Endothelial nitric oxide synthase uncoupling impairs endothelial progenitor cell mobilization and function in diabetes

Uncoupling of the endothelial nitric oxide synthase (eNOS) resulting in superoxide anion (O(2)(-)) formation instead of nitric oxide (NO) causes diabetic endothelial dysfunction. eNOS regulates mobilization and function of endothelial progenitor cells (EPCs), key regulators of vascular repair. We postulate a role of eNOS uncoupling for reduced number and function of EPC in diabetes. EPC levels in diabetic patients were significantly reduced compared with those of control subjects. EPCs from diabetic patients produced excessive O(2)(-) and showed impaired migratory capacity compared with nondiabetic control subjects. NOS inhibition with N(G)-nitro-l-arginine attenuated O(2)(-) production and normalized functional capacity of EPCs from diabetic patients. Glucose-mediated EPC dysfunction was protein kinase C dependent, associated with reduced intracellular BH(4) (tetrahydrobiopterin) concentrations, and reversible after exogenous BH(4) treatment. Activation of NADPH oxidases played an additional but minor role in glucose-mediated EPC dysfunction. In rats with streptozotocin-induced diabetes, circulating EPCs were reduced to 39 +/- 5% of controls and associated with uncoupled eNOS in bone marrow. Our results identify uncoupling of eNOS in diabetic bone marrow, glucose-treated EPCs, and EPCs from diabetic patients resulting in eNOS-mediated O(2)(-) production. Subsequent reduction of EPC levels and impairment of EPC function likely contributes to the pathogenesis of vascular disease in diabetes.     

Treatment of resistant port wine stains (PWS) with pulsed dye laser and non-contact vacuum: a pilot study

The blanching of resistant port wine stains (PWS) with a pulsed dye laser (PDL) requires a large number of treatments, resulting in substantial discomfort to patients, many of them children. Pneumatic skin flattening (PSF - Serenity Pro) is a new technology that generates a vacuum over the skin and reduces pain in laser-based treatments of the skin, while creating contact between the skin and an upper window. The same technology can be utilized to increase skin blood fraction while operated in a non-contact mode. The objective of this study was to test the enhancement in the efficacy of PWS treatment with PDL and Serenity Pro while vacuum is being utilized in the non-contact, blood-enrichment mode. Fifteen patients with resistant PWS underwent 1–4 treatments (interval of 5–20 weeks) under general anesthesia with a 595-nm PDL at 10–14 J/cm², 1.5–3 ms pulse duration, and 7-mm spot size. Lesion blanching with DCD chilling and with vacuum were photographed and compared. Better blanching of various degrees was observed on resistant PWS with the blood-enrichment technique in seven out of 11 patients who returned for follow-up. There were no cases of decrease in efficacy. Blood enrichment with the Serenity Pro non-contact vacuum technology has the potential of enhancing the capability of treating resistant port wine stains in over 50% of cases. Further studies will better quantify the number of treatments necessary for better lesion clearance. The vacuum-assisted technique may be of particular importance in view of the fact that achieving complete lesion clearance remains a challenge in PWS treatments.     

Expression of angiopoietin‐like protein 4 at the fracture site: Regulation by hypoxia and osteoblastic differentiation

Vascular disruption that occurs as a consequence of bone fracture, leads to hypoxia at the site of damage. Hypoxia regulates the expression of a number of genes that can modulate energy conservation, cell survival, tissue regeneration and angiogenesis. In this study we investigated the expression of Angiopoietin‐like 4, an adipocytokine that has additional roles in angiogenesis, at the fracture site. We demonstrate that Angptl4 mRNA expression increased early during fracture healing (day 3) returning close to baseline at day14. In the callus, Angptl4 mRNA was visualized in areas of condensing mesenchymal cells, callus cartilage and was especially high in mineralizing osteoblasts located in areas of new bone formation. In vitro, Angptl4 mRNA expression in osteoblasts increased under hypoxic conditions and in cells treated with the hypoxia mimetic desferrioxamine. Angptl4 levels were strongly induced at day 14 in differentiating MC3T3‐E1 osteoblastic cells. Exogenous ANGPTL4 increased expression of Runx2, Spp1, vegfa, and Alp mRNA in differentiating osteoblasts. We suggest that the distribution of Angptl4 in the callus may be driven by hypoxia and that Angptl4 may play a role in osteoblastic differentiation, and possibly angiogenesis via regulation of VEGF. Further studies could reveal a dual role for Angptl4 in angiogenesis and osteogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1364–1373, 2015.     

