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.

Association of the vascular endothelial damage and estrogen, progesterone / 대한산부인과학회지

OBJECTIVE: The aim of this study were to examine the serum level of estradiol, estriol, progesterone, oxidized LDL in preeclamtic patients and to evaluate the protective effects of estrogen and progesterone against lysophosphatidylcholine (LPC) induced cell death in Human umbilical vein endothelial cells (HUVECs). METHODS: We analysed the serum level of estradiol, estriol, progesterone, oxidized LDL in patients with preeclampsia and control. We used LPC to induce cell death in HUVECs. For cytotoxic assay, we did LDL assay for cell death and Resazurin assay for cell viability. HUVECs were exposed to various concentrations of LPC, LPC+estrogen, LPC+progesterone and we did cytotoxic assay. RESULTS: The serum estradiol, estriol were lower in the preeclamptic patients (P<0.05). Oxidized LDL were higher in the preeclamptic patients(P<0.05). LPC induced cell death in a concentration-dependant manner. Estrogen or progesterone inhibited LPC-induced cell death in a concentration-dependant manner (P<0.05). CONCLUSION: Estrogen and progesterone attenuated LPC-induced cytotoxicity. The results suggest that Oxidized LDL induced endothelial damage in preeclampsia may be induced by low serum estradiol, estriol and progesterone levels and prevented by estrogen and progesterone addition.

Nitric Oxide-Induced Intracellular Ca2+ Modulation in Macrovascular Endothelial Cells

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) reduces the intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells, whereas the effect of NO on [Ca2+]i in endothelial cells is still controversial. Therefore, the effect of NO on the [Ca2+]i, and its mechanism in mouse aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) were examined. MATERIALS AND METHODS: In primary cultured MAEC and HUVEC, cells were loaded with fura 2-AM and [Ca2+]i and measured using a microfluorometer. RESULTS: The NO donor, sodium nitroprusside (SNP), reduced the [Ca2+]i in 72% of the cells tested (n=100). In the remaining cells, the effect of SNP was biphasic, or the [Ca2+]i was increased. In addition, the membrane-permeable cGMP, 8-bromo cGMP, decreased the [Ca2+]i. The effects of SNP and 8-bromo cGMP were inhibited by the soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and the cGMP-dependent protein kinase inhibitor, KT5823, respectively. In contrast, in the presence of 8-bromo cGMP or ODQ, SNP increased the [Ca2+]i. CONCLUSION: These results suggest that NO inhibits the [Ca2+]i through a cGMP-dependent mechanism and increases the [Ca2+]i through a cGMP-independent mechanism.

Regulatory Mechanism of Vascular Contractility by Extracellular K+ : Effect on Endothelium-Dependent Relaxation and Vascular Smooth Muscle Contractility / 대한흉부외과학회지

BACKGROUND: Extracellular K+ concentration ([K+]o) can be increased within several mM by the efflux of intracellular K+. To investigate the effect of an increase in [K+]o on vascular contractility, we attempted to examine whether extracellular K+ might modulate vascular contractility, endothelium-dependent relaxation (EDR) and intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells (EC). MATERIAL AND METHOD: We observed isometric contractions in rabbit carotid, superior mesentery, basilar arteries and mouse aorta. [Ca2+]i was recorded by microfluorimeter using Fura-2/AM in EC. RESULT: No change in contractility was recorded by the increase in [K+]o from 6 to 12 mM in conduit artery such as rabbit carotid artery. whereas resistant vessels, such as basilar and branches of superior mesenteric arteries (SMA), were relaxed by the increase. In basilar artery, the relaxation by the increase in [K+]o from 1 to 3 mM was bigger than that by the increase from 6 to 12 mM. In contrast, in branches of SMA, the relaxation by the increase in [K+]o from 6 to 12 mM is bigger than that by the increase from 1 to 3 mM. Ba2 (30microM) did not inhibit the relaxation by the increase in [K+]o from 1 to 3 mM but did inhibit the relaxation by the increase from 6 to 12 mM. In the mouse aorta without the endothelium or treated with NG-nitro-L-arginine (30microM), nitric oxide synthesis blocker, the increase in [K+]o from 6 to 12 mM did not change the magnitude of contraction induced either norepinephrine or prostaglandin F2alpha. The increase in [K+]o up to 12 mM did not induce contraction of mouse aorta but the increase more than 12 mM induced contraction. In the mouse aorta, EDR was completely inhibited on increasing [K+]o from 6 to 12 mM. In cultured mouse aorta EC, [Ca2+]i was increased by acetylcholine or ATP application and the increased [Ca2+]i was reduced by the increase in [K+]o reversibly and concentration-dependently. In human umbilical vein EC, similar effect of extracellular K+ was observed. Ouabain, a Na+-K+ pump blocker, and Ni2 , a Na+-Ca2+ exchanger blocker, reversed the inhibitory effect of extracellular K+. CONCLUSION: In resistant arteries, the increase in [K+]o relaxes vascular smooth muscle and the underlying mechanisms differ according to the kinds of the arteries; Ba2 -insensitive mechanism in basilar artery and Ba2 -sensitive one in branches of SMA. It also inhibits [Ca2+]i increase in EC and thereby EDR. The initial mechanism of the inhibition may be due to the activation of Na+-K+ pump.

