Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

A trans-acting protein effect causes severe eye malformation in the Mp mouse.

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.

Thoracic aortic aneurysm frequency and dissection are associated with fibrillin-1 fragment concentrations in circulation.

RATIONALE: Mutations in fibrillin-1 are associated with thoracic aortic aneurysm (TAA) in Marfan syndrome. Genome-wide association studies also implicate fibrillin-1 in sporadic TAA. Fragmentation of the aortic elastic lamellae is characteristic of TAA. OBJECTIVE: Immunoassays were generated to test whether circulating fragments of fibrillin-1, or other microfibril fragments, are associated with TAA and dissection. METHODS AND RESULTS: Plasma samples were obtained from 1265 patients with aortic aneurysm or dissection and from 125 control subjects. Concentrations of fibrillin-1, fibrillin-2, and fibulin-4 were measured with novel immunoassays. One hundred and seventy-four patients (13%) had aneurysms with only abdominal aortic involvement (abdominal aortic aneurysm), and 1091 (86%) had TAA. Of those with TAA, 300 patients (27%) had chronic dissection and 109 (10%) had acute or subacute dissection. Associations of fragment concentrations with TAA (versus abdominal aortic aneurysm) or with dissection (versus no dissection) were estimated with odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, and smoking. Compared with controls, significantly higher percentages of aneurysm patients had detectable levels of fibrillin fragments. TAA was significantly more common (than abdominal aortic aneurysm) in the highest compared with lowest quartile of fibrillin-1 concentration (OR=2.9; 95% CI, 1.6-5.0). Relative to TAA without dissection, acute or subacute dissection (OR=2.9; 95% CI, 1.6-5.3), but not chronic dissection, was more frequent in the highest compared with lowest quartile of fibrillin-1 concentration. Neither TAA nor dissection was associated with fibrillin-2 or fibulin-4. CONCLUSIONS: Circulating fibrillin-1 fragments represent a new potential biomarker for TAA and acute aortic dissection.

Microenvironmental regulation by fibrillin-1.

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFß signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFß signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.

Assessing mechanical vibration-altered wall shear stress in digital arteries.

The aim of this study is to implement and validate a method for assessing acute vibration-altered Wall Shear Stress (WSS) in the proper volar digital artery of the non-exposed left forefinger when subjecting the right hand to mechanical vibration. These changes of WSS may be involved in Vibration White Finger. Hence, an experimental device was set-up to link a vibration shaker and an ultra-high frequency ultrasound scanner. The Womersley-based WSS was computed by picking up the maximum velocity from pulse Wave Doppler measurements and extracting the artery diameter from B-mode images through an in-house image processing technique. The parameters of the former method were optimised on numerical ultrasound phantoms of cylindrical and lifelike arteries. These phantoms were computed with the FIELD II and FOCUS platforms which mimicked our true ultrasound device. The Womersley-based WSS were compared to full Fluid Structure Interaction (FSI) and rigid wall models built from resonance magnetic images of a volunteer-specific forefinger artery. Our FSI model took into account the artery's surrounding tissues. The diameter computing procedure led to a bias of 4%. The Womersley-based WSS resulted in misestimating the FSI model by roughly 10% to 20%. No difference was found between the rigid wall computational model and FSI simulations. Regarding the WSS measured on a group of 20 volunteers, the group-averaged basal value was 3 Pa, while the vibration-altered WSS was reduced to 1 Pa, possibly triggering intimal hyperplasia mechanisms and leading to the arterial stenoses encountered in patients suffering from vibration-induced Raynaud's syndrome.

Adjuvant Radiotherapy After Radical Cystectomy for Patients with High-risk Muscle-invasive Bladder Cancer: Results of a Multicentric Phase II Trial.

