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Abstract

Objective:

This study was carried out to determine high pressure and pulsatile flow perfusion effects on human saphenous vein (HSV) segments obtained from diabetic and non-diabetic patients.

Methods:

The veins were perfused with oxygenated Krebs solution for 3 h, with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 or 300 × 250 mmHg. After perfusion, veins were studied by light microscopy; nitric oxide synthase (NOS) isoforms, CD34 and nitrotyrosine immunohistochemistry and tissue nitrite/nitrate (NO_x) and malondialdehyde (MDA) quantification.

Results:

Light microscopy revealed endothelial denuding areas in all HSV segments subjected to 300 × 250 mmHg perfusion pressure, but the luminal area was similar. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between groups. The endothelial nitric oxide synthase (eNOS) isoform immunostaining decreased significantly in diabetic patients’ veins independent of the perfusion pressure levels. The inducible NOS (iNOS), neuronal NOS (nNOS) and nitrotyrosine immunostaining were similar. Significant CD34 differences were observed between the diabetic 300 × 250 mmHg perfusion pressure group and the non-diabetic control group. Tissue nitrite/nitrate and MDA were not different among groups.

Conclusions:

Pulsatile flow and elevated pressures for 3 h caused morphological changes and decreased the eNOS expression in the diabetic patients’ veins.

Keywords

Human saphenous vein perfusion distension pressure pulsatile flow endothelium nitric oxide nitric oxide synthase

Introduction

The human saphenous vein (HSV) patency as a coronary artery bypass graft (CABG) is known to be inferior to that of arterial grafts.^1–3 This inferiority can be partly attributed to the normal structural and functional differences between veins and arteries, which are exposed to different hemo dynamic forces.^4,5 However, despite these disadvantages, HSVs are still widely employed as CABGs.

Although the precise mechanisms involved in graft failure remain unclear, the abnormal pressures imposed on the HSV during surgical manipulation and/or after implantation into the arterial circulation can promote pathological alterations.^5,6 Endothelium denudation exposes the underlying thrombogenic sub-intimal layer, stimulating smooth muscle cell migration and intimal proliferation.⁵ Reduced amounts of nitric oxide (NO) result in a predisposition to vasospasm, vascular smooth muscle cell proliferation, platelet aggregation and leukocyte migration and adhesion.^7–9

In the arterial circulation, vein grafts are subjected to hemodynamic changes, including increased pressure and pulsatile flow, which may result in morphological and functional changes that contribute to premature failure. In diabetic patients, saphenous vein grafts exhibit reduced endothelium-dependent vasodilatation.¹⁰ Our previous studies have shown that HSV segments perfused with non-pulsatile flow and increased pressures for 3 h exhibit greater morphological changes in the presence of higher perfusion pressures.¹¹ The present study extends our previous work by utilizing pulsatile flow for perfusion of HSV segments and by comparing veins isolated from diabetic and non-diabetic patients.

Materials and methods

Ethical approval and patients

Approval of the local ethics committee was obtained, including the informed consent of all patients. Segments of fresh, intact HSV were obtained from 24 patients (12 non-diabetics and 12 diabetics) subjected to elective CABG for atherosclerotic heart disease at Hospital das Clínicas, Ribeirão Preto Faculty of Medicine, University of São Paulo, Brazil (Table 1).

Table 1.

Characteristics of HSV segments in each group.

Group	n	Age (years)^a	Male	Female	Hypertensives
G1 control non-diabetic	6	59.50 ± 7.71	4	2	4
G2 control diabetic	6	60.66 ± 4.80	4	2	6
G3 250 × 200 non-diabetic	6	60.33 ± 8.04	2	4	3
G4 250 × 200 diabetic	6	61.66 ± 6.25	4	2	5
G5 300 × 250 non-diabetic	6	63.16 ± 7.16	4	2	4
G6 300 × 250 diabetic	6	64.16 ± 5.84	4	2	5
Total	36	61.58 ± 1.78	22 (61%)	14 (39%)	27 (75%)

G: group; HSV: human saphenous vein.

