Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

Obstruction of Small Arterioles in Patients with Critical Limb Ischemia due to Partial Endothelial-to-Mesenchymal Transition.

Critical limb ischemia (CLI) is a hazardous manifestation of atherosclerosis and treatment failure is common. Abnormalities in the arterioles might underlie this failure but the cellular pathobiology of microvessels in CLI is poorly understood. We analyzed 349 intramuscular arterioles in lower limb specimens from individuals with and without CLI. Arteriolar densities were 1.8-fold higher in CLI muscles. However, 33% of small (<20 µm) arterioles were stenotic and 9% were completely occluded. The lumens were closed by bulky, re-oriented endothelial cells expressing abundant N-cadherin that uniquely localized between adjacent and opposing endothelial cells. S100A4 and SNAIL1 were also expressed, supporting an endothelial-to-mesenchymal transition. SMAD2/3 was activated in occlusive endothelial cells and TGFß1 was increased in the adjacent mural cells. These findings identify a microvascular closure process based on mesenchymal transitions in a hyper-TGFß environment that may, in part, explain the limited success of peripheral artery revascularization procedures.

Agnoprotein Is an Essential Egress Factor during BK Polyomavirus Infection.

BK polyomavirus (BKPyV; hereafter referred to as BK) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein fail to release from host cells and do not propagate to wild-type levels. Despite this, agnoprotein is not essential for virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-soluble N-ethylmaleimide sensitive fusion (NSF) attachment protein (α-SNAP) is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.

Effectiveness and Safety of Sitagliptin in Patients with Beta-thalassaemia Major and Diabetes Mellitus: A Case Series.

Sitagliptin, a modern antidiabetic agent which is weight neutral and associated with low rate of hypoglycaemias, is being increasingly used in type 2 diabetes mellitus (DM). However, there is a paucity of data about its efficacy and safety in beta-thalassaemia major (ß-TM). This retrospective case series of five patients (mean age of 45 years) is the first study evaluating the use of sitagliptin in patients with ß-TM and DM. Four patients responded well to sitagliptin, as evidenced by a decrease in fructosamine by 77 and 96µmol/L (equivalent reduction in HbA1c of 1.5% and 1.9%) observed in two patients and reduction in the frequency of hypoglycaemia without worsening glycaemic control in two others. One patient did not respond to sitagliptin. No patients reported significant side effects. This study provides evidence that sitagliptin may be considered, with caution, for use in patients with ß-TM and DM, under the close monitoring of a Diabetologist.

New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy.

BK polyomavirus is the causative agent of several diseases in transplant patients and the immunosuppressed. In order to better understand the structure and life cycle of BK, we produced infectious virions and VP1-only virus-like particles in cell culture, and determined their three-dimensional structures using cryo-electron microscopy (EM) and single-particle image processing. The resulting 7.6-Å resolution structure of BK and 9.1-Å resolution of the virus-like particles are the highest-resolution cryo-EM structures of any polyomavirus. These structures confirm that the architecture of the major structural protein components of these human polyomaviruses are similar to previous structures from other hosts, but give new insight into the location and role of the enigmatic minor structural proteins, VP2 and VP3. We also observe two shells of electron density, which we attribute to a structurally ordered part of the viral genome, and discrete contacts between this density and both VP1 and the minor capsid proteins.

Human papillomavirus type 1 E1

UNLABELLED: The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and that HPV1 E1^E4 inhibits this phosphorylation. After mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following coexpression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4-mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts. IMPORTANCE: The HPV life cycle is tightly linked to the epithelial terminal differentiation program, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. We show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through the inhibition of a host cell kinase.

Prevalence of low bone mass and vitamin D deficiency in ß-thalassemia major.

Low bone mass, a major cause of morbidity in patients with ß-thalassemia major (ß-TM), is multifactorial. There is lack of data about the current prevalence of low bone mass in patients with ß-TM. The aims of this study are to examine the current prevalence of low bone mass in ß-TM patients and the association between demographic characteristics, markers of iron overload, endocrinopathies, glycemic status and bone mineral density (BMD) as well as to study the 25-OH-vitamin D status of the patients and its relationship with BMD. Our institution serves the largest cohort of ß-TM patients in the UK. From 99 patients (49 males, 50 females) with a mean ± standard deviation (SD) age of 36 ± 9 years, 55.5% had low BMD for their age as defined by Z-score BMD <-2.0 either at the lumbar spine (43.9%) or at the hip (25.5%). The only statistically significant association on the multivariate analysis was between hypogonadism and low BMD at the lumbar spine. In our study, 29.9% of patients had vitamin D deficiency, 65.7% had vitamin D insufficiency and 12.4% had optimal levels. No association between vitamin D status and low bone mass was found. Our study demonstrated a much lower prevalence of low bone mass in adults with ß-TM compared to previous studies. Further studies are needed to examine whether this suggests a widespread improvement across patients with ß-TM possibly due to advances in therapeutics. Most patients had suboptimal 25-OH-vitamin D levels, but no association between vitamin D status and bone mass was demonstrated.