Increase of Intracellular Ca2+ Concentration Induced by Lysophosphatidylcholine in Murine Aortic Endothelial Cells

Effects of oxidized low-density lipoprotein (ox-LDL), l-alpha-stearoyl-lysophosphatidylcholine (LPC), on intracellular Ca2+ concentration were examined in mouse endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i) with fura 2-AM and reverse transcription-polymerase chain reaction (RT-PCR). LPC increased [Ca2+]i under the condition of 1.5 mM [Ca2+]o but did not show any effect under the nominally Ca2+-free condition. Even after the store depletion with 30microM 2,5-di-tert- butylhydroquinone (BHQ) or 30microM ATP, LPC could still increase the [Ca2+]i under the condition of 1.5 mM [Ca2+]o. The time required to increase [Ca2+]i (about 1 minute) was longer than that for ATP-induced [Ca2+]i increase (10-30 seconds). LPC-induced [Ca2+]i increase was completely blocked by 1microM La3+. Transient receptor potential channel(trpc) 4 mRNA was detected with RT-PCR. From these results, we suggest that LPC increased [Ca2+]i via the increase of Ca2+ influx through the Ca2+ routes which exist in the plasma membrane.

Reoxygenation stimulates EDRF(s) release from endothelial cells of rabbit aorta

We have reported that hypoxia stimulates EDRF(s) release from endothelial cells and the release may be augmented by previous hypoxia. As a mechanism, it was hypothesized that reoxygenation can stimulate EDRF(s) release from endothelial cells and we tested the hypothesis via bioassay experiment. In the bioassay experiment, rabbit aorta with endothelium was used as EDRF donor vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F2a (3 X 10-6 M) which was added to the solution perfusing through the aorta. Hypoxia was evoked by switching the solution aerated with 95% O2/5% CO2 mixed gas to one aerated with 95% N2/5% CO2 mixed gas Hypoxia/reoxygenation were interexchanged at intervals of 2 minutes (intermittent hypoxia). In some experiments, endothelial cells were exposed to 10-minute hypoxia (continuous hypoxia) and then exposed to reoxygenation and intermittent hypoxia. In other experiments, the duration of re oxygenation was extended from 2 minutes to 5 minutes. When the donor aorta was exposed to intermittent hypoxia, hypoxia stimulated EDRF(s) release from endothelial cells and the hypoxia-induced EDRF(s) release was augmented by previous hypoxia/reoxygenation. When the donor aorta was exposed to continuous hypoxia, there was no increase of hypoxia-induced EDRF(s) release during hypoxia. But, after the donor aorta was exposed to reoxygenation, hypoxia-induced EDRF(s) release was markedly increased. When the donor aorta was pretreated with nitro-L-arginine (10-5 M for 30 minutes), the initial hypoxia-induced EDRF(s) release was almost completely abolished, but the mechanism for EDRF(s) release by the reoxygenation and subsequent hypoxia still remained to be clarified. TEA also blocked incompletely hypoxia-induced and hypoxia/reoxygenation-induced EDRF(s) release EDRF(s) release by repetitive hypoxia and reoxygenation was completely blocked by the combined treatment with nitro-L-arginine and TEA. Cytochrome P450 blocker, SKF-525A, inhibited the EDRF(s) release reversibly and endothelin antgonists, BQ 123 and BQ 788, had no effect on the release of endothelium-derived vasoactive factors. Superoxide dismutase (SOD) and catalase inhibited the EDRF(s) release from endothelial cells. From these data, it could be concluded that reoxygenation stimulates EDRF(s) release and hypoxia/reoxygenation can release not only NO but also another EDRF from endothelial cells by the production of oxygen free radicals.

