Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. OBJECTIVES: To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. METHODS: Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. RESULTS: The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. CONCLUSIONS: These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Relationship of plasma vascular endothelial growth factor to CEAP clinical stage and symptoms in patients with chronic venous disease.

OBJECTIVE: Skin damage caused by chronic venous disease (CVD) is associated with severe microangiopathic changes in the skin. The aim was to determine whether patients in various CEAP clinical stages of CVD have elevated plasma levels of VEGF compared to controls and whether this correlates with the symptoms. METHODS: One hundred and eight patients with CVD attending the vascular clinic were included and assigned to the appropriate CEAP clinical stage. Thirty healthy control subjects were also included. Patients and controls were studied after resting supine for 10 min. Blood samples were taken from a dorsal foot vein. Assay of VEGF was performed with an enzyme-linked immunosorbent assay. Volunteers' symptoms were recorded using a visual analogue scale (VAS) for heaviness, cramps, paraesthesiae and swelling. RESULTS: Plasma levels of VEGF were: control (n30) median VEGF 56 pg/ml (inter-quartile range IQR 38-85), CEAP C2 (varicose veins) 86 (39-133), C5 (healed ulcer) 88 (67-135). Median difference: control vs. C5 33 (95% confidence interval (CI) 8-61). Median differences were calculated using the Wilcoxon method. VEGF level in patients with VAS swelling score=0:54 pg/ml (IQR 31-104). VEGF level in patients with VAS swelling score >0:85 pg/ml (IQR 40-147). Difference between medians: 20 ng/ml, 95% CI for the median difference: (5-44). No differences were observed for symptoms of heaviness, cramps or paraesthesiae. CONCLUSION: There was a trend towards raised VEGF in all stages of CVD, but this only reached statistical significance in those with healed ulceration. The symptom of swelling was associated with raised VEGF levels; however, the symptoms of heaviness, cramps, and paraesthesiae were not associated with raised VEGF levels.

Deleterious effects of white cells in the course of skin damage in CVI.

Venous ulceration remains a common problem and a significant challenge to the physicians treating it. Many theories have been advanced in the past to explain its causes but there is little evidence to support tissue hypoxia as the main factor, as was once thought. In recent years attention has focussed on the inflammatory events which attend venous disease and the development of venous ulceration. It has been proposed that these form a major contribution to the development of venous leg ulcers. In the arterial system an analogous series of events appears to cause damage following severe ischemia. Massive neutrophil activation in the microcirculation following reperfusion of a tissue results in severe, ischemic damage to that tissue. A similar series of events is proposed to explain venous disease. During venous hypertension leukocytes are sequestrated in the microcirculation of the lower limb. It has been shown that these undergo activation whilst they are in the leg. The exact location of leukocyte sequestration is unclear but it is suggested that this may occur in the skin. The damage caused to the lower limb skin components can be identified by measuring plasma levels of endothelial adhesion molecules, which are shed into the circulation following a period of venous hypertension. In the long term this leads to a chronic inflammatory state in the skin in some patients where venous hypertension is sustained or there is susceptibility to venous hypertension. The resulting inflammatory process is referred to as "lipodermatosclerosis" and has a number of well known clinical features. There is proliferation of the dermal capillaries eventually leading to a "glomerulus" like appearance. In the skin and subcutaneous tissues there is fibrosis. The microcirculation in the papillary dermis is surrounded by an inflammatory cellular infiltrate. The importance of understanding the mechanisms of the development of venous ulceration is in creating new treatments for this problem. Compression treatment has been effective in healing leg ulcers for thousands of years. Surgical treatment offers a possible cure in patients where superficial venous reflux is the main problem. Deep vein reconstruction is only suitable for a few patients. Many venous ulcers can be healed by compression, only to recur within a few months. Pharmacological treatments may offer the possibility of more rapid ulcer healing and the maintenance of an ulcer-free state if the correct pathophysiological mechanisms can be identified and addressed.

