Identifying the potential causal role of insomnia symptoms on 11,409 health-related outcomes: a phenome-wide Mendelian randomisation analysis in UK Biobank.

BACKGROUND: Insomnia symptoms are widespread in the population and might have effects on many chronic conditions and their risk factors but previous research has focused on select hypothesised associations/effects rather than taking a systematic hypothesis-free approach across many health outcomes. METHODS: We performed a Mendelian randomisation (MR) phenome-wide association study (PheWAS) in 336,975 unrelated white-British UK Biobank participants. Self-reported insomnia symptoms were instrumented by a genetic risk score (GRS) created from 129 single-nucleotide polymorphisms (SNPs). A total of 11,409 outcomes from UK Biobank were extracted and processed by an automated pipeline (PHESANT) for the MR-PheWAS. Potential causal effects (those passing a Bonferroni-corrected significance threshold) were followed up with two-sample MR in MR-Base, where possible. RESULTS: Four hundred thirty-seven potential causal effects of insomnia symptoms were observed for a diverse range of outcomes, including anxiety, depression, pain, body composition, respiratory, musculoskeletal and cardiovascular traits. We were able to undertake two-sample MR for 71 of these 437 and found evidence of causal effects (with directionally concordant effect estimates across main and sensitivity analyses) for 30 of these. These included novel findings (by which we mean not extensively explored in conventional observational studies and not previously explored using MR based on a systematic search) of an adverse effect on risk of spondylosis (OR [95%CI] = 1.55 [1.33, 1.81]) and bronchitis (OR [95%CI] = 1.12 [1.03, 1.22]), among others. CONCLUSIONS: Insomnia symptoms potentially cause a wide range of adverse health-related outcomes and behaviours. This has implications for developing interventions to prevent and treat a number of diseases in order to reduce multimorbidity and associated polypharmacy.

Therapeutic Metabolic Reprograming Using microRNAs: From Cancer to HIV Infection.

MicroRNAs (miRNAs) are crucial regulators of cellular processes, including metabolism. Attempts to use miRNAs as therapeutic agents are being explored in several areas, including the control of cancer progression. Recent evidence suggests fine tuning miRNA activity to reprogram tumor cell metabolism has enormous potential as an alternative treatment option. Indeed, cancer growth is known to be linked to profound metabolic changes. Likewise, the emerging field of immunometabolism is leading to a refined understanding of how immune cell proliferation and function is governed by glucose homeostasis. Different immune cell types are now known to have unique metabolic signatures that switch in response to a changing environment. T-cell subsets exhibit distinct metabolic profiles which underlie their alternative differentiation and phenotypic functions. Recent evidence shows that the susceptibility of CD4+ T-cells to HIV infection is intimately linked to their metabolic activity, with many of the metabolic features of HIV-1-infected cells resembling those found in tumor cells. In this review, we discuss the use of miRNA modulation to achieve metabolic reprogramming for cancer therapy and explore the idea that the same approach may serve as an effective mechanism to restrict HIV replication and eliminate infected cells.

Evidence for Genetic Correlations and Bidirectional, Causal Effects Between Smoking and Sleep Behaviors.

INTRODUCTION: Cigarette smokers are at increased risk of poor sleep behaviors. However, it is largely unknown whether these associations are due to shared (genetic) risk factors and/or causal effects (which may be bidirectional). METHODS: We obtained summary-level data of genome-wide association studies of smoking (smoking initiation [n = 74 035], cigarettes per day [n = 38 181], and smoking cessation [n = 41 278]) and sleep behaviors (sleep duration and chronotype, or "morningness" [n = 128 266] and insomnia [n = 113 006]). Using linkage disequilibrium (LD) score regression, we calculated genetic correlations between smoking and sleep behaviors. To investigate causal effects, we employed Mendelian randomization (MR), both with summary-level data and individual-level data (n = 333 581 UK Biobank participants). For MR with summary-level data, individual genetic variants were combined with inverse variance-weighted meta-analysis, weighted median regression, MR-Robust Adjusted Profile Score, and MR Egger methods. RESULTS: We found negative genetic correlations between smoking initiation and sleep duration (rg = -.14, 95% CI = -0.26 to -0.01) and smoking cessation and chronotype (rg = -.18, 95% CI = -0.31 to -0.06), and positive genetic correlations between smoking initiation and insomnia (rg = .27, 95% CI = 0.06 to 0.49) and cigarettes per day and insomnia (rg = .15, 95% CI = 0.01 to 0.28). MR provided strong evidence that smoking more cigarettes causally decreases the odds of being a morning person, (RAPS) and weak evidence that insomnia causally increases smoking heaviness and decreases smoking cessation odds. CONCLUSIONS: Smoking and sleep behaviors show moderate genetic correlation. Heavier smoking seems to causally affect circadian rhythm and there is some indication that insomnia increases smoking heaviness and hampers cessation. Our findings point to sleep as a potentially interesting smoking treatment target. IMPLICATIONS: Using LD score regression, we found evidence that smoking and different sleep behaviors (sleep duration, chronotype (morningness), and insomnia) are moderately genetically correlated-genetic variants associated with less or poorer sleep also increased the odds of smoking (more heavily). MR analyses suggested that heavier smoking causally affects circadian rhythm (decreasing the odds of being a morning person) and there was some indication that insomnia increases smoking heaviness and hampers smoking cessation. Our findings indicate a complex, bidirectional relationship between smoking and sleep behaviors and point to sleep as a potentially interesting smoking treatment target.