Different mechanisms for K+-induced relaxation in various arteries

(K+)o can be increased under a variety of conditions including subarachnoid hemorrhage. The increase of (K+)o in the range of 5 ~ 15 mM may affect tensions of blood vessels and cause relaxation of agonist-induced precontracted vascular smooth muscle (K+-induced relaxation). In this study, effect of the increase in extracellular K+ concentration on the agonist-induced contractions of various arteries including resistant arteries of rabbit was examined, using home-made Mulvany-type myograph. Extracellular K+ was increased in three different ways, from initial 1 to 3 mM, from initial 3 to 6 mM, or from initial 6 to 12 mM. In superior mesenteric arteries, the relaxation induced by extracellular K+ elevation from initial 6 to 12 mM was the most prominent among the relaxations induced by the elevations in three different ways. In cerebral arteries, the most prominent relaxation was produced by the elevation of extracellular K+ from initial 1 to 3 mM and a slight relaxation wasp rovoked by the elevation from initial 6 to 12 mM. In superior mesenteric arteries, K+-induced relaxation by the elevation from initial 6 to 12 mM was blocked by Ba2+ (30 muM) and the relaxation by the elevation from 1 to 3 mM or from 3 to 6 mM was not blocked by Ba2+. In cerebral arteries, however, K+-induced relaxation by the elevation from initial 3 to 6 mM was blocked by Ba2+, whereas the relaxation by the elevation from 1 to 3 mM was not blocked by Ba2+. Ouabain inhibited all of the relaxations induced by the extracellular K+ elevations in three different ways. In cerebral arteries, when extracellular K+ was increased to 14 mM with 2 or 3 mM increments, almost complete relaxation was induced at 1 or 3 mM of initial K+ concentration and slight relaxation occurred at 6 mM. TEA did not inhibit Ba2+/-sensitive relaxation at all and NMMA or endothelial removal did not inhibit K+-induced relaxation. Most conduit arteries such as aorta, carotid artery, and renal artery were not relaxed by the elevation of extracellular K+. Among conduit arteries, trunk of superior mesenteric artery and basilar artery were relaxed by the elevations of (K+)o. These data suggest that K+-induced relaxation has two independent components, Ba2+-sensitive and Ba2+-insensitive one and there are different mechanisms for K+-induced relaxation in various arteries.

Characteristics of Ca2+ stores in rabbit cerebral artery myocytes

In a myocyte freshly isolated from rabbit cerebral artery, the characteristics of Ca2+ release by histamine or caffeine were studied by microspectrofluorimetry using a Ca2+-binding fluorescent dye, fura-2. Histamine (5 micrometer) or caffeine (10 mM) induced a phasic rise of cytoplasmic free Ca2+ concentration ((Ca2+)c) which could occur repetitively with extracellular Ca2+ but only once or twice in Ca2+-free bathing solution. Also, the treatment with inhibitor of sarcoplasmic reticulum Ca2+-ATPase suppressed the rise of (Ca2+)c by histamine or caffeine. In Ca2+-free bathing solution, short application of caffeine in advance markedly attenuated the effect of histamine, and vice versa. In normal Ca2+-containing solution with ryanodine (2 micrometer), the caffeine-induced rise of (Ca2+)c occurred only once and in this condition, the response to histamine was also suppressed. On the other hand, in the presence of ryanodine, histamine could induce repetitive rise of (Ca2+)c while the amplitude of peak rise became stepwisely decreased and eventually disappeared. These results suggest that two different Ca2+-release mechanisms (caffeine-sensitive and histamine-sensitive) are present in rabbit cerebral artery myocyte and the corresponding pools overlap each other functionally. Increase of (Ca2+)c by histamine seems to partially activate ryanodine receptors present in caffeine-sensitive pool.

Hypoxia-induced EDNO release is further augmented by previous hypoxia and reoxygenation in rabbit aortic endothelium

The present study was designed: (1) to determine whether or not hypoxia stimulates the release of endothelium-derived relaxing factors (EDRFs) from endothelial cells, and (2) to examine whether or not the hypoxia-induced EDRFs release is further augmented by previous hypoxia-reoxygenation, using vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F2alpha, (3 X 10-6 M/L), which was added to the solution perfuming through the aortic segment. Hypoxia was evoked by switching the solution aerated with 95% 02/5% CO2 mixed gas to one aerated with 95% N2/5% CO2 mixed gas. When the contraction induced by prostaglandin F2alpha reached a steady state, the solution was exchanged for hypoxic one. And then, hypoxia and reoxygenation were interchanged at intervals of 2 minutes (intermittent hypoxia). The endothelial cells were also exposed to single 10-minute hypoxia (continuous hypoxia). When the bioassay ring was superfused with the perfusate through intact aorta, hypoxia relaxed the precontracted bioassay test ring markedly. Whereas, when bioassay ring was superfused with the perfusate through denuded aorta or polyethylene tubing, hypoxia relaxed the precontracted ring slightly. The relaxation was not inhibited by indomethacin but by nitro-L-arginine or methylene blue. The hypoxia-induced relaxation was further augmented by previous hypoxia-reoxygenation and the magnitude of the relaxation by intermittent hypoxia was significantly greater than that of the relaxation by continuous hypoxia. The results suggest that hypoxia stimulates EDNO release from endothelial cells and that the hypoxia-induced EDNO release is further augmented by previous hypoxia-reoxygenation.

