Effect of perivascular low dose ethanol on rat femoral vessels: Preliminary study.

Vasospasm is one of the important causes of morbidity in free flap and replantation surgery. In secondary Raynaud's phenomenon, nearly half of the patients experience digital ulceration, pain and loss of function at least once in their lifetime. The aim of this study is to investigate the vasodilation effect of ethanol-mediated chemical denervation on peripheral vessels by topical administration. In this study, 27 Wistar albino male rats weighing 250-300 grams were used. The rats were randomly divided into three groups: saline (group S, n = 8), lidocaine (group L, n = 9) and 96% ethanol (group E, n = 9). According to group, 0.1 mL saline, 0.1 mL lidocaine and 0.1 mL ethanol were applied around the rat femoral neurovascular bundle. After the application, on the 0th day and 3th weeks, femoral artery and vein diameters were measured. After 3. weeks, histopathological samples from femoral artery, vein and nerve were evaluated. On the 0th day, the mean diameter of the femoral artery and vein was similar in group E and L and higher than group S. After three weeks, the vasodilatation effect of ethanol was increased in group E. In Group L and S, the vasodilatation effect was lost. Histopathological examination showed that ethanol significantly caused perivascular inflammation and nerve degeneration compared to other agents and did not cause endothelial damage. Vasodilatation obtained by ethanol is a rapid onset and long-lasting effect. It is also inexpensive and effective for peripheral vasodilatation.

Initial Stages of Angiogenesis after Acute Experimental Local Venous Outflow Disturbances and Application of Cell Technologies.

The initial stages of angiogenesis in rats after transcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained cell membranes in the projection of ligated femoral vein were studied by fluorescent light and confocal laser microscopy. Large clusters of brightly fluorescing elongated fibroblast-like cells were seen in the paravasal tissue and in the postoperative scar and signs of angiogenesis were noted as soon as in 4 days. The injected cells not only formed new vessels, but also integrated into vessels formed by host cells. Some injected cells were phagocytizied by macrophages and the latter started to fluoresce due to the presence of the membrane dye. These macrophages within the specified period appeared in the regional inguinal lymph nodes where they formed clusters in the lymphoid parenchyma of the cortical substance.

Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 µM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Differences between femoral artery and vein smooth muscle cells in volume-regulated chloride channels.

The purpose of the present study was to compare the differences between the role of volume-regulated Cl⁻ channels (VRCCs) in veins and arteries. We used the whole cell patch clamp and fluorescence imaging techniques to evaluate swelling-induced Cl⁻ current (I(Cl,vol)) and changes in the intracellular concentrations of Cl⁻ ([Cl⁻](i)) induced by hypotonic solutions in rat femoral artery cells (FASMCs) and vein smooth muscle cells (FVSMCs). I(Cl,vol) and [Cl⁻](i) decline induced by hypotonic solution were more prominent in FASMCs than in FVSMCs. I(Cl,vol) and the alterations in [Cl⁻](i) were gradually increased as the number of cell passages increased. However, the regulatory function of tyrosine protein phosphorylation in volume-regulated chloride movement is prominent in veins. The expression of ClC-3 was higher in FASMCs than in FVSMCs. VRCC activity is more pronounced in rat femoral arteries than in veins. VRCC activity and tyrosine protein phosphorylation regulative function increase gradually as vascular cells switch from contractile to proliferative phenotypes.

Venous graft-derived cells participate in peripheral nerve regeneration.

BACKGROUND: Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers. CONCLUSIONS/SIGNIFICANCE: These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration.

Capture of platelets to the endothelium of the femoral vein is mediated by CD62P and CD162.

