Annals of Vascular Surgery
Volume 24, Issue 6 , Pages 768-774, August 2010

Involved Intrinsic Apoptotic Pathway in the Varicocele and Varicose Veins

  • Jane-Dar Lee

      Affiliations

    • Division of Urology, Department of Surgery, Taichung Armed Forces General Hospital, Taichung, Taiwan, Republic of China
    • Central Taiwan University of Science and Technology, Taichung, Taiwan, Republic of China
    • Corresponding Author InformationCorrespondence to: Jane-Dar Lee, Department of Surgery, Taichung Armed Forces General Hospital, No 348, Sec 2, Chung-San Rd, Taiping, Taichung, Taiwan, Republic of China
    • ,
  • Wen-Kai Yang

      Affiliations

    • Division of Urology, Department of Surgery, Taichung Armed Forces General Hospital, Taichung, Taiwan, Republic of China
    • Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan, Republic of China
    • ,
  • Chin-Hu Lai

      Affiliations

    • Division of Cardiovascular Surgery, Department of Surgery, Taichung Armed Forces General Hospital, Taichung, Taiwan, Republic of China

published online 13 May 2010.

Article Outline

Background

Disordered programmed cell death may play a role in the development of venous diseases. Tissue hypoxia caused by blood stagnation and venous hypertension is the similar etiology of varicocele and varicose veins. We studied the vascular histopathology and determined whether there is the same apoptotic pathway in both venous diseases.

Methods

The study groups consisted of 1-cm venous segments obtained from 10 patients during vascular stripping surgery for varicose saphenous vein and 1 cm of internal spermatic veins obtained from 12 patients during left varicocele repair. The control samples of 1 cm internal spermatic vein were obtained from 10 male patients who underwent left inguinal herniorrhaphy. The three layers of vascular histology were measured and compared by Masson trichrome stain, and the apoptotic proteins including Bcl-2, Fas, cleaved caspase-9, cleaved caspase-8, and cleaved caspase-3 were detected. Data were analyzed using the one-way analysis of variance with Tukey's comparison test.

Results

The relative thickness of intima and adventitia layer was smaller in both study groups than in the control group. But a significant hypertrophy of media layer was observed in the varicocele and varicose veins than in the control group (p < 0.05). Overexpression of Bcl-2 and decreased expressions of cleaved caspase-9 and cleaved caspase-3 was observed in both study groups. There is no statistical difference in Fas and cleaved caspase-8 expressions in the control and study groups.

Conclusion

Our data showed vascular smooth muscle hypertrophy in the diseased vessels. The same dysregulation of apoptosis through intrinsic pathway was demonstrated in varicocele and varicose veins under tissues hypoxia. This mechanism of reduced apoptosis might contribute to the dilated and thickened walls of both venous diseases.

 

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Introduction 

Varicocele is characterized by dilated and thickened wall of the internal spermatic veins (ISVs). Although it is found in 15-20% of men,1, 2, 3 the recurrence rate of about 2-14%4 after varicocele repair is still an issue. Similarly, the prevalence of varicose veins is definitely underestimated and affects up to 40% and 51% of men and women, respectively.5 In both venous diseases, the same etiology of increased blood stasis and venous volume pressure caused tissue hypoxia and remodeling of the vessel wall as a compensatory result.3, 6, 7 The investigation of these diseased vessels is very rare. In this study, we examined whether the same histopathologic changes and molecular apoptotic pathway occurred in both venous diseases.

It has been established that apoptosis has a role in normal development and an important role in the pathogenesis of venous diseases.8 There are two pathways (intrinsic and extrinsic) that induce apoptotic cell death.9, 10 Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis, which includes a receptor for tumor necrosis factor itself and the Fas death receptor. Cells can also activate their apoptosis program from inside the cell, usually in response to injury or other stresses, such as DNA damage or lack of oxygen, nutrients, or extracellular survival signals. In vertebrate cells, such intracellular activation of the apoptotic death program occurs through the intrinsic pathway of apoptosis, which depends on the release into the cytosol of mitochondrial proteins.11 Elucidation of mechanisms involved in the disease process is the first step to improve therapeutic modulations and prevent disease recurrence.

In this research, we measured the venous thickness of intima, media, and adventitia layer to detect histologic changes by Masson trichrome stain and study the apoptotic proteins including Fas, cleaved caspase-8, Bcl-2, cleaved caspase-9, and cleaved caspase-3 expressions in normal ISV, varicocele, and varicose veins to differentiate the intrinsic or extrinsic pathway.

