Kevin M. Tharp

Enhanced survival and engraftment of transplanted stem cells using growth factor sequestering hydrogels.

We have generated a bioinspired tunable system of hyaluronic acid (HyA)-based hydrogels for Matrix-Assisted Cell Transplantation (MACT). With this material, we have independently evaluated matrix parameters such as adhesion peptide density, mechanical properties, and growth factor sequestering capacity, to engineer an environment that imbues donor cells with a milieu that promotes survival and engraftment with host tissues after transplantation. Using a versatile population of Sca-1(+)/CD45(-) cardiac progenitor cells (CPCs), we demonstrated that the addition of heparin in the HyA hydrogels was necessary to coordinate the presentation of TGFβ1 and to support the trophic functions of the CPCs via endothelial cell differentiation and vascular like tubular network formation. Presentation of exogenous TGFβ1 by binding with heparin improved differentiated CPC function by sequestering additional endogenously-produced angiogenic factors. Finally, we demonstrated that TGFβ1 and heparin-containing HyA hydrogels can promote CPC survival when implanted subcutaneously into murine hind-limbs and encouraged their participation in the ensuing neovascular response, which included blood vessels that had anastomosed with the hosts blood vessels.

Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids

Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8−/−) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through αvβ3 integrin– and αvβ5 integrin–dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.

Matrix assisted transplantation of functional beige adipose tissue

Novel, clinically relevant, approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion of brown adipose tissues that express uncoupling protein (UCP) 1 and thus can uncouple mitochondrial respiration from ATP synthesis. While expansion of UCP1-expressing adipose depots may be achieved in rodents via genetic and pharmacological manipulations or the transplantation of brown fat depots, these methods are difficult to use for human clinical intervention. We present a novel cell scaffold technology optimized to establish functional brown fat–like depots in vivo. We adapted the biophysical properties of hyaluronic acid–based hydrogels to support the differentiation of white adipose tissue–derived multipotent stem cells (ADMSCs) into lipid-accumulating, UCP1-expressing beige adipose tissue. Subcutaneous implantation of ADMSCs within optimized hydrogels resulted in the establishment of distinct UCP1-expressing implants that successfully attracted host vasculature and persisted for several weeks. Importantly, implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain, demonstrating the therapeutic merit of this highly translatable approach. This novel approach is the first truly clinically translatable system to unlock the therapeutic potential of brown fat–like tissue expansion.

Cardiac ischemia-reperfusion regulates sympathetic neuropeptide expression through gp130-dependent and independent mechanisms

Cardiac function is regulated by a balance of sympathetic and parasympathetic transmission. Neuropeptide Y (NPY) and galanin (GAL) released from cardiac sympathetic neurons inhibits parasympathetic transmission in the heart. Sympathetic peptides may contribute to autonomic imbalance, which is characterized by increased sympathetic and decreased parasympathetic transmission and contributes to life threatening cardiovascular pathologies. Several gp130 cytokines are increased in the heart after myocardial infarction (MI), and these cytokines stimulate neuropeptide expression in sympathetic neurons. We used mice whose sympathetic neurons lack the gp130 receptor (gp130(DBH-Cre/lox) mice) to ask if cytokine activation of gp130 regulated neuropeptide expression in cardiac sympathetic nerves after MI. Myocardial infarction decreased NPY mRNA through a gp130 independent mechanism and increased VIP and PACAP mRNA via gp130, while GAL mRNA was unchanged. Immunohistochemistry revealed a gp130-dependent increase in PACAP38 in cells of the stellate ganglion after MI, and PACAP was detected in pre-ganglionic fibers of all genotypes and surgical groups. VIP was identified in a few sympathetic nerve fibers in all genotypes and surgical groups. GAL and PACAP38 were not detected in sham hearts, but peptide immunoreactivity was high in the infarct three days after MI. Surprisingly, peptides were abundant in cells that co-labeled with macrophage markers F4/80 and MAC2, but were not detected in sympathetic axons. PACAP protects cardiac myocytes from apoptosis, and GAL stimulates axon regeneration in addition to inhibiting parasympathetic transmission. Thus, these peptides may play an important role in cardiac and neuronal remodeling after ischemia-reperfusion.

