Levistilide A Ameliorates NLRP3 Expression Involving the Syk-p38/JNK Pathway and Peripheral Obliterans in Rats.

BACKGROUND: Inflammation is one of the most important pathogeneses of thromboangiitis obliterans (TAO). The NLRP3 inflammasome plays a vital role in the body's immune response and disease development. It can be activated by numerous types of pathogens or danger signals. As the core of the inflammatory response, the NLRP3 inflammasome may provide a new target for the treatment of various inflammatory diseases. Levistilide A (LA) is a phthalide dimer isolated from umbelliferous plants. Its pharmacological effect is largely unknown. This study revealed the effects of LA on endothelial cell activation, NLRP3, IL-1ß, TNF-α, IL-32, and CCL-2, VCAM-1, MCP-1, and the spleen tyrosine kinase (Syk)--p38/JNK signaling axis and its effect on vasculitis in rats. RESULTS: LA inhibited endothelial activation and the expression of IL-1ß, TNF-α, IL-32, CCL-2, VCAM-1, and MCP-1. LA directly obstructed Syk phosphorylation and activity in a dose-dependent manner, inhibited the activity of p38 and JNK, and reduced the expression of NLRP3 in human umbilical vein endothelial cells and vascular tissue of rats with vasculitis. CONCLUSION: LA suppressed NLRP3 gene expression by blocking the Syk--p38/JNK pathway and reduced damage to the rats' limbs in the thromboangiitis obliterans model.

Integrated treatment for lower-limb stage II thromboangiitis obliterans by interventional therapy and oral administration of Chinese medicine: a randomized controlled clinical trial.

METHODS: Ninety lower-limb stage II or worse TAO patients were randomly divided into three groups: group A (30 cases) treated by intervention and oral administration of Chinese medicine; group B (30 cases) treated by intervention alone; and group C (30 cases) treated only with oral administration of Chinese medicine. Therapeutic effects were observed, including the cure rate; the recurrence rate after one month, three months, six months, nine months, and one year; the ankle brachial indexes; the incidence of complications; and the level of C-reactive protein and erythrocyte sedimentation rate. RESULTS: Group A had significantly better clinically curative effects, related indexes, and outcomes during the long-term follow-up survey, than that of groups B and C. CONCLUSION: Integrated treatment is more effective for treating lower-limb stage II or worse TAO. OBJECTIVE: To observe if integrated treatment is better than other therapies for lower-limb stage II thromboangiitis obliterans (TAO).

Possible involvement of Notch signaling in the pathogenesis of Buerger's disease.

PURPOSE: Under pathological conditions, the Notch signal pathway is involved in the inflammatory process in arteriosclerosis, atherosclerosis and angiogenesis under ischemic conditions. The purpose of this study was to observe whether or not Buerger's disease is associated with Notch signal activation. METHODS: All the patients were diagnosed between 1980 and 2009 at Nagoya University Hospital. Twenty-two specimens from 12 patients with Buerger's disease (TAO) and 13 specimens from nine patients with arteriosclerosis obliterans (ASO) were analyzed by immunohistochemistry for Notch1, Jagged-1 (a Notch ligand) and Hes-1 (a Notch 1 target transcription factor). RESULTS: Notch1 and Jagged-1 were highly expressed in the endothelium in the new vasa vasorum and in the smooth muscle cells in the media of specimens from both groups. These Notch-related proteins were also remarkably expressed in inflammatory cells in the intima of specimens from TAO patients. Fewer inflammatory cells expressed Notch-related proteins in atheromatous plaques (Notch1 (%): 8.4 ± 0.76 versus 1.3 ± 0.43, P < 0.001; Jagged-1(%): 9.3 ± 1.1 versus 5.2 ± 1.1, P = 0.03). Indeed, Hes-1, which is a transcription factor downstream of Notch1, was remarkably expressed in the endothelium of new capillary vessels and inflammatory cells in TAO patients. Notch1-positive mononuclear cells were also seen in the thrombus in samples from the TAO group. CONCLUSIONS: Our findings are the first demonstration that Notch signal activation in inflammatory cells may be involved in the pathophysiological mechanism underlying Buerger's disease.

