Inhibitory effect of low­intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF­1α in a carrageenan­induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

Influence of new coronavirus pandemic on behavior and awareness of young nurses and nursing students in Japan.

BACKGROUND: Although mental health disorders of health care workers in the coronavirus disease 2019 (COVID-19) pandemic have been focused, little is known about the psychological impact on nurses and the influence on their behavior and awareness, such as professionalism and views on life and death, in Japan where there are fewer cases of infection and deaths than in other countries. Moreover, the influence of the pandemic on nursing students is still unclear. METHODS: An online questionnaire survey was conducted among nurses and nursing students. Feelings during the state of emergency (at the peak of the pandemic) in Japan, changes in behavior and awareness after the rise of COVID-19, and the associated factors influencing these changes were analyzed, comparing nurses with nursing students. RESULTS: Significantly increased scores of anxiety/fear (p < .005) and voluntary restraint (p < .005) and significantly decreased score of motivation (p < .005) were observed during the state of emergency in both nurses and students. Scores of experience of discrimination (p < .005) and consideration of premature retirement (p < .01) were significantly increased in nurses. Moreover, preventive behavior (p < .005), lifestyle (p < .005), anxiety about nursing (p < .005) and views on life and death (p < .005) significantly changed after the rise of COVID-19 in both nurses and students. Only nurses reported significant damage to their professionalism (p < .01). Anxiety/fear and/or voluntary restraint and/or decreased motivation during the state of emergency were major factors associated with these changes. Also, the type of hospital, experience of care of infected patients and sex affected some of the changes. Voluntary restraint (p = .008), increased preventive behavior (p = .021) and decreased motivation (p = .005) were more marked in nurses than in students, while change in views on life and death was greater in students than in nurses (p = .002). CONCLUSION: The COVID-19 pandemic has had a psychological impact on nurses and nursing students, associated with changes in behavior and awareness even in Japan. Of note, the COVID-19 pandemic has affected nurses' professionalism and views on life and death. This study demonstrates the importance of having a coping strategy for anxiety and damaged professionalism in nurses, and education on life and death in nursing students.

Moxibustion Promotes Formation of Granulation in Wound Healing Process through Induction of Transforming Growth Factor-ß in Rats.

OBJECTIVE: To examine the effect of moxibustion on the wound healing process and its mechanism using a rat wound model. METHODS: Sixty male Sprague-Dawley rats were randomly divided into a sham-treated group (n=30, wound surgery only) and a moxibustion group (n=30, wound treated with moxibustion). Circular full-thickness skin wounds were produced in rats. Moxibustion was applied to the edge of wound and was continued on alternating days till 14 days after surgery, followed by measurement of wound size. Expression of collagens, prolyl-4-hydroxylase (P4H) and transforming growth factor-ß (TGF-ß) were evaluated by histochemical study and real-time polymerase chain reaction. RESULTS: The size of the wound lesion was significantly reduced in rats treated with moxibustion as compared to that in sham-treated rats at 4-10 days after wounding (P<0.01). Moxibustion stimulated mRNA expression of collagens at 4 days (P<0.01), but not at 7 days, accompanied by enhanced proliferation of P4H-positive fibroblasts. Of importance, expression of TGF-ß in tissue from the wound lesion treated with moxibustion was significantly increased as compared to that in sham-treated rats at 4 days (P<0.01 or P<0.05), but not at 7 days. CONCLUSIONS: The treatment with moxibustion promoted the wound healing process in the early phase through proliferation of fibroblasts and rapid formation of granulation, possibly mediated by induction of TGF-ß which is a key molecule in the physiological process of wound healing. Moxibustion can be expected to be effective as complementary treatment for intractable ulcers.

Beneficial effect of laughter therapy on physiological and psychological function in elders.

