III – FUNDAMENTALS OF VENOUS DISEASE

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ANATOMY
Basic anatomy

Chairpersons: J. T. Hobbs (UK), L. K. Moura (Brazil)

Collagen fiber networks of the human proximal long saphenous vein valve: a scanning electron microscopic study
A. KOLESNIK, T. SKADORWA (Poland)

This study used scanning electron microscopy to determine the characteristics of collagen fibers of the four saphenous venous cadaveric veins in normal subjects without signs of clinical venous insufficiency. The images obtained showed intrinsic collagen bundles, arranged in layers of parallel fascicles, crossing at different angles. The findings confirmed that the collagen fibers are organized a the way that strengthens the valve wall, especially in the area of its attachment to the venous wall and in the venous pocket. Future work should compare normal collagen architecture with the changes found in patients with venous insufficiency.

Changes in purine receptor expression in varicose veins reflect the phenotype transition of the smooth muscle
M. J. METCALFE, G. BURNSTOCK, M. TURMAINE, D. M. BAKER (UK)

This is an interesting study about purine receptors in the smooth muscle cells of the great saphenous vein. This work has demonstrated, for the first time, the changes in purine receptors in longitudinal and circular smooth muscle in the long saphenous vein in venous insufficiency. This change in purine receptors is reflected in the contractile strength of smooth muscle. The electronic microscopy findings confirm the phenotype change observed, from contractile to synthetic of smooth muscle cells in the varicose veins. Interventions targeting purine mediator pathways may be a useful approach for treating chronic venous insufficiency in the future.

Anatomy of the short saphenous vein and companion nerves: the high-risk zones for the treatment of varicose veins
J. F. UHL, C. GILLOT, P. LEMASLE, V. DELMAS (France)

This study, performed with anatomic cadaver dissection, showed that the ankle, the apex of the calf, and the popliteal level are points where the small saphenous vein is very close to various important nerves. All surgical and endovenous laser procedures undertaken at these three points must be carried out carefully to avoid nerve injuries.

Is MRI a tool for the diagnosis of soleus syndrome? Preliminary report
I. STAELENS, P. PETROONS, L. LEBRUN, S. VAN SINT JAN, X. DEMONDION (Belgium)

This report describes the MRI characteristics of soleus architecture in seven subjects described in order to study the soleus syndrome, a rare case of calf pain produced by venous entrapment in the soleus arch. The MRI results must be compared with those of phlebography in a large group of patients and normal subjects with this very infrequent syndrome in order to reach conclusion.

Anatomic variations of the great saphenous vein studied by color Doppler ultrasound
S. AMARAL, M. MATHIAS, S. LEITE (Brazil)

This study analyses the anatomic variants of the great saphenous vein by ultrasound studies. The very frequent anatomic variants can be discovered by the use of routine ultrasound to prevent problems of inadequate surgery during venous treatment procedures.

Relationship between diameter and reflux time of the saphenous veins; implication for preoperative mapping for selective stripping surgery
M. MO, C. TANAKA, M. NAKAMURA, O. KANEKO (Japan)

Venous reflux is proportional to the venous diameter. The authors of this work concluded that reflux studies can be omitted when the great saphenous vein is larger than 9 mm or smaller than 2 mm. In any other case it is necessary to perform a Doppler reflux study to test the presence of venous insufficiency. In any case, great venous insufficiency cannot be studied only in terms of venous diameter, because of the variability in height and weight of patients is related to the venous diameter.