Mean ± standard deviation

The patients included 14 men and 10 women with an average age of 61 ± 2 years. Among them, 18 patients (75%) have systemic arterial hypertension and are smokers. The systemic arterial hypertension was basically treated with diuretics (all patients), angiotensin-converting enzyme (ACE) inhibitors (11 patients), angiotensin II type 1 (ATI) blockers (7 patients) and associated beta-blockers (3 patients with metoprolol and 2 patients with atenolol). Diabetes was defined when the patient presented two measures of fasting blood glucose level above 126 mg/dL. Among the diabetic patients, eight were treated with oral agents, three with oral agents and insulin and one was treated only with insulin.

Harvesting of HSV samples

Prior to systemic administration of heparin, the great saphenous vein was dissected using open harvesting techniques by the same team. Veins with macroscopic signs of inflammation (hyperaemia and thrombosis) or varicose dilations were excluded. The side branches were ligated without any saline distension, and the dissected HSV was removed. After harvesting, a segment of the HSV distal portion was placed in Krebs solution (118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.22 mM KH₂PO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃ and 11.1 mM glucose) and transported to the laboratory, where it was attached to the perfusion system described below. All vessels were utilized within 1 h of removal from the patient.

Study design and perfusion system

Thirty-six segments of HSV were randomly allocated to one of the six groups, consisting of three groups of samples from diabetic patients and three from non-diabetic patients (n = 6 per group). Once we got a long segment of HSV, in a sufficient length for the proposed analysis, the segment was cut, then a part was perfused according to the protocol and the other was used as control (diabetic or non-diabetic). The control segment was kept in the organ bath for 3 h without perfusion (no pressure and no flow). Twelve segments, six from non-diabetic patients (group 1) and six from diabetic patients (group 2), were used as unperfused controls (0 mmHg pressure and 0 mL/min flow rate). Twenty-four segments of HSV from diabetic and non-diabetic patients were perfused with oxygenated Krebs solution for 3 h with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 mmHg (lower pressure: groups 3 and 4) or 300 × 250 mmHg (higher pressure: groups 5 and 6). The assigned experimental groups are represented in Table 1.

The perfusion system (Figure 1) consisted of a pulsatile flow pump (Harvard HABP100; Instech Laboratories, Holliston, MA, USA) that was used to propel Krebs solution from a reservoir through silicone tubes to perfuse the HSV segment mounted in an organ bath. The pulsatile flow pump used in this experimental system allows precise control of flow with adjustments in frequency and volume pumped per cycle. The frequency was maintained at 20 cycles/min and 5 mL/cycle, resulting in a flow rate of 100 mL/min.

Figure 1.

Perfusion system design: Standardized solution stored in the (1) reservoir circulated inside (2) silicone tubes, propelled by a (3) pulsatile pump, perfusing a saphenous vein segment prepared in the (4) organ bath. System pressure was controlled by a resistance in the (5) circuit placed after the vein and was measured by a (6) pressure transducer coupled with a (7) recorder for continuous registration. System temperature was maintained at 37°C by a (8) water-jacket. (9) 95% O2/5% CO2 was bubbled in the (4) organ bath and in the (1) reservoir. A gradated cylinder placed above the (1) reservoir measured the flow.

Resistance was applied to a silicone tube placed distal to the vein in order to achieve the desired pressure for each group. Pressure was measured by a transducer coupled with a record for continuing recordings (GRASS MODEL 7400 Recorder, Astro-Med Inc, West Warwick, RI, USA). A gradated cylinder, placed in series with the circuit, measured flow. The Krebs solution was maintained at 37°C and bubbled with 95% O₂/5% CO₂.

After 3 h of perfusion (or 3 h of immersion of the control segments in Krebs solution), the HSV was cut into four equal pieces and processed as described below.