AIM: In the present study we investigated the effect of laughter therapy on physiological and psychological function in older people. DESIGN: An open-label trial. METHODS: Seventeen older people who regularly attended an elderly day care centre were recruited. Stand-up comedy as laughter therapy was performed once a week for 4 weeks. Parameters of physiological and psychological function were evaluated before and after laughter therapy. RESULTS: Laughter therapy intervention resulted in a significant reduction in systolic blood pressure and heart rate, accompanied by a significant increase in plasma concentration of serotonin and a significant decrease in salivary concentration of chromogranin A. Questionnaire surveys of SF-8, GDS-15, and Vitality Index demonstrated alleviation of depression and improvement of sociability and activity in older people. Laughter therapy could be expected to become a practical treatment to improve quality of life of older people in an elderly day care centre.

Therapeutic effect of intra-articular injection of ribbon-type decoy oligonucleotides for hypoxia inducible factor-1 on joint contracture in an immobilized knee animal model.

BACKGROUND: Limited range of motion (ROM) as a result of joint contracture in treatment associated with joint immobilization or motor paralysis is a critical issue. However, its molecular mechanism has not been fully clarified and a therapeutic approach is not yet established. METHODS: In the present study, we investigated its molecular mechanism, focusing on the role of a transcription factor, hypoxia inducible factor-1 (HIF-1), which regulates the expression of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), and evaluated the possibility of molecular therapy to inhibit HIF-1 activation by ribbon-type decoy oligonucleotides (ODNs) for HIF-1 using immobilized knee animal models. RESULTS: In a mouse model, ROM of the immobilized knee significantly decreased in a time-dependent manner, accompanied by synovial hypertrophy. Immunohistochemical studies suggested that CTGF and VEGF are implicated in synovial hypertrophy with fibrosis. CTGF and VEGF were up-regulated at both the mRNA and protein levels at 1 and 2 weeks after immobilization, subsequent to up-regulation of HIF-1 mRNA and transcriptional activation of HIF-1. Of importance, intra-articular transfection of decoy ODNs for HIF-1 in a rat model successfully inhibited transcriptional activation of HIF-1, followed by suppression of expression of CTGF and VEGF, resulting in attenuation of restricted ROM, whereas transfection of scrambled decoy ODNs did not. CONCLUSIONS: The present study demonstrates the important role of HIF-1 in the initial progression of immobilization-induced joint contracture, and indicates the possibility of molecular treatment to prevent the progression of joint contracture prior to intervention with physical therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Wound healing potential of lavender oil by acceleration of granulation and wound contraction through induction of TGF-ß in a rat model.

BACKGROUND: Although previous studies have suggested that lavender oil promote wound healing, no study has examined the molecular mechanisms of its effect. In this study, we investigated the effect of lavender oil on various steps of wound healing and its molecular mechanism, focusing on transforming growth factor-ß (TGF-ß). METHODS: Circular full-thickness skin wounds were produced on rats. Control solution or lavender oil was topically applied to the wounds on alternating days for 14 days. RESULTS: The area of wounds topically treated with lavender oil was significantly decreased as compared to that of wounds of control rats at 4, 6, 8, and 10 days after wounding. Topical application of lavender oil induced expression of type I and III collagen at 4 days after wounding, accompanied by an increased number of fibroblasts, which synthesize collagen. Induced expression of type III collagen by topical application of lavender oil was reduced to control level at 7 days after wounding although increased expression of type I collagen still continued even at 7 days, suggesting rapid collagen replacement from type III to type I in wounds treated with lavender oil. Importantly, expression of TGF-ß in wounds treated with lavender oil was significantly increased as compared to control. Moreover, an increased number of myofibroblasts was observed in wounds treated with lavender oil at 4 days after wounding, suggesting promotion of differentiation of fibroblasts through induction of TGF-ß, which is needed for wound contraction. CONCLUSION: This study demonstrated that topical application of lavender oil promoted collagen synthesis and differentiation of fibroblasts, accompanied by up-regulation of TGF-ß. These data suggest that lavender oil has the potential to promote wound healing in the early phase by acceleration of formation of granulation tissue, tissue remodeling by collagen replacement and wound contraction through up-regulation of TGF-ß. The beneficial effect of lavender oil on wound healing may raise the possibility of new approaches as complementary treatment besides conventional therapy.

