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Obesity is recognized as a risk factor for adverse pregnancy outcomes. Maternal obesity prevalence has increased in parallel with that in the general population and is associated with an increase in morbidity and mortality for both mother and baby. Obese mothers are more likely to develop gestational diabetes, hypertensive disorders including preeclampsia, thromboembolic complications, miscarriage, and have an increased need for induction of labour. Babies born from obese mothers can be abnormally large (macrosomia) or small for gestational age, and have a higher risk of perinatal death and congenital malformation. Pregnancy induces marked and dynamic changes in energy metabolism, however, the direct effects of pregnancy adipose tissue biology in both normal lean and obese women is still largely unknown. The aim of this thesis was to delineate novel mechanisms by which pregnancy affects adipose tissue biology, and thus infer how obesity might adversely affect pregnancy outcomes. We used an animal model of obesity during pregnancy in which mice were given a high fat diet (HF) to make them obese. We identified that pregnancy was associated with an unexpected curtailment of visceral (mesenteric) adipose tissue mass in HF mice and with an attenuation, rather than worsening of the metabolic impairment expected from the combination of excess dietary fat and insulin resistance/glucose intolerance of pregnancy. To determine the underlying molecular mechanism contributing to this phenotype global gene expression microarray with subsequent pathway analysis and qRT-PCR validation was employed within the visceral adipose tissue. In visceral fat of HF pregnant mice, gene pathways for de novo lipogenesis and lipid storage, inflammation, retinol metabolism, insulin like growth factor and estrogenic signaling showed altered regulation. Given the known role of estrogen on adipose tissue and inflammatory cell function, a hypothesis was generated that altered estrogen receptor (ER)α expression/activation/increased estradiol presence within mesenteric fat formed a unifying molecular mechanism underlying the altered adipose biology and relative amelioration of the metabolic phenotype in HF pregnant mice. To test the ER α hypothesis, a female clonal adipocyte cell line, Chub-S7, and primary visceral and subcutaneous adipocytes from pregnant obese and lean patients were treated with the ERα selective agonist, PPT. PPT downregulated mRNA levels of key genes involved in de novo lipogenesis (ME1, FANS and SCD1 Dgat2), consistent with a direct role for ERα activation in curtailment of fat expansion. Although the primary human study lacked sufficient power to adequately address the hypothesis, PPT significantly suppressed SCD1 mRNA levels in visceral adipocytes of lean women. In parallel with the curtailment of mesenteric fat expansion, HF pregnant mice were found to have increased liver weight and liver triglyceride content. However, this “fatty liver” phenotype was not associated with increased mRNA levels of genes involved in hepatic triglyceride uptake or de novo lipogenesis. This increase in liver triglycerides may be due to an excessive influx of fatty acids from mesenteric fat through the portal vein. In conclusion, pregnancy in obese animals is associated with a beneficial curtailment in mesenteric fat expansion, normalization of metabolic disturbances and reduced adipose inflammation. Increased ERα activation within adipocytes may play a critical role in this phenotype.

Peripheral artery disease (PAD) and the consecutive build-up of an atherosclerotic plaque restricting blood flow to the lower limbs lead to critical limb ischaemia, one of the most common circulation problems in the world. Although a small number of interventions (such as surgery or revascularization treatments) are available, patients with this condition are often too ill for these procedures, giving a poor prognosis for the disease. Several strategies to promote neovascularization using different stem cell populations with angiogenic potential have been proposed as plausible therapies. Perivascular cells (PCs), key structural components of the wall of small and large blood vessels have numerous advantages over other cell types since they are highly abundant, easy to obtain from the stromal vascular fraction (SVF) of human adipose tissue (an ethically approved source) and have mesenchymal and angiogenic properties. The work described in this thesis addressed the hypothesis that PCs isolated from human white adipose tissue would promote the recovery of blood flow in an ischaemic hindlimb by increasing blood vessel number and blood perfusion to the foot. To investigate whether PCs from human white adipose tissue could rapidly increase neovascularization and, therefore, be used as a possible therapeutic treatment for PAD and critical limb ischaemia, the initial aim was to validate, characterise and demonstrate the properties of the murine equivalent of these cells, in order to establish a direct link between the injected cells and the ones natively found in the mouse. This was then followed by the use of murine models of angiogenesis to determine whether transplanted human PCs stimulate angiogenesis in vivo. Initial studies using immunohistochemistry, fluorescence-activated cell sorting (FACS) and in vitro mesodermal differentiation demonstrated that perivascular cells (namely pericytes and adventitial cells) are present in multiple mouse organs, can be sorted to purity, and have mesenchymal stem cell (MSC) properties. These cells had similar characteristics to their human counterparts, thus validating the mouse as a suitable model for determining whether transplanted human PCs could stimulate angiogenesis. Using in vitro and two in vivo (sponge implantation and hindlimb ischaemia) models, it was shown that human PCs have angiogenic properties being capable of tube formation and interaction with endothelial cells, as well as promoting angiogenesis within sponges. Contrary to expectations, PCs did not increase blood perfusion to the mouse ischaemic hindlimb, despite increasing microcirculation within the skeletal muscle and myofibre regeneration. This work showed that PCs obtained from human adipose tissue have important therapeutic implications in promoting angiogenesis and skeletal muscle regeneration but failed to increase arteriogenesis which is the key mechanism allowing the restoration of blood perfusion.

