Faculty Bibliography

Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFalpha), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFalpha in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFalpha by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases.

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Alex Schier looks back at the life and research of his graduate mentor and friend Walter Gehring.

Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans.

Vitiligo is characterized by depigmented skin patches due to localized loss of melanocytes. The etiology of vitiligo is not fully understood, particularly the mechanisms that initially trigger depigmentation. Induction of oxidative stress is thought to be a common attribute of trigger events in vitiligo, while autoimmunity contributes to disease progression. In this study we sought to identify mechanisms that link disease triggers and the spread of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions, even when cultured in vitro, is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress caused by trigger events extends to the ER where redox reactions that facilitate protein folding are disrupted and that the subsequent accumulation of misfolded peptides activates the unfolded protein response (UPR). The UPR initially signals a reduction in global protein synthesis, while promoting expression of folding chaperones in order to restore homeostasis. Sustained UPR activation has been implicated in several autoimmune disorders and may play a role in vitiligo. We used 4-tertiary butyl phenol (4-TBP) and monobenzyl ether of hydroquinone (MBEH), phenols known to trigger occupational vitiligo, to study pathways that contribute to melanocyte loss. Expression of key UPR components was increased following exposure of human melanocytes to the phenols. In addition, we observed UPRmediated increases in pro-inflammatory interleukin-6 (IL6) and IL8, which have been shown to be increased in the skin of patients with vitiligo. We next investigated UPR-regulated pathways that may contribute to cytokine production and determined that cross-talk between the UPR and the nuclear factor-kappa B (NFkB) pathway contributes to the increase in expression of IL6 and IL8 following phenol exposure. Identification of the pathways that link exposure to vitiligoinducing triggers and onset of autoimmunity may allow for the development of more effective therapies for this disfiguring di!

Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow-derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation, and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have pro-vasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Differences in epidermal melanin levels result in the wide variation in color associated with ethnic skin diversity. Ethnic differences result from variance in melanogenesis, melanin transfer, and melanosome degradation in the epidermis, but the mechanisms underlying these differences, and their contribution to the regulation of skin color, are not fully understood. In this study, we explored proteins which were differentially expressed between Caucasian- and African American donor skin- derived melanocytes using twodimensional gel electrophoresis and mass spectroscopy. We consequently identified an important role for Heat Shock Protein 70 (Hsp70 encoded by HSPA1A) in skin color determination. In contrast to a previous report suggesting negative regulation of melanogenesis, Hsp70 was found to be more abundant in melanocytes from African American donors compared to those from Caucasian donors, which also reflected protein expression in the skin. In particular, inhibition of Hsp70, using either a specific inhibitor or siRNA targeting, significantly decreased melanin content of African American donor melanocytes, consistent with previous reports that Hsp70 is an Endoplasmic Reticulum (ER) chaperone involved in post-translational modification of tyrosinase. Indeed, inhibition of Hsp70 caused a decrease in tyrosinase protein levels by affecting its maturation. Taken together, our data reveal that Hsp70 plays an essential role in regulation of melanogenesis and thereby contributes to the ethnic diversity of skin color

Vitiligo is a common disorder characterized by progressive melanocyte death. Vitiligo can be induced in an occupational setting by exposure to vitiligo-inducing phenols (VIPs) such as 4 tert-butyl phenol (4TBP) and monobenzyl ether of hydroquinone (MBEH). These VIPs are believed to specifically target melanocytes due to their structural similarity to tyrosine and competition for binding to tyrosinase, the rate-limiting enzyme for melanin synthesis. By exposing normal melanocytes to VIPs and using microarray analysis and bioinformatics approaches for gene expression profiling, we identified key signaling pathways that are involved in the melanocyte response to VIP exposure. In particular, we hypothesized that exposure of primary human melanocytes to VIPs would result in oxidative stress that triggers antioxidant responses in order to protect melanocytes from cell death. Following melanocyte exposure to VIPs, HO-1 was upregulated (4TBP exposure: 5.49-fold; MBEH exposure: 25.98- fold). HO-1 is a direct target of Nrf2, a key regulator of the Nrf2- ARE antioxidant response. Activation of Nrf2 and its targets, HO- 1 and NQO1, was confirmed by conventional Western blot analysis and quantitative RT-PCR. We are now characterizing the mechanisms that regulate Nrf2 activation in response to VIPs. Additional antioxidants including SOD2 (4TBP: 10.5-fold; MBEH: 38.5-fold), peroxiredoxin 6 (4TBP: 2.44-fold; MBEH: 15.18-fold), and Nrf2 binding partners, MafK (4TBP: 2.12-fold; MBEH: 2.50- fold) and MafF (4TBP: 2.32-fold; MBEH: 3.16-fold) were also upregulated with VIP exposure and are being investigated further. Several studies have revealed dysfunctional antioxidant responses in melanocytes from patients with vitiligo, however the underlying mechanisms that reduce their efficacy are yet to be determined. We hypothesize that investigation of VIP-induced pathways may lead to the characterization of these mechanisms and provide opportunities for development of targeted therapeutics for the treatment of vitiligo