BACKGROUND: High-risk muscle-invasive bladder cancer (MIBC) has a poor prognosis. Old trials showed that external beam radiotherapy (EBRT) after radical cystectomy (RC) decreases the incidence of local recurrences but induces severe toxicity. OBJECTIVE: To evaluate the toxicity and local control rate after adjuvant EBRT after RC delivered with volumetric arc radiotherapy. DESIGN, SETTING, AND PARTICIPANTS: This is a multicentric phase 2 trial. From August 2014 till October 2020, we treated 72 high-risk MIBC patients with adjuvant EBRT after RC. High-risk MIBC is defined as ≥pT3-MIBC ± lymphovascular invasion, fewer than ten lymph nodes removed, pathological positive lymph nodes, or positive surgical margins. INTERVENTION: Patients received 50 Gy in 25 fractions with intensity-modulated radiotherapy to the pelvic lymph nodes ± cystectomy bed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome is acute toxicity. We report on local relapse-free rate (LRFR), clinical relapse-free survival (CRFS), overall survival (OS), and bladder cancer-specific survival (BCSS). RESULTS AND LIMITATIONS: The median follow-up is 18 mo. Forty-two patients (61%) developed acute grade 2 gastrointestinal (GI) toxicity. Four patients (6%) had acute grade 3 GI toxicity. One patient had grade 5 diarrhea and vomiting due to obstruction at 1 mo. Two-year probabilities of developing grade ≥3 and ≥2 GI toxicity were 17% and 76%, respectively. Urinary toxicity, assessed in 17 patients with a neobladder, was acceptable with acute grade 2 and 3 urinary toxicity reported in 53% (N = 9) and 18% (N = 3) of the patients, respectively. The 2-yr LRFR is 83% ± 5% and the 2-yr CRFS rate is 43% with a median CRFS time of 12 mo (95% confidence interval: 3-21 mo). Two-year OS and BCSS are 52% ± 7% and 62% ± 7%, respectively. Shortcomings are the nonrandomized study design and limited follow-up. CONCLUSIONS: Adjuvant EBRT after RC can be administered without excessive severe toxicity. PATIENT SUMMARY: In this report, we looked at the incidence of toxicity and local control after adjuvant external beam radiotherapy (EBRT) following radical cystectomy (RC) in high-risk muscle-invasive bladder cancer patients. We found that adjuvant EBRT was feasible and resulted in good local control. We conclude that these data support further enrollment of patients in ongoing trials to evaluate the place of adjuvant EBRT after RC.

Microfibril structure masks fibrillin-2 in postnatal tissues.

Fibrillin microfibrils are polymeric structures present in connective tissues. The importance of fibrillin microfibrils to connective tissue function has been demonstrated by the multiple genetic disorders caused by mutations in fibrillins and in microfibril-associated molecules. However, knowledge of microfibril structure is limited, largely due to their insolubility. Most previous studies have focused on how fibrillin-1 is organized within microfibril polymers. In this study, an immunochemical approach was used to circumvent the insolubility of microfibrils to determine the role of fibrillin-2 in postnatal microfibril structure. Results obtained from studies of wild type and fibrillin-1 null tissues, using monoclonal and polyclonal antibodies with defined epitopes, demonstrated that N-terminal fibrillin-2 epitopes are masked in postnatal microfibrils and can be revealed by enzymatic digestion or by genetic ablation of Fbn1. From these studies, we conclude that fetal fibrillin polymers form an inner core within postnatal microfibrils and that microfibril structure evolves as growth and development proceed into the postnatal period. Furthermore, documentation of a novel cryptic site present in EGF4 in fibrillin-1 underscores the molecular complexity and tissue-specific differences in microfibril structure.

In vivo studies of mutant fibrillin-1 microfibrils.

In humans, mutations in fibrillin-1 result in a variety of genetic disorders with distinct clinical phenotypes. While most of the known mutations in fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type fibrillin-1. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other fibrillinopathies. To test the hypothesis that mutations in fibrillin-1 cause disorders through primary effects on microfibril structure, two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in fibrillin-1 results in stable microfibrils, demonstrating that fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae.

Latent transforming growth factor beta-binding proteins and fibulins compete for fibrillin-1 and exhibit exquisite specificities in binding sites.

Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFbeta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit "exquisite specificities," a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.

Fibrillin-1 in the Vasculature: In Vivo Accumulation of eGFP-Tagged Fibrillin-1 in a Knockin Mouse Model.

Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates: In situ analyses of epithelial and immune cell markers in rectal mucosa.

Natural-product derived lectins can function as potent viral inhibitors with minimal toxicity as shown in vitro and in small animal models. We here assessed the effect of rectal application of an anti-HIV lectin-based microbicide Q-Griffithsin (Q-GRFT) in rectal tissue samples from rhesus macaques. E-cadherin+ cells, CD4+ cells and total mucosal cells were assessed using in situ staining combined with a novel customized digital image analysis platform. Variations in cell numbers between baseline, placebo and Q-GRFT treated samples were analyzed using random intercept linear mixed effect models. The frequencies of rectal E-cadherin+ cells remained stable despite multiple tissue samplings and Q-GRFT gel (0.1%, 0.3% and 1%, respectively) treatment. Whereas single dose application of Q-GRFT did not affect the frequencies of rectal CD4+ cells, multi-dose Q-GRFT caused a small, but significant increase of the frequencies of intra-epithelial CD4+ cells (placebo: median 4%; 1% Q-GRFT: median 7%) and of the CD4+ lamina propria cells (placebo: median 30%; 0.1-1% Q-GRFT: median 36-39%). The resting time between sampling points were further associated with minor changes in the total and CD4+ rectal mucosal cell levels. The results add to general knowledge of in vivo evaluation of anti-HIV microbicide application concerning cellular effects in rectal mucosa.

Performance of carbon arc-discharge nanotubes to hydrogen energy storage.

