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.
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.
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 many in vivo characteristics.1,2 Additionally, a reliable videomicroscopy set-up with automated edge detection software allows for both qualitative and quantitative assessment of endothelium-dependent and -independent vasodilator and vasoconstrictor capacity of isolated vessels in real-time, permitting rapid physiological assessment in response to physical and pharmacological stimuli.3 Other microvascular techniques are also available such as wire myography which tends to be less time consuming and measure tension responses to various agonists by a force transducer.
Our laboratory has applied videomicroscopy to examine the relationship between obesity and vascular dysfunction, focusing on the effects of different adipose tissue domains on the vasculature. Central adiposity with accumulation of intra-abdominal visceral fat has been most closely linked to adipocytokine production, metabolic dysfunction, and cardiometabolic risk. It has been postulated that adipose tissue dysfunction with over-production of pro-atherogenic cytokines and adipokine dysregulation are strongly implicated in these processes, but specific regulatory molecules and treatment targets remain largely undiscovered.4 Moreover, at the local adipose tissue level, capillary rarefaction and impaired perfusion have been linked to adipose tissue pseudohypoxia and metabolic dysregulation. The hypothesis that cardiovascular disease may be the consequence of adipose tissue dysfunction is evolving. Pro-atherogenic mediators released from fat, particularly in association with central/visceral adiposity, likely promote endothelial dysfunction and pathogenic vascular changes that can be manifest locally in the adipose vasculature and detected using the herein described methodology.5
The functional assessment of isolated adipose tissue arterioles is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction in human obesity. To investigate mechanisms that contribute to fat depot-specific dysfunction, we have developed methods to examine endothelium-dependent and -independent vasodilatory responses of the microvasculature, and evaluate the expression of various regulatory candidates in paired visceral and subcutaneous (SC) fat tissue specimens obtained from obese subjects at the time of bariatric surgery.
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
2. Preparation of Reagents
3. Adipose Tissue Collection and Vessel Preparation
4. Assessment of Adipose Microvascular Function
NOTE: In general, adipose arteriole endothelial-dependent vasodilation can be elicited in response to physiological (flow-induced) and pharmacological stimuli (Ach-induced).
5. Data Analysis and Calculation
6. Statistical Analysis
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 Figure 1B. As described above in section 5.1, arteriolar responses can then be assessed by examining the vessel at baseline (Figure 2A), followed by pre-constriction with ET-1 to ~55% of baseline diameter (Figure 2B), and then agonist-induced vasodilation and relaxation of the vascular lumen as displayed in Figure 2C.
We and others have observed that endothelium-dependent vasodilation responses to increased flow (shear stress)11 and Ach12 were significantly blunted in visceral compared to subcutaneous adipose arterioles (Figure 3A, 3B) in human obesity. However, endothelium-independent vasodilation in response to papaverine was not differentially altered between the two depots (Figure 3C). Together, these findings suggest that vascular dysfunction in visceral domains is largely a result of dysfunction at the level of the endothelium, at least in early stages of the disease. This technique permits systematic analysis of vasodilator responses of intact small human arterioles and can also be applied to other accessible regions of the human vasculature. The ex vivo system can be manipulated using numerous pharmacological methods such as responses to insulin as an index of vascular endothelial insulin resistance6, or dose-responses studies to nitroglycerin to test vascular smooth muscle layer function.12 Biological methods such as silencing RNA can also be introduced13 to potentially develop mechanistic frameworks for understanding critical regulators that confer vascular dysfunction under specific disease conditions.
Figure 1: Videomicroscopy set-up. (A) Image depicting the components of the videomicroscopy organ chamber. (B) Image of a human adipose arteriole mounted between two glass capillary pipettes in the organ chamber. Vessel diameter changes in response to physiological and pharmacological stimuli that can be examined and quantified using specialized analysis software and an inverted camera attachment in the system. Please click here to view a larger version of this figure.
Figure 2: Illustration of microvascular vasodilation using pressure videomicroscopy. (A) resting baseline arteriolar diameter (post- pressurization diameter, no agonist or stimulation: Di) (B) pre-constricted diameter (after ET-1 induced constricted diameter: Dp), (C) Agonist-induced vasodilated diameter (after Ach was added: DT). Please click here to view a larger version of this figure.