Spatial variability of on-bicycle black carbon concentrations in the megacity of São Paulo: A pilot study.

In 2015, a controversial bicycle lane was installed on Paulista Avenue -a thoroughfare in the heart of the megacity of São Paulo with a high rate of motorised vehicles. For the first time, on-bicycle air pollution concentrations were assessed along this lane using black carbon (BC) as an indicator of fossil fuel combustion. We measured BC concentrations with a hand-held microaethalometer at a high temporal resolution, enabling the capture of fine spatial gradients along the route. Although this new link expanded the city's cycling network, our pioneering study showed that BC concentrations were large (mean 8.5 µg m-3) with extreme values reaching 24.0 µg m-3, comparable to concentrations found in many megacities. In agreement with other studies, we observed that concentrations decreased about 1.6 times on a section of the bicycle lane running through a calmer neighbourhood, which could indicate the potential to safeguard the health of cyclists by installing lanes with greater separation from main roads, such as Paulista Avenue. This pilot work paves the way to more detailed studies aiming to map out the spatial distribution of other traffic-related pollutants across the city's 458-km long bicycle network.

Role of tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and associated proteins in coronary artery disease and cardiac events.

Tumour necrosis factor alpha converting enzyme (TACE/ADAM17) is a member of the A disintegrin and metalloproteinase (ADAM) family of ectodomain shedding proteinases. It regulates many inflammatory processes by cleaving several transmembrane proteins, including tumour necrosis factor alpha (TNFα) and its receptors tumour necrosis factor alpha receptor 1 and tumour necrosis factor alpha receptor 2. There is evidence that TACE is involved in several inflammatory diseases, such as ischaemia, heart failure, arthritis, atherosclerosis, diabetes and cancer as well as neurological and immune diseases. This review summarizes the latest discoveries regarding the mechanism of action and regulation of TACE. It also focuses on the role of TACE in atherosclerosis and coronary artery disease (CAD), highlighting clinical studies that have investigated its expression and protein activity. The multitude of substrates cleaved by TACE make this enzyme an attractive target for therapy and a candidate for biomarker research and development in CAD.

A Central Bioactive Region of LTBP-2 Stimulates the Expression of TGF-ß1 in Fibroblasts via Akt and p38 Signalling Pathways.

Latent transforming growth factor-ß-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-ß1 (TGF-ß1) but appears to be implicated in the regulation of TGF-ß1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-ß1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-ß1 levels in the medium. However, the TGF-ß1 increase was due to an upregulation of TGF-ß1 expression and secretion rather than a displacement of matrix-stored TGF-ß1. The secreted TGF-ß1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-ß expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-ß1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins ß1 and αVß5 showed no reduction of LTBP-2 stimulation of TGF-ß1. However, TGF-ß1 upregulation was partially inhibited by anti-αVß3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-ß1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-ß1 signalling.

Hotspots of black carbon and PM2.5 in an urban area and relationships to traffic characteristics.

