Inflammatory Phenotypes Predict Changes in Arterial Stiffness Following Antiretroviral Therapy Initiation.

BACKGROUND: Inflammation drives vascular dysfunction in HIV, but in low-income settings causes of inflammation are multiple, and include infectious and environmental factors. We hypothesized that patients with advanced immunosuppression could be stratified into inflammatory phenotypes that predicted changes in vascular dysfunction on ART. METHODS: We recruited Malawian adults with CD4 <100 cells/µL 2 weeks after starting ART in the REALITY trial (NCT01825031). Carotid femoral pulse-wave velocity (cfPWV) measured arterial stiffness 2, 12, 24, and 42 weeks post-ART initiation. Plasma inflammation markers were measured by electrochemiluminescence at weeks 2 and 42. Hierarchical clustering on principal components identified inflammatory clusters. RESULTS: 211 participants with HIV grouped into 3 inflammatory clusters representing 51 (24%; cluster-1), 153 (73%; cluster-2), and 7 (3%; cluster-3) individuals. Cluster-1 showed markedly higher CD4 and CD8 T-cell expression of HLADR and PD-1 versus cluster-2 and cluster-3 (all P < .0001). Although small, cluster-3 had significantly higher levels of cytokines reflecting inflammation (IL-6, IFN-γ, IP-10, IL-1RA, IL-10), chemotaxis (IL-8), systemic and vascular inflammation (CRP, ICAM-1, VCAM-1), and SAA (all P < .001). In mixed-effects models, cfPWV changes over time were similar for cluster-2 versus cluster-1 (relative fold-change, 0.99; 95% CI, .86-1.14; P = .91), but greater in cluster-3 versus cluster-1 (relative fold-change, 1.45; 95% CI, 1.01-2.09; P = .045). CONCLUSIONS: Two inflammatory clusters were identified: one defined by high T-cell PD-1 expression and another by a hyperinflamed profile and increases in cfPWV on ART. Further clinical characterization of inflammatory phenotypes could help target vascular dysfunction interventions to those at highest risk. CLINICAL TRIALS NETWORK: NCT01825031.

Dimethylarginine dimethylaminohydrolase 2 regulates nitric oxide synthesis and hemodynamics and determines outcome in polymicrobial sepsis.

OBJECTIVE: Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl-L-arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl-L-arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. APPROACH AND RESULTS: Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P=0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) µmol/L (P=0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P<0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. CONCLUSIONS: Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.

Relationship between insulin resistance and circulating endothelial cells in pre-eclampsia.

Endothelial dysfunction and insulin resistance (IR) are established features of pre-eclampsia, however the cause and effect relationship between them remain unexplained. Circulating endothelial cells (CEC) are increased in pre-eclampsia and appear to correlate with the degree of endothelial dysfunction. We hypothesised that CEC count in pre-eclampsia would correlate with IR and might provide a simple measure of IR in pregnancies complicated by the disease. CEC count and IR were measured in 10 women with pre-eclampsia and 10 normal pregnant controls matched for maternal age, body mass index and gestational age during the third trimester. CEC count was determined using an established immunomagnetic bead separation method and IR was measured by the homeostasis model test. CEC count and IR were significantly increased in pre-eclampsia compared to normal pregnancy. However, there was no correlation between the CEC count and IR in pre-eclampsia. The data suggest that CEC count in pre-eclampsia is not a useful measure on its own of IR in pregnancies complicated by the disease.

Turning the tides: achieving rapid and safe glucose control in adolescents with suboptimally controlled type 1 diabetes using advanced hybrid closed loop systems.

Aim: Many adolescents with T1D experience a decline in metabolic control due to erratic eating habits and subpar adherence to treatment regimens. The objective of our retrospective observational study was to assess the effect of the Tandem Control IQ (CIQ) advanced hybrid closed-loop (AHCL) system on a cohort of adolescents with suboptimal glucose control. Methods: We retrospectively evaluated 20 non-adherent patients with T1D, who were inconsistently using Multiple Daily Injections (MDIs) and flash glucose monitoring and were subsequently started and on CIQ. Glucometrics and the Glucose Risk Index were assessed at baseline and after 2 weeks, 1 month, and 6 months of CIQ use. Results: The study included 20 adolescents with T1D (HbA1c: 10.0% ± 1.7). Time in range (TIR) increased from 27.1% ± 13.7 at baseline to 68.6% ± 14.2 at 2 weeks, 66.6% ± 10.7 at 1 month, and 60.4% ± 13.3 at 6 months of CIQ use. Time above range (TAR) >250 mg/dL decreased from 46.1% ± 23.8 to 9.9% ± 9.5 at 2 weeks, 10.8% ± 6.1 at 1 month, and 15.5% ± 10.5 at 6 months of AHCL use. Mean glucose levels improved from 251 mg/dL ± 68.9 to 175mg/dL ± 25.5 after 6 months of CIQ use. The Glucose Risk Index (GRI) also significantly reduced from 102 to 48 at 6 months of CIQ. HbA1c also improved from 10.0% ± 1.7 at baseline to 7.0% ± 0.7 after 6 months. Two patients experienced a single episode of mild diabetic ketoacidosis (DKA). Conclusions: AHCL systems provide a significant, rapid, and safe improvement in glucose control. This marks a pivotal advancement in technology that primarily benefited those who were already compliant.

