Paracrine Role of the Endothelium in Metabolic Homeostasis in Health and Nutrient Excess

The vascular endothelium traditionally viewed as a simple physical barrier between the circulation and tissue is now well-established as a key organ mediating whole organism homeostasis by release of a portfolio of anti-inflammatory and pro-inflammatory vasoactive molecules. Healthy endothelium releases anti-inflammatory signaling molecules such as nitric oxide and prostacyclin; in contrast, diseased endothelium secretes pro-inflammatory signals such as reactive oxygen species, endothelin-1 and tumor necrosis factor-alpha (TNFα). Endothelial dysfunction, which has now been identified as a hallmark of different components of the cardiometabolic syndrome including obesity, type 2 diabetes and hypertension, initiates and drives the progression of tissue damage in these disorders. Recently it has become apparent that, in addition to vasoactive molecules, the vascular endothelium has the potential to secrete a diverse range of small molecules and proteins mediating metabolic processes in adipose tissue (AT), liver, skeletal muscle and the pancreas. AT plays a pivotal role in orchestrating whole-body energy homeostasis and AT dysfunction, characterized by local and systemic inflammation, is central to the metabolic complications of obesity. Thus, understanding and targeting the crosstalk between the endothelium and AT may generate novel therapeutic opportunities for the cardiometabolic syndrome. Here, we provide an overview of the role of the endothelial secretome in controlling the function of AT. The endothelial-derived metabolic regulatory factors are grouped and discussed based on their physical properties and their downstream signaling effects. In addition, we focus on the therapeutic potential of these regulatory factors in treating cardiometabolic syndrome, and discuss areas of future study of potential translatable and clinical significance. The vascular endothelium is emerging as an important paracrine/endocrine organ that secretes regulatory factors in response to nutritional and environmental cues. Endothelial dysfunction may result in imbalanced secretion of these regulatory factors and contribute to the progression of AT and whole body metabolic dysfunction. As the vascular endothelium is the first responder to local nutritional changes and adipocyte-derived signals, future work elucidating the changes in the endothelial secretome is crucial to improve our understanding of the pathophysiology of cardiometabolic disease, and in aiding our development of new therapeutic strategies to treat and prevent cardiometabolic syndrome

IGFBP-1 in Cardiometabolic Pathophysiology—Insights From Loss-of-Function and Gain-of-Function Studies in Male Mice

We have previously reported that overexpression of human insulin-like growth factor binding protein (IGFBP)-1 in mice leads to vascular insulin sensitization, increased nitric oxide bioavailability, reduced atherosclerosis, and enhanced vascular repair, and in the setting of obesity improves glucose tolerance. Human studies suggest that low levels of IGFBP-1 are permissive for the development of diabetes and cardiovascular disease. Here we seek to determine whether loss of IGFBP-1 plays a causal role in the predisposition to cardiometabolic disease. Metabolic phenotyping was performed in transgenic mice with homozygous knockout of IGFBP-1. This included glucose, insulin, and insulin-like growth factor I tolerance testing under normal diet and high-fat feeding conditions. Vascular phenotyping was then performed in the same mice using vasomotor aortic ring studies, flow cytometry, vascular wire injury, and angiogenesis assays. These were complemented with vascular phenotyping of IGFBP-1 overexpressing mice. Metabolic phenotype was similar in IGFBP-1 knockout and wild-type mice subjected to obesity. Deletion of IGFBP-1 inhibited endothelial regeneration following injury, suggesting that IGFBP-1 is required for effective vascular repair. Developmental angiogenesis was unaltered by deletion or overexpression of IGFBP-1. Recovery of perfusion following hind limb ischemia was unchanged in mice lacking or overexpressing IGFBP-1; however, overexpression of IGFBP-1 stimulated hindlimb perfusion and angiogenesis in insulin-resistant mice. These findings provide new insights into the role of IGFBP-1 in metabolic and vascular pathophysiology. Irrespective of whether loss of IGFBP-1 plays a causal role in the development of cardiometabolic disorders, increasing IGFBP-1 levels appears effective in promoting neovascularization in response to ischemia

Progressive Development of Aberrant Smooth Muscle Cell Phenotype in Abdominal Aortic Aneurysm Disease

Abdominal aortic aneurysm (AAA) is a silent, progressive disease with a high mortality and an increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (≥5 cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of the differentiation marker miR-145 (2.2-fold, p < 0.001) and the senescence marker SIRT-1 (1.3-fold, p < 0.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, p < 0.001, and 1.8-fold, p < 0.01, respectively, vs. control cells) and displayed an aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker γH2AX (3.9-fold, p < 0.01, vs. control SMC). These features did not correlate with patients' chronological age and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at the end stage of the disease. Refinement of a porcine bioreactor model would facilitate the study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression

Preservation of Smooth Muscle Cell Integrity and Function: A Target for Limiting Abdominal Aortic Aneurysm Expansion?

