ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Plasma transforming growth factor-b₁ levels in patients with erectile dysfunction

Ji-Kan Ryu¹, Sun U. Song², Hyung-Ki Choi³, Do-Hwan Seong¹, Sang-Min Yoon¹, Seong-Jin Kim⁴, Jun-Kyu Suh¹

Asian J Androl  2004 Dec; 6: 349-353         

Keywords: erectile dysfunction; transforming growth factor; fibrosis; diagnosis

Abstract

Aim: To evaluate the plasma TGF-b₁ level in erectile dysfunction (ED) patients of various causes. Methods: Sixty-two patients with ED and 26 potent men were subjected to the study. Based on multidisciplinary work-ups, including medical history, physical examinations, blood tests with lipid profile and hormones, penile duplex Doppler ultrasonogram and neurophysiological tests, causes for ED were classified as psychogenic (n=15), neurogenic (n=16) and vasculogenic (n=31). The plasma TGF-b₁ level was measured by the ELISA method. Results: The plasma TGF-b₁ level was significantly increased in the ED group (6.7±4.9 ng/mL), compared to the control (4.0±2.1 ng/mL) (P<0.01). In the ED groups, there was a significant increase in the vasculogenic group (9.0±5.5 ng/mL), compared to the psychogenic (3.8±1.8 ng/mL) and neurogenic groups (4.8±3.2 ng/mL) (P<0.01). Of the vascular risk factors, both the smoking (7.5±4.7 ng/mL) and dyslipidemia groups (7.4±4.4 ng/mL) showed significantly increased plasma TGF-b₁ levels, compared to the non-smokers (5.5±2.8 ng/mL), and those without dyslipidemia (4.8±2.8 ng/mL) (P<0.05). Conclusion: Vascular risk factors are associated with an elevated plasma TGF-b₁ level, which may contribute to cavernous fibrosis and ED.

1 Introduction

Erectile dysfunction (ED) has many causes and vascular risk factors are the most common. Factors, including hypertension, hypercholesterolemia, smoking and diabetes, often cause atherosclerosis and finally result in ED [1, 2].

Therefore, an understanding of the factors that alter the functional smooth muscle-connective tissue balance is of paramount importance in the continuing development of therapeutic agents for the treatment of vasculo-genic ED. It is well known that vasculogenic ED results from structural changes in the corpus cavernosum and that an adequate ratio of trabecular smooth muscle to collagen is required for a successful veno-occlusion. If cavernous smooth muscle content is less than the critical amount, veno-occlusion fails regardless of the extent of smooth muscle relaxation [3].

Recently, much attention has been focused on transforming growth factor-b1 (TGF-b₁) as a fibrogenic cytokine [4]. This protein has been shown to increase collagen synthesis in the human corpus cavernosum smooth muscle cell by 2.5 to 4.5 folds [5]. Furthermore, in an animal model, TGF-b₁ causes a dose-dependent decrease in the percentage of corporal smooth muscle when administered intracorporally in a single alginate microsphere [6].

If there is a simple and effective method for detecting cavernous TGF-b₁ expression, it may have a clinical impact not only for predicting cavernous fibrosis early in its course but also for developing new therapeutics that would block TGF-b₁ signaling. Although a percutaneous biopsy may allow an evaluation of the cavernous TGF-b₁ expression through molecular or histological techniques, it cannot be applied directly to clinical situation as a form of diagnosis due to its invasive nature.

Recently, the plasma level of TGF-b₁ has been reported to be elevated in patients who smoke or have diabetes, hepatic fibrosis and in malignancies such as prostate cancer, colorectal cancer and breast cancer [7,8]; however, the plasma level in ED has not been reported yet. The aim of this study was to investigate the association of plasma TGF-b₁ level in ED patients of various aetiologies and to verify the effect of vascular risk factors on the plasma TGF-b₁ level.

