Novel proteins that regulate cell extension formation in fibroblasts.

Cell extensions are critical structures that enable matrix remodeling in wound healing and cancer invasion but the regulation of their formation is not well-defined. We searched for new proteins that mediated cell extension formation over collagen by tandem mass tagged mass spectrometry analysis of purified extensions in 3T3 fibroblasts. Unexpectedly, importin-5, ENH isoform 1b (PDLIM5) and 26 S protease regulatory subunit 6B (PSMC4) were more abundant (> 10-fold) in membrane-penetrating cell extensions than cell bodies, which was confirmed by immunostaining and immunoblotting and also observed in human gingival fibroblasts. After siRNA knockdown of these proteins and plating cells on grid-supported floating collagen gels for 6 h, formation of cell extensions and collagen remodeling were examined. Knockdown of importin-5 reduced collagen compaction (1.9-fold), pericellular collagen degradation (~ 1.8-fold) and number of cell extensions (~ 69%). Knockdown of PSMC4 reduced collagen compaction (~ 1.5-fold), pericellular collagen degradation (~ 1.7-fold) and number of cell extensions (~ 42%). Knockdown of PDLIM5 reduced collagen compaction (~ 1.6-fold) and number of cell extensions (~ 21%). Inhibition of the TGF-ß RI kinase, Smad3 or ROCK-II signaling pathways reduced the abundance of PDLIM5 in cell extensions but PSMC4 and importin-5 were reduced only by Smad3 or ROCK-II inhibitors. We conclude that these novel proteins are required for cell extension formation and their recruitment into extensions involves the Smad3 and ROCK signaling pathways.

Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs. MATERIAL AND METHODS: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription-polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), which is an AhR antagonist. RESULTS: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs. CONCLUSIONS: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking.

Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17). MATERIAL AND METHODS: Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining. RESULTS: Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 . CONCLUSION: Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.

[Successful treatment of ruptured descending thoracic aortic aneurysm associated with right tension hemothorax during operation].

A 59-year-old man was admitted to the hospital, suspected of the rupture of descending thoracic aortic aneurysm due to sudden back pain. Enhanced computed tomography revealed a ruptured descending thoracic aortic aneurysm with huge hematoma and abdominal aortic aneurysm. Based on the above diagnosis, we performed urgent operation through left thoracotomy under unilateral lung ventilation. While dissecting the aneurysm, sudden hemodynamics deterioration occurred. Although cardiopulmonary bypass was introduced immediately through femoral artery and femoral vein cannulations, hemodynamics was not improved and unilateral lung ventilation got more unmanageable. We diagnosed right tension hemothorax due to the extension of aneurysm rupture into the right thoracic cavity. After placing a cannula at the distal arch for a central perfusion, we clamped the descending thoracic aorta at both the proximal and distal sites of the aneurysm. Thereafter we opened the aneurysm and drained the right thorax through the aneursym's tear. The aneurysm was replaced with a prosthetic woven-Dacron vascular graft. The patient's postoperative condition had been stable with no significant unfavorable event. The abdominal aortic aneurysm was replaced with a bifurcated graft on 51st postoperative day. He was discharged in good condition on the 69th postoperative day.

[Successful surgical treatment of acute pulmonary thromboembolism caused by giant intra-pervic tumor; report of a case].

A 40-year-old woman was admitted to our hospital because of operation for giant intra-pervic tumor. She had a history of deep vein thrombosis. Suddenly she complained severe dyspnea while having a bath, and subsequently cardiogenic shock occurred. Immediately we set up percutaneous cardiopulmonary support (PCPS) for acute pulmonary thromboembolism. Despite thrombolytic therapy including the administration of urokinase and rt-PA, hemodinamics were not improved. Emergent pulmonary embolectomy was performed on-pump beating. Postoperative course was uneventful. The intra-pervic tumor was removed on the 24th postoperative day. She was discharged in good condition on the 38th postoperative day. Quick diagnosis and appropriate therapy are essential in patient with acute massive pulmonary thromboembolism.

[Use of a balloon occlusion catheter for descending aortic aneurysm after total arch replacement using the elephant trunk technique].

A 74-year-old man who had previously undergone prosthetic graft replacement of the total aortic arch using the elephant trunk technique and of the abdominal aorta was admitted to our hospital for surgical treatment of descending aortic aneurysm. Computed tomography (CT) on admission revealed descending aortic aneurysm of 6.5 cm in diameter, and the previously placed prosthetic graft was detected in the aneurysm. Surgery for the descending aorta was performed under femoro-femoral partial bypass. During the operation, a balloon occlusion catheter introduced through the right brachial artery into the 'elephant trunk' graft was inflated before the aneurysm was opened, then the previously placed prosthetic graft was cross-clamped and the descending aorta was replaced with a new prosthetic graft with usual fashion. The postoperative course was uneventful.

[Acute pulmonary embolism after cesarean section; report of a case].

