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

Summary

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.

Abstract

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' 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' pharmacodynamic parameters.

Introduction

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) pathway^3,4. 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.

Protocol

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

1. 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, 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 °C prior to use.
2. Prepare cold Krebs solution similarly as mentioned in step 1.1. for use as an isolation medium of IPA, as described in step 2.

2. Isolation of intrapulmonary artery (IPA)

1. 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.
2. 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 solution^10,11.
3. 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).
4. 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.
5. 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).
6. Keep the isolated IPA in cold Krebs solution until the organ bath assembly is set up (pH 7.3 and temperature 4 °C)¹¹.

3. Isolation of vascular smooth muscle cells (VSMCs)

1. 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).
2. Immerse the IPA strips in a dissociation medium (DM)^10,12 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 °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 °C for 15 min. Add 1 mg/mL collagenase type 1A in DM and further incubate at 37 °C for 5 min.
   NOTE: DM 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 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.
3. 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.

4. Organ bath technique

1. Isolate IPA as previously described in step 2. Cut the main branch of the IPA into rings of ~2 mm length (Figure 2D)¹³.
2. Affix the IPA rings in organ bath chambers (Figure 4) by threading them onto two 40 µm diameter stainless steel wires (Figure 2E)^11,13,14.
3. 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.
4. 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 glucose^10,11.
5. Assess the presence or absence of endothelium by computing the relaxation response to acetylcholine (1 x 10⁻⁵ M) in rings precontracted with phenylephrine (PE, 1 x 10⁻⁵ 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.
6. Again equilibrate the arterial rings for 30 min before the start of the test experiments.

5. ​Vasorelaxant response to plant extract

1. Investigate the relaxant effect of the plant extract by precontracting IPA rings with PE (1 x 10⁻⁵ M).
2. Then, carefully add the plant extract (1-1,000 µg/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).
3. 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).

6. Mechanism of plant extract-induced vasorelaxation via the endothelium

1. 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 10⁻⁴ M NG-nitro-L-arginine methyl ester (L-NAME, an eNOS inhibitor)⁹, 1 x 10⁻⁵ M indomethacin (a COX inhibitor)⁹, or a combination of 1 x 10⁻⁷ M apamin (a small calcium-activated potassium channel blocker) and 1 x 10⁻⁷ M charybdotoxin (an intermediate and large-conductance calcium-activated potassium channel blocker), prior to inducing contractions of IPA with 1 x 10⁻⁵ M PE.
2. Then, after the contractions to PE stabilize, add cumulative concentrations (0.1-1,000 µg/mL) of the plant extract.
3. 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.

7. Mechanism of plant extract-induced vasorelaxation via vascular smooth muscle K⁺ channels

1. Pre-incubate endothelium-denuded IPA rings for 30 min with 1 x 10⁻³ M 4-aminopyridine (4-AP), a blocker of voltage-gated potassium channel (K_V) (Figure 8A), 1 x 10⁻⁵ M glibenclamide, a blocker of ATP-sensitive potassium channel (K_ATP), or 1 x 10⁻⁷ M iberiotoxin, a blocker of large conductance Ca²⁺-activated K⁺ channels (K_Ca), prior to inducing contractions of IPA with 1 x 10⁻⁵ M PE.
2. Then, add cumulative concentrations of the plant extract.
3. 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.

8. Mechanism of plant extract-induced vasorelaxation via inhibition of extracellular calcium (Ca²⁺) influx in VSMCs

1. 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',N'-tetraacetic acid (EGTA) (Figure 9A).
2. 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).
3. Cumulatively add CaCl₂ (0.01-10 mM) to induce vasoconstriction of the IPA and construct the concentration-response curve (Figure 9A).
4. Repeat this protocol in the same IPA rings but pre-incubate them with plant extract or 1 µM nicardipine (L-type Ca²⁺ channel blocker) in Ca²⁺ containing 80 mM K⁺ solution for 10 min followed by the cumulative addition of CaCl₂⁸.
5. Lastly, compare the contractile response to the maximum contraction previously elicited by control CaCl₂ challenges (Figure 9B).

9. Mechanism of plant extract-induced vasorelaxation via inhibition of intracellular calcium (Ca²⁺) release from the sarcoplasmic reticulum (SR)

1. 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).
2. Replace the bathing solution for 10 min with Ca²⁺-free Krebs solution containing 1 mM EGTA (Figure 10A).
3. Then, challenge the IPA rings with 1 x 10⁻⁵ M PE, which activates the phospholipase C/IP₃ pathway, ultimately releasing Ca²⁺ from the SR and evoking a transient contraction of IPA (Figure 10A).
4. Repeat the same protocol to ascertain likewise transient contractions to PE.
5. 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.
6. Again, challenge the IPA rings with 1 x 10⁻⁵ M PE.
7. Compare the IPA contractions induced by PE between the condition with and without the plant extract (Figure 10B).

10. Statistical analysis

1. Express the results as mean ± SEM. Compare these values using the Student'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 < 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.

Results

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

Discussion

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)...

Materials

List of materials used in this article

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