Light microscopy

HSV segments were fixed in 10% formalin and embedded in paraffin. Transverse 5-µm sections were stained with haematoxylin-eosin and Masson’s trichrome. The amount of endothelial damage resulting from perfusion of HSV segments was assessed by computer-assisted image analysis. Using an Axioskop 2 Plus microscope and an AxioCamHRc camera (Carl Zeiss AG, Munich, Germany), images representing the entire cross section of each sample were digitalized and analyzed with AxioVision 4.6 software (Carl Zeiss AG, Munich, Germany). Two parameters were analyzed: luminal area (μm²) and percentage of luminal perimeter covered by endothelium (%).

Immunohistochemistry

HSV segments were fixed in 10% formalin, embedded in paraffin, and stained using the avidin-biotinylated peroxidase complex (ABC) method. Briefly, 3-µm sections were attached to slides pre-treated with 3-aminopropyl-triethoxysilane (Sigma–Aldrich, St. Louis, MO, USA). The sections were deparaffinized with xylene and ethanol. Endogenous peroxidase and biotin were blocked with 3% hydrogen peroxide and Pierce solution, respectively. Antigen recovery was performed in 10 mM citrate buffer (pH 6.0) under humid heat for 35 min. Non-specific binding sites were blocked with normal serum. The primary antibodies were diluted in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) at the following ratios: neuronal nitric oxide synthase [nNOS; A-11 sc-5302 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)] and iNOS [C-11 sc-7271 (Santa Cruz Biotechnology)] 1:5, endothelial nitric oxide synthase [eNOS; H-159 sc-8311 (Santa Cruz Biotechnology)] 1:25, CD34 [ICO11 sc-7324 (Santa Cruz Biotechnology)] 1:20 and nitrotyrosine [189542 (Cayman Chemical, Ann Arbor, MI, USA)] 1:10. The bound antibodies were visualized with 10 mg/mL diaminobenzene solution (Sigma–Aldrich), and the sections were stained with Harris haematoxylin. As negative controls, some sections were processed as described above but with the primary and the secondary antibodies omitted. The same equipment used for light microscopy was also used for immunohistochemical analyses. In each slide, two fields were randomly selected under 400× magnification. The numbers of positive and negative stained cells were counted in the endothelium and the whole vein wall. The results were expressed as percentages of positive cells.

Determination of tissue malondialdehyde and nitrite/nitrate

HSV segments were wrapped and promptly stored at −70°C. For analysis, the samples were homogenized, and the homogenate was clarified by centrifugation (5000 × g, 10 min, 4°C). Protein concentration was determined using the Biuret reaction. Determination of tissue levels of malondialdehyde (MDA), a product associated with oxidative stress, was performed using a spectrophotometric assay. The absorbance of the supernatant was monitored at 535 nm. The results were expressed as micromole per milligram protein.

Tissue levels of nitrite/nitrate (NO_x) were assessed by an ozone-based chemiluminescence assay. Briefly, samples were treated with ethanol (95%) at 4°C for 30 min, followed by centrifugation at 10,000 × g for 5 min. NO_x levels were measured by injecting 5 µL of the supernatant into a glass purge vessel containing 0.8% vanadium in 1 N hydrochloric acid at 95°C, which reduces NO_x to NO gas. A nitrogen stream was bubbled through the purge vessel, then through 1 N NaOH and then into a NO analyzer that detects NO released from NO_x by chemiluminescence (Sievers, model 280 NO Analyzer; Boulder, CO, USA). NO_x concentration was calculated by comparison with a standard curve of sodium nitrate (0.5, 1.5, 10 and 50 mM). NO_x levels were expressed in micromoles.

Statistical analysis

The data was expressed as mean ± standard deviation (SD). As the non-parametric test considers measuring only the variability of results indirectly applied, the Kruskal–Wallis test and Dunn’s post-test, were used for analysis of the morphological and immunohistochemical data. For analysis of variance of NO_x and MDA levels, which are directly measured, the Bonferroni adjustment of multiple comparison test was performed. GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. The p-values less than 0.05 were deemed significant.

