Faculty Bibliography

The adult hair follicle houses stem cells that govern the cyclical growth and differentiation of multiple cell types that collectively produce a pigmented hair. Recent studies have revealed that hair follicle stem cells are heterogeneous and dynamic throughout the hair cycle. Moreover, interactions between heterologous stem cells, including both epithelial and melanocyte stem cells, within the hair follicle are just now being explored. This review will describe how recent findings have expanded our understanding of the development, organization, and regeneration of hair follicle stem cells. At a basic level, this review is intended to help construct a reference point to integrate the surge of studies on the molecular mechanisms that regulate these cells.

Latent transforming growth factor beta (TGF-beta) binding proteins (LTBPs) are large extracellular glycoproteins structurally similar to fibrillins. They perform intricate and important roles in the extracellular matrix (ECM) and perturbations of their function manifest as a wide range of diseases. LTBPs are major regulators of TGF-beta bioavailability and action. In addition, LTBPs interact with other ECM proteins-from cytokines to large multi-factorial aggregates like microfibrils and elastic fibers, affecting their genesis, structure, and performance. In the present article, we review recent advancements in the field and relate the complex roles of LTBP in development and homeostasis. J. Cell. Biochem. 113: 410-418, 2012. (c) 2011 Wiley Periodicals, Inc

Introduction Atherosclerosis is characterized by the accumulation of low density lipoprotein (LDL) and recruited macrophages within arterial walls. High macrophage burden is an indicator of greater risk of atherosclerotic tissue rupture and heart attack. As computed tomography (CT) imaging is the best technique for imaging plaque in the coronary arteries, a CT contrast agent able to detect macrophages in the arteries could help identify patients at higher risk. This study investigated whether in vivo macrophage imaging using clinical scanners could be performed with gold core high density lipoprotein (Au-HDL), a macrophage targeted agent (A). Additionally, lowest effective dose and ideal imaging timeframe of AuHDL was probed. Methods & Results Dodecanethiol coated gold cores were prepared following Brust's method and subsequently coated with the phospholipid myristoyl hydroxy phosphocholine (MHPC). The nanoparticles were then purified through centrifugation to remove gold core aggregates and empty MHPC micelles. Negative stain transmission electron microscopy (TEM) images verified removal of empty MHPC micelles from solution after ultracentrifugation (B). Apolipoprotein AI (ApoAI) was added to form the final AuHDL nanoparticle. CT imaging was used to calculate gold concentrations of the samples in mg/ml. To induce atherosclerosis, male New Zealand white rabbits were fed a high fat, high cholesterol diet (4.7% coconut oil and 0.3% cholesterol enriched diet) and underwent a double balloon injury of the aorta. AuHDL was prepared such that five atherosclerotic rabbits were injected with 75 (n=2), 150 (n=2), or 300 (n=1) mg Au/kg. CT images of rabbit aortas were taken at the following three time points: pre-injection, 24 hours post-injection, and 48 hours post-injection. A custom made MATLAB program was created to measure various regions of and around the aorta. CT images of aorta walls after injection exhibited greater radiodensity compared to pre-injection images. For example, CT images taken of one rabbit injected with 150 mg Au/kg showed the radiodensity of the aorta on average to be 38 HU (Hounsfield Units) +/- 1.99 preinjection and 59 HU +/- 1.59 24 hours postinjection (C). The lowest effective dose tested was 75 mg Au/kg and best imaging timeframe tested was 24 hours postinjection. TEM images of rabbit aorta sections confirmed localization of AuHDL nanoparticles in macrophages (D). Conclusion AuHDL increased radiodensity in CT images of aortas 24 and 48 hours post-injection compared to pre-injection images. Electron microscopy showed the nanoparticles to target macrophages. Hence this agent can image macrophages using CT, and has the potential for doing so in patients. Clinical Relevance If translated clinically, AuHDL can be used to image plaques in human aortas with high macrophage burden, thus allowing identification of patients at high risk of a heart attack. In addition, the agent could be of use in studying atherosclerosis and the effect of interventions. (Figure presented)

