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Biomedical subjects

J B Sharefkin

Publications and source records attributed to J B Sharefkin.

At least 19 recordsLinked to original sources
PubMed1994

Both lisinopril and verapamil reduced platelet-derived growth factor-A chain mRNA levels in human saphenous vein endothelial cells stimulated by thrombin.

BACKGROUND: Both angiotensin-converting enzyme inhibitors and calcium channel blockers decrease postinjury intimal thickening in vivo, but their mechanisms of inhibitory action are unclear. Expression of the gene for platelet-derived growth factor (PDGF), a smooth-muscle mitogen, in endothelial cells (ECs) after vessel injury has been postulated to cause intimal thickening. In this study, we tested whether lisinopril, an angiotensin-converting enzyme inhibitor, or verapamil, a calcium channel blocker, would suppress the PDGF gene expression in stimulated human saphenous vein ECs. METHODS: Drugs were added to replicate EC cultures 30 minutes before adding 10 units/ml alpha-thrombin. Changes in PDGF-A chain mRNA levels were measured by Northern blot analysis or reverse transcription-polymerase chain reaction method. PDGF-AA homodimer in conditioned media was measured by ELISA: RESULTS: Lisinopril attenuated the induction by thrombin of PDGF-A chain mRNA levels significantly in human ECs at doses of 10(-6) mol/L and 10(-5) mol/L (p < 0.05) and appeared to decrease PDGF-AA homodimer released in conditioned medium. Verapamil also reduced thrombin induction of PDGF-A chain mRNA levels significantly at a dose of 10(-5) mol/L (p < 0.05) and appeared to reduce PDGF-AA homodimer secretion. CONCLUSIONS: These data suggest that one means by which lisinopril and verapamil both suppress intimal thickening might be inhibition of PDGF-A chain gene expression in ECs regrowing over vessel injury areas that are sites of thrombin generation.

Base Sequence↗
PubMed1993

Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells.

Increased expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A chain, and tissue plasminogen activator (tPA) by smooth muscle cells (SMC) has been postulated to mediate the progression of intimal hyperplasia. We tested whether heparin would suppress the expression of these genes in stimulated human saphenous vein SMC. Quiescent cultured human saphenous vein SMC were stimulated for 4 h with heat-inactivated fetal bovine serum (10% by vol) in the presence or absence of heparin (1 to 250 micrograms/ml). Heparin (50 micrograms/ml) attenuated the induction by serum of bFGF mRNA, tPA mRNA, and tPA secretion. Nonanticoagulant heparin also attenuated serum induction of bFGF and tPA mRNA levels. To further study the role of second messenger signaling, a more specific mode of SMC stimulation was used with thrombin (3 U/ml) in the presence or absence of dibutyryl cyclic AMP (Bu2-cAMP; 0.5 mM). In contrast to heparin, which had no effect on PDGF expression, Bu2-cAMP decreased the induction by thrombin of PDGF-A chain mRNA levels. In thrombin-stimulated SMC, Bu2-cAMP significantly decreased secretion of PDGF-AA protein. Thrombin, however, caused an increase in bFGF mRNA levels which was potentiated by Bu2-cAMP with associated potentiation by Bu2-cAMP of intracellular bFGF protein levels. The induction of tPA mRNA and tPA secretion by thrombin was sharply blocked by Bu2-cAMP. These results suggest that heparin reduces intimal hyperplasia at least partly via partial inhibition of SMC gene expression.

Bucladesine↗
PubMed1992

Enzymatic harvesting of adult human saphenous vein endothelial cells: use of a chemically defined combination of two purified enzymes to attain viable cell yields equal to those attained by crude bacterial collagenase preparations.

