The authors would like to thank the physicians and support staff of the Advanced Heart Failure and Pulmonary Hypertension service of Allegheny General Hospital for the provision of hundreds of catheter samples over several years that have facilitated the optimization of this methodology.

&#8203;All studies described in this article were approved by the Institutional Review Board of the Allegheny Health Network. All patients provided informed consent before the administration of sedative agents and were de-identified by being assigned a study number.
1. Bedside Collection
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. It is important to induce cold stasis in the cells to preserve their in situ biological phenotype. This protocol involves the handling of blood and cells obtained from humans and therefore universal precautions should be observed.
<ol>
	<li>After withdrawal of the catheter from the sheath, immediately immerse the catheter in a 50 mL centrifuge tube containing 40 mL of chilled sterile saline. Swirl the catheter several times to remove blood, and flush the line if necessary to remove blood retained within the line.</li>
	<li>Cut off the distal 5 cm of the cath tip and allow it to fall into the 50 mL centrifuge tube containing chilled saline. Immediately remove the cath tip with forceps and transfer it to a 15 mL centrifuge tube containing 15 mL of endothelial cell (EC) growth medium.</li>
	<li>Seal the tube, place it on ice and transfer to the lab to prepare for processing or shipping. It is critical that the balloon stays immersed in medium, so make sure there is minimal headspace in the 15 mL tube by filling it completely with EC growth medium.</li>
	<li>Process the sample immediately by proceeding to Step 2.1 or seal and ship the sample on wet ice overnight for processing at a laboratory skilled in this protocol. If cells are ultimately going to be used for cell culture, it is strongly recommend that the cath tip be immediately processed at the same site at which it was collected.</li>
</ol>
2.Cell Harvest
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. The cells are in cold stasis, and it is important to avoid awakening them until they are ready to be fixed or cultured. This entire protocol should be performed in a laminar flow hood, using sterile instruments, if the ultimate intent is culture of the harvested PAECs. If culture is not planned (e.g., flow cytometry analysis), it is not necessary to observe sterile precautions.
<ol>
	<li>Add 5 mL of cell detachment solution to a 15 mL centrifuge tube; place on ice.</li>
	<li>Draw 1 mL of EC media from that remaining in the bedside collection tube into the 1 mL syringe.</li>
	<li>Quickly complete steps 2.3 through 2.5. Grip the cut end of the catheter tip with hemostats.</li>
	<li>Carefully insert the needle of the 1 mL syringe into the &#34;white&#34; hole opposite the guide wire (&#34;black hole&#34;). Take great care to avoid injury with the needle.</li>
	<li>Transfer the cath tip into the 15 mL centrifuge tube containing the cell detachment solution.</li>
	<li>Inflate the balloon, being careful not to burst it; inflating the balloon with approximately 200 &#181;L of media works well.</li>
	<li>Allow the catheter to sit in cell detachment solution on ice for 5 min with the balloon inflated. If the balloon deflates on its own during the 5 min incubation, this is acceptable.</li>
	<li>Pour the contents of the 15 mL centrifuge tube into the 100 mm dish. Place the balloon in the cell detachment solution &#34;puddle&#34;.</li>
	<li>Scrape the inflated balloon with a cell scraper. The cell scraper can be drawn around the balloon in a circular motion or a brushing motion. Focus on the &#34;poles&#34; of the balloon, where it meets the catheter. Ensure that the cell scraper contacts the entire surface of the balloon at least twice. Rinse the cell scraper in the cell detachment solution &#34;puddle&#34; periodically.</li>
	<li>Deflate the balloon and scrape it with a brushing motion. Rinse the cell scraper in the cell detachment solution &#34;puddle&#34;.</li>
	<li>Discard the cath tip, needle and cell scraper in the appropriate waste container.</li>
	<li>Pour the cell detachment solution containing the cell harvest into a 50 mL centrifuge tube. Shake the collection tube and pour the remaining media from the collection tube into the collected sample as well.</li>
	<li>Centrifuge the sample at 650 x g at 4 &#176;C for 10 min. It is important to note that oftentimes a pellet will NOT be seen. It can be helpful to mark the location where a pellet would be seen, if visible, to avoid aspirating the pellet in the event it is too small to be seen. 
