B Cell Activation in Insulin Resistance and Obesity

Our group has demonstrated that inflammatory diseases such as type 2 diabetes (DM), inflammatory bowel disease (IBD), and periodontal disease (PD) are associated with altered B cell function that may contribute to disease pathogenesis. B cells were found to be highly activated with characteristics of inflammatory cells. Obesity is a pre-disease state for cardiovascular disease and type 2 diabetes and is considered a state of chronic inflammation. Therefore, we sought to better characterize B cell function and phenotype in obese patients. We demonstrate that (Toll-like receptor) TLR4 and CD36 expression by B cells is elevated in obese subjects, suggesting increased sensing of lipopolysaccharide (LPS) and other TLR ligands. These ligands may be of microbial, from translocation from a leaky gut, or host origin. To better assess microbial ligand burden and host response in the bloodstream, we measured LPS binding protein (LBP), bacterial/permeability increasing protein (BPI), and high mobility group box 1 (HMGB1). Thus far, our data demonstrate an increase in LBP in DM and obesity indicating increased responses to TLR ligands in the blood. Interestingly, B cells responded to certain types of LPS by phosphorylating extracellular-signal-regulated kinases (ERK) 1/2. A better understanding of the immunological state of obesity and the microbial and endogenous TLR ligands that may be activating B cells will help identify novel therapeutics to reduce the risk of more dangerous conditions, such as cardiovascular disease

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 µm internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions

Acute effects of vasoactive drug treatment on brachial artery reactivity

AbstractObjectivesThe goal of this study was to investigate whether concomitant therapy with vasoactive medications alters the results of noninvasive assessment of endothelial function.BackgroundUltrasound assessment of brachial artery flow-mediated dilation is emerging as a useful clinical tool. The current practice of withholding cardiac medications before ultrasound studies has unknown utility and would limit the clinical use of the methodology.MethodsTo determine whether a single dose of a vasoactive drug influences brachial reactivity, we examined flow-mediated dilation and nitroglycerin-mediated dilation in 73 healthy subjects (age 27 ± 6 years). Studies were completed at baseline and 3 h after randomized treatment with a single oral dose of placebo, felodipine (5 mg), metoprolol (50 mg), or enalapril (10 mg). To determine if holding vasoactive therapy for 24 h before study yields different results than continuation of clinically prescribed medications, we examined vascular function in 72 patients (age 57 ± 10 years) with coronary artery disease. Ultrasound studies were performed 24 h after the last dose and again 3 h after patients took their clinically prescribed medications.ResultsIn healthy subjects one dose of all three drugs lowered blood pressure, and metoprolol also lowered heart rate. However, there was no significant effect of treatment on brachial artery dilation. In patients with coronary artery disease on chronic treatment, taking prescribed medications reduced blood pressure and heart rate, but had no significant effect on brachial artery dilation.ConclusionsRecent administration of commonly used nonnitrate vasoactive drugs has no significant effect on brachial reactivity. These findings suggest that current practice of withholding cardiac medications before testing endothelial function may not be necessary, making this methodology more practical for clinical use

WNT5A regulates adipose tissue angiogenesis via antiangiogenic VEGF-A165b in obese humans

Experimental studies have suggested that Wingless-related integration site 5A (WNT5A) is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Impaired angiogenesis in fat depots has been implicated in the development of adipose tissue capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction. We have recently demonstrated that impaired adipose tissue angiogenesis is associated with overexpression of antiangiogenic factor VEGF-A165b in human fat and the systemic circulation. In the present study, we postulated that upregulation of WNT5A is associated with angiogenic dysfunction and examined its role in regulating VEGF-A165b expression in human obesity. We biopsied subcutaneous and visceral adipose tissue from 38 obese individuals (body mass index: 44 ± 7 kg/m2, age: 37 ± 11 yr) during planned bariatric surgery and characterized depot-specific protein expression of VEGF-A165b and WNT5A using Western blot analysis. In both subcutaneous and visceral fat, VEGF-A165b expression correlated strongly with WNT5A protein (r = 0.9, P \u3c 0.001). In subcutaneous adipose tissue where angiogenic capacity is greater than in the visceral depot, exogenous human recombinant WNT5A increased VEGF-A165b expression in both whole adipose tissue and isolated vascular endothelial cell fractions (P \u3c 0.01 and P \u3c 0.05, respectively). This was associated with markedly blunted angiogenic capillary sprout formation in human fat pad explants. Moreover, recombinant WNT5A increased secretion of soluble fms-like tyrosine kinase-1, a negative regulator of angiogenesis, in the sprout media (P \u3c 0.01). Both VEGF-A165b-neutralizing antibody and secreted frizzled-related protein 5, which acts as a decoy receptor for WNT5A, significantly improved capillary sprout formation and reduced soluble fms-like tyrosine kinase-1 production (P \u3c 0.05). We demonstrated a significant regulatory nexus between WNT5A and antiangiogenic VEGF-A165b in the adipose tissue of obese subjects that was linked to angiogenic dysfunction. Elevated WNT5A expression in obesity may function as a negative regulator of angiogenesis

