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Published in final edited form as: J Surg Res. 2012 Jun 23;180(1):e55–e62. doi: 10.1016/j.jss.2012.06.017

Intimal Thickness Associates with Endothelial Dysfunction in Human Vein Grafts

Fan Dong Li *,§, Kevin W Sexton *, Kyle M Hocking *, Michael J Osgood *, Susan Eagle *, Joyce Cheung-Flynn *, Colleen M Brophy *,, Padmini Komalavilas *,1
PMCID: PMC3515722  NIHMSID: NIHMS392240  PMID: 22763213

Abstract

Objectives

Intimal hyperplasia is a complex process thought to be initiated by injury and is the leading cause of vein graft failure. In this investigation we hypothesized that basal intimal thickness in human saphenous vein is a predictor of endothelial dysfunction and potentially intimal hyperplasia.

Methods

Human saphenous veins were obtained during coronary artery bypass surgery. The segments were contracted with phenylephrine, and relaxed with carbachol to determine endothelial-dependent relaxation. Vein segments were fixed in 10% buffered formalin, and cultured for 14 days in high serum and then fixed in formalin. The fixed tissues were stained with Verhoeff-Van Gieson and average intimal and medial thicknesses were calculated using light microscopy and a computerized image analysis system.

Results

Human saphenous veins displayed variable amounts of basal intimal thickness ranging from 18.80 to 241.3 µm. Endothelial-dependent relaxation of the veins was highly variable with values ranging from 0 to 27.59%. Human saphenous vein with basal intimal thickness greater than 120mm had significantly less endothelial-dependent relaxation (8.90±6.32%) than those with basal intimal thickness less than 120mm (21.97±10.64%). Endothelial dysfunction correlated with basal intimal thickness > 120µm (P = 0.02). Basal intimal thickness also correlated with increased intimal thickness after 14 days in organ culture (P=0.0001).

Conclusions

Basal intimal thickness greater than 120 µm is a predictor of endothelial dysfunction, and since greater basal intimal thickness also correlated with increased intimal thickness after organ culture, basal intimal thickness may predict vein graft failure due to intimal hyperplasia.

Keywords: Intimal thickness, Endothelial dysfunction, Endothelial-dependent relaxation, Vein graft failure, Human saphenous vein

INTRODUCTION

Human saphenous vein continues to be the most commonly used conduit for coronary artery bypass grafting and peripheral revascularization surgery [1]. Veins implanted into the arterial circulation undergo several changes and many develop neointimal hyperplasia within 4 to 6 weeks, leading to stenosis, thrombosis, and ultimately graft occlusion and graft failure [2–4]. The vein graft failure rate per patient in 1920 patients at 12 to 18 months in the Project of Ex-vivo Vein Graft Engineering via Transfection (PREVENT) IV trial was 45% [5].

Intimal hyperplasia remains the leading cause of vein graft failure [2, 3]. Intimal hyperplasia is a complex process involving migration, proliferation, and phenotypic modulation of the vascular smooth muscle cells from a contractile to a synthetic phenotype, and extracellular matrix production [6, 7]. Intimal hyperplasia in vein grafts occurs due to a combination of factors such as the vessel wall adapting to the higher intraluminal pressure of the arterial circulation, and the endothelial dysfunction associated with the harvest and surgical preparation of the vein for grafting.

Vein biopsy, angioscopy, and duplex ultrasonography have been proposed for quality assessment of human saphenous vein before implantation [8]. Histological and ultrastructural evaluation of the vein graft prior to implantation have demonstrated that morphological changes of the graft wall correlate significantly with early postoperative complications [9]. Preexisting conditions such as low endothelial cell coverage, stenotic lesions of the lumen and thickness of the intima and media of the graft walls have been considered responsible for the early occlusion of grafts [9]. In fact, approximately 20% of human saphenous vein conduits used as vascular grafts are angioscopically normal, yet contain histological lesions such as atheromatous plaques, fibrous strands, and thickened valve cusps [8, 10].