CHRONIC VENOUS DISEASE
SERVIER/UIP Fellowships

Chairperson: C. Allegra (Italy)

Results of the project by the previous winner (2003/2005) of the SERVIER/UIP Fellowships:
Elastin dysregulation in varicose veins
M. G. PASCUAL GONZALEZ (Spain)

The aim of this study was to examine the in vivo and in vitro expression of lysin oxidase, like proteins (LOXL) and tropoelastin and explore their association with tissue growth factor â1 (TGFâ1) as a marker of tissue remodeling. Also, the elastic components of the vein wall (BA4, fibrillin 1 and 2) and differentiation state of the muscle component of normal and varicose veins (vimentin, desmin, myosin, and á-actin) were evaluated and correlated with age of patients with varicose veins.
Results:
– The markers involved in the synthesis of vein elastic components (LOXL/tropoelastin) in the vein wall diminished with age. In varicose veins, they showed an inverse relationship (LOXL decreased, tropoelastin increased)
– The previously established reduction in the level of elastin in the varicose condition may be related, at least in part, to the observed reduction in lysin oxidase like-1 (LOXL-1) levels which decreased the spontaneous covalent crosslinking of elastin
– The varicose pathology induces dysregulation of the expression of TGFâ1
– Altered TGFâ1 could be a good marker of vein wall damage. The dysregulation of both its active and latent TGFâ1 forms detected in the varicose specimens could play an important role in the fibrous process, representing the end point of venous insufficiency.

Project by the last winner (2005/2007) of the SERVIER/UIP Fellowship:
Physiological and morphological characterization of a porcine model of superficial varicose veins
G. T. JONES (New Zealand)

In this project, the creation of an arteriovenular fistulae in the pig is an experimental model which produces superficial varicose veins (VV) with valve changes which appear to mimic that observed in human patients. Rather than produce acute distension of the overlying superficial veins, the veins dilate slowly after an initial lag period of 1 to 2 weeks. Interestingly, in the model, the superficial VV that develop are more pronounced when the animals are placed in an erect posture. Also of great interest in this model is the formation of similar changes within the superficial veins of the contralateral limb which may allow more extensive valve remodeling before becoming incompetent, a hypothesis which the author hopes to test by measuring levels of the tissue remodeling enzymes, the MMPs.
The main objectives of the study are the following:
– Determine the physiological basis of the superficial varicose veins that develop in this porcine model: intravenous blood pressure and velocities at baseline, 6, and 12 weeks; quantify postural reflux into the superficial veins
– Measure the venous tissue levels of matrix metalloproteinases: determine correlations between MMP levels and vein wall structure, diameter, and physiology.

Bauerfeind/UIP Fellowships

Chairperson: H. Partsch (Austria)

Results of the project by the previous winner (2003/2005) of the BAUERFEIND/UIP Fellowship:
Apoptosis-related proteins, extracellular matrix molecules (ECM) and microangiopathy of the skin: their role in chronic venous insufficiency
S. HEISING ET AL (Germany)

The epidermis is continuously renewed and, this process is ensured by cell-cycle-related proteins. During wound epithelization, cell-cycle regulatory proteins having a blocking (bcl-2)or promoting (bax, caspace 3) play a role in this process.
Main conclusions:
– Not only patches of skin in the perimalleolar area, but even perfusion of proximal calf is impaired
– Loss of bcl-2 may be a signal for upward migration of cells within the epidermis.

Project by the latest winner (2005/2007) of the BAUERFEIND/UIP Fellowship:
Gastrocnemius vein follow-up under compression and correlation to clinical symptoms
C. JEANNERET-GRIS, I. VON PLANTA (Switzerland)

Main study objectives:
– To study the influence of compression stockings on venous diameters of the gastrocnemius veins in patients with leg symptoms (VCS – score >2)
– To study the correlation between gastrocnemius vein diameters and quality of life measurements.

Is primary chronic venous disease an evolving disease?

