JPM Vol. 28, No. 3

November 1992:149-157

Removal of Venous Endothelium With Air Dale E. Bjorling, Ricardo Saban, Mark W. Tengowski, Suzann M. Gruel, and V e n k a t K. R a o

Department of Surgical Sciences (D.E.B., R.S., M. W.T.), School of Veterinary Medicine and Department of Surgery (S.M.G., V.K.R.), School of Medicine, University of Wisconsin

Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or enzymatic processes. It has been observed that the technique used for the removal of arterial endothelium may profoundly affect smooth muscle function and release of prostanoids by the vessel wall. The function and patterns of regeneration of arterial endothelium have been extensively described, but there is a relative paucity of information about the venous endothelium, due in part to the difficulty of its removal. We developed a technique for removal of the endothelium of rabbit femoral veins by passing a stream of air through the lumen of the vessel to dry and remove the endothelium. The effectiveness of endothelium removal was verified by the lack of in vitro reactivity to endothelium-dependent relaxing substances, examination of frozen sections of vessels, labeled with fluorescent-tagged acetylated low-density lipoprotein, with fluorescent light microscopy and scanning electron microscopy of vessel segments. Air drying effectively removed the endothelium and abolished mechanical responses to endothelium-dependent vasodilators but did not affect the function of the smooth muscle. We propose the use of air to remove endothelium from veins to be used to study endothelium-derived factors since this method achieves complete removal of endothelium without causing detectable damage (morphological or functional) to the remainder of the vessel wall.

Keywords: Venous endothelium; EDRF.

Introduction Since Furchgott and Zawadzki (1980) first reported the integral role of endothelium in mediating acetylcholine-induced relaxation of rabbit aorta, endotheliumderived relaxing factor (EDRF) has been extensively investigated (Ignarro et al., 1987; Palmer et al., 1987). Most experimental work has focused on arterial endothelium. Venous musculature has the capacity to respond to EDRF, but it has been suggested that venous endothelium produces significantly lower quantities of EDRF in response to substances such as acetylcholine and calcium ionophore A23187 which trigger release of EDRF by arterial endothelium (Seidel and La Rochelle, 1987). Other investigators have reported that basal release of EDRF by venous endothelium is actually greater than that from arterial endothelium from Address reprint requests to Dale E. Bjorling, D.V.M., M.S., Department of Surgical Science, School of Veterinary Medicine, 2015 Linden Dr. West, Madison, Wisconsin 53706, U.S.A. Journal of Pharmacological and ToxicologicalMethods 28, 149-157 (1992) © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

corresponding vascular beds (McGrath et al., 1990). The differences in observation of function of venous endothelium may, in part, be due to the techniques used to remove endothelium from the relatively delicate veins. When denuded of endothelium, both arterial and venous musculature retain the ability to respond to EDRF released from another vessel with intact endothelium (Seidel et al., 1987). The disparity between the reactivity of arteries and veins to compounds that trigger the release of EDRF appears to be due to differences in the ability of their endothelia to release EDRF rather than an inability of the smooth muscle to respond to EDRF (Seidel and La Rochelle, 1987). Veins from various locations also exhibit heterogeneous endothelium-dependent relaxation responses within the same species (Vedernikov et al, 1988). Other factors, such as modulation of endothelial function by hemodynamic variables, may explain why endothelium-dependent responses to acetylcholine in animals and humans are considerably blunted in peripheral

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JPM Vol, 28, No. 3 November 1992:149-157

veins compared with those in arteries (Vanhoutte, 1989). A variety of techniques to remove endothelium from vessels for in vitro study have been developed, including simply rubbing the inner surface of the isolated vessels (Furchgott and Zawadzki, 1980; Vedernikov et al., 1988), enzymatic removal of the endothelium (Furchgott, 1984), laser irradiation (Kovacs et al., 1975), or perfusion with saponin (Samata et al., 1986). For in vivo study of the effects of removal of arterial endothelium, investigators have used balloon catheters (Clowes et al., 1986; Shimokawa et al., 1989), a catheter and bronchial lavage brush (Cox et al., 1989), a loop of surgical suture (Lindner et al., 1989), surface drying and balloon catheter passage (Goffet al., 1988), or infusion of air bubbles during perfusion of the vessel with blood at a high flow rate (Ralevic et al., 1989). In vitro removal of the endothelium from rabbit aorta by vigorous rubbing with a cotton swab caused a reduction in maximum contractile response to potassium chloride when compared with tissues in which the endothelium was gently removed (Rosolowsky et al., 1991). When challenged with the calcium ionophore A23187, these tissues produced more hydroxyeicosatetraenoic acid (HETEs) than did control vessels from which endothelium was removed gently. Examination of these vessels with electron microscopy also demonstrated slight degeneration of smooth muscle cells. The fragile nature of the venous wall has limited the use of mechanical removal of venous endothelium in vitro. We have found that small veins collapse and are difficult to cannulate for mechanical removal of endothelium without damaging the vessel wall. Fishman et al. (1975) developed a model to examine endothelial regeneration after in vivo endothelium removal by briefly passing a gentle stream of air through the lumen of the vessel. An advantage of this model is that the endothelium is completely removed with minimal injury to the media at the time of endothelium removal, as shown by an absence of inflammatory cells in the area. We investigated this technique of endothelium removal in isolated rabbit femoral veins to determine whether the endothelium could be completely removed without damaging the subendothelial layers of the veins. This method of endothelium removal was evaluated morphologically as well as functionally.

