Temporal and quantitative profiles of growth factors and metalloproteinases in acute wound fluid after mastectomy

Seventy-three samples of acute wound fluid were collected from 47 patients during the first 3 postoperative days (POD) following mastectomy for cancer ( n =47 on POD-1, n =19 on POD-2, and n =7 POD-3). Samples were analyzed by enzyme-linked immunosorbent assay for growth factor levels (epidermal [EGF], platelet-derived [PDGF], basic fibroblast [bFGF], transforming growth factor-β1 [TGF-β1], vascular endothelial [VEGF]), interleukin-6 (IL-6), matrix metalloproteinases (MMPs-2, -3, -9), and the tissue inhibitor of metalloproteinase 1 (TIMP-1). The levels of EGF, bFGF, PDGF, and interleukin-6 peaked on POD-1, with a significant decrease by POD-3, while total and active MMP-2, MMP-3, and tissue inhibitor of metalloproteinase 1 showed a progressive and significant increase from days 1 to 3. The wounds that later developed an infection (11%) were found to have a significantly lower PDGF and EGF on day 1 (PDGF, median 169 pg/mL [range, 86–2,595]) than the noninfected wounds (2,098 [17–66,506] p <0.05, Mann–Whitney U -test). Sixty-two percent patients developed a seroma and the levels of bFGF were significantly less in these patients (441 pg/mL [45–4,108]) than in those patients where there was no seroma (807 [245–3,133] p <0.05). The levels of certain growth factors in acute wound fluid may be important markers for wound outcomes.     

Profiles of matrix metalloproteinases and their tissue inhibitors in intraperitoneal drainage fluid: Relationship to wound healing

Matrix degradation and remodeling occurs during wound healing, thereby aiding tissue repair, angiogenesis, and cell migration. It is dependent on the balance between proteinases and their inhibitors, namely the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Acute wound fluid samples (n = 58 patients) were collected daily from the intraperitoneal drain placed after colorectal surgery from the first postoperative day until drain removal. Three laboratory techniques were performed: enzyme linked immunosorbent assays (MMP-1, MMP-3, TIMP-1, TIMP-2), gelatinase activity assays (MMP-2, MMP-9), and quenched fluorescent substrate hydrolysis (total MMP activity). Levels were correlated with each postoperative day, wound healing, and surgical outcome (p < 0.05, Spearman's correlation). Significant negative (MMP-9, MMP-3, MMP-8, TIMP-2, total MMP activity) and positive (MMP-2, TIMP-1) correlations were observed with the postoperative day, e.g., total MMP-9: day 1, median, 121 (range, 12-189) ng/ml; day 3, 46 (8-179); day 5, 31 (0-155), day 7, 20 (6-58). Differences were also observed with the type of operation, estimated blood loss, and length of operation and with postoperative complications. MMPs and TIMPs are involved in wound healing after elective colorectal cancer surgery and their levels in drain fluid may act as markers of wound healing and surgical outcome.     

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.     

Cellular sources and inducers of cytokines present in acute wound fluid

Acute wounds contain many biological active molecules, including several cytokines and growth factors. However, the cellular sources of each molecule, as well as the stimuli inducing them, are poorly characterized. We quantified the levels of 27 cytokines, chemokines, and growth factors in acute wound fluid in a luminex‐based assay. The acute wound fluid contained particularly high levels of IL‐6 and IL‐8, as well as elevated levels of MCP‐1, IL‐1RA, PDGF, IP‐10, IFN‐γ, and TNF‐α. Surprisingly, the amounts of IL‐1β and IL‐10 were relatively low. To characterize the cellular sources of these molecules, we analyzed supernatants from monocytes, neutrophils, keratinocytes, fibroblasts, and endothelial cells stimulated with pro‐ and anti inflammatory cytokines, and different Toll‐like receptor (TLR) ligands. The different cell types showed overlapping but distinct patterns of production of signal molecules, as well as sensitivity to ligands. Among pro‐inflammatory cytokines, IL‐1β was the most potent inducer of signal molecule production. Furthermore, keratinocytes and endothelial cells were in particular responsive to the Toll‐like receptor‐3 ligand polyI:C. New interactions between cytokines and growth factors were revealed, which may have important roles in wound healing, including IL‐1β‐induced IFN‐γ and IL‐10‐induced VEGF.     

