Hrev_master Veins and Lymphatics 2012; volume 1:e6 [page 18] [Veins and Lymphatics 2012; 1:e6] Ulcerated hemosiderinic dyschromia and iron deposits within lower limbs treated with a topical application of biological chelator Eugenio Brizzio,1 Marcelo Castro,2 Marina Narbaitz,3 Natalia Borda,2 Claudio Carbia,2 Laura Correa,4 Roberto Mengarelli,5 Amalia Merelli,2 Valeria Brizzio,2 Maria Sosa,6 Biagio Biancardi,7 Alberto Lazarowski2 1International Group of Compression and Argentina Medical Association, Buenos Aires, Argentina; 2Department of Clinical Biochemistry, Institute of Pathophysiology and Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina; 3Pathology Department - Hematology - National Academy of Medicine, Buenos Aires, Argentina; 4Favaloro’s Foundation, Buenos Aires, Argentina; 5Pontifical Catholic University of Argentina, Buenos Aires, Argentina; 6Italian Hospital of Buenos Aires, Argentina; 7Faculty of Pharmacy, University of Naples, Italy Abstract The ulcerative haemosiderinic dyschromia of chronic venous insufficiency is difficult to heal and presents a high accumulation of iron. Lactoferrin, a potent natural iron chela- tor, could help to scar this ulcerative haemosi - derinic dyschromia. The objective of this study was to determine whether the topical application of a liposomal gel with Lactoferrin favors scarring/degradation of the brown col- ored spot typical of ulcerative haemosiderinic dyschromia. Nine patients with severe chron- ic venous insufficiency and ulcerative haemosiderinic dyschromia (CEAP-C6), with a natural evolution of over 12 months, were included in the study. Hemo chromatosis gene mutations were investigated. The levels of serum ferritin, transferrin saturation and blood cell counts were analyzed. The presence of hemosiderin was investigated through periulcerous and ulcer fundus biopsies car- ried out at baseline and 30 days after treat- ment with Lactoferrin. The severity of the injuries (CEAP classification) was evaluated at the beginning of and throughout the whole 3-month treatment period. No patient had received compression treatment during the three months previous to this therapy. Significant improvement in these injuries, with a reduction in the dimensions of the brown spot (9 of 9) at Day 90, and complete scarring with a closure time ranging from 15 to 180 days (7 of 9) were observed. The use of topical lactoferrin is a non-invasive therapeu- tic tool that favors clearance of hemosiderinic dyschromia and scarring of the ulcer. The success of this study was not influenced either by the hemochromatosis genetics or the iron metabolism profile observed. Introduction Chronic venous insufficiency (CVI) is one of the most significant health problems in devel- oped countries. Though the pathogenesis of skin changes and venous ulcers is not com- pletely understood, they occur as a late conse- quence of chronic ambulatory venous hyper- tension, caused by outflow obstruction and reflux due to superficial or deep venous valve incompetence. Ethological theories including fibrin cuffs or leukocyte entrapment by chron- ic inflammation have been suggested.1 Haemosiderinic dyschromia (HD) of CVI is a pathological entity that features a brown colored spot resulting from the deposit of free iron within leg tissues. Iron is a highly irrita- tive element capable of stimulating free-radi- cal release and of causing leg ulcers, thus pro- ducing an ulcerated hemosiderinic dyschro- mia (UHD). Since it has been recognized as a grade IV cause of skin dyschromia according to CEAP classification, and taking into account that these effects can be self-pro- duced or generated by stimulation of melanin, there is an increasing interest in the role of iron tissue deposits caused by chronic venous disorders. According to this, a brown discol- oration of the skin near the injury can be con- sidered to be a typical sign of venous disease. It occurs when blood cells leak out of blood vessels. The hemoglobin from red blood cells is broken down into hemosiderin that is then permanently stored within the tissues. This can take place after a significant injury in the leg and is often worsened by an underlying venous problem.2,3 Since extravasated blood cells with hemo- globin are phagocyted by tissue macrophages called siderophages, the accumulation of hemosiderin within the injury area is a char- acteristic feature of the disease, resulting in the brownish color of the skin.2 Furthermore, urinary hemosiderin could be a biological marker for the clinical follow up of chronic venous insufficiency with haemosiderinic dyschromia.4 Nearly 25% of absorbed iron is normally eliminated from the body by exfoliation of epi- dermal cells;5 therefore, iron accumulation in the skin should be secondary to any mecha- nism that may increase iron deposits before carrying out this exfoliation. Iron is thought to be a co-factor or mediator of skin toxicity in a variety of pathological situations, including sunburn,6 porphyria cutanea tarda,7 inflamma- tion,8 and skin cancer,9 as well as in hereditary hemochromatosis (HH).10 It is important to distinguish HD in CVI from hereditary HH because individual differences could be genet- ically determined by genes related to HH (H63D, S65C