We present a case of acute pulmonary embolism (APE) after cesarean section. A cesarean section was performed on a 27-year old woman with normal course. However, one day after operation, she suddenly developed syncope and dyspnea. Soon after the symptom, she developed hypotension 60 mmHg. As a result of various examinations, her illness was diagnosed as APE with right ventricular dysfunction after cesarean section. She was consulted to our hospital for treatment. Soon after her arrival, we treated her for both APE and cardiogenic shock. The combined with antithrombotic therapy using heparin sodium, was successfully treated the patient from cardiogenic shock due to APE with right ventricular dysfunction after cesarean section.

Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein.

Chymase may play an important role in vascular proliferation, as shown by in-vitro experiments, but the role of chymase in vivo has been unclear. In this study, we investigated the effect of a novel chymase inhibitor, NK3201, on this proliferation in dog grafted veins. NK3201 inhibited human and dog chymases, but not rabbit ACE. NK3201 suppressed the Ang I-induced vascular contraction in isolated dog arteries in the presence of an ACE inhibitor, and the IC50 value of chymostatin and NK3201 in dog artery was 320 nM. In dog, the concentration of NK3201 in blood was about 10 microM at 24 h after oral administration of the drug (5 mg/kg). In the group treated with NK3201, each dog was administered orally 5 mg/kg per day from 5 days before to the day before the removal of the grafted veins. Each dog underwent right common carotid artery bypass grafting with the ipsilaterial external jugular vein. By 28 days after grafting, a significant vascular proliferation was observed in the grafted veins and the chymase activity was also increased significantly. Treatment with chymase inhibitor significantly suppressed the proliferation of the grafted veins and the increased chymase activity. In this study, we demonstrate for the first time that oral administration of a specific chymase inhibitor, NK3201, appears useful for preventing vascular proliferation.

Significance of chymase-dependent angiotensin II-forming pathway in the development of vascular proliferation.

BACKGROUND: Vascular tissues of humans and dogs contain chymase as an angiotensin II-forming enzyme. In this study, we investigated whether chymase-dependent angiotensin II formation plays a crucial role in the development of vascular proliferation in dog grafted veins. METHODS AND RESULTS: The right external jugular vein of dogs was grafted to the ipsilateral carotid artery. As a control group, the right external jugular veins in dogs that had not received grafts were used. In the chymase inhibitor-treated group, the vein was infiltrated with 10 micromol/L Suc-Val-Pro-Phe(P)(OPh)(2) and was grafted to the carotid artery. In the placebo-treated group, ACE activity in the grafted veins was significantly lower than that in the control veins up to 7 days after the operation, whereas chymase activity was increased significantly. After 7 days, the mRNA levels of collagen I, collagen III, and fibronectin, all of which are induced by an increase of angiotensin II action, were significantly increased in the grafted veins, and the intima-media ratio of the grafted veins was also increased. In the chymase inhibitor-treated group, the chymase activity in the grafted veins 7 days after the operation was suppressed to 12.1%. The elevated mRNA levels of fibronectin, collagen I, and collagen III in the grafted veins were significantly suppressed by treatment with the chymase inhibitor, and the intima-media ratio was also decreased significantly. CONCLUSIONS: We demonstrate for the first time that chymase-dependent angiotensin II formation plays an important role in the development of vascular proliferation in the grafted veins.

Chymase-dependent angiotensin II formation in the saphenous vein versus the internal thoracic artery.

OBJECTIVES: The great saphenous vein graft is known to be less patent than the internal thoracic artery graft. Recently, we reported that chymase-dependent angiotensin II formation plays an important role in the development of intimal hyperplasia in dog grafted veins. In this study we investigated the levels of angiotensin II-forming enzymes, angiotensin-converting enzyme, and chymase in human saphenous veins and internal thoracic arteries. METHODS: The saphenous vein and internal thoracic artery specimens were obtained from coronary artery bypass grafts of patients during surgical procedures (saphenous vein, n = 16; internal thoracic artery, n = 16). Activities of angiotensin-converting enzyme and chymase were determined by using the extract from the saphenous vein or internal thoracic artery. Sections of the saphenous vein or internal thoracic artery were stained with van Gieson's elastin stain and were immunostained with anti-human chymase antibody. RESULTS: The activities of angiotensin-converting enzyme in the saphenous vein and internal thoracic artery were 0.34 +/- 0.12 and 0.32 +/- 0.17 mU/mg protein, respectively, and the difference was not significant. The chymase activity in the saphenous vein was significantly higher than that in the internal thoracic artery (saphenous vein, 10.1 +/- 0.81 mU/mg protein; internal thoracic artery, 6.21 +/- 1.86 mU/mg protein). Chymase-positive cells in the saphenous vein were located in both the media and adventitia, and those in the internal thoracic artery were located only in the adventitia. The number of chymase-positive cells in the saphenous vein was about 2.6 times that in the internal thoracic artery. CONCLUSION: The chymase activity, but not the angiotensin-converting enzyme activity, was significantly higher in the saphenous vein, suggesting that the high levels of chymase activity may be related to the poorer performance of the saphenous vein for use as a bypass conduit.

Cilostazol suppresses intimal formation in dog grafted veins with reduction of angiotensin II-forming enzymes.

