Fibrinolytic proteins in apoptotic human umbilical vein endothelial cells

Endothelial cells express fibrinolytic proteins including: urokinase (u-PA) and tissue type (t-PA) plasminogen activators, type-1 (PAI-1) and 2 (PAI-2) plasminogen activator inhibitors, and u-PA receptor (u-PAR). Apoptotic endothelial cells detach, potentially forming both local and circulating microthrombi in vivo. In this article, apoptotic human umbilical vein endothelium was obtained by serum starvation and compared with nonapoptotic cells rescued from death with fresh medium containing serum. Antigen levels for t-PA, PAI-1, PAI-2, and u-PAR were reduced greatly in apoptosis (p< 0.05). In contrast, u-PA levels were similar in apoptotic as compared with rescued cells (p<0.05). Radioactive amino acids were used to determine absolute levels of protein synthesis and degradation in these cells. Reduced antigen levels likely were due to proteolysis as there was 98% total protein degradation and very little protein synthesis in apoptotic endothelial cells. Also, u-PA levels in apoptotic endothelial cells were not affected by the protein synthesis inhibitor cycloheximide. Endothelial cells in inflammatory sites are exposed to cytokines, which increase both apoptosis and u-PA levels. Data from this article support the idea that maintained u-PA levels in apoptotic endothelium may protect from micro-thrombosis in inflammatory sites as well as in the circulation.     

Characterization of endothelin receptors in the human umbilical artery and vein

The aim of the present study was to characterize pharmacologically endothelin receptors that are present in human umbilical vessels. 2. Endothelin-1 (ET-1) and endothelin-2 (ET-2) are potent stimulants of both the human umbilical artery (pEC50 7.9 and 7.5) and vein (pEC50 8.1 and 8.0). Endothelin-3 (ET-3) is inactive on the artery but contracts the vein (pEC50 7.6). IRL1620 is inactive in both vessels. The order of potency of agonists is suggestive of a typical ET(A) receptor in the artery (ET-1 = ET-2 > > ET-3) and a mixture of ET(A) and ET(B) receptors in the vein (ET-1 = ET-2 > or = ET-3). 3. The selective ET(A) receptor antagonist, BQ123, competitively inhibits the effect of ET-1 in the human umbilical artery (pA2 6.9), while in the vein, only a mixture of BQ123 and BQ788 (a selective ET(B) antagonist) weakly displaces to the right of the cumulative concentration-response curve to ET-1. Contractions induced by ET-3 in the vein are inhibited by BQ788 (pA2 7.6), but not by BQ123. 4. Inhibition of Ca2+ channels by nifedipine (0.1 microM) is accompanied by a significant decrease of the maximal response to ET-1 by 40% in the artery and by 30% in the vein. The response of the vein to ET-3 is almost abolished by nifedipine. 5. The results indicate that: (i) endothelins contract the human isolated umbilical artery via stimulation of an ET(A) receptor type; (ii) the contraction induced by ET-1 in the vein is mediated by both ET(A) and ET(B) receptors, while ET-3 stimulates the ET(B) receptor; (iii) the contribution of Ca2+ channels to the contraction mediated by the ET(B) receptor appears to be more important than to that mediated by the ET(A) receptor.     

Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Recanalized umbilical vein in portal hypertension

Experience with splenoportography suggests that patency of the umbilical vein occurs in about 9% of the patients with portal hypertension. A widely patent umbilical vein might serve as a decompressive portosystemic shunt. Percutaneous transhepatic portography was performed in 107 patients with cirrhosis of the liver and portal hypertension. A patent umbilical vein was found in 28 patients (26%). This finding significantly paralleled the number and size of other collateral veins, apart from gastroesophageal varices. No significant relation was found between umbilical vein patency and portal pressure, extrahepatic shunting, variceal bleeding, or ascites. It is concluded that a large patent umbilical vein does not effectively relieve portal hypertension, prevent gastroesophageal varices, or protect against variceal bleeding or ascites.     