Methods Care and handling of the animals used in this research was in compliance with t h e " Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the Na-

tional Institutes of Health (NIH Publication No. 80-23, revised 1978). Albino New Zealand rabbits (2.5-3.5 kg) were anesthetized with ketamine hydrochloride (60 mg/kg, i.m.), xylazine (6 mg/kg, i.m.), and acepromazine maleate (1.5 mg/kg i.m.). Surgery was performed using a double-headed operating microscope (Zeiss Universal S-3). A 2- to 3-cm segment of femoral vein was harvested and placed in physiological salt solution (PSS) of the following composition (mM): NaC1, 119; KC1 4.7; NaH2PO4, 1.0; MgCI2, 0.5; CaCI2, 2.5; NaHCO3, 25; glucose, 11; and pH 7.4. The distal end was cannulated (PE-50 Clay Adams, Parsippany, New Jersey, U.S.A.), and a gentle stream of air (25 mL/min) was passed through the lumen for 3 min while the adventitial surface was kept moistened with PSS (37°C). This procedure was adapted from one described by Fishman et al. (1975). After passage of air, the luminal surface was gently rinsed with PSS several times, the cannulated portion of the harvested vessel was discarded, and the vein was studied within minutes of exposure to air. An adjacent segment of the same vein with endothelium preserved was also placed in PSS and used as a control. Venous segments (control and endothelium denuded) were divided and randomly selected for in vitro functional experiments, labelling with the fluorescent probe 1,1 '-dioctadecyl- 1-3,3' ,3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL, Biomedical Technologies Inc.), or fixation with glutaraldehyde for scanning electron microscopy (SEM).

Labelling with DiI-Ac-LDL The internalization of Ac-LDL has been used as an indicator of the presence of metabolically active endothelium (Netland et al., 1985; Voyta et al., 1984). Segments of the blood vessels were placed in 2 mL of aerated (95% O2:5% CO2) PSS (37°C), and DiI-AcLDL (20 IxL) was added and kept in contact with the tissues for 4 hr, with replacement of PSS and DiI-AcLDL after the first 2 hr. After 4 hr, the excess DiI-AcLDL was removed by rinsing with PSS. Samples were frozen, sectioned (4 txm) on a cryostat, evaluated by a combination of differential interference contrast (DIC) and epifluorescence, and photographed.

Scanning Electron Microscopy Each femoral vein was canulated with PE-50 polyethylene tubing (Clay Adams) and tied with 5-0 braided polyester suture before removal. Veins were flushed via canula with PSS to remove blood components. Air and PSS were passed through the vein as previously described. Vessels were perfused via canula with 4%

D. E. BJORLING ET AL. REMOVAL OF VENOUS ENDOTHELIUM WITH AIR

glutaraldehyde and 2.5% formaldehyde in 0.2 M cacodylate buffer, pH 7.2 for 75 min at 4°C, while immersed in the same solution. Vessels were rinsed in 0.2 M cacodylate buffer (pH 7.2) for three 10-min exchanges. Post-fixation was in 1% osmium tetroxide buffered with 0.1 M cacodylate buffer (pH 7.2) for 60 min at room temperature. Vessels were again rinsed in 0.2 M cacodylate buffer (pH 7.2) for three 10-min exchanges before dehydration through increasing concentrations of ethanol and drying by the critical point method (Pawley and Albrecht, 1988). Veins were then opened on their longitudinal axis to expose the lumen. The dried specimens were mounted to silicon chips with colloidal carbon and ion beam rotary platinum coated (1-2 nm) in a vacuum system. The specimens were examined and photographed with the Hitachi S-900 high resolution low-voltage scanning electron microscope.