Paediatric Wolfram syndrome Type 1: should gonadal dysfunction be part of the diagnostic criteria?

Aims: Wolfram Syndrome Spectrum Disorder (WFS1-SD), in its "classic" form, is a rare autosomal recessive disease with poor prognosis and wide phenotypic spectrum. Insulin dependent diabetes mellitus (DM), optic atrophy (OA) diabetes insipidus (DI) and sensorineural deafness (D) are the main features of WFS1-SD. Gonadal dysfunction (GD) has been described mainly in adults with variable prevalence and referred to as a minor clinical feature. This is the first case series investigating gonadal function in a small cohort of paediatric patients affected by WFS1-SD. Methods: Gonadal function was investigated in eight patients (3 male and 5 female) between 3 and 16 years of age. Seven patients have been diagnosed with classic WFS1-SD and one with non-classic WFS1-SD. Gonadotropin and sex hormone levels were monitored, as well as markers of gonadal reserve (inhibin-B and anti-Mullerian hormone). Pubertal progression was assessed according to Tanner staging. Results: Primary hypogonadism was diagnosed in 50% of patients (n=4), more specifically 67% (n=2) of males and 40% of females (n=2). Pubertal delay was observed in one female patient. These data confirm that gonadal dysfunction may be a frequent and underdiagnosed clinical feature in WFS1-SD. Conclusions: GD may represent a frequent and earlier than previously described feature in WFS1-SD with repercussions on morbidity and quality of life. Consequently, we suggest that GD should be included amongst clinical diagnostic criteria for WFS1-SD, as has already been proposed for urinary dysfunction. Considering the heterogeneous and elusive presentation of WFS1-SD, this clinical feature may assist in an earlier diagnosis and timely follow-up and care of treatable associated diseases (i.e. insulin and sex hormone replacement) in these young patients.

Impact of sub-optimal HIV viral control on activated T cells.

OBJECTIVE: HIV viral load (VL) monitoring is generally conducted 6-12 monthly in low- and middle-income countries, risking relatively prolonged periods of poor viral control. We explored the effects of different levels of loss of viral control on immune reconstitution and activation. DESIGN: Two hundred and eight participants starting protease inhibitor (PI)-based second-line therapy in the EARNEST trial (ISRCTN37737787) in Uganda and Zimbabwe were enrolled and CD38 + /HLA-DR + immunophenotyping performed (CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC; centrally gated) in real-time at 0, 12, 48, 96 and 144 weeks from randomization. METHODS: VL was assayed retrospectively on samples collected every 12-16 weeks and classified as continuous suppression (<40 copies/ml throughout); suppression with transient blips; low-level rebound (two or more consecutive VL >40, <5000 copies/ml); high-level rebound/nonresponse (two or more consecutive VL >5000 copies/ml). RESULTS: Immunophenotype reconstitution varied between that defined by numbers of cells and that defined by cell percentages. Furthermore, VL dynamics were associated with substantial differences in expression of CD4 + and CD8 + cell activation markers, with only individuals with high-level rebound/nonresponse (>5000 copies/ml) experiencing significantly greater activation and impaired reconstitution. There was little difference between participants who suppressed consistently and who exhibited transient blips or even low-level rebound by 144 weeks ( P  > 0.2 vs. suppressed consistently). CONCLUSION: Detectable viral load below the threshold at which WHO guidelines recommend that treatment can be maintained without switching (1000 copies/ml) appear to have at most, small effects on reconstitution and activation, for patients taking a PI-based second-line regimen.

Efficacy and Safety of Transarterial Chemoembolization with DC Beads LUMI in the Treatment of HCC: Experience from a Tertiary Centre.

