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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Streszczenie

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Wprowadzenie

Cardiovascular disease is the leading cause of morbidity and mortality in the world, including the United States where it accounts for over 780,000 deaths annually.1 Coronary artery calcification and aortic calcification are hallmarks of atherosclerotic disease and serve as strong predictors of cardiovascular events.2-4 Two main types of vascular calcification have been reported in adults: intimal calcification, associated with atherosclerosis, and medial (also known as Mönckeberg) calcification, associated with chronic kidney disease and diabetes.5 Intimal calcification occurs in the setting of lipid accumulation and macrophage infiltration into the vessel wall.5,6 Medial wall calcification occurs independently of intimal calcification, localizes to elastin fibers or smooth muscle cells, and is not associated with lipid deposition or macrophage infiltration.5,7,8 Studies on the molecular mechanisms of vascular calcification have relied on cell-based and animal model systems. Rodent models for atherocalcific disease include mice deficient in either apolipoprotein E (ApoE)9,10 or low-density lipoprotein receptor (LDLR)11 fed a high-fat diet, while models for medial calcification include mice with matrix Gla protein (MGP) deficiency12 or rats that develop uremia either by near total nephrectomy (the 5/6th nephrectomy model) or by exposure to a high-adenine diet.13

Here, the model of medial vascular calcification associated with MGP deficiency is focused on. MGP is an extracellular protein that inhibits arterial calcification.12 Mutations in the MGP gene have been identified in Keutel syndrome, a rare human disease characterized by diffuse cartilage calcification in addition to brachytelephalangy, hearing loss, and peripheral pulmonary stenosis.14-18 Although not often observed,19 concentric calcification of multiple arteries has been described in Keutel syndrome.20 Common polymorphisms in the human MGP gene are associated with increased risk for coronary artery calcification,21-23 while higher circulating levels of uncarboxylated, biologically inactive MGP predict cardiovascular mortality.24 Unlike humans with Keutel syndrome, MGP-deficient mice develop a severe vascular phenotype consisting of spontaneous widespread arterial calcification starting at two weeks of age and die 6-8 weeks after birth due to aortic rupture.12

Unlike ApoE-/- and LDLR-/- mice fed a high-fat diet, which develop intimal vascular calcification with associated macrophage-induced inflammation, MGP-/- mice develop medial vascular calcification in the absence of macrophage infiltration.11,25 Although these findings suggest different underlying stimuli for intimal and medial calcification, there is overlap in the signaling mechanisms that mediate both forms of calcification.26 Multiple signaling pathways have been identified that contribute to vascular calcification including inflammatory mediators such as tumor necrosis factor-α and IL-1 and pro-osteogenic factors such as Notch, Wnt, and bone morphogenetic protein (BMP) signaling.27,28 These signaling pathways increase expression of the transcription factors runt-related transcription factor 2 (Runx2) and osterix, which in turn increase expression of bone-related proteins (e.g., osteocalcin, sclerostin, and alkaline phosphatase) in the vasculature that mediate calcification.28-30 We and others have demonstrated that the vascular calcification observed in ApoE-/- and LDLR-/- mice fed a high-fat diet and the spontaneous vascular calcification observed in MGP-/- mice all depend on bone morphogenetic protein (BMP) signaling, and it is this pathway that is focused on here.11,25,31 BMPs are potent osteogenic factors required for bone formation and are known to exhibit increased expression in human atherosclerosis.32-34In vitro studies have implicated BMP signaling in regulating the expression of osteogenic factors such as Runx2.35-37 Overexpression of the BMP ligand, BMP-2, accelerates the development of vascular calcification in ApoE-deficient mice fed a high fat diet.38 Moreover, the use of specific BMP signaling inhibitors such as LDN-193189 (LDN)39,40 and/or ALK3-Fc prevents the development of vascular calcification in both LDLR-/- mice fed a high-fat diet and MGP-deficient mice.11,25

Vascular smooth muscle cells (VSMCs) have a critical role in the development of vascular calcification.30,41,42 The medial vascular calcification that develops in MGP-deficient mice is characterized by a transdifferentiation of VSMCs to an osteogenic phenotype. Loss of MGP results in decreased expression of VSMC markers including myocardin and alpha smooth muscle actin, with a concomitant rise in osteogenic markers such as Runx2 and osteopontin. These changes coincide with the development of vascular calcification.25,43,44