	NOTE: Normally, a pellet is only seen when the sample has been contaminated by blood. Blood contamination is immaterial to the ultimate harvest product because blood fails to bind the CD146 beads and washes through the column. Extensive testing has demonstrated that the specific brand of 1.5 mL microcentrifuge tubes referenced in the Table of Materials minimizes cell loss relative to other tubes utilized during protocol optimization.</li>
	<li>Carefully aspirate media, and avoid disturbing or aspirating the pellet.</li>
</ol>
3. Cell Purification
NOTE: Everything in this protocol should be done on ice, using pre-chilled solutions. The cells are in cold stasis, and it is important to avoid awakening them until they are ready to be fixed or cultured.
<ol>
	<li>Resuspend the pellet in 1 mL of chilled red blood cell lysis solution, and transfer it to a 1.5 mL microfuge tube; gently rock for 10 min at 4 &#176;C.</li>
	<li>Centrifuge for 5 min at 500 x g and 4 &#176;C. Aspirate the RBC lysis solution.</li>
	<li>Resuspend the pellet in 60 &#956;L of PBS, 20 &#956;L of FcR blocker, and 20 &#956;L of anti-human CD146 microbeads. Incubate for 15 min at 4 &#176;C.</li>
	<li>Add 1 mL of chilled PBS. Centrifuge for 5 min at 500 x g and 4 &#176;C. Aspirate the supernatant and resuspend the pellet in 1 mL of chilled PBS.</li>
	<li>Place a magnetic LS column on a magnetic rack (see Table of Materials for specific commercial identities of these items) at room temperature. Prime column by adding 1 mL of PBS and allow the entire volume to flow through. Discard the flow-through.</li>
	<li>Place the sample in the magnetic column. Discard the flow-through.</li>
	<li>Wash column with 3 mL of chilled PBS three times. Discard the flow-through from each wash.</li>
	<li>Add 3 mL of chilled PBS to a 15 mL centrifuge tube. Remove the magnetic column from magnet, pour the 3 mL of PBS from the 15 mL centrifuge tube into the column and quickly place the column in the centrifuge tube to catch the flow-through. Immediately plunge with the plunger supplied with column. The sample is now purified, ready for fixation/staining, or culture.</li>
</ol>

<ol>
	<li>Galie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+ESC/ERS+Guidelines+for+the+diagnosis+and+treatment+of+pulmonary+hypertension:+The+Joint+Task+Force+for+the+Diagnosis+and+Treatment+of+Pulmonary+Hypertension+of+the+European+Society+of+Cardiology+(ESC)+and+the+European+Respiratory+Society+(ERS):+Endorsed+by:+Association+for+European+Paediatric+and+Congenital+Cardiology+(AEPC),+International+Society+for+Heart+and+Lung+Transplantation+(ISHLT).">2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT).</a> European Respiratory Journal. 46 (4), 903-975 (2015).</li><li>Pollett, J. B., Benza, R. L., Murali, S., Shields, K. J., Passineau, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvest+of+pulmonary+artery+endothelial+cells+from+patients+undergoing+right+heart+catheterization.">Harvest of pulmonary artery endothelial cells from patients undergoing right heart catheterization.</a> Journal of Heart Lung Transplantation. 32 (7), 746-749 (2013).</li><li>Benza, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+expression+of+Bcl-2+in+pulmonary+artery+endothelial+cells+associates+with+pulmonary+arterial+hypertension+relative+to+heart+failure+with+preserved+ejection+fraction.">In situ expression of Bcl-2 in pulmonary artery endothelial cells associates with pulmonary arterial hypertension relative to heart failure with preserved ejection fraction.</a> Pulmonary Circulation. 6 (4), 551-556 (2016).</li><li>Bull, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+cells+in+pulmonary+hypertension.">Circulating endothelial cells in pulmonary hypertension.</a> Thrombosis and Haemostasis. 90 (4), 698-703 (2003).</li><li>Kaneko, F. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+reaction+products+of+nitric+oxide+as+quantitative+markers+of+primary+pulmonary+hypertension.">Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.</a> American Journal of Respiratory and Critical Care Medicine. 158 (3), 917-923 (1998).</li><li>Rothman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvenous+procurement+of+pulmonary+artery+smooth+muscle+and+endothelial+cells+using+a+novel+endoarterial+biopsy+catheter+in+a+canine+model.">Transvenous procurement of pulmonary artery smooth muscle and endothelial cells using a novel endoarterial biopsy catheter in a canine model.</a> Journal of the American College of Cardiology. 27 (1), 218-224 (1996).</li><li>Savale, L., Guignabert, C., Weatherald, J., Humbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+medicine+and+personalising+therapy+in+pulmonary+hypertension:+seeing+the+light+from+the+dawn+of+a+new+era.">Precision medicine and personalising therapy in pulmonary hypertension: seeing the light from the dawn of a new era.</a> European Respiratory Reviews. 27 (148), (2018).</li><li>Kanwar, M., Raina, A., Passineau, M., Benza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+Pulmonary+Arterial+Hypertension:+Evolving+Therapeutic+Strategies.">Idiopathic Pulmonary Arterial Hypertension: Evolving Therapeutic Strategies.</a> Seminars in Respiratory and Critical Care Medicine. 38 (5), 606-618 (2017).</li><li>Elshal, M. F., Khan, S. S., Takahashi, Y., Solomon, M. A., McCoy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD146+(Mel-CAM),+an+adhesion+marker+of+endothelial+cells,+is+a+novel+marker+of+lymphocyte+subset+activation+in+normal+peripheral+blood.">CD146 (Mel-CAM), an adhesion marker of endothelial cells, is a novel marker of lymphocyte subset activation in normal peripheral blood.</a> Blood. 106 (8), 2923-2924 (2005).</li><li>Elshal, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+population+of+effector+memory+lymphocytes+identified+by+CD146+having+a+distinct+immunophenotypic+and+genomic+profile.">A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile.</a> BMC Immunolology. 8, 29 (2007).</li><li>Newman, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PECAM-1+(CD31)+cloning+and+relation+to+adhesion+molecules+of+the+immunoglobulin+gene+superfamily.">PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.</a> Science. 247 (4947), 1219-1222 (1990).</li></ol>

MJP and RLB are inventors on patents claiming portions of the methods described.

The technique described in this article is best appreciated in the context of previous experimental paradigms for studying the pulmonary vascular endothelium. Previous attempts to obtain PAECs from humans have been limited to explanted/cadaveric lungs5 and a short-lived attempt to develop a catheter capable of pulmonary artery biopsy6. As noted in a recent review7, the present technique is distinguished from lung tissue harvest by its ability to obtain PAECs from a living human, and to do so at multiple time points in the progression of the disease and/or therapy. However, it should be noted that a companion technique has recently been reported whereby induced pluripotent stem cells (IPSCs) can be transdifferentiated into PAECs4,5. This technique produces abundant cultures of patient-specific PAECs and allows study of the effects of patient-specific genetic background on PAEC biology. Despite these advantages, these IPSC-derived PAECs cannot offer any information about the interaction between local microenvironment and patient-specific genetic background. Our own group has presented the view that the "cath tip" and IPSC techniques are likely to be synergistic in advancing the field8.
Within this present and historical context, the limitations of this technique should be noted. First, it is important to note that yields of PAECs derived from this method can vary considerably. Presumably, patient-, disease state- and physician-specific factors contribute to this variability, as the protocol does not contain steps that would be expected to result in major loss of source material. Table 1 shows representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators. Second, cell yields are overall low, and their viability for culture has proven to be lower yet in our hands. While we have previously grown cultures of PAECs using this method1, we assume those results represent rescue of a subset of progenitor cells following die-off of most of the sample and is only intermittently successful in our hands. For example, it is not unusual to find only a few dozen adherent PAECs in culture the morning after a harvest has been plated. Often these micro-cultures do not survive to expansion. Further work is needed to optimize this protocol in a manner that will sustain the viability of PAECs and their conditions in culture to achieve the desired aim of propagation of patient-specific PAEC harvests. Finally, the CD146 positive selection we have adapted cannot exclude contamination with other CD146+ cells, such as circulating endothelial cells4, pericytes, smooth muscle cells and rare CD146+ T cells9,10. Nevertheless, these cells are presumed to be very few in number in samples collected in this manner, and CD146 is considerably more specific for this protocol than other cell markers of endothelial cells such as CD31 (which is also present on a variety of blood-borne cells including platelets11). Any future improvements aimed at improving specificity should be balanced against their potentially negative effect on yield.

