Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

Summary

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Abstract

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.

Introduction

Videomicroscopy is a useful technique utilized to examine the vasomotor function of small arterioles removed from living human subjects ex vivo. Our laboratory has focused on dissecting out tiny microvessels from different adipose tissue compartments to characterize the effects of various adipose microenvironments on the microvasculature. A major advantage of this technique is that blood vessels removed from the human body remain functional and can be examined readily within minutes to hours following biopsy. Physiological conditions are mimicked and steady transmural pressure maintained in the intraluminal space via micro-glass cannulas which recapitulate ma....

Protocol

The protocol and examples described here were approved by Boston University School of Medicine Institutional Review Board (IRB, protocol #H-25644) and were conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent before participation.

1. Preparation of Solutions and Micro-glass Cannulas

1. Prepare the 4-2-hydrosyethyl-1-piperazineethanesulfonic acidic saline (HEPES) solution. For 1 L, dissolve 8.059 g NaCl, 0.298 g KCl, 0.296 g MgSO₄•7H₂O, 0.235 g CaCl₂•2H₂O, 0.16 g KH₂PO₄, 0.01 g EDTA, 1.081 g D-Glucose, an....

Results

Our laboratory has used videomicroscopy to examine endothelium-dependent and -independent vasodilation, as well as vasocontractile function of adipose tissue arterioles isolated from subcutaneous and visceral fat of obese humans. The characteristic experimental set-up is displayed in Figure 1A. Adipose tissue arterioles are suspended between two glass capillary pipettes and secured in place with sutures within the organ chamber as shown in

Discussion

The dissection and isolation of adipose arterioles from surrounding tissues can be a time-consuming and labor-intensive process with careful attention to detail and technical protocol. The microdissection procedure requires meticulous skills and specialized dissection utensils to prevent potential damage to the smooth muscle or endothelial cell layers of the microvasculature. Even tiny accidental punctures in the arteriolar wall can prevent intraluminal pressurization and results in an unsuccessful experiment. Additional.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemical Name
Acetylcholine	Sigma Aldrich	A6625
Calcium chloride (CaCl2)	Sigma Aldrich	223506
D-(+)-Glucose	Sigma Aldrich	G5767
Endothelin-1	Sigma Aldrich	E7764
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetra acetic acid (EGTA)	Sigma Aldrich	E3889
Ethylene diamine tetra acetic acid (EDTA)	Sigma Aldrich	E9884
HEPES	Sigma Aldrich	H3784
Nw-nito-L-arginine methyl ester hydrochloride	Sigma Aldrich	N5751
Magnesium sulfate (MgSO4)	Sigma Aldrich	M7506
Potassium chloride (KCL)	Sigma Aldrich	P3911
Potassium phosphate (KH2PO4)	Sigma Aldrich	P5655
Papaverine	Sigma Aldrich	P3510
Sodium bicarbonate (NaHCO3 )	Sigma Aldrich	S6014
Sodium chloride (NaCl)	Sigma Aldrich	S7653
Sodium phosphate monobasic monohydrate (NaH2Po4)	Sigma Aldrich	S9638
Name	Company	Catalog Number	Comments
Equipment
Forceps	Finescience tools	15000-08
Inverted microscope	Zeiss Achromat
Laboratory tubing	Euro-Pharm	250100306F999
Needle/pippette puller	David kopf instruments	720
Ophthalmic monofilament nylon suture	Surgical specialties	A7756N
Scissors	Finescience tools	150000-08
Vessel Chamber	DMT	VAS v.2.1