The authors would like to acknowledge the National Research Council of Thailand, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), and the International Research Network (IRN61W0005) for providing financial support, and the Department of Physiology Faculty of Medical Science, Naresuan University, for research facility support.

The experiments performed in this study were approved by the Ethics Committee of Naresuan University Animal Care and Use Committee (NUACUC), protocol number NU-AE620921, for the care and use of animals for scientific purposes.
1. Composition of physiological solutions
<ol>
	<li>Formulate Krebs solution by dissolving chemicals in distilled water to achieve the final concentrations as follows: 122 mM NaCl, 10 mM HEPES, 5 mM KCl, 0.5 mM KH2PO4, 0.5 mM NaHPO4,&#160;1 mM MgCl2, 1.8 mM CaCl2, and 11 mM glucose8. Adjust the pH of the solution to 7.3 with 1 M NaOH and preheat to 37 &#176;C prior to use.</li>
	<li>Prepare cold Krebs solution similarly as mentioned in step 1.1. for use as an isolation medium of IPA, as described in step 2.</li>
</ol>
2. Isolation of intrapulmonary artery (IPA)
<ol>
	<li>Anesthetize 8-week-old male Wistar rats with an intraperitoneal injection of sodium thiopental (100 mg/kg)9. Check the rats for their reaction to painful stimuli in deep sleep by using forceps clamped to their feet, then ensure that the rats do not have a foot pull-back reaction before euthanizing them in step 2.2.</li>
	<li>Cut open the middle thorax of the rat and the heart terminal with scissors (size 14 cm). Then, locate the root of the lung with scissors (size 14 cm). Harvest the entire lung by cutting with scissors and immerse it in cold Krebs solution10,11.</li>
	<li>Slice a single lobe of the lung with scissors (size 11 cm) and place on a Petri dish (size 9 cm) with the medial side/root of the lung facing upward (Figure 2A, B). Observe and identify the alignment of the vein, bronchia, and artery from top to bottom (Figure 2B).</li>
	<li>Cut open the bronchus longitudinally with scissors (size 11 cm). Then, use the forceps (size 11 cm) to grab the tip of the bronchus. Gently dissect and remove the bronchus and veins out of the lung. Note that the IPA is always anatomically aligned beneath the bronchus.</li>
	<li>After that, the main IPA can be visualized (Figure 2C). Use the forceps (size 11 cm) to grab the tip of the IPA and carefully dissect it out of the lung tissue with scissors (size 11 cm).</li>
	<li>Keep the isolated IPA in cold Krebs solution until the organ bath assembly is set up (pH 7.3 and temperature 4 &#176;C)11.</li>
</ol>
3. Isolation of vascular smooth muscle cells (VSMCs)
<ol>
	<li>Isolate IPA as previously described in step 2. Cut open the main branch of the IPA longitudinally with scissors (size 11 cm) and cut into small strips (2 mm) (Figure 3A).</li>
	<li>Immerse the IPA strips in a dissociation medium (DM)10,12&#160;containing 110 mM NaCl, 5 mM KCl, 0.5 mM KH2PO4, 0.5 mM NaH2PO4, 10 mM NaHCO3, 10 mM HEPES, 0.03 mM phenol red, 10 mM taurine, 0.5 mM EDTA, 2 mM MgCl2, 11 mM glucose, and 0.16 mM CaCl2, and adjust the pH to 7.0 with 1 M NaOH. Incubate the strips for 1 h at 4 &#176;C in DM containing 1 mg/mL papain, 0.04% bovine serum albumin (BSA), and 0.4 mM 1,4- dithiothreitol (DTT), and further incubate at 37 &#176;C for 15 min. Add 1 mg/mL collagenase type 1A in DM and further incubate at 37 &#176;C for 5 min. 
	NOTE: DM&#160;is a solution used to preserve cell viability. Papain and collagenase type 1A are enzymes that break down the extracellular matrix proteins to isolate single cells. BSA is a serum albumin protein used for the stabilization of enzymes during storage and enzymatic reactions. DTT&#160;is a reducing agent used to stabilize and promote the activity of enzymes during the cell isolation process. Taurine is amino sulfuric acid used for stabilizing the cell membrane and cell integrity.</li>