Characterization of synovial fluid cytokine profiles in chronic meniscal tear of the knee

Concentrations of pro‐ and anti‐inflammatory cytokines in synovial fluid samples collected from patients with chronic meniscal tears were investigated. An acute inflammatory response is generally reported 24–48 h after knee injury, but the largest body of data available in literature concerns anterior cruciate ligament injury and very little information is available about the balance of soluble factors in the synovial fluid of knees with chronic meniscal tears. Sixty‐nine patients (46 males and 23 females) with meniscal tear that occurred more than 3 months earlier were enrolled. According to cartilage integrity assessment by arthroscopic examination, patients were assigned to one of the following groups: (i) no chondral damage (n = 18); (ii) chondral damage graded from I to II (n = 15); and (iii) chondral damage graded from III to IV (n = 37). In all groups, levels of IL‐10 and inflammatory cytokines IL‐6, TNF‐α, and IL‐8 where greater compared with those reported in the intact population; by contrast, levels of IL‐1ra and IL‐1β were significantly lower. Interestingly, IL‐6 levels were higher in female than male patients. Cytokine levels did not correlate with degree of chondral damage. IL‐6 and IL‐1ra levels positively correlated with IL‐1β, and negatively correlated with TNF‐α. Interestingly, levels of IL‐1β and TNF‐α were inversely correlated. Our data demonstrate increased levels of pro‐inflammatory cytokines (IL‐6, IL‐8, and TNF‐α) in the chronic phase of meniscal trauma. This pro‐inflammatory state is maintained in the joint from the time of initial injury to several months later and could be a key factor in hampering cartilage regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:340–346, 2017.

     

Comparison of oxidative stress biomarker profiles between acute and chronic wound environments

Increasing evidence implicates excessive reactive oxygen species (ROS) generation and ROS-derived degradation products in the pathogenesis of many skin diseases. While numerous attempts have been made to identify prognostic biomarkers of wound healing in skin, these have met with limited success. This study examined the profiles of various oxidative stress biomarkers, namely total protein carbonyl content (from protein oxidation), malondialdehyde content (from lipid peroxidation), and the total antioxidant capacities, in acute wound fluid (n= 10) and chronic wound fluid (n= 12), using a rapid, noninvasive collection technique. Protein carbonyl content was quantified spectrophotometrically and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting, following 2,4-dinitrophenylhydrazine derivitization. Malondialdehyde levels were similarly quantified, following N-methyl-2-phenylindole derivitization. Total antioxidant capacity was determined via wound fluid inhibition of cytochrome C reduction by a superoxide radical flux. Acute wound fluid contained higher protein carbonyl content than chronic wound fluid, particularly evident following sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis under nonreducing and reducing conditions (p < 0.001 and p < 0.02, respectively), related to significantly higher protein levels (p = 0.0005) in acute wound fluid. Human serum albumin ( approximately 66 kDa) was identified as the most prominent protein oxidized in both acute and chronic wound fluid, which may contribute to the reduced albumin and total protein levels in chronic wound fluid. No significant difference (p > 0.1) in malondialdehyde levels or total antioxidant capacities were determined between acute and chronic wound fluids, although chronic wound fluid exhibited significantly higher total antioxidant capacities (p < 0.005), accounting for variations in wound fluid protein content. These findings suggest an adaptation in the antioxidant profiles of chronic wound fluid to counteract the loss of consumed antioxidants in the chronic wound environment. This study highlights the roles of ROS/antioxidants in skin wound healing, their possible involvement in chronic wounds and the potential value of ROS-induced biomarkers in wound healing prognosis.     

Value of pretransplantation cytokine profiles for predicting acute rejection in renal transplant recipients

