A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

Plasma Sphingolipid Profile Associated With Subclinical Atherosclerosis and Clinical Disease Markers of Systemic Lupus Erythematosus: Potential Predictive Value.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects females more than males, with African Americans developing more severe manifestation of the disease. SLE patients are at increased risk for cardiovascular disease (CVD), and SLE women 35-44 years old have 50 fold the incidence rate of CVD. Because SLE patients do not follow the typical age and gender pattern for CVD, but instead an accelerated disease course, the traditional biomarkers of elevated LDL and total cholesterol levels do not accurately assess their CVD risk. Recently, we have reported that African American SLE patients had higher ceramide, hexosylceramide, sphingosine and dihydrosphingosine 1-phosphate levels compared to their healthy controls, and those with atherosclerosis had higher sphingomyelin and sphingoid bases levels than those without (PLoS One. 2019; e0224496). In the current study, we sought to identify sphingolipid species that correlate with and pose the potential to predict atherosclerosis severity in African American SLE patients. Plasma samples from a group of African American predominantly female SLE patients with well-defined carotid atherosclerotic plaque burden were analyzed for sphingolipidomics using targeted mass spectroscopy. The data demonstrated that at baseline, plaque area and C3 values correlated inversely with most lactoceramide species. After one-year follow-up visit, values of the change of plaque area correlated positively with the lactoceramide species. There was no correlation between LDL-C concentrations and lactoceramide species. Taken together, lactocylcermide levels may have a 'predictive' value and sphingolipidomics have an added benefit to currently available tools in early diagnosis and prognosis of African American SLE patients with CVD.

Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins.

Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.

Race disparity in blood sphingolipidomics associated with lupus cardiovascular comorbidity.

Systemic lupus erythematous (SLE) is a chronic multi-organ autoimmune disease. Genetic and environmental factors contribute to disease onset and severity. Sphingolipids are signaling molecules involved in regulating cell functions and have been associated with multiple genetic disease processes. African-Americans are more likely to suffer from SLE morbidity than Whites. The Medical University of South Carolina has banked plasma samples from a well-characterized lupus cohort that includes African-Americans and Whites. This study examined the influence of race on plasma sphingolipid profiles in SLE patients and association of sphingolipid levels with comorbid atherosclerosis and SLE disease activity. Mass spectrometry revealed that healthy African-Americans had higher sphingomyelin levels and lower lactosylcermide levels compared to healthy Whites. SLE patients, irrespective of race, had higher levels of ceramides, and sphingoid bases (sphingosine and dihydrosphingosine) and their phosphates compared to healthy subjects. Compared to African-American controls, African-American SLE patients had higher levels of ceramides, hexosylceramides, sphingosine and dihydrosphingosine 1-phosphate. Compared to White controls, White SLE patients exhibited higher levels of sphingoid bases and their phosphates, but lower ratios of C16:0 ceramide/sphingosine 1-phosphate and C24:1 ceramide/sphingosine 1-phosphate. White SLE patients with atherosclerosis exhibited lower levels of sphingoid bases compared to White SLE patients without atherosclerosis. In contrast, African-American SLE patients with atherosclerosis had higher levels of sphingoid bases and sphingomyelins compared to African-American SLE patients without atherosclerosis. Compared to White SLE patients with atherosclerosis, African-American SLE patients with atherosclerosis had higher levels of select sphingolipids. Plasma levels of sphingosine, C16:0 ceramide/sphingosine 1-phosphate ratio and C24:1 ceramide/sphingosine 1-phosphate ratio significantly correlated with SLEDAI in the African-American but not White SLE patients. The C16:0 ceramide/sphingosine 1-phosphate ratio in SLE patients, and levels of C18:1 and C26:1 lactosylcermides, C20:0 hexosylceramide, and sphingoid bases in SLE patients with atherosclerosis could be dependent on race. Further ethnic studies in SLE cohorts are necessary to verify use of sphingolipidomics as complementary diagnostic tool.

Sphingolipids as Biomarkers of Disease.

