Cell death pattern of a varicose vein organ culture model

Abstract

The study aimed to investigate the viability of a varicose vein (VV) organ culture model by assessing cell death pattern. To assess pattern of cell death with time, VV organ cultures were incubated for up to 14 days with regular medium changed. To assess viability, cell death of VV organ cultures treated with sodium azide and their untreated counterparts was assayed. Increased cell death was measured in VV organ cultures from day 0 to 2. Cell death decreased gradually after day 2 and plateaued from day 8 to 14. VV organ cultures treated with sodium azide demonstrated significantly more cell death in tissue (P = 0.001). Cell death measured in cultures treated with sodium azide continued to increase until day 7. In conclusion, this study demonstrated the viability of a VV organ culture model with most cell death occurred within the first two days and then declined to a relatively low level.

Keywords

varicose vein organ culture apoptosis necrosis cell death

Introduction

Varicose vein (VV) affects about one-third of the adult population.¹ Despite this, the pathophysiology of VV remains uncertain.¹ Previously, valvular dysfunction causing reflux was thought to be the primary cause of VV.¹ Recently, evidence suggests that vein wall changes and dilation may be the primary events whereas valvular reflux occurs as an epiphenomenon in VV pathogenesis.^1,2 Various structural and biochemical changes in VV wall including reduction of elastin, alteration of collagen content, imbalances of matrix metalloproteases (MMPs) and their tissue inhibitors, activation of endothelium, intimal hyperplasia, increased smooth muscle cell proliferation, abnormal angiogenesis, dysregulated apoptosis, reduced vascular tone and upregulation of the hypoxia-inducible factor pathway.^1–4 These changes in vein wall are likely multifactorial in origin including genetic predisposition, increasing age, pregnancy and hormonal changes.^1,2 Several abnormal hemodynamic and physiological stresses such as hypoxia, low shear stress and venous hypertension have also been postulated.¹

Studying the biochemical, physiological and structural changes in VV wall, and their potential contributing factors and exogenous stimuli is important in understanding the mechanisms of the disease formation, as well as identifying potential therapeutic targets.¹ Organ cultures have often been used to study the effects of various exogenous factors and stimuli including potential pathological stresses and therapeutic agents on the organ or tissue of interest including in diseases of venous tissues.^5–11 In the study of VV disease, organ cultures of varicosities have been used, and they were often relatively similar, with some modifications, to a non-varicose saphenous vein organ culture model that was initially developed for the study of neointimal hyperplasia in vein grafts.^{3–5,7,12,13} Although the original non-varicose saphenous vein organ culture model was well validated,^14,15 the viability of the VV organ culture model has never been reported. It is unwise to extrapolate results and assume that VV and non-VV organ culture have similar viability despite similarities in technical set-up of the organ culture since VVs are different structurally, physiologically and biochemically from non-VVs. Therefore, the aim of the study is to investigate the viability of a VV organ culture model that may be used in in vitro studies of VV wall behaviour and changes by assessing the organ culture cell death pattern.

Materials and methods

Patient recruitment and tissue collection

Ethical approval for the study was obtained from the Riverside Research Ethics Committee (RREC3092), London, United Kingdom.

Primary VVs were retrieved from patients who underwent phlebectomies with or without open surgery or endovenous treatment. The VVs used were refluxing, hence diseased truncal veins or their tributaries in any location of the superficial venous system of the leg. All patients with VVs were scanned with duplex ultrasonography in the standing position and refluxing veins were marked by vascular scientists preoperatively. Reflux was defined as retrograde flow lasting for more than 0.5 seconds. Patients with a history of secondary VVs including deep venous thrombosis and congenital VV, connective tissue disease, vasculitis and rheumatoid arthritis were excluded from the study. The demographic data, previous medical history, drug history, and the clinical, etiological, anatomical and pathophysiological (CEAP) classification of patients recruited were collected. Tissues collected were transported in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories GmbH, Pasching, Austria) to the laboratory. Vein organ cultures were prepared within one hour from the time of retrieval from patient.