History of myocardial iron loading is a strong risk factor for diabetes mellitus and hypogonadism in adults with ß thalassemia major.

Endocrinopathies are common complications of transfusional hemosiderosis among patients with ß thalassemia major (TM). Previous studies had shown associations between some endocrinopathies and iron overload of the myocardium, liver and/or endocrine organs as assessed by MRI techniques. This retrospective analysis of 92 patients with TM (median age 36 yr) from a tertiary adult thalassemia unit in UK aimed to determine independent risk factors associated with endocrinopathies among these patients. Unlike previous studies, longitudinal data on routine measurements of iron load [worst myocardial and liver T2* values since 1999, worst LIC by MRI-R2 since 2008 and average 10-yr serum ferritin (SF)] up to April 2010 together with demographic features and age of initiating chelation were analyzed for associations with endocrinopathies. The most common endocrinopathies in this cohort were hypogonadism (67%) and diabetes mellitus (DM) (41%), and these were independently associated with myocardial T2* <20 ms (P < 0.001 and P = 0.008, respectively) and increased age (P = 0.002 and P = 0.016, respectively). DM and hypogonadism were independently associated with average SF >1250 µg/L (P = 0.003) and >2000 µg/L (P = 0.047), respectively. DM was also associated with initial detection of abnormal myocardial T2* at an older age (30 yr vs. 24 yr, P = 0.039). An abnormal myocardial T2* may therefore portend the development of DM and hypogonadism in patients with TM.

Joint diabetes thalassaemia clinic: an effective new model of care.

Diabetes is a significant complication of ß-thalassemia major (ß-TM) and most patients receive fragmented diabetes care. In 2005, we developed a unique Joint Diabetes Thalassaemia Clinic, based at the Department of Diabetes, Whittington Health, London, UK, where patients were reviewed jointly by a multidisciplinary team, including Consultant Diabetologist and Hematologist. Study of the Joint Diabetes Thalassaemia Clinic (2005-2009) showed improvement in glycemic control with fructosamine reduction from 344 umol/L to 319 umol/L over a 1-year period as well as improvement in lipid profiles. The proportion of patients attending the Joint Clinic who achieved metabolic targets compared to the National Diabetes Audit for England was higher for glycemic control (73.0 Joint Diabetes Thalassaemia Clinic vs. 63.0% nationally), blood pressure control (58.0 Joint Diabetes Thalassaemia Clinic vs. 30.0% nationally) and cholesterol control (81.0 Joint Diabetes Thalassaemia Clinic vs. 78.0% nationally). Five patients (22.7%) had microvascular complications. A significant proportion of our patients had endocrinopathies (86.0% hypogonadism, 18.0% hypothyroidism, 23.0% hypoparathyroidism). The unique partnership of our Joint Diabetes Thalassaemia Clinic, allowed these very complex patients to be managed effectively.

Merkel cell polyomavirus small T antigen targets the NEMO adaptor protein to disrupt inflammatory signaling.

Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKß-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aß, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell.

Normalized left ventricular volumes and function in thalassemia major patients with normal myocardial iron.

PURPOSE: To determine the reference range in thalassemia major (TM) for left ventricular (LV) function. MATERIALS AND METHODS: We used cardiovascular magnetic resonance (CMR) to measure heart volumes and function in 81 TM patients with normal myocardial T2* measurements (T2* > 20 msec) and by inference without excess myocardial iron. Forty age- and gender-matched healthy controls were also studied. RESULTS: Resting LV volumes and function normalized to body surface area differed significantly between TM patients and controls. The lower limit and the mean for ejection fraction (EF) were higher in TM patients (males 59 vs. 55%, mean 71% vs. 65%; females 63 vs. 59%, mean 71% vs. 67%; both P < 0.001). The upper limit and mean for end-diastolic volume index were higher in TM patients (males 152 vs. 105 mL/m(2), mean 97 vs. 84 mL/m(2); females 121 vs. 99 mL/m(2), mean 87 vs. 79 mL/m(2); both P < 0.05). In TM patients the cardiac index (P < 0.001) was increased. CONCLUSION: At rest, TM patients with a normal myocardial T2* have different "normal" values for LV volume and function parameters compared to controls, and this has the potential to lead to a misdiagnosis of cardiomyopathy. We present new reference "normal" ranges in TM to alleviate this problem.

Development of thalassaemic iron overload cardiomyopathy despite low liver iron levels and meticulous compliance to desferrioxamine.

It is believed that myocardial iron deposition and the resultant cardiomyopathy only occur in the presence of severe liver iron overload. Using cardiovascular magnetic resonance, it is now possible to assess myocardial and liver iron levels as well as cardiac function in the same scan, allowing this supposition to be examined. We describe a patient with progressive myocardial iron deposition and the development of early iron overload cardiomyopathy despite excellent compliance to standard subcutaneous desferrioxamine, minimal liver iron and well-controlled serum ferritin levels. These indirect markers remained far below the thresholds conventionally believed to be associated with increased cardiac risk.