Effect of oral micronized purified flavonoid fraction treatment on leukocyte adhesion molecule expression in patients with chronic venous disease: a pilot study.

PURPOSE: The purpose of this study was to determine the effects of a micronized purified flavonoid fraction treatment on surface expression of leukocyte adhesion molecules in chronic venous disease (CVD). METHODS: Twenty patients with chronic venous disease were assessed with the use of clinical and Duplex scanning criteria. Consenting patients were treated for 60 days with a micronized purified flavonoid fraction treatment (500 mg twice daily). Blood was collected from a foot vein immediately before the start of treatment and within 1 week after the treatment was stopped. Neutrophil and monocyte surface adhesion molecule expression was determined by flow cytometry using the monoclonal antibodies to CD11b and CD62L. RESULTS: Neutrophil CD11b (248:212), monocyte CD11B (204:190), neutrophil CD62L (130:97 [P =.002]), and monocyte CD62L (170:121 [P =.03]) were determined, respectively, before and after treatment. All values are arbitrary units and represent median values. CONCLUSION: Micronized purified flavonoid fraction treatment for 60 days seems to decrease the surface expression of CD62L by neutrophils and by monocytes. The clinical significance of this finding needs to be explored further. It is feasible to use changes in the levels of these molecules as a marker for response to therapy in chronic venous disease.

Plasma VEGF as a marker of therapy in patients with chronic venous disease treated with oral micronised flavonoid fraction - a pilot study.

AIM: to study the effect on plasma vascular endothelial growth factor (VEGF) levels of oral purified flavonoid fraction treatment for sixty days in patients with chronic venous disease (CVD). MATERIAL AND METHODS: twenty patients <> (clinical stage 2-4) with chronic venous disease were included. Duplex ultrasonography was used to assess the venous disease. Patients were treated for 60 days with Daflon(R) 500 mg twice daily. Blood was collected from a foot vein immediately before starting treatment and within one week of stopping treatment. Plasma VEGF levels were determined using a sandwich ELISA method. RESULTS: VEGF levels decreased in patients with stage C4 CVD after treatment with purified micronised flavonoid fraction treatment (98 pg/ml to 57 pg/ml). The levels of VEGF in patients with skin changes were significantly higher (98 pg/ml) than those with normal skin (9 pg/ml p=<0.001 Wilcoxon). CONCLUSIONS: plasma VEGF levels were much higher in patients who have skin changes of CVD. Plasma VEGF protein decreases in patients with CVD-induced dermatologic changes following treatment with purified micronised flavonoid fraction.

Endothelial activation response to oral micronised flavonoid therapy in patients with chronic venous disease--a prospective study.

BACKGROUND: Endothelial activation is important in the pathogenesis of skin changes due to chronic venous disease (CVD). Purified micronised flavonoid fraction has been used for symptomatic treatment of CVD for a considerable period of time. The exact mode of action of these compounds remains unknown. AIM: To study the effects of micronised purified flavonoidic fraction (Daflon 500 mg, Servier, France) treatment on plasma markers of endothelial activation. MATERIALS AND METHODS: Twenty patients with chronic venous disease were treated for 60 days with DAFLON 500 mg twice daily. Duplex ultrasonography and PPG was used to assess the venous disease. Blood was collected from a foot vein immediately before starting treatment and within 1 week of stopping treatment. Plasma markers of endothelial activation were measured using commercial ELISA kits. RESULTS: Reduction in the level of ICAM-1, 32% (141 ng/ml: 73 ng/ml) and VCAM 29% (1292 ng/ml: 717 ng/ml) was seen. Reduction in plasma lactoferrin (36% decrease, 760 ng/ml: 560 ng/ml) and VW factor occurred in the C4 group only. CONCLUSIONS: Micronised purified flavonoidic fraction treatment for 60 days seems to decrease the levels of some plasma markers of endothelial activation. This could ameliorate the dermatological effects of (CVD). This could also explain some of the pharmacological actions of these compounds. Our study demonstrates the feasibility of using soluble endothelial adhesion molecules as markers for treatment.