PURPOSE: The aim of this study was to describe the safety and efficacy profiles of TACE using DC Beads LUMI. MATERIALS AND METHODS: We retrospectively analyzed 90 patients with HCC who underwent TACE with DC Bead LUMI™ between November 2018 and November 2020 at Fondazione IRCCS Cà Granda Policlinico Hospital in Milan, Italy. Patient- and tumour-related factors were registered, including the number of treated lesions, dose of DC Beads LUMI™, dose of Epirubicin, DC Beads LUMI™ target tumour coverage (LC) according to the percentage of target nodule involvement (LC1-0%-25%, LC2-25%-50%, LC3-50%-75%, LC4 75%-100%). Treatment efficacy was obtained through reviewing the follow-up imaging for evidence of response in target lesion(s), according to modified response criteria in solid tumours (mRECIST) criteria with the following outcomes: complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). Safety assessment was based on the quantitative and qualitative recording of the adverse events, classified according to CIRSE classification. RESULTS: Seventy-two patients were enrolled, and 95 procedures were carried out. We observed a target tumour response rate at 1 month with CR in 68%, PR in 10.3% 11.8%, SD in 13%, PD in 7.2%, and an overall tumour(s) (whole liver) response at 1 month with CR in 58.9%, PR in 12.6%, SD in 10.5% and PD in 18%. We found a significant association (p < 0.01) between tumour response CR or CR + PR and the number of the target lesion(s). CIRSE classification grade I and grade II complications were recorded, respectively, in 11 (11.6%) and 6 (6.3%) procedures. No grade III-IV-V complications occurred. CONCLUSION: TACE using DC Beads LUMI is a safe and effective treatment option for patients with HCC.

A comparative study using insulin pump therapy and continuous glucose monitoring in newly diagnosed very young children with type 1 diabetes: it is possible to bend the curve of HbA1c.

AIMS: The target of metabolic control (HbA1c < 7% or 53 mmol/mol) recommended by the ADA and ISPAD is attained by 30% of children with Type 1 Diabetes (T1D). Advances in technologies for T1D aim to improve metabolic outcomes and reduce complications. This observational study assesses the long-term outcomes of advanced technologies for treatment of T1D compared to conventional approach started at onset in a group of very young children with T1D. METHODS: 54 patients with less 4 years old at onset of T1D were enrolled and followed for up to 9 years after diagnosis. 24 subjects started continuous subcutaneous insulin (CSII) treatment and 30 subjects received MDI therapy from onset. Auxological data, HbA1c and total daily insulin dose (TDD/kg) have been collected at admission and every 4 months. HbA1cAUC>6%, rates of acute complications, glycemic variability indices and glucometrics were also recorded. RESULTS: Patients with CSII therapy had significantly lower mean HbA1c values compared to subjects receiving MDI treatment. CSII approach also recorded lower mean HbA1cAUC>6% and TDD/kg than MDI therapy. At the last download data, the time in range (TIR) was higher in patients with CSII and hyperglycemia events were lower. Better glycemic variability indices have been described during CSII therapy, including mean glycemia, standard deviation, coefficient of variation (CV), glycemia risk index (GRI) and high blood glucose index (HBGI). There was no statistically significant difference between frequency of severe hypoglycemia and ketoacidosis episodes between groups. CONCLUSIONS: Early initiation of diabetes technologies is safe and able to determine a better long term glycemic control in young children with T1D. It also allows to flatten the trajectory of HbA1c, probably reducing microvascular, macrovascular and neurological complications of diabetes in this very peculiar age group.

An Italian case series' description of thiamine responsive megaloblastic anemia syndrome: importance of early diagnosis and treatment.

BACKGROUND: Individuals with thiamine-responsive megaloblastic anemia (TRMA) mainly manifest macrocytic anemia, sensorineural deafness, ocular complications, and nonautoimmune diabetes. Macrocytic anemia and diabetes may be responsive to high-dosage thiamine treatment, in contrast to sensorineural deafness. Little is known about the efficacy of thiamine treatment on ocular manifestations. CASES PRESENTATION: Our objective is to report data from four Italian TRMA patients: in Cases 1, 2 and 3, the diagnosis of TRMA was made at 9, 14 and 27 months. In 3 out of 4 subjects, thiamine therapy allowed both normalization of hyperglycemia, with consequent insulin suspension, and macrocytic anemia. In all Cases, thiamine therapy did not resolve the clinical manifestation of deafness. In Cases 2 and 3, follow-up showed no blindness, unlike Case 4, in which treatment was started for megaloblastic anemia at age 7 but was increased to high doses only at age 25, when the genetic diagnosis of TRMA was performed. CONCLUSIONS: Early institution of high-dose thiamine supplementation seems to prevent the development of retinal changes and optic atrophy in TRMA patients. The spectrum of clinical manifestations is broad, and it is important to describe known Cases to gain a better understanding of this rare disease.