In the framework of the SERVIER Symposium.
Chairpersons: C. Allegra (Italy), A. N. Nicolaides (Cyprus)

Venous hypertension and the inflammatory cascade: major manifestations and trigger mechanisms.
G.W. Schmid-Schönbein*; L. Pascarella(USA)

Chronic venous disease and the endothelium- leukocyte interaction: from early symptoms to open ulceration.
A. N. Nicolaides (Cyprus)

MPFF* and venous valve damage induced by venous hypertension.
G.W. Schmid-Schönbein (USA)

MPFF*, symptoms and edema: clinical update.
G. Jantet (France)

MPFF* and venous leg ulcer: new results from a meta-analysis.
P. D. Coleridge-Smith (UK)

Rapidly advancing understanding of the mechanisms involved in chronic venous disease (CVD) progression might allow the identification of targets for pharmacological intervention aimed at preventing future morbidity.
Recent histological and immunocytochemical evidence of venous leg ulcers supports the hypothesis that lesions observed at different stages of chronic venous disease may be associated and possibly caused by an inflammatory process. Primary venous insufficiency is associated with venous hypertension, largely as a result of reflux through failed venous valves. One of several theories put forward as a possible cause of CVD progression is that primary valvular dysfunction is acquired. This view is supported by findings such as activated leukocytes that have been shown to migrate into the endothelium of proximal surfaces of the vein valves and to promote remodeling of the valves with consequent valvular insufficiency. Schmid-Schönbein and his team have sought to weigh the responsibility of triggering mechanisms such as genetic risk factors, hormonal impregnation, prolonged hydrostatic load, and abnormal fluid shear stress in the process that leads to a cascade associated with such inflammation. They have also obtained evidence that venous valve deficiency may be associated with leukocyte infiltration into valve leaflets, and hypothesize that a key event in the inflammatory cascade is the enzymatic degradation of the valve leaflets and venous wall. Accordingly, they examined, in a rat femoral fistula model with venous hypertension, the metalloproteinases (MMP) in veins exposed to elevated pressures for up to 6 weeks. Zymography shows increased activity of Pro-MMP 2 at 3 and 6 weeks. MMP 2 and MMP 9 activity was predominantly observed at day 7 and 21 after creation of the fistula. The degree of extracellular matrix remodeling correlates with the morphologic finding of macroscopic lesions. They conclude that MMP 2 and MMP 9 activation is present in veins already days after exposure to elevated blood pressure, and coincides with periods of early alterations of the valve morphology and early forms of reflux.
In a pharmacological model set up by the same team, chronic elevation of venous pressure was associated with an inflammatory reaction in venous valves, a process that may lead to their dysfunction, reflux, and upstream elevation of venous pressure. These effects were mitigated by the antiinflammatory action of MPFF* in a dose-dependent manner, as evidenced by J. J. Bergan et al.
As pointed out by A. N. Nicolaides, we are just beginning to understand the pathophysiology of venous disorders, and the optimum management of their various manifestations remains to be improved. It is generally agreed that primary chronic venous disease, with its diversity of symptoms and signs, is related to venous hypertension.1,2 Not only elevation of pressure, but also a cascade of biochemical events cause venous valve deterioration, and vein wall remodeling and restructuring with microcirculatory dysfunction which lead to reflux, varicose veins, and finally to skin changes and ulceration. These underlying processes prevail throughout the progression of chronic venous disease. Among the many proposed mechanisms linking venous hypertension to macroscopic and microcirculatory changes, “leukocyte-endothelium interaction” is currently the most credible. This mechanism involves valve and venous wall infiltration by monocytes and macrophages, leading to valve destruction and varicose remodeling. In the microcirculation, the accumulation of leukocytes in the lower extremity under conditions of high venous pressure is thought to be largely due to leukocyte adhesion to the endothelium and migration through small vessels, especially postcapillary venules.
It is now agreed that abnormalities in the microcirculation greatly contribute to the etiology of varicose veins as well as to the chronic skin changes of CVD. Work on animal and human models has shown that MPFF* modulates leukocyte adhesion, prevents endothelial damage, and activates the microlymphatic system. As concluded by G. Jantet, such treatment is useful first-line for edema as well as other symptoms of CVD. It continues to be effective in all subsequent stages of the disease, including leg ulceration.
The results of a recently published meta-analysis of a randomized prospective study using MPFF*, presented by P. Coleridge-Smith, confirm that venous leg ulcer healing is accelerated by adding MPFF* to conventional treatment. Medical literature databases and manufacturer’s records were searched for relevant clinical trials to be included in the analysis. Five prospective, randomised, controlled studies, in which 723 patients with venous ulcers were treated between 1996 and 2001, were identified. Conventional treatment (compression and local care) in addition to MPFF* was compared with conventional treatment plus placebo in two studies (N=309), or with conventional treatment alone in three studies (N=414). The primary end point was complete ulcer healing at 6 months. The results were expressed as reduction of the relative risk (RRR) of healing with 95% confidence intervals (CI). At 6 months, the chance of healing ulcer was 32% better in patients treated with adjunctive MPFF* than in those managed by conventional therapy alone (RRR: 32%; CI, 3%- 70%). This difference was present from month (RRR= 44%; CI, 7%-94%), and was associated with a shorter time to healing (16 weeks vs 21 weeks; P=0.0034).
The benefit of MPFF* was found in the subgroup of ulcers between 5 and 10 cm2 in area (RRR: 40%; CI, 6%-87%), as it was in patients with ulcers of 6 to 12 months, duration (RRR: 44%; CI, 6%-97%). Larger ulcers and long-lasting ulcers were found to benefit most from such treatment. These ulcers tend to heal more slowly, and an adjunctive treatment may be of advantage in such circumstances. In summary, it is now acknowledged that it may be possible to prevent worsening of CVD. This can be drawn from the experience in Italy, where preventative measures, including patient education and prophylactic treatment, have demonstrated both clinical effectiveness and cost-effectiveness. This symposium highlighted the fact that better understanding of the etiology of venous disorders and better prevention will lead to better care, allowing alleviation of the considerable suffering of patients in the long term.