Duplex scanning and effect of multisegmental arterial disease on its accuracy in lower limb arteries.

AIM: To assess the accuracy of duplex in assessment of peripheral arterial disease and determine the effect of multisegmental disease on the accuracy duplex as opposed to single lesion. PATIENTS AND METHODS: One hundred and seventy-seven lower limbs were examined in 90 patients who presented with lower limb arterial disease, (59 male, 31 female, median age 68 years--81 with intermittent claudication, eight rest pain, one ulceration). Patients were examined with duplex US, and arteriography (IA DSA). Two radiologists and two technologists were involved in this double-blind study. Patients were classified into five groups; groups with single stenotic lesions, single occlusions, multiple stenotic lesions or occlusions, and multiple mixed disease. Duplex accuracy was determined in each group. RESULTS: Duplex was able to differentiate between normal and disease arterial segment with a sensitivity of 92%, specificity 99%, PPV 91%, and NPV 100% and Kappa 0.87. Sixty-six limbs were found to have single lesions, and 68 multisegmental disease. Duplex showed accuracy with a sensitivity of 87%, and specificity of 99%, for single stenotic lesion and 95%, 96% respectively for multisegmental. For single occlusions duplex accuracy showed sensitivity 92% and specificity 100%, and for multisegmental occlusions, sensitivity 97%, and specificity 99%. For mixed multisegmental pathology (stenosis and occlusion), sensitivity 94% and specificity 97%. CONCLUSION: Duplex is an accurate tool in diagnosis of lower limb arterial disease and multisegmental pathology does not adversely effect this accuracy.

Increased plasma vascular endothelial growth factor among patients with chronic venous disease.

Skin damage in the presence of chronic venous disease is partially mediated through leukocytes. The endothelium is activated and exhibits proliferation in the skin. Up-regulation of vascular endothelial growth factor (VEGF) expression in the skin of patients with chronic venous disease has been demonstrated with immunohistologic techniques. Abnormal VEGF expression can have local deleterious effects. The aim of this study was to determine whether patients with chronic venous disease have elevated plasma levels of VEGF. We conducted a prospective study with 30 patients with varicose veins of clinical, etiologic, anatomic, and pathologic class C3 (normal skin, n = 15) and C4 (trophic skin changes, n = 15) and 25 control subjects with no clinical evidence of venous or arterial disease of the lower limb. Blood samples were collected from a foot vein of each subject before and after a period of experimental venous hypertension produced by means of standing. Assay of VEGF protein was performed with a sandwich enzyme-linked immunosorbent assay. Plasma VEGF level was elevated in both groups of patients with venous disease compared with the control group. The median VEGF levels among patients were 81 pg/mL (interquartile range [IQR] 56 to 122) supine and 98 pg/mL (IQR 63 to 153) after standing for 30 minutes. Median VEGF levels among control subjects were 52 pg/mL (IQR 35 to 71) lying supine and 60 pg/mL (IQR 39 to 105) after standing for 30 minutes. Experimental venous hypertension caused a small rise in VEGF levels among the patients but not the control subjects. Further studies are required to determine whether increased VEGF expression contributes to tissue injury in chronic venous disease.

Comparison of duplex imaging and arteriography in the evaluation of lower limb arteries.