Adsorption properties of gram-scale samples of different kind of arc discharge nanotubes were studied, namely: (A) raw collaret collected on the cathode, (B) raw soots collected on the lateral reactor wall, (C) thermally treated soot, and (D) thermally then chemically treated soot. The morphology, structure, and composition of these materials were characterized by SEM, TEM, TGA, and BET. In addition, hydrogen adsorption isotherms were recorded experimentally for A, B, and D samples over the pressure range of 0 to 55 bar at ambient temperature. Our experiments indicated a maximum-yet weak-hydrogen storage at room temperature of approximately 0.13 H2 wt% for the purified product (D).

Substantial improvement of nanotube processability by freeze-drying.

As-produced carbon nanotubes often contain a fraction of impurities such as metal catalysts, inorganic supports, and carbon by-products. These impurities can be partially removed by using acidic dissolution. The resulting nanotube materials have to be dried to form a powder. The processability of nanotubes subjected to regular (thermal vaporisation) drying is particularly difficult because capillary forces pack and stick the nanotubes irreversibly, which limits their dispersability in polymeric matrices or solvents. We show that this dramatic limitation can be circumvented by using freeze-drying instead of regular-drying during nanotube purification process. In this case, the nanotubes are trapped in frozen water which is then sublimated. As a result the final powder is significantly less compact and, more important, the nanotubes can be easily dispersed with no apparent aggregates, thereby greatly enhancing their processability, e.g., they can be used to make homogeneous composites and fibers. Results from coagulation spinning from water-based dispersions of regularly-dried and freeze-dried nanotubes are compared. We also show that freeze-dried materials, in contrast to regularly-dried materials, can be dissolved in organic polar solvents using alkali-doped nanotubes. High resolution TEM and XRD analysis demonstrate that the nanotube structure and quality are not affected at the nanoscale by freeze-drying treatments.

Medication adherence in chronic disease: issues in posttransplant immunosuppression.

Poor medication adherence is a widespread problem that undermines the potential benefits of medical treatment. Typical adherence rates among chronic disease patients are approximately 50%, and these low adherence rates have a substantial economic impact, estimated at $100 to $300 billion annually. Nonadherence to immunosuppressants among transplant recipients is surprisingly frequent, and the consequences are serious. Among adult renal transplant patients, the median rate of nonadherence is approximately 22% and is associated with acute rejection episodes and approximately 36% of all graft losses. In the United States, nonadherence results in an estimated 903 episodes of acute rejection and 1319 renal transplants failures annually, costing approximately $15 million and $100 million, respectively. Drug regimen complexity is known to impact adherence. Research demonstrates an inverse relationship between dosing frequency and medication adherence in various chronic diseases, with once-daily dosing resulting in the highest adherence rates. Reducing the dosing frequency may positively impact both clinical and patient-reported outcomes, as well as health care costs. However, the increased costs of less frequently administered drugs must be outweighed by the net savings achieved through improved adherence rates and better health outcomes. If trends among patients with chronic diseases apply, once-daily dosing regimens may improve adherence rates by approximately 6% to 14% among renal transplant patients and could substantially reduce the number of acute rejection episodes and graft failures, although the exact economic impact is difficult to estimate. Further research into adherence issues in transplant patients and the potential clinical and economic benefits of once-daily dosing of immunosuppressants is warranted.

Surface plasmon resonance investigations of human epidermal growth factor receptor 2.

This investigation utilizes surface plasmon resonance (SPR) spectroscopy to detect and quantify human epidermal growth factor receptor 2 (HER-2), an oncogene product that is over-expressed in some aggressive forms of breast cancer. Specifically, the HER-2 trans-membrane protein p185 and its extra cellular fragment p105 are analytes targeted in this work by using a gold-based biosensor slide on which an anti-HER-2 antibody has been immobilized by attachment to Protein G that is fixed to the gold film. A detection limit of > or =11 ng/mL for p185 resulted when trastuzumab was used as the anti-HER-2 antibody on the biosensor slide. Experiments with semi-purified p105 revealed that it binds weakly and reversibly to trastuzumab, therefore complicating its detection and quantification. Results of studies that reacted a 13-amino-acid peptide (PP13) from the HER-2 kinase domain with its specific antibody were critically different than p185 and p105 studies. Spectral analysis of the reflectivity at constant bulk buffer refractive index revealed a progressive negative SPR shift over time. A negative shift suggests that a loss of protein mass from the anti-PP13 antibody-Protein G biosensor is occurring. Several possibilities that may explain these negative SPR shifts are discussed.

Post-heparin lipolytic activity with no hepatic triacylglycerol lipase involved in a mammalian species.