Despite the advancements in modern medicine, there are still difficulties in diagnosing common illnesses. The invasiveness and price of the tests used to follow up certain diseases can be a barrier to proper patient follow-up. Sphingolipids are a diverse category of lipids. They are structural molecules in cell membranes and signaling molecules involved in the regulation of crucial cell functions, including cell growth, differentiation, proliferation and apoptosis. Recent research has shown that abnormal sphingolipid metabolism is associated with genetic and metabolic disease processes. Given their crucial role to maintain homeostasis within the body, sphingolipids have been investigated as potential biomarkers to predict disease in the population. Here we discuss how sphingolipids levels are altered in different diseases, thus illustrating their possible use as diagnostic and prognostic biomarkers for disease.

Oridonin inhibits aberrant AKT activation in breast cancer.

Aberrant activation of phosphatidylinosito-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) signaling in cancer has led to pursuit of inhibitors for targeting this pathway. However, inhibitors of PI3K and AKT have failed to yield efficacious results without adverse effects. Here, we screened a library containing 441 authenticated traditional chinese medicine (TCM) plant extracts by examining their effect on cell viability of a human mammary epithelial cell line HMEC-PIK3CAH1047R, which expresses mutant PIK3CAH1047R and has constitutively active AKT signaling. We found that Oridonin, an extract from Rabdosia rubescens, reduced cell viability to the greatest extent. Oridonin binds to AKT1 and potentially functions as an ATP-competitive AKT inhibitor. Importantly, Oridonin selectively impaired tumor growth of human breast cancer cells with hyperactivation of PI3K/AKT signaling. Moreover, Oridonin prevented the initiation of mouse mammary tumors driven by PIK3CAH1047R. Our results suggest that Oridonin may serve as a potent and durable therapeutic agent for the treatment of breast cancers with hyperactivation of PI3K/AKT signaling.

Yogic breathing when compared to attention control reduces the levels of pro-inflammatory biomarkers in saliva: a pilot randomized controlled trial.

BACKGROUND: Self-report measures indicate that Yoga practices are perceived to reduce stress; however, molecular mechanisms through which YB affects stress are just beginning to be understood. While invasive sampling such as blood has been widely used to measure biological indicators such as pro-inflammatory biomarkers, the use of saliva to measure changes in various biomolecules has been increasingly recognized. As Yoga practice stimulates salivary secretion, and saliva is considered a source of biomarkers, changes in salivary cytokines before and after Yogic breathing exercise as specified in an ancient Tamil script, Thirumanthiram, were examined using a Cytokine Multiplex to compare to Attention Control (AC) group. METHODS: Twenty healthy volunteers were randomized into two groups stratified by gender (N = 10 per YB and AC groups); The YB group performed two YB exercises, each for ten minutes, for a total of twenty minutes in a single session as directed by a trained Yoga instructor. The AC group read a text of their choice for 20 min. Saliva was collected immediately after YB training at 0, 5, 10, 15 and 20 min and analyzed by Multiplex enzyme linked immunosorbent assay (ELISA). RESULTS: The levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein -1 (MCP-1) were significantly reduced in YB group when compared to AC group. The level of reduction of IL-8 was significant at all time points tested, whereas IL-1ß showed reduction at 15 and 20 min time points (p < 0.05), and MCP-1 level was marginally different at 5-20 min. There were no significant differences between YB and AC groups in the salivary levels of IL-1RA, IL-6, IL-10, IL-17, IP-10, MIP-1b, and TNF-α. CONCLUSIONS: These data are the first to demonstrate the feasibility of detecting salivary cytokines using multiplex assay in response to a Yoga practice. This study was registered in Clinical Trials.gov # NCT02108769.

Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development.

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1.

The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane.

Fibulin1C peptide induces cell attachment and extracellular matrix deposition in lung fibroblasts.