BACKGROUND: The aim of this study was to assess the accuracy of duplex imaging, compared with arteriography, in detecting arterial disease distal to the renal arteries. METHODS: Some 177 legs were studied in 90 patients (59 men, 31 women; median age 68 years) with peripheral arterial disease. Each patient had conventional intra-arterial angiography and duplex ultrasonography. Each leg was divided into 17 arterial segments, and the disease in each segment was quantified by measuring the peak systolic velocity ratio across any arterial lesion that was identified. RESULTS: Compared with angiography, duplex imaging was able to detect arterial disease with an overall sensitivity of 92 per cent, specificity of 99 per cent, positive predictive value 91 per cent and negative predictive value 100 per cent, and with a kappa value of 0.87 (95 per cent confidence interval (c.i.) 0.81-0.93). Duplex was able to determine the length of the arterial lesion with a sensitivity of 89 per cent and a specificity of 98 per cent with a kappa value of 0.88 (95 per cent c.i. 0.86-0.90). When the surgeon's final management decision concerning each vascular lesion was used as the reference, duplex and arteriography were equivalent, with an accuracy of 84 per cent and 85 per cent respectively in identifying the management used. CONCLUSION: Duplex ultrasonography is reliable in detecting arterial lesions in peripheral arteries and could be used routinely in the initial evaluation of patients with lower limb arterial disease.

Endothelial activation in patients with chronic venous disease.

OBJECTIVES: Leukocyte trapping due to leukocyte-endothelial activation has been implicated as the cause of lipodermatosclerosis and ulceration in patients with chronic venous disease. We investigated endothelial activity in normal controls and patients subjected to short-term venous hypertension. METHODS: Twenty-five normal volunteers and 30 patients with chronic venous disease divided into two groups: varicose veins with skin changes (LDS, n = 15); and varicose veins without skin changes (VVs, n = 15) were studied. Blood samples were taken from a foot vein before and after experimental venous hypertension. Plasma levels of ELAM-1 (endothelial leukocyte adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and von Willebrand factor (vWf) was measured by an ELISA. RESULTS: There was a significant rise in the plasma concentration of ELAM-1, ICAM-1 and VCAM-1 in patients and normal controls in response to venous hypertension. Basal levels of plasma VCAM-1 and vWf were higher in patients with LDS compared to patients with VVs. The magnitude of rise of VCAM-1 was greater in patients with LDS compared to patients with VVs (p = 0.01, Mann-Whitney U-test). There was no difference in the basal levels or in the magnitude of change in plasma ICAM-1 and ELAM-1 between the two patient groups. CONCLUSION: Venous hypertension results in endothelial activation which may aid endothelial-leukocyte adhesion. Patients with LDS exhibit increased VCAM-1, which is a counterligand for receptors expressed by monocytes and lymphocytes signifying that these cells may be more important in the development of skin changes.

Leukocyte activity in the microcirculation of the leg in patients with chronic venous disease.

PURPOSE: It has been suggested that leukocyte trapping and activation in the microcirculation of the leg skin causes lipodermatosclerosis and ulceration in patients with chronic venous disease. Ambulatory venous hypertension is accepted as the physiologic factor that leads to ulceration. We investigated leukocyte endothelial adhesion in patients who were subjected to short-term venous hypertension. METHODS: Two groups of patients with venous disease were studied: group 1, varicose veins with skin changes (n = 15); and group 2, varicose veins without skin changes (n = 15). Blood samples were taken from a foot vein before and after standing for 30 minutes to raise the venous pressure in the lower limb, and after lying supine again for 10 minutes. The samples were analyzed for leukocyte surface CD11b and L-selectin (CD62L) expression using a flow cytometer. Plasma-soluble L-selectin was also measured using an enzyme-linked immunosorbent assay. RESULTS: In patients with skin changes, median neutrophil CD11b levels fell from 4.66 to 3.83 arbitrary units (p = 0.005, Wilcoxon) after 30 minutes of venous hypertension, Median monocyte CD11b levels fell from 7.65 to 5.8 arbitrary units (p = NS, Wilcoxon) after venous hypertension and then fell further to 5.43 arbitrary units (p = 0.02 vs baseline; Wilcoxon) when the venous hypertension was removed. Neutrophil and monocyte L-selectin levels also fell in response to venous hypertension, remaining low even after venous hypertension was removed. A similar pattern was seen in patients with uncomplicated varicose veins. There was a rise in soluble L-selectin in the plasma of both groups of patients after venous hypertension, reflecting leukocyte adhesion to endothelium. In the group of patients with skin changes the level of soluble L-selectin rose from 695 ng/ml to 836 ng/ml (p = 0.02, Wilcoxon), and in the group without skin changes the rise was from 700 ng/ml to 801 ng/ml (p = 0.02, Wilcoxon). CONCLUSION: Venous hypertension results in sequestration of the more activated population of neutrophils and monocytes in the microcirculation of the leg in patients with venous disease. These cells bind to the endothelium, releasing L-selectin, and do not emerge from the limb when venous hypertension is reversed. These findings do not differ between patients with varicose veins and those with skin changes.

