Kou Gi Shyu

Intramyocardial injection of naked DNA encoding HIF-1α/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat

OBJECTIVES The therapeutic utility of hypoxia-inducible factor-1 (HIF-1) transcriptional regulatory system for ischemic hindlimb has been demonstrated. It is not yet known whether this transcriptional regulatory system can be used as a therapeutic strategy to enhance collateral vessel formation in myocardial tissues, where acute hypoxia occurs due to inadequate perfusion. We aimed to test the hypothesis that exogenous administration of HIF-1alpha/VP16 could enhance collateral vessel formation in a rat acute myocardial infarction model. METHODS Sprague-Dawley rats received ligation of the proximal left anterior descending coronary artery to induce acute myocardial infarction. Immediately after the ligation, 50 microg total plasmid DNA (control, plasmid encoding human vascular endothelial growth factor (pVEGF(165)), or pHIF-1alpha/VP16) was injected into the infarct area at three locations. RESULTS Reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of HIF-1alpha and VEGF mRNA in the myocardium, but not in other organs at days 3 and 7. The infarct size significantly decreased from 37+/-4% (control) to 24+/-2% in the VEGF-treated group and 23+/-2% in the HIF-1alpha/VP16 treated group (P<0.05). Capillary density also significantly increased from 550+/-75/mm(2) (control) to 850+/-75/mm(2) in the VEGF group and 850+/-50/mm(2) in the HIF-1alpha/VP16-treated group (P<0.01). Combined therapy with HIF-1alpha/VP16 and VEGF resulted in higher capillary density (1230+/-50/mm(2)) than treatment with either therapy alone. Regional myocardial blood flow was also higher in the treated groups than in the control. Plasma levels of VEGF were also significantly higher in the HIF-1alpha/VP16 and VEGF-treated group than in the control group. CONCLUSIONS The HIF-1alpha/VP16 hybrid transcription factor is able to reduce infarct size and enhance neovascularization in an acute ischemic myocardium. The potency of VEGF and HIF-1alpha/VP16 hybrid as therapeutic angiogenic factors in acute hypoxic myocardium is similar.

Intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia

We sought to investigate the safety and efficacy of intramuscular gene therapy with vascular endothelial growth factor (VEGF) in patients with chronic critical leg ischemia.Gene transfer was performed in 24 limbs of 21 patients with rest pain, some of whom also had nonhealing ischemic ulcers (n = 16) due to occlusive peripheral arterial disease. Between 400 microg and 2000 microg of phVEGF(165) (400 microg, n = 2; 800 microg, n = 4; 1200 microg, n = 4; 1600 microg, n = 6; and 2000 microg, n = 8) was injected directly into the muscles of the ischemic limb; the same dose was injected 4 weeks later. The ratio of blood pressures at the ankle and brachial artery was measured before and after treatment. Mean (+/- SD) plasma levels of VEGF increased significantly from 26 +/- 31 pg/mL to 63 +/- 56 pg/mL (P <0.005), and the ankle-brachial index improved significantly from 0.58 +/- 0.24 to 0.72 +/- 0.28 (P <0.001). Magnetic resonance angiography showed qualitative evidence of improved distal flow in 19 limbs (79%). Ischemic ulcers healed or improved markedly in 12 limbs (75%). Rest pain was relieved or improved markedly in 20 limbs (83%). Amputation was performed in two limbs because of wound infection. Complications were limited to transient leg edema in six limbs. Intramuscular gene therapy with VEGF(165) for patients with chronic critical leg ischemia is safe, feasible, and effective.

Serum levels of intercellular adhesion molecule-1 and E-selectin in patients with acute ischaemic stroke

Abstract To investigate the role of circulating intercellular adhesion molecule-1 (ICAM-1) and E-selectin in ischaemic stroke, serum levels of ICAM-1 and E-selectin were measured by ELISA in 51 patients with acute ischaemic stroke within 24 h, and in 25 age-matched healthy controls and 10 young healthy volunteers. Carotid Doppler ultrasonography showed a significant stenosis (>50%) of the carotid or vertebrobasilar artery in 11 of 51 stroke patients. Serum levels of ICAM-1 [mean (SE)] were higher (P <0.01) in patients with ischaemic stroke [381 (30) ng/ ml] than in age-matched controls [271 (27) ng/ml] and young controls [246 (6) ng/ml]. There was no significant difference in serum E-selectin levels [mean (SE)] among stroke patients, age-matched and young controls [47 (6), 39 (3), and 41 (3) ng/ ml, respectively; P = NS]. The leucocyte count [mean (SD)] was higher (P < 0.01) in patients with ischaemic stroke [8310 (2800)] than in age-matched controls [6040 (930)]. Serum levels of ICAM-1 and E-selectin did not significantly differ between patients with or without abnormal carotid or vertebrobasilar artery disease. In conclusion, serum ICAM-1 level and leucocyte count were elevated in acute ischaemic stroke within 24 h, while the E-selectin level did not change significantly. This finding suggests that adhesion molecules may play an important role in the post-rolling process of leucocyte-endothelial cell interaction in acute ischaemic stroke.