and C282Y).11 Lactoferrin (LFR) is a glycoprotein belong- ing to the family of transferrins, capable of binding to iron. Both human and bovine LFR show a wide antimicrobial spectrum, against positive and negative Gram bacterias, and cer- tain viruses and fungi.12 The studies on LFR have focused on its ability to chelate iron in cases of hemosiderinic iron accumulation (ecchymosis, post sclerotherapy, CVI).12 The results of recent studies indicate that it is a powerful regulator of dermal fibroblasts, and that it promotes cutaneous wound healing; 13,14 Correspondence: Eugenio Brizzio and Alberto Lazarowski, San Martín 965, 1st floor (Zip code 1004) Buenos Aires, Argentina. Tel. +54.11.4311.5559. E-mail: eugeniobrizzio@ciudad.com.ar; ebrizzio@fibertel.com.ar; eugeniobrizzio@gmail.com; alazarowski@gmail.com Key words: ulcerated haemosiderinic dyschro- mia, liposomal Lactoferrin, scarring, hemo- siderin-ferritin. Acknowledgments: the authors would like to thank P. Girimonte for her assistance with the statistical analysis of the results. We also wish to thank the patients who made this study possible. Conference presentation: part of the present study has been presented at the following con- gresses: i) XX Argentine Congress of Hematology, October 18-22, 2011, Mar Del Plata, Argentina (submitted to the Poster Section); ii) III World Symposium of advances in Phlebology and Lymphology, November 10-12, 2011, Buenos Aires, Argentina (awarded); iii) Latin American Congress of Angiology and Vascular Surgery, November 24th, 2011, Cartagena, Colombia. Received for publication: 26 July 2012. Revision received: 19 September 2012. Accepted for publication: 26 September 2012. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright E. Brizzio et al., 2012 Licensee PAGEPress, Italy Veins and Lymphatics 2012; 1:e6 doi:10.4081/vl.2012.e6 No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e6] [page 19] however, this has been poorly researched. There is currently no efficient treatment for HD and associated ulcer in patients with CVI. In a recent systematic survey and meta-analy- sis, eight randomized clinical trials were iden- tified comparing treatment with stockings and bandages. Five studies revealed an advantage in the use of stockings over bandages, while three other assays showed no difference.15 Our aim was to study the effect of liposomat- ed bovine Lactoferrin, locally applied on the surface of UHD, in 9 patients with long-lasting evolution of refractory CVI, selected from our previous study,16 and to evaluate its potential relation to the iron metabolism profile and mutations of HH genes.17 Study design This was a prospective controlled pilot study performed on 9 selected patients with severe and persistent UHD of CVI, carried out accord- ing to the inclusion and exclusion criteria described below. All patients gave their signed individual con- sent to treatment with topical application of liposomated LFR and to undergo biopsy of two lesions. There were three evaluation time points: at 30, 90 and 180 days. The therapeutic protocol was presented for review by the inde- pendent court of ethics on March 5th 2010 and was accepted on April 23th 2010; the protocol was approved in accordance with the princi- ples of the Declaration of Helsinki. The study used a database consistent with the results obtained during a 6-month follow- up period of 9 patients with recalcitrant venous ulcers. Only one of them presented bilateral ulcers with similar severity of lesions in both legs. This was a pilot study and the data collect- ed should be considered in this light. The main parameters controlled before and after treatment were: i) stratification of CVI (Ecodoppler), and leg goniometry and edema; ii) severity of ulcerous injuries (color, ulcer areas, rate of scarring time); iii) pain and quality of life; iv) hemosiderin staining in biopsies and blood iron metabolism parame- ters; v) hematologic profiles; vi) iron profiles; vii) the presence of HH mutations as potential predictive parameters of evolution. Inclusion criteria The main inclusion criteria were: i) patients over 18 years old; ii) unilateral or bilateral ulcers at the anteromedial part of the calf, of proved venous origin, confirmed by Ecodoppler ultrasound; iii) surface larger than 3 cm² and smaller than 25 cm²; iv) presence of associated periulcerative haemosiderinic dyschromia; v) pre-existing ulcer with at least two months of evolution; vi) patients accepting to undergo treatment according to protocol; vii) each patient received a written report and signed their consent. Exclusion criteria The main inclusion criteria were: i) pres- ence of occlusive arterial pathology with a more than 0.8 arm/ankle index; ii) known allergies; iii) pregnancy; iv) life expectancy less than 12 months; v) severe diseases co- existing simultaneously with venous patholo- gies, e.g. cardiac or mental disorders, renal or hepatic insufficiencies, tumors, etc.; vi) symp- tomatic peripheral neuropathy, e.g. diabetic neuropathy; vii) patients with motor disabili- ties; viii) diabetes; ix) severe joint disease of the ulcerated leg, besides the ankle stiffness caused by venous ulceration. Materials and Methods Patients Nine patients were selected (3 males, 