Levels of circulating endothelial cells and colony-forming units are influenced by age and dyslipidemia.

BACKGROUND: The balance between endothelial injury and repair in childhood is poorly understood. We examined this relationship in healthy children, in adults, and in children with familial hypercholesterolemia (FH). METHODS: Circulating endothelial cells (CECs) were measured as a marker of vascular injury, with vascular repair assessed by counting colony-forming units (CFUs), also known as endothelial progenitor cells. RESULTS: CEC number increased with age. Children with FH had elevated CECs as compared with healthy children, with similar levels numerically to those found in healthy adults. CFU numbers were higher in healthy children than either healthy adults or children with FH. Endothelium-dependent vascular function, measured by flow-mediated dilatations, was positively associated with CFU number, even after adjustment for confounding risk variables. CONCLUSION: Levels of CECs increase and CFUs decrease with age. In childhood, before the onset of clinically detectable cardiovascular dysfunction, children with a major risk factor for atherosclerotic disease have levels of these indexes of vascular injury and repair approaching those seen in adults.

Non-Occlusive Mesenteric Ischemia in Children With Diabetic Ketoacidosis: Case Report and Review of Literature.

Introduction: Despite the use of technology, recurrent diabetic ketoacidosis (DKA) prevention remains an unmet need in children and adolescents with T1D and may be accompanied by life-threatening acute complications. We present a rare case of non-occlusive mesenteric ischemia (NOMI) with overt manifestation after DKA resolution and a discussion of recent literature addressing DKA-associated NOMI epidemiology and pathogenesis in children and adolescents. Case Presentation: A 13-year-old female with previously diagnosed T1D, was admitted at our emergency department with hypovolemic shock, DKA, hyperosmolar state and acute kidney injury (AKI). Mildly progressive abdominal pain persisted after DKA correction and after repeated ultrasound evaluations ultimately suspect for intestinal perforation, an intraoperative diagnosis of NOMI was made. Conclusion: The diagnosis of DKA-associated NOMI must be suspected in pediatric patients with DKA, persistent abdominal pain, and severe dehydration even after DKA resolution.

The impact of viraemia on inflammatory biomarkers and CD4+ cell subpopulations in HIV-infected children in sub-Saharan Africa.

OBJECTIVE: To determine the impact of virological control on inflammation and cluster of differentiation 4 depletion among HIV-infected children initiating antiretroviral therapy (ART) in sub-Saharan Africa. DESIGN: Longitudinal cohort study. METHODS: In a sub-study of the ARROW trial (ISRCTN24791884), we measured longitudinal HIV viral loads, inflammatory biomarkers (C-reactive protein, tumour necrosis factor alpha, interleukin 6 (IL-6), soluble CD14) and (Uganda only) whole blood immunophenotype by flow cytometry in 311 Zimbabwean and Ugandan children followed for median 3.5 years on first-line ART. We classified each viral load measurement as consistent suppression, blip/post-blip, persistent low-level viral load or rebound. We used multi-level models to estimate rates of increase or decrease in laboratory markers, and Poisson regression to estimate the incidence of clinical events. RESULTS: Overall, 42% children experienced viral blips, but these had no significant impact on immune reconstitution or inflammation. Persistent detectable viraemia occurred in one-third of children and prevented further immune reconstitution, but had little impact on inflammatory biomarkers. Virological rebound to ≥5000 copies/ml was associated with arrested immune reconstitution, rising IL-6 and increased risk of clinical disease progression. CONCLUSIONS: As viral load testing becomes more available in sub-Saharan Africa, repeat testing algorithms will be required to identify those with virological rebound, who need switching to prevent disease progression, whilst preventing unnecessary second-line regimen initiation in the majority of children with detectable viraemia who remain at low risk of disease progression.

Inflammatory pathways amongst people living with HIV in Malawi differ according to socioeconomic status.