*Registered as Ardium®, Alvenor®, Arvenum® 500 mg, Capiven®, Daflon® 500 mg, Detralex®, Elatec®, Flebotropin®, Variton®, Venitol®.

LYMPHATIC DISEASE
Lymphology

Chairpersons: P. Poredos (Slovenia), S. Nieto (Argentina)

Peripheral lymph low-molecular-weight proteins in normal subjects and patients with lymph stasis
B. INTEREWICZ (Poland), W. L. OLSZEWSKI (Poland), L. V. LEAK (USA), E. F. PETRICOIN (USA), S. ROSS (USA), L. A. LIOTTA (USA)

Lymph contains a great number of proteins that are similar to blood plasma, as it is generally referred to as an ultrafiltrate of plasma. The identification and validation of tissue fluid and lymph proteins under normal conditions and associated with particular diseases have so far been carried out only sporadically. In the absence of protein databases for efferent lymph in comparison with those well established for plasma there is a need for rapid multiparametric and reproducible method for identification and analysis of lymph proteins.
The aim of this study was to define lymph and plasma peptide profile collected from healthy subjects and patients obstructive lymphedema. Lymph and plasma samples (10 uL each) were directly applied to a weak cation exchange (WCX2) protein chip and analyzed using a PBS II Ciphergen SELDI-TOF -MS. The protein profile patterns of normal lymph were similar in all samples. Only minor differences were seen between the plasma samples. Thirteen proteins were detected in plasma and seven in lymph. Five identical proteins, although of different relative intensities, were found in lymph and plasma. The protein profile patterns in inflamed lymph discriminate them from normal ones while the plasma profiles are similar. Seven proteins were found in all examined lymph samples of lymphedema patients.
Low-molecular-weight proteins produced in tissues are identified in lymph but not in serum due to their dilution. On the other hand, there are lowmolecular- weight proteins in serum that are not filtered to tissue fluid probably due to binding to high-molecular-weight proteins. The function of the low-molecular-weight protein remains to be determined.
The obtained data will be correlated with clinical observations by the authors to establish their validity for understanding of the inflammatory process in tissues deprived of lymphatic circulation.