Three instrumented bicycles were used to measure black carbon (BC) and PM2.5 concentrations in a midsized city in southern Brazil. The objective of this study was to map the spatial distribution of BC and PM2.5, to identify air pollution hotspots and to assess factors that may affect the concentrations of these pollutants, e.g. traffic volume, number of heavy-duty diesel vehicles (HDDV), position of traffic signals and street incline. The cyclists collected data in the city centre along streets of different traffic density during nine sampling sessions in the weekday morning and afternoon rush hours, between March 13 and April 28, 2015. The sampling by bicycle covered an area of 2.70 km2, over variable elevation, and travelled a total distance of 215 km. BC and PM2.5 exhibited a large spatial variability on a scale of tens of metres and the concentrations were positively correlated with traffic counts, but exhibited a stronger relationship with the number of HDDV. These results imply that older buses and diesel-powered trucks may be the main driver behind the high pollution levels in the city's inner core. We observed a strong relationship between BC concentrations at junctions managed by traffic signals and the quantity of HDDV. The mean BC concentration was found to be 8.10 µg m-3 near traffic signals located on an inclined street (HDDV > 100 vehicles h-1) compared to traffic signals on flat terrain (6.00 µg m-3), which can be attributed to the higher acceleration required at the start of motion. This pattern was less evident for PM2.5 concentrations.

The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche.

The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFß family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvß3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFß signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.

Impacts of air cleaners on indoor air quality in residences impacted by wood smoke.

Residential wood combustion is an important source of ambient air pollution, accounting for over 25% of fine particulate matter (PM2.5) emissions in Canada. In addition to these ambient contributions, wood smoke pollutants can enter the indoor environment directly when loading or stoking stoves, resulting in a high potential for human exposure. A study of the effectiveness of air cleaners at reducing wood smoke-associated PM2.5 of indoor and outdoor origin was conducted in 31 homes during winter 2009-10. Day 1, the residents' wood burning appliance operated as usual with no air cleaner. Days 2 and 3, the wood burning appliance was not operational and the air cleaner was randomly chosen to operate in "filtration" or "placebo filtration" mode. When the air cleaner was operating, total indoor PM2.5 levels were significantly lower than on placebo filtration days (p = 0.0001) resulting in a median reduction of 52%. There was also a reduction in the median PM2.5 infiltration factor from 0.56 to 0.26 between these 2 days, suggesting the air cleaner was responsible for increased PM2.5 deposition on filtration days. Our findings suggest that the use of an air cleaner reduces exposure to indoor PM2.5 resulting from both indoor and ambient wood smoke sources.

Ecological risk assessment of nonylphenol in coastal waters of China based on species sensitivity distribution model.

Nonylphenol (NP) is an endocrine disruptor and causes feminization and carcinogenesis in various organisms. Consequently, the environmental distribution and ecological risks of NP have received wide concern. China accounts for approximately 10% of the total NP usage in the world, but the water quality criteria of NP have not been established in China and the ecological risks of this pollutant cannot be properly assessed. This study thus aims to determine the predicted no effect concentration (PNEC) of NP and to assess the ecological risks of NP in coastal waters of China with the PNEC as water quality criteria. To obtain the HC5 (hazardous concentration for 5% of biological species) and PNEC estimates, the species sensitivity distributions (SSDs) models were built with chronic toxicity data of NP on aquatic organisms screened from the US Environmental Protection Agency (USEPA) ECOTOX database. The results showed that the PNEC for NP in freshwater and seawater was 0.48 µg L(-1) and 0.28 µg L(-1), respectively. The RQ (risk quotient) values of NP in coastal waters of China ranged from 0.01 to 69.7. About 60% of the reported areas showed a high ecological risk with an RQ value ≥ 1.00. NP therefore exists ubiquitously in coastal waters of China and it poses various risks to aquatic ecosystems in the country. This study demonstrates that the SSD methodology can provide a feasible tool for the establishment of water quality criteria for emergent new pollutants when sufficient toxicity data is available.

Impact of biogas digesters on wood utilisation and self-reported back pain for women living on rural Kenyan smallholder dairy farms.