Figure 3: Endothelium-dependent, and -independent vasodilation in subcutaneous vs. visceral adipose arterioles in obese humans. Endothelium-dependent vasodilation was significantly attenuated in visceral compared to subcutaneous adipose tissue arterioles when assessed by (A) increased flow (paired subcutaneous and visceral vessels, n = 20), or (B) acetylcholine dose response with/without L-NAME (paired subcutaneous and visceral vessels, n = 16). (C) Endothelium-independent vasodilation was not differentially altered between visceral and subcutaneous arterioles when assessed by papaverine dose response (paired subcutaneous and visceral vessels, n = 8). *Denotes group differences between depots; †Denotes depot-specific differences between acetylcholine with L-NAME compared to acetylcholine alone; NS: not significant. Data are presented as mean± SEM, p <0.05. Please click here to view a larger version of this figure.
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. Additionally, cannulation of arterioles utilizing sutures is crucial to secure the arterioles between the glass capillary pipette tips at each end of the vessel.
Human isolated microvessels can develop spontaneous myogenic tone after 30 – 40 min of pressurization at 60 mmHg. However, we found that human adipose arterioles generally sized 50 – 350 µm of internal luminal diameter are relatively small with sparse layers of vascular smooth muscle, and tend not to develop significant myogenic tone compared to skeletal muscle arterioles. We thus induce pre-constriction of these small arterioles using ET-1 before exposure to vasodilators. We find that ET-1 exerts a more predictable vasoconstrictor response while in some cases there may be variability in responses to alpha receptor agonists such as phenylephrine. It is imperative to choose the appropriate agonists to induce a desired ~55% pre-constriction; however it is also critical to select the correct amount of pre-constricting agent to prevent over dosage and toxic damage to the vessel. We achieve this by starting with the lower range dose and up-titrating to effect as described in section 4.1.2.
Arterioles in adipose tissue are sensitive to the changes in pH and temperature;14 therefore, it is crucial to monitor and control the appropriate pH (7.4) and temperature (37 °C) during the experimental process. While videomicroscopy is a tightly regulated temperature controlled system, it is imperative to monitor the pH of the KREBS solution throughout the experiment to avoid shifts that may skew results. Continuous bubbling of the KREBS solution and replacement of solution every 15 min during pressurization and washing steps will reduce pH variations over time.
While videomicroscopy is a useful technique to examine the functional properties of microvessels, the technique has a substantial learning curve and may be labor intensive particularly during the process of vascular isolation. Depending on the type of tissue that is biopsied, our experience with adipose tissue microvessels has demonstrated that the cannulation procedure may take significant amount of time and patience. Additionally, there is inherent time sensitivity to the experiments as vessels outside the body tend to lose their physiological and functional properties over time. Thus, the experiments need to be performed within the viable window of the vessels of minutes to hours after surgical biopsy prior to eventual tissue decay.
The clinical relevance and rationale of this experimental model utilizing videomicroscopy for studying arterioles is supported by our ability to directly probe pathophysiology in intact whole segments of human blood vessels removed from living subjects, which cannot be replicated by non-invasive imaging. In fact, our ability to directly access and examine dysfunctional human blood vessels in, for example an obese body, can provide us with opportunities to gain insight into pathways that are differentially altered in disease conditions and discover novel therapeutic targets. Moreover, published data from our laboratory and others show that ex vivo assessment of adipose arteriolar function correlates with in vivo systemic endothelial function responses in other vascular beds within an individual, and associate with cardiovascular risk factors including hypertension, smoking, diabetes, and inflammation.12,15,16 We have also published data demonstrating a significant correlation between immunohistochemical findings relevant to nitric oxide biology in visceral adipose endothelial cells and brachial arterial flow-mediated vasodilation suggesting parallel abnormalities in adipose and systemic circulations.17 Given the systemic nature of endothelial dysfunction, we believe that this technique represents a pragmatic approach to study not only human vascular tissue but also potentially utilize this method in animals and clinical longitudinal studies to examine treatment effects. Other techniques to examine the microvasculature are also available such as wire myography, which tends to be less technically difficult to perform and allows for measures of isometric tension of isolated vessels to various agonists using a force transducer. While this method also provides information regarding physiologic properties of microvessels, the set-up does not utilize an intra-luminal pressured system which may render it less apt to recapitulate in vivo conditions. An advantage of videomicroscopy is the ability to induce a relatively constant intraluminal pressure minimizing changes in shear in the lumen. The method can be applied to similar sized microvessels in both humans and animals.9,12,18 Finally, it should be noted that a number of commercially available videomicroscopy systems are available for purchase and set-up; however the overall physiological concepts are fairly uniform across all platforms.2,6,18
The authors have nothing to disclose.
The authors would like to thank the volunteers for their participation in these studies and the surgical staff at the Boston Medical Center for providing adipose tissue biopsies. Dr. Gokce is supported by National Institutes of Health (NIH) grants HL081587, HL114675, and HL126141. Dr. Farb is supported by NIH grant K23 HL135394.
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 |