6 females); average age 63 years. A total of 10 ulcerated legs were studied (unilateral ulcers, n=8; bilateral ulcers, n=2). Ecodoppler A SonoScape® colour Ecodoppler S6 (SonoScape Co. Ltd., Shenzhen, China) was used to confirm the venous vascular etiology of the ulcer and the stratification of patients according to the type of reflux observed. Only one baseline control was performed at the moment of admission. Stratification of patients was carried out according to the type of reflux observed, such as superficial, perfo- rating, deep, or their combined forms as super- ficial + perforating, perforating + deep, super- ficial + perforating + deep. Lesion evaluation Time from the onset of the CVI, the evolu- tion of the hemosiderinic dyschromia, and the ulcer development were all recorded. Since skin pigmentation as a brown discol- oration near the lesion is a typical feature of HD in case of venous ulcers, a visual scale of brown color was used to follow up treatment. Baseline and weekly controls were carried out. We used an analogical visual arbitrary num- bered scale of brown (Figure 1) that allowed us to build a follow-up chart and to identify any improvement in HD. Wound size is a basic parameter used to evaluate the success of treatment. The plani- metric Visitrak® Smith and Nephew device (Smith and Nephew, Hull, UK) was used. Volume was obtained through perimeters measured at 4 segments of the leg: 12 cm from hallux extremity, and 10, 20 and 30 cm from the floor (Figure 2A and B).18-19 Goniometry was measured using the model described by Cleusa Belczak (Figure 3). 20 Pain was measured at baseline and after four weeks of treatment using the arbitrary numerical Likert scale from 1 to 5, where 1 Indicates the lowest intensity of pain and 5 the highest one.21 The quality of life questionnaire in chronic lower limb venous insufficiency (CIVIQ) was evaluated as previously described.22 Venous blood samples for determining hematimetric parameters as well as molecular studies were drawn in 2 separate collection tubes containing potassium ethylene diamine tetraacetic acid (K3-EDTA), while those for determining biochemical parameters were drawn in tubes with serum separators. Serum was freshly separated from venous blood sam- ples by centrifugation at 1800 g for 10 min at room temperature. All fractioned serum sam- ples and those for molecular studies were stored frozen at -20°C for three months before assaying. Hematimetric parameters were eval- uated on fresh samples immediately after blood sample collection; full blood cell counts were studied by SYSMEX XT-1800 (Roche, Penzberg, Germany). Serum iron (SFe, mg/dL), total iron-binding capacity (TIBC, mg/dL), transferrin saturation (sat-Trf %) and serum ferritin (SF, ng/mL) were assayed using a Cobas 6000 autoanalyzer system (Roche). Figure 1. Arbitrary identification of color scale used. No n- co mm er cia l u se on ly Article [page 20] [Veins and Lymphatics 2012; 1:e6] HFE genotyping Samples: five drops of 25 uL of anticoagulat- ed blood with K3-EDTA were collected on Whatman filter paper no. 1 (5¥5 cm) and stored at room temperature in a paper envelope DNA extraction: DNA was extracted by the modified Boom method.23 Two drops of dried blood from filter paper of each sample were cut with a scalpel and placed on 4.5 mL of lysis buffer. After 4 h of gentle shaking, the paper was carefully discarded and the DNA was extracted in the supernatant as previously described.23 DNA extracts were stored at -20°C. Amplification and detection: the polymerase chain reaction (PCR) mixture was prepared in separate tubes for the study of mutations in exons 4 and 2, respectively, at a final volume of 50 uL and a final concentration of 1X Taq Buffer, 0.2 mM dNTP, 2.5 mM MgCl2, 0.5 U of Taq poly- merase (Invitrogen Corp., Carlsbad, CA, USA) and 0.2 uM of each primers (Invitrogen). We used 5 uL of DNA extract per sample. The sequences of the primers used were: i) exon 4 (Cys282Tyr mutation) Forward: 5’TGGCAAGGGTAAACAGATCC Reverse 5’CTCAGGCACTCCTCTCAACC (390 bp); ii) exon 2 (mutations His63Asp and Ser65Cys) Forward: 5’ACATGGTTAAGGCCTGTTGC Reverse 5’GCCACATCTGGCTTGAAATT (208 bp). For both constructions of primers, amplifi- cation conditions were 35 cycles with an annealing temperature of 63°C. Then 15 uL of PCR products were digested with 2 U of Rsa I for codon 282, Bcl I for codon 63 and Hinf I for codon 65 (New England Biolabs, Ipswich, MA, USA) overnight, according to the manufactur- er’s instructions. The digestion products were run on 3% agarose gel (Invitrogen) for 2 h at 120 Volts, with ethidium bromide for viewing under UV light. This allowed us to distinguish wild-type (WT), heterozygous (HT) and homozygous (HO) genotypes for each muta- tion with the following cutting patterns:24,25 - Rsa I (Cys282Tyr): WT 250 pb/140 pb, HO 250 pb/111 pb/29 pb, HT 250 pb/140 pb/111 pb/29 pb; - Bcl I (His63Asp): WT 138 pb/70 Pb, HO 208 pb, HT 208 pb/138 pb/70 pb; - Hinf I (Ser65Cys): WT 147 pb/60 Pb, HO 207 pb, HT 207 pb/147 pb/60 pb. Biopsy handling: all patients were treated with subcutaneous local administration of 1% sterile xilocaine. After 10 min, an approximate- ly 12 mm long and 3 mm wide rectangle of tis- sue was excised to include the surrounding