BACKGROUND: Non-communicable diseases (NCDs) are increased amongst people living with HIV (PLWH) and are driven by persistent immune activation. The role of socioeconomic status (SES) in immune activation amongst PLWH is unknown, especially in low-income sub-Saharan Africa (SSA), where such impacts may be particularly severe. METHODS: We recruited Malawian adults with CD4<100 cells/ul two weeks after starting ART in the REALITY trial (NCT01825031), as well as volunteers without HIV infection. Clinical assessment, socioeconomic evaluation, blood draw for immune activation markers and carotid femoral pulse wave velocity (cfPWV) were carried out at 2- and 42-weeks post-ART initiation. Socioeconomic risk factors for immune activation and arterial stiffness were assessed using linear regression models. RESULTS: Of 279 PLWH, the median (IQR) age was 36 (31-43) years and 122 (44%) were female. Activated CD8 T-cells increased from 70% amongst those with no education to 88% amongst those with a tertiary education (p = 0.002); and from 71% amongst those earning less than 10 USD/month to 87% amongst those earning between 100-150 USD/month (p = 0.0001). Arterial stiffness was also associated with higher SES (car ownership p = 0.003, television ownership p = 0.012 and electricity access p = 0.029). Conversely, intermediate monocytes were higher amongst those with no education compared to a tertiary education (12.6% versus 7.3%; p = 0.01) and trended towards being higher amongst those earning less than 10 USD/month compared to 100-150 USD/month (10.5% versus 8.0%; p = 0.08). Water kiosk use showed a protective association against T cell activation (p = 0.007), as well as endothelial damage (MIP1ß, sICAM1 and sVCAM1 p = 0.047, 0.026 and 0.031 respectively). CONCLUSIONS: Socioeconomic risk factors for persistent inflammation amongst PLWH in SSA differ depending on the type of inflammatory pathway. Understanding these pathways and their socioeconomic drivers will help identify those at risk and target interventions for NCDs. Future studies assessing drivers of inflammation in HIV should include an SES assessment.

Microvascular function in pre-eclampsia is influenced by insulin resistance and an imbalance of angiogenic mediators.

In preeclampsia, maternal microvascular function is disrupted and angiogenesis is dysfunctional. Insulin resistance that occurs in some pregnancies also pathologically affects microvascular function. We wished to examine the relationship of angiogenic mediators and insulin resistance on microvascular health in pregnancy. We performed a nested, case-control study of 16 women who developed preeclampsia with 17 normal pregnant controls. We hypothesized that the impaired microvascular blood flow in preeclamptic women associated with an increased ratio of the antiangiogenic factors; (s-endoglin [sEng] and soluble fms-like tyrosine kinase-1 [sFlt-1]) and proangiogenic molecule (placental growth factor [PlGF]) could be influenced by insulin resistance. Serum samples taken after 28 weeks of gestation were measured for the angiogenic factors, insulin, and glucose alongside the inflammatory marker; tumor necrosis factor-α and endothelial activation, namely; soluble vascular cell adhesion molecule 1, intercellular adhesion molecule-1, and e-selectin. Maternal microvascular blood flow, measured by strain gauge plethysmography, correlated with ratios of pro- and antiangiogenic mediators independently of preeclampsia. Decreased microvascular function measured in preeclampsia strongly correlated with both the antiangiogenic factor (sFlt-1 + sEng): PlGF ratio and high levels of insulin resistance, and combining insulin resistance with antiangiogenic factor ratios further strengthened this relationship. In pregnancy, microvascular blood flow is strongly associated with perturbations in pro- and antiangiogenic mediators. In preeclampsia, the relationship of maternal microvascular dysfunction with antiangiogenic mediators is strengthened when combined with insulin resistance.

all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase.

all-trans-Retinoic acid (atRA) has important effects on the developing and mature cardiovascular system. Nitric oxide (NO) production has been associated with the atRA-induced differentiation of neuronal cells, and we hypothesized that NO may also mediate certain actions of atRA in the cardiovascular system. We studied the effects of atRA on NO production by endothelial cells and determined whether regulation of enzymes responsible for metabolism of asymmetric dimethylarginine (ADMA) contributed to the effects seen. Murine endothelioma (sEnd.1) cells were incubated with or without atRA. Nitrite production was determined using the Griess reaction. The expression of NO synthase (NOS) and dimethylarginine dimethylaminohydrolase (DDAH) genes was determined by Northern blotting. A reporter gene assay was also used to study the effect of atRA on the DDAH II promoter. atRA significantly increased nitrite production by sEnd.1 cells despite no increase in eNOS expression. atRA also increased DDAH II gene expression and promoter activity and reduced the ratio of ADMA to symmetric dimethylarginine (SDMA) in culture medium. The DDAH inhibitor 4124W significantly reduced the induction of NO synthesis by atRA. The present study demonstrates that atRA increases NO synthesis in endothelial cells without increasing eNOS expression. atRA also increases the expression of DDAH II, the predominant DDAH isoform in endothelial cells. Our data suggests that the induction of NO synthesis by atRA may be facilitated by DDAH II. This pathway may help to explain some of the effects of atRA on the cardiovascular system.