Identification of S100A8/A9 involved in thromboangiitis obliterans development using tandem mass tags-labeled quantitative proteomics analysis.

Thromboangiitis obliterans (TAO) is characterized by inflammation and obstruction of small-and medium-sized distal arteries, with limited pharmacotherapies and surgical interventions. The precise pathogenesis of TAO remains elusive. By utilizing the technology of tandem mass tags (TMT) for quantitative proteomics and leveraging bioinformatics tools, a comparative analysis of protein profiles was conducted between normal and TAO rats to identify key proteins driving TAO development. The results unveiled 1385 differentially expressed proteins (DEPs) in the TAO compared with the normal group-comprising 365 proteins with upregulated expression and 1020 proteins with downregulated expression. Function annotation through gene ontology indicated these DEPs mainly involved in cell adhesion, positive regulation of cell migration, and cytosol. The principal signaling pathways involved regulation of the actin cytoskeleton, vascular smooth contraction, and focal adhesion. The roles of these DEPs and associated signaling pathways serve as a fundamental framework for comprehending the mechanisms underpinning the onset and progression of TAO. Furthermore, we conducted a comprehensive evaluation of the effects of S100A8/A9 and its inhibitor, paquinimod, on smooth muscle cells (SMCs) and in TAO rats. We observed that paquinimod reduces SMCs proliferation and migration, promotes phenotype switching and alleviates vascular stenosis in TAO rats. In conclusion, our study revealed that the early activation of S100A8/A9 in the femoral artery is implicated in TAO development, targeting S100A8/A9 signaling may provide a novel approach for TAO prevention and treatment.

Toll-like receptor signaling pathway involved in pathogenesis of thromboangiitis obliterans through activating of NF-κB.

OBJECTIVES: The pathogenic mechanisms of Thromboangiitis Obliterans (TAO) are not entirely known and autoimmune inflammation plays a vital role in the initiation and continuance of TAO activity. The authors investigated in this study the role of the TLR signaling pathway in the pathogenesis of TAO. METHODS: First, the authors detected the expressions of MyD88, TRIF and NF-κB in vascular walls of 46 patients with TAO and 32 patients with trauma and osteosarcoma by western blot assay. Second, the authors detected the cellular localization of MyD88, TRIF and NF-κB in vascular walls of patients with TAO by immunofluorescent assay. RESULTS: The protein expressions of MyD88, TRIF and NF-κB were much higher in vascular walls of TAO patients (p < 0.05). Higher expressions of MyD88 and NF-κB were detected both on vascular endothelial and vascular smooth muscle cells of TAO patients. However, higher expression of TRIF was just detected on vascular smooth muscle cells of TAO patients. CONCLUSIONS: These dates suggest that the TLR signaling pathway might play an important role in the pathogenesis of TAO, it might induce vasospasm, vasculitis and thrombogenesis to lead to the pathogenesis and progression of TAO.

Si-Miao-Yong-An Decoction alleviates thromboangiitis obliterans by regulating miR-548j-5p/IL-17A signaling pathway.