intact skin, the ulcer edge, and the ulcer base. In order to compare changes on both anatomic and hemosiderin staining patterns, biopsies were taken at two different contiguous sites for initial and end-point samples, respectively.26 Biopsy histochemistry: each biopsy was immediately divided lengthways. One half was fixed in cold 4% paraformaldehyde in phos- phate buffered saline and the other was snap- frozen in optimal cutting temperature over liq- uid nitrogen for immunocytochemistry. After 24 h, fixed tissues were embedded in paraffin wax and sectioned at 4 m. Hematoxylin & Eosin and Perls’ Prussian blue methodswere used to stain alternate sections.27 Operational protocol: the product, a gel con- taining bovine LFR liposomated to 3%, placed on a 15¥20 cm sterile paraffin dressing (tulle gras) made of open weave gauze, with 0.5% chlorhexidine acetate, an antiseptic with a broad spectrum (Bactigras® plaque, Smith and Nephew), was locally applied every day for four weeks (first end point). It was applied twice a week during the sec- ond and third months using an additional com- pression by means of a multilayer bandage from the very first day, assembled with 3 short 8 cm and 10 cm elastic bandages (Fisiodur®, Zuccari srl, Trento, Italy). The multilayer band- age was applied in a figure-of-eight with turns that regularly crossed one another, with a rest- ing pressure of 40 mmHg and 5-7 mmHg of static stiffness, as previously described.28 At each medication session, the ulcer was cleaned using gauzes impregnated with Prontosan® solution (B. Braun Medical Inc., Bethlehem PA, USA) followed by administra- tion of simple occlusive medication. Results Patients Nine patients were selected from a cohort of 55 patients who had previously completed the aforementioned comparative study of low- strength medical compression stockings with bandages for treating recalcitrant venous ulcers,16 after a long period without showing any improvement in the ulcers’ features (e.g. dimensions, color intensity of dyschromia), as well as pain and CIVIQ. Of the 9 independent cases analyzed, 5 corresponded to primary (P) ulcers and the remaining 4 to recidivating (R) ulcers. Ninety percent of the patients included in this study presented dynamic static alter- ations of plantar support, 4 patients with pes cavus (grades II and III), another 4 with flat feet (grades II and III), and the remaining patient without any foot disorder. There was equal dis- tribution of lower limb ulcers between both legs (right n=5, left n=5), taking into account that one patient had bilateral ulcers that both pre- sented the same degree of severity (Table 1). Stratification of refluxes through ecodoppler Ecodoppler confirmed the venous vascular etiology of the ulcer (Figure 4). No patients presented purely deep reflux. One case exhibited an altered reflux due to the com- bination of the three systems associated with a severe hemodynamic condition (Table 2). Lesion evaluation Range of duration of CVIs was 48-560 months, of hemosiderinic dyschromia 15-240 months, and of ulcers 2-54 months (Table 1). In order to perform an evolutive control of the hemosiderinic dyschromia, we used an analogi- cal and arbitrary visual numbered scale of the color brown, and built a chart of color evolution. Therefore, the real color intensity of the skin was compared to the intensities of the afore- mentioned color scale (Table 3). This scale Figure 2. (A) Patient’s leg with (B) schematic representation of cylindrical volume of each area (1-4). Modified from Rossi et al.29 Figure 3. Belczak’s model of goniometry. No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e6] [page 21] showed a starting point with an average inten- sity of 18.7 and decreased to an average intensi- ty of 7.2 at week 12 (P<0.001) (Figure 5). An approximately 50% decrease in color intensity was observed at Week 4. Measurement of the area of the ulcer The area of the ulcer and the scarring rate were checked at study start and at weekly inter- vals, and observations were recorded. During the initial control, dimensions of ulcers ranged from 3.3 to 23.6 cm. In 8 of 9 patients (9 of 10 ulcers), the area involved decreased significant- ly (P<0.001) (Table 4 and Figure 6), and in 7 patients, the healing rate reached 80% of the ulcers at the 6-month follow up (Table 5). Only one patient, who had abandoned the trial, showed a transient increase in the size of the surface of the ulcer, which returned to baseline at Month 6. Volumetric perimeter: control of edema Perimetral control of foot and leg was trans- formed to volume in cm3 as previously described.18-20 With the exception of case no. 3437 (corresponding to the patient with bilat- eral ulcers) whose values were approximately 9500 cm3, the remaining cases presented val- ues ranging from 3000 cm3 to 5000 cm3 and a mild decrease in the edema volume was observed (Table 6). Goniometry was measured at baseline and after 30 days to check flexion (flx) and exten- sion (ext) movements. A favorable evolution of the tibioastragaline joint could be observed at the expense of an increase in flexor and exten- sor excursions without any additional treat- ment (Table 7). Pain and chronic lower limb venous insuffi- ciency controls