VV organ culture

Organ cultures of VV were prepared based on a model previously described by Soyombo et al.¹⁵ for saphenous (non-varicose) vein organ cultures for the study of intimal hyperplasia in bypass graft with some modifications. Briefly, VVs were cut open longitudinally and transversely to expose the endothelial surface and dissected into segments measuring 0.5 cm × 0.5 cm. Each segment was then pinned out with A1 minuten pins (Watkins & Doncaster, Kent, UK) at the four corners with the endothelial surface uppermost, onto a polyester mesh (Sericol Limited, Kent, UK) resting on sylgard resin (Dow Corning, Seneffe, Belgium) in a glass Petri dish (DURAN® Group GmbH, Mainz, Germany). The culture medium used was 5 mL of DMEM high glucose (4.5 g/L) with l-glutamine, and sodium pyruvate (PAA Laboratories GmbH) plus 100 U/mL penicillin and 0.1 mg/mL streptomycin (PAA Laboratories GmbH) plus 10% fetal bovine serum (Gibco™, Invitrogen Life Sciences, Paisley, UK). The organ culture was incubated at 37℃ and 5% CO₂.

Two separate experiments were carried out to assess the viability of a VV organ culture model; firstly to investigate the cell death pattern of VV cultured for 14 days, and secondly to study the effects of sodium azide on cell death pattern of VV organ culture.

Cell death pattern of VVs cultured for 14 days

To assess the pattern of cell death with time, VV organ cultures were incubated at 37℃ and 5% CO₂ for up to 14 days with culture medium changed every 48 hours. VV specimens were obtained from a total of fourteen patients for analysis. Vein obtained from each patient was used to prepare five vein organ cultures. All the VV organ cultures were incubated at 37℃ and 5% CO₂. Culture medium was changed every 48 hours. Culture medium that had been used was collected and stored in −80℃ for cell death assay later. VV organ cultures from each patient were sampled at random and snap frozen on days 2, 4, 8, 10 and 14 for cell death assay later.

The effects of sodium azide on cell death pattern of VV organ culture

To demonstrate that the VV organ culture model contained viable cells, the cell death of VV organ cultures treated with sodium azide 10 mmol/L and their untreated counterparts were assayed and compared. Sodium azide causes cell death through apoptosis and/or necrosis.^16–19 VV specimens obtained from every patient (total of four patients) were used to prepare seven vein organ cultures. All the organ cultures were incubated at 37℃ and 5% CO₂. On day 1, one organ culture selected at random from each patient was snap frozen, and the culture medium was collected; both tissue and culture medium were stored at −80℃. The remaining organ cultures of that patient were divided into two groups; treated and untreated with sodium azide. The culture medium of the organ cultures was changed on days 1, 3 and 5 with or without sodium azide containing medium depending on the group they belonged to. On days 3, 5 and 7, one VV organ culture from each group was sampled at random and snap frozen, and the culture medium was also collected; both tissue and culture medium were stored at −80℃.

Cell death assay

Preparation of VV tissue lysate

VV tissues stored in the −80℃ freezer were thawed in room temperature. Vein tissues were homogenized by cutting it into tiny pieces. They were then crushed (3–5 times) gently with the rubber end of a plunger from a 1-mL syringe. The crushed tissue of each vein was transferred into a 1.5-mL Eppendorf before the addition of the lysis buffer that came with the Cell Death Detection ELISA Plus® (Roche Diagnostics GmbH, Mannheim, Germany) kit; 200 μL of lysis buffer per 6.6 mg of VV tissues was used. The tissues were further crushed and homogenized in the lysis buffer. The homogenates were left for 30 minutes at 4℃, and then were centrifuged at 13,000 g for 20 minutes at 4℃. The supernatant (tissue lysate) of the homogenates were transferred into new 1.5-mL Eppendorfs for further use. Please note that cutting and crushing the tissues to prepare tissue lysate and then homogenates would not affect cell death analysis since all the cells would have been killed and all biological processes would have been stopped instantly when the tissues were snap frozen. Similar method of tissue lysate preparation for this type of cell death assay had previously been reported.²⁰

Preparation of culture medium lysate

Culture medium that was stored in the −80℃ freezer was thawed in room temperature. Then, 1 mL of culture medium from each specimen was transferred into a Falcon® 24-well tissue culture plate (Becton Dickinson Labware, NJ, USA). The culture medium was centrifuged at 1200 rpm for 10 minutes with the Napco 2028R multifunction centrifuge. The supernatant was then discarded, leaving the pellets containing cellular debris including the mono- and oligonucleosomes. The pellet was then treated with 200 μL of lysis buffer that came with the Cell Death Detection ELISA Plus® (Roche Diagnostics GmbH) kit for 30 minutes. The mixture was then centrifuged at 1200 rpm for 10 minutes. The supernatant (culture medium lysate) were transferred into new 1.5-mL Eppendorfs for further use.