Fibulin-1 is an extracellular matrix (ECM) protein, levels of which are elevated in serum and lung tissue from patients with idiopathic pulmonary fibrosis compared to healthy volunteers. Inhibition of fibulin-1C, one of four fibulin-1 isoforms, reduced proliferation and wound healing in human airway smooth muscle (ASM) cells. This study identified the bioactive region/s of fibulin-1C which promotes fibrosis. Seven fibulin-1C peptides were synthesized and used to pre-coat tissue culture plates before lung derived ASM cells and fibroblasts from patients with pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) or neither disease (Control) were plated. Peptide effects on in vitro measures of fibrosis: cell attachment, proliferation and viability, and ECM deposition, were examined. Among these peptides, peptide 1C1 (FBLN1C1) enhanced ASM cell and fibroblast attachment. FBLN1C1 increased mitochondrial activity and proliferation in fibroblasts. In addition, FBLN1C1 stimulated fibulin1 deposition in PF and COPD fibroblasts, and augmented fibronectin and perlecan deposition in all three groups. Peptides FBLN1C2 to FBLN1C7 had no activity. The active fibulin-1C peptide identified in this study describes a useful tool for future studies. Ongoing investigation of the role of fibulin-1 may reveal the mechanisms underlying the pathphysiology of chronic lung diseases.

A novel intracellular fibulin-1D variant binds to the cytoplasmic domain of integrin beta 1 subunit.

Fibulin-1 is a member of a growing family of proteins that includes eight members and is involved in cellular functions such as adhesion, migration and differentiation. Fibulin-1 has also been implicated in embryonic development of the heart and neural crest-derived structures. It is an integral part of the extracellular matrix (ECM) and has been shown to bind to a multitude of ECM proteins. However, fibulin-1 was first identified as a protein purified from placental extracts that binds to the cytoplasmic domain of integrin ß1. Human fibulin-1 is alternatively spliced into four different isoforms namely A-D. These isoforms share a common N-terminus sequence that contains a secretion sequence but differ in their carboxy-terminal fibulin-1 module. In this report we identify a new splice variant of fibulin-1 that differs from all other fibulin-1 variants in the N-terminus sequence and has a similar carboxy-terminus sequence as fibulin-1D. This variant that we named fibulin-1D prime (fibulin-1D') lacks a secretion sequence and the anaphlatoxin region of fibulin-1 variants. The protein has an apparent molecular weight of 70.5kDa. Herein we show that fibulin-1D' binds to the intracellular domain of integrin ß1 as well as to integrin α5ß1. The protein was localized intracellularly in CHO cells transfected with a pEF4 plasmid containing full-length coding sequence of fibulin-1D'. We also localized the protein in human placenta. We propose that the fibulin-1D' variant might play a role in early embryo development as well as in modulating integrin ß1 functions including adhesion and motility.

Elevated transferrin saturation, health-related quality of life and telomere length.

We sought to examine the relationship between elevated transferrin saturation (TS) and measures of health status (telomere length and patient-reported health-related quality of life) to assess whether elevated TS is associated with negative patient outcomes beyond increased risk for morbidity and mortality, using a cross-sectional analysis of the Hemochromatosis and Iron Overload Screening Study supplemented with assays for leukocyte telomere length in adults ≥25 years old (n = 669). Among individuals with elevated TS (≥45 % for women and ≥50 % for men), who also had a usual source of care, only 5.2 % reported ever being told by a doctor that they had an elevated iron condition. In a fully adjusted general linear regression model controlling for demographic characteristics as well as health conditions associated with iron overload, elevated TS versus non-elevated TS was associated with worse general health status (60.4 vs. 63.8, P < 0.05), mental health status (76.5 vs. 82.2, P < 0.0001) and shorter telomere length (241.4 vs. 261.3, P < 0.05). Increased surveillance of elevated TS may be in order as elevated TS is associated with decreased health status and very few patients with elevated TS are aware of their condition.

Cellularized microcarriers as adhesive building blocks for fabrication of tubular tissue constructs.

To meet demands of vascular reconstruction, there is a need for prosthetic alternatives to natural blood vessels. Here we explored a new conduit fabrication approach. Macroporous, gelatin microcarriers laden with human umbilical vein endothelial cells and aortic smooth muscle cells were dispensed into tubular agarose molds and found to adhere to form living tubular tissues. The ability of cellularized microcarriers to adhere to one another involved cellular and extracellular matrix bridging that included the formation of epithelium-like cell layers lining the lumenal and ablumenal surfaces of the constructs and the deposition of collagen and elastin fibers. The tubular tissues behaved as elastic solids, with a uniaxial mechanical response that is qualitatively similar to that of native vascular tissues and consistent with their elastin and collagen composition. Linearized measures of the mechanical response of the fabricated tubular tissues at both low and high strains were observed to increase with duration of static culture, with no significant loss of stiffness following decellularization. The findings highlight the utility of cellularized macroporous gelatin microcarriers as self-adhering building blocks for the fabrication of living tubular structures.