Seeding vascular prostheses with enzymatically harvested endothelial cells can create endothelial linings that improve small-caliber prosthetic patency. But crude bacterial collagenases used for endothelial harvest contain cytotoxic nonspecific proteases and clostridial cell wall debris which might limit their clinical usefulness. We therefore compared the endothelial cell harvest efficiency of crude bacterial collagenase with that of purified bacterial collagenase alone, purified trypsin alone, and combinations of purified bacterial collagenase and trypsin using concentrations of pure collagenase equal in collagenolytic activity to the crude bacterial collagenase material. The efficiency of harvest from human saphenous vein segments was measured by a microtiter well-growth curve assay of the number of living endothelial cells capable of attachment to fibronectin and subsequent growth obtained per unit area of saphenous vein lumen. Whereas pure collagenase and purified trypsin alone both harvested less than 5% of the baseline endothelial cell density on the veins, a combination of purified collagenase and 0.01% w/v purified trypsin was found to harvest 22% +/- 10% (SD) (n = 8 veins) of the approximately 1.3 x 10(5) endothelial cells/cm2 available on normal saphenous veins. This figure was not statistically different from the harvest efficiency of 19% +/- 10% (N = 4 veins) (p greater than 0.05) obtained by use of 0.1% w/v crude collagenase alone. This result suggests that endothelial harvesting can be done with a defined mixture of pure enzymes which would be clinically preferable to presently used crude extracts of clostridial cultures as a standardized preparation for graft seeding.

Adult↗
PubMed1992

Hantaan virus infection of human endothelial cells.

The primary pathophysiologic finding of the viral disease known as Korean hemorrhagic fever, the etiological agent of which is Hantaan virus (HTV), is vascular instability. To investigate whether HTV was able to infect cells derived from human vascular tissue and alter their behavior, we infected in vitro primary adult human endothelial cells from saphenous veins (HSVEC). We were able to detect the presence of viral antigens in infected cells both by immunofluorescence and by Western blot (immunoblot) analysis as early as day 1 postinfection. HSVEC infected with HTV produce infectious virus during the first 3 days of infection but, at later times (days 4 to 8), show decreasing yields of virus. This contrasts with the HTV growth pattern observed for the permissive simian CV-7 cell line, which generates infectious virus up to day 12 after infection. Further investigation showed that the late decrease in viral production in HSVEC is the result of the induction of beta interferon and can be reversed by the addition of anti-beta interferon serum to the culture medium. At no time during the course of infection of HSVEC with HTV was any obvious cytopathic effect observed. When tests for changes in mRNA levels of other cytokines and endothelial cell gene products following HTV infection of HSVEC were done by reverse transcription and polymerase chain reaction methods, no significant changes were observed in the levels of interleukin 1, interleukin 6, or von Willebrand factor mRNA. We hypothesize that, while HTV can replicate in human vascular endothelial cells, the mechanism of microvascular damage seen with Korean hemorrhagic fever is not likely to be a direct effect of virus replication but may conceivably be the consequence of an immune-mediated endothelial injury triggered by viral infection.

Antigens, Viral↗
PubMed1991

Evaluation of the role of Shiga and Shiga-like toxins in mediating direct damage to human vascular endothelial cells.

Infection with Shiga toxin- and Shiga-like toxin-producing strains of Shigella dysenteriae and Escherichia coli, respectively, can progress to the hemolytic-uremic syndrome. It has been hypothesized that circulating Shiga toxin, Shiga-like toxins, and endotoxins may contribute to the disease by directly damaging glomerular endothelial cells. The effects of these toxins on HeLa, Vero, and human vascular endothelial cells (EC) were examined. Confluent EC were sensitive to Shiga toxin but were at least 10(6)-fold less sensitive to the toxins than were Vero cells. Shiga toxin was the predominant cytotoxic factor. Lipopolysaccharides were not cytotoxic and did not augment Shiga toxin-mediated toxicity. Lower doses of Shiga toxin caused cytotoxicity when coincubated with tumor necrosis factor. The relative resistance of EC to Shiga toxin and Shiga-like toxins may be due to reduced toxin binding, as low levels of globotriaosylceramide (Gb3), the toxin-specific receptor, were found in EC membranes.

Animals↗
PubMed1991

Effects of aspirin, dipyridamole, and dibutyryl cyclic adenosine monophosphate on platelet-derived growth factor A chain mRNA levels in human saphenous vein endothelial cells and smooth muscle cells.