Continuous infusion of angiotensin II modulates hypertrophic differentiation and apoptosis of chondrocytes in cartilage formation in a fracture model mouse.

Although components of the renin-angiotensin system (RAS) are reported to be expressed in cultured chondrocytes and cartilage, little is known about the precise function of Angiotensin II (Ang II) in chondrocytes. In this study, we employed a rib fracture model mouse to investigate the effect of Ang II on chondrocytes. Ang II type 1 receptor (AT1R) was expressed in chondrocytes in the growth plate of mouse tibia. Continuous infusion of Ang II to rib-fractured mice resulted in a significant increase in the volume of cartilage, suggesting Ang II-induced hypertrophic differentiation of chondrocytes. It was also confirmed by a significant increase in the mRNA expression of Sox9 and runt-related transcription factor 2 (Runx2), which are genes related to chondrocyte differentiation, and type X collagen, matrix metalloproteinase (MMP)-13 and Indian hedgehog (Ihh), which are hypertrophic chondrocyte-specific molecular markers. Chondrocyte hypertrophy with upregulation of these genes was attenuated by administration of olmesartan, an AT1R blocker, but not by hydralazine. Moreover, Ang II infusion significantly suppressed apoptosis of chondrocytes, accompanied by significant induction of mRNA expression of bcl-2 and bcl-xL. Olmesartan, but not hydralazine, significantly attenuated the reduction of apoptotic cells and the increase in anti-apoptotic genes induced by Ang II infusion. Overall, the present study demonstrated that Ang II promoted hypertrophic differentiation of chondrocytes and reduced apoptosis of hypertrophic chondrocytes independently of high blood pressure. The present data indicate the role of Ang II in cartilage, and might provide a new concept for treatment of cartilage diseases.

Effect of angiotensin II receptor blocker, olmesartan, on turnover of bone metabolism in bedridden elderly hypertensive women with disuse syndrome.

AIMS: Although recent studies suggest that several antihypertensive drugs could reduce the risk of bone fracture, it is still unclear how these drugs act on bone remodeling, especially in elderly women with severe osteoporosis with disuse syndrome. In the present study, we investigated the effects of a calcium channel blocker (CCB) and an angiotensin II receptor blocker (ARB) on bone metabolism in elderly bedridden women with hypertension and disuse syndrome. METHODS: Elderly bedridden women (aged >75 years) receiving antihypertensive therapy treated with CCB were recruited in the present study. The participants were divided into two groups--CCB group and ARB group--and followed up to 12 months. RESULTS: Markers of bone resorption were markedly increased, suggesting accelerated bone resorption in the participants of the present study. In the follow-up period, the patients treated with a CCB showed a significant decrease in bone mineral density in a time-dependent manner, accompanied by a significant increase in bone resorption markers, whereas treatment with olmesartan inhibited bone loss, associated with attenuation of increased bone resorption markers. Bone mineral density of femoral neck in the CCB group was significantly lower than that in the ARB group at 6 months. CONCLUSION: The present study showed inhibitory effects of an ARB on bone resorption in hypertensive patients with accelerated bone resorption, such as elderly bedridden women, and indicated an important role of the renin-angiotensin system in bone metabolism. In elderly hypertensive patients, ARB might be expected to have additional beneficial potential to maintain bone health in bedridden patients.

Therapeutic effect of ribbon-type nuclear factor-κB decoy oligonucleotides in a rat model of inflammatory bowel disease.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) involves local expression of inflammatory cytokines, some of which are coordinated by nuclear factor-κB (NF-κB). Several reports documented the therapeutic potential of double-stranded phosphorothioated decoy oligonucleotides (S-ODNs) targeting NF-κB in IBD models. However, S-ODNs are easily degraded by endonucleases. In this study, we employed newly developed nonchemically modified ribbon-type NF-κB decoy ODNs (R-ODNs) with loop ends that increase the stability, and investigated their therapeutic effect in rats with dextran sulfate sodium (DSS)-induced colitis. METHODOLOGY/PRINCIPAL FINDINGS: We administered R-ODN, S-ODN, or scrambled ODN (Scr-ODN) to rats with DSS-induced IBD using ultrasound with contrast microbubbles to enhance the transfection efficiency of ODN. Until day 10 after DSS treatment, the rats showed a decrease in body weight and survival rate and an increase in the disease activity index (DAI). In rats treated with S-ODN or R-ODN, the survival rate, colon length, and DAI were significantly improved. In addition, DSS-induced expression of tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß was significantly decreased. Of importance, treatment with R-ODN was more effective to improve disease conditions as compared to S-ODN. CONCLUSIONS: These data suggest that intracolonic administration of R-ODN may be effective to treat DSS-induced colitis.