	<li>Transfer the tissues into fresh DM and disperse by gentle trituration using a glass Pasteur pipette (Figure 3B). Keep triturating until isolated VSMCs become visible in the bathing solution under the microscope (Figure 3C). 
	NOTE: Freshly isolated VSMCs can be used for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. However, the present study focuses only on the vasorelaxation of isolated IPA using the organ bath technique.</li>
</ol>
4. Organ bath technique
<ol>
	<li>Isolate IPA as previously described in step 2. Cut the main branch of the IPA into rings of ~2 mm length (Figure 2D)13.</li>
	<li>Affix the IPA rings in organ bath chambers (Figure 4) by threading them onto two 40 &#181;m diameter stainless steel wires (Figure 2E)11,13,14.</li>
	<li>Attach stainless steel wires mounted with IPA rings to the isometric force transducers connected to the data acquisition device and the computer system installed with the suitable physiological software for data recording and analysis, then gently raise the tension of the IPA ring to 1 g11.</li>
	<li>Allow the vessel segments to equilibrate for about 45 min at a resting tension of 1 g. During the equilibration period, ensure that the Krebs solution is regularly changed every 15 min. After this period of equilibration, test the viability of the vessels by measuring their vasoconstriction to high extracellular K+ (80 mM) solution containing 47.4 mM NaCl, 80 mM KCl, 10 mM HEPES, 0.5 mM KH2PO4, 0.5 mM NaHPO4, 1.8 mM CaCl2, 1 mM MgCl2, and 11 mM glucose10,11.</li>
	<li>Assess the presence or absence of endothelium by computing the relaxation response to acetylcholine (1 x 10&#8722;5 M) in rings precontracted with phenylephrine (PE, 1 x 10&#8722;5 M) (Note that vascular contractions remain stable for 1 h after adding PE). Consider the rings as endothelium-intact if they quantify for more than 70% relaxation (Figure 5A). If they have less than 10% relaxation, consider the rings as endothelium-denuded13 (Figure 5B). Mechanically remove the endothelium by gently rubbing inside the vessel with a small wire to induce denudation.</li>
	<li>Again equilibrate the arterial rings for 30 min before the start of the test experiments.</li>
</ol>
5. &#8203;Vasorelaxant response to plant extract
<ol>
	<li>Investigate the relaxant effect of the plant extract by precontracting IPA rings with PE (1 x 10&#8722;5 M).</li>
	<li>Then, carefully add the plant extract (1-1,000 &#181;g/mL) cumulatively to endothelium-intact rings and endothelium-denuded rings to induce vasorelaxation (Figure 6A, B) and to acquire a concentration-dependent response curve (Figure 6C).</li>
	<li>Make certain that the effect of dimethyl sulfoxide (DMSO) used as a solvent is also evaluated similarly to serve as a negative control (Figure 6C).</li>
</ol>
6. Mechanism of plant extract-induced vasorelaxation via the endothelium
<ol>
	<li>Evaluate the vasorelaxant mechanism of action of the plant extract via endothelial nitric oxide synthase (eNOS), cyclo-oxygenase (COX)13, and endothelium-derived hyperpolarizing factor (EDHF) pathways by incubating the endothelium-intact IPA rings for 30 min with the following inhibitors (Figure 7A): 1 x 10&#8722;4 M NG-nitro-L-arginine methyl ester (L-NAME, an eNOS inhibitor)9, 1 x 10&#8722;5 M indomethacin (a COX inhibitor)9, or a combination of 1 x 10&#8722;7 M apamin (a small calcium-activated potassium channel blocker) and 1 x 10&#8722;7 M charybdotoxin (an intermediate and large-conductance calcium-activated potassium channel blocker), prior to inducing contractions of IPA with 1 x 10&#8722;5 M PE.</li>
	<li>Then, after the contractions to PE stabilize, add cumulative concentrations (0.1-1,000 &#181;g/mL) of the plant extract.</li>
	<li>Present the effects of plant extract as percentage relaxation of the IPA rings in the presence of inhibitors compared to the response of the IPA rings without inhibitors (Figure 7B-D) and construct the concentration-response curve.</li>
</ol>