Aortic calcification and inflammation in mice are typically assessed utilizing histochemical techniques such as alkaline phosphatase activity for early calcification and osteogenic activity, von Kossa and Alizarin red staining for late calcification, and immunohistochemical protocols that target macrophage protein markers (e.g., CD68, F4/80, Mac-1, Mac-2, Mac-3).9,45 However, these standard imaging techniques require processing of aortic tissues into cross-sections, which is time consuming and imperfect due to sampling bias, and are limited in their ability to quantify inflammation and calcification in the whole aorta. This protocol describes a method to visualize and quantify whole aortic and medium-sized arterial calcification and macrophage accumulation utilizing near-infrared fluorescent (NIR) molecular imaging ex vivo. Also provided is a method for harvesting and culturing primary aortic VSMCs from mice and inducing the calcification of murine and human VSMCs in vitro in order to determine the molecular mechanisms underlying vascular calcification. These techniques provide the investigator with both in vivo and in vitro methods for studying atherocalcific disease.

Protokół

All studies with mice were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Housing and all procedures involving mice described in this study were approved by the Institutional Animal Care and Use Committees of Massachusetts General Hospital (Subcommittee on Research Animal Care). All procedures were performed with care to minimize suffering.

1. Preparation of Reagents

  1. Near-Infrared Fluorescence Imaging of Whole Aortas
    Note: A bisphosphonate-derived, near-infrared fluorescent imaging probe can be used to mark osteogenic activity in the vasculature by binding to hydroxyapatite.46,47 A cathepsin-activated fluorescent imaging probe can serve as a marker for macrophage proteolytic and elastolytic activity in the vasculature.9 To permit the simultaneous use of both fluorescent probes, it is important to use probes that are spectrally distinct. The notation calcium NIR will be used to indicate the calcification-specific near-infrared fluorescent imaging probe and cathepsin NIR to indicate the cathepsin activity-specific near-infrared fluorescent imaging probe.
    1. Prepare the solutions of calcium NIR and cathepsin NIR. As per manufacturer's protocols, add 1.2 ml of 1x phosphate buffered saline (PBS) to the vial containing 24 nmol of calcium NIR or cathepsin NIR and shake gently.
      Note: According to the manufacturer, once reconstituted with PBS, the calcium NIR and cathepsin NIR solutions remain stable for 14 days when stored in the dark at 2-8 °C.
  2. Isolation and Calcification of Murine Aortic VSMCs
    1. Aortic Digestion Solution:
      1. Prepare a fresh solution (~3-5 ml per aorta harvested) with Hank's Balanced Salt Solution (HBSS) containing 175 U/ml type 2 collagenase and 1.25 U/ml elastase. Sterilize the solution with a 0.22 µm vacuum-driven filtration system and keep the solution on ice until use.
    2. Cell Media:
      1. Supplement 500 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Warm the media to 37 °C prior to use.
    3. Calcification Media:
      1. Calcification Media A (NaPhos; used in mouse cell lines):
        1. Supplement 100-500 ml of DMEM (volume as needed) with 10% fetal bovine serum, 2 mM sodium phosphate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Warm the media to 37 °C prior to use.

          OR
           
      2. Calcification Media B (βGP/Asc/DEX; used in either mouse or human cell lines):
        1. Supplement 100-500 ml of DMEM (volume as needed) with 10% fetal bovine serum, 10 mM β-glycerophosphate disodium, 50 µg/ml L-ascorbic acid, 10 nM dexamethasone, 100 units/ml penicillin, and 100 µg/ml streptomycin. Warm the media to 37 °C prior to use.

2. Tail Vein Injection

  1. Before tail injection, warm the mice under a mild heating lamp for 5 min.
  2. Restrain the mouse in a tube rodent holder. Disinfect the tail with an alcohol swab.
  3. Utilize a 30 G needle for tail vein injection. Tail veins are located laterally.
    1. Apply a gentle amount of forward pressure on the syringe as the needle is advanced into the tail. The vein is accessed once the resistance to injection is no longer present.
    2. Inject a volume of 100 µl of calcium NIR and/or 100 µl of cathepsin NIR at a steady rate. At the end of injection, after a 5 sec pause, withdraw the needle.
  4. Harvest the aortas (see section 3) 3-24 hr after injection.

