Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

University of Illinois at Chicago

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes.

Implication of mitochondrial cytoprotection in human islet isolation and transplantation.

The present study describes a simple and cost-effective islet isolation procedure. Using this method, allogeneic islets reverse diabetes in cynomolgus monkeys.

Implementation of a simplified method of islet isolation for allogeneic islet transplantation in cynomolgus monkeys.

Endothelial inwardly rectifying K (Kir2.1) channels regulate flow-induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries. Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance. Flow-induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K channels. This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow.

Inwardly rectifying K channels are major contributors to flow-induced vasodilatation in resistance arteries.

Hypercholesterolemia-induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K (Kir) channels and that Kir2.1 is an upstream mediator of flow-induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia-induced inhibition of flow-induced NO production and flow-induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions.

Hypercholesterolemia-Induced Loss of Flow-Induced Vasodilation and Lesion Formation in Apolipoprotein E-Deficient Mice Critically Depend on Inwardly Rectifying K Channels.

To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type-Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery.

Impairment of Flow-Sensitive Inwardly Rectifying K Channels via Disruption of Glycocalyx Mediates Obesity-Induced Endothelial Dysfunction.

Vascular stiffening of the arterial walls is well-known as a key factor in aging and the development of cardiovascular disease; however, the role of endothelial stiffness in vascular dysfunction is still an emerging topic. In this review, the authors discuss the impact of dyslipidemia, oxidized lipids, substrate stiffness, age and pro-atherogenic disturbed flow have on endothelial stiffness. Furthermore, we investigate several mechanistic pathways that are key contributors in endothelial stiffness and discuss their physiological effects in the onset of atherogenesis in the disturbed flow regions of the aortic vasculature. The findings in this chapter describe a novel paradigm of synergistic interaction of plasma dyslipidemia/oxidized lipids and pro-atherogenic disturbed shear stress, as well as aging has on endothelial stiffness and vascular dysfunction.

Mechanisms of endothelial stiffening in dyslipidemia and aging: Oxidized lipids and shear stress.

Dyslipidemia-induced endothelial dysfunction is an important factor in the progression of cardiovascular disease; however, the underlying mechanisms are unclear. Our recent studies demonstrated that flow-induced vasodilation (FIV) is regulated by inwardly rectifying K channels (Kir2.1) in resistance arteries. Furthermore, we showed that hypercholesterolemia inhibits Kir2.1-dependent vasodilation. In this study, we introduced 2 new mouse models: (1) endothelial-specific deletion of Kir2.1 to demonstrate the role of endothelial Kir2.1 in FIV and (2) cholesterol-insensitive Kir2.1 mutant to determine the Kir2.1 regulation in FIV under hypercholesterolemia. FIV was significantly reduced in endothelial-specific Kir2.1 knock-out mouse mesenteric arteries compared with control groups. In cholesterol-insensitive Kir2.1 mutant mice, Kir2.1 currents were not affected by cyclodextrin and FIV was restored in cells and arteries, respectively, with a hypercholesterolemic background. To extend our observations to humans, 16 healthy subjects were recruited with LDL (low-density lipoprotein)-cholesterol ranging from 51 to 153 mg/dL and FIV was assessed in resistance arteries isolated from gluteal adipose. Resistance arteries from participants with >100 mg/dL LDL (high-LDL) exhibited reduced FIV as compared with those participants with <100 mg/dL LDL (low-LDL). A significant negative correlation was observed between LDL cholesterol and FIV in high-LDL. Expressing dominant-negative Kir2.1 in endothelium blunted FIV in arteries from low-LDL but had no further effect on FIV in arteries from high-LDL. The Kir2.1-dependent vasodilation more negatively correlated to LDL cholesterol in high-LDL. Overexpressing wild-type Kir2.1 in endothelium fully recovered FIV in arteries from participants with high-LDL. Our data suggest that cholesterol-induced suppression of Kir2.1 is a major mechanism underlying endothelial dysfunction in hypercholesterolemia.

Cholesterol-Induced Suppression of Endothelial Kir Channels Is a Driver of Impairment of Arteriolar Flow-Induced Vasodilation in Humans.

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity. We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx.

Cholesterol is a major regulator of multiple types of ion channels. Although there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues-V265 and H222-and high throughput electrophysiology.

Sang Joon Ahn

Division of Pulmonary,
Critical Care,
Sleep and Allergy,
Department of Medicine,
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine

University of Illinois at Chicago

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Sang Joon Ahn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Pulmonary,Critical Care,Sleep and Allergy,Department of Medicine,Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine

University of Illinois at Chicago

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Division of Pulmonary,
Critical Care,
Sleep and Allergy,
Department of Medicine,
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine