This work was financially support by the grants from Research Grant Council of Hong Kong [17124718 and 17121714], Hong Kong Health and Medical Research Fund [13142651 and 13142641], Collaborative Research Fund of Hong Kong [C7055-14G], and the National Basic Research Program of China [973 Program 2015CB553603].

All animals used for the following study were provided by the Laboratory Animal Unit of the Faculty of Medicine, The University of Hong Kong. Ethical approval was obtained from the departmental Committee on Use of Laboratory Animals for Teaching and Research (CULATR, no.: 4085-16).
1. Preparations
<ol>
	<li>Preparation of drugs
	<ol>
		<li>Store drugs appropriately as stated in the Material Safety Data Sheet (MSDS) immediately after receiving them. Dissolve the drugs in powd...

<ol>
	<li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetylcholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.</a> Nature. 288 (5789), 373-376 (1980).</li><li>Furchgott, R. F., Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+and+contracting+factors.">Endothelium-derived relaxing and contracting factors.</a> The FASEB Journal. 3 (9), 2007-2018 (1989).</li><li>Feletou, M., Kohler, R., Vanhoutte, P. 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Apart from the endothelial cells, signals derived from PVAT play an important role in the regulation of smooth muscle tone reactivity30. Healthy PVAT releases NO and anti-inflammatory adiponectin to exert an anti-contractile effect on arteries, which is lost under pathological conditions such as obesity and metabolic syndrome31,32. In disease states, PVAT contributes to the development of endothelial dysfunction and other cardiovascular ab...

Dilatations and constrictions of arteries are achieved by relaxations and contractions, respectively, of their vascular smooth muscle cells. Changes in vascular responsiveness of small arteries contribute to the homeostatic regulation of arterial blood pressure by autonomic nerves and hormones present in the blood (e.g., catecholamines, angiotensin II, serotonin, vasopressin). At the local level, the vascular responses of smooth muscle cells are modulated by signals from both the endothelial cells of the intima and the adipose tissue surrounding the arteries (Figure 1).
The endothelium is not only a passive barrier, but also serves as a surface to exchange signals between the blood and the underlying vascular smooth muscle cells. By releasing various vasoactive substances, the endothelium plays a critical role in the local control of vascular tone responses1. For example, in response to acetylcholine, endothelial nitric oxide synthase (eNOS) is activated in the endothelium to produce nitric oxide (NO), which induces relaxation of the underlying vascular smooth muscle by activating soluble guanylyl cyclase (sGC)2. Other vasoactive substances include the products of cyclooxygenases (e.g., prostacyclin and thromboxane A2), lipoxygenase (e.g., 12-hydroxyeicosatetraenoic acids, 12-HETE), and cytochrome P450 monooxygenases (HETEs and epoxyeicosatrienoic acids, EETs), reactive oxygen species (ROS), and vasoactive peptides (e.g., endothelin-1 and angiotensin II), and endothelium-derived hyperpolarizing factors (EDHF)3. A delicate balance between endothelium-derived vasodilators and vasoconstrictors maintain the local vasomotor tone4,5.
Endothelial dysfunction is characterized by the impairment in endothelium-dependent vasodilatation6, a hallmark of vascular aging7. With age, the ability of endothelium to promote vasodilatation is progressively reduced, due largely to a decreased NO bioavailability, as well as the abnormal expression and function of eNOS in the endothelium and sGC in the vascular smooth muscle cells8,9,10. Reduced NO bioavailability potentiates the production of endothelium-dependent vasoconstrictors11,12. In aged arteries, endothelial dysfunction causes hyperplasia in the media, as reflected by the marked increases in wall thickness, number of medial nuclei, which are reminiscent of the arterial thickening in hypertension and atherosclerosis observed in human patients13,14. In addition, pathophysiological conditions such as obesity, diabetes or hypertension accelerate the development of endothelial dysfunction15,16.
Perivascular adipose tissue (PVAT) releases numerous adipokines to regulate vascular structure and function17. The anti-contractile effect of PVAT is mediated by relaxing factors, such as adiponectin, NO, hydrogen peroxide and hydrogen sulphide18,19,20. However, depending on the location and pathophysiological condition, PVAT also can enhance contractile responses&#160;in various arteries21. The pro-contractile substances produced by PVAT include angiotensin-II, leptin, resistin, and ROS22,23.&#160; In most of the studies on isolated blood vessels, PVAT has been considered as a simple structural support for the vasculature and thus removed during the preparation of blood vessel ring segments. Since adipose dysfunction represents an independent risk factor for hypertension and associated cardiovascular complications24, the PVAT surrounding the blood vessels should be considered when investigating the vascular responsiveness of different arteries.
The multi wire myograph systems have been widely used to investigate the vasomotor functions of a variety of blood vessels, including the aorta, mesenteric, renal, femoral, cerebral and coronary arteries25,26. The protocols described herein will use wire myography to evaluate vascular responsiveness in mesenteric arteries isolated from genetically modified mouse models, with a special focus on the modulation by PVAT.