3. Mouse Dissection

  1. Euthanize mouse with a 200 mg/kg intraperitoneal pentobarbital injection.
  2. Lay the animal supine on the dissection board and stabilize by taping each paw to the board. Using a dissecting microscope and small scissors, make a midline incision extending from the lower abdomen to the upper thorax.
  3. Peel back the skin with forceps and remove the peritoneum, revealing the abdominal organs. Remove the gastrointestinal organs, taking care not to transect the aorta.
  4. Make a lateral incision in the anterior diaphragm and continue the incision across the abdomen. Using dissection scissors, release the ribcage by cutting through the sides of the ribs and removing the soft tissue adherent to the superior portion of the sternum. Remove the ribcage, revealing the lungs.
  5. Leave the heart in place initially (to aid in identifying and dissecting the proximal aorta) and carefully remove the lungs. Remove the thymus, trachea, and esophagus with care, ensuring that the aorta remains intact.
  6. Using straight fine forceps and micro-dissecting scissors, remove the soft tissue surrounding the aorta from the iliac bifurcation to the aortic arch, paying careful attention when removing the peri-aortic fat (Figure 1A). Remove the remaining fat and soft tissue surrounding the large branches of the aortic arch (i.e., brachiocephalic, common carotid and subclavian arteries, Figure 1B).
    Note: It is important to remove the fat from the aorta because fat can increase the background signal when performing fluorescence imaging.
  7. Remove the heart from the thoracic cavity, carefully detaching it from the proximal aorta, and discard. Transect the distal aorta at the iliac bifurcation. Using an insulin needle, inject normal saline into the aorta from the aortic arch to wash out remaining blood cells. Detach the aorta along with the aortic arch vessels, completely removing it from the body.
  8. Place the aorta in normal saline solution on ice until ready for imaging.

4. Aortic Imaging

  1. Image aortas ex vivo immediately after harvest by near-infrared fluorescence reflectance imaging.25
    1. Set the fluorescence imager at the appropriate multichannel wavelengths to quantify fluorescence signal intensities from the aortas of calcium NIR and cathepsin NIR-injected mice, as previously described.25 According to the manufacturer, calcium NIR can be excited by ~ 650-678 nm light with a maximal emission in the ~ 680-700 nm range. Cathepsin NIR can be excited by ~ 745-750 nm light with a maximal emission at ~770 nm.

5. Isolation of Primary Murine Aortic Vascular Smooth Muscle Cells

  1. Perform steps 3.1-3.7 as described above.
  2. Place aortas in cold HBSS until the dissections are complete. Carefully cut away any remaining periaortic fat and soft tissue, leaving only the aorta.
  3. Under a sterile tissue culture hood, transfer the aortas to the Aortic Digestion Solution in 35 mm x 10 mm tissue culture dishes. Place in an incubator at 37 °C for 30 min with gentle intermittent rocking. After digestion, the aortas exhibit a stretched or frayed appearance.
  4. With the dissection microscope and sterile forceps, remove the outer adventitial layer of the aorta while keeping the medial layer intact. One technique for removing the adventitia is to peel away the outer layer of the aorta at one end and remove it from the underlying medial layer like a sock could be peeled back and removed.
  5. Once the adventitial layer has been removed, place the remaining aorta into a new tissue culture dish with cell culture media and store at 37 °C with 5% CO2 for 2-4 hr.
  6. Under a sterile hood and using sterile 3 mm micro-dissection scissors, cut the aorta into 1-2 mm wide rings.
  7. Place these rings in a new tissue culture dish with Aortic Digestion Solution and incubate at 37 °C with gentle intermittent rocking for 120 min. Pipette the solution up and down several times during this incubation to resuspend cells.
  8. Add 5 ml of warm cell culture media to the digestion solution and transfer to a 15 ml conical tube.
  9. Centrifuge the tube for 5 min at 200 x g.
  10. Aspirate the media and resuspend cells in the desired volume of cell culture media (e.g., 5 ml).
  11. Plate the entire amount of isolated cells from each aorta in a 25 cm2 cell culture flask and incubate at 37 °C with 5% CO2. Propagate cells using standard techniques, as previously described.25,48 During the initial 7-10 days of incubation, change the media every 72-96 hr. As the cells approach confluence, replenish media more frequently (every 48 hr).
    Note: It may take many weeks to grow a sufficient quantity of cells.
  12. Once confluent, passage cells with trypsin that is warmed to 37 °C.
    1. Add 0.5-1.0 ml of trypsin to each culture flask and incubate for 3-5 min; gently tap the side of the flask every 30-60 sec as needed to detach cells from the surface.
    2. Once the cells detach from the bottom of the flask, add 10 ml of cell media to the cells in trypsin. Centrifuge the cells at 200 x g for 5 min. Aspirate the media and trypsin from the cell pellet. Resuspend the cells in the desired amount of fresh cell media (e.g., 5-10 ml) and transfer to a new flask (with some cells transferred to a chamber slide).
  13. At the first passage of cells, confirm the smooth muscle cell lineage with standard immunocytochemistry techniques, as previously described,49 using an antibody directed against α-smooth muscle actin.