Rationale: Inflammation drives progression and destabilization of atherosclerotic plaques. Statins constitute the backbone for strategies to lower cardiovascular risk because of their potent cholesterol lowering capability. Whereas preclinical studies have shown that statins also have anti-inflammatory effects, the clinical relevance is hampered by the limited bioavailability of orally administered statins. To enhance the anti-inflammatory effects we developed statin-loaded reconstituted high-density lipoprotein nanoparticle ([s]-rHDL). The advantages of [s]-rHDL comprise its long half-life in plasma and the targeting to macrophages in atherosclerotic plaques. Methods & Results: To focus on anti-inflammatory effects, we used ApoE KO mice, whose cholesterol level is unaffected by statins. First, to evaluate macrophage targeting by [s]-rHDL, Gd-DTPA labeled [s]-rHDL was administered intravenously to ApoE KO mice (n=3). In vivo T1-weighted MR imaging (9.4 T Bruker MRI scanner) revealed strong signal enhancement in the abdominal aortic wall (Suppl. Fig a-d). Moreover, accumulation of [s]-rHDL was observed in the aortic valve and branching areas in the mice administered with Cy5.5 labeled [s]-rHDL by NIRF imaging (Suppl. Fig e, f) and specific uptake of [s]-rHDL by macrophages was revealed by fluorescence microscopy (Suppl. Fig g-l). Furthermore, flow cytometry confirmed that macrophages robustly took up rHDL in plaques, and the more differentiated macrophages took up more rHDL than less differentiated macrophages (Suppl. Fig m-r). Second, to assess the anti-inflammatory effects of [s]-rHDL, mice (n=62) were put on a high fat diet from 4 weeks of age onwards. At 14 weeks after diet initiation, mice were randomized to receive either placebo (n=15), oral simvastatin (10 mg/kg per day; n=15), intravenous rHDL (10 mg/kg ApoAI twice per week; n=16), or intravenous [s]-rHDL (15 mg/kg simvastatin with 10 mg/kg ApoAI twice a week; n=16) for 12 weeks. In vivo MR imaging of abdominal aorta was performed in 8 mice of each group at baseline, 6, and 12 weeks after randomization. Progression of vessel wall thickness was significantly inhibited in [s]-rHDL-treated animals compared to oral simvastatin, rHDL, and placebo groups (panel a). To objectively and quantitatively analyze histological sections (n4000), we built an automated Matlab procedure. Histology results at termination showed that plaque size (hematoxylin phloxine saffron staining) in the [s]-rHDL treated group was significantly reduced compared to rHDL and placebo groups (panel b). Importantly, the macrophage positive area (anti-CD68 immunostaining) in the [s]- rHDL treated group was profoundly reduced compared to all other groups (panel c). Conclusion: [s]-rHDL successfully delivers simvastatin to macrophages in atherosclerotic plaques as revealed by in vivo MRI imaging, ex vivo imaging, and histology. As a consequence, the [s]-rHDL formulation improves the anti-inflammatory effects of statins, which can be expected to improve its atheroprotective effects compared to oral statin therapy. These data warrant further studies in patients at increased cardiovascular risk. (Figure presented)

ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.

Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNgamma) CGS vs. IFNgamma alone, n = 4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 +/- 2%; p < 0.5113, inhibition vs. IFNgamma alone, n = 4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1