2 Materials and methods

A total of 62 patients with ED, aged 30 - 72 (mean 51.8) years, participated in this study. The control group consisted of 26 potent men, aged 28 - 72 (mean 54.7) years, of whom 10 suffered from benign prostate hyper-plasia, 8 from chronic prostatitis, 4 from infertility and 4 from premature ejaculation. Informed consent was obtained from all the patients. Detailed medical and sexual histories were obtained. A physical examination and a diagnostic assessment were performed; these included a complete blood count, serum fasting glucose, haemoglobin A1C, lipid profile and testosterone determinations. Neurologic tests including penile biothesiometry, bulbocavernosus reflex latency and somatosensory evoked potential from pudendal nerve and penile duplex Doppler ultrasound scanning were performed. We also verified the presence of risk factors for vasculogenic ED such as smoking, hypertension, diabetes mellitus, dyslipidemia and atherosclerosis. The smoking group consisted of those who had smoked more than 1 pack/day for over a year. Dyslipidemia was defined as repeated elevation of total cholesterol, LDL-cholesterol or triglyceride level or decrease in HDL-cholesterol. Atherosclerosis was diagnosed when an obstruction of more than half of the arterial lumen was confirmed by a coronary or femoral angi-ography.

The penile arterial system was evaluated using a 10 MHz color Doppler ultrasonography. An initial real-time examination in both the transverse and longitudinal planes was performed. Measurements were made at 5, 10, 15 and 20 minutes after an intracavernous injection of 10 µg prostaglandin E₁ (PGE₁). Patients with a negative response to a single injection of PGE1 were asked to perform manual genital self-stimulation without ejaculation for 5 minutes. A peak systolic velocity of ≥30 cm/s was considered normal, 25-29 cm/s, borderline and <25 cm/s, arterial insufficiency; an end-diastolic velocity of >5 cm/s was accepted as evidence of venous leakage, although this is not an absolute criterion.

A detailed medical history, including diabetes, multiple sclerosis, epilepsy and spinal trauma was obtained as well as the neurologic examinations, included deep tendon reflexes, perineal sensation, bulbocavernosus and creamasteric reflexes and the tone of the rectal sphincter. Penile biothesiometry was performed and bulbocavernosus reflex latency and somatosensory evoked potential from pudendal nerve were measured. A diagnosis of neurogenic ED was made when both the medical history and the physical examination showed evidences which suggest neurological disease. The vibratory sensation at the glans and shaft was significantly decreased than normal age-adjusted normograms and neurophysiologic tests revealed prolonged pudendal nerve conduction and impaired sensory evoked potential.

The diagnosis of psychogenic ED was made when a patient showed different sexual responses as indicated by his sexual partner or had psychological problems or factors that contributed to ED but did not suffer from any chronic medical diseases or alcoholism. The patient also had normal findings on penile duplex Doppler ultrasonography and neurologic tests.

Venous blood (5 mL) was immediately placed on ice after collected in precooled EDTA-containing tubes. The samples were centrifuged at 2 500 ×g at 4 ℃ for 30 minutes to remove platelets; the top 0.6 mL of the platelet-poor plasma was preserved at -80 ℃ until assay. The plasma TGF-b₁ concentration was determined using quantitative sandwich enzyme immunoassay specific for TGF-b₁ (R & D systems, Inc., Minneapolis, USA) according to the manufacturer's instructions. To activate the latent TGF-b₁ into the immunoreactive form, the samples were activated by acid and neutralized thereafter [9]. 0.5 mL sample was briefly acidified with 0.5 mL 1.25 mol/L acetic acid/10 mol/L urea plus 50 mg of phenylmethyl-sulfonyl fluoride; 5 samples were neutralized with 0.5 mL of 2.7 mol/L NaOH/0.1 mol/L HEPES free acid. Serially diluted standards and samples with calibrator diluents in the immunoassay kit were incubated in a 96-well plate coated with TGF-b receptor type II. After washing away any unbound protein, a polyclonal antibody specific for TGF-b₁ conjugated to horseradish peroxidase was added to the wells to sandwich the TGF-b₁, which was immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and the color development at 450 nm was measured. The TGF-b₁ content was determined by extrapolation of a standard curve.