The endothelium is the primary regulator of vessel wall homeostasis, controlling vascular tone, the coagulation cascade, leukocyte recruitment, and angiogenesis. The term endothelial dysfunction has now become synonymous with reduced nitric oxide production [11, 12]. Loss of endothelial nitric oxide production by denudation predisposes to vasospasm, vascular smooth muscle cell proliferation, platelet aggregation, leukocyte migration, and adhesion [13, 14]. Endothelial cells play an important role in regulating intimal growth through a number of tonic growth-inhibitory mechanisms and the loss of endothelial layer markedly attenuates these growth-modulating effects [6, 15]. Impaired brachial artery endothelial function has been demonstrated to predict long term cardiovascular events in patients with peripheral arterial disease [16]. Thus preservation of the endothelial layer during vein harvesting and preparation is of primary importance to reduce intimal hyperplasia.

To date, few studies have simultaneously evaluated basal thickness of the intima histologically and endothelial functional viability of human saphenous vein physiologically. In this study we explored the relationships between pre-existing basal intimal thickness, endothelial function, and intimal thickening in organ culture, an in vitro model system of vein graft intimal hyperplasia. We hypothesized that the basal intimal thickness could be used to predict endothelial dysfunction of human saphenous vein and the subsequent development of intimal hyperplasia.

MATERIALS AND METHODS

Chemicals and reagents

All chemicals were purchased from Sigma Aldrich (St. Louis, Mo) unless specified otherwise.

Procurement of human saphenous veins

Human saphenous vein samples were collected after obtaining approval of the Institutional Review Boards of the Vanderbilt University Medical Center and the VA Tennessee Valley Healthcare System, Nashville, TN. Forty-one unidentified segments of human saphenous veins and 5 Left Internal Mammary Arteries were obtained from patients that underwent coronary artery bypass graft surgery. For endothelial-dependent relaxation experiments, human saphenous vein segments (n=20) were collected immediately after surgical harvest without any further intraoperative manipulation (‘back table’ preparation, such as marking and manual distention) and tested within 2 hrs of surgical procurement. The human saphenous veins were harvested by open or minimally invasive endoscopic technique according to surgeon discretion and were stored in heparinized Plasmalyte (140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 mEq gluconate, [Baxter Healthcare Corporation Deerfield, IL]) solution in the operating room. Upon gross inspection of the segments, regions of the grafts that were damaged intraoperatively by forceps or clamps were discarded. Only regions that were without damage or branches were used for physiological analysis since complete rings without branches gives consistent responses to contractile agonists and relaxants. All human saphenous vein segments were cut into sequential rings that were fixed in 10% buffered formalin immediately for basal intimal thickness. Additional rings were cut and placed into organ culture for 14 days prior to fixation in formalin. Human saphenous vein segments were then dissected free of fat and connective tissue for determination of endothelial function in an organ bath.

Physiologic measurements of human saphenous veins

One-millimeter rings from the human saphenous vein segments were weighed and their lengths recorded. Rings were suspended in a muscle bath containing a bicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3, pH 7.4), equilibrated with 95% oxygen and 5% carbon dioxide at 37°C. Each ring was progressively stretched to its optimal resting tension (approximately 1 g) that would produce a maximal response to contractile agonists as determined previously, then maintained at the resting tension and equilibrated for a minimum of 2 hours [17]. Force measurements were obtained using a Radnoti Glass Technology (Monrovia, CA) force transducer (159901A) interfaced with a Powerlab data acquisition system and Chart software (ADInstruments, Colorado Springs, CO). The rings were contracted first with 110 mM KCl (with equimolar replacement of NaCl in bicarbonate buffer) to determine functional viability of the smooth muscle. Any tissue failing to contract with KCl was considered non-functional and was not used in further experiments. Viable tissues were allowed to equilibrate in the bicarbonate solution for 30 minutes and were then exposed to the contractile agonist phenylephrine (10−6 M). Endothelial-dependent relaxation was determined by treating the pre-contracted veins with 5 × 10−7 M carbachol. In the absence of a functional endothelial layer carbachol will induce contraction instead of relaxation of human saphenous vein. Force was converted to stress using the equation [105Newtons (N)/m2] = force (g) × 0.0987 / area, where area is equal to the wet weight [(mg) / length (mm at maximal length] divided by 1.055. Percent relaxation was measured as the change in stress compared to the maximal tension induced by phenylephrine as described previously [17].

