Kazuhiro Okahara

Upregulation of Prostacyclin Synthesis–Related Gene Expression by Shear Stress in Vascular Endothelial Cells

Prostacyclin (prostaglandin I2, PGI2) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI2 production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI2 upregulation, we examined the gene expression of the enzymes involved in PGI2 production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm2) loading. The production of 6-keto-PGF1alpha, a stable metabolite of PGI2, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A2 expression, as detected by reverse transcription-polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI2 synthase was also found to be upregulated. These results suggest that the elevated PGI2 production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI2 synthase.

Shear stress induces expression of CNP gene in human endothelial cells

To elucidate the effect of blood flow on gene transcription of C‐type natriuretic peptide (CNP), human umbilical vein endothelial cells were exposed to shear stress in a cone‐plate viscometer. Expression of CNP mRNA, evaluated by reverse transcription‐polymerase chain reaction, was markedly increased by exposure to shear stress of 24 dyne/cm2 at 3 h. The CNP mRNA level was maintained until 12 h. Thus, the present study demonstrated for the first time that shear stress induces expression of CNP gene in human endothelial cells.

Shear stress down‐regulates gene transcription and production of adrenomedullin in human aortic endothelial cells

Vascular endothelial cells are potent modulators of vascular tone in response to shear stress. Levels of vasoactive peptides such as adrenomedullin (AM), endothelin‐1 (ET‐1), C‐type natriuretic peptide (CNP), and nitric oxide (NO) are affected by fluid shear stress. AM, a potent vasodilator and suppressor of smooth muscle cell proliferation, contains the shear stress responsive element (SSRE) “GAGACC” in its promoter region. To examine the role of AM in the shear stress response, cultured human aortic endothelial cells (HAoECs) were exposed to fluid shear stresses of 12 and 24 dynes/cm2 in a cone‐plate shear stress loading apparatus for various time periods, and the levels of AM gene expression and peptide secretion from HAoECs were measured by Northern blotting analysis and radioimmunoassay (RIA), respectively. Both AM gene transcription and AM peptide levels were down‐regulated by fluid shear stress in a time‐ and magnitude‐dependent manner. Our results demonstrate that the normal level of arterial shear stress down‐regulates AM expression in HAoECs, suggesting that AM participates in the modulation of vascular tone by fluid shear stress. J. Cell. Biochem. 71:109–115, 1998.

Possible Involvement of m-Calpain in Vascular Smooth Muscle Cell Proliferation

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.

Hemostasis activation during sclerotherapy of lower extremity varices

The influence of compression sclerotherapy upon hemostasis activation was investigated in 41 consecutive patients with lower extremity varices by serial measurement of thrombin-antithrombin III complexes (TAT), D-dimer, fibrinogen and C-reactive protein (CRP). Blood sampling was carried out before operation and on the 7th and 28th post-operative day in patients randomly assigned to either the control group (n = 18), in which high ligation of sapheno-femoral junction and local excision of varices were performed, or the sclerotherapy group (n = 23) in which the comparable surgical intervention and compression sclerotherapy using hypertonic saline were performed simultaneously. In both groups, the TAT, D-dimer and fibrinogen concentrations at day 7 were significantly elevated compared to the value before operation while CRP showed no significant change during the observation period. In the sclerotherapy group, higher incidence of superficial thrombosis was observed and the TAT concentration at day 7 was significantly higher than that in the control group (p < 0.01), and the TAT at day 28 was still significantly elevated compared to the pre-operative level (p < 0.05). However, no relationship between TAT and D-dimer concentrations and the extent of superficial thrombosis was observed. We conclude that compression sclerotherapy for lower extremity varices causes latent activation of coagulation system and can be a risk factor for venous thromboembolism.

Expanded Polytetrafluoroethylene (ePTFE) Vascular Graft Loses Its Thrombogenicity Six Months after Implantation

Although expanded polytetrafluoroethylene (ePTFE) has been believed to be an inert material for vascular prosthesis, it shows less tendency of graft maturation by means of endothelialization. The aim of this study is to evaluate long-term alteration in thrombogenicity of ePTFE grafts after implantation. Serial levels of thrombin-antithrombin III complex (TAT), D-dimer, C-reactive protein (CRP), and prothrombin time (PT) were examined in 77 patients following ePTFE Y-graft implantation for up to five years. TAT showed biphasic elevation after implantation; TAT increased from 16.4+/-8.6 ng/ml to 27.4+/-10.5 ng/ml at one day, decreased to 18.5+/-4.5 ng/ml at one week, and increased again to 25.3+/-8.5 ng/ml at two weeks. Elevated TAT gradually decreased after the second peak to reach a lower level than that before surgery after six months. There was no significant difference in TAT level after six months due to the difference in diagnosis or anti-thrombotic therapy. We suggest that ePTFE grafts lose their thrombogenicity six months after implantation, after which anti-thrombotic therapy might be unnecessary.

