Role of MyD88 and Toll-like receptors 2 and 4 in the sensing of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88(-/-), TLR4(-/-), and TLR2(-/-) mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

An angiotensin II- and NF-kappaB-dependent mechanism increases connexin 43 in murine arteries targeted by renin-dependent hypertension.

AIMS: Connexins (Cxs) play a role in the contractility of the aorta wall. We investigated how connexins of the endothelial cells (ECs; Cx37, Cx40) and smooth muscle cells (SMCs; Cx43, Cx45) of the aorta change during renin-dependent and -independent hypertension. METHODS AND RESULTS: We subjected both wild-type (WT) mice and mice lacking Cx40 (Cx40(-/-)), to either a two-kidney, one-clip procedure or to N-nitro-l-arginine-methyl-ester treatment, which induce renin-dependent and -independent hypertension, respectively. All hypertensive mice featured a thickened aortic wall, increased levels of Cx37 and Cx45 in SMC, and of Cx40 in EC (except in Cx40(-/-) mice). Cx43 was up-regulated, with no effect on its S368 phosphorylation, only in the SMCs of renin-dependent models of hypertension. Blockade of the renin-angiotensin system of Cx40(-/-) mice normalized blood pressure and prevented both aortic thickening and Cx alterations. Ex vivo exposure of WT aortas, carotids, and mesenteric arteries to physiologically relevant levels of angiotensin II (AngII) increased the levels of Cx43, but not of other Cx. In the aortic SMC line of A7r5 cells, AngII activated kinase-dependent pathways and induced binding of the nuclear factor-kappa B (NF-kappaB) to the Cx43 gene promoter, increasing Cx43 expression. CONCLUSION: In both large and small arteries, hypertension differently regulates Cx expression in SMC and EC layers. Cx43 is selectively increased in renin-dependent hypertension via an AngII activation of the extracellular signal-regulated kinase and NF-kappaB pathways.

Isolation of cardiovascular precursor cells from the human fetal heart.

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model.

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies.

Immunohistochemical analysis of bone morphological protein signaling pathway in human myometrium.

We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.

The impact of spatial resolution and respiratory motion on MR imaging of atherosclerotic plaque.

PURPOSE: To examine the impact of spatial resolution and respiratory motion on the ability to accurately measure atherosclerotic plaque burden and to visually identify atherosclerotic plaque composition. MATERIALS AND METHODS: Numerical simulations of the Bloch equations and vessel wall phantom studies were performed for different spatial resolutions by incrementally increasing the field of view. In addition, respiratory motion was simulated based on a measured physiologic breathing pattern. RESULTS: While a spatial resolution of > or = 6 pixels across the wall does not result in significant errors, a resolution of < or = 4 pixels across the wall leads to an overestimation of > 20%. Using a double-inversion T2-weighted turbo spin echo sequence, a resolution of 1 pixel across equally thick tissue layers (fibrous cap, lipid, smooth muscle) and a respiratory motion correction precision (gating window) of three times the thickness of the tissue layer allow for characterization of the different coronary wall components. CONCLUSIONS: We found that measurements in low-resolution black blood images tend to overestimate vessel wall area and underestimate lumen area.

Pulmonary involvement in Fabry disease: Overview and perspectives.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of alpha-galactosidase A, which leads to storage of sphingolipids in virtually all human cells and consequently to organ dysfunction. Pulmonary involvement is still debated. But, obstructive lung disease is up to ten times more prevalent in patients with FD compared to general public. Also, an accelerated decline in forced expiratory volume in one second (FEV1) over time was observed in these patients. Lysosomal storage of glycosphingolipids is considered leading to small airway disease via hyperplasia of the bronchiolar smooth muscle cells. Larger airways may become involved with ongoing disease process. There is no evidence for involvement of the lung interstitium in FD. The effect of enzyme replacement therapy on respiratory involvement remains to be determined in large, prospective controlled trials.

The use of external mesh reinforcement to reduce intimal hyperplasia and preserve the structure of human saphenous veins.