We used the numerical Likert scale, most commonly seen as a 5�point scale (0=no pain, 5=worst possible pain). In 5 patients, the max- imum level of pain (grade 5) was observed at the initial control; this was later reduced to grade 1 after four weeks of treatment. As far as the remaining patients are concerned, the level of the Likert scale diminished 1 point dur- ing the same period (Table 8). The survey of quality of life (CIVIQ), the baseline control and the tests performed during Weeks 4, 8 and 12 showed an improvement at all levels. It could be observed that, at all times, the average scor- ing of the CIVIQ scale (total score) in patients with ulcers closed at Week 12 was lower than the average scoring in patients with unclosed ulcers (P<0.05) (Table 9). Blood tests: hematimetric and iron metabo- lism profiles Normal results of basal control (1st) and after four weeks (2nd) of treatment on hematimetric parameters and platelet counts were observed in all cases (data not shown), and iron metabo- lism profile (Table 10). Interestingly, while transferrin sat (%) decreased in 7 of 9 cases, ferritin increased in 6 of 9. HFE genotyping Only one of the 9 patients (case no. 3334) was heterozygous for the mutant H63D gene (Table 10). Coincidentally, the patient present- ed a high level of ferritin (222 and 244 µg/mL). Table 1. Main features of patients’ lesions. No patients were previously treated by surgical or sclerotherapy procedures. Case no. Laterality Duration CVI Duration Interval Duration Primary Cavus BMI (months) dyschromia (months) ulcer age or recurrent and/flat feet (months) (months) 3446 R 180 22 20 2 P FC Normal 3437R R 180 60 24 36 R FC OB.3 3437L L 180 60 30 30 R FC OB.3 3283 L 336 36 34 2 R FC OB.1 3334 R 276 48 46 2 R FC Over weight 3161 L 192 84 36 48 R Normal Normal 3441 L 60 54 --- 54 P FF Normal 3451 R 48 15 2 13 P FF OB.2 3274 L 560 60 57 3 P FF OB.2 3449 R 360 240 236 4 R FC Normal R, right; L, left; CVI, chronic venous insufficiency; P, primary; R, recurrent; BMI, body mass index; OB, obesity. Figure 4. Color ecodoppler: reflux in great saphenous vein. No n- co mm er cia l u se on ly Article [page 22] [Veins and Lymphatics 2012; 1:e6] Ecodoppler for this patient evidenced reflux on the 3 systems, superficial, perforating and deep reflux mentioned above. Biopsy histochemistry Biopsy features of borders and ulcer bed: sam- ples of two control biopsies (initial and after 4 weeks of treatment) were obtained from the periulcerous area in 10 ulcers, and additional samples from the ulcer bed were obtained in another 2. Histological features (Figure 7) and hemosiderin staining (Figure 8) were evaluated before and after treatment. Before treatment, the histological analysis showed the presence of fibrin cuffs, small vessels, and extravasation of red blood cells, fibrosis and a chronic inflamma- tory pattern (Figure 7A). Perls’ Prussian blue staining showed superficial and deep high cumuli of hemosiderin in the border and fundus of the ulcer (Figure 8A). After four weeks of treatment, extravasated red blood cells and fibro- sis were still present; however, certain neovas- cular structures as well as a repairing inflamma- tory pattern were observed (Figure 7B). In some cases, Perls’ Prussian blue staining seemed to have decreased (Figure 8B). Discussion Ochre dermatitis is a secondary pigmentary disorder of venous stasis in which the increase in intravascular pressure and endothelial alterations cause extravasations of erythro- cytes, hemosiderin-laden macrophages, and melanin deposits. It is associated with long- term and high care costs, with an equally high incidence of recurrence, and a significant pro- portion of negative patient outcomes.29 In our study, all 9 patients were selected from a previ- ous study because they had ulcers and hemo- siderinic dyschromia, both associated to refractory ulcer. Wound repair depends on neoangiogenesis and activation of a local immune response, as well as on the presence of growth factors, including epidermal growth factor, transform- ing growth factor b, and basic fibroblast growth factor.30,31 It has been recently suggest- ed that systemic or topical drugs acting in the wound repair and regeneration processes could be promising and useful agents in the treatment of chronic venous ulcers.32 However, in a previously reported systematic review per- formed by Bradley et al., 16 randomized con- trolled trials were identified that compared topic agents (growth factors, cell suspensions, free-radical scavengers) versus placebo for treating CVI ulcers, concluding that there was insufficient evidence to recommend any par- ticular agent.33 The main finding was that topic application of liposomated LFR allowed a fast and progressive Table 2. Stratification of refluxes through ecodoppler (Type) and frequencies (No.) of pure or mixed forms of reflux. Systems Type No. Superficial reflux only One system 1 Perforating reflux only One system 3 Deep reflux only One system 0 Mixed superficial and perforating reflux Two systems 3 Mixed perforating and deep reflux Two systems 1 Mixed superficial, perforating and deep reflux Three systems 1 Table 3. Brown color scale: colorimetric evolution. Weeks Case no. Baseline 1 2 3 4 8 12 3446 