Cell Death Detection ELISA Plus®

Cell death was assayed using the Cell Death Detection ELISA Plus® (Roche Diagnostics GmbH). The Cell Death Detection ELISA Plus® was based on a quantitative and semi-quantitative sandwich-enzyme-immunoassay principle, using mouse monoclonal antibodies directed against DNA and histones, respectively. The assay allowed the specific detection and quantitation of histone-complexed DNA (mono- and oligonucleosomes) that are released into the cytoplasm and surrounding medium of cells undergoing apoptosis and necrosis, respectively. Therefore, the cell death assayed from tissue and culture medium lysate represented apoptosis and necrosis, respectively. Lysate (20 μL) from each specimen (tissue and culture medium), positive control (provided in the kit) and negative control (incubation buffer only) were analyzed on the microplate provided. Each sample was analyzed in duplicates. Immunoreagent was prepared by mixing of 1/20 volume anti-DNA-POD and 1/20 volume anti-histone-biotin with 18/20 volumes incubation buffer, and 80 μL of this was added to each lysate. The microplate was then covered with an adhesive plate cover and left on a plate shaker (Orbit 300, Labnet International Incorporation, Edison, NJ, USA) at 300 rpm at room temperature for two hours. The plate was then washed three times with 250 μL per well of incubation buffer. Then 100 μL of the ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) solution (1 tablet of ABTS in 5 mL of substrate buffer) was added into each well and absorbance was measured at 405 nm using a plate reader.

Statistical analysis

Statistical analyses were performed with GraphPad Prism version 5.00 (GraphPad Software, San Diego CA, USA). Data were expressed as mean ± SEM. Two-way analysis of variance with Bonferroni post-tests was used to test the differences between the cell death of VV organ cultures treated and untreated with sodium azide. P < 0.05 was considered significant.

Results

Cell death pattern of VVs cultured for 14 days

The demographic details of the 14 patients from whom the VVs were harvested and used for the analysis of cell death pattern of VVs cultured for 14 days are summarized in Table 1.

Table 1

Demographic data of patients whose varicose veins were used for the assessment of cell death of varicose veins cultured for 14 days

Number of patients	14
Mean age (range)/year	46.0 (21–70)
Sex	5 Males
	9 Females
Smoking	11 Non-smokers
	1 Ex-smokers
	2 Smoker
Laterality	7 Right
	3 Left
	4 Bilateral
Past medical history
Deep vein thrombosis	0
Cardiac disease	0
Diabetes mellitus	1
Peripheral arterial disease	0
Hypertension	1
Drugs
Aspirin	0
Statin	1
Clinical CEAP
C₂	6
C₃	5
C_4a	2
C_4b	1
Sites of reflux
GSV	11
SSV	4
Perforator	2
Popliteal	1
Tributaries only	1
Recurrence	5

GSV, great saphenous vein; SSV, small saphenous vein; CEAP, clinical, etiological, anatomical and pathophysiological classification

Figure 1 demonstrates the pattern of cell death (apoptosis) measured by the Cell Death Detection ELISA Plus® of tissue lysate of VVs cultured for 14 days. Increased cell death (apoptosis) was measured in the tissue lysate of VV organ cultures from days 0 to 2. The amount of cell death (apoptosis) decreased rapidly from days 2 to 4. After this period cell death (apoptosis) continued to decrease less rapidly and then plateaued off from day 8 until at least day 14. Figure 2 shows the pattern of cell death (necrosis) represented as optical density, per gram of VV tissue measured in the culture medium of the VV organ cultures over 14 days using Cell Death Detection ELISA Plus®. Similar to tissue lysate, the cell death (necrosis) measured in the culture medium increased rapidly from days 0 to 2. The amount of cell death (necrosis) decreased rapidly from day 2 onwards and seemed to plateau off to relatively little cell death (necrosis) from day 8 onwards until day 14.