Thromboangiitis obliterans (TAO) is a rare, chronic, progressive, and segmental inflammatory disease characterized by a high rate of amputation, significantly compromising the quality of life of patients. Si-Miao-Yong-An decoction (SMYA), a traditional prescription, exhibits anti-inflammatory, anti-thrombotic, and various other pharmacological properties. Clinically, it was fully proved to be effective for TAO therapy, but the specific therapeutic effect of SMYA on TAO has been unknown. Thus, deep unveiling the mechanism of SMYA in TAO for identifying clinical therapeutic targets is extremely important. In this study, we observed elevated levels of IL-17A in the peripheral blood mononuclear cells (PBMCs) of TAO patients, whereas the expression of miR-548j-5p was significantly decreased. A negative correlation between the levels of miR-548j-5p and IL-17A was also demonstrated. In vitro experiments showed that overexpression of miR-548j-5p led to a decrease in IL-17A levels, whereas downregulation of miR-548j-5p showed the opposite effect. Using a dual luciferase assay, we confirmed that miR-548j-5p directly targets IL-17A. Furthermore, serum containing SMYA effectively decreased IL-17A levels by increasing the expression of miR-548j-5p. More importantly, the results of in vivo tests indicated that SMYA mitigated the development of TAO by inhibiting IL-17A through the upregulation of miR-548j-5p in vascular tissues. In conclusion, SMYA significantly enhances the expression of miR-548j-5p, thereby reducing the levels of the target gene IL-17A and alleviating TAO. Our research not only identifies novel targets and pathways for the clinical diagnosis and treatment of TAO but also advances the innovation in traditional Chinese medicine through the elucidation of the SMYA/miR-548j-5p/IL-17A regulatory axis in the pathogenesis of TAO.

High-mobility-group box protein 1A box reduces development of sodium laurate-induced thromboangiitis obliterans in rats.

OBJECTIVE: High-mobility-group box protein 1 (HMGB1), as a late mediator of inflammation, plays a key role in inflammatory responses by inducing and extending the production of proinflammatory cytokines. The effect of HGMB1 in the inflammatory disease thromboangiitis obliterans (TAO) is unknown. We aimed to investigate the role of HMGB1 in sodium laurate-induced TAO in rats. METHODS: Male Wistar rats were randomly divided into five groups (n=8 each) for treatment: normal, sham-operated, TAO model, and low-dose (15 mg/kg) or high-dose (30 mg/kg) recombinant A box (rA box) infection (administered intraperitoneally once daily for 15 days). The TAO model was induced by sodium laurate and graded by gross appearance on day 15 after femoral artery injection. Histologic changes were measured by histopathology in rat femoral arteries. Plasma levels of HMGB1, thromboxane B2, 6-keto-prostaglandin F1-α, and blood cell counts and blood coagulation levels were measured. Expression of HMGB1, receptor for advanced glycation end-products (RAGE), interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was assessed by immunohistochemistry and immunofluorescence, Western blot analysis, and quantitative reverse-transcription polymerase chain reaction. RESULTS: The typical signs and symptoms of TAO were observed on day 15 after sodium laurate injection. The expression of HMGB1, RAGE, interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was markedly increased in rat femoral arteries. Plasma levels of HMGB1 and thromboxane B2 were elevated, but the level of 6-keto-prostaglandin F1-α was decreased. Blood was in a hypercoagulable state, and prothrombin, thrombin, and activated partial thromboplastin times were all significantly shortened, whereas fibrinogen level was increased in TAO rats compared with sham-operated rats. These effects were terminated by the HMGB1 antagonist rA box. CONCLUSIONS: HMGB1 is involved in the inflammatory state in a model of TAO induced by sodium laurate in rats, probably via its receptor RAGE. As the antagonist of HMGB1, rA box can attenuate the development of TAO, which may be a potential therapeutic target for the treatment of TAO.

Reduced circulating endothelial progenitor cells in thromboangiitis obliterans (Buerger's disease).

To determine the role of endothelial progenitor cells (EPCs) in the pathogenesis of thromboangiitis obliterans (TAO), EPC numbers and colony-forming units, migratory function and tubular structure formation in vitro were compared between 13 young male TAO patients and two age-matched healthy control groups: 11 smokers and 12 non-smokers. TAO patients had significantly lower numbers of EPCs and EPC colonies compared to both non-smokers [190 (97.0-229) vs 528 (380-556), p < 0.001 for EPCs and 0.80 (0.53-1.00) vs 2.80 (2.08-4.00) per mm(2), p = 0.001 for EPC colonies] and smokers [190 (97.0-229) vs 272 (229-326), p = 0.012 for EPCs and 0.80 (0.53-1.00) vs 2.80 (1.80-3.93) per mm(2), p = 0.001 for EPC colonies]. However, there were no significant differences in migratory function or tube formation between the three groups. These results suggest that TAO patients have an intrinsic decrease in EPCs not entirely associated with smoking, which may be the cause of endothelial dysfunction seen in TAO patients leading to the development of this disease at early ages.