Results

Light microscopy

Light microscopy showed areas of endothelial denuding associated with peeling and the presence of luminal slits in all segments subjected to a perfusion pressure of 300 × 250 mmHg, including samples from both diabetic and non-diabetic patients. Some veins subjected to 250 × 200 mmHg perfusion pressures also exhibited these changes, which were not observed in segments from the control group (Figure 2(a) to (d)). Luminal area did not differ significantly among groups. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between diabetic and non-diabetic groups subjected to a perfusion pressure of 300 × 250 mmHg in comparison to control groups (Figure 3).

Figure 2.

(a) Photomicrograph of a group 5 (300 × 250 mmHg) saphenous vein, where luminal slits can be seen in this vessel (shown by arrows →). Lumen (L) muscle tunica (M). Masson’s trichrome, 100×. (b) Photomicrograph of a group 1 (control) saphenous vein, with normal endothelial cells indicated by arrows. Muscle tunica (M). Masson’s trichrome, 400×. (c) Photomicrograph of a group 6 (300 × 250 mmHg) saphenous vein, showing endothelial cells peeling (greater arrow) and endothelial denudation (smaller arrows). The small arrows indicate a luminal slit. Masson’s trichrome, 400×. (d) Photomicrograph of a group 6 (300 × 250 mmHg) saphenous vein, showing inflammatory infiltrate in adventitia tunica (A) from a blood vessel in the presence of neutrophils within the blood vessel and connective tissue (arrows). Lumen (L): HE, 100×.

Figure 3.

(a) Graphical representation of percentage of luminal circumference covered by endothelium in human saphenous veins in studied groups. Results were expressed as mean ± standard error; (b) graphical representation of human saphenous veins’ luminal area in studied groups. Results were expressed as mean ± standard deviation. *, ○, • and Δ indicate significant difference (p < 0.05).

Immunohistochemical analysis of NOS, CD34 and nitrotyrosine expression

Immunohistochemistry revealed that eNOS levels decreased significantly in the three vein layers of HSVs from diabetic patients subjected to perfusion pressures of 250 × 200 mmHg and 300 × 250 mmHg in comparison to diabetic and non-diabetic control groups (Figures 4(a) and 5(a) and (b)). Immunohistochemical expression of the inducible nitric oxide synthase (iNOS; Figures 4(b) and 6(a) and (b)) and nNOS (Figures 4(c) and 7(a) and (b)) isoforms were observed in all vein layers, and no significant differences in expression of these isoforms were observed among the groups.

Figure 4.

(a) Photomicrograph of eNOS expression in a group 4 (250 × 200 mmHg) saphenous vein sample, showing in detail the marking of some smooth muscle cells of media tunica (M) and some endothelial cells (arrows), 400×. (b) Photomicrograph of iNOS expression in a group 4 saphenous vein sample. Greater cytoplasmic staining can be observed in smooth muscle fibres of the media (M), being lower in the endothelium, 100×. (c) Photomicrograph of nNOS expression in a group 5 saphenous vein sample, showing in detail some smooth muscle cells marked in the media (M), 400×. (d) Photomicrograph of CD34 expression in a group 4 saphenous vein sample, showing in detail the labelling of some endothelial cells (arrows), 400×.

Figure 5.

(a) Graphical representation of the percentage of positive eNOS-labelled cells in the endothelium of human saphenous veins. (b) Graphical representation of the percentage of positive eNOS-labelled cells in the three tunica of human saphenous veins. The results were expressed as mean ± standard deviation. *, ○, • and Δ indicate significant difference (p < 0.05).

Figure 6.

(a) Graphical representation of the percentage of positive iNOS-labelled cells in the endothelium of human saphenous veins. (b) Graphical representation of the percentage of positive iNOS-labelled cells in three tunica of human saphenous veins. The results were expressed as mean ± standard deviation (no significant differences; p > 0.05).

Figure 7.

(a) Graphical representation of the percentage of positive nNOS-labelled cells in the endothelium of human saphenous veins. (b) Graphical representation of the percentage of positive nNOS-labelled cells in the three tunica of human saphenous veins. The results were expressed as mean ± standard deviation (no significant differences; p > 0.05).