Introduction The study of nanoparticle-nanoparticle and nanoparticle-cell interactions is of paramount importance to better understand biological processes and to improve the design of nanomaterials to be applied for molecular imaging. Forster resonance energy transfer (FRET) between a nanoparticle core and its coating allows studying the aforementioned phenomena with a variety of optical techniques. In the current study we applied to lipoproteins, natural nanoparticles comprised of lipids and apolipoproteins that transport fats throughout the body. We developed a high-density lipoprotein (HDL) based nanoparticle that consists of a quantum dot (QD) core and Cy5.5 labeled lipidic coating. The methodology allows judicious tuning of the QD/Cy5.5 ratio, which enabled us to optimize FRET between the QD core and the Cy5.5 labeled coating (Fig.1A). FRET allowed us to study lipoprotein-lipoprotein interactions, lipid exchange dynamics and the influence of apolipoproteins on these processes. Moreover, we were able to study HDL-cell interactions and exploit FRET to visualize HDL uptake by and dissociation in live macrophage cells. Methods and Results Exceptionally stable CdSe-CdS-ZnS core-shell-shell (CSS) QDs were synthesized, capped with oleic acid and coated with an appropriate mixture of Cy5.5 labeled and unlabeled phospholipids. Subsequently, ApoA-I was incorporated in the lipid corona to render stable dual labeled hybrid nanoparticles that consist of one QD core per particle, schematically depicted in Fig. 1A. To investigate the occurrence of FRET we acquired emission spectra and life time measurements of QD-HDL nanoparticles that contained varying amounts of Cy5.5 in their lipid coating (Fig. 1B). Next, we studied the particle-particle interactions through FRET facilitated optical measurements. Particle-cell interactions with cell membranes (Fig. 1C-D) were studied using the same methodology and ultimately, we performed a proof-ofprinciple study where we aimed to visualize FRET of QD-HDL-Cy5.5 nanoparticles in living adhered J774A.1 macrophages via fluorescence microscopy. This allowed us to study and visualize the temporal fate of QD-HDL once associated with macrophages (Supplemental Data). We observed the lipid coating to very differently interact with the cells from the QD core The fluorescence from the lipids primarily integrated in the cell membrane, while the QD core was predominantly found in the cytoplasm, suggesting a dissociation of the HDL nanoparticle upon association with the macrophage cells. Lastly, FRET fluorescence microscopy convincingly corroborated the aforementioned and proved to be a valuable tool to study the disassembly of the HDL nanoparticle. Conclusion Both the increasing interest in lipid-coated nanocrystals and the need to better understand HDL biology in detail inspired us to develop a hybrid nanoarchitecture that resembles HDL. We show that these lipid-coated and dye labeled QDs represent a versatile probe to study FRET and also allow the study of fundamental and biological processes via FRET, including lipid-exchange between nanoparticles and nanoparticle uptake by cells. (Figure presented)

Background/purpose: Wound healing is a complex process that involves multiple intercellular and intracellular processes and extracellular interactions. Explanted human skin has been used as a model for the re-epithelialization phase of human wound healing. The currently used standard technique uses a circular punch biopsy tool to make the initial wound. Despite its wide use, the geometry of round wounds makes it difficult to measure them reliably. Methods: Our group has designed a linear wounding tool, and compared the variability in ex vivo human linear and circular wounds. Results: An F test for differences in variances demonstrated that the linear wounds provided a population of wound size measurements that was 50% less variable than that obtained from a group of matched circular wounds. This reduction in variability would provide substantial advantages for the linear wound technique over the circular wound punch technique, by reducing the sample sizes required for comparative studies of factors that alter healing. Conclusion: This linear wounding tool thus provides a method for wounding that is standardized, provides minimal error in wound gap measurements, and is easily reproducible. We demonstrate its utility in an ex vivo model for the controlled investigation of human skin wounds

OBJECTIVES: Miltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to have macrophage-activating functions that aid parasite killing. We investigated the type of immunological responses generated in miltefosine-treated Leishmania donovani-infected hamsters, which simulate the clinical situation of human kala-azar. METHODS: Twenty-five-day-old infected hamsters, treated with miltefosine at 40 mg/kg for 5 consecutive days, were euthanized on days 30 and 45 post treatment (p.t.) and checked for parasite clearance and for real-time analysis of mRNAs of the Th1/Th2 cytokines interferon-gamma (IFN-gamma), interleukin-12 (IL-12), tumour necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), IL-4, IL-10 and transforming growth factor-beta (TGF-beta), nitric oxide (NO) production, the lymphocyte transformation test (LTT) and antibody responses. Responses were compared with the normal and Leishmania-infected groups at the same time points. RESULTS: By day 45 p.t. there was a significant increase in the mRNA expression of iNOS, IFN-gamma, IL-12 and TNF-alpha, whereas there were significant decreases in IL-4, IL-10 and TGF-beta in cured hamsters as compared with their infected counterparts. In vitro stimulation of lymphocytes with concanavalin A and soluble Leishmania donovani antigen showed a maximum LTT response and there was a gradual increase in the NO level ( approximately 7-fold compared with infected counterparts). Anti-Leishmania IgG and IgG1 levels, found to be elevated in the infected group, decreased significantly after treatment but there was a significant increase in IgG2 isotype. CONCLUSIONS: Treatment of Leishmania-infected hamsters with miltefosine reverses the Th2-type response into a strong Th1-type immune response.