Sesamin induces nitric oxide and decreases endothelin-1 production in HUVECs: Possible implications for its antihypertensive effect

Objective Sesamin has been proved to be antihypertensive. Nitric oxide (NO) is the most important vascular relaxing factor that is regulated in endothelium. Endothelin-1 (ET-1) is characterized as a potent vasoconstrictor and is also regulated in endothelium. Alterations in the endothelial production of NO and ET-1 are known to correlate with hypertension. This study investigated the effect of sesamin on NO and ET-1 in the human umbilical vein endothelial cells (HUVECs). Design The concentrations of NO and ET-1 in the medium of HUVECs treated by sesamin were measured. The mRNA and protein expressions of nitric oxide synthase (NOS), endothelin converting enzyme-1 (ECE-1), and endothelin-1 (ET-1) were also investigated. Other than the mRNA and protein expression, NOS activity and cyclic GMP (cGMP) were detected. Methods The NO concentration was detected by colorimetric assay. The cGMP and ET-1 were analyzed by EIA. The eNOS, ECE-1, and ET-1 mRNA expressions were assayed by Northern blot. The eNOS and ECE-1 protein expressions were analyzed by Western blot. The NOS activity was assayed by detecting the level of [H3]-1-citrullin transformed from [H3]-1-arginine. Results Sesamin not only increased the NO concentration in the medium of HUVECs in a dose-dependent manner after 24 h, but also induced eNOS mRNA and protein expressions. NOS activity in the HUVECs was also induced by sesamin. The content of cGMP was induced by sesamin through NO signaling. On the other hand, the ET-1 concentration in the medium of HUVECs treated by sesamin was suppressed in a dose-dependent manner after 24 h. The ECE-1 protein and mRNA expressions were also inhibited by sesamin. However, the mRNA expression of prepro ET-1 was not influenced by sesamin. Conclusion From the above results, it is suggested that sesamin may improve hypertension by its ability to induce NO and inhibit ET-1 production from endothelial cells. The increase of NO by sesamin is through the induction of eNOS gene expression. The decrease of ET-1 by sesamin is through the inhibition of ECE gene expression, but is not through the inhibition of prepro ET-1 gene expression.

The diagnostic accuracy of 256-row computed tomographic angiography compared with invasive coronary angiography in patients with suspected coronary artery disease

AIMS To assess the diagnostic accuracy of 256-row computed tomographic angiography (CTA) in patients with suspected coronary artery disease (CAD). Non-invasive imaging of the coronary artery by CTA has increasingly been used in recent years. The accuracy of 256-row CTA has not yet been studied. We sought to assess the accuracy of 256-row CTA compared with invasive coronary angiography (ICA) in the diagnosis and assessment of CAD. METHODS AND RESULTS We prospectively evaluated 104 consecutive individuals who accepted CTA and then underwent ICA. The presence of stenosis > or =50% was considered obstructive. The diagnostic accuracy of CTA for detecting obstructive stenosis was compared with that of ICA. The area under the receiver-operating-characteristic curve (AUC) was used to evaluate the diagnostic accuracy of CTA relative to ICA. A total of 86 patients had obstructive CAD. The patient-based analysis of CTA for detecting stenosis > or =50% according to ICA revealed an AUC of 0.744 [95% confidence interval (CI), 0.572-0.916], with a sensitivity of 98.8%, a specificity of 50%, a positive predictive value (PPV) of 92.4%, and a negative predictive value (NPV) of 87.5%. The segment-based analysis revealed an AUC of 0.915 (95% CI, 0.847-0.982), with a sensitivity of 93.5%, a specificity of 95%, a PPV of 77.6%, and an NPV of 98.7%. The vessel-based analysis revealed an AUC of 0.887 (95% CI, 0.808-0.966), with a sensitivity of 94.3%, a specificity of 87.3%, a PPV of 82.7%, and an NPV of 95.9%. CONCLUSION 256-Row CTA is a highly sensitive test of CAD and has a high predictive value. 256-Row CTA may be a potential alternative to detect coronary artery stenosis and rule out CAD in suspected patients.