CD34 expression was observed by immunohistochemistry in all vein layers, with predominant staining in the endothelium. A significant increase in the CD34 expression was observed between HSVs from non-diabetic patients subjected to a perfusion pressure of 300 × 250 mmHg and the non-diabetic control group (Figures 4(d) and 8(a) and (b)). Nitrotyrosine expression was not observed by immunohistochemistry in the vein wall.

Figure 8.

(a) Graphical representation of the percentage of positive CD34 immunostained cells in the endothelium of human saphenous veins. (b) Graphical representation of the percentage of positive CD34 immunostained cells in three tunica of human saphenous veins. The results were expressed as mean ± standard deviation.* indicates significant difference.

MDA and NO_x levels

No significant differences in tissue levels of MDA were observed between treatment groups (Figure 9(a)). In addition, no significant differences in tissue levels of nitrite/nitrate were observed between treatment groups (Figure 9(b)).

Figure 9.

(a) Graphical representation of tissue concentration of MDA in human saphenous vein. (b) Graphical representation of tissue concentration of nitrite/nitrate (NO_x) in human saphenous vein. The results were expressed as mean ± standard deviation (no significant differences, p > 0.05).

Discussion

The experimental model

It is essential to understand that the model compares the results of arterial conditions over the endothelium and venous wall (higher pressures and pulsatile flow), not the effects of pressures applied during surgical preparation as was done by a previous model in along the same research line.^12,13 Three investigative questions deserve to be clarified: (1) why the vein segments were perfused with crystalloid solution rather than blood, (2) why the perfusion pressures tested were much higher than physiological pressures and (3) why the veins were perfused for 3 h. The perfusion with blood was considered but due to the possibility of interference in the results due to circulating factors (vasoactive substances and cytokines), we decided to use crystalloid solution to perfuse the vein segments. Concerning the second question, originally, we planned to perfuse HSV with lower pressures (200 × 150 mmHg and 120 × 80 mmHg), but some facts motivated us to use higher pressure, including the difficulty to obtain vein segments with the adequate length for the desired analysis. Our previous studies with a non-pulsatile flow did not find alterations with reduced perfusion pressures (50 mmHg and 100 mmHg) and found alterations with higher perfusion pressures (200 mmHg and 300 mmHg). Therefore, considering it appropriate to compare the effects of pulsatile flow, we utilized higher pressures instead of lower pressures, even far from physiological conditions, to identify potential effects of cyclic strain due to pulsatile flow. Finally, according to some authors, 3 h was the minimum time to result in endothelial morphology alterations due to shear stress action.⁴ Our previous study with a non-pulsatile flow perfused HSV segments during this period.¹¹ Otherwise, alterations in the eNOS messenger RNA (mRNA) expression were observed in endothelial cells submitted to shear stress, in vitro, during 3 h.¹⁴ In this study, light microscopy revealed endothelial lesions in all segments subjected to the higher pulsatile perfusion pressures, and in some segments subjected to the lower pressures, including samples from both diabetic and non-diabetic patients. Areas of endothelial denuding associated with peeling and luminal slits were present. Similar findings have been observed in previous investigations.^11–13 In addition, many authors described the presence of endothelial denudation and venous lumen dilation under distention pressure without flow.^5,7,10

The histological data

Unlike previous studies that reported increased luminal area⁹ and increased inner diameter of the saphenous vein,¹⁰ the luminal area in the present study did not differ among experimental groups. The area and lumen diameter increased when the saphenous vein was distended.¹² In addition, the venous wall deforms when subjected to hydrostatic pressure alone or in combination with shear stress. The results of the present study suggest that the presence of cyclic stress due to pulsatile flow could exert a protective effect by preventing such dilation. As observed in previous studies, flow shear stress is a stimulus for endothelial eNOS expression, which may explain the levels of eNOS maintained even in the presence of visible morphological endothelial injury.^14,15 When we started our research programme on the pressure effects in HSV endothelial dysfunction, we were wondering how morphological endothelial injury would be possible without endothelial dysfunction. We observed this fact in studies of in vitro HSV vascular reactivity (‘organ chambers’). Thus, we assumed that the remnant cells were functionally able to, at least in the short term, maintain endothelial pharmacological functionality. We think this is an attractive idea because it goes against the possible fragility of the endothelial cell.^11,17,18

