Hung-I Yeh

Synthesis, Characterization, and Bioconjugation of Fluorescent Gold Nanoclusters toward Biological Labeling Applications

Synthesis of ultrasmall water-soluble fluorescent gold nanoclusters is reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Specific staining of cells and nonspecific uptake by living cells is demonstrated.

Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system.

Gap junctions play essential roles in the normal function of the heart and arteries, mediating the spread of the electrical impulse that stimulates synchronized contraction of the cardiac chambers, and contributing to co‐ordination of activities between cells of the arterial wall. In common with other multicellular systems, cardiovascular tissues express multiple connexin isotypes that confer distinctive channel properties. This review highlights how state‐of‐the‐art immunocytochemical and cellular imaging techniques, as part of a multidisciplinary approach in gap junction research, have advanced our understanding of connexin diversity in cardiovascular cell function in health and disease. In the heart, spatially defined patterns of expression of three connexin isotypes—connexin43, connexin40, and connexin45—underlie the precisely orchestrated patterns of current flow governing the normal cardiac rhythm. Derangement of gap junction organization and/or reduced expression of connexin43 are associated with arrhythmic tendency in the diseased human ventricle, and high levels of connexin40 in the atrium are associated with increased risk of developing atrial fibrillation after coronary by‐pass surgery. In the major arteries, endothelial gap junctions may simultaneously express three connexin isotypes, connexin40, connexin37, and connexin43; underlying medial smooth muscle, by contrast, predominantly expresses connexin43, with connexin45 additionally expressed at restricted sites. In normal arterial smooth muscle, the abundance of connexin43 gap junctions varies according to vascular site, and shows an inverse relationship with desmin expression and positive correlation with the quantity of extracellular matrix. Increased connexin43 expression between smooth muscle cells is closely linked to phenotypic transformation in early human coronary atherosclerosis and in the response of the arterial wall to injury. Current evidence thus suggests that gap junctions in both their guises, as pathways for cell‐to‐cell signaling in the vessel wall and as pathways for impulse conduction in the heart, contribute to the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease. Microsc. Res. Tech. 52:301–322, 2001.

Individual Gap Junction Plaques Contain Multiple Connexins in Arterial Endothelium

Gap-junctional intercellular communication in endothelial cells is implicated in the coordination of growth, migration, and vasomotor responses. Up to 3 connexin types, connexin40 (Cx40), Cx37, and Cx43 may be expressed in vascular endothelium according to vascular site, species, and physiological conditions. To establish how these connexins are organized at the level of the individual endothelial gap junction, we used affinity-purified connexin-specific antibodies raised in 3 different species to permit double and triple immunolabeling in combination with confocal and electron microscopy. Using HeLa cells transfected with Cx37 and Cx40 for characterization, the anti-Cx37 antibody (raised in rabbit) and the anti-Cx40 antibody (raised in guinea pig) were shown to recognize single bands of 37 and 40 kDa, respectively, on Western blots and to give prominent punctate labeling at the cell borders, specifically in the corresponding transfectant. By applying these antibodies together with mouse monoclonal anti-Cx43 for double and triple immunofluorescence labeling at confocal microscopy, rat aortic and pulmonary arterial endothelia were found to express all 3 connexin types, whereas coronary artery endothelium expressed Cx40 and Cx37 but lacked Cx43. High-resolution en face confocal viewing of the aortic endothelium after double labeling demonstrated frequent colocalization of connexins, with distinct variation in the expression pattern within a given cell, where it made contact with different neighbors. Triple immunogold labeling at the electron-microscopic level revealed that aortic endothelial gap junctions commonly contain all 3 connexin types. This represents the first definitive demonstration of any cell type in vivo expressing 3 different connexins organized within the same gap-junctional plaque.

Connexin45, a Major Connexin of the Rabbit Sinoatrial Node, Is Co-expressed with Connexin43 in a Restricted Zone at the Nodal-Crista Terminalis Border

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

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.

Upregulation of Connexin43 Gap Junctions Between Smooth Muscle Cells After Balloon Catheter Injury in the Rat Carotid Artery

Phenotypic transformation of smooth muscle cells (SMCs) to the synthetic state in vitro and in human coronary atherosclerosis is reported to be associated with upregulation of connexin43 gap junctions. To determine whether cellular interactions mediated by gap junctions participate in the phenotypic transformation of SMCs in arterial injury and disease in general and to establish the spatial and temporal pattern of any such change in relation to neointimal development, we investigated SMC connexin43 gap junction expression during vascular healing in the rat carotid artery after balloon catheter injury. Quantitative immunoconfocal microscopy was applied to localize and to quantify connexin43 gap junctions 1, 3, 9, and 14 days after injury. Parallel studies were conducted by electron microscopy (direct morphological demonstration of SMC gap junctions) and immunoconfocal microscopy (localization of altered actin expression). Synthetic-state SMCs in the neointima (first apparent from 9 days postinjury) revealed abundant expression of gap junctions, with levels of immunodetectable connexin43 threefold greater than those of medial cells. However, the first detectable changes were found in the media, before neointimal formation; at 1 to 3 days postinjury, an increase in SMC gap junction expression was apparent in the innermost (subluminal) zone, the major site from which the cells subsequently found in the neointima are recruited. We conclude that upregulation of connexin43 gap junctions is intimately linked to SMC phenotypic transition and that interactions mediated by gap junctions may be a hitherto unrecognized contributor to the cellular mechanisms underlying the vascular response to injury.

Gap Junction Localization and Connexin Expression in Cytochemically Identified Endothelial Cells of Arterial Tissue

Vascular endothelial cells interact with one another via gap junctions, but information on the precise connexin make-up of endothelial gap junctions in intact arterial tissue is limited. One factor contributing to this lack of information is that standard immunocytochemical methodologies applied to arterial sections do not readily permit unequivocal localization of connexin immunolabeling to endothelium. Here we introduce a method for multiple labeling with specific endothelial cell markers and one or more connexin-specific antibodies which overcomes this limitation. Applying this method to localize connexins 43, 40, and 37 by confocal microscopy, we show that the three connexin types have quite distinctive labeling patterns in different vessels. Whereas endothelial cells of rat aorta and coronary artery characteristically show extensive, prominent connexin40, and heterogeneous scattered connexin37, the former, unlike the latter, also has abundant connexin43. The relative lack of connexin43 in coronary artery endothelium was confirmed in both rat and human using three alternative antibodies. In the aorta, connexins43 and 40 commonly co-localize to the same junctional plaque. Even within a given type of endothelium, zonal variation in connexin expression was apparent. In rat endocardium, a zone just below the mitral valve region is marked by expression of greater quantities of connexin43 than surrounding areas. These results are consistent with the idea that differential expression of connexins may contribute to modulation of endothelial gap junction function in different segments and subzones of the arterial system.

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.