MiR-223 alleviates thrombus and inflammation in thromboangiitis obliterans rats by regulating NLRP3.

OBJECTIVE: The aim of this study was to observe the regulatory effects of micro ribonucleic acid (miR)-223 on thromboangiitis obliterans (TAO) rats, and to explore the potential regulatory mechanism. MATERIALS AND METHODS: Online database TargetScan was used to predict the downstream regulatory targets of miR-223. A total of 45 Sprague Dawley (SD) rats were randomly divided into three groups, including sham operation group (Sham group), Model group, and miR-223 agonist group (miR-223 mimic group). TAO model was successfully established in rats through the injection of lauric acid via the femoral artery. The content of serum thromboxane B2 (TXB2) and endothelin (ET) was measured via enzyme-linked immunosorbent assay (ELISA). The pathological changes in the left hind limb were detected via hematoxylin-eosin (HE) staining. Moreover, the expressions of interleukin-6 (IL-6) and IL-1ß in the tissues of the rat left hind limb were determined via immunohistochemistry. In addition, the protein expression of Nod-like receptor protein 3 (NLRP3) in tissues was determined using Western blotting. RESULTS: TargetScan database predicted that NLRP3 was the downstream target gene of miR-223. Compared with the Sham group, Model group exerted significantly higher content of serum TXB2 and ET, severe lesions in the rat left hind limb, as well as significantly increased expressions of IL-6 and IL-1ß and protein expression of NLRP3 in tissues of the rat left hind limb (p<0.05). Besides, compared with the Model group, miR-223 mimic group showed remarkably lower content of serum TXB2 and ET, improved lesions in the rat left hind limb, as well as decreased expressions of IL-6 and IL-1ß and protein expression of NLRP3 in the tissues of the rat left hind limb (p<0.05). CONCLUSIONS: MiR-223 agonist can alleviate thrombus and inflammatory response in TAO rats. The possible underlying mechanism may be related to targeted regulation on NLRP3 inflammasome expression.

Effect of bradykinin on rats with thromboangiitis obliterans through PI3K/Akt signaling pathway.

OBJECTIVE: To explore the effect of bradykinin on rats with thromboangiitis obliterans (TAO) through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) signaling pathway. MATERIALS AND METHODS: The female Wistar rats were injected with lauric acid via the femoral artery to establish the TAO model, and they were randomly divided into control group (healthy rats), model group (TAO rats) and bradykinin group (TAO rats injected with bradykinin B2 receptor-specific inhibitor). The control was set in each group before the operation. The level of serum bradykinin in each group was detected via enzyme-linked immunosorbent assay (ELISA), and the reactive oxygen species (ROS) level, Caspase-3 activity and PI3K/Akt protein concentration in vascular tissues were measured via ELISA, Western blotting, ROS assay, and Caspase-3 activity assay, respectively. Moreover, the specific therapeutic mechanism of bradykinin was analyzed. RESULTS: In control group, the intima of the lower extremity venous tissues was smooth, the extima had no evident changes, and there was no inflammatory cell invasion around the arteries and veins. In model group, there was massive inflammatory cell invasion into the lower extremity venous tissues. In bradykinin group, fibrosis and atrophy occurred in venous tissues, the extima was thickened without fibrosis, and there was phagocytosis of neutrophils and mononuclear macrophages around the arteries and veins, as well as massive inflammatory infiltration. The PI3K/Akt protein concentration in lower extremity venous tissues was the highest in control group and the lowest in bradykinin group, and there were statistically significant differences (p<0.01). At 24 h after administration of doxorubicin (DOX), the level of ROS in lower extremity venous tissues was higher in bradykinin group than that in model group (p<0.05), and it was also higher in model group than that in control group (p<0.05). Besides, the activity of Caspase-3 in lower extremity venous tissues was significantly increased in bradykinin group compared with that in model group and control group, while it was slightly higher in model group than that in control group (p<0.05). CONCLUSIONS: The low expression of bradykinin can promote TAO in rats by the mechanism that it inhibits the PI3K/Akt signaling pathway to raise the oxidative stress level, thereby aggravating TAO.