6. Inducing Calcification of Cultured Smooth Muscle Cells

  1. Plate cells obtained from 5.12 in a 6-well format. Note: Starting with 1 x 105 cells/well in a total volume of 2.0 ml of cell media per well is recommended.
  2. Allow cells to grow in Calcification Media A or B for at least 7 days in a 6-well plate format. Incubate cells at 37 °C with 5% CO2.
  3. Change cell media every 48 hr.

7. Assessing VSMC Calcification Using the von Kossa Staining Method

Note: The von Kossa method for measuring extracellular matrix calcification of tissues or cultured cells is based on the substitution of phosphate-bound calcium ions with silver ions.50 In the presence of light and organic compounds, the silver ions are reduced and visualized as metallic silver. Any unreacted silver is removed by treatment with sodium thiosulfate.50 The protocol for von Kossa staining is as follows:

  1. Aspirate media from cell culture plates.
  2. Fix cells by placing them in 1 ml of 10% formalin at room temperature for 20 min.
  3. Remove the formalin and wash the fixed cells with distilled water for 5 min.
  4. Incubate cells in 1 ml of 5% silver nitrate solution under a 60-100 W bulb for 1-2 hr.
  5. Aspirate the silver nitrate solution and wash with distilled water for 5 min.
  6. Remove unreacted silver by placing the cells in 1 ml of 5% sodium thiosulfate (w/v) solution in distilled water for 5 min.
  7. Rinse cells with distilled water for 5 min. Repeat washes 3x. The von Kossa stain is ready for imaging with standard inverted light microscopy.
  8. Optional Step: Counterstain with 1ml of nuclear fast red for 5 min. Follow this with three washes with distilled water (5 min each).

OR

8. Assessing VSMC Calcification with Near-infrared Fluorescent Imaging

Note: Similar to its ability to identify calcification within mouse aortas, calcium NIR readily binds calcific mineral deposited by cultured cells. Using this technique, fluorescent microscopy and plate readers with long wavelength filters can image and quantify in vitro calcification, respectively. The long wavelength emission of the calcium NIR allows for the simultaneous utilization of lower wavelength emitting fluorophores to detect other features. The protocol for calcium NIR staining is as follows:

  1. As described in Section 1.1.1, add 1.2 ml of 1x PBS to the vial containing 24 nmol of calcium NIR.
  2. Dilute the calcium NIR stock 1:100 in the appropriate calcification or control media.
  3. Aspirate cell media from culture plates and replace with the calcium NIR-containing culture media.
  4. Incubate the culture plates with calcium NIR media overnight at 37 °C.
  5. Aspirate the media from the wells and wash the wells once with PBS.
    Note: At this point, the original culture media can be added to the wells, and the cells can be imaged live. Otherwise, proceed to the steps below.
  6. Fix cells by placing them in 1 ml of 10% formalin at room temperature for 20 min.
  7. Remove the formalin and wash the fixed cells with distilled water for 5 min. Repeat washes 3x.
  8. Optional step: Perform immunofluorescence staining or other counterstains for proteins of interest.8,9
  9. Image or detect the calcium NIR stain using appropriate fluorescence excitation wavelengths (e.g., calcium NIR can be excited by 650-678 nm light) and emission filters (~ 680-700 nm).