Functional Studies Adjacent vessel segments 4-5 mm long were suspended as rings between two stainless-steel stirrups in water-jacketed (37°C) tissue baths. The tissue baths contained I0 mL of PSS gassed continuously with 95% O2:5% CO2. Mechanical responses were measured isometrically and recorded on a chart recorder (Grass) via isotonic force transducers (Grass FT-03). Data was acquired and stored with a Tecmar LabMaster board in an IBM-compatible PC computer using Data Collection Program V1.0 (developed by Paul Kaarakka, Department of Surgical Sciences, UW-Madison). All vessel segments were stretched to the tension found to maximize responses (usually 1.5 to 2.0 × relaxed diameter) and allowed to equilibrate for 1.5 hr before any manipulation. The vessels were washed with fresh PSS every 15 min during the period of equilibration. Cumulative concentration-response curves were determined for phenylephrine by increasing the concentration by a factor of about 3 after maximal contraction developed to the previous concentration (van Rossum, 1963). The concentration of phenylephrine that produced 30% of the maximal contractile response (EC3o) was calculated by probit analysis for each individual preparation. After washing the tissues for 60 rain with PSS to remove phenylephrine, tone was induced with phenylephrine (EC3o). In separate experiments, it was found that phenylephrine produced a consistent contraction of sufficient duration to reliably obtain a cumulative relaxation-response curve in response to the addition of vasodilators. Endothelium-dependent vasodilators were added to the tissue bath in a cumulative fashion (van Rossum, 1963). Only one substance was tested in each tissue. Substance P (10 - 6 M) was added at the end of the experiment to confirm the pres-

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ence or absence of functional endothelium, because it has been observed previously (Bolton and Clapp, 1986) that absence of relaxation in response to this peptide confirms the absence of functional endothelium. In separate experiments, sodium nitroprusside was used to generate concentration-response curves to evaluate endothelium-independent relaxation. Papaverine (10 -3 M) was added at the end of the experiment to produce maximal relaxation of the veins.

Drugs Used DiI-Ac-LDL was purchased from Biomedical Technologies, Inc, Stoughton, Massachusetts, U.S.A. Phenylephrine hydrochloride, substance P acetate salt, Ach chloride, arachidonic acid, adenosine 5'-triphosphate disodium salt (ATP), sodium nitroprusside, osmium tetroxide, papaverine hydrochloride, and indomethacin were purchased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. Substance P was dissolved in saline containing sodium metabisulfite (0.05%). Stock solutions of arachidonic acid were made in ethanol, gassed with argon, stored at -20°C, and further dilutions were prepared each day with 0.9% sodium chloride and stored on ice. Other substances were dissolved and diluted in 0.9% sodium chloride. Statistical analysis of the results was performed with StatWork software utilizing analysis of variance and Student's t test for paired or unpaired data (Bernard, 1986). All values are expressed as mean ___ standard error of the mean, and a p value less than 0.05 was considered to indicate the presence of significant differences.

Results

Morphological Data Scanning electron microscopy of control veins [Figure l(a)] exhibited a normal cohesive endothelium with tightly arranged, spindle-shaped cells and luminally projecting nuclei. Veins exposed to air [Figure l(b)] demonstrated complete removal of the endothelium without damage to the subendothelial layer. The washed, denuded surface was devoid of endothelial cells and cell fragments. Stereo microscopy [Figure 1(c)] of the vessel lumen distal to instillation of air demonstrated the demarcation between endothelium, smooth muscle, and intimal support. Fluorescence microscopy of fresh, frozen vein cross-sections highlighted the presence of endothelium in control vessels with internalized (metabolized) DiIAc-LDL [Figure 2(a)]. Endothelial fluorescence averaged 4.55 p.m (range: 1.24-11.61 Ixm). This thickness of endothelial cell layer is due to folding and tilting

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JPM Vol. 28, No. 3 November 1992; 149-157

A

B

Figure 1. Scanning electron micrographs of control femoral vein (a) and femoral vein after endothelium removal by exposure to a gentle stream of air (b). Air passage successfully removes endothelium, yet no trauma results to subendothelium. Stereo pair (c) of the luminal surface of a vein. Arrow A indicates the separation of endothelium and subendothelial smooth muscle. Arrow B identifies the collagen support of vessel intima external to smooth muscle layer. Scale bar = 10 i~m for (a) and (b); 2 Izm for (c).

during sectioning. In separate experiments, arterial segments labelled in the same manner had a thin and clear monolayer of endothelial cells. Figure 2(b) is a combination of transmitted light and fluorescence microscopy of the same tissues viewed in Figure 2(a), demonstrating that fluorescence is predominantly limited to the endothelial monolayer, with some background fluorescence. The removal of endothelium by

passage of air was confirmed in experimental veins which did not label with D i I - A c - L D L [Figure 2(c)].