Protein C activation and factor Va inactivation on human umbilical vein endothelial cells

The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10(5) cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg506 and Arg306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg506 was accelerated in the presence of HUVECs, while cleavage at Arg306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg506, generating an M(r) 75,000 species. Further proteolysis at Arg306 generated an M(r) 30,000 product. When protein C (0.5 mumol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10(-14) mol/min per cell was observed. With APC generated in situ, cleavage at Arg506 on the HUVEC surface is followed by cleavage at Arg306, generating M(r) 75,000 and M(r) 30,000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M(r) 97,000 fragment, which is further processed by APC to generate an M(r) 43,000 fragment. NH2-terminal sequence analysis of the M(r) 97,000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M(r) 97,000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg506 followed by a second cleavage at Arg306. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.     

Effects of estrogen on tight junctional resistance in cultured human umbilical vein endothelial cells

OBJECTIVE: To study the effects of estrogen on transendothelial paracellular permeability in women. METHODS: Human umbilical vein endothelial cells (HUVEC) obtained from women were grown on filters. The paracellular permeability characteristics were determined in terms of changes in the permeability to the polar acid pyranine (Ppyr) and as changes in the transendothelial electrical resistance (RTE). Tight junctional resistance characteristics were assayed by lowering luminal NaCl and measuring the dilution potential, and were expressed as the ratio of monoion mobility uCl/uNa (cation selectivity). RESULTS: Low extracellular calcium and hyperosmolarity increased Ppyr and decreased RTE. The former but not the latter condition abolished the endothelium-specific cation selectivity. Treatment with 10 nM of estradiol-17 beta had no effect on RTE, but it increased the cation selectivity. The effect of estradiol required 1-6 hours' incubation with the hormone; it was dose dependent and saturable, with a median effective concentration of estradiol of 1 nM. Diethylstilbestrol, but not estriol, could mimic the effect of estradiol, and the estrogen receptor antagonist ICI-182, 780 blocked it. CONCLUSION: Cultured HUVEC cells form patent tight junctions. Estrogens increase the cation selectivity across HUVEC cultures. The effect of estrogen may be mediated by an estrogen receptor. These effects may be important for vasculoprotection in cases of sudden changes in ions levels across the capillary wall, such as ischemia or reperfusion.     

Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia

OBJECTIVE: To determine whether stretch-activated cation channels (SAC) are present in intact human umbilical vein endothelium (HUVE) and in an endothelial cell line (EA.hy) and whether they act as endothelial mechanosensors, and to determine whether endothelial SAC in HUVE from women with pregnancies complicated by preeclampsia undergo functional changes compared with those in HUVE from women with normotensive pregnancies. METHODS AND RESULTS: By use of the patch-clamp technique we identified a SAC in intact HUVE and in an endothelial cell line. The SAC had mean conductances of 29+/-5 pS (n = 38) for K+ and 12+/-2 pS (n = 4) for Ca2+. Administration of 50 micromol/I gadolinium, a blocker of mechanosensitive ion channels, completely blocked activity of this channel. We found from single-channel recordings that influx of Ca2+ through SAC directly activated high-conductance Ca2+-dependent potassium channels, proving that a significant influx of Ca2+ through SAC occurs at physiologic concentrations of Ca2+. In a comparative study, apparent channel density of SAC (percentage of patches with SAC activity) in HUVE from women with pregnancies complicated by preeclampsia (36.2 +/- 4.3%) was twofold higher than that in HUVE from women with normal pregnancies (17.9+/-2.9%, P< 0.01). Channel conductance and sensitivity to stretching of SAC were not altered by preeclampsia. CONCLUSIONS: Since SAC are capable of acting as endothelial mechanosensors, the greater than normal density of SAC associated with preeclampsia might reflect an alteration of mechanotransduction.     