Metabolism of asymmetric dimethylarginines is regulated in the lung developmentally and with pulmonary hypertension induced by hypobaric hypoxia.

BACKGROUND: Nitric oxide (NO) plays an important part in lowering pulmonary vascular resistance after birth, and in persistent pulmonary hypertension of the newborn (PPHN), NO-mediated dilation is dysfunctional. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) circulates in plasma, and its concentrations are elevated in certain cardiovascular diseases, including pulmonary hypertension. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), the activity of which regulates ADMA concentrations and provides a mechanism for modulating NO synthase in vivo. We investigated the changes in expression and activity of the 2 isoforms of DDAH in lungs from newborn piglets both during normal development and in PPHN. METHODS AND RESULTS: Using Western blotting, we showed that DDAHI expression did not change in the normal developing lung; however, DDAHII increased after birth and reached a peak at 1 day. This was reflected in an increase in total DDAH activity according to an L-citrulline assay. With pulmonary hypertension, no changes in DDAHI expression were observed, but DDAHII expression was markedly decreased compared with age-matched controls. Total DDAH activity was similarly reduced. CONCLUSIONS: These results indicate that each DDAH isoform is differentially regulated during both lung development and PPHN. Suppression of DDAHII isoform expression may be a mechanism underlying PPHN.

Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy.

Mutations in prominin 1 (PROM1) have been shown to result in retinitis pigmentosa, macular degeneration and cone-rod dystrophy. Because of the putative role of PROM1 in hippocampal neurogenesis, we examined two kindreds with the same R373C PROM1 missense mutation using our established paradigm to study brain structure and function. As the protein encoded by PROM1, known as CD133, is used to identify stem/progenitor cells that can be found in peripheral blood and reflect endothelial reparatory mechanisms, other parameters were subsequently examined that included measures of vascular function, endothelial function and angiogenic capacity. We found that aspects of endothelial function assayed ex vivo were abnormal in patients with the R373C PROM1 mutation, with impaired adhesion capacity and higher levels of cellular damage. We also noted renal infections, haematuria and recurrent miscarriages possibly reflecting consequences of abnormal tubular modelling. Further studies are needed to confirm these findings.

Developmental regulation of GTP-CH1 in the porcine lung and its relationship to pulmonary vascular relaxation.

Nitric oxide (NO) plays an important role in lowering pulmonary vascular resistance after birth. However, in persistent pulmonary hypertension of the newborn (PPHN) NO-mediated dilation is dysfunctional. GTP-cyclohydrolase 1 (GTP-CH1) is the rate-limiting enzyme for the biosynthesis of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) an essential cofactor for nitric oxide synthase (NOS) activity. Suboptimal levels of BH4 may result in NOS uncoupling and the subsequent generation of harmful superoxide anions. We therefore investigated the functional effects of supplementing BH4 and/or a superoxide dismutase mimetic (MnTMPyP) in porcine intrapulmonary arteries from normal animals and from a porcine model of PPHN. We investigated whether any functional effects of BH4 could be explained by changes in GTP-CH1 expression. Supplementation of BH4 significantly improved endothelium-dependent relaxations in arteries from 3- and 14-d-old healthy animals, whereas no effect was seen in vessels from younger animals and adults. GTP-CH1 protein expression was lowest at 3 and 14 d, suggesting a rate limitation of BH4 at this time. BH4 supplementation alone did not improve the relaxant response to acetylcholine in arteries obtained in a porcine model of PPHN. Furthermore, GTP-CH1 protein expression was normal for age. However, co-treatment with both BH4 and MnTMPyP restored endothelial function. GTP-CH1 is developmentally regulated in the pulmonary vasculature of neonates and this results in a functionally significant limitation of BH4. Although GTP-CH-1/BH4 levels alone do not explain the profound endothelial dysfunction seen in PPHN, increasing NO bioavailability by supplementing BH4 and quenching superoxide may prove to be therapeutically beneficial.

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.