The saphenous vein is the conduit of choice in bypass graft procedures. Haemodynamic factors play a major role in the development of intimal hyperplasia (IH), and subsequent bypass failure. To evaluate the potential protective effect of external reinforcement on such a failure, we developed an ex vivo model for the perfusion of segments of human saphenous veins under arterial shear stress. In veins submitted to pulsatile high pressure (mean pressure at 100 mmHg) for 3 or 7 days, the use of an external macroporous polyester mesh 1) prevented the dilatation of the vessel, 2) decreased the development of IH, 3) reduced the apoptosis of smooth muscle cells, and the subsequent fibrosis of the media layer, 4) prevented the remodelling of extracellular matrix through the up-regulation of matrix metalloproteinases (MMP-2, MMP-9) and plasminogen activator type I. The data show that, in an experimental ex vivo setting, an external scaffold decreases IH and maintains the integrity of veins exposed to arterial pressure, via increase in shear stress and decrease wall tension, that likely contribute to trigger selective molecular and cellular changes.

Epithelioid soft tissue tumors.

Epithelioid neoplasms are generally carcinomas. As confirmation that every rule is meant to be broken, some sarcomas demonstrate epithelioid morphology, and can even express cytokeratins. These sarcomas have unique behavior, for example, a much higher rate of lymph node metastasis than other sarcomas. This group of sarcomas also presents diagnostic and therapeutic challenges to those clinicians who help patients contend with these difficult tumors. In this review, some of the major categories of epithelioid soft tissue tumors are described, with clinical data reported as available. Some of these tumors provide excellent opportunities to examine newer protein-targeted agents in investigational settings.

Interplay between connexin40 and nitric oxide signaling during hypertension.

Connexins (Cxs) and endothelial nitric oxide synthase (eNOS) contribute to the adaptation of endothelial and smooth muscle cells to hemodynamic changes. To decipher the in vivo interplay between these proteins, we studied Cx40-null mice, a model of renin-dependent hypertension which displays an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels. These mice, which were either untreated or subjected to the 1-kidney, 1-clip (1K1C) procedure, a model of volume-dependent hypertension, were compared with control mice submitted to either the 1K1C or the 2-kidney, 1-clip (2K1C) procedure, a model of renin-dependent hypertension. All operated mice became hypertensive and featured hypertrophy and altered Cx expression of the aorta. The combination of volume- and renin-dependent hypertension in Cx40-/- 1K1C mice raised blood pressure and cardiac weight index. Under these conditions, all aortas showed increased levels of Cx40 in endothelial cells and of both Cx37 and Cx45 in smooth muscle cells. In the wild-type 1K1C mice, the interactions between Cx40 and Cx37 with eNOS were enhanced, resulting in increased NO release. The Cx40-eNOS interaction could not be observed in mice lacking Cx40, which also featured decreased levels of eNOS. In these animals, the volume overload caused by the 1K1C procedure resulted in increased phosphorylation of eNOS and in a higher NO release. The findings provide evidence that Cx40 and Cx37 play an in vivo role in the regulation of eNOS.

Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation.

Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated.

1C.06: Ambulatory pulse pressure is negatively associated with excretions of urinary caffeine and its metabolites

