Overexpression of urokinase by plaque macrophages causes histological features of plaque rupture and increases vascular matrix metalloproteinase activity in aged apolipoprotein e-null mice.

BACKGROUND: The mechanisms of atherosclerotic plaque rupture are poorly understood. Urokinase-type plasminogen activator (uPA) is expressed at elevated levels by macrophages in advanced human plaques. Patients with evidence of increased plasminogen activation have an elevated risk of major cardiovascular events. We used atherosclerotic mice to test the hypothesis that increased macrophage uPA expression in advanced plaques would cause histological features similar to those in ruptured human plaques. METHODS AND RESULTS: Bone marrow from transgenic mice with increased macrophage uPA expression or nontransgenic controls (all apolipoprotein E-null [Apoe(-/-)]) was transplanted into 35-week-old Apoe(-/-) recipients, and innominate lesions and aortas were examined 8 to 13 weeks later. Donor macrophages accumulated in innominate lesions adjacent to plaque caps and in aortas, increasing uPA expression at both sites. Recipients of uPA-overexpressing macrophages had an increased prevalence of intraplaque hemorrhage (61% versus 13%; P=0.002) as well as increased lesion fibrin staining and fibrous cap disruption (P=0.06 for both). Transplantation of uPA-overexpressing macrophages increased aortic matrix metalloproteinase activity (40%; P=0.02). This increase was independent of matrix metalloproteinase-9. CONCLUSIONS: In advanced plaques of Apoe(-/-) mice, macrophage uPA overexpression causes intraplaque hemorrhage and fibrous cap disruption, features associated with human plaque rupture. uPA overexpression also increases vascular matrix metalloproteinase activity. These data provide a mechanism that connects macrophage uPA expression, matrix metalloproteinase activity, and plaque rupture features in mice. The data also suggest that elevated plaque plasminogen activator expression and plasminogen activation in humans may be causally linked to plaque rupture and cardiovascular events.

Factors predicting growth of vestibular schwannoma in neurofibromatosis type 2.

We retrospectively reviewed characteristics of patients with neurofibromatosis type 2 to identify factors predicting further growth of bilateral vestibular schwannomas. Subjects comprised 27 neurofibromatosis type 2 patients with 54 vestibular schwannomas, followed for 24-204 months (mean, 86 months). This study investigated factors predictive of vestibular schwannoma growth in neurofibromatosis type 2. Features distinguishing actively growing from quiescent VS were determined for untreated course (28 vestibular schwannomas) and posttreatment course (including either resection or radiosurgery; 33 vestibular schwannomas). A general estimation equation was used to identify factors affecting tumor growth. During the untreated course, 19 vestibular schwannomas showed growth and 9 vestibular schwannomas were stable. No factors predictive of growth were shown. During the posttreatment course (23 surgical resections, ten radiosurgeries), ten treatments were followed by growth and 23 were followed by stability, with growth showing an association with onset at an early age (p = 0.007). Multivariate analysis identified no factors predictive of growth. After treatment, close follow-up is warranted for patients with onset at an early age.

Transmission of specific genotype streptomycin resistant strains of Mycobacterium tuberculosis in the Tokyo Metropolitan Area in Japan.

BACKGROUND: From 2003 through to 2004, an outbreak of tuberculosis was identified at a university campus in Yokohama City, located in the southern part of the Tokyo Metropolitan Area (TMA). All Mycobacterium tuberculosis (M. tuberculosis) strains detected with regards to this outbreak turned out to be Streptomycin resistant with matched patterns of 14 IS6110 bands of Restriction Fragment Length Polymorphism (RFLP). The M. tuberculosis bacilli, which had the matched IS6110 band patterns with resistance to Streptomycin to those of bacilli isolated in the outbreak, were also concurrently detected through either the population-based or the hospital-based DNA fingerprinting surveillance of M. tuberculosis either in Shinjuku City or in Kawasaki City respectively. The aim of the present study is to describe the spread of the specific genotype strains of M. tuberculosis in the TMA as observed in the above incident, and to identify the possible transmission routes of the strains among people living in urban settings in Japan. METHODS: We applied Variable Numbers of Tandem Repeats (VNTR) analysis to all M. tuberculosis isolates which were resistant to Streptomycin with a matched IS6110-RFLP band pattern (M-strains). They were isolated either from cases related to the tuberculosis outbreak that happened at a university, or through DNA fingerprinting surveillance of M. tuberculosis both in Shinjuku City and in Kawasaki City. For VNTR analysis, 12MIRU loci, 4ETR loci, seven loci by Supply, four loci by Murase (QUB15, Mtub24, VNTR2372, VNTR3336) were selected. RESULTS: Out of a total of 664 isolates collected during the study period, 46 isolates (6.9%) were identified as M-strains. There was a tendency that there was a higher proportion of those patients whose isolates belonged to M4-substrains, with four copies of tandem repeat at the ETR-C locus, to have visited some of the internet-cafés in the TMA than those whose isolates belonged to M5-substrains, with five copies at the ETR-C locus, although statistically not significant (38.1% vs. 10.0%, Exact p = 0.150). CONCLUSION: Although firm conclusions could not be reached through the present study, it suggested that we have to take into consideration that tuberculosis can be transmitted in congregated facilities like internet cafés where tuberculosis high-risk people and general people share common spaces.