Proteases in human peripheral lymph in normal subjects and inflammatory conditions
B. INTEREWICZ, W. L. OLSZEWSKI, A. DOMINIA (Poland)

Long-lasting edema of the skin and subcutaneous tissue of limbs is caused by inflammation. Controlled degradation of the extracellular matrix (ECM) is required for the removal of damaged components and to allow cell migration and angiogenesis. A key step in the degradation of the ECM is the extracellular secretion of metalloproteinases (MMPs). The proteolytic MMPs profile of human normal and inflamed lymph has so far not been evaluated. The aim of this study was to determine proteolytic environment of lymph and plasma collected from healthy subjects and patients with obstructive lymphedema. Twelve subjects in each group had their leg lymphatic vessels cannulated, and lymph samples were collected. Proteolytic profiles were examined using gelatin and casein zymography. Samples of lymph and plasma-derived clarified serum were subjected to polyacrylamide SDS gel electrophoresis under nonreducing conditions. After electrophoresis enzymes separat- ed on gels were renaturated and activated.
The gelatin zymography of the normal lymph samples consistently showed 6 bands of 53, 55, 60, 72, and 88 kDa characteristic for activated MMP-9. In lymph of patients with rheumatoid arthritis the proteolytic profile was similar, but without one band of 70 kDa (MMP-2). In lymph from lymphedematous limbs there were detected: up to 11 bands 32, 35, 38, 48, 50, 53, 55, 57, 58, 72, and 88 kDa with maximal intensities for bands 38 and 48 kDa. There were no differences in the proteolytic profile of plasma samples. The casein zymograms showed a different pattern of hydrolysis, with band formation at 150, 100, 65, and 29 kDa, without any significant difference between the samples.

Ultrasound volumeter and limb meter 3D: a new future for phlebolymphology volumetry?
L. TESSARI (Italy)

Searching for a valid method of volume measurement of the limbs in lymphedema is indispensable to: i) highlight the clinical status of the disease, ii) to evaluate the improvements obtained with the adopted therapeutic procedures, and iii) to monitor the trend of the pathology over time. There is still not a validated and worldwide recognized method for the measurement of edema and for the volumetric study of lymphedema; the principal current reference methods are water displacement volumetry and measurement of the circumference of the limbs at several levels. The water volumeter has been considered the reference method, though it has some routine applicability limitations and cannot be used in open wounds and infected cases; furthermore it does not allow one to analyze the distribution of lymphedema along the limb. The measurement of the circumference of the limb at different levels is strictly operator-dependent with a high probability of intrainterobserver errors due to: a) the variable tension of the device which is used to take measurements on the skin of the limb; b) the reproducibility of the points of reference on the limb; c) the position of the measuring device in relation to the limb. Moreover, this technique does not take into account the volume of the limb. On the basis of the flaws mentioned above, the authors developed a project for a new volumeter and a new method of measurement of the limbs, based on the use of ultrasound. In fact, on one hand it is possible to measure the change of volume in an ultrasound field in the presence or absence of a limb within the same ultrasound irradiation. On the other hand it is possible to use the echo effect of ultrasound, which is emitted by a system with spiroidal movement for the measurement of the limbs.

FROM SKIN CHANGES TO ULCERATION

LECTURE
The eclipse effect in ulcer pathogenesis: the overlapping of primary CVD and the C282y gene mutation
P. ZAMBONI (Italy)

The author presents this study after the observation of a relationship between chronic venous leg ulceration and disturbances in iron metabolism. He studied a cohort of 238 selected cases rated as CEAP C4-6 looking for the HFE C282Y mutation, the most commonly recognized genetic defect in iron metabolism in Northern Europeans. Among patients affected by primary CVD, this mutation increased the risk of developing chronic venous ulcers more than three times when compared with healthy controls. This finding may lead to the development of new strategies in the prevention and treatment of severe CVD.