Figure 1

Cell death (apoptosis) pattern of tissue lysate of varicose veins cultured for 14 days, assayed with Cell Death Detection ELISA Plus®. Varicose veins were obtained from 14 patients. Culture medium was changed every 48 hours. Cell death was measured as optical density, A_405nm. Data represent mean ± SEM

Figure 2

Cell death (necrosis) pattern of culture medium lysate sampled from varicose vein organ cultures cultured for 14 days, assayed with Cell Death Detection ELISA Plus®. Varicose veins were obtained from 14 patients. Culture medium was changed every 48 hours. Cell death was measured as optical density, A_405nm per gram of tissue. Data represent mean ± SEM

The effects of sodium azide on cell death pattern of VV organ culture

The demographic details of the four patients from whom the VVs were harvested and used to study the effects of sodium azide on cell death of VV organ culture are summarized in Table 2.

Table 2

Demographic data of patients whose varicose veins were used for the assessment of the effects of sodium azide on cell death on varicose vein organ culture

Number of patients	4
Age/year	35, 38, 38, and 63
Sex	1 Males
Sex	3 Females
Smoking	3 Non smokers
Smoking	1 Smoker
Laterality	1 Right
	1 Left
	2 Bilateral
Past medical history
Deep vein thrombosis	0
Cardiac disease	0
Diabetes mellitus	0
Peripheral arterial disease	0
Infections	0
Drugs
Aspirin	0
Statin	0
Clinical CEAP
C₂	3
C_4b	1
Sites of reflux
GSV	4
SSV	1
Recurrence	1

GSV, great saphenous vein; SSV, small saphenous vein; CEAP, clinical, etiological, anatomical and pathophysiological classification

Figures 3 and 4 compare the cell death measured in tissue lysate (apoptosis) and culture medium (necrosis), respectively, of VV organ cultures treated and untreated with sodium azide. Overall, VV organ cultures that were treated with sodium azide on day 1 onwards demonstrated significantly more cell death (apoptosis) in the tissue lysate (P = 0.001). The cell death (apoptosis) measured in tissue lysate of VV organ cultures treated with sodium azide on day 1 onwards continued to increase until day 7. Whereas, the cell death (apoptosis) measured in tissue lysate of VV organ cultures in untreated samples did not seem to increase after day 1. The cell death (necrosis) measured in culture medium of VV organ cultures both treated and untreated with sodium azide appeared similar, i.e. increased from days 1 to 3 and then declined from days 3 to 7.

Figure 3

Cell death (apoptosis) pattern of tissue lysate of varicose vein organ culture untreated (full line) and treated (dotted line) with 10 mmol/L sodium azide, assayed with Cell Death Detection ELISA Plus®. Varicose veins were obtained from four patients. Cell death was measured as optical density, A_405nm. Data represents mean ± SEM. Varicose vein organ cultures were cultured at 37℃ and 5% CO₂ for seven days. Culture medium was changed on day 1 and then every 48 hours up to day 7. In the sodium azide group, the varicose vein organ cultures were treated with 10 mmol/L sodium azide from day 1 onwards. Significantly more cell death (apoptosis) was measured in varicose vein organ cultures treated with sodium azide than those untreated (P = 0.001; two-way analysis of variance with Bonferroni post-tests)

Figure 4

Cell death (necrosis) pattern of culture medium lysate of varicose vein organ culture untreated (full line) and treated (dotted line) with 10 mmol/L sodium azide, assayed with Cell Death Detection ELISA Plus®. Varicose veins were obtained from four patients. Cell death was measured as optical density, A_405nm per gram of tissue. Data represents mean ± SEM. Culture medium was changed on day 1 and then every 48 hours up to day 7. In the sodium azide group, the varicose vein organ cultures were treated with sodium azide 10 mmol/L from day 1 onwards. No difference in cell death (necrosis) was measured between varicose vein organ cultures treated with sodium azide 10 mmol/L and those untreated with two-way analysis of variance with Bonferroni post-tests