Functional Data The effectiveness of endothelium removal was verified by the lack of in vitro reactivity of the vein to endothelium-dependent relaxing substances, including

D. E. BJORLING ET AL. REMOVAL OF VENOUS ENDOTHELIUM WITH AIR

153

Figure 2. Photomicrographs of frozen sections (4 p.m) of control (a and b) and endothelium-denuded (c) femoral veins labelled in vitro with DiI-Ac-LDL. When viewed by epifluorescence microscopy, the endothelium is seen as a continuous fluorescent layer. When viewed with a combination of differential interference contrast microscopy and epifluorescence microscopy, a merged image of a control femoral vein incubated with DiI-Ac-LDL is produced which clearly shows the intact endothelium. This layer is not seen in veins from which the endothelium was removed which were subsequently examined in the same manner (c).

substance P (data not shown), after removal of endothelium. The contractile response of venous tissue to phenylephrine was not altered by endothelium removal (Figure 3). The maximal response was 2.5 g in both groups, and the ECso values were not altered (1.2 x 10 -6 M and 1.3 x 10 -6 M for control and endothelium removed veins, respectively). These results confirmed that the subendothelial layers o f the vein remained intact, and the ability of the veins to contract was not altered by removal o f the endothelium; however, endo-

thelium-dependent relaxation in response to Ach ( 1 0 - s to 10-4 M) was abolished in denuded vessels (Figure 4). The responses to A T P (10 - s to 10 -4 M), another endothelium-dependent relaxant substance, were substantially reduced but not completely abolished by endothelium removal (Figure 5). F u r t h e r m o r e , denuded vessels exhibited a trend towards contraction in contrast to relaxation observed in control veins when exposed to arachidonic acid (I0 - s to 10 -4 M) in the presence of indomethacin (Figure 6). Sodium nitroprus-

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Figure 2. (Continued)

side, an endothelium-independent relaxant substance, induced similar relaxation in control and denuded vessels (Figure 7). Papaverine (data not shown) induced the same responses in control and denuded vessels and completely reversed tone induced by phenylephrine. The fact that control and denuded veins responded in the same manner to these endothelium-independent vasodilators confirmed that this technique of endothelium removal did not alter the sub-endothelial layers of the vein, maintaining the smooth muscle reactivity to endothelium-independent vasodilators.

Discussion Removal of venous endothelium by passage of air was found to be effective and reliable. Air drying and

subsequent washing of the luminal surface of the vein removed the endothelial layer, leaving the smooth muscle layer morphologically and functionally intact. Smooth muscle bundles, elastic fibers, and collagen are present in the intermixed tunica adventitia of large veins. Their arrangement probably accounts for the lack of destruction of structure in this layer by the air stream. Denuded venous segments demonstrated the same responses as control veins to endothelium-independent substances such as phenylephrine and sodium nitroprusside. Dependence on the presence of functional endothelium for relaxant responses to the addition of Ach, ATP, and arachidonic acid was clearly demonstrated and correlated well with morphological findings.

D. E. BJORLING ET AL. REMOVAL OF VENOUS ENDOTHELIUM WITH AIR

155

Figure 2. (Continued)

Morphologic evaluation of the tissues in this study demonstrated that air removal of venous endothelium did not affect the subendothelial layers. The alteration in responses to potassium chloride observed after in vitro mechanical removal of endothelium from rabbit aorta was attributed to a loss of smooth muscle mass (Rosolowsky et al., 1991). In that study (Rosolowsky et al., 1991), the in vitro contractile response of rabbit aorta to norepinephrine was decreased by mechanical removal of the endothelium, regfirdless of whether this was done gently or vigorously, while we found that in vitro removal of venous endothelium with air failed to affect the response of the tissues to phenylephrine. Labelling of endothelial cells with DiI-Ac-LDL has distinct advantages over scanning electron microscopy

for evaluating the presence or absence of functional endothelium. DiI-Ac-LDL is specifically metabolized by monocytes, macrophages, and endothelial cells via a receptor that does not recognize unmodified low-density lipoproteins (LDL) (Basu et al., 1976; Goldstein et al., 1979; Via et al., 1982). The specificity of DiI-AcLDL uptake by endothelium has been demonstrated, and DiI-Ac-LDL has been attached to several labels to facilitate critical identification of functional endothelium by microscopy (Stein and Stein, 1980; Pitas et al., 1985; Voyta et al., 1984, Netland et al., 1985). Other advantages of DiI-Ac-LDL labelling are that the samples are easy to handle and fixation of tissues is not required. Figure 2 demonstrates the versatility of this technique. Different images of the same frozen section