Human saphenous vein organ culture and morphometric analyses

Two rings of human saphenous vein from each patient were placed in 10% neutral buffered formalin to measure the basal intimal thickness. To measure intima development in vitro, two rings were placed in 8-well chamber slides, and maintained in RPMI 1640 medium supplemented with 30% FBS (Gibco, Carlsbad, CA), 1% L-glutamine, and 1% penicillin/streptomycin for 14 days at 37°C in an atmosphere of 5% CO2 in air. The culture medium was replaced every 2–3 days. After 14 days rings were fixed in 10% formalin, and sent to the Pathology Histochemistry Core at Vanderbilt University or Wax-it Histology Services Inc. (Vancouver, BC, Canada) for histological preparation. The rings were embedded in paraffin, sectioned (5 µm) and multiple sections were stained using Verhoeff-Van Gieson to allow the visualization of the internal elastic lamina. Measurements of intimal and medial thickness were made on transverse sections of each vessel using a Zeiss Axiovert 200M microscope (Carl Zeiss, Thornwood, N.Y., USA) with a computerized image analysis system (Zeiss software and Adobe Photoshop). Intima was defined as tissue on the luminal side of the internal elastic lamina and the medial layer was contained within the intimal layer and the external elastic lamina. Four measurements were made in each image, one from each quadrant, for 3 vein sections for a total of 12 measurements made on each vein section. The mean intimal thickness was the average of 24 measurements on 6 histological sections from 2 vein rings from a single human saphenous vein sample.

Statistical Analysis

Data are reported as mean responses ± standard deviation. Unpaired t tests and correlation of intimal thickness to endothelial-dependent relaxation or post 14 day culture intimal thickening analysis were conducted using Graph Pad Prism software and the P values are reported (LaJolla, CA).

RESULTS

Variability in the pre-existing intimal thickness of human saphenous vein

Forty-one vein segments were collected in heparinized plasmalyte and were fixed in formalin stained using Verhoeff-Van Gieson stain and the pre-existing basal intimal thickness was measured. The basal intimal thickness of veins was highly variable with an average thickness of 85.96±53.00 µm and a range of 18.80 to 241.3 µm (n=41, Figure 1 and 2). When compared to human saphenous vein, left internal mammary artery had significantly lower basal intimal thickness (average 23.010±14.37 mm and a range of 4.8 mm to 37.93 mm, n=4, Figure 2).

FIGURE 1.

FIGURE 1

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Basal intimal thickening in human saphenous veins and left internal mammary artery. Human saphenous vein and left internal mammary artery rings were fixed in formalin, sectioned and stained with Verhoeff-Van Gieson stain and examined by microscopy. Representative images of two saphenous vein segments with thin (A, 5× image and B, 40× image) and thick (C, 5× image and D, 40× image) intima and a left internal mammary artery (E, 5X image and F, 40X image) stained with Verhoeff-Van Gieson. In the image: L=lumen, IEL=internal elastic lamina (arrow), M=media, white line=thickness of intima. Scale bar=100 µm for 5× images and 50 µm for 40X images.

FIGURE 2.

FIGURE 2

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Variability of intimal thickening in Human saphenous veins and left internal mammary artery: Human saphenous vein (HSV, n=41) and left internal mammary artery (LIMA, n=4) rings were fixed in formalin, sectioned and stained with Verhoeff-Van Gieson stain and examined by microscopy. Scatter plot demonstrating the variability of basal intimal thickness measured as an average from two vein segments from each patient as described in the methods section.