Episodes of acute rejection may represent an important risk factor for the development of chronic allograft nephropathy. Various studies have shown that pretransplant cytokine profiles in recipient blood are associated with transplant outcome. Serum samples were collected 24 hours before transplantation from 57 patients (38 men and 19 women of age 36 +/- 5 years) receiving kidneys from unrelated living donors. Additional samples were collected at 1 and 2 weeks after transplantation, as well as during every rejection episode. The immunosuppression consisted of a cyclosporine, prednisolone, and mycophenolate mofetil. Among the transplanted patients, 19 (33.3%) individuals experienced an acute rejection episode based on an increased level of serum creatinine and blood urea nitrogen during the first 14 days after transplantation. TGF-beta, IL-2 and IFN-gamma serum levels were determined by an ELISA method using Bindermed system kits. The mean concentration of TGF-beta before transplantation tended to be lower among patients with acute rejection episodes compared to those with stable graft (75,265 versus 85,394 pg/mL; P = .34) and at 1 week after transplantation (77,558 versus 84,390 pg/mL), although the differences were not significant. Among patients with rejection the mean IL-2 concentration was significantly higher before, at 1 week, and at 2 weeks after transplantation (15.0 versus 6.8 pg/mL, P = .005; 19.0 versus 4.9 pg/mL, P = .001; and 21.1 versus 4.7 pg/mL, P = .0001). The mean concentration of IFN-gamma was significantly higher pre- and at 1 and 2 weeks posttransplantation in patients with acute rejection episodes (161.1 versus 65.2, 175.6 versus 66.5 and 173.7 versus 77.1 pg/mL, all P < .001). In conclusion, evaluation of Th1 cytokines before transplantation may represent valuable predictive marker for an acute rejection episode.     

Effect of cytokine growth factors on the prevention of acute wound failure

Cytokine growth factor treatment of chronic wounds has met with mixed results. The chronic wound presents a hostile environment to peptides such as growth factors. Cytokine growth factors have not been studied extensively in acute wounds. However, incisional hernias are a major example of acute wound failure that has not been solved by various mechanical approaches. A biological approach to acute wound failure by use of cytokine growth factors may offer a new strategy. A rodent incisional hernia model was used. Seventy‐six rats underwent 3‐cm midline celiotomies and were closed with fine, fast‐absorbing sutures to induce intentional acute wound failure. Group 1 received no other treatment. The midline fascia in Groups 2–10 was infiltrated with 100 µl of vehicle alone or vehicle containing various test cytokine growth factors. Necropsy was performed on postoperative day 28 and the wounds were examined for herniation. Incisional hernias developed in 83 percent (13/16) of untreated incisional and 88 percent (7/8) and 83 percent (5/6) of the two vehicle‐treated incisions (PBS and carboxymethylcellulose). Hernia incidences were decreased by priming of the fascial incision with transforming growth factor‐β₂ (12%, 1/8), basic fibroblast growth factor (25%, 2/8) and interleukin‐1β (50%, 3/6) (p < 0.05). Aqueous platelet‐derived growth factor, becaplermin, insulin‐like growth factor, and granulocyte macrophage‐colony stimulating factor did not significantly decrease the incidence of acute wound failure (p > 0.05). A biological approach to acute wound failure as measured by incisional hernia formation can be useful in reducing the incidence of this complication. Transforming growth factor‐β₂, basic fibroblast growth factor, and interleukin 1β all eliminated or significantly reduced the development of incisional hernias in the rat model.     

Growth factor profiles in intraperitoneal drainage fluid following colorectal surgery: Relationship to wound healing and surgery

Cytokines and growth factors are important at each stage of wound healing. This study aims to determine the changing profiles of these factors in intraperitoneal drainage, acute wound fluid, following colorectal surgery, and to correlate levels to wound healing and surgical outcomes. Acute wound fluid samples (n = 52 patients) were collected daily from postoperative day 1 until drain removal. Levels of cytokines (interleukins-6 and -1beta and tumor necrosis factor-alpha) and epidermal growth factor, platelet-derived growth factor, vascular endothelial derived growth factor, basic fibroblast growth factor, and transforming growth factor-beta1 were determined by enzyme-linked immunosorbent assay. A significant negative correlation emerged between the levels of interleukin-6, epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor and the postoperative day, e.g., basic fibroblast growth factor : day 1, 695, median (29-2,806, range) pg/ml; day 2, 249 (1-1,784); day 3, 94 (0-722); day 7, 22 (0-326) (p < 0.05, Spearman's correlation). Levels appeared to relate to the stage of wound healing. Several factors, in particular interleukin-1beta and tumor necrosis factor-alpha levels, correlated with surgical outcomes such as the need for a defunctioning stoma and/or postoperative complications. Cytokines and growth factors are involved in normal wound healing, and their levels in acute wound fluid may act as markers of wound healing and surgical outcome.     

Cefoxitin concentration in wound fluid

The concentration of cefoxitin was determined in fluid obtained from human surgical wounds during the first postoperative day. Intravenous administration of cefoxitin sodium at a dosage of 1 or 2 g every six hours rapidly produced wound-fluid concentrations greater than the minimal inhibitory concentration for most susceptible organisms. After three hours, wound-fluid concentrations surpassed the serum concentrations of cefoxitin. The higher dosage resulted in higher wound-fluid levels.     