Insulin Status and Vascular Responses to Weight Loss in Obesity

ObjectivesThe aim of this study was to determine whether the effects of weight loss on arterial function are differentially modified by insulin status.BackgroundClinical studies suggest that plasma insulin levels may predict the extent of cardiovascular benefit achieved with weight loss in obese individuals, but mechanisms are currently unknown.MethodsWe prospectively followed 208 overweight or obese patients (body mass index [BMI] ≥25 kg/m2) receiving medical/dietary (48%) or bariatric surgical (52%) weight-loss treatment during a median period of 11.7 months (interquartile range: 4.6 to 13 months). We measured plasma metabolic parameters and vascular endothelial function using ultrasound at baseline and following weight-loss intervention and stratified analyses by median plasma insulin levels.ResultsPatients age 45 ± 1 years, with BMI 45 ± 9 kg/m2, experienced 14 ± 14% weight loss during the study period. In individuals with higher baseline plasma insulin levels (above median >12 μIU/ml; n = 99), ≥10% weight loss (compared with <10%) significantly improved brachial artery macrovascular flow-mediated vasodilation and microvascular reactive hyperemia (p < 0.05 for all). By contrast, vascular function did not change significantly in the lower insulin group (≤12 μIU/ml; n = 109) despite a similar degree of weight loss. In analyses using a 5% weight loss cut point, only microvascular responses improved in the higher insulin group (p = 0.02).ConclusionsInsulin status is an important determinant of the positive effect of weight reduction on vascular function with hyperinsulinemic patients deriving the greatest benefit. Integrated improvement in both microvascular and macrovascular function was associated with ≥10% weight loss. Reversal of insulin resistance and endothelial dysfunction may represent key therapeutic targets for cardiovascular risk reduction in obesity

WNT5A-JNK regulation of vascular insulin resistance in human obesity

Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (pWNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p

Obesity accelerates endothelial-to-mesenchymal transition in adipose tissues of mice and humans

IntroductionVascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive an endothelial-to-mesenchymal transition (EndoMT), where endothelial cells undergo a phenotypic switch to mesenchymal-like cells that are pro-inflammatory and pro-fibrotic. In this study, we sought to determine whether obesity can promote EndoMT in adipose tissue.MethodsMice in which endothelial cells are lineage-traced with eYFP were fed a high-fat/high-sucrose (HF/HS) or Control diet for 13, 26, and 52 weeks, and EndoMT was assessed in adipose tissue depots as percentage of CD45−CD31−Acta2+ mesenchymal-like cells that were eYFP +. EndoMT was also assessed in human adipose endothelial cells through cell culture assays and by the analysis of single cell RNA sequencing datasets obtained from the visceral adipose tissues of obese individuals.ResultsQuantification by flow cytometry showed that mice fed a HF/HS diet display a time-dependent increase in EndoMT over Control diet in subcutaneous adipose tissue (+3.0%, +2.6-fold at 13 weeks; +10.6%, +3.2-fold at 26 weeks; +11.8%, +2.9-fold at 52 weeks) and visceral adipose tissue (+5.5%, +2.3-fold at 13 weeks; +20.7%, +4.3-fold at 26 weeks; +25.7%, +4.8-fold at 52 weeks). Transcriptomic analysis revealed that EndoMT cells in visceral adipose tissue have enriched expression of genes associated with inflammatory and TGFβ signaling pathways. Human adipose-derived microvascular endothelial cells cultured with TGF-β1, IFN-γ, and TNF-α exhibited a similar upregulation of EndoMT markers and induction of inflammatory response pathways. Analysis of single cell RNA sequencing datasets from visceral adipose tissue of obese patients revealed a nascent EndoMT sub-cluster of endothelial cells with reduced PECAM1 and increased ACTA2 expression, which was also enriched for inflammatory signaling genes and other genes associated with EndoMT.DiscussionThese experimental and clinical findings show that chronic obesity can accelerate EndoMT in adipose tissue. We speculate that EndoMT is a feature of adipose tissue dysfunction that contributes to local inflammation and the systemic metabolic effects of obesity.

Stochastic last mile relief network design with resource reallocation

We study a last mile distribution network design problem for situations where there exist local distribution centers (LDCs) with pre-positioned supplies. Given the information on the existing pre-disaster relief network, the problem determines the locations and capacities of LDCs and points of distribution in the relief network, while capturing the uncertain aspects of the post-disaster environment. We introduce a new accessibility metric and develop a two-stage stochastic programming model that would allow more accessible and equitable distribution of relief supplies. Since solving the proposed stochastic optimization model is computationally challenging, we employ a scenario decomposition-based branch-and-cut algorithm. We perform a computational study -- based on the real-world data from the 2011 Van earthquake in Turkey -- to provide insights about the model and demonstrate the effectiveness of the solution method