Wyniki

Aortic calcification in MGP-/- and wild-type mice was measured using imaging of calcium NIR fluorescence. No calcium NIR signal was detected in the aortas from wild-type mice, indicating the absence of calcification (Figure 2). A strong calcium NIR signal was detected in the aortas from MGP-deficient mice, which is consistent with advanced vascular calcification. Tissue sections of aortas from wild-type and MGP-/- mice were stained with Alizarin red<...

Dyskusje

Arterial calcification is an important risk factor for cardiovascular disease in humans and may contribute directly to the pathogenesis of cardiovascular events.1,5,52 Intimal calcium deposition in the thin fibrous caps of atherosclerotic disease has been proposed to increase local biomechanical stress and contribute to plaque rupture.53,54 Medial calcification impacts clinical outcomes by increasing arterial stiffness, which can induce cardiac hypertrophy and affect cardiac function.55 T...

Ujawnienia

Massachusetts General Hospital has applied for patents related to small molecule inhibitors of BMP type I receptors and the application of ALK3-Fc to treat atherosclerosis and vascular calcification, and MD, PBY, KDB, and RM may be entitled to royalties.

Podziękowania

This work was supported by the Sarnoff Cardiovascular Research Foundation (MFB and TET), the Howard Hughes Medical Institute (TM), the Ladue Memorial Fellowship Award from Harvard Medical School (DKR), the START-Program of the Faculty of Medicine at RWTH Aachen (MD), the German Research Foundation (DE 1685/1-1, MD), the National Eye Institute (R01EY022746, ESB), the Leducq Foundation (Multidisciplinary Program to Elucidate the Role of Bone Morphogenetic Protein Signaling in the Pathogenesis of Pulmonary and Systemic Vascular Diseases, PBY, KDB, and DBB), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR057374, PBY), the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK082971, KDB and DBB), the American Heart Association Fellow-to-Faculty Award #11FTF7290032 (RM), and the National Heart, Lung, and Blood Institute (R01HL114805 and R01HL109506, EA; K08HL111210, RM).

Materiały

NameCompanyCatalog NumberComments
15 ml conical tubeFalcon352096
30 G needleBD305106
Alpha smooth muscle actin antibodySigmaSAB2500963
Chamber slideNunc Lab-Tek154461
Collagenase, Type 2 WorthingtonLS004176
DexamethasoneSigmaD4902
Dulbecco's Modified Eagle MediumLife Technologies11965-084
Dulbecco's Phosphate Buffered Saline, no calciumGibco14190-144
ElastaseSigmaE1250
Fetal bovine serumGibco16000-044
Forceps, fine pointRobozRS-4972
Forceps, full curve serratedRobozRS-5138
Formalin (10%)Electron Microscopy Sciences15740
Hank's Balanced Salt SolutionGibco14025-092
Human coronary artery smooth muscle cellsPromoCellC-12511
Insulin syringe with needleTerumoSS30M2913
L-ascorbic acidSigmaA-7506
Micro-dissecting spring scissors (13 mm)RobozRS-5676
Micro-dissecting spring scissors (3 mm)RobozRS-5610
NIR, cathepsin (ProSense-750EX)Perkin ElmerNEV10001EX
NIR, osteogenic (OsteoSense-680EX)Perkin ElmerNEV10020EX
Normal SalineHospira0409-4888-10
Nuclear fast redSigma-AldrichN3020
Odyssey Imaging SystemLi-CorOdyssey 3.0
Penicillin/StreptomycinCorning30-001-CI
Silver nitrate (5%)Ricca Chemical Company6828-16
Sodium phosphate dibasic heptahydrateSigma-AldrichS-9390
Sodium thiosulfateSigmaS-1648
ß-glycerophosphate disodium salt hydrateSigmaG9422
Tissue culture flask, 25 cm2Falcon353108
Tissue culture plate (35 mm x 10 mm)Falcon353001
Tissue culture plate, six-wellFalcon353046
TrypsinCorning25-053-CI
Tube rodent holderKent ScientificRSTR551
Vacuum-driven filtration systemMilliporeSCGP00525

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