Aspirin and dipyridamole have frequently failed to control intimal hyperplasia in vascular grafts in animal and clinical trials. These trials were based on the concept that the smooth muscle mitogen, platelet-derived growth factor (PDGF) released from platelets, was a major cause of intimal hyperplasia. Both endothelial and smooth muscle cells (ECs and SMCs), however, can also release PDGF-like SMC mitogens that might cause intimal hyperplasia. We therefore tested whether aspirin and dipyridamole alone or together can affect PDGF-A chain mRNA levels in cultured human saphenous vein ECs and SMCs. Cultures were exposed for 72 hours to 3 x 10(-5) mol/L aspirin and/or 5 x 10(-6) mol/L dipyridamole. Cellular RNA was then extracted, and PDGF-A chain mRNA signal levels were measured by a reverse transcription/polymerase chain reaction method. mRNA for glyceraldehyde-3-phosphate dehydrogenase was used as a constitutively expressed control RNA species. Signal strength on Southern blots of amplified polymerase chain reaction products was measured by densitometry. Neither aspirin nor dipyridamole alone or together reduced the ratio (PDGF-A chain signal/glyceraldehyde-3-phosphate dehydrogenase signal) below that of control cultures. PDGF-A chain expression was not a constitutive artifact of culture because dibutyryl cyclic AMP (5 x 10(-4) mol/L) reduced PDGF-A chain signal from a control index of 1.0 to 0.5 +/- 0.1 (mean +/- SE) (n = 3; p less than 0.05) in EC cultures and to 0.2 (mean) (n = 2) in SMC cultures. These data may explain why aspirin and dipyridamole fail to reduce intimal hyperplasia in some animal and clinical trials despite effective inhibition of platelet aggregation.

Aspirin↗
PubMed1991

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells.

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1990

Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress.

Fluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type-1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvest of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (greater than 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.

Base Sequence↗
PubMed1990

Recombinant DNA and surgery.

We have the ability to isolate DNA from tissue, determine its base-pair sequence, and ask if a gene of interest is present. DNA strands can be isolated from one type of cell or organism, cleaved, and inserted (recombined) with DNA from another cell or organism. Recombinant DNA techniques have already improved health care by providing clinically useful quantities of pure human protein hormones such as erythropoietin, insulin, and growth hormone. Furthermore these techniques may increase our understanding of cellular growth control mechanisms to a level that was previously unattainable. They will also increase our knowledge of the development of major diseases and provide a means of specific nontoxic therapies for these diseases. Surgeons will need to understand basic DNA research terminology to keep up with the revolution in medical therapies that these techniques will cause. Our purpose is to begin the process of linking surgery to DNA.

DNA Probes↗
PubMed1990

Rapid cellular luminal coverage of Dacron inferior vena cava prostheses in dogs by immediate seeding of autogenous endothelial cells derived from omental tissue: results of a preliminary trial.

Endothelial cell seeding methods might reduce the high failure rates of venous vascular prostheses, but low flow rates in venous vascular prostheses impose a need to protect early patency and to attain early endothelial cell coverage without waiting several weeks for relatively small endothelial cell innocula from autologous veins to form confluent linings. To obtain large number of autologous endothelial cells for high-density seeding, canine omental microvascular endothelial cells were harvested by collagenase digestion and density gradient centrifugation, with yields of 1.34 +/- 0.24 (SD) X 10(6) cells/gm of omentum (N = 8 harvests). Primary culture of a subfraction from each harvest showed the cell population to be dominated by factor VIII-related antigen-positive endothelial cells with only a few nonstaining cells (estimated to be 10% or less of total cell number) visible. Freshly harvested omental cells were seeded onto double velour knitted Dacron prostheses at densities of 5 X 10(5) cells/cm2 of graft luminal surface in an autologous plasma suspension by use of prior preclotting with cell-free autologous plasma, followed by endothelial cell seeding in autologous plasma, with plasma recalcification during endothelial cell instillation. Six seeded and two control (sham-seeded) vascular prostheses 5 cm long with 10 mm inner diameter were used as inferior vena cava interposition grafts. A distal arteriovenous fistula and aspirin (300 mg) and dipyridamole (50 mg orally every day) starting 3 days before surgery were used to protect early patency of all grafts. Seeded venous vascular prostheses were explanted for study at intervals of 1,5, and 10 days after surgery (N = 2 prostheses at each time); the two control venous vascular prostheses were explanted at 10 days. All venous vascular prostheses were patent at time of removal. In seeded venous vascular prostheses, light, scanning, and transmission electron microscopy showed emergence of numerous flattened endothelial cell-like cells on the luminal surface 24 hours after surgery, followed by formation of a confluent cellular lining without adherent platelets by 5 to 10 days after surgery. Control venous vascular prostheses, in contrast, remained covered by an irregularly thickened fibrin and red cell thrombus, which sometimes encroached on the lumen. Our results suggest that (1) omental tissue can furnish endothelial cells for high-density immediate seeding of venous vascular prostheses, and (2) that the method we used to combine features of both so-called high density "seeding" and "sodding" techniques offers both more rapid prosthesis coverage than the former and shorter intraoperative times for cell attachment to prostheses than the latter.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1989

Detection of fibronectin on vascular flow surfaces by enzyme-linked immunosorbent assay.