Endothelial–dependent relaxation of human saphenous vein is highly variable

We next examined whether the intimal thickness had an effect on the functional viability of the human saphenous vein, particularly the endothelial function. One of the most reliable methods to assess endothelial function is endothelial-dependent relaxation which can be determined in a muscle bath; hence, endothelial-dependent relaxation was measured in each segment. Human saphenous vein segments were pre-contracted with phenylephrine (10−6 M) then treated with carbachol (5×10−7 M) and maximal relaxation was determined (Figure 3A). The majority of the human saphenous vein segments collected as surgical remnants after ‘back table’ preparation (surgical preparation of the vein segment after harvest involving marking with a surgical marker to orient the vein and distention to locate the branches to prepare for grafting) demonstrated very little to no endothelial-dependent relaxation (−2.12±1.886 % data not shown). Thus, 20 human saphenous vein segments were collected immediately after harvest without any marking, distention, or other surgical preparation on the ‘back table’ to preserve the endothelial function. These human saphenous vein segments demonstrated various endothelial-dependent relaxation to carbachol with an average relaxation of 16.28±8.11 % and a range from 0 to 27.59 % relaxation, (n=20, Figure 3B). Endothelial-dependent relaxation of human saphenous vein was significantly lower than the left internal mammary artery, with a mean relaxation of 63.89±12.73% (n=5, Figure 3B).

FIGURE 3.

FIGURE 3

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Variability of endothelial-dependent relaxation in human saphenous veins. Human saphenous vein and left internal mammary artery segments were collected immediately after harvest and subjected to physiologic measurement in a muscle bath. Endothelial-dependent relaxation was measured by contracting with 10−6 M phenylephrine (PE) and relaxing with 5×10−7 M carbachol (Cch). A) Representative tracing of pre-contracted HSV relaxed with carbachol to 24.22%. B) Endothelial-dependent relaxation variability for saphenous veins (HSV) and left internal mammary artery (LIMA).

Basal intimal thickness inversely correlates with endothelial-dependent relaxation

Linear regression analysis of basal intimal thickness versus endothelial-dependent relaxation had a significantly non-zero slope of −0.07264±0.028 (P=0.02 R2=0.2634, Figure 4A), indicating a linear relationship between basal intimal thickness and endothelial-dependent relaxation. There was a sharp decline in endothelial function when basal intimal thickness exceeded 120 µm (Figure 4B). Human saphenous vein with intimal thickness greater than 120 µm had significantly less endothelial-dependent relaxation (8.90±6.32 % relaxation, n=6) than those with intimal thickness less than 120 µm (21.97±10.64 % relaxation n=14, P=0.0119, Figure 4B).

FIGURE 4.

FIGURE 4

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Endothelial-dependent relaxation of human saphenous vein correlates negatively with increase in basal intimal thickness. Human saphenous vein segments obtained from coronary artery bypass surgery were subjected to physiologic measurement of endothelial-dependent relaxation to carbachol in a muscle bath. Histological examination using the Verhoeff-Van Gieson stain was used for the visualization of the internal elastic lamina and the basal intimal thickening was measured. A) A linear regression of the percent endothelial-dependent relaxation as a function of basal intimal thickness was run yielding R2=0.2634, (P=0.02, n=20). B) Vein segments with basal intimal thickening greater than 120 µm had impaired endothelial dependent relaxation (P=0.0119, n = 20).

Basal intimal thickness correlates with intimal hyperplasia in organ culture

Since intimal hyperplasia is a leading cause of vein graft failure, we investigated whether the basal intimal thickness of the human saphenous vein segments had an effect on the development of intimal hyperplasia in vitro in an organ culture model. Human saphenous vein segments had an average basal intimal thickness of 73.82 µm and this thickness increased to 111.6 µm after 14 days in culture. There was a positive correlation with basal intimal thickness and the intimal thickening developed during organ culture (Figure 5A). A linear regression between basal intimal thickness and intimal thickness after organ culture demonstrated a significant non-zero slope of 1.127±0.1375 (p<0.0001, n=21 R2=0.7794, Figure 5A). Greater basal I/M ratio also correlated with greater I/M ratio after organ culture, non-zero slope of 1.109±0.1435 with P<0.0001 (n=21 R2=0.7587, Figure 5B).