7. Mechanism of plant extract-induced vasorelaxation via vascular smooth muscle K+ channels
<ol>
	<li>Pre-incubate endothelium-denuded IPA rings for 30 min with 1 x 10&#8722;3 M 4-aminopyridine (4-AP), a blocker of voltage-gated potassium channel (KV) (Figure 8A), 1 x 10&#8722;5 M glibenclamide, a blocker of ATP-sensitive potassium channel (KATP), or 1 x 10&#8722;7 M iberiotoxin, a blocker of large conductance Ca2+-activated K+ channels (KCa), prior to inducing contractions of IPA with 1 x 10&#8722;5 M PE.</li>
	<li>Then, add cumulative concentrations of the plant extract.</li>
	<li>Present the effects of plant extract as percentage relaxation of the IPA rings with inhibitor compared to the IPA rings without inhibitor (Figure 8B-D) and construct the concentration-response curve.</li>
</ol>
8. Mechanism of plant extract-induced vasorelaxation via inhibition of extracellular calcium (Ca2+) influx in VSMCs
<ol>
	<li>Pre-incubate endothelium-denuded IPA rings for 30 min in Ca2+-free Krebs solution containing 1 mM ethylene glycol-bis (2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA) (Figure 9A).</li>
	<li>Then, replace the bathing solution with Ca2+ free-80 mM K+ solution for 10 min to depolarize the VSMCs, which then open the VOCCs (Figure 9A).</li>
	<li>Cumulatively add CaCl2 (0.01-10 mM) to induce vasoconstriction of the IPA and construct the concentration-response curve (Figure 9A).</li>
	<li>Repeat this protocol in the same IPA rings but pre-incubate them with plant extract or 1 &#181;M nicardipine (L-type Ca2+ channel blocker) in Ca2+ containing 80 mM K+ solution for 10 min followed by the cumulative addition of CaCl28.</li>
	<li>Lastly, compare the contractile response to the maximum contraction previously elicited by control CaCl2 challenges (Figure 9B).</li>
</ol>
9. Mechanism of plant extract-induced vasorelaxation via inhibition of intracellular calcium (Ca2+) release from the sarcoplasmic reticulum (SR)
<ol>
	<li>Expose endothelium-denuded IPA rings to 80 mM K+ solution for approximately 5 min, depolarizing the VSMCs, opening the VOCCs, and ultimately generating the Ca2+ loading into the SR (Figure 10A).</li>
	<li>Replace the bathing solution for 10 min with Ca2+-free Krebs solution containing 1 mM EGTA (Figure 10A).</li>
	<li>Then, challenge the IPA rings with 1 x 10&#8722;5 M PE, which activates the phospholipase C/IP3 pathway, ultimately releasing Ca2+ from the SR and evoking a transient contraction of IPA (Figure 10A).</li>
	<li>Repeat the same protocol to ascertain likewise transient contractions to PE.</li>
	<li>Challenge the IPA rings again with 80 mM K+ solution for about 5 min, then replace the bathing solution with Ca2+-free Krebs solution containing 1 mM EGTA with or without the plant extract for 10 min.</li>
	<li>Again, challenge the IPA rings with 1 x 10&#8722;5 M PE.</li>
	<li>Compare the IPA contractions induced by PE between the condition with and without the plant extract (Figure 10B).</li>
</ol>
10. Statistical analysis
<ol>
	<li>Express the results as mean &#177; SEM. Compare these values using the Student&#39;s t-test or analyze by an analysis of variance (ANOVA) followed by the Tukey-Kramer post hoc test using suitable statistical software. Consider the differences at p &#60; 0.05 to be statistically significant. Here, there were n = 6 rats/experimental protocol. 
	NOTE: The recording of vasoconstriction and vasorelaxation can be assessed with suitable software installed on the computer. For example, Figure 5A shows that, when the blood vessels are stimulated by the PE causing contraction, this could be observed from the increased tension of the original tracing. The tracing will stabilize in 20-30 min, which is considered a 100% contraction. After that, the ACh stimulates the blood vessels, causing relaxation, which could be observed from the decreased tension of the original tracing. Thus, the reduced tension is calculated as the percentage compared to 100% contraction.</li>
</ol>

