Cheng-Ho Tsai

Fluorescent Gold Nanoclusters as a Biocompatible Marker for In Vitro and In Vivo Tracking of Endothelial Cells

We have been investigating the fluorescent property and biocompatibility of novel fluorescent gold nanoclusters (FANC) in human aortic endothelial cells (HAEC) and endothelial progenitor cells (EPC). FANC (50-1000 nmol/L) was delivered into cells via the liposome complex. The fluorescence lasted for at least 28 days with a half-life of 9 days in vitro. Examination of 12 transcripts regulating the essential function of endothelial cells after a 72 h delivery showed that only the vascular cell adhesion molecule 1 and the vascular endothelial cadherin were down-regulated at high concentration (500 nmol/L). In addition, no activation of caspase 3 or proliferating cell nuclear antigens was detected. 3-[4,5-Dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) assay demonstrated that, unlike the markedly suppressed viability in cells treated with quantum dots, FANC had minimal effect on the viability, unless above 500 nmol/L, at which level a minor reduction of viability mainly caused by liposome was found. Tube formation assay showed no impaired angiogenesis in the EPC treated with FANC. In vivo study using hindlimb ischemic mice with an intramuscular injection of FANC-labeled human EPC showed that the cells preserved an angiogenic potential and exhibited traceable signals after 21 days. These findings demonstrated that FANC is a promising biocompatible fluorescent probe.

Age-related alteration of gap junction distribution and connexin expression in rat aortic endothelium.

We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and ≥20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week (p < 0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until ≥20 months of age (≥20 months vs 28 days, p < 0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages (p < 0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.

Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions

Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery (P<0.05). Double-label analysis showed that even though colocalization of connexins to the same gap-junctional spot is a common feature, the extent of colocalization was time dependent (>80% in the intact artery at postinjury day 28 and <70% at postinjury days 7 and 14, P<0.01). We conclude that distinct alterations in expression of the 3 connexins are associated with regeneration of the arterial endothelium in situ, implying different intercellular communication requirements during the various phases of the healing process.

Reduced expression of endothelial connexin37 and connexin40 in hyperlipidemic mice: recovery of connexin37 after 7-day simvastatin treatment.

Objective—We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results—Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P <0.01). After simvastatin treatment, Cx40 remained depressed, but Cx37 recovered to 94% of the level found in non–cholesterol-fed animals (P <0.01). Electron microscopy demonstrated that gap junctions were smaller in animals fed the CED compared with those given simvastatin and with controls fed NC (P <0.01). Endothelial connexins were rare in the atherosclerotic plaques of apoE-deficient mice. Conclusions—Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.

Connexin37 gene polymorphism and coronary artery disease in Taiwan.

BACKGROUND A recent study shows that a C1019T polymorphism of the gene encoding the gap junction protein connexin37 contributes to the genesis of atherosclerotic plaques in human carotid artery. However, whether such a polymorphism can be used as a prognostic marker in atherosclerotic disease of other arterial sites, such as coronary artery disease, is not known. METHODS We analyzed the allelic status in 177 subjects with coronary artery disease (age, 61+/-11 years; male/female, 120/57) and 102 controls (60+/-11 years; male/female, 70/32). Both groups were matched, before genotype analysis, for a variety of other traditional risk factors, including body mass index, smoking status, levels of blood pressure, sugar, creatinine, and lipid profiles, in addition to age and sex. RESULTS The T allele was less frequently seen in the control group, compared to the disease group (10.7 vs. 20.1%, TT+TC vs. CC, P<0.01). Subsequent analysis demonstrated that a significant difference existed in the male (9.2 vs. 22.8%, TT+TC vs. CC, P<0.005), but not in the female. Another finding was that the T allele frequency in all participants was less than 15%, markedly lower than that reported in non-Taiwanese. CONCLUSIONS The observation indicates that the polymorphism in the connexin37 gene potentially plays a role in the manifestation of coronary atherosclerosis in Taiwan.

Epicardial adipose tissue relating to anthropometrics, metabolic derangements and fatty liver disease independently contributes to serum high-sensitivity C-reactive protein beyond body fat composition: a study validated with computed tomography.