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Materials and Methods 

Patients and Tissue Samples 

One study group consisted of 12 patients with mean age 23 ± 1.5 years suffering from left varicocele (grade 3) for many months who received varicocele repair because of scrotal dragging pain. Varicocele was graded in 1970 according to Dubin and Amelar.12 To prevent interobserver bias, all physical examinations were performed by one physician. The other study group consisted of 10 patients (five men, five women) with mean age 56.5 ± 2.5 years who were undergoing stripping surgery for varicose saphenous veins after venous duplex ultrasound study. The operative indications were leg pain, edema, or venous stasis disease. All patients were characterized as having primary varicosities, and their symptoms persisted for more than 6 months. According to the CEAP classification for chronic lower-extremity venous disease,13 most of the patients were in class 2 (n = 6) and others in class 3 (n = 4). There were three patients with hypertension, three with diabetes mellitus, and two were known to abuse tobacco. A 1-cm venous segment was obtained to compare with the control group. The control group consisted of 10 male volunteers with mean age 23 ± 1.8 years suffering from indirect left inguinal hernia, for whom the possibility of varicocele was ruled out (ISV diameter <2 mm)14 and there was no venous disease by duplex ultrasound study. The same left inguinal surgical incision was performed in patients with hernia and varicocele, the ISV specimen was resected and stored at −80 °C for histopathologic study by Masson trichrome stain, and immunoblotting of Fas, cleaved caspase-8, Bcl-2, cleaved caspase-9, and cleaved caspase-3 were detectedr. All specimens were removed only after written informed consent was obtained from the patients. This study was approved by the Institutional Review Board of our hospital.

Masson Trichrome Stain and Vascular Thickness Detection 

Formalin-fixed and paraffin-embedded sections (5 μm) of vessels from three groups were deparaffinized and Masson trichrome stain was performed. Deparaffinized sections were washed two times for 5 minutes each in deionized water (ddH2O). The sections were incubated in mordant solution containing 5% (wt/vol) trichloroacetic acid and 5% (wt/v) potassium dichromate, Carazzi's hematoxylin, 0.75% orange G solution, Masson B stain solution mixing 2% ponceau xylidine or 2% acid fuchsin (v/v: 2/1) in acetic acid, 2.5% phosphotungstic/phosphomolybdic (v/v: 1/1) acid solution, and aniline blue solution containing 0.4% aniline blue and 8% acetic acid in proper order. Finally, the sections were dehydrated through alcohol, cleared by xylene, and stored hermetically. Then all sections were observed using an Olympus BX50 light microscope and photographed with a Nikon CP5000 digital camera.

The thickness of vein walls was obtained randomly from an average of at least eight transections of each vein thickness and quantified by Image-Pro Plus software (Image-Pro Plus version 4.5.0.29; Media Cybernetics, Bethesda, MD). The thickness of walls was divided into four parts, that is, intima, media, adventitia, and total thickness.

Antibodies 

Six primary antibodies were used in the present study: (1) Fas: a rabbit polyclonal antibody (sc-7886; Santa Cruz, Santa Cruz, CA) (2) caspase-8: a mouse monoclonal antibody (9746; Cell Signaling, Beverly, MA); (3) Bcl-2: a mouse monoclonal antibody (sc-7382, Santa Cruz); (4) caspase-9: a rabbit polyclonal antibody (9502; Cell Signaling); (5) cleaved caspase-3: a rabbit monoclonal antibody (9664; Cell Signaling); (6) β-actin: a mouse monoclonal antibody (8226; Abcam, Cambridge, MA).

The secondary antibodies for Western blot were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (0031430; Pierce, Hercules, CA; for detection of Bcl-2, caspase-8, and β-actin) or goat anti-rabbit IgG (0031460, Pierce; for detection of Fas, caspase-9 and cleaved caspase-3).

Immunoblotting 

The method used was modified from our previous studies.15, 16 Each sample stored at −80 °C was homogenized on ice and then centrifuged at 13,000g, at 4 °C for 20 minutes. The supernatants were used for determination of protein concentrations or immunoblotting. Protein concentrations were identified by reagents from BCA Protein Assay Kit (Prod No. 23225; Pierce), using bovine serum albumin as a standard (Prod No. 23209; Pierce).