Comparative Finite Element Model Analysis of Ascending Aortic Flow in Bicuspid and Tricuspid Aortic Valve

In bicuspid aortic valve (BAV) disease, the role of genetic and hemodynamic factors influencing ascending aortic pathology is controversial. To test the effect of BAV geometry on ascending aortic flow, a finite element analysis was undertaken. A surface model of aortic root and ascending aorta was obtained from magnetic resonance images of patients with BAV and tricuspid aortic valve using segmentation facilities of the image processing code Vascular Modeling Toolkit (developed at the Mario Negri Institute). Analytical models of bicuspid (antero‐posterior [AP], type 1 and latero‐lateral, type 2 commissures) and tricuspid orifices were mathematically defined and turned into a volumetric mesh of linear tetrahedra for computational fluid dynamics simulations. Numerical simulations were performed with the finite element code LifeV. Flow velocity fields were assessed for four levels: aortic annulus, sinus of Valsalva, sinotubular junction, and ascending aorta. Comparison of finite element analysis of bicuspid and tricuspid aortic valve showed different blood flow velocity pattern. Flow in bicuspid configurations showed asymmetrical distribution of velocity field toward the convexity of mid‐ascending aorta returning symmetrical in distal ascending aorta. On the contrary, tricuspid flow was symmetrical in each aortic segment. Comparing type 1 BAV with type 2 BAV, more pronounced recirculation zones were noticed in the latter. Finally, we found that in both BAV configurations, maximum wall shear stress is highly localized at the convex portion of the mid‐ascending aorta level. Comparison between models showed asymmetrical and higher flow velocity in bicuspid models, in particular in the AP configuration. Asymmetry was more pronounced at the aortic level known to be more exposed to aneurysm formation in bicuspid patients. This supports the hypothesis that hemodynamic factors may contribute to ascending aortic pathology in this subset of patients.     

Tetralogy of Fallot correction with transannular patch reconstruction in a patient with an anomalous right coronary artery and an unusual course of the right ventricular branch

Anomalous coronary arteries occur in as many as 12% of patients with tetralogy of Fallot (TOF). In patients with this condition, pulmonary hypoplasia can be prohibitive in performing a valve-sparing repair, subsequently resulting in various techniques to preserve the anomalous coronary artery. The management strategy is often complex in such a situation. We report on a case of TOF with an anomalous right coronary artery crossing the right ventricular outflow tract, with an unusual course of the right ventricular (RV) branch, which precluded placement of a valved conduit. In this case, we performed a successful repair with mobilization of the anomalous coronary artery and reconstruction of the right ventricular outflow tract with a limited transannular patch.     

BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival

Previous studies have shown frequent mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) or NRAS (neuroblastoma RAS viral [V-ras] oncogene homolog) genes in cutaneous melanoma, but the relationship between these alterations and tumor cell proliferation has not been examined in human melanoma. In our study of 51 primary nodular melanomas and 18 paired metastases, we found mutations in BRAF (codon 600, previously denoted 599) in 15 primary tumors (29%) and eight metastases (44%). The figures for NRAS mutations were 27% and 22%, respectively. Mutations in BRAF and NRAS genes were mutually exclusive in all but one case, and were maintained from primary tumors through their metastases. Mutations, however, were not associated with tumor cell proliferation by Ki-67 expression, tumor thickness, microvessel density, or vascular invasion, and there were no differences in patient survival. Although BRAF and NRAS mutations are likely to be important for the initiation and maintenance of some melanomas, other factors might be more significant for proliferation and prognosis in subgroups of aggressive melanoma.     

Migration of V delta 1 and V delta 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV-1-infected patients: competition by HIV-1 Tat

We show that HIV-1-infected patients have increased concentrations of circulating V delta 1 T cells (2.2%-9.0% of T lymphocytes; healthy donors, 1.0%-2%) and, in some instances, V delta 2 T cells (3.5%-4.8% vs 2.0%-3.3%). In these patients, both V delta 1 and V delta 2 T cells are CXCR3+CXCR4+, whereas in healthy donors CXCR4 was preferentially expressed on V delta 1 T lymphocytes. gamma delta T cells transmigrated across endothelial monolayers, in response to interferon-gamma-inducing protein-10 (IP-10/CXCL10), stromal cell-derived factor-1 (SDF-1/CXCL12), or both, according to the expression of the specific receptors CXCR3 and CXCR4. Interestingly, 6Ckine/SLC/CCL21 was more effective than IP-10/CXCL10 on V delta 1 CXCR3+ cells, whereas V delta 2 CXCR3+ cells were driven more efficiently by IP-10/CXCL10. IP-10/CXCL10- and SDF-1/CXCL12-induced transmigration was dependent on phosphoinositide-3 kinase (PI-3K), as demonstrated by the use of the specific blockers wortmannin and LY294002 and by the activation of the downstream serine kinase Akt/PKB on ligation of CXCR3 and CXCR4. Occupancy of CXCR3, but not of CXCR4, led to CAMKII activation; accordingly, the CAMKII inhibitors KN62 and KN93 decreased IP-10/CXCL10- but not SDF-1/CXCL12-driven transmigration. Finally, HIV-1 Tat, which is present in the serum of HIV-1-infected patients, interferes with the chemotactic activity of these chemokines because of the cysteine-rich domain of the protein, which contains CXC and CC chemokine-like sequences.     

Why patients sign out against medical advice (AMA): factors motivating patients to sign out AMA

A retrospective review of the against medical advice (AMA) discharges revealed that the majority of the patients left AMA for personal reasons, i.e., sickness or death in the family; reconciliation with spouse, girl friend, or family members; financial problems; and legal issues such as a court date. Strategies to reduce AMA discharges and increase patient retention in treatment are suggested.