TGF-[beta]1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein E-null mice.

OBJECTIVE: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 in hyperlipidemic mice affects atherogenesis and aortic dilation. METHODS AND RESULTS: We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. CONCLUSIONS: When overexpressed in the heart and plasma, TGF-beta1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Mechanisms of TGF-beta1-induced intimal growth: plasminogen-independent activities of plasminogen activator inhibitor-1 and heterogeneous origin of intimal cells.

Transforming growth factor (TGF)-beta(1) is a potent stimulator of intimal growth. We showed previously that TGF-beta(1) stimulates intimal growth through early upregulation of plasminogen activator inhibitor-1 (PAI-1) and, subsequently, PAI-1-dependent increases in cell migration and matrix accumulation. We also showed that PAI-1 negatively regulates TGF-beta(1) expression in the artery wall. Here we use plasminogen-deficient mice to test whether TGF-beta(1)-stimulated, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are mediated through inhibition of plasminogen activation by PAI-1. We also use lineage tracing to investigate the origin of cells in TGF-beta(1)-induced intimas. Surprisingly, both TGF-beta(1)-induced, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are independent of plasminogen. Moreover, approximately 50% of cells that migrate into the intima of TGF-beta(1)-overexpressing arteries carry a smooth muscle lineage marker, <1% carry a bone marrow lineage marker, and the remaining cells carry neither marker. Therefore, PAI-1 stimulates intimal growth and suppresses TGF-beta(1) expression through activities other than inhibition of plasminogen activation. In addition, contrary to widely held models, our results do not support a role for plasmin (or thrombospondin) in TGF-beta(1) activation in the artery wall. Further identification of the molecular targets through which PAI-1 stimulates intimal formation and suppresses TGF-beta(1) expression in the artery wall may reveal new approaches for inhibiting intimal formation. Our studies also discount bone marrow as an important source from which TGF-beta(1) recruits intimal cells and suggest instead that TGF-beta(1) induces substantial cell migration either from the adventitia or from an extravascular, but nonhematopoietic source.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator.

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.

Transforming growth factor beta 1 induces neointima formation through plasminogen activator inhibitor-1-dependent pathways.

OBJECTIVE: The mechanisms through which transforming growth factor (TGF)-beta1 promotes intimal growth, and the pathways through which TGF-beta1 expression is regulated in the artery wall, are incompletely understood. We used a mouse model to investigate mechanisms of TGF-beta1-induced intimal growth. METHODS AND RESULTS: Adenovirus-mediated overexpression of TGF-beta1 in uninjured carotid arteries of wild-type mice induced formation of a cellular and matrix-rich intima. Intimal growth appeared primarily due to cell migration and matrix accumulation, with only a negligible contribution from cell proliferation. Overexpression of TGF-beta1 also stimulated expression of plasminogen activator inhibitor type 1 (plasminogen activator inhibitor [PAI]-1) in the artery wall. To test the hypothesis that PAI-1 is a critical downstream mediator of TGF-beta1-induced intimal growth, we transduced carotid arteries of PAI-1-deficient (Serpine1(-/-)) mice with the TGF-beta1-expressing vector. Overexpression of TGF-beta1 in Serpine1(-/-) arteries did not increase intimal growth, matrix accumulation, cell migration, or proliferation. Moreover, TGF-beta1-transduced arteries of Serpine1(-/-) mice secreted 6- to 10-fold more TGF-beta1 than did arteries of wild-type mice that were infused with the same concentration of the TGF-beta1-expressing vector. CONCLUSIONS: PAI-1 is both a critical mediator of TGF-beta1-induced intimal growth and a key negative regulator of TGF-beta1 expression in the artery wall.

Age at symptom onset and long-term survival in patients with neurofibromatosis Type 2.

