation, sustained for 10 min, and was abolished by the co-treatment with apocynin, but not allopurinol or rotenone. Phenylephrine-induced MLC20 phosphorylation was dose-dependently decreased by apocynin. Furthermore, apocynin inhibited phenylephrine-stimulated RhoA translocation to plasma membrane and phosphorylation of both myosin phosphatase regulatory subunit MYPT1Thr855 and myosin phosphatase inhibitor CPI-17Thr38. CONCLUSIONS: ROS, probably derived from NADPH oxidase and mitochondria, partially regulate alpha1-adrenoceptor-activated smooth muscle contraction by altering myosin phosphatase-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways.

Effects of oral estrogen on aortic ROS-generating and -scavenging enzymes and atherosclerosis in apoE-deficient mice.

The effect of hormone replacement therapy (HRT) on cardiovascular diseases remains controversial. Studies conducted on postmenopausal women indicate that oral HRT increases risk factors that may counteract the atheroprotective effect of estrogen. However, the effects of estrogen on atherosclerosis have been examined using subcutaneous estrogen in most animal studies, which points to the need for evaluating the effect of oral estrogen. Reactive oxygen species (ROS) have emerged as critical factors in the pathogenesis of atherosclerosis. This study examined the effect of long-term oral estrogen treatment on aortic oxidative stress and atherosclerosis in female apoE(-/-) mice to mimic HRT in humans. Ovariectomized apoE(-/-) mice were given 6 microg/day of oral 17beta-estradiol (E(2)) or control vehicle for 12 weeks. Estrogen treatment reduced atherosclerotic lesions by 38% (E(2): 0.20 +/- 0.01 mm(2)/section; control vehicle: 0.32 +/- 0.02 mm(2)/section) and intima by 32% (E(2): 0.44 +/- 0.02 mm(2)/section; control vehicle: 0.65 +/- 0.04 mm(2)/section) in the aortic root. Serum levels of total and low-density lipoprotein cholesterol were significantly decreased after estrogen treatment. Aortic superoxide anion levels and the expression of NAD(P)H oxidase subunit p22(phox) markedly decreased, and two ROS scavenging enzymes, Cu/ZnSOD and MnSOD, were upregulated after estrogen treatment. Estrogen at physiological concentration inhibited tumor necrosis factor-alpha-stimulated NAD(P)H oxidase activity in both cultured smooth muscle cells and peritoneal macrophages. These results showed that long-term oral estrogen treatment reduces ROS levels and atherosclerosis progression in apoE(-/-) mice. Oral estrogen alters ROS-generating and -scavenging enzyme expression, suggesting that anti-oxidative actions in the vessel wall contribute to atheroprotective effects of estrogen.

Nestin serves as a prosurvival determinant that is linked to the cytoprotective effect of epidermal growth factor in rat vascular smooth muscle cells.

Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H(2)O(2) treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3 and PARP were activated in nestin-depleted VSMCs following H(2)O(2) treatments, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well known that EGF serves as a survival factor in rat VSMCs. Here, we show that the cytoprotective effect of EGF was prevented by nestin RNAi. In addition, the inhibition of Cdk5 prevented Bcl-2 phosphorylation and enhanced H(2)O(2)-induced caspase-3 activation as well as subsequent DNA fragmentation. Taken together, these results provide evidence for another cytoprotective role of EGF in that it is mediated through its stimulation of nestin expression which leads to the prevention of caspase activation by Cdk-5-induced Bcl-2 phosphorylation in rat VSMCs.

Thrombin induces nestin expression via the transactivation of EGFR signalings in rat vascular smooth muscle cells.

Regulation of nestin gene expression is largely unknown despite that it is widely used as a progenitor cell marker. In this study, we showed that nestin expression is regulated by the thrombin-mediated EGFR transactivation in serum-deprived primary cultures of rat vascular smooth muscle cells (VSMCs). This resulted from the direct binding of thrombin to PAR-1 rather than indirectly affecting through the binding to thrombomodulin, as demonstrated by thrombomodulin RNAi. In this process, the PAR-1-induced c-Src plays a critical role through two routes; one was the direct intracellular phosphorylation of EGFR and the other was the extracellular activation of the MMP-2-mediated shedding of HB-EGF. The transactivated EGFR then led to the downstream Ras-Raf-ERK signaling axis, but not the p38 or JNK pathways. In addition, the EMSA experiment showed that the transcriptional factor Sp1 is critical for the thrombin-induced nestin expression in rat VSMCs. Furthermore, RNAi of nestin attenuated the thrombin-induced cell proliferation, indicating that thrombin-induced nestin expression and cell proliferation share the same EGFR transactivation mechanism. This study also suggested that nestin may play an important role in cell proliferation induced by the thrombin-mediated EGFR transactivation.