Long-term follow-up evaluation of results from clinical trial using hepatocyte growth factor gene to treat severe peripheral arterial disease.

OBJECTIVE: As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. METHODS AND RESULTS: Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. CONCLUSIONS: Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.

Systemic administration of ribbon-type decoy oligodeoxynucleotide against nuclear factor κB and ets prevents abdominal aortic aneurysm in rat model.

Currently, there is no effective clinical treatment to prevent abdominal aortic aneurysm (AAA). To develop a novel therapeutic approach, we modified decoy oligodeoxynucleotide (ODN) against nuclear factor κB (NFκB) and ets, to a ribbon-shaped circular structure without chemical modification, to increase its resistance to endonuclease for systemic administration. Intraperitoneal administration of ribbon-type decoy ODNs (R-ODNs) was performed in an elastase-induced rat AAA model. Fluorescent isothiocyanate (FITC)-labeled R-ODNs could be detected in macrophages migrating into the aneurysm wall, and NFκB and ets activity were simultaneously inhibited by chimeric R-ODN. Treatment with chimeric R-ODN significantly inhibited aortic dilatation, whereas conventional phosphorothioate decoy ODN failed to prevent aneurysm formation. Significant preservation of elastic fibers was observed with chimeric R-ODN, accompanied by a reduction of secretion of several proteases from macrophages. Activation of matrix metalloproteinase (MMP)-9 and MMP-12, but not MMP-2, was suppressed in the aneurysm wall by chimeric R-ODN, whereas recruitment of macrophages was not inhibited. Treatment with chimeric R-ODN also inhibited the secretion of cathepsin B and K from macrophages. Overall, the present study demonstrated that systemic administration of chimeric R-ODNs prevented aneurysm formation in a rat model. Further modification of the decoy strategy would provide a means of less invasive molecular therapy for human AAA.

Phase I/IIa clinical trial of therapeutic angiogenesis using hepatocyte growth factor gene transfer to treat critical limb ischemia.

OBJECTIVE: To evaluate the safety and feasibility of intramuscular gene transfer using naked plasmid DNA-encoding hepatocyte growth factor (HGF) and to assess its potential therapeutic benefit in patients with critical limb ischemia. METHODS AND RESULTS: Gene transfer was performed in 22 patients with critical limb ischemia by intramuscular injection of HGF plasmid, either 2 or 4 mg, 2 times. Safety, ankle-brachial index, resting pain on a 10-cm visual analog scale, wound healing, and walking distance were evaluated before treatment and at 2 months after injection. No serious adverse event caused by gene transfer was detected over a follow-up of 6 months. Of particular importance, no peripheral edema, in contrast to that seen after treatment with vascular endothelial growth factor, was observed. In addition, the systemic HGF protein level did not increase during the study. At 2 months after gene transfer, the mean ± SD ankle-brachial index increased from 0.46 ± 0.08 to 0.59 ± 0.13 (P<0.001), the mean ± SD size of the largest ischemic ulcers decreased from 3.08 ± 1.54 to 2.32 ± 1.88 cm (P=0.007), and the mean ± SD visual analog scale score decreased from 5.92 ± 1.67 to 3.04 ± 2.50 cm (P=0.001). An increase in ankle-brachial index by >0.1, a reduction in ulcer size by >25%, and a reduction in visual analog scale score by >2 cm at 2 months after gene transfer were observed in 11 (64.7%) of 17 limbs, 18 (72%) of 25 ulcers, and 8 (61.5%) of 13 limbs, respectively. CONCLUSIONS: Intramuscular injection of naked HGF plasmid is safe and feasible and can achieve successful improvement of ischemic limbs as sole therapy.