Gestational hypertension plasma and human umbilical vein endothelial cells: an in vitro study

A case of metastatic dermatofibrosarcoma protuberans (DFSP) in a 47-year-old woman is presented. Dermatofibrosarcoma protuberans occasionally recurs, but rarely metastasizes. The patient underwent local removal of the nuchal tumor by a general practitioner, followed by a rapid recurrence. She underwent total removal of the tumor and a diagnosis of spindle cell sarcoma was made after an incisional biopsy was performed. This lesion had both a typical DFSP-like area and a fibrosarcoma (FS)-like area. After 7 years, an abnormal lung shadow was observed and a segmental lung resection was performed. Histologically, the lung tumor was similar to the FS-like area in the nuchal tumor. Confirming CD34 expression in the tumor cells, this lung tumor was diagnosed as metastatic DFSP. Usually CD34 expression is unique to DFSP but almost negative in FS-like areas. In the present case, the FS-like area in the nuchal tumor showed decreased CD34 reactivity, as previously reported, but the FS-like area in the metastatic tumor still widely preserved CD34 expression. The presented case suggests that the FS-like area in DFSP is histogenetically different from typical FS or malignant fibrous histiocytoma.     

Late occlusion of microvascular vein grafts in replantation

Two cases are described in which patients presented 16 and 17 years, respectively, after complete or incomplete amputation/replantation of the arm. In case 1, the patient complained of coldness, pain, and tingling in the replanted arm in the previous 24 hours and noticed that his fingers had gone white. Arteriography and subsequent surgery revealed obliteration of the vein graft (inserted in the distal brachial artery) by neointimal thickening and atherosclerotic plaque, which was confirmed in a subsequent morphologic examination. In case 2, the patient presented with discomfort and a pulsatile swelling on the inner aspect of his upper arm. Arteriography and surgery revealed an aneurysm in the previously inserted vein graft in the brachial artery, with some atherosclerotic degeneration. Both vein grafts were successfully replaced with a fresh autologous vein graft and the patients remain well several years later. The 2 cases suggest that as part of replantation surgery of a limb, it is essential to maintain postoperative clinical monitoring for signs of graft degeneration in all patients with long-term vein graft insertion.     

Surgical treatment of threatened reversed infrainguinal vein grafts

PURPOSE: Current information concerning the results of surgical revision of threatened infrainguinal vein grafts is largely limited to in situ conduits. Infrainguinal grafts may be threatened by intrinsic graft lesions or significant stenosis in the adjacent inflow or outflow arteries. To assess the results of operative revision of infrainguinal reversed vein grafts, we reviewed our experience with surgical revision of threatened infrainguinal reversed vein grafts identified through a program of postoperative clinical and vascular laboratory graft surveillance. METHODS: All patients who underwent surgical revision of a threatened but patent infrainguinal reversed vein graft from January 1987 through April 1993 were identified through review of our vascular registry. Data were analyzed for type of vein used, date of original reversed vein graft, clinical and vascular laboratory findings leading to reversed vein graft revision, results of preoperative angiography, patient risk factors, operative techniques and complications, and long-term assisted primary graft patency and limb salvage. RESULTS: Ninety-six patients with 100 infrainguinal reversed vein grafts (69) femoral-popliteal, 31 femoral-tibial) underwent 117 surgical vein graft revisions or inflow procedures during the study period. Eighty-one percent of the original reversed vein grafts consisted of a single segment of greater saphenous vein. All revised grafts had at least a 50% stenosis in the graft itself or the proximal or distal artery. A single revision was performed in 85 grafts, two revisions in 13 grafts, and three revisions in two grafts. There were nine (8%) isolated inflow procedures, eight (7%) vein patch angioplasties, 62 (53%) interposition vein grafts, and 29 (25%) vein graft extensions to a new distal anastomotic site. The remaining nine (8%) procedures consisted of combinations of the above. Median time to primary graft revision after initial graft implantation was 15 months (range 2 days to 316 months). Mean time to secondary revision after primary revision was 21 months. Operative mortality was 0.9%. Cumulative assisted primary patency of the original grafts revised for stenotic lesions was 99%, 96%, and 92% at 1, 3, and 5 years, respectively. Limb salvage was 99%, 97%, and 97% at 1, 3, and 5 years, respectively. CONCLUSIONS: Although surgical revision of reversed vein graft requires much use of alternative vein sources, these procedures can be performed with minimum mortality and provide excellent assisted primary graft patency and limb salvage.     