The Sense of Smell Impacts Metabolic Health and Obesity

Olfactory inputs help coordinate food appreciation and selection, but their role in systemic physiology and energy balance is poorly understood. Here we demonstrate that mice upon conditional ablation of mature olfactory sensory neurons (OSNs) are resistant to diet-induced obesity accompanied by increased thermogenesis in brown and inguinal fat depots. Acute loss of smell perception after obesity onset not only abrogated further weight gain but also improved fat mass and insulin resistance. Reduced olfactory input stimulates sympathetic nerve activity, resulting in activation of β-adrenergic receptors on white and brown adipocytes to promote lipolysis. Conversely, conditional ablation of the IGF1 receptor in OSNs enhances olfactory performance in mice and leads to increased adiposity and insulin resistance. These findings unravel a new bidirectional function for the olfactory system in controlling energy homeostasis in response to sensory and hormonal signals.

Bioengineering Beige Adipose Tissue Therapeutics.

Unlocking the therapeutic potential of brown/beige adipose tissue requires technological advancements that enable the controlled expansion of this uniquely thermogenic tissue. Transplantation of brown fat in small animal model systems has confirmed the expectation that brown fat expansion could possibly provide a novel therapeutic to combat obesity and related disorders. Expansion and/or stimulation of uncoupling protein-1 (UCP1)-positive adipose tissues have repeatedly demonstrated physiologically beneficial reductions in circulating glucose and lipids. The recent discovery that brown adipose tissue (BAT)-derived secreted factors positively alter whole body metabolism further expands potential benefits of brown or beige/brite adipose expansion. Unfortunately, there are no sources of transplantable BATs for human therapeutic purposes at this time. Recent developments in bioengineering, including novel hyaluronic acid-based hydrogels, have enabled non-immunogenic, functional tissue allografts that can be used to generate large quantities of UCP1-positive adipose tissue. These sophisticated tissue-engineering systems have provided the methodology to develop metabolically active brown or beige/brite adipose tissue implants with the potential to be used as a metabolic therapy. Unlike the pharmacological browning of white adipose depots, implantation of bioengineered UCP1-positive adipose tissues offers a spatially controlled therapeutic. Moving forward, new insights into the mechanisms by which extracellular cues govern stem-cell differentiation and progenitor cell recruitment may enable cell-free matrix implant approaches, which generate a niche sufficient to recruit white adipose tissue-derived stem cells and support their differentiation into functional beige/brite adipose tissues. This review summarizes clinically relevant discoveries in tissue-engineering and biology leading toward the recent development of biomaterial supported beige adipose tissue implants and their potential for the metabolic therapies.

Differential requirement for de novo lipogenesis in cholangiocarcinoma and hepatocellular carcinoma of mice and humans.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most prevalent types of primary liver cancer. These malignancies have limited treatment options, resulting in poor patient outcomes. Metabolism reprogramming, including increased de novo lipogenesis, is one of the hallmarks of cancer. Fatty acid synthase (FASN) catalyzes the de novo synthesis of long‐chain fatty acids from acetyl‐coenzyme A and malonyl‐coenzyme A. Increased FASN expression has been reported in multiple tumor types, and inhibition of FASN expression has been shown to have tumor‐suppressing activity. Intriguingly, we found that while FASN is up‐regulated in human HCC samples, its expression is frequently low in human ICC specimens. Similar results were observed in mouse ICC models induced by different oncogenes. Ablating FASN in the mouse liver did not affect activated AKT and Notch (AKT/Notch intracellular domain 1) induced ICC formation in vivo. Furthermore, while both HCC and ICC lesions develop in mice following hydrodynamic injection of AKT and neuroblastoma Ras viral oncogene homolog oncogenes (AKT/Ras), deletion of FASN in AKT/Ras mice triggered the development almost exclusively of ICCs. In the absence of FASN, ICC cells might receive lipids for membrane synthesis through exogenous fatty acid uptake. In accordance with the latter hypothesis, ICC cells displayed high expression of fatty acid uptake‐related proteins and robust long‐chain fatty acid uptake. Conclusion: Our data demonstrate that FASN dependence is not a universal feature of liver tumors: while HCC development is highly dependent of FASN and its mediated lipogenesis, ICC tumorigenesis can be insensitive to FASN deprivation; our study supports novel therapeutic approaches to treat this pernicious tumor type with the inhibition of exogenous fatty acid uptake. (Hepatology 2016;63:1900‐1913)

Altered norepinephrine content and ventricular function in p75NTR-/- mice after myocardial infarction.