Women living on rural Kenyan dairy farms spend significant amounts of time collecting wood for cooking. Biogas digesters, which generate biogas for cooking from the anaerobic decomposition of livestock manure, are an alternative fuel source. The objective of this study was to quantify the quality of life and health benefits of installing biogas digesters on rural Kenyan dairy farms with respect to wood utilisation. Women from 62 farms (31 biogas farms and 31 referent farms) participated in interviews to determine reliance on wood and the impact of biogas digesters on this reliance. Self-reported back pain, time spent collecting wood and money spent on wood were significantly lower (p < 0.01) for the biogas group, compared to referent farms. Multivariable linear regression showed that wood consumption increased by 2 lbs/day for each additional family member living on a farm. For an average family of three people, the addition of one cow was associated with increased wood consumption by 1.0 lb/day on biogas farms but by 4.4 lbs/day on referent farms (significant interaction variable - likely due to additional hot water for cleaning milk collection equipment). Biogas digesters represent a potentially important technology that can reduce reliance on wood fuel and improve health for Kenyan dairy farmers.

Seasonal variation of nonylphenol concentrations and fluxes with influence of flooding in the Daliao River Estuary, China.

Nonylphenol is an endocrine disruptor with harmful effects including feminization and carcinogenesis on various organisms. This study aims to investigate the distribution and ecological risks of nonylphenol in the Daliao River Estuary, China. Nonylphenol, together with other phenolic endocrine disruptors (bisphenol A, 4-t-butylphenol, 4-t-octylphenol, and 2,4-dichlorophenol), was detected in surface water and sediment on three cruises in May 2009, June 2010, and August 2010, respectively. A large flooding occurred during our sampling campaign in August and its effect on nonylphenol concentrations and fluxes in the estuary was therefore evaluated. The results showed that nonylphenol with a concentration range between 83.6-777 ng l(-1) and 1.5-456 ng g(-1) dw in surface water and sediment was the most abundant among the phenolic compounds, accounting for 59.1-81.0 and 79.9-92.1 % of the total phenolic concentration in surface water and sediment, respectively. The concentrations recorded in May and June were comparable, whereas those in August were considerably higher, mainly due to the flush of flooding. The flooding also caused a 50 times increase in nonylphenol flux from the estuary into the adjacent Bohai Sea. Nonylphenol concentrations in the estuary have exceeded the threshold level of undesirable effects with a potential risk of harm to local species, especially benthic organisms.

Association of 25-hydroxy-vitamin D levels with semen and hormonal parameters.

Vitamin D levels have been linked to various health outcomes including reproductive disorders. The purpose of this study was to explore the association between serum vitamin D level (25-hydroxy-vitamin D, or 25OHD) and semen and hormonal parameters. This is a cross-sectional study that included 170 healthy men recruited for the study of spermatogenesis from the general population. Men completed general and reproductive health questionnaires, and donated blood and semen samples. The main measures were hormonal (total and free testosterone, sex hormone-binding globulin, estradiol, follicle-stimulating hormone and luteinizing hormone) and semen parameters, adjusted (n=147) for age, body mass index (BMI), season, alcohol intake and smoking, in relation to categories of vitamin D levels, determined a priori. The mean age of the study population was 29.0±8.5 years and mean BMI was 24.3±3.2 kg m(-2). The mean 25OHD was 34.1±15.06 ng ml(-1). BMI showed a negative association with 25OHD. Sperm concentration, sperm progressive motility, sperm morphology, and total progressively motile sperm count were lower in men with '25OHD≥50 ng ml(-1)' when compared to men with '20 ng ml(-1)≤25OHD<50 ng ml(-1)'. Total sperm count and total progressive motile sperm count were lower in men with '25OHD<20 ng ml(-1)' when compared to men with '20 ng ml(-1)≤25OHD<50 ng ml(-1)'. The adjusted means of various hormonal parameters did not show statistical difference in the different categories of 25OHD. In conclusion, serum vitamin D levels at high and low levels can be negatively associated with semen parameters.

An evaluation on combination effects of phenolic endocrine disruptors by estrogen receptor binding assay.

Phenolic compounds are widely distributed in the natural environment, typically existing as a mixture at the nanomole or micromole per liter level. Among the phenolic compounds, 4-nonylphenol, 4-t-octylphenol, bisphenol A and 2,4-dichlorophenol attract the most concern due to their abundance and risks in the natural environment. The former three are known as endocrine disruptors causing feminization in various organisms, whereas the latter requires further clarification for its estrogenic effect. This study aims to evaluate the combination effects of these chemicals with estrogen receptor binding as an endpoint. An ELISA based receptor binding assay was employed to avoid radioactive pollution in the traditional assay. The results showed that all these chemicals could bind with estrogen receptor with a relative binding affinity of bisphenol A>4-t-octylphenol>4-nonylphenol>2,4-dichlorophenol. The four chemicals were further mixed in two ways, at an equipotent ratio and at an equal environmental level ratio, and their combination effects on receptor binding were evaluated with both the toxicity units method and concentration addition model. The resulting effects of both mixtures showed an antagonistic mode, which was assumed to be a general mode of action with estrogen receptor binding assay due to competitive ligand binding on receptors.