FIGURE 5.

FIGURE 5

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Intimal thickening in the basal state predicts degree of thickening in organ culture. Human saphenous vein segments were obtained from coronary artery bypass surgery and were cultured in RPMI medium with 30% serum for 14 days (post culture). Rings were fixed, sectioned, stained with Verhoeff-Van Gieson and analyzed by microscopy and intimal thickness was measured and compared to the intimal thickness before culture. A) Linear Regression of the change in intimal thickness from basal to post 14 day culture R2 = 0.7794 (P<0.0001, n=21) shows a positive correlation where a higher basal intimal thickening leads to a higher intimal thickening in culture. B) Linear Regression of the change in intimal to medial ratio from basal to post 14 day culture R2=0.7587 (P<0.0001, n=21) also showing a positive correlation between basal intimal thickening and intimal thickening in culture.

DISCUSSION

Failure of human saphenous vein bypass conduits due to intimal hyperplasia remains a major limitation of aortocoronary and peripheral vascular bypass procedures [5]. Studies have demonstrated that 20% of venous grafts occlude during the first year after bypass and 50% of venous grafts occlude 10 years later, while the remaining 50% had significant atheromatous lesions [18, 19]. Several factors are considered responsible for the early occlusion of grafts and preoperative quality assessment of human saphenous vein for pre-existing wall changes has been proposed to predict later graft failure [10, 20]. In this study we examined basal intimal thickness, endothelial function, and the effect of basal intimal thickness on intimal thickening in vitro on conduits used for coronary artery bypass grafting. We demonstrated that the pre-existing intimal thickness of the conduits used for revascularization was highly variable with a range from 18 mm to 241mm (Figure 2) for the 41 vein segments analyzed. Using histologic assessment, Kanellaki-Kyparissi et al, reported that prior to implantation, 91% of vein grafts have varying degrees of histological lesions such as local thickening of the vessel wall especially the intima, which was accompanied by a decrease of endothelial coverage only in the stenotic part of the vessel [8]. By histological and ultrastructural evaluation of saphenous vein grafts before implantation Kokkona et al reported that patients with early postoperative complications had a mean intimal thickness of 206.56 ± 32.29 mm, while patients who did not have postoperative complications had a mean intimal thickness of 67.44 ± 10.17 mm [9]. To evaluate the viability and quality of human saphenous vein used in coronary artery bypass operation, a functional method was used since the presence of an intact structure of the vessel wall, as obtained by morphological studies, does not necessarily imply normal function of the tissue [21].

Intimal hyperplasia in vein grafts is thought to be a response to injury. Several factors have been shown to cause injury to the vein segments. Harvest and intraoperative handling of vein grafts have been shown to decrease the expression of endothelial nitric oxide synthase and nitric oxide production when compared to ‘no-touch’ method of vein harvest [22]. We have recently demonstrated that mechanical stretch and the use of surgical skin markers for labeling markedly reduce the functional viability of human saphenous vein [17, 23]. In this study, we found that the majority of the vein segments that were collected after the ‘back table’ preparation had little to no detectable endothelial-dependent relaxation. In contrast, those collected immediately after harvest and prior to any further graft preparation displayed a significantly higher endothelial-dependent relaxation suggesting that the endothelial dysfunction is further exacerbated by the current, commonly employed surgical graft preparation methods. When the histological morphology was compared to the endothelial-dependent relaxation we observed a negative correlation, implying that preexisting thickness of the intima can influence the functional state of the vein graft. Human saphenous vein with intimal thickness greater than 120 µm correlated with impaired endothelial–dependent relaxation (Figure 4). A link between increased basal intimal thickness and postoperative complications has been reported earlier [9]. Our study demonstrates that increased basal intimal thickness also decreases endothelial-dependent relaxation suggesting that endothelial dysfunction associated with increased thickness may partly be responsible for the postoperative complications affecting their graft patency. However, this needs to be confirmed with long term studies. Evaluation of vein grafts for basal intimal thickness may be useful to identify candidate conduits for coronary artery bypass surgery and may reduce postoperative complications.