Venous ulcers: clinical research

Chairpersons: S. Hoshino (Japan), J. A. Puentes (Uruguay)

Microcirculatory flow abnormalities in patients with healed and active leg ulcer
A. JAWIEN, P. BRAZIS, A. MIGDALSKI, T. GRZELA, M. SZEWCZYK, R. PIOTROWICZ, M. CIECIERSKI (Poland)

Chronic venous insufficiency (CVI) is associated with a great number of microcirculatory abnormalities, but there are a limited number of studies demonstrating correlation of microcirculatory flow failure with different stages of CVI severity. The aim of the present study was to compare microcirculatory flow characteristics in patients with healed and active venous ulcerations (stages C5, C6 of CEAP classification) against a control group. Thirtynine patients were enrolled in the study (45 legs): 14 patients (15 legs) with healed ulcer (C5 group); 14 patients (15 legs) with active ulcer (C6 group) and, as a control group, 11 patients (15 legs) without any symptoms and signs of CVI. All patients underwent duplex scan investigation, and the microcirculation was assessed by laser Doppler flowmeter. The following parameters were investigated in the study (supine and standing position): skin blood flux (SBF), concentration and velocity of moving blood cells (CVMBC); venoarteriolar response (VAR) values; and thermal stimulation test values. The statistical analysis was performed by the Mann-Whitney test. The significant difference in calf SBF values (supine and standing position) between all tested groups (P<0.01) was observed. The calf SBF reached the highest level in the C6 group and the lowest level in the control group. The values for CVMBC in supine position at the calf level were higher in the C5 and C6 group vs the control group (P<0.001). Statistically significant decreased intensity of the VAR on the toe in C5 and C6 group vs the control group was also observed (P<0.05). Impairment of thermal stimulation test values was found in the C5 and C6 group in comparison with the control group (P<0.001). It seems that advanced stages of CVI (C5,C6) are associated with significant microcirculatory flow abnormalities at the calf level, which are expressed mainly by: increased skin blood flux, increased concentration and velocity of moving blood cells, and impairment of thermal stimulation test values.

Cell cycle-related proteins and apoptosis in epidermis of human chronic leg ulcers
H. GALKOWSKA, W. L. OLSZEWSKI, U. WOJEWODZKA (Poland)

The epidermis is continuously being renewed, and this process is ensured by antigen p63+ CD29+ epidermal stem cells in the basal layer. Terminally differentiated keratinocytes expressing cytokeratine 10 undergo cellular renewal or apoptosis. Both mechanisms are controlled by similar molecular regulators. During wound epithelialization, keratinocytes (KC) undergo many changes, including cell migration, proliferation, and differentiation. Cells become activated and express PCNA and p53, the cell cycle regulatory proteins. KC apoptosis and terminal differentiation are regulated by proteins having a blocking (bcl2) or promoting (bax, caspase3) effect on DNA fragmentation. In order to evaluate the role of regulatory proteins in the pathogenesis of protracted epithelialization of leg ulcers we characterized immunohistochemically the expression of these proteins in the epidermis.
The aim of this study was to evaluate the role of epidermal apoptosis and cell cycle-related proteins in the pathogenesis of protracted reepithelialization of calf venous and diabetic foot ulcers.
Studies were carried out in 12 patients with diabetic foot and 10 patients with varicose ulcers of the calf. Skin biopsies were obtained under local anesthesia from the border area of ulcers and from corresponding sites of normal subjects (orthopedic patients).
KC at the edge of both types of ulcer and in the distal areas expressed cytokeratin 16 and 17, but not cytokeratin 10. KC were p63+, CD29+, PCNA+, p53-. The intensity of bcl2 staining was higher at the edge of foot ulcers compared to calf ulcers, whereas the intensity of bax staining was the same. The intensity of caspase 3 staining was higher in foot than in calf ulcers. The intensity of DNA break staining (TUNEL) was lower at the edges of both types of ulcers compared with controls. This means that impaired epithelialization of chronic ulcers is not caused by impaired epidermal stem cell proliferation, differentiation, and apoptosis. This may reflect a distorted organization of the ulcer bed, hampering keratynocyte migration from the ulcer edge.

