Gregory M. Hendricks

Similarity of mouse perivascular and brown adipose tissues and their resistance to diet-induced inflammation

Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and the metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea, and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared with white adipose tissue (WAT), PVAT and BAT from C57BL6/J mice fed a high-fat diet for 13 wk had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80 and CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b(+)/CD11c(+) macrophages in BAT (1.0%) compared with WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from inflammatory stress.

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

Intracellular bacillary burden reflects a burst size for Mycobacterium tuberculosis in vivo

We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ∼25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b− CD11c+/hi, CD11b+/lo CD11clo/−, CD11b+/hi CD11c+/hi) and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b+/hi CD11c+/hi mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b+/lo CD11clo/− cells assumed that role by ten weeks. Alveolar macrophages (CD11b− CD11c+/hi) were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest that burst size cytolysis is a major cause of infection-induced mononuclear cell death in tuberculosis.

Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-gamma-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.

Inhibition of experimental neointimal hyperplasia by recombinant human thrombomodulin coated ePTFE stent grafts

OBJECTIVES The goal of this study was to evaluate the ability of recombinant human thrombomodulin (rTM) to inhibit neointimal hyperplasia when bound to expanded polytetrafluoroethylene (ePTFE) stent grafts placed in a porcine balloon injured carotid artery model. METHODS The left carotid artery of male pigs, weighing 25 to 30 Kg, was injured with an angioplasty balloon. Two weeks later either a non-coated standard ePTFE stent graft (Viabahn, 6 x 25 mm, W. L. Gore & Associates) or a rTM coated stent graft was implanted into the balloon-injured segment using an endovascular technique. Carotid angiography was performed at the time of the balloon injury, two weeks later and then at 4 weeks to assess the degree of luminal stenosis. One month after stent graft deployment, the grafts were explanted following in situ perfusion fixation for histological analysis. The specimens were then cross-sectioned into proximal, middle and distal segments, and the residual arterial lumen and intimal to media (I/M) ratios were calculated with computerized planimetry. RESULTS rTM binding onto ePTFE-grafts was confirmed by functional activation of protein C and histopathology with immuno-scanning electron microscopy, backscatter electron emission imaging and x-ray microanalysis. All seven of the rTM coated stent grafts and six of the seven uncoated stent grafts were patent at the time of explantation. The mean luminal diameter of the rTM coated stents was 93% +/- 2.0% of the original diameter, compared with 67% +/- 23% (P = .006) in the control group. Histological analysis demonstrated that the area obliterated by intimal hyperplasia at the proximal portion of the rTM stent was -27% compared with the control group: (2.73 +/- 0.69 mm(2), vs 3.47 +/- 0.67 mm(2), P <.05). CONCLUSIONS Neointimal hyperplasia is significantly inhibited in ePTFE stent grafts coated with rTM compared with uncoated grafts, as documented by improved luminal diameter by angiography and by computerized planimetry measurements of residual lumen area. These findings suggest that binding of recombinant human thrombomodulin onto ePTFE grafts may improve the long-term patency of covered stents grafts. CLINICAL RELEVANCE Decrease of neointimal hyperplasia of the magnitude observed in this study could significantly improve blood flow and patency of small caliber prosthetic grafts. If the durability of these results can be confirmed by long-term studies, this technique may prove useful in preventing graft stenosis and arterial thrombosis following angioplasty or vascular bypass procedures.

Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria

1 Presynaptic function was investigated at K+‐stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μm), oligomycin (8 μg ml−1) or CCCP and oligomycin together. 2 Miniature endplate currents (MEPCs) were recorded at ‐150 mV with two‐electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mm), MEPC frequencies initially increased to values > 350 s−1, but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. 3 Staining with FM1‐43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP‐ or oligomycin‐treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. 4 The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1‐43 in preparations exposed to either CCCP or oligomycin. 5 We propose that the decline in K+‐stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin‐treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy‐dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including zeta(cyt) and CD3epsilon(cyt) all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of zeta(cyt) and CD3epsilon(cyt) ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of zeta(cyt) and CD3epsilon(cyt) is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of zeta(cyt) and CD3epsilon(cyt) depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of zeta(cyt) and CD3epsilon(cyt) to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for zeta(cyt) and CD3epsilon(cyt) depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

Interleukin 18 binding protein (IL18-BP) inhibits neointimal hyperplasia after balloon injury in an atherosclerotic rabbit model.

OBJECTIVES Interleukin 18 (IL18) is an interferon (IFN)-gamma-inducing factor and a proinflammatory and proatherogenic cytokine. IL18 binding protein (IL18-BP) functions as an IL18 inhibitor. This study was designed to investigate whether systemic administration of IL18-BP could inhibit neointimal hyperplasia and arterial lipid deposition. METHODS New Zealand white, male rabbits were fed with a 21% fat, 0.15% cholesterol diet. The left superficial femoral artery (SFA) was de-endotheliazed with a 2F arterial embolectomy catheter. IL18-BP (5 microg, 10 microg, or 25 microg), or 0.9% saline (control) was administered by i.v. bolus during surgery. Rabbits were followed-up at 2 and 4 weeks. Intima-media (I/M) and lumen-whole artery (L/A) area ratios, and luminal areas were measured. Serum lipid levels, liver enzymes, and kidney function were evaluated. Inflammatory cells were quantified and further verified with immunohistofluorescence staining. The extent of lipid deposition in the artery wall was quantified with Oil Red O (ORO) staining employing Zeiss AxioVision 4.6.3. Image analysis software. Lipid laden cells including macrophages were evaluated by transmission electron microscopy (TEM). RESULTS Intravenous IL18-BP 5 microg, 10 microg, and 25 microg significantly reduced I/M ratios compared with the control group at both 2 and 4 weeks. There was no significant difference between the 5 microg and 10 microg dose groups. However, at 10 microg, IL18-BP significantly increased L/A ratio more than either the 5 microg IL18-BP or control groups. The high fat diet caused significant elevation of serum lipids at 4 and 6 weeks. IL18-BP had no effect on blood lipid levels. Lipid deposit in the thoracic aorta of the control group at 6 weeks was more than at 4 weeks (P = .025). Administration of IL18-BP inhibited the lipid deposition at 4 weeks (not significant) and 6 weeks (P = .012 to .008) compared with its control group. Lipid laden macrophages (foam cells), as well as endothelial cells and smooth muscle cells were seen in the descending thoracic aorta after 6 weeks of a high fat diet by ORO, immunohistofluorescence staining, and TEM. The lipid laden cells were not seen in either of IL18-BP groups. IL18-BP 10 microg significantly inhibited mono/macro adherence and infiltration in the SFA after balloon-injury at 2 weeks after surgery. CONCLUSION A single intravenous dose of IL18-BP significantly decreased arterial neointimal hyperplasia, improved lumen to artery ratio after balloon-injury and also prevented arteriosclerosis progression. CLINICAL RELEVANCE A single intravenous dose of IL18BP decreased neointimal hyperplasia and improved arterial L/A ratios in an atherosclerotic balloon-injury animal model. These preliminary results suggest that IL18BP may be a promising molecular approach to inhibit neointimal hyperplasia and arteriosclerosis progression following coronary and peripheral angioplasty.

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots, and mixtures of empty and fully packaged virions with variable ratios of empty virions. The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP (enhanced green fluorescent protein) or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin (hA1AT)) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ALT levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side effects of rAAV8 EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.