Cardiac sympathetic neurons stimulate heart rate and the force of contraction through release of norepinephrine. Nerve growth factor modulates sympathetic transmission through activation of TrkA and p75NTR. Nerve growth factor plays an important role in post-infarct sympathetic remodeling. We used mice lacking p75NTR to examine the effect of altered nerve growth factor signaling on sympathetic neuropeptide expression, cardiac norepinephrine, and ventricular function after myocardial infarction. Infarct size was similar in wildtype and p75NTR-/- mice after ischemia-reperfusion surgery. Likewise, mRNAs encoding vasoactive intestinal peptide, galanin, and pituitary adenylate cyclase activating peptides were identical in wildtype and p75NTR-/- cardiac sympathetic neurons, as was expression of the TrkA neurotrophin receptor. Norepinephrine content was elevated in the base of the p75NTR-/- ventricle compared to wildtype, but levels were identical below the site of occlusion. Left ventricular pressure, dP/dt(MAX), and dP/dt(MIN) were measured under isoflurane anesthesia 3 and 7 days after surgery. Ventricular pressure decreased significantly 3 days after infarction, and deficits in dP/dt(MAX) were revealed by stimulating beta receptors with dobutamine and release of endogenous norepinephrine with tyramine. dP/dt(MIN) was not altered by genotype or surgical group. Few differences were observed between genotypes 3 days after surgery, in contrast to low pressure and dP/dt(MAX) previously reported in control p75NTR-/- animals. Seven days after surgery ventricular pressure and dP/dt(MAX) were significantly lower in p75NTR-/- hearts compared to WT hearts. Thus, the lack of p75NTR did not enhance cardiac function after myocardial infarction.

Consumption of Clarified Grapefruit Juice Ameliorates High-Fat Diet Induced Insulin Resistance and Weight Gain in Mice

To determine the metabolic effects of grapefruit juice consumption we established a model in which C57Bl/6 mice drank 25–50% sweetened GFJ, clarified of larger insoluble particles by centrifugation (cGFJ), ad libitum as their sole source of liquid or isocaloric and sweetened water. cGFJ and control groups consumed similar amounts of liquids and calories. Mice fed a high-fat diet and cGFJ experienced a 18.4% decrease in weight, a 13–17% decrease in fasting blood glucose, a three-fold decrease in fasting serum insulin, and a 38% decrease in liver triacylglycerol values, compared to controls. Mice fed a low-fat diet that drank cGFJ experienced a two-fold decrease in fasting insulin, but not the other outcomes observed with the high-fat diet. cGFJ consumption decreased blood glucose to a similar extent as the commonly used anti-diabetic drug metformin. Introduction of cGFJ after onset of diet-induced obesity also reduced weight and blood glucose. A bioactive compound in cGFJ, naringin, reduced blood glucose and improved insulin tolerance, but did not ameliorate weight gain. These data from a well-controlled animal study indicate that GFJ contains more than one health-promoting neutraceutical, and warrant further studies of GFJ effects in the context of obesity and/or the western diet.

α8β1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism.

Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders. In this work, we demonstrate a critical role for the RGD-binding integrin α8β1 in promoting nutrient absorption through regulation of gastrointestinal motility. Smooth muscle-specific deletion and antibody blockade of α8 in mice result in enhanced gastric antral smooth muscle contraction, more rapid gastric emptying, and more rapid transit of food through the small intestine leading to malabsorption of dietary fats and carbohydrates as well as protection from weight gain in a diet-induced model of obesity. Mechanistically, ligation of α8β1 by the milk protein Mfge8 reduces antral smooth muscle contractile force by preventing RhoA activation through a PTEN-dependent mechanism. Collectively, our results identify a role for α8β1 in regulating gastrointestinal motility and identify α8 as a potential target for disorders characterized by hypo- or hyper-motility. DOI: http://dx.doi.org/10.7554/eLife.13063.001