The immunohistochemical data

With respect to the immunohistochemical expression of NOS isoforms, positive staining associated with eNOS, iNOS and nNOS was observed in all vein layers and in all groups, confirming the findings of previous studies.^7,13,18,19

In the present study, expression of eNOS in the endothelium was not decreased, confirming the findings of Dalio et al.¹¹ Shear stress in the presence of flow is a stimulus for eNOS endothelial expression, resulting in consequential synthesis and release of NO in the remaining endothelium.^14,15 In addition, expression of eNOS was not reduced in the venous walls of HSVs from non-diabetic patients. However, in diabetic patients, a significant decrease in eNOS expression was observed in the three venous wall layers after perfusion with the lower and higher pressures. These findings could explain the increased risk of occlusion of saphenous vein grafts observed in diabetics. Although diabetic patients do not show a decrease in endothelial expression of eNOS, as observed in this study, they may indicate alterations in NOS regulation and a consequent reduction in endothelial production of NO. Lower eNOS expression in the venous wall of HSVs from diabetic patients subjected to high perfusion pressures could explain the lower vasodilatation due to reduced NO production.

Immunohistochemical staining of inducible NOS was higher in the tunica media, and no difference in expression in the endothelium and venous wall was observed among the treatment groups. Inducible NOS expression was observed even in non-perfused veins. This finding is consistent with data from previous studies showing that iNOS was expressed in saphenous veins harvested conventionally without perfusion.^11,19 Although iNOS expression was increased in the endothelium of the perfused groups, compared to controls, the difference was not significant. This difference could be due to the use of a perfusion time of 3 h, which may not be sufficient to cause an increase in iNOS expression.

Similar to iNOS, nNOS expression was not affected by perfusion pressures, consistent with previous studies.^11,13 Although, not significant, the present study found evidence of slightly higher nNOS expression in endothelium undergoing perfusion, suggesting the existence of induction by shear stress and pulsatile flow that could be better observed after a longer perfusion time. Consistent with other studies, in all groups, higher nNOS expression was found in smooth muscle fibres of the middle layer.^11,17

In the present study, immunohistochemistry revealed CD34 expression in the three layers of the veins in all groups. Expression in the venous endothelium was significantly different between the non-diabetic control group and the non-diabetic group subjected to 300 × 250 mmHg perfusion pressures, with higher expression in the perfusion group. This difference was not observed in the diabetic group subjected to the same perfusion pressures or in groups undergoing 250 × 200 mmHg pressures. Despite the distinct morphological lesions described in this study, with areas of endothelial denudation, shear stress generated by flow could protect the remaining endothelium from the effects of hydrostatic pressure, as suggested by Dalio et al.¹¹ This protective effect may occur in the presence of both continuous and pulsatile flows. The CD34 expression increased in non-diabetic patients with high pressure. On the other hand, we could not explain why the CD34 expression remained unchanged in non-diabetic patients with low pressure group, and in all groups of diabetic patients (perfused and control).

Tissue nitrite/nitrate

Tissue levels of nitrite/nitrate did not differ significantly among treatment groups, similar to findings from our previous studies.^11,12 Although, a significant reduction in eNOS staining was observed in the three venous wall layers under 300 × 250 and 250 × 200 mmHg perfusion pressures in diabetic patients, this reduction was not sufficient to reduce NO_x tissue levels during the 3 h of perfusion. As eNOS’s endothelial expression did not differ among the study groups, it is reasonable to assume that NO production was not affected over the observation period. Although not significant, there was a trend towards increased NO_x tissue levels in groups subjected to perfusion pressures and pulsatile flow compared with control groups. This observation could reflect an increase in tissue levels of NO due to stimulation by shear stress and cyclic stress caused by pulsatile flow, even in diabetic patients and in the presence of a morphologically injured endothelium.^14,15