Inhibition of development of experimental aortic abdominal aneurysm in rat model by atorvastatin through inhibition of macrophage migration.

Recently, atherosclerosis has been considered to be the result of inflammation. Interestingly, hydroxymethylglutaryl-coenzyme (HMG-Co) A inhibitors (statins), which are clinically used as lipid-lowering agents, have been reported to have various anti-inflammatory effects. As abdominal aortic aneurysm (AAA) is a common degenerative condition associated with atherosclerosis, this study was designed to investigate the inhibitory effect of a statin, atorvastatin, on aneurysm formation apart from its lipid-lowering effect. We employed an elastase-induced rat AAA model, as statins do not lower cholesterol in rats. Mean aneurysm diameter was significantly smaller in the atorvastatin treatment group as compared to control at 4 weeks after surgery (P<0.05). Interestingly, atorvastatin inhibited the expression of ICAM and MCP-1, followed by the suppression of macrophage recruitment into the aortic wall at 1 week after operation. A significant reduction in MMP-12, but not MMP-2, -3 and -9, expression was also observed by treatment with atorvastatin at 1 week after surgery. In addition, synthesis of collagen and elastin in the vascular wall were significantly increased by atorvastatin. Here, the present study demonstrated a direct effect of atorvastatin to inhibit the progression of aortic aneurysm, independent of its lipid-lowering effect. This study suggests new therapeutic aspects of statins to inhibit the progression of aneurysms.

Inhibition of experimental abdominal aortic aneurysm in a rat model by the angiotensin receptor blocker valsartan.

Angiotensin (Ang) II exerts direct effects on the arterial wall to influence atherosclerosis and aneurysm development with the induction of vascular inflammation. Therefore, we examined the hypothesis that the inhibition of Ang II would decrease the expansion of abdominal aortic aneurysm (AAA) in a rat model. We used the Ang II receptor blocker (ARB) valsartan to inhibit the effect of Ang II. Additionally, we employed a dosage of valsartan (1 mg/ kg/day) that does not affect blood pressure, to avoid the effect of blood pressure lowering. Notably, progression of elastase-induced AAA was inhibited in rats treated with valsartan (P< or =0.05). To clarify the mechanism, we focused on matrix metalloproteases (MMPs) and inflammatory related factors. Western blot analysis demonstrated that the expression of MMPs was significantly decreased in an AAA model treated with continuous ARB infusion compared to an AAA model treated with vehicle (P< or =0.05), through suppression of nuclear factor kappaB activation (P< or =0.05). Consistently, valsartan significantly inhibited infiltration of macrophages into the aortic wall, accompanied by a reduction of protein expression of intercellular adhesion molecule-1. Importantly, the inhibitory effect of valsartan on MMP-2 and MMP-9 expression was also confirmed using isolated peritoneal macrophages from a rat AAA model. Moreover, treatment with valsartan protected against the destruction of elastic fibers. Overall, the present study demonstrated that treatment with valsartan, significantly prevented the progression of experimental AAA in a rat model. These data suggest that blockade of Ang II has an inhibitory effect on the development of AAA, independent of its antihypertensive effect.

Inhibition of anastomotic intimal hyperplasia using a chimeric decoy strategy against NFkappaB and E2F in a rabbit model.