OBJECTIVE: Systolic blood pressure (BP) has been associated with urinary caffeine and its metabolites such as paraxanthine and theophylline. Caffeine and caffeine metabolites could influence arterial pulse pressure (PP) via sympathomimetic effects, smooth muscle relaxation, and phosphodiesterase inhibition. The purpose of this analysis was to explore the association of ambulatory PP with urinary caffeine and its related metabolites in a large population-based sample. DESIGN AND METHOD: Families were randomly selected from the general population of three Swiss cities (2009-2013). Ambulatory BP monitoring was conducted using validated Diasys Integra devices. PP was defined as the difference between the systolic and diastolic ambulatory BP. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24 h urine using ultra-high performance liquid chromatography tandem mass spectrometry. Urinary excretions were log-transformed to satisfy regression assumptions. We used linear mixed models to explore the associations of urinary caffeine and caffeine metabolite excretions with 24-hour, day- and night-time PP while adjusting for major confounders. RESULTS: The 836 participants (48.9% men) included in this analysis had mean (±SD) age of 47.8 (±17.5), and mean 24-hour systolic and diastolic BP of 120.1 mmHg (±13.9) and 78.0 (±8.6). Except theobromine, log transformed urinary caffeine and caffeine metabolite excretions were associated negatively with 24-hour, daytime and night-time ambulatory PP. 24-hour, daytime, and night-time ambulatory PP decreased by -0.804 mmHg (SE, 0.209), -0.749 (0.215), and -0.968 (0.243) (all P values <0.005), for each doubling excretion of caffeine. Strong negative associations with night-time ambulatory PP were observed for paraxanthine and theophylline.(Figure is included in full-text article.) CONCLUSIONS: : The negative associations of PP with caffeine, paraxanthine, and theophylline excretions suggest that caffeine and its metabolites do lower BP, possibly by modifying arterial stiffness.

Lung CD57+ cell density is increased in very severe COPD

Among all inflammatory cells involved in COPD, those with a cytolytic or elastolytic activity are thought to play a key role in the pathogenesis of the disease. However, there is no data about the infiltration of cells expressing the CD57 marker in small airways and parenchyma of COPD patients. In this study, surgical specimens from 43 subjects undergoing lung resection due to lung cancer (9 non-smokers, 18 smokers without COPD and 16 smokers with moderate COPD) and 16 patients undergoing double lung transplantation for very severe COPD were examined. CD57+ cells, neutrophils, macrophages and mast cells infiltrating bronchioles (epithelium, smooth muscle and connective tissue) and parenchymal interstitium were localized and quantified by immunohistochemical analysis. Compared to the other groups, the small airways of very severe COPD patients showed a significantly higher density of CD57+ cells, mainly infiltrated in the connective tissue (p=0.001), and a significantly higher density of neutrophils located characteristically in the epithelium (p=0.037). Also, the density of neutrophils was significantly higher in parenchyma of very severe COPD patients compared with the rest of the groups (p=0.001). Finally, there were significant correlations between the bronchiolar density of CD57+ cells and the FEV1 values (R=-0.43, p=0.022), as well as between the parenchymal density of neutrophils and macroscopic emphysema degree (R=0.43, p=0.048) in COPD groups. These results show that CD57+ cells may be involved in COPD pathogenesis, especially in the most severe stages of the disease.

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.

Perivascular epithelioid cell tumor (PEComa) of the uterus: A case report

Background. Perivascular epithelioid cell tumors (PEComas) are a rare family of mesenchymal tumors arising in a wide array of anatomic locations and characterized by coexpression of melanocytic and muscle markers. The uterus accounts for around one-fourth of the overall PEComa cases reported in the literature. Methods. We report a case of PEComa of the uterus with multiple malignancy features. Results. A uterine mass suspect for leiomyosarcoma was found in a 53-year-old woman with post-menopausal bleeding. Total hysterectomy and bilateral adnexectomy was performed. The tumor measured 7 cm in diameter, was unique, well-circumscribed, nodular, and whiteyellow without haemorrhage or necrosis. Microscopically, two populations of cells could be seen: small fusiform cells growing in fascicles resembling a smooth muscle tumor, and large epithelioid cells with abundant pale vacuolated cytoplasm growing in a diffuse pattern. Cytologic atypias were marked and mitoses numerous and often atypical in the second component. The tumor infiltrated into the myometrium with lymphovascular invasion. Immunostains showed positivity for MelanA, HMB45, smooth muscle actin, CD10, TFE3 and cathepsin K. Conclusions. This PEComa case presents several of the recently precised criteria for malignancy (Schoolmeester JK et al. Perivascular epithelioid cell neoplasm (PEComa) of the gynecologic tract: Clinicopathologic and immunohistochemical characterization of 16 cases. Am J Surg Pathol 2014; 38:176-188).