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The authors have nothing to disclose.

In this manuscript, we describe the technique for the isolation of rat IPA and VSMCs. Several experimental protocols have been employed to investigate the vascular response of IPA in vitro, which can be used to characterize the pharmacological effect and mechanistic basis of IPA vasorelaxation induced by plant extract.
Regarding the endothelium-dependent vasodilator action of the plant extract, various blockers such as L-NAME (eNOS), indomethacin (COX), and apamin+charybdotoxin (EDHF)...

The pulmonary vasculature is a low-pressure vascular system in which the main function is to deliver deoxygenated blood to the gas exchanging area of the lungs. The pulmonary arteries in the lungs are arranged in branches parallel to the bronchial tree, ultimately forming an extensive network of capillaries that is continuous over several alveoli and, finally, coming together into venules and veins. The vascular tone of the pulmonary artery is controlled by several factors, involving the interaction between the endothelium and vascular smooth muscle cells (VSMCs)1.
In this study, we focus on the endothelium-dependent and -independent vasorelaxation of the intrapulmonary artery (IPA). With regard to the endothelium-dependent vasorelaxation, various mechanisms occurring on the surface of endothelial cells could increase intracellular Ca2+ concentration (e.g., acetylcholine [ACh] binds with muscarinic receptor [M3]), leading to the formation of nitric oxide (NO), prostacyclin (PGl2) and endothelium-derived hyperpolarizing factor (EDHF) (Figure 1). NO is the main endothelium-derived relaxing factor synthesized from L-arginine by endothelial nitric oxide synthase (eNOS)2, which then dissociates out of the endothelial cells to VSMCs (Figure 1) and stimulates the soluble guanylyl cyclase (sGC) enzyme; this enzyme changes guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), which activates protein kinase G (PKG) and reduces cytosolic Ca2+ levels, thus causing vasorelaxation (Figure 1). PGl2 is synthesized by endothelial cells via the cyclo-oxygenase (COX) pathway3,4. It binds with the prostacyclin receptor (IP) on VSMCs and stimulates the adenylyl cyclase (AC) enzyme, which then converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) (Figure 1)3,4. cAMP activates protein kinase A (PKA), reducing cytosolic Ca2+ levels and causing vasorelaxation5 (Figure 1). The EDHF pathway also participates in endothelium-dependent vasorelaxation via various endothelial mediators and electrical events. The activation of the EDHF pathway leads to the hyperpolarization of VSMCs, thus closing voltage-operated Ca2+ channels (VOCCs), reducing intracellular Ca2+ levels, and inducing vasorelaxation6. The endothelium-independent vasorelaxation occurs directly on VSMCs via several mechanisms, such as the reduction of intracellular Ca2+ level, the inhibition of myosin light chain kinase (MLCK), the activation of myosin light chain phosphatase (MLCP), and the reduction of Ca2+ sensitivity to the contractile machinery of VSMCs. In this study, we focus on the vasorelaxation caused by the opening of various K+ channels, the blockade of VOCCs, and the inhibition of Ca2+ release from the sarcoplasmic reticulum7, which leads to the reduction of intracellular Ca2+ levels, thus decreasing VSMC myosin light chain phosphorylation and myosin-actin binding or cross-bridge formation, respectively, ultimately resulting in vasorelaxation.
The technique for evaluating vasoconstriction and vasorelaxation measurements in isolated IPA is well established for rodents, but the data varied depending on the experimental protocols. The present study describes the method used to evaluate the vascular reactivities of rat IPA preparations in vitro, which were made in the absence of external factors modulating vascular response in vivo, such as nerve signals, hormones, cytokines, blood pressure, etc.
We employed several experimental protocols using the plant extract as an example for studying the vascular reactivities of IPA. Various blockers (Figure 1) were utilized to identify the mechanisms of endothelium-dependent and -independent vasorelaxation induced by the plant extract. Nevertheless, the same protocols can be adapted to evaluate the vascular responses of IPA to any drugs, extracts or phytochemicals used for the treatment of various pulmonary pathologies.