It was found that lipolytic activity in bovine post-heparin plasma differed from that of other mammalian species by the fact that intravenous heparin induced the release of lipoprotein lipase but not hepatic triacylglycerol lipase. Initially, this fact was strongly suspected when no remaining lipolytic activity could be found after whole bovine post-heparin plasma had been tested with either 1 M NaCl or antiserum against lipoprotein lipase. This was further confirmed by using heparin-Sepharose affinity chromatography when the entire lipolytic activity was eluted with 1.5 M NaCl but none with 0.4 or 0.7 M NaCl. The active fraction had lipoprotein lipase characteristics, i.e. it required serum activators to produce optimum activity and was fully inhibited by NaCl of high molarity and by anti-lipoprotein lipase antiserum. Neither the different doses of heparin nor the various times of sampling altered the results. This raises the question whether hepatic triacylglycerol lipase is absent from the bovine liver or whether this enzyme is present but cannot be released by heparin.

Interferon-gamma inhibits lipoprotein lipase in human monocyte-derived macrophages.

Lipoprotein lipase (LPL) (EC 3.1.1.34) hydrolyzes triacylglycerols of very low density lipoproteins and chylomicrons. It is produced by several cell types, including macrophages, which are frequent in atherosclerotic lesions. The atherosclerotic plaque also contains activated T lymphocytes. We therefore investigated the possible regulatory effect of the T lymphocyte-derived lymphokine interferon-gamma (IFN-gamma) on macrophage LPL. Human monocyte-derived macrophages were treated with recombinant IFN-gamma or conditioned medium from activated peripheral blood mononuclear cells for 3 days. LPL activity was thereafter measured in the culture medium and in cell homogenates. The enzyme protein was detected at a cellular level by immunocytochemistry and immunopredicipitation. Recombinant IFN-gamma caused a profound decrease in macrophage LPL secretion. The IFN-gamma-treated cells, however, still contained immunodetectable enzyme and the decrease in secretion was apparently only partly due to an inhibited synthesis. Conditioned medium from activated peripheral blood mononuclear cells also drastically decreased the macrophage LPL secretion. When the conditioned medium was treated with antibodies against IFN-gamma, its down-regulating effect on macrophage LPL was totally removed. The data indicate that IFN-gamma is inhibiting macrophage LPL at least in part via a reduction of LPL synthesis. A local release of IFN-gamma may be important in the pathogenesis of atherosclerosis by affecting the lipid accumulation in the lesion.

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells.

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.

Antibody against rat adipose tissue lipoprotein lipase.

To facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation, a high titre polyclonal antibody was raised against purified rat adipose tissue lipoprotein lipase (in a goat). The first stage of the purification of the lipoprotein lipase was carried out with heparin-Sepharose affinity chromatography. In the second stage we took advantage of the binding property of lipoprotein lipase to ampholytes. These ampholytes, used during this second step, do not have to be eliminated prior to injecting the enzyme preparation into the animal. They have neither toxic nor antigenic effects on the animal; moreover, their presence does not affect the antigenic potency of the lipoprotein lipase. When pre-incubated with a constant amount of adipose tissue lipoprotein lipase (8 mU/75 microliter), an equal volume of the antiserum raised either pure or diluted up to 1/50 resulted in complete inhibition of enzyme activity, and half maximal inhibition was observed at a dilution of 1/800. The antibody was effective in inhibiting rat heart lipoprotein lipase but not salt-resistant hepatic lipase. Immunodiffusion revealed a single line of precipitation between this antibody and the adipose tissue lipoprotein lipase.

Fibrillin-1 and fibrillin-2 in human embryonic and early fetal development.

The extracellular glycoproteins fibrillin-1 and fibrillin-2 are major components of connective tissue microfibrils. Mutations in the fibrillin-1 and fibrillin-2 genes are responsible for the phenotypical manifestations of Marfan syndrome and congenital contractural arachnodactyly respectively, which emphasizes their essential roles in developmental processes of various tissues. Consistent with this last notion, organ culture experiments have indirectly suggested morphogenic roles for fibrillins in lung and kidney development. In order to contribute to the understanding of the roles of fibrillins in developmental and morphogenetic events, we have investigated the distribution of fibrillin-1 and fibrillin-2 in human embryonic and early fetal tissues between the 5th and the 12th gestational week, i.e. at the beginning of organogenesis. Fibrillin-1 and fibrillin-2 were localized immunohistochemically using specific monoclonal antibodies, mAb 69 and mAb 48, respectively. Both fibrillins are widely distributed in various human anlagen, from early developmental stages. In most embryonic and early fetal human organs such as skin, lung, heart, aorta, central nervous system anlage, nerves, and ganglia, fibrillin-1 and fibrillin-2 follow the same temporo-spatial pattern of distribution. However, in other organs such as kidney, liver, rib anlagen, notochord fibrillin-1 and fibrillin-2 are distributed differentially. The present paper is focused on this aspect. These results suggest different roles for fibrillin-1 and -2 in the development of these structures.