The IL-6/STAT3 pathway regulates adhesion molecules and cytoskeleton of endothelial cells in thromboangiitis obliterans.

Thromboangiitis obliterans (TAO) (also known as Buerger's disease) is an inflammatory vascular disease that predominantly affects small- and medium-sized blood vessels of extremities. Endothelial cells play critical roles in the initiation and progression of this disease, but the underlying mechanisms remain unclear. In the present study, we demonstrate that patients with TAO had significantly higher levels of interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in their plasmas, and the involved arterial tissues expressed higher levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), ICAM-1 and VCAM-1. In exploring the molecular mechanisms with human aortic endothelial cells (HAECs), we found that recombinant IL-6 activated the STAT3 pathway, leading to the upregulation and overproduction of ICAM-1 and VCAM-1. RhoA (Ras homolog family member A), eNOS (endothelial nitric oxide synthase) and MMP-9 (matrix metalloproteinase-9) participated in this cellular signaling, and their interaction regulated the expression of ICAM-1 and VCAM-1. The activated STAT3 pathway by IL-6 also modulated the cytoskeleton of HAECs by regulating phosphorylation of focal adhesion kinase (FAK) and acetylation of α-tubulin through interplaying with RhoA. In summary, the present results indicate that activation of the IL-6/STAT3 pathway contributes to the pathogenesis of TAO by regulating cellular adhesion molecules and cytoskeleton of vascular endothelial cells, suggesting that targeting this pathway may provide a potential approach for the management of TAO.

[Indices of antioxidant system of blood in differential diagnosis of obliterating atherosclerosis and obliterating thromboangiitis of the lower limbs].

The work was aimed at elucidating the role of the indices of the antioxidant system (AOS) of the blood and nitrogen oxide content in differential diagnosis of atherosclerosis obiiterans (AO) and thromboangiitis obiiterans (TO) of the lower extremities. Presented herein are the findings of examining a total of one hundred and thirteen 30-to-45-year-old patients (of these, 60 had TO and 53 had AO) with various level of occlusion (from the femoropoplietal segment to the aortoiliac zone) and the stage of chronic arterial insufficiency (CAI IIA-IV). The control group was composed of 30 apparently healthy age-matched male subjects. Besides the clinical, laboratory and special instrumental methods of study, we determined the parameters of the AOS of blood and the content of nitrogen oxide degradation products. It was determined that the directedness and pronouncedness of deviations in the parameters of the AOS and nitrogen oxide from the physiological norm depended on not only the aetiology of the disease involved, but on the degree of tissue hypoxia predetermined by the stage of CAI of the lower limbs. For differential diagnosis between AO and TO, the most informative should be considered the coefficient: SOD activity/catalase activity, index of peroxide-luminol-dependent chemiluminescence and the content of nitrogen oxide degradation products.

[Correlation of expressions of ER and CD59 in Buerger disease].