An infection-resistant PTFE vascular graft; spiral coiling of the graft with ofloxacin-bonded PTFE thread

OBJECTIVE To develop an infection-resistant polytetrafluoroethylene (PTFE) vascular graft for potential clinical use in grafting in sites of bacterial contamination and in replacement of the infected grafts. SETTING Experimental study in rabbits. MATERIALS AND METHODS An antibiotic ofloxacin (OFLX) was bonded to a sheet of PTFE by impregnation, which was cut and twisted into fine threads. The in-vitro antibacterial activity of OFLX-PTFE thread was determined by measuring the zone of growth inhibition against Escherichia coli. The thread was spirally coiled around a ridged outerwall PTFE to make the OFLX-PTFE graft. OFLX-PTFE graft or control graft was interposed in the inferior vena cava (IVC) of rabbits and the entire graft was covered with fibrin containing a fixed number of E. coli. Three or 7 days after the grafting, the grafts with perigraft tissue were harvested and subjected to bacteriological studies. RESULTS In spite of early phase rapid elution of OFLX, a significant antibacterial activity was retained for more than 2 weeks. The antibacterial activity of OFLX-PTFE threads implanted in the subcutaneous space of rabbits decreased to 48% after 24 h and to approximately 1% after a week. The swab culture of all the control grafts was positive, while only one of 13 PTFE-OFLX grafts was positive. The number of viable bacteria in the perigraft tissue of OFLX-PTFE grafts was remarkably low in comparison with that of control grafts. Thus, the OFLX-PTFE grafts exhibited a marked in-vivo antibacterial activity. CONCLUSION By a unique method, it was possible to furnish PTFE graft with an excellent infection-resistant property, without affecting the original biological behaviour.

Subendothelial layer of pseudointima of polytetrafluoroethylene graft is formed by transformation of fibroblasts migrated from extravascular space

A well organised pseudointima is formed in polytetrafluoroethylene (PTFE) grafts within 4 weeks after implantation into inferior vena cava (IVC) of rabbits. To investigate the process of the subendothelial organisation of pseudointima, an animal experiment was conducted. The outer wall of PTFE graft (30 microns fibril length, 3 mm inner diameter, 3 cm long) was coated with 10 um silicon film in the following ways to prevent cellular ingrowth from the extravascular space: non-coating; full-length coating; half-length coating; and full-length coating excluding 5 mm midportion. These grafts were implanted into rabbit IVC and were harvested 4 weeks later. All the grafts were patent but the lumen of the non-coated area was narrowed by pseudointimal hyperplasia. The degree of the hyperplasia estimated by dried tissue deposit was inversely proportional to the length of the coating. The coverage of the luminal surface with endothelial-like cells was noted at anastomotic areas and also at the surface corresponding to the non-coated area. Light microscopy and immunostaining studies on the non-coated midportion revealed the presence of fibroblasts in the interstices of PTFE and smooth muscle cells and myofibroblasts in the pseudointima. Transmission electron microscopy confirmed the presence of myofibroblasts in the midportion of the non-coated area. No transmural capillary ingrowth was observed in the midportion by histological and immunohistochemical analysis. These observations suggest that the subendothelial layer of pseudointima in PTFE grafts is formed by proliferation and transformation of fibroblasts migrating from the extravascular space and that endothelial-like cells may also be derived from such transformation.

Expression of Platelet-Activating Factor Receptor Transcript-2 Is Induced by Shear Stress in HUVEC

Platelet-activating factor (PAF) is an important lipid involved in inflammation reaction and circulation regulation. The receptor for human PAF is synthesized from two spliced transcripts of the same gene. Our observation in the present study shows that HUVEC express transcript-1 only in the static condition. Shear stress induces the expression of transcript-2 in these cells but not transcript-1, resulting in increased PAF receptor expression as measured by FACS analysis. These results suggest that shear stress may increase the susceptibility of endothelial cells to PAF by inducing transcript-2 expression.

Healing of Polytetrafluoroethylene Vascular Grafts Analyzed by Anti-Smooth Muscle Myosin Heavy Chain Isoforms

The purpose of this study was to characterize the cellular contributions to the formation of the pseudointima (PI) using immuno-histochemistry with antibodies to specific smooth muscle myosin isoforms, SM1, SM2, SMemb, and to alpha-smooth muscle actin. The outer wall of polytetrafluoroethylene tube grafts were coated with a 10-microns silicon film except for 5 mm of the midportion and the grafts were implanted into the inferior vena cava of rabbits. The PI in the anastomotic area was mainly formed by dedifferentiation and redifferentiation of medial smooth muscle cells of the host vessel, while PI in the midgraft was formed by differentiation of myofibroblasts into smooth muscle cells in the internodal space.