Sensitivity of rabbit cerebral artery to serotonin is increased with the moderate increase of extracellular K+

(K+)O can be increased under a variety of conditions including subarachnoid hemorrhage. The increase of (K+)O in the range of 5~15 mM may affect tensions of blood vessels and can change their sensitivity to various vasoactive substances. Therefore, it was examined in the present study whether the sensitivity of cerebral arteries to vasoactive substances can be changed with the moderate increase of (K+)O, using Mulvany-type myograph and (Ca2+)c measurement. The contractions of basilar artery and branch of middle cerebral artery induced by histamine were not increased with the elevation of (K+)O from 6 mM to 9 mM or 12 mM. On the contrary, the contractions induced by serotonin were significantly increased with the elevation of (K+)O. The contractions were also significantly increased by the treatment with nitro-L-arginine (10-4 M for 20 minutes). In the nitro-L-arginine treated arteries, the contractions induced by serotonin were significantly increased with the elevation of (K+)O from 6 mM to 12 mM. K+-induced relaxation was evoked with the stepwise increment of extracellular K+ from 0 or 2 mM to 12 mM by 2 mM in basilar arterial rings, which were contracted by histamine. But (K+)O elevation from 4 or 6 mM to 12 mM by the stepwise increment evoked no significant relaxation. Basal tension of basilar artery was increased with (K+)O elevation from 6 mM to 12 mM by 2 mM steps or by the treatment with ouabain and the increase of basal tension was blocked by verapamil. The cytosolic free Ca2+ level was not increased by the single treatment with serotonin or with the elevation of (K+)O from 4 mM to 8 or 12 mM. In contrast to the single treatment, the Ca2+ level was increased by the combined treatment with serotonin and the elevation of (K+)O. The increase of free Ca2+ concentration was blocked by the treatment with verapamil. These data suggest that the sensitivity of cerebral artery to serotonin is increased with the moderate increase of (K+)O and the increased sensitivity to serotonin is due to the increased (Ca2+)i induced by extracellular Ca2+ influx.

Effects of arachidonic acid on the calcium channel current (I|B|a) and on the osmotic stretch-induced increase of I|B|a in guinea-pig gastric myocytes

We employed the whole-cell patch clamp technique to investigate the effects of arachidonic acid (AA) on barium inward current through the L-type calcium channels (IBa) and on osmotic stretch-induced increase of IBa in guinea-pig antral gastric myocytes. Under isosmotic condition, AA inhibited IBa in a dose-dependent manner to 91.1 +/- 1.4, 72.0 +/- 3.2, 46.0 +/- 1.8, and 20.3 +/- 2.3% at 1, 5, 10, 30 mM, respectively. The inhibitory effect of AA was not affected by 10 micrometer indomethacin, a cyclooxygenase inhibitor. Other unsaturated fatty acids, linoleic acid (LA) and oleic acid (OA) were also found to suppress IBa but stearic acid (SA), a saturated fatty acid, had no inhibitory effect on IBa. The potency sequence of these inhibitory effects was AA (79.7 +/- 2.3%) > LA (43.1 +/- 2.7%) > OA (14.2 +/- 1.1%) at 30 mM. On superfusing the myocyte with hyposmotic solution (214 mOsm) the amplitude of IBa at 0 mV increased (38.0 +/- 5.5%); this increase was completely blocked by pretreatment with 30 mM AA, but not significantly inhibited by lower concentrations of AA (1, 5 and 10 micrometer) (P > 0.05). Unsaturated fatty acids shifted the steady-state inactivation curves of IBa to the left; the extent of shift caused by AA was greater than that caused by LA. The activation curve was not affected by AA or LA. The results suggest that AA and other unsaturated fatty acids directly modulate L-type calcium channels and AA might modulate the hyposmotic stretch-induced increase of L-type calcium channel current in guinea-pig gastric smooth muscle.

Acting mechanisms of extracellular Ca2+ and Ca2+-antagonists on endothelium-derived relaxing factor in rabbit aorta / 대한흉부외과학회지

No abstract available.