(1) Abdominal aortic aneurysm (AAA) is a silent, progressive disease with significant mortality from rupture. Whilst screening programmes are now able to detect this pathology early in its development, no therapeutic intervention has yet been identified to halt or retard aortic expansion. The inability to obtain aortic tissue from humans at early stages has created a necessity for laboratory models, yet it is essential to create a timeline of events from EARLY to END stage AAA progression. (2) We used a previously validated ex vivo porcine bioreactor model pre-treated with protease enzyme to create “aneurysm” tissue. Mechanical properties, histological changes in the intact vessel wall, and phenotype/function of vascular smooth muscle cells (SMC) cultured from the same vessels were investigated. (3) The principal finding was significant hyperproliferation of SMC from EARLY stage vessels, but without obvious histological or SMC aberrancies. END stage tissue exhibited histological loss of α-smooth muscle actin and elastin; mechanical impairment; and, in SMC, multiple indications of senescence. (4) Aortic SMC may offer a therapeutic target for intervention, although detailed studies incorporating intervening time points between EARLY and END stage are required. Such investigations may reveal mechanisms of SMC dysfunction in AAA development and hence a therapeutic window during which SMC differentiation could be preserved or reinstated

Inhibition of plasmin-mediated TAFI activation may affect development but not progression of abdominal aortic aneurysms

Objective: Thrombin-activatable fibrinolysis inhibitor (TAFI) reduces the breakdown of fibrin clots through its action as an indirect inhibitor of plasmin. Studies in TAFI-deficient mice have implicated a potential role for TAFI in Abdominal Aortic Aneurysm (AAA) disease. The role of TAFI inhibition on AAA formation in adult ApoE-/- mice is unknown. The aim of this paper was to investigate the effects of TAFI inhibition on AAA development and progression. Methods: Using the Angiotensin II model of AAA, male ApoE-/- mice were infused with Angiotensin II 750ng/kg/min with or without a monoclonal antibody inhibitor of plasmin-mediated activation of TAFI, MA-TCK26D6, or a competitive small molecule inhibitor of TAFI, UK-396082. Results: Inhibition of TAFI in the Angiotensin II model resulted in a decrease in the mortality associated with AAA rupture (from 40.0% to 16.6% with MA-TCK26D6 (log-rank Mantel Cox test p = 0.16), and 8.3% with UK-396082 (log-rank Mantel Cox test p = 0.05)). Inhibition of plasmin-mediated TAFI activation reduced the incidence of AAA from 52.4% to 30.0%. However, late treatment with MA-TCK26D6 once AAA were already established had no effect on the progression of AAA in this model. Conclusions: The formation of intra-mural thrombus is responsible for the dissection and early rupture in the angiotensin II model of AAA, and this process can be prevented through inhibition of TAFI. Late treatment with a TAFI inhibitor does not prevent AAA progression. These data may indicate a role for inhibition of plasmin-mediated TAFI activation in the early stages of AAA development, but not in its progression

Cixutumumab reveals a critical role for IGF-1 in adipose and hepatic tissue remodelling during the development of diet-induced obesity

High fat diet (HFD)-induced obesity leads to perturbation in the storage function of white adipose tissue (WAT) resulting in deposition of lipids in tissues ill-equipped to deal with this challenge. The role of insulin like growth factor-1 (IGF-1) in the systemic and organ-specific responses to HFD is unclear. Using cixutumumab, a monoclonal antibody that internalizes and degrades cell surface IGF-1 receptors (IGF-1 R), leaving insulin receptor expression unchanged we aimed to establish the role of IGF-1 R in the response to a HFD. Mice treated with cixutumumab fed standard chow developed mild hyperinsulinemia with no change in WAT. When challenged by HFD mice treated with cixutumumab had reduced weight gain, reduced WAT expansion, and reduced hepatic lipid vacuole formation. In HFD-fed mice, cixutumumab led to reduced levels of genes encoding proteins important in fatty acid metabolism in WAT and liver. Cixutumumab protected against blunting of insulin-stimulated phosphorylation of Akt in liver of HFD fed mice. These data reveal an important role for IGF-1 R in the WAT and hepatic response to short-term nutrient excess. IGF-1 R inhibition during HFD leads to a lipodystrophic phenotype with a failure of WAT lipid storage and protection from HFD-induced hepatic insulin resistance

Endothelial IGF‐1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity

Changes in composition of the intestinal microbiota are linked to the development of obesity and can lead to endothelial cell (EC) dysfunction. It is unknown whether EC can directly influence the microbiota. Insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) are critical for coupling nutritional status and cellular growth; IGF-1R is expressed in multiple cell types including EC. The role of ECIGF-1R in the response to nutritional obesity is unexplored. To examine this, we use gene-modified mice with EC-specific overexpression of human IGF-1R (hIGFREO) and their wild-type littermates. After high-fat feeding, hIGFREO weigh less, have reduced adiposity and have improved glucose tolerance. hIGFREO show an altered gene expression and altered microbial diversity in the gut, including a relative increase in the beneficial genus Akkermansia. The depletion of gut microbiota with broad-spectrum antibiotics induces a loss of the favourable metabolic differences seen in hIGFREO mice. We show that IGF-1R facilitates crosstalk between the EC and the gut wall; this crosstalk protects against diet-induced obesity, as a result of an altered gut microbiota

Species Accumulation Curves and Incidence-Based Species Richness Estimators to Appraise the Diversity of Cultivable Yeasts from Beech Forest Soils

Background: Yeast-like fungi inhabit soils throughout all climatic zones in a great abundance. While recent estimations predicted a plethora of prokaryotic taxa in one gram of soil, similar data are lacking for fungi, especially yeasts. Methodology/Principal Findings: We assessed the diversity of soil yeasts in different forests of central Germany using cultivation-based techniques with subsequent identification based on rDNA sequence data. Based on experiments using various pre-cultivation sample treatment and different cultivation media we obtained the highest number of yeasts by analysing mixed soil samples with a single nutrient-rich medium. Additionally, several species richness estimators were applied to incidence-based data of 165 samples. All of them predicted a similar range of yeast diversity, namely 14 to 16 species. Randomized species richness curves reached saturation in all applied estimators, thus indicating that the majority of species is detected after approximately 30 to 50 samples analysed. Conclusions/Significance: In this study we demonstrate that robust species identification as well as mathematical approaches are essential to reliably estimate the sampling effort needed to describe soil yeast communities. This approach has great potential for optimisation of cultivation techniques and allows high throughput analysis in the future

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas