Isolation and In Vitro Culture of Vascular Endothelial Cells from Mice

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidate the disease mechanism. Although the mouse model has many advantages for in vivo and in vitro research, efficient procedures for the isolation and propagation of primary mouse EC have been problematic. We describe a high yield process for isolation and in vitro culture of primary EC from mouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle of Willis). Mouse arteries were carefully dissected without damage under a light microscope, and small pieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growth supplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetal calf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity of the cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized by a monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers for von-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Our simple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 microM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca2+ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca2+ concentration ([Ca2+]i), and the increase of [Ca2+]i by OxLDL or by LPC was inhibited by La3+ or heparin. LPC failed to increase [Ca2+]i in the presence of an antioxidant tempol. In addition, store-operated Ca2+ entry (SOC), which was evoked by intracellular Ca2+ store depletion in Ca2+-free solution using the sarcoplasmic reticulum Ca2+ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca2+]i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La3+ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca2+ to 1 micrometer activated large-conductance Ca2+-activated K+ (BKCa) current spontaneously, and this activated BKCa current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca2+-permeable Ca2+-activated NSC current and BKCa current simultaneously, thereby increasing SOC.

Human Caliciviruses in Korea: A New Prevalent Group Defined by RNA-Dependent RNA Polymerase Diversity

Human caliciviruses (HuCVs) cause sporadic cases and outbreaks of acute gastroenteritis (AGE). Three maior genogrovps of HuCVs have been described including the Norwalk virus (NV)-, the Snow Mountain virus (SMA)-, and the Sapporo-genogroups. This study describes the detection and genetic variation of HuCVs from hospitalized infants with AGE in Korea by RT-PCR and sequencing. The cDNA fragments of 206 to 470bp corresponding to the region of 3 primer pairs (36/35, 35/51 or 3/51) in the polymerase region of NV were generated. Of 185 stools screened, 8% were positive by RT-PCR and their sequences showed that all strains contained the GLPSG and YGDD motifs which are conserved for HuCVs. Amino acid (aa) sequence analysis showed that these strains can be divided into 3 maior genogroups. High conservation was observed in that one strain shares 100% of as sequence with Southampton virus, another shares 99% with the Sapporo virus, and six strains share 90 to 95% with Snow Mountain virus. However, significant sequence variation was also found in other strains. This study indicates that all maior genogroups of HuCVs are circulating in Korea.

Genotypic Variations among Human Calciviruses in Korea: 1987-1994

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8Flla/68/US, with exceptions of HuCV185/87/Korea and HuCV1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV185/87/Korea and HuCV1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.