Platelets contribute to blood coagulation at sites of vascular injury and to the recruitment of leukocytes at sites of inflammation. Under pathological conditions, platelets are involved in numerous diseases and clinical complications, such as deep venous thrombosis, embolism and atherosclerosis. But so far, little is known about the mechanisms of inflammation in large veins and the role of platelets in inflamed large veins. For this purpose, we investigated primary and secondary interactions between platelets, leukocytes and endothelial cells in the femoral vein in vivo with special regard to the role of CD62P (P-selectin) and CD162 (PSGL-1). Mice were challenged with lipopolysaccharide (LPS)/D-galactosamine (D-gal) and either CD162 or CD62P was blocked by intravenous administration of a corresponding antibody at the time point of LPS/D-gal injection. Four hours after LPS/gal injection, intravital fluorescence microscopy of the femoral vein was performed and primary and secondary platelet-leukocyte-endothelial cell-interactions were visualized after in vivo platelet and leukocyte staining with rhodamine 6G. Analysis of intravital fluorescence microscopy revealed that LPS/D-gal caused a strong inflammatory reaction of the venous endothelium with significant induction of platelet and leukocyte tethering, rolling and adhesion. Secondary interactions of platelets to adherent or rolling platelets or leukocytes were also increased after LPS/D-gal-injection. Immunoneutralization of either CD162 or CD62P significantly decreased platelet primary and secondary capture as well as leukocyte rolling and adhesion. CD162 and CD62P play a central role in mediating inflammatory primary and secondary interactions of platelets and leukocytes to the endothelium in inflamed large veins in vivo. Thus, blocking CD162 or CD62P might be an attractive tool for preventing platelet and leukocyte-driven venous diseases.

Central role of rho kinase in lipopolysaccharide-induced platelet capture on venous endothelium.

BACKGROUND: Platelet-endothelial cell interactions play a key role in hemostasis and pathological coagulation and are dependent on different surface molecules that are expressed upon activation (eg, mediated by lipopolysaccharide [LPS]). Recently, we have shown that Rho kinase plays a central role in LPS-mediated leukocyte-endothelial cell interactions. We investigated the role of Rho-kinase in inflammatory interactions between platelets and the endothelium in femoral veins in vivo. METHODS: Mice were injected intravenously with LPS (0.5 mg/kg)/D-galactosamine (900 mg/kg), and Rho kinase was blocked with fasudil 15 minutes before LPS application. Four hours after LPS administration, intravital fluorescence microscopy of the femoral vein was performed, and primary and secondary platelet-endothelial cell interactions were visualized after in vivo platelet staining with rhodamine 6G. RESULTS: Intravital microscopy showed a significant increase in platelet tethering, rolling, and firm adhesion as well as platelet secondary capture in LPS-treated mice. Rho-kinase inhibition by fasudil significantly reduced platelet tethering, rolling, and firm adhesion. Interestingly, functional blockade of Rho kinase was also able to diminish secondary platelet capture by 79%. CONCLUSIONS: From our data, we conclude that Rho-kinase signaling plays a central role in the regulation of LPS-induced platelet-endothelial cell interactions in large veins in vivo. Thus, Rho-kinase inhibition might be useful in prevention or treatment of pathological inflammation and endotoxin-mediated hypercoagulation.

Treatment with raloxifene and 17beta-estradiol differentially modulates nitric oxide and prostanoids in venous endothelium and platelets of ovariectomized pigs.

Oral treatment with raloxifene, a synthetic estrogen receptor modulator (SERM), or 17beta-estradiol (E2) increases risk for venous thrombosis in women. Acute application of either substance releases endothelium-derived factors from isolated femoral veins but it is not known how their chronic use affects venous functions or the interaction of platelets with veins. This study tested the hypothesis that treatment of ovariectomized animals with oral raloxifene or E2 would increase release of proaggregatory factors from venous endothelium and platelets. Ovariectomized (OVX) pigs were either untreated or treated with oral raloxifene (60 mg/day) or E2 (2 mg/day) for 4 weeks. Plasma concentrations of nitric oxide were comparable in both treatment groups and greater than in OVX pigs. Ratio of plasma thromboxane to prostacyclin was twofold greater in raloxifene compared to E2-treated pigs. In isolated femoral veins, NG-monomethyl-L-arginine (L-NMMA; 10(-4) M) augmented endothelium-dependent relaxations to adenosine diphosphate in veins from E2-treated pigs but inhibited relaxations in veins from raloxifene-treated pigs. Addition of indomethacin (10(-5) M) reversed these effects. Endothelium-dependent relaxations to thrombin were inhibited by L-NMMA only in OVX and raloxifene-treated pigs. Autologous platelets contracted veins in all groups; the magnitude of contractions depended upon the number of platelets and existing tone. Basal release of thromboxane from platelets was greatest in raloxifene compared to OVX or E2-treated pigs. Raloxifene treatment compared to E2 increased production of contractile and proaggregatory prostanoids from venous endothelium and platelets. These differences, if found in humans, may contribute to varying degrees of thrombotic risk with the SERM compared to the natural hormone.

The tissue-engineered vascular graft using bone marrow without culture.