Cilostazol prevents neointimal formation, but its mechanism has remained unclear. We investigated whether intimal formation in dog grafted veins is suppressed by cilostazol, and studied the effect of cilostazol on angiotensin II-forming enzymes. The external jugular vein was grafted to the carotid artery, and cilostazol (60 mg/kg/day) was administered orally. By 28 days after the surgery, the intimal cross-sectional area of the grafted vein was reduced to 16.7% by treatment of cilostazol, and the activities of angiotensin II-forming enzymes were suppressed significantly. The inhibitory effect of cilostazol in intimal formation may be dependent on inhibition of angiotensin II-forming enzymes.

Angiotensin II receptor antagonist, L-158,809, prevents intimal hyperplasia in dog grafted veins.

We investigated the levels of the angiotensin II-forming enzymes, chymase and angiotensin converting enzyme (ACE), in dog grafted veins, and studied the effect of an angiotensin II type 1 receptor antagonist, L-158,809, on vascular proliferation in the grafted veins. The right external jugular vein was grafted to the ipsilaterial carotid artery. In the group treated with L-158,809, the drug (10 mg/kg per day, p.o.) were administered orally from 7 days before the operation to 28 days after it, while the others were administrated placebo. In the placebo-treated group, the chymase activity in the grafted veins was increased about 10-fold and the ACE activity was doubled. The areas of intima and media were significantly increased in the grafted veins in the placebo-treated group. L-158,809 significantly reduced the intimal area of the grafted veins. An angiotensin II receptor antagonist, L-158,809, prevented the vascular proliferation in the grafted veins, and the development of the proliferation may depend on activation of local angiotensin II formation.

Inhibition of chymase reduces vascular proliferation in dog grafted veins.

We investigated the effect of a chymase inhibitor Suc-Val-Pro-Phe(P)(OPh)(2) on the proliferation of the grafted vein in dog. By 28 days after the operation, the mean intimal area of the grafted vein in the placebo group was 3.24+/-0.32 mm(2). The intimal area of the grafted vein in the chymase inhibitor-treated group was reduced to 63.9%. In the placebo group, the activities of chymase and angiotensin-converting enzyme in grafted vein were significantly increased 15- and 2-fold, respectively. In the chymase inhibitor-treated group, chymase activity in the grafted veins was decreased significantly. These findings suggest that inhibition of chymase appears useful for preventing vascular proliferation.

[A case report of infective endocarditis caused by MRSA and characterized by pedicled vegetation on the posterior wall of left atrium].

We report here a case of active infective endocarditis caused by Methicilin-Resistant Staphylococcus aureus (MRSA). A 24-year-old woman was admitted to the Osaka Medical Collage Hospital with continuous fever. After admission, MRSA was detected by blood culture and chemotherapy with Vancomycin was started. However, after 1 week, her condition had not improved. Moreover, a pedicled vegetation on the posterior wall of the left atrium and mitral regurgitation due to prolapse of the anterior leaflet were revealed by transesophageal echocardiography. The vegetation grew to about 2 cm in diameter and prolapsed into the left ventricle during diastole. We performed an early operation although the infection was still active due to its rapid growth and the risk of embolism. There was a large pedicled vegetation on the posterior wall of the left atrium as shown by preoperative echocardiography, but the mitral valve appeared to be intact. Therefore, the vegetation was completely removed and the mitral annulus was plicated by Kay's method to treat the associated mitral regurgitation. Postoperatively, we administered VCM 2 g/day for 24 days. The course was uneventful. The patient was discharged from the hospital on the 31st postoperative day.

[A case report of coronary artery bypass grafting in a patient with ankylosing spondylitis].

We reported a case of ankylosing spondylitis who successfully underwent coronary artery bypass grafting (CABG) for unstable angina pectoris. A 67-year-old man was admitted with symptom of anginal pain. Selective coronary angiography revealed coronary artery stenoses; 90% in seg 6, 90% in seg 11, proximal 75%, distal 90% in seg 3, 99% in 4 PD and 99% with delay in 4 AV. The left internal thoracic artery was anastomosed to seg 7 and saphenous vein (SVG) to PL-2, PL-1 sequentially, and another SVG to 4 PD. His postoperative course was uneventful. Cardiac lesions accompanied by ankylosing spondylitis are rare in Japan. Perioperative problems of these lesions therefore, are discussed.

[Rupture of thoracic aorta due to blunt trauma: a report of three cases].

Three cases of the thoracic aortic rupture due to blunt trauma were successfully treated. All of the cases were young male motorcyclists. The chest X-ray on admission showed either a widened mediastinum or an apical extrapleural cap sign. Ruptures were confirmed by an enhanced CT clearly showing the presence of a psuedoaneurysm and a hematoma around the isthmus of the aorta. One of them was operated upon urgently and others in whom heparin could not be used because of associated injuries were operated upon electively two and three months after the traffic accident, respectively. In two of them, aneurysms were removed and replaced with dacron grafts and in the remaining one, the aneurysm was resected and repaired directly under the femoro-femoral bypass. Post-operative courses were uneventful. They discharged and are doing well after the treatment for associated injuries. In view of the high early mortality of aortic rupture, an early diagnosis and treatment is important. But in the case who has stable hemodynamics and contraindication for heparinization, a delayed operation may be recommended.