OBJECTIVE: To inquire into the expression conditions of ER, CD59 and IgG in Buerger disease arteria vessels and probe their correlation. METHODS: Two groups of arteria vascular endothelial cells were investigated. The cells in Buerger group were derived from 30 male Buerger disease patients; the cells in the other group were derived from 30 normal men. The levels of ER, CD59 and IgG expression were detected by immunohistochemistry staining. RESULTS: The levels of ER and CD59 expression in Buerger disease group were higher than those of control group (P<0.05); the level of IgG expression on arteria intima in Buerger group was lower than that of control group (P<0.05); the level of IgG expression under arteria intima in Buerger group was higher than that of control group (P<0.05). Spearman rank correlation analysis of the Buerger group showed that ZD59 expression and IgG expression are significantly correlated and ER expression and IgG expression are significantly correlated, too. CONCLUSION: The expressions of CD59 and ER are up-regulated, which are probably

Mechanisms of thrombosis, available treatments and management challenges presented by thromboangiitis obliterans.

Thromboangiitis obliterans (TAO) is a thrombotic-occlusive and an inflammatory peripheral arterial disease with unidentified aetiology. Thrombotic events can lead to limb loss in TAO patients, who are typically young male smokers of low socioeconomic status. It is still unknown whether the initial process is thrombosis or inflammation, so it is difficult to ascertain whether managing inflammation or thrombosis improves the outcome of the disease. In this review, the possible mechanisms of thrombosis in TAO are evaluated; the treatments, based on the discussed mechanisms of thrombosis in TAO, are then reviewed and the challenges and limitations associated with the management of TAO are discussed.

Expression of ICAM-1, VCAM-1, E-selectin and TNF-alpha on the endothelium of femoral and iliac arteries in thromboangiitis obliterans.

Immunohistochemical light and electron microscopical analysis of surgical biopsies obtained from femoral and iliac arteries of patients with thromboangiitis obliterans (TAO) were performed to investigate the presence of tumour necrosis factor-alpha (TNF-alpha) and expression of the endothelial cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Expression of ICAM-1, VCAM-1 and E-selectin was increased on endothelium and some inflammatory cells in the thickened intima in all TAO patients. Ultrastructural immunohistochemistry revealed contacts between mononuclear blood cells and ICAM-1-, and E-selectin-positive endothelial cells. These endothelial cells showed morphological signs of activation. The present data indicate that endothelial cells are activated in TAO and that vascular lesions are associated with TNF-alpha secretion by tissue-infiltrating inflammatory cells, ICAM-1-, VCAM-1- and E-selectin expression on endothelial cells and leukocyte adhesion via their ligands. The preferential expression of inducible adhesion molecules in microvessels and mononuclear inflammatory cells suggests that angiogenesis contributes to the persistence of the inflammatory process in TAO.

Transcutaneous PO2 response to transient arterial occlusion in peripheral occlusive disease.

Transcutaneous oxygen tension response to transient arterial occlusion (tcPO2T/2) was examined at the medial mid-calf and dorsum of the foot in 20 patients with thromboangiitis obliterans or arteriosclerosis obliterans, and in normal subjects in order to accurately assess the rate of skin oxygenation. Though the mean value of transcutaneous oxygen tension (tcPO2) correlated with the mean value of tcPO2T/2 before and after arterial reconstruction, a discrepancy between the tcPO2 and tcPO2T/2 values was demonstrated in some cases. The tcPO2T/2 values manifested the severity of the peripheral skin oxygenation more clearly than tcPO2 values because changes in the tcPO2T/2 value paralleled improvement in symptoms after arterial reconstruction. It was concluded that the tcPO2T/2 value was useful in grading the severity of disease, in detecting the site of the arterial occlusion and in evaluating changes in symptoms before and after arterial reconstruction.

[Changes in oxygen free radical and prostacyclin in thromboangiitis obliterans and its relationship with syndrome differentiation].