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can be used to complement each other to determine the extent of the endothelial layer within the wall of the femoral vein. Merged photomicrographs [Figure 2(b)] clearly identify the exact location of endothelium, regardless of artifact produced by sectioning frozen tissues. The lack of DiI-Ac-LDL labelling of denuded veins demonstrates an absence of endothelium after air drying [Figure 2(c)] confirms observations made with SEM. This study demonstrates that removal of venous endothelium by exposure to air is an effective method to prepare tissues for functional and morphological studies. This technique completely removes the endothelium with minimal injury to the subendothelial layers of the vessel wall.

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D. E. BJORLING ET AL. REMOVAL OF VENOUS ENDOTHELIUMWITH AIR

The authors thank Dr. Victoria E. Centonze for her excellent technical assistance with confoeal microscopy. This study was supported by grant NIH NSS-2-S07-RR-05912-07. The Integrated Microscopy Resource, UW-Madison was supported by grant NIH D-RR-570.

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thelium. Denudation with minimal trauma leads to complete endothelial cell regrowth. Lab Invest 61:556-563. McGrath GC, Monaghan S, Templeton AG, Wilson VG (1990) Effects of basal and acetylcholine-induced release of endotheliumderived relaxing factor on contraction to alpha-adrenoceptor agonists in a rabbit artery and corresponding veins. Br J Pharmacol 99:77-86. Netland PA, Zetter BR, Via DP, Voyta JC (1985) In situ labelling of vascular endothelium with fluorescent acetylated low density lipoprotein. Histochem J 17:1309-1320. Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526. Pawley J, Albrecht RM (1988) Imaging colloidal gold labels in LVSEM. Scanning Microsc 10:184-189. Pitas RE, Boyles J, Mahley RW, Bissell DM (1985) Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells. J Cell Biol 100:103-117. Ralevic V, Kristek F, Hudlicka O, Burnstock G (1989) A new protocol for removal of the endothelium from the perfused rat hindlimb preparation. Circ Res 64:1190-1196. Rosolowsky M, Pfister SI, Buja LM, Clubb FJ-Jr, Campbell WB (1991) Method of removal of aortic endothelium affects arachidonic acid metabolism and vascular reactivity. Eur J Pharmacol 193:293-300. Samata K, Kimura T, Satoh S, Watanabe H (1986) Chemical removal of the endothelium by saponin in the isolated dog femoral artery. Eur J Phamacol 128:85-91. Seidel CL, LaRochelle J (1987) Venous and arterial endothelia: different dilator abilities in dog vessels. Circ Res 60:626-630. Shimokawa H, Flavahan NA, Shepherd JT, Vanhoutte PM (1989) Endothelium-dependent inhibition of ergonovine-induced contraction is impaired in porcine coronary arteries with regenerated endothelium. Circulation 80:643-650. Stein O, Stein Y (1980) Bovine aortic endothelial cells display macrophage--like properties towards acetylated [I-125]-labeled low density lipoprotein. Biochim Biophys Acta 620:631-635. Vanhoutte PM (1989) Endothelium and control of vascular function. State of the art lecture. Hypertension 13:658-667. van Rossum JM (1963) Cumulative dose-response curves. II. Techniques for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch lnt Pharmacodyn Ther 143:299-330. Vedernikov YP, Gr~iser T, Tiedt N, Vikhert AM (1988) Heterogeneity of the response of venous smooth muscle to arterial endothelium-derived relaxing factor (EDRF) in respect to the role of nitric oxide. Basic Res Cardiol 83:122-127. Via DP, Dresel HA, Gotto AM Jr (1982) Isolation and characterization of the murine macrophage acetyl-LDL receptor. Arteriosclerosis 2:414a (Abstr.). Voyta JC, Via DP, Butterfield CE, Zetter BR (1984) Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034-2040.

Removal of venous endothelium with air.

Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or e...
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