archives@tulane.edu / Background: Individuals with human immunodeficiency virus (HIV) and undergoing antiretroviral therapy (ART) exhibit high levels of circulating inflammatory cytokines and proteins, which are strongly correlated with shortened time to death and disease. To target damaging inflammation at the source, the drivers of inflammation must be identified. Adipose tissue is a massive organ that contains adipocytes and immune cells capable of producing pro-inflammatory mediators. Dysregulated adipose tissue is implicated in the pathogenesis of obesity and related diseases, such as type 2 diabetes, that are likewise reported in persons with chronic HIV infection. Adipose tissue was therefore explored as a contributor to circulating inflammation in patients with HIV using the rhesus macaque model. Simian immunodeficiency virus (SIV) closely models HIV regarding pathogenesis, including CD4+ T cell depletion, induction of a viral reservoir, and development of opportunistic infections before succumbing to Acquired Immunodeficiency Syndrome (AIDS) and death. Methods: Subcutaneous adipose tissue (SQAT) from SIV-infected rhesus macaques was characterized using confocal microscopy to describe the major immune cell subsets. Adipose tissue homogenates and plasma were analyzed for expression of genes and proteins related to inflammatory processes using antibody and RNA-based fluorescent multiplex bead technology for protein and gene quantitation, respectively. The functions of adipose tissue immune cells during SIV infection were measured with stimulation and phagocytosis assays. / 1 / Marissa Fahlberg

Purpose: The present study investigates the potential anti-inflammatory effects of in vivo oxytocin (OT) infusion on adipose tissue inflammation in the Watanabe Heritable Hyperlipidimic Rabbits (WHHL). Methods: Twenty-eight 3-month-old WHHL were surgically implanted with osmotic minipumps containing OT (n = 14, infusion rate 250 ng/kg/hr) or vehicle (n = 14). Blood samples were taken at baseline, midpoint, and endpoint for lipids and C-reactive protein (CRP). After 16 weeks, animals were sacrificed and samples of adipose tissue (epididiymal, retroperitoneal, mesenteric, pericardial, and subcutanous) were collected and analyzed for pro-inflammatory cytokine (IL-6, TNF-α, and MCP-1) and anti- inflammatory adipokine (adiponectin and IL-10) expression levels by Real Time- Polymerase Chain Reaction. Adipose tissue was also immunohistologically analyzed for macrophage infiltration. Aortas were dissected, formalin-fixed, and stained with oil-red O for en face quantification of lesion area. Student’s t-tests were used to compare group means for all measures. Results: Endpoint OT levels were significantly different (p < .05) between the control ( M = 11.28 pg/ml, SEM = 2.5) and treatment group (M = 132.35 pg/ml, SEM = 8.5). Plasma lipids were not altered by OT infusion. OT-treatment significantly decreased plasma CRP, a marker of systemic inflammation, at midpoint and endpoint compared to controls (p = 0.05). OT-treated animals displayed significantly less atherosclerosis in the thoracic aorta (p < 0.05); a finding similar to our previously published study in a mouse model of atherosclerosis. In some fat depots, there was a trend suggesting adiponectin gene expression increased in the OT-treatment group. There were no significant differences or trends regarding macrophage infiltration in adipose tissue. Conclusions: Oxytocin infusion attenuated thoracic aortic atherosclerosis, plasma CRP, and may affect inflammatory cytokine expression in adipose tissue in the WHHL model.

Macrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulinresistance. miR-223-/- mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell-mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow-derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic pro-inflammatory pathways and enhances the alternative anti-inflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. For the first time, this study demonstrates that miR-223 acts to inhibit Pknox 1,suppressing pro-inflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.

Maintenance of body temperature is one of the first and most important physiological processes that must be initiated after birth. Failure to sustain homeothermy leads to hypothermia and death. Indeed, in sheep, 40% of non-predator lamb deaths are attributed to cold and cold-related causes. Brown adipose tissue (BAT) is an essential mediator of thermogenesis in many species and is responsible for 50% of the heat generated in the newborn lamb despite comprising only 2% of body weight. Previously, we found that maternal arginine supplementation increased fetal peri-renal BAT by 62%. This observation led us to test the hypothesis that increased the amount of fetal BAT will enhance neonatal thermogenesis at birth and thus combat the effects of cold stress. Thirty-one multiparous Suffolk ewes gestating singletons and twins were assigned to receive either intravenous injections of L-arginine (27 mg/kg bodyweight; n=17) or sterile saline (n=14) three times daily from Day 75 to Day 125 of gestation (term=147). Following parturition lambs were removed from their dams, placed in a thermoneutral environment, and fed artificial colostrum on a per body weight basis. At 4 hours of age, lambs were cold challenged at 0 degrees C for 2 hours. Rectal temperatures were recorded at 15 minute intervals. At 6 hours of age all singletons and one lamb of each twin pair was sacrificed. The remaining twin lamb was challenged again at 22 hours of age for an additional 2 hours prior to necropsy. Rectal temperature was greater for the duration of both cold challenges in lambs from arginine-treated ewes than lambs from saline-treated ewes (P<0.050). Interestingly, at time of necropsy, BAT weight did not differ (P>0.10) between treatments. UCP1 mRNA levels were not affected by treatment or age (P>0.10). However, TEK, PPARGC1A, NRF1, NRF2, PPARG, ADRB3, ARG2, RPS6KA1, EIF4EBP1, ODC1 were not affected by treatment (P>0.10) but were upregulated (P<0.05) by age; being greater at 24 hours of age versus 6 hours of age. Results indicate that maternal arginine treatment results in increased neonatal thermogenesis after birth. Although the underlying mechanisms remain to be elucidated, the data reported herein represent the first step in improving neonatal survival in response to cold.