Telomere length and elevated iron: the influence of phenotype and HFE genotype.

Elevated body iron stores are associated with morbidity and mortality due to oxidative stress. Hereditary hemochromatosis, a common condition caused by HFE gene mutations, can lead to excess iron storage and disease but clinical penetrance of HFE gene mutations is low and many people with elevated iron stores lack HFE mutations. We analyzed data from the Hemochromatosis and Iron Overload Screening Study to assess the relationship among HFE genotype (individuals with either homozygous or compound heterozygous status for C282Y and/or H63D HFE mutations were defined as genotype positive, or G+), elevated iron phenotype (individuals exceeding gender-specific transferrin saturation and serum ferritin threshold levels were considered phenotype positive, or P+), and leukocyte telomere length, a marker of biological aging and cumulative oxidative stress. In unadjusted analyses in comparison to individuals who were G-P-, G+P- were not significantly different (OR 0.74; 95% CI 0.26-2.04), while the G+P+ (OR 2.03; 95% CI 1.15-3.56), and G-P+ (OR 2.24; 95% CI 1.5-3.29) had increased risk of short telomeres (<=25th percentile) rather than long telomeres (>=75th percentile). In analyses adjusting for age, gender, and race/ethnicity, the effect of individuals with elevated iron phenotypes having short telomeres persisted with G+P+ individuals (OR 1.94; 95% CI 1.02-3.72), and G-P+ individuals (OR 2.17; 95% CI 1.39-3.39) being significantly different from the G-P- group. In conclusion, elevated iron phenotype, but not HFE genotype, was associated with shortened telomeres. Further studies will be needed to determine whether telomere length provides a marker for morbidities specifically associated with iron overload.

Association of elevated cytokines with childhood adversity in a sample of healthy adults.

OBJECTIVE: Childhood trauma has been associated adult stress-related disorders. However, little is known about physiologic alterations in adults with a history of early life trauma that do not have current psychiatric or medical diagnoses. In this study, the relationships between childhood adversity and cytokine and C-reactive protein (CRP) levels in healthy adults were examined. METHOD: Participants included men (n = 18) and women (n = 20) who did not meet DSM-IV criteria for Axis I psychiatric disorders or any major medical illness. Cytokine and CRP levels were obtained from baseline blood samples. Subjects completed the Early Trauma Inventory Self Report (ETI-SR). The primary outcomes included serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL1-ß), and CRP levels. In addition, the mean numbers of traumatic experiences (sexual, physical, emotional, general, and the summed total) were measured. RESULTS: Significant positive associations were found between the total ETI score and IL-6 (p = 0.05), IL1-ß (p < 0.05), and TNF-α (p = 0.01). Significant positive correlations were found between the number of general traumas and IL1-ß (p < 0.05), TNF-α (p < 0.05), and IL-6 (p < 0.01). Neither the total number of traumas nor any of the trauma subscales were significantly associated with CRP levels. CONCLUSIONS: The positive association between childhood trauma and basal cytokine levels supports the extant literature demonstrating the long-term impact of childhood trauma and stress on homeostatic systems. Importantly, this association was found in healthy adults, suggesting that these alterations may precede the development of significant stress-related psychiatric disorder or disease.

Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus.

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.

A disintegrin and metalloenzyme (ADAM) 17 activation is regulated by α5ß1 integrin in kidney mesangial cells.

BACKGROUND: The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5ß1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation. METHODOLOGY/PRINCIPAL FINDINGS: We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5ß1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5ß1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5ß1 integrin interaction. In cell-free assays, we showed that purified α5ß1 integrin and ß1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of ß1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of ß1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing ß1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT. CONCLUSIONS/SIGNIFICANCE: Our data show for the first time that ß1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5ß1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

Fibulin-1 is required during cardiac ventricular morphogenesis for versican cleavage, suppression of ErbB2 and Erk1/2 activation, and to attenuate trabecular cardiomyocyte proliferation.