Discussion

Organ culture is an in vitro model involving keeping a whole organ or tissue in conditions that enable the tissue or organ to remain viable and behave as close to its in vivo characteristics as possible. Using such an in vitro model has the advantage of being able to control the experimental conditions more easily compared with an in vivo whole-body model. Therefore, like cell culture, organ culture can be used to study the effects of various exogenous stimuli including drugs, radiation, cytokines, growth factors, hormones and physical strains such as mechanical stretch.²¹ Furthermore, organ culture model has been developed as a disease model to study a particular pathology.¹⁵ However, one major limitation of an in vitro model is the loss or compromised of real-life representation.²¹ Compared with cell culture, organ culture has the advantage of preserving the structural relationship and interactions between the different cell types and with the extracellular matrix. However, organ culture is generally less predictable than cell culture, and the interpretation of the results may be more complex due to the presence of various factors involving different cell types and extracellular matrix.

Organ cultures of venous tissues have been reported in many vascular biology studies.^4,7,14,15 The most commonly used vein organ culture model was the non-varicose saphenous vein organ culture described by Soyombo and colleagues, for which the VV organ culture used in this study was based on with some modifications. The original model was developed to study the pathophysiology of neointimal hyperplasia development in saphenous vein graft.^8,14,15 The non-varicose saphenous vein organ culture model had been used and validated in many occasions, and it appeared to be reproducible by different research groups.^7,14,15,22 Porter et al.²³ used this model to demonstrate that the development of neointimal hyperplasia was associated with increased production of MMP-9. Besides, this model has also been used to study the effects of various growth factors (e.g. platelet-derived growth factor)¹¹ and drugs (e.g. simvastatin and marimastat)^8–10 on neointimal thickening in saphenous vein graft. VV organ cultures have also been used not uncommonly to study the pathophysiology and potential treatment of the disease. Matagne and Gilles⁵ used VV organ cultures to study the oxidative metabolism and glucose transport and the effects of O-(beta-hydroxyethyl)-rutoside on lactate dehydrogenase in VVs. Recently, Alda and colleagues demonstrated the in vitro protective effect of calcium dobesilate on oxidative stress in VVs. In another study, VV organ cultures was used to assess the effect of micronized purified flavonoid fraction (Daflon® 500 mg) on noradrenaline induced contraction of the veins.⁶ Nomura et al.⁷ used a VV organ culture model similar to the non-varicose saphenous vein organ culture with few modifications to study the effect of statins on MMPs in VVs. Similar VV organ culture model has also been used by us to study the hypoxia-inducible factor pathway and potential therapeutic agents in VVs.^4,13

Almost all the patients with VV in our unit were treated with endovenous treatment with or without phlebectomies rather than open surgery. Therefore, to increase the sample size, VV removed through phlebectomies were used. Duplex scanning were performed preoperatively to mark and ensure that the veins removed were refluxing, hence diseased. Furthermore, since the use of endovenous treatment is increasing, it is likely that future studies of VV will more likely to use varices obtained through phlebectomies rather than open surgery.

In the original non-varicose saphenous vein model described by Soyombo and colleagues, the viability of the organ culture was checked with adenosine diphosphate (ADP) to adenosine triphosphate (ATP) ratio assay, measurement of [³H]-thymidine incorporation into DNA, and histological analysis with light and scanning electron microscopy. The ATP concentration in cultured veins after 7 and 14 days was found to be approximately 30 and 20% lower than in freshly isolated vein, respectively. The ATP/ADP ratio was found to have increased by 25 and 45% after seven and 14 days in culture, respectively. The DNA concentration declined by about 20% after seven days but recovered by 14 days in culture. Scanning electron microscopy of freshly isolated veins prepared for culture showed that endothelial cells were largely undamaged in the central portion but appeared injured and loss near the cut edges of segments. Light microscopy of silver-stained segments showed that about 70% of the total surface was covered by endothelial cells in the freshly isolated veins prepared for culture compared with 80% of those cultured for 14 days.^14,15,22,24