Numerous variables are involved in the attachment of endothelial cells to a substrate. Quantifying these factors both on native vessels and on synthetic substrates is important in determining the success of endothelial cell attachment, retention, and growth on these substrates. Fibronectin is an important cell attachment molecule and is likely to be key to the successful attachment of endothelial cells to any substrate. For this reason we have developed an enzyme-linked immunosorbent assay for interrogation of the luminal surface of native and synthetic vessels for the presence of fibronectin. A plexiglass chamber was designed with two blocks, an upper block with wells and a lower supporting block. The chamber was then assembled with a vessel between the two blocks, forming the bottom of the well. This luminal surface was then interrogated by conventional enzyme-linked immunosorbent assay. Native vessels, collagenase-digested vessels, acellular matrices and PTFE preclotted with whole blood were assayed to determine the quantity of fibronectin present. These results were correlated with a bioassay developed to determine the quantity of fibronectin necessary for cell attachment. It was concluded that all of the samples assayed had ample fibronectin for cell attachment and that other factors must be responsible for successful maintenance of a cell monolayer.

Acid Phosphatase↗
PubMed1989

Reduced reproductive capacity of freshly harvested endothelial cells in smokers: a possible shortcoming in the success of seeding?

In an attempt to explain the failure of first clinical trials of autologous endothelial seeding in smokers, the initial reproductive capacity of saphenous vein endothelial cells from smokers and nonsmokers was studied by a replicate microwell technique. Endothelial cells were enzymatically harvested from saphenous vein segments of patients with coronary bypasses (21 smokers and 18 nonsmokers). After 15 minutes (group A) and 7 minutes (group B) of collagenase exposure, the endothelial cell harvest from donors who smoked was 41% (p less than 0.02) lower for group A and 30% (p less than 0.2) lower for group B than that from nonsmokers. In analogy, the viable cell yield was 32% (p less than 0.04) and 29% (p less than 0.05) lower for groups A and B, respectively, in cultures from donors who smoked. Daily cell counts over an ensuing 10-day period also revealed a significant difference in the proliferative behavior of endothelial cells from smokers and nonsmokers. Whereas endothelial cells from nonsmokers regularly entered the exponential phase of proliferation on day 4.4 +/- 1.8 (group A) and day 4.6 +/- 1.3 (group B), endothelial cells from smokers reached the logarithmic growth phase either with delay (day 6.8 +/- 2.1, group A) or remained completely quiescent (group B). Lower harvest efficiency and suppressed reproductive capacity of endothelial cells in smokers--on top of an already critically low inoculum in single-staged endothelial cell seeding--might explain the failure of initial clinical trials.

Cell Count↗
PubMed1988

Expression of genes for platelet-derived growth factor in adult human venous endothelium. A possible non-platelet-dependent cause of intimal hyperplasia in vein grafts and perianastomotic areas of vascular prostheses.

Neointimal fibromuscular hyperplasia (NFH) in vein grafts and perianastomotic zones of vascular prostheses has been attributed to the effects of platelet-derived growth factor (PDGF) released by platelets interacting with bypass conduits. But inhibition of platelet aggregation often fails to prevent NFH, and recurrent growth of intact, platelet-free endothelium over perianastomotic areas where NFH occurs is inconsistent with the concept of sustained PDGF release from platelets causing NFH progression at late times after surgical procedures. Cultured bovine aortic endothelial cells (ECs) and human umbilical vein ECs have been shown to release a PDGF-like molecule. We report that confluent cultured fourth passage adult human saphenous vein ECs (AHSVECs) grown in the presence of heparin (100 micrograms/ml) and retina-derived growth factor (RDGF) studied by Northern blotting transcribed a messenger ribonucleic acid (mRNA) of 3.9 kb, strongly hybridizing to PDGF B chain probes, and two species of 2.0 and 2.6 kb hybridizing to PDGF A chain probes. Withdrawal of RDGF and heparin from these cultures for 48 hours before mRNA extraction amplified the scanning densitometric mRNA signal per cell by 8.0 +/- 7.6 fold (mean +/- SD) (N = 4 cultures) for B chain mRNA and 5.2 +/- 3.6 fold (N = 3 cultures) for A chain mRNA. In addition, AHSVEC cultures released a PDGF-like substance, because 50% vol/vol AHSVEC-conditioned serum-free medium increased tritiated thymidine uptake elevenfold in PDGF receptor-bearing 3T3 cells whereas an excess (50 micrograms/ml) of nonspecific goat anti-human-PDGF antibody significantly reduced this increase by a mean of 30% to 7.0 +/- 3.4 fold (N = 6 trials, p less than 0.001). Flow cytometry determined AHSVEC cultures to be proliferating with a mean of 6.2% +/- 1.9% (N = 3 culture lines) of ECs in S phase even at confluence when deprived of EC mitogens for 48 hours. Adult human ECs, which proliferate on bypass conduits and host vessels after perioperative injury, may play a role in causing NFH by stimulating proliferation of adjacent smooth muscle cells. Prevention of NFH may require not only antiplatelet agents but also ways to prevent EC release of smooth muscle cell mitogens in response to perioperative EC injury.