The microcirculation in venous hypertension.

Venous ulceration is a common problem in western countries and results in large costs to healthcare systems. A number of hypotheses of the mechanisms of development of venous ulceration have been advanced, but this question has not been fully resolved. In recent years research effort has focused on the microcirculation of the skin and many methods of investigation have been employed to study this. Some of the principal findings described in published work are reviewed in this article. It seems unlikely from the available evidence that venous ulceration is attributable solely to failure of diffusion of oxygen and other small nutritional molecules to the tissues of the skin. The microvascular changes in the skin are characterised by activated endothelium and perivascular inflammatory cells. It is much more likely that leucocytes attach themselves to the cutaneous microcirculation, become activated and produce endothelial injury. Repeated over many months or years, this chronic inflammatory process leads to be tissues changes of lipodermatosclerosis. Although there is evidence of leucocyte involvement in the pathogenesis of venous ulceration, the exact mechanisms remain to be resolved. Improved treatment for patients may be devised once a better understanding of the basic causes of this condition has been reached.

Effects of compression and type of bed surface on the microcirculation of the heel.

OBJECTIVE: To assess the effects of compression on the skin microcirculation of the heel using laser Doppler fluxmetry. DESIGN: Parallel groups comparing patients with control groups. SETTING: Department of Surgery, University College London Medical School, London. SUBJECTS AND MATERIALS: Ten patients at risk of developing pressure ulceration, 10 age- and sex-matched healthy subjects and 10 young, healthy volunteers. An acrylic indenter with a slot to accommodate a laser Doppler probe was used to apply compression to the heel region. A pressure sensor was used to measure the applied compression. OUTCOME MEASURES: The resting laser Doppler flux was measured with the subject lying supine. Compression forces were then applied in increments from 50 g to 1500 g and the corresponding interface pressure (IP) and laser Doppler flux (LDF) recorded. The IP and LDF were also measured from the heel while the subject was lying on a low air-loss system and then on an NHS conventional hospital bed. RESULTS: The resting LDF is lower in the patient group compared to the control groups (p < 0.05). Compression of the heel caused a progressive decrease in LDF in all groups. Compression greater than 50 mmHg as well as lying on an NHS bed reduced the LDF signal to a minimal value (biological zero). On the low air-loss system, the median LDF was 17% of the resting value in the age-matched control group and 32% in the patient group. CONCLUSIONS: The results indicate that the heel microcirculation is vulnerable to compression. The low air-loss system maintained the IP sufficiently low to prevent complete cessation of the heel microcirculation.

Duplex ultrasonography for assessment of venous valvular function of the lower limb.