Ets-1 mediates platelet-derived growth factor-BB-induced thrombomodulin expression in human vascular smooth muscle cells.

AIMS: Thrombomodulin (TM), a potent anticoagulant, is not detected in quiescent vascular smooth muscle cells (VSMCs). In diseased vessels, VSMC expresses TM, but the mechanisms are unclear. This study examined molecular mechanisms for TM expression in VSMCs. METHODS AND RESULTS: Platelet-derived growth factor-BB (PDGF-BB) induced TM expression in cultured human aortic VSMCs. PDGF-induced TM is functional in activating protein C. TM induction was eliminated by inhibitors of Src kinase, phosphatidylinositol 3-kinase (PI3-kinase), and mammalian target of rapamycin (mTOR) and by expressing dominant-negative Akt while expressing active Akt-stimulated TM expression. PDGF-BB activated the TM promoter, and the deletion of a sequence segment -394/-255 drastically reduced TM promoter activity. Transcription factor E26 transformation-specific sequence-1 (Ets-1) was upregulated by PDGF-BB in a PI3-kinase- and mTOR-dependent manner. RNA interference of Ets-1 inhibited PDGF induction of TM, and overexpressing Ets-1 increased TM expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay detected increased Ets-1 binding to the TM promoter after PDGF treatment. Following carotid artery ligation of C57/BL6 mice, PDGF-BB and TM were co-expressed in the media and neointima. CONCLUSION: In VSMCs, PDGF-BB stimulates TM expression that is mainly mediated by Ets-1 via the Src kinase/PI3-kinase/Akt/mTOR signalling pathway. Furthermore, PDGF-BB may regulate TM expression in VSMCs during vascular remodelling.

Epidermal growth factor up-regulates the expression of nestin through the Ras-Raf-ERK signaling axis in rat vascular smooth muscle cells.

The contractile-synthetic phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event during atherosclerosis progression. Although many studies have reported possible cytokines and growth factors implicated to this process, the critical factors affecting the VSMC phenotype remain unclear due to the lack of early de-differentiation marker identifications. In this study, we showed that nestin, an intermediate filament protein, is expressed in primary cultures of rat VSMCs representing the synthetic phenotype and its expression is diminished as these cells re-differentiate after serum deprivation. However, the regulation of nestin expression was never reported despite its common usage as an early differentiation marker. Herein, we showed that nestin expression is regulated by epidermal growth factor (EGF) via de novo RNA and protein synthesis. Furthermore, signaling analyses revealed that the EGF-induced nestin re-expression is mediated through the activation of the Ras-Raf-ERK signaling axis. This is the first report to show that nestin expression is regulated by an extracellular signaling molecule.

Tie2-R849W mutant in venous malformations chronically activates a functional STAT1 to modulate gene expression.

Tie2 is an endothelial receptor tyrosine kinase. An amino-acid substitution of tryptophan for arginine at residue 849 (Tie2-R849W) leads to a ligand-independent activation of its kinase activity. This mutation has been associated with familial venous malformations (VMs), manifested by variable thickness or lack of smooth-muscle cells in the veins of patient lesions. The underlying mechanism for Tie2-R849W action in endothelial cells remains elusive. In this study, we used adenoviral infection to differentiate the effects of ectopic Tie2 (wild type, kinase-dead K855A, or constitutively active R849W) expression on endothelial cellular behaviors and Tie2-mediated downstream targets. Ectopic Tie2 reduced endothelial cell proliferation and serum withdrawal-induced apoptosis, while stimulating migration. When comparing R849W with K855A and its wild-type counterpart, a functional tyrosine kinase activity was required only for migration, and constitutively active Tie2-R849W conferred highest resistance to serum-induced apoptosis, but lowest ability to maintain tube-like structures formed on Matrigel. We further demonstrated that Tie2-R849W chronically induced STAT1 tyrosine phosphorylation and the promoter activity of STAT1-responsive IFN-regulatory factor 1 (IRF1). Although STAT1 phosphorylation required JNK and p38MAPK activation, only JNK activation was essential for IRF1 promoter activation by Tie2-R849W. Additional studies are needed to study the role of STAT1 activation in VMs.

High-dose morphine impairs vascular endothelial function by increased production of superoxide anions.