Chronic leg ulcers: innate antimicrobial peptide β defensin-2
expression and Langerhans cell density in epidermis

H. GALKOWSKA, U. WOJEWODZKA, J. MIJAL, W. L. OLSZEWSKI (Poland)

Normal human skin is resistant to penetration by micro-organisms that routinely colonize its surface. Skin epidermal antimicrobial peptides and Langerhans cells are the most prominent factors in the defensive responses. Two major classes of dermal peptides, cathelicidins and â-defensins expressing antibacterial activity, are produced by keratinocytes. Epidermal expression of â-defensin-1 is systemic, whereas expression of â-defensin-2 is upregulated by the inflammatory process in human skin. â-defensin-2 has also chemotactic and – in vivo – activating effect on dendritic cells. In vivo recruitment of epidermal dendritic cell precursor from blood into skin and Langerhans cell mutation can also be influenced by other keratinocyte- derived factors.
The aim of this study was to investigate immunohistochemically whether chronic skin ulceration can influence hBD-2 expression, Langerhans cell (LC) density, and activation of epidermis in diabetic foot and venous leg ulcers. Studies were carried out in 10 patients with diabetic foot ulcer and 10 patients with varicous ulcers of the calf, without clinical signs of infection of adjacent tissues. Control biopsies were taken from corresponding sites of 10 normal subjects (orthopedic patients).
There was significantly higher hBD-2 staining in sole skin epidermis, both in normal and diabetic foot ulcers (P<0.05) compared with normal calf skin. There was no difference in defensin staining between ulcerative and normal epidermis. Studies of CD1a+ LC densityrevealed lack of these cells in neoepidermis at the edge of calf ulcers compared with normal epidermis (P<0.05). In contrast, there was increased density of CD1a+ LC and DR+ LC in epidermis from diabetic foot ulcers compared with normal, nondiabetic foot sole skin (P<0.05). Moreover, there was significant reduction of CD1a+ LC in normal foot sole epidermis compared with calf skin (P<0.05). Significantly higher expression of GM-CSF was observed in diabetic foot epidermis compared with normal sole skin and calf epidermis (P<0.05). Overexpression of CD1a+ LC and GM-CSF in epidermis from diabetic foot ulcers suggests in vivo GM-CSF contribution to the observed LC upregulation in diabetes.

Topical treatment of phlebostatic ulcers with recombinant human growth factor with Sulfadiazina de plata (Ag Sulfadiazin)
M. QUIÑONES CASTRO, M. BORRÁS MIGUES, L. RODRIGUEZ VILLALONGA, L. CHIRINO DÍAZ, S. GARCÍA PELEGRÍ (Cuba)

Chronic venous insufficiency is the main cause of the appearance of the leg ulcer, giving rise to a medical-social problem that can be extended to lifelong incapacity for the patient. Many topical treatments have been used and reported in the worldwide medical literature aimed at achieving faster healing of the ulcerous lesions. The proposal of the topical use of growth factor cream with Sulfadiazina de plata (Ag Sulfadiazin) in patients with phlebostatic ulcers was performed. Ninety patients with 6- to10- cm diameter ulcerous lesions not healed over 1 year of the treatment were included in this research work. They were divided into three groups: Group A (control group): Antiseptic warming; Group B: Ag sulfadiazin, cream 1%; Group C: Recombinant human growth factor cream with Ag Sulfadiazin. The general treatment was the same for all groups. Two parameters were used to measure the treatment success: healing time given by hospitalization period and percentage of healed ulcers.
In group A, the medium hospitalization period was 93.8 days and a good result – total ulcer healing – was observed in 23.3% of treated patients. Nonhealing ulcer was observed in 40% of patients from this group. In group B, the medium hospitalization period was 54.4 days, and total healing was observed in 76.7% of treated patients. In group C, the medium hospitalization period was 38.8 days, with 90% of cases having good results. The results suggest that recombinant human growth factor together with other antiseptic vehicles can lead to total wound healing in ulcer resistant to previous treatment.