Results are expressed as mean±SD. The means of two groups were compared using Student's t-test. The means of more than three groups were compared by analysis of variance (ANOVA). The statistical significance of differences between two groups was assessed by the Scheffe test. Probability values less than 5 % were considered significant. All statistical analyses were performed using the SPSS-Win 10.0 package.

Of the 62 ED patients, 15 had psychogenic ED, 16 neurogenic ED and 31 vasculogenic ED. The plasma TGF-b₁ level was significantly higher in the ED group (6.7±4.9 ng/mL) than that in the control (4.0±2.1 ng/mL, P<0.01). Furthermore, the plasma TGF-b₁ concentration was significantly higher in patients with vasculogenic ED than those with psychogenic ED or neurogenic ED (P<0.01) (Figure 1).

Figure 1. Plasma TGF-b₁ level was significantly higher in patients with vasculogenic ED than that in control, psychogenic or neurogenic ED (P<0.01).

Significantly higher plasma TGF-b₁ level was observed in patients with either smoking or dyslipidemia than in those without the risk factors. Patients with atherosclerosis also showed increased plasma TGF-b₁ level, compared to those without, but it was not statistically significant (Table 1). It was also identified that haemoglobin A1C level correlated well with the plasma TGF-b₁ level (g = 0.78; P<0.05).

Table 1. Plasma TGF-b₁ level according to vascular risk factors in patients with erectile dysfunction.

4 Discussion

Many kinds of cells produce TGF-b₁, including fibroblasts, endothelial cells, lymphocytes and macro-phages, bone and liver cells, which may contribute to circulating levels of TGF-b₁ [10]. Platelets are in charge of storing a large amount of TGF-b₁ in alpha-granule [11]. Therefore, particular care was taken to avoid damage to the platelets in this study. In addition, we used only the platelet-poor plasma to eliminate potential contamination of platelet TGF-b₁. To minimize contamination with platelet-derived TGF-b₁, other precautions, such as taking blood samples without placing a tourniquet, were also recommended [12].

In this study, the level of plasma TGF-b₁ was significantly increased in patients with vasculogenic ED, while in the controls the level was similar to those reported previously for healthy subjects [7]. Our recent study also showed that cavernous fibrosis and the TGF-b₁ expression was more prominent in patients with vasculo-genic ED than those with other causes for ED and the extent of fibrosis was well correlated with the TGF-b₁ expression in the corpus cavernosum [13]. The increase in TGF-b₁ level in patients with vasculogenic ED may result from a vicious cycle, i.e., cavernous hypoxia from a compromised blood supply induces TGF-b₁ synthesis, which then increases collagen synthesis and resultant cavernous fibrosis; the latter in turn further increases the production of TGF-b₁. Under continuous ischemic conditions, TGF-b₁ induces synthesis of its own mRNA, leading to a further increase in TGF-b₁ synthesis that reinforces the development of severe fibrosis [10]. The ability of TGF-b₁ to induce its own production may be the key in the development of scarring and fibrosis into chronic, progressive conditions that eradicate the normal tissue structure. The plasma level of TGF-b₁ has been reported to be elevated in patients with other diseases, including diabetes, smoking and several malignancies [7,8].

In this study, patients with atherosclerosis showed a higher TGF-b₁ level than patients without this risk factor, although it was not statistically significant. Atherosclerosis, a pathologic vascular phenomenon covering a variety of diseases, is the most common cause of vasculogenic ED [14] and frequently occurs when more than half of the lumen of the internal pudendal, common penile or cavernosal arteries is obstructed [15]. Grainger and his coworkers [16] found most of circulating plasma TGF-b₁ to be in the active form in individuals without coronary artery disease, but almost completely in the latent form in patients with coronary artery disease. Based on this study it was suggested by these authors that TGF-b₁ might have a preventive role in the pathogenesis of atherosclerosis. However, TGF-b₁ level in our patients with both atherosclerosis and ED is somewhat different from that in patients with atherosclerosis without ED. Although TGF-b₁ may have a preventive role in the pathogenesis of atherosclerosis, once atherosclerosis is established, hypoxia-induced overexpression of TGF-b₁ may contribute to elevated plasma TGF-b₁ level and causes cavernous fibrosis.