BACKGROUND: The effects of high-dose morphine on vascular endothelial function have not been previously shown. The authors hypothesized that the pro-oxidant effect of high-dose morphine impairs vascular endothelial function. METHODS: Mice were subjected to placebo or morphine (20 mg/kg intraperitoneal) injection for consecutive 14 days. Aortas were harvested for assessment of vasomotor function by isometric force recordings. Protein expression p47phox was determined by Western blotting. Generations of superoxide anions were detected under a confocal microscope. RESULTS: Compared with controls, contraction response to phenylephrine was significantly enhanced in the aorta of mice treated with high-dose morphine (maximal contractions were 150 +/- 26 vs. 261 +/- 32 mg, respectively; n = 5 or 6, P = 0.04). Endothelium-dependent relaxations to acetylcholine (10 to 10 m) were significantly reduced in morphine-treated animals but were normalized by superoxide scavenging. Fluorescent densities of dihydroethidium were increased in the aorta of morphine-treated mice. Aorta of mice treated with morphine expressed higher levels of p47phox (a major subunit of nicotinamide adenine dinucleotide phosphate oxidase). In cultured endothelial cells, morphine enhanced production of reactive oxygen species. CONCLUSIONS: Collectively, the authors' results showed that high-dose morphine impairs vascular endothelial function via attenuation of biologic activity of endothelium-derived nitric oxide. Chemical antagonism between superoxide anions generated by nicotinamide adenine dinucleotide phosphate oxidases may be the molecular mechanism responsible for the inactivation of endogenous nitric oxide after treatment with high-dose morphine.

The effect of statin on the aortic gene expression profiling.

BACKGROUND: Beyond lipid lowering, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) has been found to have anti-inflammatory and anti-thrombotic effects. However, the genetic expression pattern changes in atherosclerotic lesions produced by statin are rarely studied. METHODS: Cholesterol-fed apolipoprotein (Apo) E-deficient mice were examined for the treatment effect of statin on aortic gene expression. ApoE-deficient mice were fed with a hypercholesterolemic diet started at 8 weeks of age for a total of 22 weeks. In the statin treatment group (n=25), the ApoE-deficient mice were treated with pravastatin (80 mg/kg/day) dissolved in water by daily oral inoculation from 25 to 30 weeks of age. For the control group (n=25), the ApoE-deficient mice were orally inoculated with water only for the same period of time. The aortic gene expression affected by pravastatin was identified using oligonucleotide microarray technology with Agilent gene chips. RESULTS: The total cholesterol and atherosclerotic lesion/total aortic area were significantly lower in the pravastatin treatment group. Microarray analysis of the expression of 20,281 murine genes in the aortas between the two groups indicated that 94 genes were significantly regulated. Thirty genes were up-regulated and 64 genes were down-regulated. The most up-regulated genes were troponin T3, actin alpha1, tubulin alpha1, regulator of G-protein signaling 5 (Rgs5), stathmin-like 2 and myosin light chain kinase. Most of them are related with cytoskeleton organization, while Rgs5 is a G-protein signal transduction molecule. The most down-regulated genes were adenosine deaminase, atrial natriuretic peptide, troponin T2, FXYD domain-containing ion transport regulator 3, and glutathione S-transferase alpha4. CONCLUSIONS: The beneficial effect of the 6-week statin treatment in ApoE-deficient mice is largely dependent on its influence on the cytoskeleton organization. Our study results might provide insight into the clinical benefits of chronic statin treatment.

Rho-kinase-mediated regulation of receptor-agonist-stimulated smooth muscle contraction.

Rho kinase was shown to regulate smooth muscle contraction through modulating myosin phosphatase (MLCP) activity, but the in vivo mechanism remains to be clarified. This study examined the effects of Rho kinase inhibition on the phosphorylation time course of MLCP subunit MYPT1 at Thr697 and Thr855 and MLCP inhibitory protein CPI-17 at Thr38 and on actin polymerization during the contraction of rat tail artery (RTA) smooth muscle. Rho kinase inhibitor Y27632 suppressed force activated by alpha(1)-adrenergic agonist phenylephrine or thromboxane A(2) analog U46619 with concomitant decreases in MLC(20) phosphorylation. Phenylephrine and U46619 significantly increased MYPT1(Thr855) phosphorylation that was eliminated by Y27632 pretreatment, whereas MYPT1(Thr697) phosphorylation was not stimulated. Phenylephrine increased CPI-17(Thr38) phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor Ro 31-8220; in contrast, U46619 did not stimulate CPI-17 phosphorylation. Both agonists increased actin polymerization that was diminished by Y27632 under phenylephrine but not U46619 activation. These results demonstrated a temporal correlation between MYPT1(Thr855) phosphorylation, MLC(20) phosphorylation, and contraction in a Rho-kinase-dependent manner for both phenylephrine and U46619 stimulation, suggesting that Rho kinase regulates MLCP activity through MYPT1(Thr855) phosphorylation during RTA smooth muscle contraction. Furthermore, Rho kinase regulates actin polymerization activated by alpha(1)-adrenoceptors but is less significant in thromboxane receptor stimulation.