For immunoblotting, the antibodies of Fas, cleaved caspase-8, Bcl-2, cleaved caspase-9, cleaved caspase-3, and β-actin revealed molecular weights of about 48, 43, 26, 35-37, 17-19, and 44 kDa, respectively. The blots were cut into upper and lower portions at feasible sites for incubation, incubated at 4 °C overnight with the diluted primary antibodies, and then incubated with diluted secondary antibody for 1 hour. Finally, the immunoreactive bands were analyzed by using MCID software version 7.0 (Imaging Research, Ontario, Canada). The results were converted to numerical values (normalized relative to β-actin expression) to compare the relative protein abundance of the immunoreactive bands.

Statistical Analysis 

Data were analyzed using the one-way analysis of variance and Tukey's comparison tests with p < 0.05 considered a statistically significant difference among three groups.

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Results 

First, the relative thickness of intima and adventitia layers in both study groups was smaller than the control group, but the significant hypertrophy of muscular layer (media) was observed in the varicocele and varicose veins as compared with the control group (p < 0.05) by Masson trichrome stain (Table I; Figs. 1 and 2). Second, the immunoblots of Bcl-2 (at 26 kDa), Fas (at 48 kDa), cleaved caspase-9 (at 35-37 kDa), cleaved caspase-8 (at 43 kDa), cleaved caspsae-3 (at 17-19 kDa), and relative intensity were seen in all patients (Table II). There was no statistical difference in Fas and cleaved caspase-8 expressions among the control and study groups (Table II, Figs. 3 and 4). Overexpression of Bcl-2 and significantly decreased expression (6.8–9.5 folds) of cleaved caspase-9 and cleaved caspase-3 (p < 0.05) in both study groups compared with the control group (Table II, Fig. 5, Fig. 6, Fig. 7, respectively), which meant that reduced apoptosis through intrinsic pathway occurred in these diseased vessels.

Table I. Relative thickness of intima (Int), media (Med), and adventitia (Adv) of three venous groups
Tunica\VeinsHerniaVaricose veinVaricocele
Int7.26 ± 0.40a2.77 ± 1.12b2.01 ± 0.28b
Med73.73 ± 1.78a86.67 ± 0.52b88.28 ± 0.25b
Adv19.01 ± 1.51a10.55 ± 0.60b9.71 ± 0.53b

Dissimilar letters indicate the significant differences among three venous groups. Mean ± SD, one way ANOVA with Tukey's comparison, p < 0.05.

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  • Fig. 1 

    Micrographs of representative ISV sections after Masson trichrome staining for hernia (A; 100×), varicose vein (B; 40×), or varicocele (C; 100×). The vein walls are divided into three parts: intima (Int.), media (Med.), and adventitia (Adv.).

Table II. Relative abundance of apoptotic proteins in three venous groups
Proteins\VeinsHerniaVaricose veinVaricocele
Fas47.64 ± 9.2527.46 ± 2.4230.99 ± 3.00
Bcl-238.09 ± 4.63a84.87 ± 13.43b91.64 ± 11.30b
Cleaved caspase-881.43 ± 27.6133.30 ± 12.6652.13 ± 16.94
Cleaved caspase-9245.43 ± 45.57a35.80 ± 18.17b25.78 ± 2.67b
Cleaved caspase-3142.53 ± 18.89a80.75 ± 5.78b73.74 ± 3.31b

Dissimilar letters indicate the significant differences among three venous groups. Mean ± SD, one way ANOVA with Tukey's comparison, p < 0.05.

  • View full-size image.
  • Fig. 3 

    Representative immunoblot and relative intensities of Fas protein in three venous groups. β-actin was used as the loading control. Values of Fas expression are normalized relative to β-actin expression.

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  • Fig. 4 

    Representative immunoblot and relative intensities of cleaved caspase-8 protein in three venous groups. β-actin was used as the loading control. Values of cleaved caspase-8 expression are normalized relative to β-actin expression.

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  • Fig. 5 

    Representative immunoblot and relative intensities of Bcl-2 protein in three venous groups. β-actin was used as the loading control. Dissimilar letters indicated significant differences among various groups. Values of Bcl-2 expression are normalized relative to β-actin expression.