Both legs of 29 patients with venous disease and those of 15 controls without venous disease were assessed by duplex ultrasonography. The duration of reverse flow after release of manual calf compression was measured in the common femoral, long saphenous, popliteal and short saphenous veins. Before undertaking the study, the reproducibility of the technique was evaluated in six subjects by repeating the examination over 3 consecutive days; the coefficient of variation of the test was 7.3 per cent. The 95 per cent confidence interval (c.i.) of the median (0.16 s) of all measurements in the normal limbs was 0.12-0.18 s. The 95 per cent c.i. for the 95th percentile of all measurements in normal limbs was 0.32-0.52 s. In limbs with clinical evidence of venous disease at least one of the sites examined was found to have reverse flow lasting longer than 0.5 s. These data suggest that the measurement of reverse flow after release of manual calf compression is a reproducible technique. While the method records some reverse flow in normal veins, its duration is unlikely to exceed 0.5 s; significant reflux is therefore defined as reverse flow exceeding 0.5 s.

Effect of leg elevation on the skin microcirculation in chronic venous insufficiency.

PURPOSE: Leg elevation is advised in the treatment of venous disease associated with edema. We have used laser Doppler fluxmetry to assess the effects of leg elevation on the skin microcirculation. METHODS: Fifteen patients with lipodermatosclerosis caused by chronic venous insufficiency and 15 control subjects were studied. Measurements were made from the liposclerotic skin of patients and 8 cm above the medial malleolus in control subjects. Laser Doppler flux, blood cell velocity, and concentration of moving blood cells were recorded with the subject lying in the supine position and after elevating the foot 30 cm above the heart level. RESULTS: In subjects in the horizontal position, the resting laser Doppler flux was significantly higher in patients with lipodermatosclerosis than in control subjects (median difference 63 arbitrary units; 95% confidence interval: 36, 108). This difference was due to a higher concentration of moving blood cells in the patient group (median difference 6.5 arbitrary units; 95% confidence interval: 3.4, 9). The blood cell velocity was not statistically significant between the two groups. On leg elevation, there was a substantial increase in the laser Doppler flux in the patient group; the median percentage increase in flux was 45% (p < 0.01). This was due to an increase in blood cell velocity; the median percentage increase was 41% (p < 0.01). There was no corresponding change in the concentration of moving blood cells. The results in the control group showed a similar trend but have not reached statistical significance. CONCLUSION: We conclude that limb elevation enhanced the microcirculatory flow velocity in liposclerotic skin of patients with chronic venous insufficiency.

Saphenous vein reflux without incompetence at the saphenofemoral junction.

Duplex ultrasonography was used to assess patients with primary varicose veins to determine whether varicosities of the long saphenous vein (LSV) occurred without saphenofemoral junction (SFJ) incompetence. Some 167 consecutive patients with the clinical diagnosis of primary varicose veins were investigated. Of 190 limbs with LSV reflux 63 had no SFJ incompetence, of which only five had incompetent perforators; these were midthigh perforators in two limbs and medial calf perforators in three. LSV reflux often occurs in the presence of a competent SFJ. This indicates that, in such circumstances, saphenofemoral ligation alone is unlikely to control varices associated with LSV reflux. It also suggests that the development of primary varicose veins may be an ascending rather than a descending phenomenon.

Stripping of the long saphenous vein in the treatment of primary varicose veins.

Eighty-nine legs with long saphenous vein (LSV) reflux and saphenofemoral junction incompetence were treated by saphenofemoral ligation and multiple avulsions; patients were randomized to undergo additional stripping of the LSV from groin to upper calf (n = 43) or no additional treatment (n = 46). At a median of 21 months after surgery recurrence was evaluated by duplex ultrasonography, photoplethysmography, clinical examination and patient assessment. Fewer persisting incompetent LSVs in the calf were found (21 versus 38) and median (interquartile range) photoplethysmographic refilling times were longer (20 (13-27) versus 14 (11-21) s) when the LSV was stripped than after saphenofemoral ligation alone (both P < 0.1). More patients were completely satisfied (65 versus 37 percent and were recurrence-free (65 versus 17 per cent) when the LSV had been stripped compared with saphenofemoral ligation alone (P < 0.05 and P < 0.001 respectively). The addition of LSV stripping to saphenofemoral ligation and multiple avulsions results in a better overall outcome.