Left ventricular diastolic function compared with T2* cardiovascular magnetic resonance for early detection of myocardial iron overload in thalassemia major.

PURPOSE: To compare left ventricular (LV) diastolic function with myocardial iron levels in beta thalassemia major (TM) patients, using cardiovascular magnetic resonance (CMR). MATERIALS AND METHODS: We studied 67 regularly transfused patients with TM and 22 controls matched for age, gender, and body surface area. The early peak filling rate (EPFR) and atrial peak filling rate (APFR) were determined from high-temporal-resolution ventricular volume-time curves. Myocardial iron estimation was achieved using myocardial T2* measurements. RESULTS: Myocardial iron loading was found in 46 TM patients (69%), in whom the EPFR correlated poorly with T2* (r = -0.20, P = 0.19). The APFR (r = 0.49, P < 0.001) and EPFR/APFR ratio (r = -0.62, P < 0.001) correlated better with T2*. The sensitivity of the diastolic parameters for detecting myocardial iron loading ranged from 4% (EPFR and APFR) to 17% (EPFR/APFR ratio). CONCLUSION: Myocardial iron overload results in diastolic myocardial dysfunction, but low sensitivity limits the use of a single estimation for early detection of iron overload, for which T2* has a superior categorical limit of normality.

Myocardial iron clearance during reversal of siderotic cardiomyopathy with intravenous desferrioxamine: a prospective study using T2* cardiovascular magnetic resonance.

Heart failure from iron overload causes 71% of deaths in thalassaemia major, yet reversal of siderotic cardiomyopathy has been reported. In order to determine the changes in myocardial iron during treatment, we prospectively followed thalassaemia patients commencing intravenous desferrioxamine for iron-induced cardiomyopathy during a 12-month period. Cardiovascular magnetic resonance assessments were performed at baseline, 3, 6 and 12 months of treatment, and included left ventricular (LV) function and myocardial and liver T2*, which is inversely related to iron concentration. One patient died. The six survivors showed progressive improvements in myocardial T2* (5.1 +/- 1.9 to 8.1 +/- 2.8 ms, P = 0.003), liver iron (9.6 +/- 4.3 to 2.1 +/- 1.5 mg/g, P = 0.001), LV ejection fraction (52 +/- 7.1% to 63 +/- 6.4%, P = 0.03), LV volumes (end diastolic volume index 115 +/- 17 to 96 +/- 3 ml, P = 0.03; end systolic volume index 55 +/- 16 to 36 +/- 6 ml, P = 0.01) and LV mass index (106 +/- 14 to 95 +/- 13, P = 0.01). Iron cleared more slowly from myocardium than liver (5.0 +/- 3.3% vs. 39 +/- 23% per month, P = 0.02). These prospective data confirm that siderotic heart failure is often reversible with intravenous iron chelation with desferrioxamine. Myocardial T2* improves in concert with LV volumes and function during recovery, but iron clearance from the heart is considerably slower than from the liver.

Comparison of effects of oral deferiprone and subcutaneous desferrioxamine on myocardial iron concentrations and ventricular function in beta-thalassaemia.

BACKGROUND: Despite the introduction of the parenteral iron chelator desferrioxamine more than 30 years ago, 50% of patients with thalassaemia major die before the age of 35 years, predominantly from iron-induced heart failure. The only alternative treatment is oral deferiprone, but its long-term efficacy on myocardial iron concentrations is unknown. METHODS: We compared myocardial iron content and cardiac function in 15 patients receiving long-term deferiprone treatment with 30 matched thalassaemia major controls who were on long-term treatment with desferrioxamine. Myocardial iron concentrations were measured by a new magnetic-resonance T2* technique, which shows values inversely related to tissue iron concentration. FINDINGS: The deferiprone group had significantly less myocardial iron (median 34.0 ms vs 11.4 ms, p=0.02) and higher ejection fractions (mean 70% [SD 6.5] vs 63% [6.9], p=0.004) than the desferrioxamine controls. Excess myocardial iron (T2* <20 ms) was less common in the deferiprone group than in the desferrioxamine controls (four [27%] vs 20 [67%], p=0.025), as was severe (T2* <10 ms) iron overload (one [7%] vs 11 [37%], p=0.04). The odds ratio for excess myocardial iron in the desferrioxamine controls versus the deferiprone group was 5.5 (95% CI 1.2-28.8). INTERPRETATION: Conventional chelation treatment with subcutaneous desferrioxamine does not prevent excess cardiac iron deposition in two-thirds of patients with thalassaemia major, placing them at risk of heart failure and its complications. Oral deferiprone is more effective than desferrioxamine in removal of myocardial iron.