OBJECTIVE: To overcome the shortcomings of current vascular grafts, tissue-engineering methods have been applied to cardiovascular regions. We previously reported the creation of a tissue-engineered vascular graft by using vascular mixed cells. However, the cost and manpower for harvesting and culturing the cells was too burdensome. To overcome these drawbacks, we have developed a new method for creating a tissue-engineered vascular graft by using bone marrow cells, which can be obtained easily and used immediately, without cell culture. METHODS: Biodegradable polymers seeded with different types of cells (group V, cultured venous cells; group B, bone marrow cells without culture; and group C, non-cell-seeded graft [as control]) were implanted into the inferior venae cavae of dogs. The grafts were explanted at 4 weeks and assessed histologically and biochemically. RESULTS: In the histologic examination, a regular layer of Masson-staining collagen fiber and a layer of factor VII-stained endothelial and ant-alpha-smooth muscle cell antigen-immunoreactive cells stained in groups V and B like native vascular tissue, whereas no such stained regular lining was detected in group C. A 4-hydroxyproline assay in group C showed significantly lower levels than in groups V and B or native tissue ( P < .05). The DNA content of the tissue-engineered vascular graft tended to be higher in group C than in groups V and B or in native tissue. CONCLUSIONS: In the creation of tissue-engineered vascular grafts, the method of using bone marrow cells seems to be useful and superior to that of using vascular cells because bone marrow cells can be used directly, without culture.

Healing of implanted expanded polytetrafluoroethylene vascular access grafts with different internodal distances: a histologic study in dogs.

OBJECTIVE: We assessed characteristics of healing, over time, of two types of expanded polytetrafluoroethylene grafts. STUDY DESIGN: An experimental histological study in dogs. METHODS: The graft types studied had the same internal diameter (5 mm) but different internodal distances. In one, the internodal distance was 60 microm in the external surface and 20 microm in the luminal surface. In the other, the internodal distance was 30 microm throughout the material. Sixteen grafts of each type were implanted between the femoral artery and vein in 16 dogs; explanted 1, 2, 4 or 12 weeks later; and examined histologically. RESULTS: In both graft types, infiltrating-cell density and maximum cell-penetration depth increased significantly between 1 and 2 weeks after implantation, but no significant increases occurred after 2 weeks. The number of inflammatory cells peaked 1 week after implantation and decreased significantly by 2 weeks. Subsequently, there were no significant changes in inflammatory cell numbers, suggesting that the inflammatory phase was over by 2 weeks after implantation and the grafts had become attached to surrounding tissue. There were no significant differences between the two graft types in cell density, cell-penetration depth, or number of inflammatory cells at any assessment time. CONCLUSION: Our results provide histologic support for guidelines recommending that synthetic vascular grafts for hemodialysis access should not be cannulated until 2 weeks after implantation. Since increasing the internodal distance to 60 microm in the external surface had no effect on graft healing, methods other than manipulation of internodal distance should be used in developing a graft suitable for early cannulation.

Cellular remodeling of depopulated bovine ureter used as an arteriovenous graft in the canine model.

BACKGROUND: One of the greatest challenges in hemodialysis access surgery is improving the durability of prosthetic grafts caused by structural deterioration. The depopulated bovine ureter SynerGraft (SG) (CryoLife, Inc) is a tissue-engineered vascular graft processed to remove the xenograft cells while maintaining an unfixed connective tissue matrix capable of autologous cell repopulation by the recipient. STUDY DESIGN: Nineteen 6-mm diameter bovine ureter SG conduits were implanted in 12 dogs as arteriovenous grafts between the carotid artery and jugular vein (n = 11) or between the femoral artery and vein (n = 8). Performance of these biologic conduits was compared with that of 15 IMPRA (Bard) ePTFE grafts implanted in 9 dogs, including 9 arteriovenous grafts between the carotid artery and jugular vein and 6 femoral artery to femoral vein grafts. After 14 days, the grafts were accessed once weekly. Histologic and immunohistochemical analyses were performed on grafts explanted between 10 to 60 weeks. RESULTS: The 6- and 12-month primary patency rates of the bovine SG were 72.6% and 58.6%, respectively, compared with 6- and 12-month primary patency for ePTFE conduits of 57.4% and 57.4%, respectively. None of the bovine SG grafts became infected, but synthetic conduits became infected within 54 days of implantation. At 10 weeks, bovine ureter SG conduit showed fibroblast cell migration and proliferation with incorporation into the surrounding subcutaneous tissue, and elongated cells expressing the contractile protein smooth muscle actin were also observed. After 24 weeks, procollagen synthesis was demonstrated in the fully colonized graft matrix. The ePTFE grafts had no evidence of cellular ingrowth and an absence of endothelium. CONCLUSIONS: The bovine SG was appropriately remodeled to its host environment through an organized process of recellularization and neovascularization. The absence of infection, similar patency rates, and cell repopulation of the matrix warrant further investigation.