BACKGROUND Epicardial adipose tissue (EAT) measured by echocardiography has been proposed to be associated with metabolic syndrome and increased cardiovascular risks. However, its independent association with fatty liver disease and systemic inflammation beyond clinical variables and body fat remains less well known. METHODS The relationships between EAT and various factors of metabolic derangement were retrospectively examined in consecutive 359 asymptomatic subjects (mean age, 51.6 years; 31% women) who participated in a cardiovascular health survey. Echocardiography-derived regional EAT thickness from parasternal long-axis and short-axis views was quantified. A subset of data from 178 randomly chosen participants were validated using 16-slice multidetector computed tomography. Body fat composition was evaluated using bioelectrical impedance from foot-to-foot measurements. RESULTS Increased EAT was associated with increased waist circumference, body weight, and body mass index (all P values for trend = .005). Graded increases in serum fasting glucose, insulin resistance, and alanine transaminase levels were observed across higher EAT tertiles as well as a graded decrease of high-density lipoprotein (all P values for trend <.05). The areas under the receiver operating characteristic curves for identifying metabolic syndrome and fatty liver disease were 0.8 and 0.77, with odds ratio estimated at 3.65 and 2.63, respectively. In a multivariate model, EAT remained independently associated with higher high-sensitivity C-reactive protein and fatty liver disease. CONCLUSIONS These data suggested that echocardiography-based epicardial fat measurement can be clinically feasible and was related to several metabolic abnormalities and independently associated fatty liver disease. In addition, EAT amount may contribute to systemic inflammation beyond traditional cardiovascular risks and body fat composition.

Down-regulating effect of nicotine on connexin43 gap junctions in human umbilical vein endothelial cells is attenuated by statins

We investigated the effect of nicotine on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). We also evaluated whether the effect requires activation of acetyl choline receptors sensitive to nicotine (nAChRs) and is altered by statins. The results showed that expression of Cx43 protein is reduced by nicotine in a dose-dependent manner (6 x 10(-4) M nicotine vs control, 33% reduction, p < 0.01), though Cx43 mRNA is up-regulated (6 x 10(-4) M nicotine vs control, 36% increase, p < 0.01). Concomitantly, the communication function, determined by fluorescence recovery after photobleaching, is decreased (6 x 10(-4) M nicotine vs control, 38% reduction, p < 0.05). Such a down-regulation of Cx43 gap junctions by nicotine disappears in the presence of the nAChRs antagonist, dihydro-beta-erythroidine, and protease inhibitors leupeptin plus N-acetyl-Leu-Leu-Norleu-al (ALLN). Similarly, the effect of nicotine is attenuated by statins, including fluvastatin, lovastatin, pravastatin, and simvastatin, even at the presence of mevalonate. We concluded that i) nicotine down-regulates Cx43 expression and gap-junctional communication in HUVEC via post-transcriptional modification, which involves enhancement of Cx43 proteolysis; ii) the effect of nicotine is mediated via activation of nAChRs; and iii) the effect of nicotine is attenuated by statins through mechanisms outside the hypolipidemic pathway.

Reduction of connexin43 in human endothelial progenitor cells impairs the angiogenic potential

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.

G protein β3 subunit variant and essential hypertension in Taiwan — a case–control study

Recent studies have shown that a C825T polymorphism of the gene encoding the G protein β3 subunit contributes to the genesis of essential hypertension. However, the link between the gene and blood pressure is not consistently found in different populations. The aim of the present study is to investigate this issue in Taiwan. We analyzed the allelic status in 302 hypertensive (age, 60±11 years; male/female, 136/166) and 199 normotensive subjects (62±15 years; male/female, 90/109). Our result showed that the T allelic was more frequently seen in the hypertensive group than the normotensive, but the difference did not reach statistic significance (56.5 vs. 54.3%, P>0.1). Subsequent analysis demonstrated a similar trend in the female (58.7 vs. 53.7%, P>0.1) but a reverse trend in the male (53.7 vs. 55%, P>0.1). Another finding was that the T allele frequency in all the groups was over 50%, markedly higher than those reported in whites. In conclusion, the observation suggests that the polymorphism in the G protein gene is not likely to play an important role in the manifestation of high blood pressure in Taiwan.

Late Development of Renal Arteriovenous Fistula Following Gunshot Trauma A Case Report

A 43-year-old man presenting with symptoms of congestive heart failure, cardiomegaly, and impaired left ventricular (LV) function was diagnosed as having a huge left renal arteriovenous (AV) fistula. The AV fistula might be attributed to a gunshot wound suffered during his military service twenty years ago. Percutaneous transcatheter arterial embolization utilizing multiple spring coils in conjunction with cyanoacrylic glue success fully occluded the fistula, with subsequent improvement of LV function and reduction of LV size on his serial echocardiographic follow-up.