Oxidative stress

Similar to other published studies, no immunohistochemical expression of nitrotyrosine in the venous wall was observed.^11,12 NO can react with the superoxide anion (O₂⁻) to produce peroxynitrite (ONOO⁻),²⁰ which can cause nitration of tyrosine residues in proteins. Therefore, the absence of nitrotyrosine staining provides indirect evidence that oxidative stress does not have a crucial role.²¹ In addition, no differences in tissue MDA levels were observed among groups in this study, consistent with results from Dalio et al. and Viaro et al.^11,12 This result suggests that lipid peroxidation did not occur in the vein wall under conditions of higher pressure and pulsatile flow perfusion. Upregulation of reactive oxygen species was also not observed in previous studies,^11,12 suggesting that this detrimental mechanism is not acutely triggered under the present experimental conditions. Therefore, in addition to further studies, using the present pulsatile flow experimental system, we also need to consider other forces to which vein grafts can be subjected, including twisting and longitudinal stretching.

Clinical implications

Finally, a few words about possible clinical implications of studies of mechanical forces on HSV graft patency. As well known, HSV is prone to chronic degeneration (aneurysmal changes/degeneration in HSV grafts), which is a much rarer complication than occlusive complications in the early post-operative period. Discovering the factors, which predispose graft failure (such as reduced production of NO), may contribute to new therapies to improve graft patency. Maybe we can assume that even at higher pressures, the HSV preserves a functional reserve, suggesting a pivotal role of atherosclerosis risk factors on long-term venous graft patency.

Study limitations

One limitation of this study is that we evaluated only the distal portion of the HSV found close to the internal maleolus of the right leg. It could be argued that differences in endothelial function probably exist based on the area from which the vein was harvested. However, a recent publication failed to find functional or morphological differences between proximal and distal saphenous vein segments, suggesting that there is no advantage in using one of these preparations over the other as a CABG.²²

The correlation between diabetics and non-diabetics is a matter for criticism since different ‘types’ of diabetic patients were enrolled (i.e. some of the patients were on insulin, some others on oral medications and some others on both). The profound impacts of insulin on endothelial function, of different degrees of insulin resistance and of some sulphonyl urea agents are well known. Therefore, the diabetic group would also be considered heterogeneous to be representative of a broad ‘diabetic condition’ from an ‘endothelial’ point of view.

Footnotes

Authors’ contributions

All authors (1) made substantial contributions to conception and design,acquisition of data and/or analysis and interpretation of data;(2) participated in drafting the article or revising it critically for important intellectual content and (3) gave final approval of the version to be submitted and any revised version to be published. Roles of each of the authors in the study are as follows: (1) Designing the study (Paulo Roberto Evora);(2) collecting,analyzing and interpreting the data (all authors);(3) writing the report (Marina Rosique and Paulo Roberto Evora) and (4) making the decision to submit for publication (all authors).

Conflict of interest

None declared.

Funding

This study was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP),Fundação de Apoio ao Ensino,Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA/HCFMRP/USP),São Paulo,Brazil and Conselho Nacional de Pesquisa (CNPq),Brazil.

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Effects of elevated perfusion pressure and pulsatile flow on human saphenous veins isolated from diabetic and non-diabetic patients

Abstract

Objective:

Methods:

Results:

Conclusions:

Keywords

Introduction

Materials and methods

Ethical approval and patients

Harvesting of HSV samples

Study design and perfusion system

Light microscopy

Immunohistochemistry

Determination of tissue malondialdehyde and nitrite/nitrate

Statistical analysis

Results

Light microscopy

Immunohistochemical analysis of NOS, CD34 and nitrotyrosine expression

MDA and NOx levels

Discussion

The experimental model

The histological data

The immunohistochemical data

Tissue nitrite/nitrate

Oxidative stress

Clinical implications

Study limitations

Footnotes

Authors’ contributions

Conflict of interest

Funding

References

MDA and NO_x levels