Urinary cytokine profiles in unilateral congenital hydronephrosis

Background

We aimed to evaluate possible clinical application of urinary monocyte chemotactic protein-1 (MCP-1), osteopontin (OPN), and regulated upon activation, normal T-cell expressed and secreted (RANTES) chemokine as useful noninvasive markers in children with congenital hydronephrosis (HN) caused by ureteropelvic junction obstruction (UPJO).

Methods

The study cohort consisted of surgical cases (study group 1), comprising 15 children with severe HN who required surgery (median age 1.03?years), conservative cases (study group 2), comprising 21 patients with mild, nonobstructive HN, and control group, comprising 19 healthy children. All children had normal renal function. Urinary (u) concentrations of MCP-1, OPN, and RANTES were measured using immunoenzymatic enzyme-linked immunosorbent assay (ELISA) commercial kits and were expressed in nanograms per milligram creatinine. Increased levels of MCP-1 and OPN were found in children with HN in comparison with study group 2 and controls (p?<?0.05). UMCP-1/Cr correlated with half-time (T_1/2) of the elimination phase of tracer excretion of technetium-99m-mercaptoacetyltriglycine (^99mTc-MAG-3) (p?<?0.05).

Results

Receiver operator characteristic (ROC) analyses revealed good diagnostic profile for uMCP-1 only in identifying children (<40?%) with abnormal differential renal function (DRF) [area under the curve (AUC) 0.862], and in detecting kidney injury in all examined children (AUC 0.704).

Conclusions

Additional studies with larger number of patients are required to confirm a potential application of uMCP-1 as a promising parameter for early identification of kidney obstruction.     

T cell cytokine profiles in childhood asthma

BACKGROUND: An imbalance of T cell subsets in asthma with a predominance of Th2 type cells has been proposed. The aim of this study was simultaneously to detect surface markers and intracellular production of cytokines in T cells from the airways of children with and without asthma. METHODS: Bronchoalveolar lavage (BAL) fluid was obtained by wedging a suction catheter into the distal airway immediately before elective surgery. Cells were stimulated with phorbol 12-myristrate 13-acetate (PMA) and ionomycin and intracytoplasmic cytokine retention was achieved using monensin. The cells were stained with the relevant antibodies and analysed by flow cytometry. RESULTS: No statistical difference was observed between children with atopic asthma, atopic non-asthmatic subjects, and normal controls in the percentage of CD3+ cells producing interleukin (IL)-2 or IL-4. Interferon (IFN)gamma+ T cells were, however, present in a much higher percentage than either IL-2 or IL-4 positive cells. The percentage of IFNgamma+ T cells was significantly increased in subjects with atopic asthma (median 71.3%, interquartile range (IQR) 65.1-82.2, n=13) compared with both atopic non-asthmatic subjects (51.9%, IQR 37.2-70.3, n=12), p<0.05 and normal controls (58.1%, IQR 36.1-66.1, n=23), p<0.01. CONCLUSIONS: These findings indicate that IFNgamma producing T cells are more abundant in the airways of children with atopic asthma than in atopic non-asthmatic subjects and controls. The proinflammatory activities of IFNgamma may play an important role in the pathogenesis of childhood asthma and may suggest that asthma is not simply a Th2 driven response.     

基质金属蛋白酶及其组织抑制剂与慢性创面的形成

基质金属蛋白酶家族（matrix-metalloproteinases，MMPs）是一类活性依赖于金属锌离子的蛋白酶。它以酶原形式被分泌，经激活后参与胶原等细胞外基质的降解。MMPs现有二十余种不同的蛋白酶。根据它们的结构和降解底物分为胶原酶、明胶酶、基质水解酶、膜型基质金属蛋白酶和其他基质金属蛋白酶五大类。MMPs的表达受生长因子等许多因素的调控。     

Studies in fetal wound healing. IV. Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid.

Recent clinical and experimental evidence suggests that the fetus responds to injury in a fashion fundamentally different from that of the adult. Our initial experience with human open fetal surgery reinforces experimental observations that the fetal wounds heal without the scarring, inflammation, and contraction that often accompany adult wounds. In this study we examine fetal wound fluid in an attempt to elucidate the control mechanisms that endow the fetus with unique healing properties. The extracellular matrix of fetal wounds is rich in hyaluronic acid, a glycosaminoglycan found in high concentrations whenever there is tissue proliferation, regeneration, and repair. We establish that wound fluid from the fetus contains high levels of hyaluronic acid-stimulating activity that may underlie the elevated deposition of hyaluronic acid in the fetal wound matrix. In contrast there was no hyaluronic acid-stimulating activity present in adult wound fluid. Hyaluronic acid, in turn, fosters an extracellular environment permissive for cell motility and proliferation that may account for the unique properties observed in fetal wound healing.     

