Name: assessment of human adipose tissue microvascular function using videomicroscopy
Uploaded: 2017-09-29T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy Video at JoVE.com

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.

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
<ol>
	<li>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 MgSO...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Microcirculation:+Regulation+of+Flow+and+Beyond.">The Human Microcirculation: Regulation of Flow and Beyond.</a> Circ Res. 118 (1), 157-172 (2016).</li><li>Fuster, J. J., Ouchi, N., Gokce, N., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity-Induced+Changes+in+Adipose+Tissue+Microenvironment+and+Their+Impact+on+Cardiovascular+Disease.">Obesity-Induced Changes in Adipose Tissue Microenvironment and Their Impact on Cardiovascular Disease.</a> Circ Res. 118 (11), 1786-1807 (2016).</li><li>Farb, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+adipose+tissue+inflammation+represents+an+intermediate+cardiometabolic+phenotype+in+obesity.">Reduced adipose tissue inflammation represents an intermediate cardiometabolic phenotype in obesity.</a> J Am Coll Cardiol. 58 (3), 232-237 (2011).</li><li>Farb, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNT5A-JNK+regulation+of+vascular+insulin+resistance+in+human+obesity.">WNT5A-JNK regulation of vascular insulin resistance in human obesity.</a> Vasc Med. 21 (6), 489-496 (2016).</li><li>Durand, M. J., Phillips, S. A., Widlansky, M. E., Otterson, M. F., Gutterman, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+renin-angiotensin+system+contributes+to+blunted+vasodilation+induced+by+transient+high+pressure+in+human+adipose+microvessels.">The vascular renin-angiotensin system contributes to blunted vasodilation induced by transient high pressure in human adipose microvessels.</a> Am J Physiol Heart Circ Physiol. 307 (1), H25-H32 (2014).</li><li>Farb, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase+inhibition+improves+endothelial+vasomotor+dysfunction+of+visceral+adipose+arterioles+in+human+obesity.">Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity.</a> Obesity (Silver Spring). 22 (2), 349-355 (2014).</li><li>Park, S. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+muscle+feed+arteries+studied+in+vitro:+the+effect+of+temperature+on+alpha(1)-adrenergic+responsiveness.">Human skeletal muscle feed arteries studied in vitro: the effect of temperature on alpha(1)-adrenergic responsiveness.</a> Exp Physiol. 96 (9), 907-918 (2011).</li><li>Grizelj, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+flow-and+acetylcholine-induced+dilations+in+visceral+compared+to+subcutaneous+adipose+arterioles+in+human+morbid+obesity.">Reduced flow-and acetylcholine-induced dilations in visceral compared to subcutaneous adipose arterioles in human morbid obesity.</a> Microcirculation. 22 (1), 44-53 (2015).</li><li>Farb, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriolar+function+in+visceral+adipose+tissue+is+impaired+in+human+obesity.">Arteriolar function in visceral adipose tissue is impaired in human obesity.</a> Arterioscler Thromb Vasc Biol. 32 (2), 467-473 (2012).</li><li>Tanner, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-Related+Protein+1+Mediates+Low+Glucose-Induced+Endothelial+Dysfunction+in+Human+Arterioles.">Dynamin-Related Protein 1 Mediates Low Glucose-Induced Endothelial Dysfunction in Human Arterioles.</a> Am J Physiol Heart Circ Physiol. , (2016).</li><li>Ives, S. 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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...

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Chemical Name</td>
			<td style="width:159px;"></td>
			<td style="width:132px;"></td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acetylcholine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">A6625</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride (CaCl2)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td>223506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">D-(+)-Glucose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">G5767</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Endothelin-1</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E7764</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetra acetic acid (EGTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E3889</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Ethylene diamine tetra acetic acid (EDTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E9884</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">HEPES</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">H3784</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:352px;">Nw-nito-L-arginine methyl ester hydrochloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">N5751</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">M7506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride (KCL)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3911</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Potassium phosphate (KH2PO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P5655</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Papaverine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3510</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Sodium bicarbonate (NaHCO3 )</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S6014</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium chloride (NaCl)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S7653</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Sodium phosphate monobasic monohydrate (NaH2Po4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:127px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Finescience tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td>Zeiss Achromat</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laboratory tubing</td>
			<td>Euro-Pharm</td>
			<td>250100306F999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle/pippette puller</td>
			<td>David kopf instruments</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic monofilament nylon suture</td>
			<td>Surgical specialties</td>
			<td>A7756N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Finescience tools</td>
			<td>150000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vessel Chamber</td>
			<td>DMT</td>
			<td>VAS v.2.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 &#181;m 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.

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 <strong class="xfig...

Watch this Scientific Journal Video about Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy Video at JoVE.com

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy Video (Video) | JoVE

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.

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. ...

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