26 17 13 11 9 7 7 3437R 23 23 22 11 9 8 8 3437L 25 18 17 14 11 10 9 3283 17 10 9 8 7 - - 3334 19 18 17 14 11 10 8 3161 8 7 5 5 5 5 5 3441 18 17 14 14 13 8 5 3451 16 15 14 11 10 8 7 3274 18 16 14 11 10 8 7 3449 17 15 14 13 13 11 9 R, right; L, left. Table 4. Evolution of ulcerous area in cm2 during the first six months of follow up. Case no. Study start Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 3446 3.3 0.0 - - - - - 3437R 6.5 0.1 0.1 0.0 - - - 3437L 12.8 10.7 8.8 8 5.7 3.2 0.0 3283 4.5 5.8 7.2 8.3 4.5 Deserted 3334 3.1 0.5 0.2 0.1 0.0 - - 3161 6.0 5.1 7.5 6.4 4.7 2.7 0.0 3441 19.9 8.8 0.6 0.0 - - - 3451 23.6 11 3.5 2.1 0.0 - - 3274 3.6 0.8 1.8 0.3 0.5 0.2 0.0 3449 9.6 6.2 6.7 6.2 5.2 4.9 00 R, right; L, left. Table 5. Time (months) taken to achieve complete closure of the 10 lesions from 9 patients. Time (months) Number closed lesions Total 1 1/10 1 3 2/10 3 6 6/10* 9 *One patient left the study. No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e6] [page 23] reduction in the dimensions of the area of the ulcer in 9 of 9 patients and complete closure in 7 of 9 cases. The 90 days of evolution evidenced an important improvement in the injuries, with a reduction in the intensity of the brown color of the spot (9 of 9) and time to complete scarring ranging from 15 to 180 days (7 of 9). It is impor- tant to emphasize that the patients belonged to the group of refractory cases included in the pre- vious study already mentioned.16 This assay showed that 50% of ulcers showed complete clo- sure using medical compression stockings, and 67% of complete closure with multilayer bandag- es, after 180 days. One of the most remarkable findings was the significant decrease, in all cases, of the brown color of the HD and the size of the ulcer- ous areas (Figures 5 and 6), with a concomi- tant goniometric improvement (Table 7), and complete closure of lesions in 7 cases after six months of treatment. The rate of healing was independent of baseline or recurrent ulcers (Figure 5). In all patients, clinical improvement of the wounds (10 ulcers) was associated with a signif- icant decrease in pain and improvement in qual- ity of life, except in one case (*case no. 3161) due to a domestic accident on the lesion, which showed no clinical improvement and led to the patient discontinuing treatment (Figure 6, Tables 8 and 9). All biopsies showed changes in cytological patterns (Figure 7). In several cases, a decrease was seen in the high level of stain- Figure 5. Variation in brown color scale values during the treatment. Figure 6. Ulcer’s areas diminution during six weeks of follow up. Table 6. Edema control in cm3: volumetric variation in leg edemas. Case no. Basal Week 1 Week 2 Week 3 Week 4 Week 8 Week 12 3446 4227 4127 4089 4072 3958 3932 3886 3437R 9457 9409 9678 9623 9562 9549 9605 3437L 9963 9856 9777 9670 9581 9588 9540 3283 4215 4122 4092 4061 4011 3999 3980 3334 4747 4680 4565 4548 4495 4424 4366 3161 3163 3056 3431 3214 3125 3008 2998 3441 4149 4011 4020 3977 3959 3884 3892 3451 4480 4314 4241 4204 4115 4097 4161 3274 4261 4008 3878 4129 4114 4059 3993 3449 4737 4512 4498 4451 4388 4275 4174 R, right; L, left. Table 7. Baseline-final goniometry. Goniometric values before and after 30 days of treatment. Case no. BG (flx) FG (flx) BG (ext) FG (ext) 3446 12° 12° 30° 42° 3437R 10° 19° 22° 25° 3447L 9° 10° 22° 24° 3283 5° 12° 25° 35° 3334 10° 12° 30° 35° 3161 10° 12° 19° 28° 3441 3° 10° 37° 40° 3451 9° 12° 22° 24° 3274 10° 11° 20° 23° 3449 9° 10° 15° 30° R, right; L, left; BG, baseline; FG, final goniometry; flx, flexion; ext, extension. No n- co mm er cia l u se on ly Article [page 24] [Veins and Lymphatics 2012; 1:e6] ing for HS in periulcerous and ulcer fundus biopsies present during the initial control (Figure 8) and this associated with a significant improvement in the edema and ulcerous areas (Figure 9) after treatment. Iron deposits in the skin of patients with CVI cause readily visible HD (brown colored der- mal areas) that always surrounds ulcers. The origin of increased iron loads in these lesions lies in the extravasations of red blood cells dur- ing significant venous stasis. Erythrocytes are degraded by resident dermal macrophages, and iron is incorporated into ferritin which, in time, changes to HS according to progressive iron overload.30,32 Furthermore, the urinary excretion of hemosiderin described in these patients4,33 suggests that the phenomenon of leg hemosiderin deposits could be of signifi- cance on the entire body.34,35 However, in con- trast with this hypothesis, in 1988 Ackermann found a 20-fold higher average concentration of iron in lower limbs affected by venous ulcers as compared to the upper arms of the same subjects.2 The distribution of high levels of ferritin staining in leg ulcers of patients with CVI were reported to be located intra and extracellular in the matrix, as compared with normal skin tis- sue with considerably less alterations or non- evident alterations at all.11 However, the sys- temic parameters of iron metabolism observed in our study (Table 7) did not seem to influence either the severity of HD nor the evolution of treatment with local LFR. Furthermore, the abnormal levels of ferritin observed in some patients did not