Our study showed that patients with dyslipidemia have significantly increased plasma TGF-b₁ level than patients without this risk factor. The association between hyperlipidemia and ED was originally attributed to atherosclerosis in the hypogastric-cavernosal arterial bed, with a subsequent deficiency in penile arterial inflow. In an animal model of hypercholesterolemia, ultrastructural assessments showed atherosclerosis-like processes in focal areas of the cavernosal sinusoids [17]. These changes were not thought to be the primary cause of ED but more likely precursors of more complex atherosclerotic lesions.

Our study also showed that patients who smoke have significantly increased plasma TGF-b₁ levels than patients without this risk factor. It has been reported that cigarette smoke condensate induces an increase in cell adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in human endothelial cells through protein kinase C, which is a common pathway for TGF-b synthesis [18].

There was no difference in the plasma TGF-b₁ level in patients with and without diabetes mellitus, although there is clear evidence that glucose stimulates the production of TGF-b₁ by several cell types in vitro [19], most likely by stimulating the production of protein kinase C via formation of diacylglycerol. However, hemoglobin A1C level showed positive correlation with the plasma TGF-b₁ level, although total number of the subject is small in this study. No difference was observed in the plasma TGF-b₁ level, either in the presence or absence of hypertension, although an epidemiological study [20] showed that hypertension significantly correlates with ED. It has been inferred that a vascular compromise, as reflected in hypertension, shares common determinants with vascular impairment of the erectile mechanism.

Although no significant increase of plasma TGF-b₁ level was shown in our diabetes and hypertension groups, it is likely that these factors also play important roles in cavernous fibrosis as these are major risk factors for atherosclerosis. The observed results in this study could possibly be due to the following reasons. First, severity of diabetes or hypertension in this study group was mild, therefore did not have an effect on the systemic plasma TGF-b₁ level. Second, the number of patients with those risk factors was also relatively small, thereby making it even more difficult to detect a difference. Third, these factors may cause ED via a mechanism that has minimal involvement of the TGF-b signal pathway.

However, at this juncture, it is not easy to explain clearly whether the elevated plasma TGF-b₁ level is a systemic or local phenomenon. In the former, systemic conditions such as smoking, dyslipidemia and atherosclerosis elevate the plasma TGF-b₁ level and may result in cavernous fibrosis; in the latter, cavernous fibrosis is secondary to vascular risk factors in causing a local production of TGF-b₁ and may contribute to the elevated plasma level. It is very much unlikely for the penis to be the discrete source of high systemic TGF-b₁ considering its size to the body proportion. A prospective study that compares the plasma TGF-b₁ levels between potent patients with vascular risk factors and ED patients with vascular risk factors may provide an answer.

In conclusion, plasma TGF-b₁ level was significantly increased in patients with ED, especially those with a vasculogenic cause. Among the vascular risk factors, the smoking and dyslipidemia groups showed an elevated plasma TGF-b₁ level. However, a clinical study with simultaneous analysis of blood TGF-b₁ and cavernosal fibrosis in a large patient population will be required to definitely identify whether the increase in TGF-b1 is the cause or effect of cavernosal fibrosis and ED.

This project was supported by Grant No. R01-2002-000-00009-0 (2002) from the Basic Research Program of the Korea Science and Engineering Foundation.

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Correspondence to: Dr. Jun-Kyu Suh, Department of Urology, University Hospital, Inha University School of Medicine, Incheon, Korea, 7-206 3rd ST., Sinhung-Dong, Jung-Gu, Incheon, 400-103, Korea.
Tel: +82-32-890 3441, Fax: +82-32-890 2363
E-mail: jksuh@inha.ac.kr
Received 2004-04-02   Accepted 2004-08-23