Counteracting effect of head-up tilt on increased femoral venous compliance after simulated weightlessness in rabbits.

BACKGROUND: The increased femoral venous compliance is one of the factors of orthostatic intolerance after space flight. The purpose of this study was to observe the effect of daily head-up tilt (HUT) on stress-strain relationship of femoral vein during simulated weightlessness in rabbits. METHODS: Head-down tilt (HDT) 20 degrees rabbit model was used to simulate weightlessness. Twenty-four healthy male New-Zealand rabbits were randomly divided into three groups: simultaneous control, 21 d HDT, and 21 d HDT plus daily 2 h HUT 45 degrees, with 8 in each. Twenty-one days later, stress-strain relationship of femoral vein was examined. RESULTS: Under the same stress, the circumferential strain both in the 21 d HDT group and the 21 d HDT plus daily 2 h HUT group increased significantly than those in the control group. There were no significant differences between these two groups. There were no significant changes in the longitudinal strain among three groups under the same stress. CONCLUSIONS: The femoral venous compliance of rabbits increase significantly after 21 days simulated weightlessness. Daily 2 h HUT 45 degrees could not prevent increase of femoral venous compliance in rabbits induced by 21 days simulated weightlessness.

Acute effects of 17beta -estradiol on femoral veins from adult gonadally intact and ovariectomized female pigs.

Our experiments were designed to determine the acute effects of 17beta-estradiol on femoral veins from intact and ovariectomized female pigs. Rings of femoral veins with or without endothelium were suspended in organ chambers for measurement of isometric force. Concentration-response curves to 17beta-estradiol (10(-9) to 10(-5) M) were obtained in veins contracted with prostaglandin F(2alpha) in the absence and presence of inhibitors of either estrogen receptors (ICI-182780; 10(-5) M), nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 10(-4) M], soluble guanylate cyclase (1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10(-5) M), or potassium channels (tetraethylammonium; 10(-2) M). Estrogen receptors were identified with the use of Western blotting and immunostaining in veins of both groups. 17beta-Estradiol caused acute endothelium-dependent relaxations in both groups. Relaxations to 17beta-estradiol were inhibited by l-NMMA and 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but not ICI-182780. Tetraethylammonium inhibited relaxations only in veins with endothelium from intact females. Results indicate that 17beta-estradiol causes acute endothelium-dependent relaxations in femoral veins. The relative contribution of nitric oxide and K(+) channels as mechanisms involved in relaxations to 17beta-estradiol in femoral veins is modulated by ovarian status.

[Effects of simulated weightlessness on pressure-volume relationships of femoral vein of New Zealand Rabbits].

Objective. To observe the changes of pressure-volume relationships of rabbit femoral veins and their structural changes caused by simulated weightlessness. Method. Head-Down Tilt (HDT) -20 degrees rabbit model was used to simulate weightlessness. Twenty four healthy male New Zealand Rabbits were randomly divided into 21 d HDT group,10 d HDT group and control group, (8 in each group). Pressure-volume (P-V) relationship of rabbits femoral veins was measured and the microstructure of the veins was observed. Result. The femoral vein P-V relationship curves of HDT groups showed a larger volume change ratio than that of control group. This change was that 21 d HDT group was even more obvious than that of HDT-10 d group. B1 and B2 in quadratic equations of 21 d HDT group were significantly higher than the values of both 10 d HDT group and control group during expansion (inflow) and collapse (outflow) (P<0.01). The result of histological examination showed that the contents and structure of femoral vein wall of HDT-rabbits changed significantly. Endothelial cells of femoral vein became short and columnar or cubic, some of which fell off. Smooth muscle layer became thinner. Conclusion. Femoral venous compliance increased after weightlessness-simulation and the femoral venous compliance in 21 d-HDT rabbits increased more obviously than that in 10 d-HDT rabbits. The structure of femoral vein wall had changed obviously.

Normal and perturbed endothelial cells from canine femoral arteries and femoral veins exhibit heterogeneity in hemostatic properties and growth characteristics.