limit the previously mentioned improvement in the ulcers. However, it is important to note that potential co-morbidities could be associated to systemic iron overload. One case that presented an altered reflux caused by the combination of the three systems associated with a severe hemodynamic condi- tion was also a carrier of an HFE gene mutation (heterozygous), evidencing high levels of serum ferritin (222 and 244 ng/mL), and suf- fered a sudden death. In another case with ele- vated serum ferritin (405 and 355 ng/mL) the patient experienced heart insufficiency and later stroke. Finally, the patient with bilateral ulcers also exhibited high serum ferritin values (233 and 251 ng/mL). We still do not know with certainty if ferritin could constitute a prognostic evolutive param- eter, but its association to the clinical evolu- tion observed in 3/9 patients suggests that it should be included in follow-up protocols. Because all parameters studied including ulcer lesion features, as well as quality of life (CIVIQ) and pain (Likert’s scale) were improved after treatment, topic application of liposomed LF could be a new therapeutic strat- egy, particularly in patients with refractory ulcers and HD associated secondary to chronic venous insufficiency. Figure 7. (A) Histological features before treatment: presence of fibrin sleeves, small ves- sels, extravasations of red blood cells, fibrosis, chronic inflammatory pattern. (B) Histological features after treatment (4 weeks): presence of new vascular structures, extravasations of red blood cells, fibrosis and granulation tissue-granulation, chronic repairing inflammatory pattern. Figure 8. Baseline sample (A) showed high hemosiderin concentration (local iron over- load) with (B) an evident reduction in hemosiderin staining after four weeks of treatment. Table 9. Positive chronic lower limb venous insufficiency score: variation between baseline and final values were observed in all cases except in one patient (*) who discontinued treatment. Case no. Baseline Final B-F 3446 35 33 2 3437 83 75 8 3283 66 59 7 3334 53 47 6 3161* 49 53 -4 3441 73 60 13 3451 89 80 9 3274 67 60 7 3449 71 66 5 B, baseline; F, final. *One patient left the study. Table 8. Pain control using the Likert scale comparing baseline values with those obtained at Week 4. Case no. Baseline Week 4 3446 1 1 3437R 5 1 3437L 5 1 3283 5 3 3334 3 2 3161* 2 3 3441 5 2 3451 5 1 3274 2 1 3449 5 1 R, right; L, left. *One patient left the study. No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e6] [page 25] As regards its iron binding properties, LFR differs from serum transferrin in its higher iron binding affinity and unique ability to retain iron over a broad pH range.1 The protective effects of topic LFR on induced dermatological allergic process was demonstrated experimentally.36 Similar results were obtained in a study carried out on human volunteers, treated by topical administration of the contact allergen and using purified recombinant human LFR.37 Although originally identified as an abundant protein in milk secretions, LFR is mainly expressed by surface epithelia and secreted into the mucosal environment. However, further research is needed to clarify whether local iron mobilization, free radical scavenging and induc- tion to tissue repair are simultaneously staged by the multiple properties of LF. Conclusions Our results suggest that the topical use of LFR could be a potential non-invasive thera- peutic tool that favors clearance of HD and a faster closure of ulcers, with concomitant relief or disappearance of pain, and conse- quent improvement in quality of life in patients with chronic venous insufficiency. Further research is needed to confirm these results by prospective randomized controlled studies. References 1. Raju S, Neglen P. Chronic venous insuffi- ciency and varicose veins. N Engl J Med 2009;360:2319-27. 2. Ackerman Z, Seidenbaum M, Loewenthal E, Rubinow A. Overload of iron of patients with varicose ulcers: possible contributing role of iron accumulation in progression of the dis- ease. Arch Dermatol 1988;124:1376-8. 3. Allegra C, Antignani PL, Bergan JJ, et al. International Union of Phlebology Working Group. The “C” of CEAP: suggested defini- tions and refinements: an International Union of Phlebology conference of experts. J Vasc Surg 2003;37:129-31. 4. Piotrowicz R, Grzela T, Jawień A, Kuligowska-Prusińska M. Urine haemo - siderin: a marker of chronic venous insuf- ficiency. Acta Angiol 2009;15:101-7. 5. Weintraub L, Demis D, Conrad M, Crosby W. Iron excretion by the skin selective localization of iron in epithelia cells. Am J Pathol 1965;46:121-7. 6. Bissett DL, Chatterjee R, Hannon DP. Chronic ultraviolet radiation induced increase in skin iron and the photo protec- tive effects of topically applied iron chela- tors. Photochem Photobiol 1991;54:215-23. 7. Takeshita K, Takajo T, Hirata H, et al. In vivo oxygen radical generation in the skin of the protoporphyria model mouse with visible light exposure: an L-band ESR study. J Invest Dermatol 2004;122:1463-70. 8. Gira AK, Casper KA, Otto KB, et al. Induction of interferon regulatory factor 1 expression in human dermal endothelial cells by interferon-gamma and tumor necrosis factor-alpha is transcriptionally regulated and requires iron. J Invest Dermatol 2003;121:1191-6. 