BACKGROUND: Trabeculation is an integral component of cardiac ventricular morphogenesis and is dependent on the matrix metalloproteinase, ADAMTS1. A substrate of ADAMTS1 is the proteoglycan versican which is expressed in the developing ventricle and which has been implicated in trabeculation. Fibulin-1 is a versican and ADAMTS1-binding extracellular matrix protein required for ventricular morphogenesis. Here we investigated the involvement of fibulin-1 in ADAMTS1-mediated cleavage of versican in vitro, and the involvement of fibulin-1 in versican cleavage in ventricular morphogenesis. RESULTS: We show that fibulin-1 is a cofactor for ADAMTS1-dependent in vitro cleavage of versican V1, yielding a 70-kDa amino-terminal fragment. Furthermore, fibulin-1-deficiency in mice was found to cause a significant reduction (>90%) in ventricular levels of the 70-kDa versican V1 cleavage product and a 2-fold increase in trabecular cardiomyocyte proliferation. Decreased versican V1 cleavage and augmented trabecular cardiomyocyte proliferation in fibulin-1 null hearts is accompanied by increased ventricular activation of ErbB2 and Erk1/2. By contrast, versican deficiency was found to lead to decreased cardiomyocyte proliferation and reduced ventricular trabeculation. CONCLUSION: We conclude that fibulin-1 regulates versican-dependent events in ventricular morphogenesis by promoting ADAMTS1 cleavage of versican leading to suppression of trabecular cardiomyocyte proliferation mediated by the ErbB2-Map kinase pathway.

ALTERED BLOOD SPHINGOLIPIDOMICS AND ELEVATED PLASMA INFLAMMATORY CYTOKINES IN COMBAT VETERANS WITH POST-TRAUMATIC STRESS DISORDER.

Patients with post-traumatic stress disorder (PTSD) have greater risk of developing cardiovascular disease (CVD). While chronically elevated plasma cholesterol and pro-inflammatory cytokines levels increase CVD risk, several studies have shown that cholesterol reduction does not reduce CVD risk. Acid sphingomyelinase (ASMase) activation has been implicated in both CVD and major depressive disorder. We investigated plasma pro-inflammatory cytokine levels, ASMase activity, and changes in sphingolipids in PTSD patients compared to healthy controls. Levels of interleukin 6, interleukin 10, interferon-γ and tumor necrosis factor-α were higher in PTSD patients than controls. Plasma ASMase activity and sphingosine 1-phosphate were higher in the PTSD group (1.6-fold and 2-fold, respectively; p<0.05). The results suggest that CVD risk factors in PTSD patients remain high despite cholesterol reduction.

Fibulin-1 is a marker for arterial extracellular matrix alterations in type 2 diabetes.

BACKGROUND: Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined. METHODS: Mammary artery specimens from 17 men with type 2 diabetes and 18 nondiabetic individuals were used for microarray expression profiling, quantitative real-time PCR, immunoassay, and immunohistochemical analyses. A derived candidate marker, fibulin-1, which is an elastin-associated matrix molecule, was measured immunochemically in plasma from (a) 70 patients scheduled for vascular surgery, (b) 305 patients with type 2 diabetes examined with carotid ultrasonography and echocardiography, and (c) 308 patients with type 2 diabetes, followed for 15 years. RESULTS: The most upregulated transcript in nonatherosclerotic arterial tissue from patients with type 2 diabetes encoded the extracellular matrix protein, fibulin-1. Higher concentrations of fibulin-1-protein were present in artery extracts from patients with diabetes than extracts from individuals without diabetes, and increased fibulin-1 immunostaining was apparent around the external elastic lamina of diabetic arteries. Patients with diabetes displayed increased plasma concentrations of fibulin-1 (P = 0.006). Plasma fibulin-1 concentrations correlated with hemoglobin A(1c) (P < 0.001), arterial stiffness indices including pulse pressure (P < 0.001), and carotid compliance (P = 0.004), as well as plasma N-terminal pro-B-type natriuretic peptide concentrations (P < 0.001) and were predictive of 15-year mortality (P = 0.013). CONCLUSIONS: Fibulin-1 accumulates in the arterial wall and in plasma of patients with type 2 diabetes, and appears to be a factor associated with arterial extracellular matrix changes in type 2 diabetes.