BACKGROUND: We sought to examine the heterogeneity of endothelial cells from the same anatomic site but different vascular systems and described von Willebrand factor (vWF) release and morphological change in response to injury-associated factor in femoral vessels from canine in vitro. METHODS: Levels of hemostatic factors (vWF, plasminogen activator inhibitor type 1(PAI-1), antithrombin III (ATIII), in tissue sections and cultured endothelial cells of canine femoral arteries and canine femoral veins were compared by the immunohistochemistry technique. In addition to comparing cell growth density and cell protein contents, cultured femoral arterial endothelial cells (FAECs) and cultured femoral venous endothelial cells (FVECs) were incubated with a series concentration of basic fibroblast factor (bFGF) (1, 10, 100 ng/ml) for up to 48 hours to test the amount of vWF secretion and morphological change. RESULTS: Both in tissue sections and cultured cells, the levels of vWF are higher in FVECs than in FAECs. We were unable to differentiate the level of PAI-1 and ATIII difference between FAECs and FVECs. bFGF (10 ng/ml) significantly increased vWF secretion from cultured FAECs but not from FVECs. The size of cultured FAECs is smaller than of FVECs; however, FAECs have higher amounts of protein contents than FVECs. CONCLUSIONS: These comparative studies provide evidence indicating that the characteristics of FVECs differ from those of FAECs. These differences may be indicated heterogeneity with either inherited or acquired thrombotic disease.

Adenoviral expression of a urokinase receptor-targeted protease inhibitor inhibits neointima formation in murine and human blood vessels.

BACKGROUND: Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha(2)-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. METHODS AND RESULTS: The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x10(9) pfu/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9+/-2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. CONCLUSIONS: These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.

Apparent blood stream origin of endothelial and smooth muscle cells in the neointima of long, impervious carotid-femoral grafts in the dog.

The purpose of this study was to determine whether endothelial and smooth muscle cells originating from the blood stream contribute to the endothelialization of impervious, small-caliber, long Dacron grafts used as extraanatomical bypasses in dogs. We implanted silicone-rubber-coated, permanently impervious grafts 64 to 77 cm long and 6 mm in diameter, made of externally supported knitted Dacron as unilateral carotid-femoral bypasses with distal femoral arteriovenous fistulae in 10 dogs for 3 months; sides were alternated between cases. Subjects received 162 mg/day of aspirin, and its effectiveness on platelet aggregation (PA) was evaluated and expressed as a PA score. Graft healing was studied by stereomicroscopy with silver nitrate staining, by light microscopy with hematoxylin-eosin and immunocytochemical staining for endothelial and smooth muscle cells, and by scanning and transmission electron microscopy. Five grafts were patent for 3 months and could be included in the healing study; the five occluded grafts thrombosed within 14 days. Although there was no transinterstitial tissue ingrowth from perigraft tissues into the impervious Dacron grafts, scattered islands of endothelial cells were conclusively demonstrated on graft flow surfaces 3 months after implantation. Average endothelial-like cell coverage of the flow surfaces was 15.6% +/- 3.8%, and alpha-actin-positive smooth muscle cells and microvessels were found beneath some of the endothelial islands. These findings suggest that blood stream-derived endothelial and smooth muscle cells play a role in the healing of the inner wall of Dacron grafts in the dog.

Re-endothelialisation in autogenous vein grafts.

OBJECTIVES: To clarify the course of re-endothelialisation (Re-E) in an entire graft and to establish the effect of immersion media for the preservation of endothelial cells. METHODS: Autogenous femoral veins of dogs were immersed in heparinised saline solution (n = 18) or heparinized autogenous blood (n = 18). After immersion, the grafts were implanted into bilateral femoral artery, and were retrieved 1 day to 4-8 weeks after implantation. RESULTS: For the grafts immersed in the heparinised saline solution, the values for % area of endothelial cell coverage before implantation, and at 1 day, 1 week, and 4 weeks after implantation were 44.9%, 6.2%, 14.5%, and 81.3%, respectively. For the grafts immersed in heparinised autogenous blood, the values were 73.5%, 20.6%, 79.2% and 95.5%, respectively. However, such relatively rapid speed of Re-E slowed down considerably after 1 week following implantation in this group. CONCLUSIONS: The use of heparinized autogenous blood is strongly recommended as a preparation media for autogenous vein grafts. Almost all of the endothelial cells fall away in the earlier period after implantation and regenerate multifocally and irregularly. Re-E is incomplete even at 8 weeks after surgery, and we suggest that the area of incomplete Re-E may develop into intimal hyperplasia.