Apoptosis is characterized by cell shrinkage, fragmentation into membrane-enclosed apoptotic bodies and phagocytosis by macrophages. Apoptosis has long been considered as a highly regulated and efficient cell death process with the level of ATP in the apoptotic cells being maintained and inflammation being avoided. In contrast, necrosis is characterized by loss of plasma membrane integrity, cellular and organellar swelling and marked inflammation.²⁵ Necrosis has long been described as a result of extreme physicochemical stress, such as heat, osmotic shock, mechanical strain, freeze–thawing and high concentration of hydrogen peroxide. In these conditions, cell death occurs rapidly due to the direct effect of the stress on the cell, and therefore necrosis has been described as accidental and uncontrolled. However, many different cellular stimuli including tumor necrosis factor, double-stranded RNA, interferon-γ, hypoxia and ATP depletion have been shown to induce a programmed necrosis.²⁶ Both apoptosis and programmed necrosis are mediated by distinct, but highly overlapping central pathways including the death receptor (extrinsic) and mitochondrial (intrinsic) pathways.²⁵ In this study, we did not intend, and hence were not able to determine the factors that affected cellular apoptosis and necrosis in the organ culture. However, both processes were detected by the cell death assay used. Cell death assay of VV organ culture demonstrated that most cell death, both apoptosis and necrosis, occurred within the first two days of the culture. The cell death then declined to a relatively low level and remained more or less the plateau until at least day 14. We did not perform any histological analysis to identify the cell type of the vein organ culture that underwent cell death. However, based on the findings from previous study, the spike of cell death observed within the first two days was likely to represent the trauma caused by the surgical retrieval and organ culture preparation that involved tissue handling, dissection and cutting.¹⁵ This was consistent with the endothelial injury and loss at the edges demonstrated in the original model of non-varicose saphenous vein organ culture. In the non-varicose saphenous vein model, surgically preparation of veins has also been shown to cause damage to the tissues leading to more neointimal hyperplasia development.^15,24 The relatively low level of cell death measured in the VV organ culture after day 2 was also consistent with the viability demonstrated in the original non-varicose saphenous vein organ culture which was maintained at least up to 14 days.^15,22,24 The relatively high level of cell death in the first 48 hours of culture suggested that the VV organ culture model should be used as early as possible, for example, within 4–6 hours. Although we demonstrated that the VV organ culture model contained living cells for up to 14 days, it was likely that the longer the VV were cultured, the more the in vitro tissues deviated from the in vivo characteristics, losing the real-life representation.

Sodium azide is known to cause cell death through apoptosis and/or necrosis. It inhibits oxidative phosphorylation by preventing electron transfer between cytochrome c oxidase or mitochondrial complex IV and oxygen.^16–19 It also reversibly increases intracellular Ca²⁺, at least partially involving NMDA glutamate receptor activation.^19,27 Treatment of VV organ culture with sodium azide was expected to cause increases in cell death either by apoptosis or necrosis, or both. This was clearly shown in this study when treatment of VV organ culture with sodium azide from day 1 of culture onwards caused the cell death to continue to rise to at least day 7. Whereas, the cell death of those untreated did not increase after day 1. This finding also supported that the VV organ culture preparations contained living cells. Meanwhile, the cell death measured in the culture medium of VV organ cultures treated and untreated with sodium azide appeared similar. This finding suggested that sodium azide caused cell death in VV organ culture through apoptosis, not necrosis.

One of the limitations of this study was no non-VV organ culture was studied alongside with VV organ culture model. The non-varicose saphenous vein organ culture model had been previously well validated including in terms of viability and cell death by previous studies.^14,15 Although the findings from this study seemed to agree with previous studies of non-varicose saphenous vein organ culture model, it is important to stress that these findings should be interpreted with caution.

Conclusion

In conclusion, this study demonstrated the viability of a VV organ culture model with most cell death occurred within the first two days of the culture. The cell death then declined to a relatively low level and remained more or less the plateau until at least day 14.

Footnotes

Competing interest: None.

Funding: CSL was awarded a Research Fellowship from the Royal College of Surgeons of England and Rosetrees’ Trust, and received research grants from the European Venous Forum, Graham-Dixon Charitable Trust and European Society for Vascular Surgery.

Ethical approval: Ethical approval for the study was obtained from the Riverside Research Ethics Committee (RREC3092), London, UK.

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