  • View full-size image.
  • Fig. 6 

    Representative immunoblot and relative intensities of cleaved caspase-9 protein in three venous groups. β-actin was used as the loading control. Dissimilar letters indicated significant differences among various groups. Values of cleaved caspase-9 expression are normalized relative to β-actin expression.

  • View full-size image.
  • Fig. 7 

    Representative immunoblot and relative intensities of cleaved caspase-3 protein in three venous groups. β-actin was used as the loading control. Dissimilar letters indicated significant differences among various groups. Values of cleaved caspase-3 expression are normalized relative to β-actin expression.

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Discussion 

Both varicocele and varicose veins are venous diseases characterized by tortuous, dilated, and thickened vein walls as a compensatory result of increased blood stasis and venous volume pressure.5, 7, 8, 17 Both vascular diseases affect millions of people but the investigation of apoptotic pathway in these diseased vessels is still very poor. We had reported a 7-fold increase of hypoxia-inducible factor-1α in the ISV of patients with varicocele15, which indicated that hypoxia-related pathophysiologic changes have occurred in the diseased vessels. Similarly, the varicose veins occurrence because of poor venous return and blood stagnation causing tissue hypoxia is associated with remodeling of vessel wall. So we examined whether the same histopathologic changes and molecular apoptotic pathway in both venous diseases compared with normal vessels.

Blood stasis will provoke an ischemia, thus decreasing oxygen availability to tissues. Under hypoxia, the vascular endothelium is the starting point of cascade of events leading to the disorganization of the vessel wall typical of these pathologies.6, 7 Apoptosis (programmed cell death) plays a major role in tissue homeostasis and maintenance. In the human apoptotic pathway cascade, 14 caspases (cysteinyl aspartate-specific proteinases) have been found to date.9, 10 Two pathways can induce apoptotic cell death: the intrinsic (or mitochondrial) pathway is induced by cellular stress, which involves Bcl-2 (anti-apoptotic protein), mitochondrial outer-membrane permeability, and caspase-9 protein; and the extrinsic (or death receptor) pathway is induced by specific ligands that engage death receptors, which involves Fas and activation of caspase-8 protein.9, 10 Our data of the present study showed overexpression of Bcl-2 and significantly decreased expressions of cleaved caspase-9 and cleaved caspase-3, which meant that reduced apoptosis through intrinsic pathway occurred in both venous diseases.

Because the ISV became dilated and thickened in varicocele, it is hypothesized that hypoxic stress may upregulate Bcl-2 expression, which leads to apoptosis less than the control group. Studies on the varicose veins showed that many neutrophils adhered to the endothelium of vein incubated in hypoxic conditions rather than in normoxia.7 It had been reported that the hypoxia-activated endothelial cells secrete growth factors that trigger smooth muscle cell proliferation.17, 18, 19, 20 Hence, hypoxia may be one of the factors responsible for Bcl-2 regulation, because Bcl-2 protein expression was increased in different cells under hypoxic conditions to protect cells against apoptosis.20, 21 Bcl-2 overexpression may decrease the vascular cell apoptosis in the hypoxic condition and lead to vascular cell proliferation (predominant in muscular layer) causing dilated and thickened wall of varicose veins.22, 23 The same situation and histopathologic changes occurred in varicocele veins. In addition, this research demonstrated the decreased expressions of cleaved caspase-9 and cleaved caspase-3 that revealed decrease of cellular apoptosis in varicocele and varicose veins through intrinsic pathway. Both venous diseases have the same dysregulation of apoptotic pathway under tissues hypoxia and this mechanism might contribute to the dilated and thickened vein walls. Further study is needed to investigate whether the hyperbaric oxygenation is effective to decrease the recurrence after varicocelectomy or vascular stripping surgery.

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Conclusions 

The results showed vascular smooth muscle hypertrophy in the diseased vessels. The same dysregulation of apoptosis through intrinsic pathway was demonstrated in varicocele and varicose veins under tissues hypoxia. This mechanism of reduced apoptosis might contribute to the dilated and thickened walls of both venous diseases.

We believe that elucidating the molecular mechanism and apoptotic pathway of diseases could provide a better treatment and prevent the recurrence in future.

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This study was supported by a grant from Taichung Armed Forces General Hospital, Taiwan, Republic of China.

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References 

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PII: S0890-5096(10)00087-7

doi:10.1016/j.avsg.2010.02.018

Annals of Vascular Surgery
Volume 24, Issue 6 , Pages 768-774, August 2010

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