Since intimal hyperplasia is associated with vein graft stenosis, we also examined the growth of intimal layer in a 14-day organ culture model. This in vitro model of intimal hyperplasia has been reported as a representative model of the changes that occur in vivo after vein graft transplantation [24], and had been used previously in our laboratory. We compared the basal intimal thickness to the subsequent increase in intimal thickening to determine the effect of basal intimal thickness on intimal hyperplasia formation. We observed a direct correlation between high basal intimal thickness and increased rate of intimal thickening in vitro when the vein segments were grown in culture with high serum (Figure 5). Our results demonstrated that higher basal intimal thickness of the vein potentiate the increase of intimal thickness in culture, suggesting that higher basal intimal thickness may predispose the graft to develop intimal hyperplasia more rapidly compared to vein grafts with lower basal intimal thickness. These results suggest that basal wall thickening of human saphenous vein could be a precursor for the formation of intimal hyperplasia in the arterialized graft.

Preservation of endothelium-dependent relaxation plays an important role in inhibiting the development of intimal hyperplasia of vein graft. There is a direct link between the degree of preservation of nitric oxide function in vein grafts and the magnitude of intimal hyperplasia formation [25]. Analysis of retrieved vein grafts (ranged from 5–17 years) from patients undergoing repeat coronary artery bypass grafting demonstrated that grafts with the most pronounced intimal hyperplasia exhibited the least amount of endothelium-dependent relaxation. Taken together, it is plausible that the loss of endothelial nitric oxide synthase expression in the grafts is the central contributor to the loss of endothelial-dependent relaxation along with increased intimal thickness.

The limitations of this study are that tissues were de-identified and the demographic information of the patients was not available, hence we were not able to determine the effect of basal intimal thickness on graft patency in these patients. Also, intimal thickness could not be correlated to drug use in these patients. We did not measure endothelial function along the entire length of the conduit; therefore we did not determine whether the correlation of intimal thickness to endothelial-dependent relaxation was uniform along the conduit or unique to the region that we tested. Since the majority of the veins were harvested by endoscopy we have not directly compared the endothelial dependent relaxation and intimal thickness of veins harvested conventionally versus endoscopically in this investigation. Vein grafts harvested endoscopically undergo more injury and have inferior patency compared to veins harvested by open method [26]. Besides intimal thickening other factors such as harvesting techniques, intrinsic vasospasm, degree of heart failure, peripheral vessel disease and patient demographics may also account for the variability in endothelial-dependent relaxation. Even though evaluation of the vein grafts may help identify ideal conduits for coronary artery bypass grafting and for lower extremity arterial grafts, this study does not provide a "real-time" method of assessing conduits.

In summary these data suggest that basal intimal thickness greater than 120 µm is a predictor of human saphenous vein endothelial dysfunction. Greater basal intimal thickness also leads to increased intimal hyperplasia formation in organ culture suggesting that higher basal intimal thickness may predispose the vein graft to develop intimal hyperplasia more rapidly compared to vein grafts with lower basal intimal thickness. Further studies are needed to determine optimal methods of preoperative vein assessment. The mechanism of the reduction of endothelial-dependent relaxation due to increased intimal thickening is not known. Future studies are also needed to determine if any correlation exists between basal intimal thickness and clinical demographics or clinical outcomes.

ACKNOWLEDGEMENT

This material is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Biomedical Laboratory Research and Development Grant and a National Institute of Health grant RO1HL70715 to CM Brophy, American Heart Association grant in aid to S. Eagle and a Vanderbilt’s CTSA grant NCRR/NIH ULIRR024975 to J Cheung-Flynn. This material is the result of work supported in part with resources and the use of facilities at the VA Tennessee Valley Healthcare System.

Footnotes

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DISCLOSURE

The authors CB, KH, SE, JCF and PK have financial interest in the subject matter or materials discussed in the manuscript, as there is a license on a patent related to this work. CB owns stock and is a consultant for the company, Moerae Matrix Inc., which has licensed technology related to this work.

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