Release of cytokines from human umbilical vein endothelial cells treated with platinum compounds in vitro

Endothelial cells (EC) produce cytokines, such as interleukin (IL)-1, IL-6, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF). These cytokines have an important role in the proliferation and differentiation of hematopoietic progenitor cells. On the other hand, anticancer agents generally cause hematopoietic disorders. However, little is known about the effects of chemotherapeutic agents on the secretion of cytokines from EC. Therefore, we investigated if treatment with platinum compounds may stimulate EC to secrete cytokines. EC newly isolated from a human umbilical vein were exposed to cisplatin, carboplatin, or TRK-710 for 80 min, then the cells were washed and placed in fresh medium. The levels of cytokines in the fresh medium were measured by the ELISA method, the levels of intracellular hydrogen peroxide (H2O2) were measured by flow cytometry, and the rhodamine 123-stained live mitochondria of the EC were observed under a confocal laser microscope. Platinum compounds induced cytokine production in human EC: cisplatin most prominently induced the release of IL-1 and IL-6, and TRK-710 had the greatest ability to induce the release of GM-CSF. Intracellular H2O2 production and IL-8 release were transiently induced immediately after treatment with platinum compounds, leading to IL-1 release when H2O2 production was eliminated. These results may provide new insights into the hematological toxicity induced by anticancer agents and the role of IL-1 and IL-6 secreted from EC in this toxicity.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca2+ ([Ca2+]i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca2+, mobilised Ca2+ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca2+ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow "pacemaker" and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca2+]i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca(2+)-free solution, but could be returned to a higher level if the cell was exposed to external Ca2+. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca(2+)-containing medium reactivated responses. In the absence of external Ca2+, continuous application of histamine activated a series of transient increases in intracellular Ca2+, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca2+ stores and their sensitivity to cytoplasmic Ca2+ and intrasarcoplasmic reticulum Ca2+.     

Composite polytetrafluroethylene/vein bypass grafts: conventional distal vein segment or vein cuff?

Faecal samples from asymptomatic dairy cows and calves from a farm on the Island Falster, Denmark, were examined by a sucrose gradient flotation technique. Giardia cysts were found in 7.6% of the 92 samples, and estimated cyst excretion rates ranged from 50-200 cysts per gram faeces. Given that Giardia has the potential to cause clinical disease in cattle and to be transmitted to other animal species and humans, finding the parasite in cattle may be of major epidemiological significance. Future work should focus on elucidating the pathogenicity, transmission patterns and the genetic structure of Giardia populations in cattle in Denmark.     

Heat shock provides delayed protection against oxidative injury in cultured human umbilical vein endothelial cells

During both mild and severe ischemia, vascular endothelial cells lining large and small vessels of the ischemic organ are exposed to oxygen-derived free radicals resulting in oxidative damage to the organ. Heat shock has been shown to induce thermotolerance and also protect against ischemic injury, possibly via increased synthesis of heat shock proteins (HSPs). We hypothesized that heat shock preconditioning may protect human endothelial cells against oxidative damage. Cultured human umbilical vein endothelial cells (HUVEC) were subjected to heat shock (42 degrees C, 1 h) and allowed to recover for 2 or 20 h, at which times the cells were oxidatively stressed for 1 h by exposing them to 100-200 mumol/l of hydrogen peroxide (H2O2). Cellular damage was assessed immediately and 18 h later by morphology and release of lactate dehydrogenase (LDH). No protection of HUVEC was seen using the 2-hour recovery interval, but a significant protection (P < 0.05) was observed after the 20-hour delay. Northern blot analysis at 1 and 2 h after heating showed induction of HSP-70 mRNA. Western blot analysis demonstrated a significant increase in HSP-72 protein after 2 as well as 20 h of recovery from heat shock, although the amounts of protein at the two times were not significantly different. Furthermore, no differences in the activity of the antioxidant enzyme catalase were observed between heated and unheated HUVEC at 2 and 20 h after heat preconditioning. Thus, heat shock preconditioning induces delayed protection against oxidative injury in HUVEC, and the mechanism of protection appears to involve more than the expression of HSP-72 or activity of catalase.