Venous ulcers

Chairpersons: L. Norgren (Sweden), P. Mendonça (Brazil)

Changes in P2X and P2Y receptor expression reflect keratinocyte function on skin epidermis during chronic venous insufficiency
M. J. METCALFE, G. BURNSTOCK, D. M. BAKER (UK)

Purines are extracellular nucleotides that have long-term effects on keratinocyte proliferation (via P2Y 1 and P2Y 2 receptors), differentiation (via P2X 5 receptors), and death (via P2X 7 receptors). The authors studied the changes in expression of these P2 receptors on epidermal keratinocytes in chronic venous insufficiency (CVI), a condition leading to skin changes including lipodermatosclerosis and ulceration. The study was performed in a small group – 5 patients with venous leg ulcer. Significant increase of P2Y 1 and P2Y 2 receptor expression (representing increased keratinocite proliferation) in basal and spinosal layers of the epidermis in all CVI patients was observed. P2X 5 receptor expression in CVI was increased too, largely in the spinosal layer.

Basic Science

Chairpersons: J. Strejcek (Czech Republic), C. Canedo (Brazil)

With the participation of:
M. J. METCALFE, G. BURNSTOCK, D. M. BAKER (UK), Y. HEROUY, E. SCHOEPF, L. BRUCKNERTUDERMAN (Germany), S. BRAUN, M. JUENGER, A. OSTROWITZKY, F. RIEDEL, E. MANSFELD, H. HAASE, E. KAUTSCHKE; J. FANGHANEL, J. GIEBEL (Germany), J. NORGAUER (Germany), B. ARAVIND, T. NAVIN, B. SHARP, C. MONACO, E. PALEOLOG, A. H. DAVIES (UK), S. R. REWERK, C. D. DUCZEK, A. G. GRUBER, M. W. WINKLER, H. N. NULLEN, G. W. H. HALLER, S. FREUDENBURG (USA/Germany)

This session consisted of a selection of presentations which addressed the pathophysiology of leg ulceration. The underlying mechanism by which venous and capillary hypertension leads to trophic skin damage is inflammation. Inflammation results in a migration of neutrophils, macrophages, and lymphocytes followed by a proliferation of endothelial cells and fibroblasts. Secreted proteins influence different enzymatic functions at the cellular and extracellular levels.
Proteolysis of cell surface-and extracellular matrix molecules is intrinsically linked to cell function. Apoptosis of dermal and epidermal cells results in venous leg ulcers.
Alteration of concentration of several proteins such as Fas, Fas-L, bcl-2, collagen, elastin, tenascin, fibronectin, and changes in the proteolytic activity of enzymes (metalloproteinases and its inhibitors) were compared in healthy skin and lesional skin. Besides clinical investigation, methods used by the presenters included epiluminescence microscopy, laser Doppler fluxmetry, transcutaneous oxygen measurements, protein expression in skin biopsies determined by immunohistochemistry, zymography, Western-Blot assay, and TUNEL assay (DNA fragmentation).
Extracellular matrix remodeling by metalloproteinases and its inhibitors in the vein wall can result in structural weakness with varicose vein formation. High levels of MMPI have been formed in atrophic areas of varicose veins, and VEGF expression was higher in recurrent varicose veins.
The data from studies on the skin microcirculation provide evidence that venous leg ulcers could be the result of an apoptotic pathway. The entry of keratinocytes into the apoptopic pathway may be regulated by bcl-2 in congested areas and at the ulcer edge.
Tenascin and fibronectin deposition is rising in the damaged skin region. Soluble and membrane-bound metalloproteinases may favor enhanced turnover of the extracellular matrix in the lesional skin.
These results suggest that controlling programmed cell death may have therapeutic potential in preventing venous leg ulceration. For example, Factor XIII is an effective antifibrinolytic drug for the topical treatment of ulcers because it regulates the activity of matrixmetalloproteinases by influencing their conversion into the enzymatically active form.