Faculty Bibliography

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.

BACKGROUND: Chronic wounds are a major source of morbidity for patients and represent a significant health burden. Implementing noninvasive techniques that accelerate healing of these wounds would provide great benefit. Ultrasound appears to be an effective modality for the treatment of chronic wounds in humans. MIST Therapy is a noncontact, low-frequency ultrasound treatment delivered through a saline mist. A variety of mechanisms have been proposed to explain the efficacy of ultrasound therapy, but the underlying molecular and cellular pathways impacted by this technique remain unclear. The in vivo effect of noncontact, low-frequency ultrasound was therefore examined in a humanized excisional wound model. METHODS: The treatment group received noncontact, low-frequency ultrasound therapy three times per week, whereas the control group received a standard dressing change. Wounds were photographed at regular intervals to calculate healing kinetics. Wound tissue was harvested and processed for histology, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: The MIST group demonstrated significantly accelerated wound healing, with 17.3 days to wound closure compared with 24 days in the controls (p < 0.05). This improvement became evident by day 9, with healing evidenced by significantly decreased mean wound area relative to original size (68 percent versus 80 percent; p < 0.01). Expression of markers of neovascularization (stromal cell-derived factor 1, vascular endothelial growth factor, and CD31) was also increased in the wound beds of noncontact, low-frequency ultrasound-treated mice compared with controls. CONCLUSION: Noncontact, low-frequency ultrasound treatment improves neovascularization and wound closure rates in excisional wounds for diabetic mice, likely because of the stimulated release of angiogenic factors.

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.

BACKGROUND: The American Heart Association recommends targeting 7 cardiovascular (CV) health metrics to reduce morbidity and mortality. Control of these targets in patients undergoing CV intervention is uncertain. METHODS: We prospectively studied patients undergoing elective percutaneous coronary or peripheral intervention from November 2010 to May 2012. We recorded data on patient demographics, clinical characteristics, and social history. Risk factor control was categorized as ideal, intermediate, or poor according to the 7 American Heart Association-defined CV health metrics (smoking status, body mass index, physical activity, diet, cholesterol, blood pressure, and metabolic control). Linear regression model was used to evaluate the association between baseline characteristics and poor CV health. RESULTS: Among 830 consecutive patients enrolled, mean age is 67.3 +/- 10.8 years, 74.2% are male, and 62.1% are white. The adequacy of achievement of ideal CV health is suboptimal in our cohort; the mean number of ideal CV metrics is 2.15 +/- 1.06. Less than 1 in 10 (9.7%) met >/=4 ideal CV health metrics. After multivariate analysis, male sex (P = .04), nonwhite race (P = .01), prior coronary artery disease (P < .01), prior peripheral arterial disease (P < .01), and history of depression (P = .01) were significantly associated with poor CV health. CONCLUSIONS: Among patients referred for elective CV intervention, achievement of ideal CV health is poor. Elective interventions represent an opportunity to identify and target CV health for risk factor control and secondary prevention.

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1alpha (IRE1alpha),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1alpha in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1alpha inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1alpha-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1alpha C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin-epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1alpha expression in chondrogenesis. The expression of IRE1alpha is depended on GEP signaling, and IRE1alpha expression is hardly detectable in GEP-/- embryos. In addition, IRE1alpha inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1alpha in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1alpha may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.

BACKGROUND:: Scarring represents a significant biomedical burden in clinical medicine. Mechanomodulation has been linked to scarring through inflammation, but until now a systematic approach to attenuate mechanical force and reduce scarring has not been possible. METHODS:: We conducted a twelve month, prospective, open label, randomized, multi-center clinical trial to evaluate abdominoplasty scar appearance following post-operative treatment with the embrace device to reduce mechanical forces on healing surgical incisions. Incisions from 65 healthy adult subjects were randomized to receive embrace treatment on one half of an abdominoplasty incision and control treatment (surgeon's optimal care methods) on the other half. The primary endpoint for this study was the difference between assessments of the scar appearance for the treated and control sides using the Visual Analogue Scale (VAS) scar score. RESULTS:: Final twelve month study photos were obtained from 36 subjects who completed at least five weeks of dressing application. The mean VAS score for embrace-treated scars (2.90) was significantly improved compared to control-treated scars (3.29) at 12 months (difference = 0.39, 95% confidence interval [0.14, 0.66], p = 0.027). Both subjects and investigators found that embrace-treated scars demonstrated significant improvements in overall appearance at 12 months using the Patient and Observer Scar Assessment Scale (POSAS) evaluation (p = 0.02 and p < 0.001, respectively). No serious adverse events were reported. CONCLUSION:: These results demonstrate that the embrace device significantly reduces scarring following abdominoplasty surgery. To our knowledge, this represents the first level one evidence for post-operative scar reduction.

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!

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