OBJECT: Neurofibromatosis Type 2 (NF2) is an intractable disorder predisposing to multiple, recurrent tumors of the central nervous system (CNS). To clarify the survival rate and characteristics that predict poor survival, we retrospectively reviewed clinical data in cases of NF2. METHODS: From among 283 patients with neurofibromatosis who had been registered in a nationwide study in Japan between 1986 and 1987, 74 patients with bilateral vestibular schwannomas were analyzed. The mean duration of follow up after diagnosis was 121 months (range 2-287 months). Results of a Kaplan-Meier product-limit analysis indicated that overall 5-, 10-, and 20-year patient survival rates following diagnosis of NF2 were 85, 67, and 38%, respectively. Early onset of the initial symptom significantly compromised survival; 5-, 10-, and 20-year survival rates in patients with symptom onset at an age younger than 25 years were 80, 60, and 28%, respectively, whereas in patients with symptom onset at an age of 25 years or older the rates were 100, 87, and 62%, respectively. Patients with small vestibular schwannomas at diagnosis (< 2 cm in diameter) had better rates of survival. Other variables such as sex, additional tumors in the CNS, or dermal abnormalities did not significantly affect survival. CONCLUSIONS: This first report of long-term follow-up results concerning the survival of patients with NF2 indicates an adverse effect of early symptom onset.

Effect of heat stimulation on viability and proteoglycan metabolism of cultured chondrocytes: preliminary report.

Thermotherapy has been applied to various joint diseases and injuries, but its direct effects on articular cartilage have remained unclear. The present study examined the effects on cell viability and metabolism by using the chondrocyte-like cell line HCS-2/8. The temperatures and durations of heat stimulation were 39 degrees, 41 degrees, 43 degrees, and 45 degrees C for 15 or 30 min. After heat stimulation of 41 degrees C or lower for 15 or 30 min, cell viability increased and proteoglycan metabolism was accelerated, whereas after stimulation at 43 degrees C or higher for 30 min the viability and metabolism decreased. These results indicate that appropriate heat stimulation positively affects cell viability and the proteoglycan metabolism of articular cartilage, whereas too much heat stimulation produces negative effects. Clinical efficacy is therefore determined by the overall thermal dose. When the appropriate combination of temperature and duration is found, thermotherapy for diseases and injury of articular cartilage can be highly useful in clinical practice.

De novo cerebral aneurysms manifesting as repeated subarachnoid hemorrhage and cerebral ischemic stroke--case report.

A 29-year-old man suffered repeated subarachnoid hemorrhage and cerebral ischemic stroke over a period of 6 years. Cerebral angiography at each episode disclosed development of multiple de novo aneurysms at the bilateral middle cerebral arteries (MCAs), internal carotid arteries, right anterior cerebral artery, and right vertebral artery. Two of the ruptured aneurysms were treated by surgical and endovascular treatment, but he died of the effects of rupture of a de novo right MCA aneurysm. Histological examination at autopsy disclosed marked degenerative changes in all layers of the cerebral vessels, which were probably congenital in origin.

Experimental model of dissecting aneurysms.

BACKGROUND AND PURPOSE: The pathogenesis and optimal treatment for arterial dissection are still unclear. We devised an experimental model of arterial dissection and observed the morphologic changes with angiography. METHODS: Sixty-four experimental dissections were created in the common carotid arteries of 34 mongrel dogs. After a small incision was made in the arterial adventitia, it was dissected from the media. Elliptical defects (2, 4, 6, and 8 mm in groups I-A, I-B, I-C, and I-D, respectively; n = 47) or longitudinal incisions (4, 6, and 8 mm in groups II-A, II-B, and II-C, respectively; n = 17) were made in the intima distal to the adventitial incision to serve as an entry zone for dissection. RESULTS: Immediately after the lesions were created, the influx of blood into the dissected cavity produced massive subadventitial hematomas, resulting in stenotic changes in all of the arteries, including seven with occlusion. Follow-up (1-week) angiograms demonstrated complete healing, with normal arterial calibers in 11 (79%) of 14 I-A lesions and aneurysm formation in nine (69%) of 13 I-B lesions. All 10 I-D lesions had complete arterial occlusion. Persistent stenosis was observed in all 10 I-C lesions; six of these developed aneurysms. Pathologic examination of the freshly dissected cavities revealed a clot-filled cleft between the media and adventitia. Mature aneurysms, evaluated 3 mo later, had endothelialization within the aneurysmal dome. CONCLUSION: Morphologic changes after arterial dissection are closely related to the size of the intimal entry zone, which may determine whether a dissecting aneurysm forms.