Regulation of Discoidin Domain Receptor 2 by Cyclic Mechanical Stretch in Cultured Rat Vascular Smooth Muscle Cells

Discoidin domain receptor 2 (DDR2) plays potential roles in the regulation of collagen turnover mediated by smooth muscle cells in atherosclerosis. How mechanical stretch affects the regulation of DDR2 in smooth muscle cells is not fully understood. We sought to investigate the cellular and molecular mechanisms of regulation of DDR2 by cyclic stretch in smooth muscle cells. Rat vascular smooth muscle cells grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased DDR2 protein and mRNA expression after stretch. Cyclic stretch also significantly increased DNA–protein binding activity of Myc-Max. Addition of SB203580, transforming growth factor-β1 (TGF-β1) monoclonal antibody, p38 small interfering RNA (siRNA), and c-myc siRNA 30 minutes before stretch inhibited the induction of DDR2 protein and abolished the DNA–protein binding activity induced by cyclic stretch. Cyclic stretch increased, whereas SB203580 abolished the phosphorylated p38 protein. Conditioned medium from stretched smooth muscle cells and exogenous administration of angiotensin II and TGF-β1 recombinant proteins to the nonstretched cells increased DDR2 protein expression similar to that seen after stretch. In conclusion, cyclic mechanical stretch enhances DDR2 expression in cultured rat smooth muscle cells. The stretch-induced DDR2 is mediated by angiotensin II and TGF-β1, at least in part, through p38 mitogen-activated protein kinase and Myc pathway.

Mechanism of the inhibitory effect of atorvastatin on endoglin expression induced by transforming growth factor‐β1 in cultured cardiac fibroblasts

Transforming growth factor‐β1 (TGF‐β1) and endoglin play a causal role in promoting cardiac fibrosis. Atorvastatin has been shown to have an inhibitory effect on cardiac fibroblasts in vitro. However, the effects of statins on TGF‐β1 and endoglin are poorly understood. We therefore sought to investigate the molecular mechanisms of atorvastatin on endoglin expression after TGF‐β1 stimulation in cardiac fibroblasts.

Acute hypoxia enhances proteins' S-nitrosylation in endothelial cells

Hypoxia-induced responses are frequently encountered during cardiovascular injuries. Hypoxia triggers intracellular reactive oxygen species/nitric oxide (NO) imbalance. Recent studies indicate that NO-mediated S-nitrosylation (S-NO) of cysteine residue is a key posttranslational modification of proteins. We demonstrated that acute hypoxia to endothelial cells (ECs) transiently increased the NO levels via endothelial NO synthase (eNOS) activation. A modified biotin-switch method coupled with Western blot on 2-dimensional electrophoresis (2-DE) demonstrated that at least 11 major proteins have significant increase in S-NO after acute hypoxia. Mass analysis by CapLC/Q-TOF identified those as Ras-GTPase-activating protein, protein disulfide-isomerase, human elongation factor-1-delta, tyrosine 3/tryptophan 5-monooxygenase activating protein, and several cytoskeleton proteins. The S-nitrosylated cysteine residue on tropomyosin (Cys 170) and beta-actin (Cys 285) was further verified with the trypsic peptides analyzed by MASCOT search program. Further understanding of the functional relevance of these S-nitrosylated proteins may provide a molecular basis for treating ischemia-induced vascular disorders.

Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes.

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [3H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

Acute hypoxia to endothelial cells induces activating transcription factor 3 (ATF3) expression that is mediated via nitric oxide

Endothelial cells (ECs) play an important role in hypoxia-induced vascular disorders. We investigated the acute hypoxia effect on endothelial expression of activating transcription factor 3 (ATF3), a stress-inducible transcription factor playing significant roles in cellular responses to stress. Bovine aortic ECs were subjected to acute hypoxia (1% O(2), pO(2)=8 mmHg) and ATF3 expression was examined. ECs exposed to hypoxia transiently induced ATF3 expression. A transient increase in the activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in ECs was observed; however, only ECs pretreated with a specific inhibitor to JNK suppressed the hypoxia-induced ATF3 expression. ECs exposed to acute hypoxia transiently increased endothelial nitric oxide (eNOS) activity. Pre-treating ECs with a specific inhibitor to eNOS (l-NAME) or PI3-kinase significantly inhibited the hypoxia-induced JNK activation and ATF3 expression. ATF3 induction has been shown to inhibit matrix metalloproteinase-2 (MMP-2) expression. Consistently, ECs exposed to hypoxia attenuated the MMP-2 expression. This hypoxia-attenuated MMP-2 expression can be rescued by pre-treating ECs with an inhibitor of eNOS. These results suggest that the ATF3 induction by acute hypoxia is mediated by nitric oxide and the JNK pathway in ECs. Our findings provide a molecular basis for the mechanism in which ECs respond to acute hypoxia.