Malondialdehyde (MDA) and 6-keto-PGF1 alpha levels in plasma and erythrocyte superoxide dismutase activities (Ery-SODA) were observed in 56 cases of thromboangiitis obliterans (TAO). The results showed that: (1) Ery-SODA and 6-keto-PGF1 alpha levels lowered and MDA raised significantly in TAO (P < 0.01), compared with that in control. (2) 6-keto-PGF1 alpha levels were markedly related with Ery-SODA and MDA in TAO (P < 0.01). (3) Ery-SODA and 6-keto-PGF1 alpha levels were lower and MDA higher in III phase of TAO than that in II phase. (4) Ery-SODA and 6-keto-PGF1 alpha levels markedly declined and MDA contents elevated significantly in Dampness-Heat (DH) and Heat-Toxin (HT) group, compared with that in Yin-Cold (YC) group and Blood-Stasis (BS) group, respectively (P < 0.05, P < 0.01); all above substances between YC and BS group or between DH and HT group had no significant differences (P > 0.05). (5) Ery-SODA and 6-keto-PGF1 alpha levels were lower and MDA higher in Heat Syndrome than that in Cold Syndrome (P < 0.01). It suggested that oxygen free radical and lipid peroxide response that might participate in vascular endothelial cell injury in TAO markedly increased and the detection of these substances might provide complementary evidences for syndrome differentiation of TAO.

Immunohistochemical properties in the patients with Buerger's disease--possible role of plasminogen activator inhibitor-1 for preservation of vessel wall architecture.

BACKGROUND: The architecture of the arterial wall affected with Buerger's disease has been known to be preserved in all three layers, while the one affected by arteriosclerosis obliterans (ASO) is degenerated and destroyed. We analyzed affected arteries with immunohistochemical methods to clarify the differences between Buerger's disease and ASO. MATERIALS AND METHODS: Crural arteries obtained from 13 patients with Buerger's disease and 6 patients with ASO at our institute were studied. In addition, we examined seven specimens from six patients who were thought to be normal (without Buerger's disease or ASO) as negative control. Immunohistochemical studies were performed on paraffin-embedded tissues. The primary antibodies were urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-3 (MMP-3). Both are known to play an important role of extracellular proteolysis and to activate each other. Additionally, plasminogen activator inhibitor-1(PAI-1) was also examined. RESULTS: In Buerger's disease, PAI-1 was well expressed along the internal elastic lamina. Urokinase-type plasminogen activator and MMP-3 were slightly positive in intima and media. In ASO, a slight amount of PAI-1 was recognized on vessel walls, and both uPA and MMP-3 were strongly positive in media. In addition, in the control group, PAI-1, uPA, and MMP-3 were well expressed in media. CONCLUSION: In Buerger's disease, PAI-1 was strongly expressed around the internal elastic lamina, while both uPA and MMP-3 were slightly recognized on vessel walls. These findings could be one of the reasons the general architecture of vessel walls in Buerger's disease is preserved.

Transfection of human HGF plasmid DNA improves limb salvage in Buerger's disease patients with critical limb ischemia.

AIM: Hepatocyte growth factor is a potent angiogenic agent. This study investigated the efficacy and safety of intramuscular injection of naked plasmid DNA encoding the human hepatocyte growth factor gene in Japanese patients with Buerger's disease and critical limb ischemia. METHODS: An open-label clinical study was performed at eight hospitals in Japan from May 2004 to April 2008. Ten patients were enrolled. They had Buerger's disease with ischemic ulcers, were not candidates for revascularization, and were unresponsive to conventional drug therapy. Treatment consisted of 8 injections (total dose: 4 mg) of hepatocyte growth factor plasmid, which were administered into the calf muscles and/or distal thigh muscles of the ischemic limbs under ultrasound guidance. Administration was done twice at an interval of 4 weeks. If there was no improvement after 2 doses, a 3rd dose could be administered. The response to treatment was evaluated from the reduction of ischemic ulcer size. RESULTS: The size of ischemic ulcers showed a decrease in 6/9 (66.7%) patients and the ulcers healed completely in 5/9 (55.6%) patients after gene therapy. Major amputation was not required. There were no deaths and no major safety concerns. CONCLUSION: Hepatocyte growth factor gene therapy is safe and effective for critical limb ischemia in patients with Buerger's disease.