<table>
	<tbody>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625</td>
			<td>Stock concentration: 10-1 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>L-NAME (N&#969;-nitro-L-arginine methyl ester)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751</td>
			<td>Stock concentration: 3 x 10-2 M 
			Working concentration: 10-4 M</td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126</td>
			<td>Stock concentration: 10-2 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>U46619 (9,11-dideoxy-9&#945;,11&#945;methanoepoxy prostaglandin F2&#945;)</td>
			<td>Enzo</td>
			<td>BML-PG023-0001</td>
			<td>Stock concentration: 10-5 M 
			Working concentration: 1-3 x 10-8 M</td>
		</tr>
		<tr>
			<td>Multiwire myograph</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>620M</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab 4/26</td>
			<td>ADInstruments</td>
			<td>ML848</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart7</td>
			<td>ADInstruments</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipo-SIRT1 wild type mice</td>
			<td>Laboratory Animal Unit, The University of Hong Kong</td>
			<td>CULATR NO.: 4085-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon-coated Petri dishes</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wires</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>300331</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of vascular tone responsiveness using isolated mesenteric arteries with a focus on modulation by perivascular adipose tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

Examination of the length/tension relationships to obtain the normalization factor k 
The amount of stretch applied to a vessel segment influences the extent of the actin-myosin interaction and hence the maximal active force developed. Thus, for every type of blood vessel, determining the amount of stretch needed for maximal active force is required for proper myography studies. Here, normalization of t...