Ethnic variation in estradiol metabolism in reproductive age Asian and white women treated with transdermal estradiol.

Asian women have significantly higher serum E(2) levels during treatment with transdermal E(2) compared with white women. This finding suggests altered metabolic clearance of this steroid hormone.

Linkage of regulators of TGF-ß activity in the fetal ovary to polycystic ovary syndrome.

Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-ß activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-ß bioactivity in tissues by binding latent TGF-ß binding proteins. We therefore examined expression of fibrillins 1-3, latent TGF-ß binding proteins 1-4, and TGF-ß 1-3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-ß pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.

Sleep apnea, reproductive hormones and quality of sexual life in severely obese men.

The effect of sleep apnea on the reproductive function of obese men is not entirely elucidated. The objective of this study was to define the effect of sleep apnea on the reproductive hormones and sexual function in obese men. This study included 89 severely obese men with BMI ≥35 kg/m2 considering gastric bypass surgery. Anthropometrics (weight, and BMI), reproductive hormones, and sleep studies were measured. The sexual quality of life was assessed using the Impact of Weight on Quality of Life-Lite questionnaire (IWQOL-Lite). The mean age of our patients was 46.9 ± 11.0 years, the mean BMI was 47.8 ± 8.7 kg/m2 and the mean weight was 337.7 ± 62.4 lb. After correction for age and BMI, means of free testosterone per severity group of sleep apnea were as follows: no or mild sleep apnea 74.4 ± 3.8 pg/ml, moderate sleep apnea 68.6 ± 4.2 pg/ml, and severe sleep apnea 60.2 ± 2.92 pg/ml, P = 0.014. All other parameters of sleep apnea including hypopnea index, percent time below a SpO2 of 90%, and percent time below a SpO2 of 80% were also negatively correlated with testosterone levels after correction for age and BMI. BMI and presence of coronary artery disease decreased the sexual quality of life. Sleep apnea was associated with reduced sexual quality of life. In summary, sleep apnea negatively affects testosterone levels independent of BMI. Severely obese men had decreased sexual quality of life.

Association of aromatase (TTTAn) repeat polymorphism length and the relationship between obesity and decreased sperm concentration.

BACKGROUND: Obesity in men is associated with low sperm count, however, this finding is inconsistent. Here, we describe length of the short tandem repeat aromatase (CYP19A1) polymorphism and its relationship to increased weight and sperm count. METHODS: A cohort of 215 men was recruited from the community and BMI, hormone levels and sperm parameters were determined at enrollment. Men (196) were genotyped for length of the tetranucleotide TTTA repeats polymorphism (TTTA(n)), defined as short (S ≤ 7 repeats) or long (L > 7 repeats). Genotypes were categorized using allele combinations as 'low repeats' = S-S, or 'high repeats' = S-L/L-L. Weight and sperm parameters were examined in relation to size of TTTA(n) repeat. RESULTS: Mean (±SD) age was 29.8 ± 8.6 years and mean BMI was 25.6 ± 4.6 kg/m(2). Men with high repeats had higher estradiol (E(2)) levels (98.0 ± 33.36 pmol/l) than men with low repeats (85.9 ± 26.61 pmol/l; P= 0.026). Lower FSH levels tended to be present in men with high repeats versus men with low repeats (P= 0.052). After stratification by genotype, a negative correlation between BMI and sperm count (Pearson's coefficient = 0.406) was seen only among men with high repeats (P= 0.019). Only men with high repeats exhibited increased E(2) with increased weight. A decrease in testosterone: E(2) ratio with increasing BMI was more pronounced in men with high versus low, repeats (R(2) = 0.436 versus 0.281). CONCLUSIONS: Higher TTTA repeat numbers (>7 repeats) in the aromatase gene are associated with a negative relationship between obesity and sperm count. The effect of obesity on E(2) and sperm count appears to be absent in men with low (≤7) repeats.

LTBP-2 has multiple heparin/heparan sulfate binding sites.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca(2+) ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca(2+) dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG-LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.