AIMS: Neointimal formation remains a major limitation after arterial reconstruction. To overcome this problem, we focused on two important transcription factors, nuclear factor-kappaB (NFkappaB) and E2F. The purpose of this study was to determine the effects of simultaneous inhibition of these transcription factors on the formation of neointimal hyperplasia. METHODS AND RESULTS: We employed chimeric decoy oligodeoxynucleotides (ODN) to inhibit both NFkappaB and E2F simultaneously, and examined the effects of chimeric decoy ODN on the proliferation and migration of cultured vascular cells and on the formation of neointimal hyperplasia using prosthetic graft placement in a rabbit hypercholesterolemia model. Our in vitro study demonstrated that transfection of chimeric decoy ODN inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of vascular smooth muscle cells, whereas endothelial cell proliferation was not inhibited. In an in vivo study, treatment with chimeric decoy ODN significantly inhibited proximal and distal anastomotic intimal hyperplasia, and accelerated re-endothelialization. alpha-Smooth muscle actin (alpha-SMA)-positive cell proliferation was inhibited at the anastomotic sites. Expression of PDGF-BB and PDGF receptor-beta was also suppressed by chimeric decoy ODN, resulting in a reduction of alpha-SMA-positive cell accumulation. In addition, chimeric decoy ODN treatment inhibited macrophage accumulation, which was accompanied by a reduction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 gene expression. CONCLUSION: The present study demonstrates the feasibility of chimeric decoy ODN treatment for preventing neointimal formation. This strategy might be useful to improve the clinical outcome after arterial reconstruction.

Long-term follow up of initial clinical cases with NF-kappaB decoy oligodeoxynucleotide transfection at the site of coronary stenting.

BACKGROUND: An essential transcription factor for inflammation, nuclear factor-kappa B (NF-kappaB), plays a pivotal role for restenosis after percutaneous coronary intervention (PCI). To evaluate the safety and effectiveness of NF-kappaB decoy oligodeoxynucleotides (ODN) to prevent restenosis, we initiated an open-label phase I/IIa clinical trial. METHODS: Seventeen patients who were suffering from angina with organic coronary stenosis were treated with NF-kappaB decoy ODN after PCI using bare metal stents. RESULTS: Although the average coronary stenosis was 90.8 +/- 7.0% before the stent implantation, the stenosis improved to 1.4 +/- 5.9% after the intervention. Serum MCP-1 levels were significantly suppressed in NF-kappaB decoy ODN treated patients compared to those in non-treated patients on day 3 after the PCI. Ticlopidine treatment was employed, since clopidogrel was not launched in Japan. Six months after the PCI and decoy ODN transfection, significant restenosis was found in only 1 of the 17 patients, and the average restenosis rate was 39.6 +/- 22.3%. No in-stent thrombosis was found and no significant systemic adverse effect occurred in any of the patients in this observation period. CONCLUSIONS: These results suggest the clinical usefulness and safety of the NF-kappaB decoy ODN transfer after PCI, although further placebo control trials are necessary.

Regression of abdominal aortic aneurysms by simultaneous inhibition of nuclear factor kappaB and ets in a rabbit model.

Because current therapy to treat abdominal aortic aneurysm (AAA), and particularly to manage small AAA, is limited to elective surgical repair, we explored less invasive molecular therapy by simultaneous inhibition of the transcription factors nuclear factor (NF)kappaB and ets using a decoy strategy. Both NFkappaB and ets were shown to be markedly activated in human AAA. In addition, NFkappaB- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NFkappaB and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NFkappaB and ets binding sites to treat AAA. Inhibitory effects of chimeric decoy ODNs on matrix metalloproteinase-1 and -9 expression were confirmed by ex vivo experiments using a human aorta organ culture. To examine the regressive effect in a rabbit already-formed AAA model, transfection by wrapping a delivery sheet containing chimeric decoy ODNs around the aneurysm was performed 1 week after incubation with elastase. Importantly, treatment with chimeric decoy ODNs significantly decreased the size of AAA. Interestingly, significant preservation of elastic fibers was observed with chimeric decoy ODN treatment, accompanied by a reduction of matrix metalloproteinase-2 and -9 and induction of macrophage apoptosis. Regression of AAA was also associated with an increase in elastin and collagen type I and III synthesis in the aneurysm wall. Minimally invasive molecular therapy targeted to the inhibition of NFkappaB and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.

Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase.

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor kappaB and Ets.

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor kappaB (NFkappaB) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3 , -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NFkappaB and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NFkappaB and ets. Inhibition of NFkappaB and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.