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Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Very strict protocol is required so that we can validly compare preparations from the same animals or from different animals or arteries in the presence or absence of the fat that surrounds them or in the presence or absence of the endothelium. The main advantage of this experimental technique is that we can compare rings of the same artery under isometric condition and thus validly make conclusions concerning the impact of the experimental factors that we investigate. This protocol provides insights into modulation of vascular responsiveness by perivascular adipose tissues of different genetically modified animals.Adipose tissue dysfunctions represent an independent risk factor for hypertension and its associated cardiovascular complications. Our myograph involves multiple complicated steps that require extreme caution at each point to avoid damaging the vessel, especially during mounting, and to ensure that the vessel responds appropriately to various stimuli. Begin this procedure with preparations and dissection of the mesenteric arterial rings as described in the text protocol.Switch on the computer and open the data recording software. Save the experiment as a lab chart data file with a new name to avoid overwriting the original setting file. Open the normalization settings window and set the K factor to one.Accept the default values for IPs calibration, target pressure, online averaging time, and delay time. Click okay to save the settings. Select channels of interest and input the wire diameter, tissue endpoints, and initial micrometer reading in the normalization window.To start the normalization procedure, apply the first passive stretch to the blood vessel by turning the micrometer screw counterclockwise. After waiting three minutes for the vessel to stabilize, input the new micrometer reading in the normalization window. The wall tension is automatically calculated and shown as a point on the graph.After each passive stretch, replace the control krebs buffer with an iso-osmotic high potassium krebs containing 150 millimolar potassium chloride. When the contraction reaches a plateau at about three minutes, record the active force by subtracting the passive force at each stretch from the potassium activated force. Calculate the wall tension, as well as the internal circumference values.Remove the high potassium condition by replacing the high potassium buffer with fresh krebs buffer. Repeat washing for three times over five minutes. Repeat the normalization steps by inducing passive stretches followed by active contraction in alternate turns until the active tension starts to decrease.Thoroughly wash out the high potassium krebs buffer and equilibrate the preparations for another 30 to 45 minutes. Reset the basal tensions to zero so that only active contractile responses will be recorded during the subsequent experiment. Prepare and mount paired arterial rings as described in the text protocol from the adjacent sections of each artery.Prepare one arterial ring with PVAT intact and the other with PVAT removed. After normalization, precontract the arterial segments with high potassium krebs buffer by adding 115 millimolar potassium chloride solution to the chamber containing krebs. Following contraction to the plateau period, wash out the high potassium and replace the buffer with fresh aerated krebs buffer.Repeat washing three times over five minutes. Repeat the potassium chloride stimulation and washing three times and record the maximal contractile response to potassium chloride by subtracting the baseline tension from the tension due to potassium chloride stimulation. After the last contraction and washing, refill the chamber with warm, aerated krebs buffer and allow the artery to recover for about 30 minutes before performing the next task.To each chamber, add cumulative amounts of phenylephrine to induce the concentration-dependent increases and isometric tension of the quiescent preparations. After adding the last dose of agonist, wash out the drug thoroughly and refill the chamber with fresh krebs buffer. Plot the concentration dependent responses as increasing percentages of the potassium chloride induced maximal contractions.To assess the contribution of nitric oxide, incubate the preparations with the nitric oxide synthase inhibitor L-NAME for 30 minutes prior to the addition of phenylephrine. For performing a second concentration response curve sequentially, wash the chamber completely and repeatedly to remove all of the previous agonists until no further changes in tone are observed. Normalization of the length-tension relationship was performed for mesenteric arteries isolated from mouse models fed with standard chow or a high-fat diet.A passive length-tension relationship was established by incremental stretching of the artery segments until an internal circumference corresponding to 100 millimeters of mercury transmural pressure, or IC100, was obtained. After each stretch, 115 millimolar potassium chloride was applied to stimulate contractions. The active length tension curves were plotted from the active force data on the Y-axis and the calculated IC values from the micrometer data on the X-axis.An IC value lying within the peak plateau is IC1. The normalization factor, K, was calculated as the ratio of IC1 and IC100, which could then be applied to samples of the same vessel type in the subsequent experiment. Shown here are output recordings of the vasoconstrictor responses to phenylephrine in mesenteric arteries with or without surrounding PVAT.Cumulative concentrations of phenylephrine were applied to stimulate the contractions of mesenteric arteries collected from 16-week-old adiposet one mice. The contractile responses were recorded and calculated as a percentage of 150 millimolar potassium chloride induced maximal contraction. The area under the contraction curves were plotted for comparison.Note that PVAT from adiposet one mice elicited an anti-contractile effect on the response to phenylephrine. When obtaining KCL-induced contractions, the responses have to be stable and steady. If the response is still increasing after three incremental administrations of KCL, KCL stimulation may be needed.To achieve optimal conditions of the tensions, the normalization step is very important, as this determines the force to open the blood vessels. If it's not done properly, it will affect the whole experiment. This protocol can be applied to other settings in addition to phenylephrine-induced contraction or relaxation.For example, in the presence or absence of perivascular adipose tissue, autonomic nerves, or endothelium. With this protocol, researchers can examine small blood vessels from diseased animal models under different pathophysiological conditions, such as obesity, diabetes, and cardiovascular diseases.

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