Animals↗
PubMed1987

Platelet survival and serotonin content after placement of arterial prostheses in dogs: effects of neointimal coverage and high- and low-dose aspirin.

Because prosthetic neointima produces much less prostacyclin (PGI2) than arterial intima and may be more susceptible to cyclooxygenase inhibition, aspirin treatment might enhance surface thrombogenesis. To test this hypothesis, aortic prostheses were placed in eight dogs and measurements of platelet survival and platelet serotonin (5HT) were made under conditions of no treatment and treatment with low-dose (2mg/kg) and high-dose (30 mg/kg) aspirin. These doses equally suppressed platelet function. Measurements were performed preoperatively, 6 to 8 weeks postoperatively (when little neointima was present), and 28 to 32 weeks postoperatively (neointima fully developed). Platelet survival and 5HT levels were markedly reduced 6 to 8 weeks postoperatively and returned to normal at 28 to 32 weeks after implantation. At all times, low-dose aspirin improved platelet survival and this effect was most apparent 6 to 8 weeks postoperatively. Treatment with either aspirin dose decreased platelet 5HT levels at the 28 to 32 week postoperative period but not at other times. At recovery of prostheses, 90% of the luminal surface was covered with endothelialized neointima. Neointimal production of PGI2 was one half to one third that of aortic production. Despite this, low- and high-dose aspirin equally suppressed PGI2 production from both neointima and aorta. Furthermore, aspirin did not increase labeled platelet uptake on neointima. We conclude that (1) aspirin treatment does not render prosthetic neointima thrombogenic and (2) aspirin alters platelet survival and 5HT levels by mechanisms other than inhibition of platelet and neointima cyclooxygenase.

Animals↗
PubMed1986

The cytotoxic effect of surgical glove powder particles on adult human vascular endothelial cell cultures: implications for clinical uses of tissue culture techniques.

Clinical use of autogenous endothelial cell (EC) seeding of vascular prostheses (VP) would require reliable methods for EC harvest for immediate seeding or primary culture in a hospital or operating room setting. Observation of glove powder particles (GPP) in failed primary adult human saphenous vein EC (AHSVEC) cultures led us to study the effect of surgical GPP on cultured AHSVEC. Addition of GPP to the culture medium of growing ASHVEC cultures reduced the cell counts in a dose-dependent fashion; the mean concentration of GPP required to produce a greater than 50% decrease in cell number was 1.5 +/- 0.8 (SD) X 10(4) GPP/ml (N = 10 experiments), equivalent to a mean dose of 36 micrograms glove powder per milliliter. The effect was seen within 24 hr of addition of GPP and was not due to interference with EC attachment and spreading or to changes in medium osmolality, pH, glucose, electrolyte, Ca2+, or Mg2+ content. Instead, the effect appeared to be due to a filterable toxin added during the final rubber-vulcanizing stage of glove manufacture, since pure cornstarch particles and epichlorhydrin-treated pure cornstarch did not prevent culture growth, whereas 0.2 micron filtrates of medium incubated with GPP taken directly from gloves were lethal. We conclude that filterable cytotoxic substances from GPP may be an avoidable cause of failure in EC seeding of VP, and may affect surgical wound healing as well.

Cell Adhesion↗