9. Bhasin G, Kausar H, Sarwar Alam M, Athar M. Progressive iron overload enhances chemically mediated tumor promotion in murine skin. Arch Biochem Biophys 2003;409:262-73. 10. Chevrant-Breton J, Simon M, Bourel M, Ferrand B. Cutaneous manifestations of Figure 9. Comparison of severity of lesion before (A, C) and after (B, D) treatment. (A and B) Size of ulcers and (C and D) edema. Intensity of hemosiderin staining. Decreasing dimensions of edema and ulcers correlate with a lower staining hemosiderin observed after treatment (A and B). Table 10. Iron metabolism profile. Case no. Iron m/dL TIBC m/dL Transferrin Ferritin sat. (%) ng/mL 1° 2° 1° 2° 1° 2° 1° 2° 3446 38 53 361 253 10.5 20.1 25 23.8 3437 61 58 302 306 20.1 19 233 251 3283 60 74 263 265 23.5 27.9 117 126 3334 73 53 325 382 22.4 18.9 222 244 3161 57 48 297 311 19 15.4 82 72.9 3441 52 44 309 325 16.8 13.5 26 31.3 3451 73 70 198 212 36.8 33 405 355 3274 128 120 296 301 43 40 197 201 3449 72 66 250 251 28.8 26.2 112 117 TIBC, total iron binding capacity. No n- co mm er cia l u se on ly Article [page 26] [Veins and Lymphatics 2012; 1:e6] idiopathic hemochromatosis. Study of 100 cases. Arch Dermatol 1977;113:161-5. 11. Zamboni P, Tognazzo S, Izzo M, et al. Hemochromatosis C282Y gene mutation increases the risk of venous leg ulceration. J Vasc Surg 2005;42:309-14. 12. Valenti P, Berlutti F, Conte MP, et al. Lactoferrin functions: current status and perspectives. J Clin Gastroenterol 2004;38 Suppl 6:S127-9. 13. Tang L, Wu JJ, Ma Q, et al. Human Lactoferrin stimulates skin keratinocyte function and wound re-epithelialization. Br J Dermatol 2010;163:38-47. 14. Engelmayer J, Blezinger P, Varadhachary A. Talactoferrin stimulates wound healing with modulation of inflammation. J Surg Res 2008;149:278-86. 15. Brizzio E, Blättler W, Rossi G, et al. Healing venous ulcers with different modalities of leg compression. Unexpected findings of a pilot study. Phlebologie 2006;35:249-55. 16. Brizzio E, Amsler F, Lun B, Blättler W. Comparison of low-strength compression stockings with bandages for the treatment of recalcitrant venous ulcers. J Vasc Surg 2010;51:410-6. 17. Blättler W, Lüscher D, Brizzio E, et al. Healing of chronic venous leg ulcers could be affected by an interaction of the hemochromatosis gene polymorphism HFE H63D with the strength of compres- sion treatment - a re-analysis of patients from previous studies. Wound Repair Regen 2012;20:120-4. 18. Perrin M, Guex JJ. Edema and leg volume: methods of assessment. Angiology 2000;51:9-12. 19. Rossi G, Idiazabal G. Mathematical model to obtain the volume of the lower limb. Int Angiol 2005:3 Suppl 1:110. [Abstract]. 20. Belczak CEQ. Fisiologia do sistema venoso. In: Thomaz JB, Belczak CEQ (eds.). Tratado de flebología y linfología. Rio de Janeiro: Editorial Rubio; 2006. pp 37-70. 21. Likert R. A technique for the measurement of attitudes. Archiv Psychol 1932;140:1-55. 22. Launois R, Reboul-Marty J, Henry B. Construction and validation of a quality-of- life questionnaire in chronic lower limb venous insufficiency (CIVIQ). Qual Life Res 1996;5:539-54. 23. Boom R, Sol C, Salimans M, et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495-503. 24. Baptista-González HA, Rosenfeld-Mann F, Trueba-Gómez R, et al. Association of HFE mutations (C282Y and H63D) with iron overload in blood donors from Mexico City. Ann Hepatol 2007;6:55-60. 25. Oliveira VC, Caxito FA, Gomes KB, et al. Frequency of the S65C mutation in the hemochromatosis gene in Brazil. Genet Mol Res 2009;8:794-8. 26. Herrick SE, Sloan P, McGurk M, et al. Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am J Pathol 1992;141:1085-95. 27. Bancroft J, Gamble M (eds.). Theory and practice of histological techniques ban- croft & gamble. 6th edition. London: Churchill-Livingstone, Elsevier Health Sciences; 2008. 28. Brizzio E, Idiazabal G. Multilayer system. Rev Acta Flebol 2003;3:65-8. 29. Beebe-Dimmer JL, Pfeifer JR, Engle JS, Schottenfeld D. The epidemiology of chronic venous insufficiency and varicose veins. Ann Epidemiol 2005;15:175-84. 30. Eming SA, Kieg T, Davidson JM. Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol 2007;127:514-25. 31. Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835-70. 32. Palfreyman S, King B, Walsh B. A review of the treatment for venous leg ulcers. Br J Nurs 2007;16:S6-14. 33. Bradley M, Cullum N, Nelson EA, et al. Systematic reviews of wound care man- agement: (2) Dressings and topical agents used in the healing of chronic. Health Technol Assess 1999;3:1-35. 34. Zamboni P, Scapoli G, Lanzara V, et al. Serum iron and MMP-9 variations in limbs affected by chronic venous disease and venous leg ulcers. Dermatol Surg 2005; 31:644-9. 35. Zamboni P, Izzo M, Tognazzo S, et al. The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogene- sis. Free Radic Biol Med 2006;40:1869-73. 36. Zweiman B, Kucich U, Shalit M, et al. Release of lactoferrin and elastase in human allergic skin reactions. J Immunol 1990;144:3953-60. 37. Griffiths CE, Cumberbatch M, Tucker SC, et al. Exogenous topical Lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans. Br J Dermatol 2001;144:715-25. No n- co mm er cia l u se on ly