Antibiotics in human hematoma and wound fluid.

Using the total hip replacement patient, we measured the level of antibiotic in the wound at the time of surgery, the wound drainage for 36 to 48 hours after surgery and over a 10 day period in incubated surgical debris obtained at the time of total hip replacement. The antibiotics sodium oxacillin, lincomycin hydrochloride and sodium cephalothin were administered in standard doses at 6 hour intervals. In one group of patients the antibiotic was begun at the time of surgery, in a second group the antibiotic was begun 24 hours after the completion of surgery. Sodium oxacillin, lincomycin hydrochloride and sodium cephalothin produce antibiotic levels in the venous serum consistently above the level obtained in the simultaneous would drainage specimens. Sodium oxacillin and lincomycin hydrochloride used as described in this study are capable of bathing the area of the surgical wound with concentrations of antibiotics above the minimal inhibitory concentrations. This is evidenced by the antibiotic levels above minimal inhibitory concentrations found in the wound drainage. Sodium cephalothin levels in the wound drainage were below measurable levels in 3 of 4 patients. Sodium oxacillin and lincomycin hydrochloride antibiotic levels in the surgical wound debris and simultaneous venous clotted specimens diminished progressively in a similiar manner indicating no additional effects of the surgical wound debris on the antibiotic's biologic effectiveness. Sodium cephalothin dropped below measurable levels in three days. Measurable levels of antibiotics appeared in the wound aspirate well above the minimal inhibitory concentration in patients receiving their initial dose of sodium oxacillin 24 hours after surgery.     

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.     

Chemokine and inflammatory cytokine changes during chronic wound healing.

Wound healing is a complex process resulting from an interplay of processes including coagulation, inflammation, angiogenesis, and epithelialization. The chemokine family has been shown to contain members that are potent regulators of many of these pathways. Because we have previously shown that chemokines "pool" in biologic wound dressings, we studied the levels of CXC and CC chemokines, along with key inflammatory mediators, serially from a group of patients undergoing therapy for chronic venous leg ulcers. After 8 weeks, all patients had marked clinical healing of their ulcers (median 63.3% reduction in size) with two of 10 completely healed. Wound fluids extracted from dressings showed high levels of platelet factor-4 and interferon-gamma-inducible protein, with a trend toward increases in the ratio of the sums of the angiogenic versus angiostatic CXC chemokines (p = 0.082) in the tissues collected from the center of the ulcers during wound closure. Neutrophil-activating peptide-2 and interleukin-8 accounted for the most changes in wound fluid angiogenic chemokines, with significant differences both as compared with baseline levels and with patients' plasma level noted at various time points between weeks 0 and 8. The level of angiostatic chemokines, interferon-y inducible protein 10 and platelet-activating-4, fell most significantly between weeks 0 and 3 as compared with plasma levels. The observed shift toward angiogenic CXC chemokines suggests that effective healing in chronic venous insufficiency ulcers appears to "move" the ulcer bed toward a state more conducive to epithelialization,characteristic of the proliferative phase of wound healing. CC chemokines were also elevated at baseline in the wound fluid samples as compared with the patients' plasma levels. Macrophage inflammatory protein-1 (3 and regulated on activation, normal T expressed and secreted (RANTES) levels decreased with healing, whereas there were significant increases in the tissue levels of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 a over the first 4 weeks of therapy (p< or = 0.05 for both). Coincident with these changes was a steady increase in the ratio of interleukin-1 R/interleukin-1 receptor antagonist protein in the ulcer center tissues, which also correlated with healing (p < 0 .05) as compared with a decreasing ratio at the ulcer edge, and a biphasic response in the wound fluids. These findings suggest that advanced wound care techniques help move the ulcer from a chronic inflammatory state into one more characteristic of the late inflammatory/early proliferative phase of wound healing. Chemokines may play a critical role in the pathogenesis of chronic venous ulcers through their effects on angiogenesis and/or the progression of inflammatory reactions at the site of injury.