<table><tbody><tr><td>1,4-dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D0632&lt;br/&gt; CAS NO. 348-12-3</td><td></td></tr><tr><td>4-aminopyridine (4-AP)</td><td>Aldrich Chemical</td><td>A78403&lt;br/&gt; CAS NO. 504-24-5</td><td></td></tr><tr><td>Acetylcholine</td><td>Sigma-Aldrich</td><td>A6625&lt;br/&gt; CAS NO. 60-31-1</td><td></td></tr><tr><td>Apamin</td><td>Sigma-Aldrich</td><td>A9459&lt;br/&gt; CAS NO. 24345-16-2</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153&lt;br/&gt; CAS NO. 9048-46-8</td><td></td></tr><tr><td>Calcium choride</td><td>Ajax Finechem</td><td>AJA960&lt;br/&gt; CAS NO. 1707055184</td><td></td></tr><tr><td>Charybdotoxin</td><td>Sigma-Aldrich</td><td>C7802&lt;br/&gt; CAS NO. 95751-30-7</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Sigma-Aldrich</td><td>C9891&lt;br/&gt; CAS NO. 9001-12-1</td><td>From Clostridium histolyticum</td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Millipore Corporation</td><td>K50876942 924&lt;br/&gt; CAS NO. 14431-43-7</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D4540&lt;br/&gt; CAS NO. 67-68-5</td><td></td></tr><tr><td>Ethylene glycol-bis (2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E3889&lt;br/&gt; CAS NO. 67-42-5</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E9884&lt;br/&gt; CAS NO. 60-00-4</td><td></td></tr><tr><td>Forceps 11 cm.</td><td>Rustless Dumoxel</td><td>-</td><td></td></tr><tr><td>Forceps 14 cm.</td><td>Rustless Dumoxel</td><td>-</td><td></td></tr><tr><td>Glibenclamide</td><td>Sigma-Aldrich</td><td>G6039&lt;br/&gt; CAS NO. 16673-34-0</td><td></td></tr><tr><td>GraphPad Prism program</td><td>Software version 5.0 (San Diego, CA, USA)</td><td></td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375&lt;br/&gt; CAS NO. 7365-45-9</td><td></td></tr><tr><td>Iberiotoxin</td><td>Sigma-Aldrich</td><td>I5904&lt;br/&gt; CAS NO. 1002546960</td><td>recombinant from Mesobuthus tamulus</td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378&lt;br/&gt; CAS NO. 53-86-1</td><td></td></tr><tr><td>Labchart Program</td><td>Software version 7.0 (A.D. Instrument, Castle Hill, Australia).</td><td></td><td></td></tr><tr><td>Magnesium chloride</td><td>Ajax Finechem</td><td>296&lt;br/&gt; CAS NO. 1506254995</td><td></td></tr><tr><td>Male Wistar rats</td><td>Nomura Siam International Co. Ltd., Bangkok, Thailand</td><td></td><td></td></tr><tr><td>NG-nitro-L-arginine methyl ester (L-NAME)</td><td>Sigma-Aldrich</td><td>N5751&lt;br/&gt; CAS NO. 51298-62-5</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma-Aldrich</td><td>N7510&lt;br/&gt; CAS NO. 54527-84-3</td><td></td></tr><tr><td>Organ bath 15 mL.</td><td>-</td><td>-</td><td>Specific order by the researchers</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>P4762&lt;br/&gt; CAS NO. 9001-73-4</td><td>FromPapaya Latex</td></tr><tr><td>Phenal red</td><td>Sigma-Aldrich</td><td>P5530&lt;br/&gt; CAS NO. 34487-61-1</td><td></td></tr><tr><td>Phenylephrine</td><td>Sigma-Aldrich</td><td>P6126&lt;br/&gt; CAS NO. 61-76-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Kemaus</td><td>KA383&lt;br/&gt; CAS NO. 7447-40-7</td><td></td></tr><tr><td>Potassium dihydrogenphosphate</td><td>Aldrich Chemical</td><td>EC231-913-4&lt;br/&gt; CAS NO. 7778-77-0</td><td></td></tr><tr><td>S+A2:E36odium chloride</td><td>Kemaus</td><td>KA465&lt;br/&gt; CAS NO. 7647-14-5</td><td></td></tr><tr><td>Scissors 11 cm.</td><td>Spall Stainless</td><td>-</td><td></td></tr><tr><td>Scissors 14 cm.</td><td>Spall Stainless</td><td>-</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Ajax Finechem</td><td>475&lt;br/&gt; CAS NO. 912466</td><td></td></tr><tr><td>Sodium dihydrogenphosphate</td><td>Aldrich Chemical</td><td>33,198-8&lt;br/&gt; CAS NO. 7558-80-7</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Ajax Finechem</td><td>482&lt;br/&gt; CAS NO. 1506196602</td><td></td></tr><tr><td>Sodium thiopental</td><td>Anesthal</td><td>JPN3010002&lt;br/&gt; CAS NO. 1C 314/47</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625&lt;br/&gt; CAS NO. 107-35-7</td><td></td></tr><tr><td>Waterbath WBU 45</td><td>Memmert</td><td>2766&lt;br/&gt; CAS NO. -</td><td></td></tr></tbody></table>

isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

The protocol in the present study has been developed to determine the optimal experimental conditions for measuring physiological phenomena observed in the vascular responses of isolated IPA preparations. The pilot experiments were performed to describe the potential outcomes that aid the understanding of the vascular effects and mechanistic basis of the vasorelaxant action of the plant extract, as follows.
Vasorelaxant effect of the plant extract 
As shown in <strong cla...

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Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

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JoVE Journal

Medicine

3.9K Views.  Naresuan University. Deze studie beschrijft het isoleren van intrapulmonale slagader- en vasculaire gladde spiercellen van ratten. Verschillende experimentele protocollen werden gebruikt, zoals het gebruik van de orgaanbadtechniek om de vasculaire respons van de intrapulmonale slagader te onderzoeken. Intrapulmonale slagaders en vasculaire gladde spiercellen zijn uitstekende modellen om vasculaire fysiologie en pathofysiologie te bestuderen.Aanpassingen van deze techniek kunnen vasculaire reacties van gene...

Video: Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biology

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Revealing Murine Pulmonary Veins Myocardial Architecture

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

Author Spotlight: Developing a Translational Model for Atrial Fibrillation Research Across Species 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

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