Current U.S. and European guidelines require right heart catheterization for the diagnosis of pulmonary hypertension1. Physicians and scientists seeking to understand this disease desire biological samples from the pulmonary vasculature to complement the hemodynamic data that is gathered during RHC. Unfortunately, the risk associated with pulmonary vascular biopsy is very high, and therefore biological samples of the pulmonary vasculature are rarely if ever obtained from living patients undergoing RHC. While much insight has been gained from pulmonary vascular tissue obtained from cadaveric or explanted lungs, these samples are unable to reflect the biology of the PH disease process as it is unfolding in a living patient. In 2013, we first reported that pulmonary artery endothelial cells (PAECs) are denuded from the branches of the pulmonary artery by physical contact with the balloon tip of the Swan-Ganz catheter during RHC, and can be recovered in small numbers from the catheter tips after the procedure2.
This article illustrates our technique for the removal and purification of pulmonary artery endothelial cells (PAECs) from the balloon tips of Swan-Ganz catheters following right heart catheterization. Heretofore, the methodology for performing this harvest technique has been described in insufficient detail in the literature to ensure uniform results between laboratories performing the technique. This article is intended to overcome this deficiency, offering visualized demonstration of the technique, from bedside collection of catheter tips to the final product of purified CD146+ endothelial cells. This technique should aid in advancing research into the pathobiology of various etiologies of PH, and possibly result in the development of new diagnostic assays to complement hemodynamic data gathered during RHC.
The importance of this technique to the field of pulmonary vascular medicine and research is that it provides access to a snapshot of the biology of the pulmonary endothelium in situ and in contrast to earlier studies on explanted or cadaveric lungs, does not represent an endpoint for the subject&#39;s lung. Recently, we have shown that an anti-apoptotic biomarker in these cells may have usefulness in more precisely phenotyping related varieties of pulmonary hypertension3. The yield of cells obtained is relatively small, ~5,000-25,000 PAECs per sample, and these cells are difficult (but not impossible) to culture. We believe that clever clinicians and researchers will find synergies between these companion techniques in order to advance pulmonary vascular research, diagnostics and prognostics.

<table border="0" cellpadding="0" cellspacing="0" style="width:659px;" width="659">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">100mm culture dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:123px;">NC9385690</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">15 mL Centrifuge Tubes</td>
			<td style="width:145px;">Midsci</td>
			<td style="width:123px;">C15R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">16% Formaldehyde</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:123px;">PI28908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">50 mL centrifuge tubes</td>
			<td style="width:145px;">Midsci</td>
			<td style="width:123px;">C50R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Accutase</td>
			<td style="width:145px;">Thermo</td>
			<td style="width:123px;">A1110501</td>
			<td>cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Cell Scraper</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:123px;">08-100-241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Endothelial Cell Growth media</td>
			<td style="width:145px;">Cell Applications</td>
			<td style="width:123px;">211-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Forceps</td>
			<td style="width:145px;">any</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Hemostat</td>
			<td style="width:145px;">any</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">LoBind microfuge tube</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:123px;">13-698-794&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Miltenyi CD146 beads</td>
			<td style="width:145px;">Miltenyi</td>
			<td style="width:123px;">130-093-596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Miltenyi FCR blocker</td>
			<td style="width:145px;">Miltenyi</td>
			<td style="width:123px;">130-059-901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Miltenyi LS columns</td>
			<td style="width:145px;">Miltenyi</td>
			<td style="width:123px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Miltenyi magnet</td>
			<td style="width:145px;">Miltenyi</td>
			<td>MidiMACS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Miltenyi RBC lysis buffer</td>
			<td style="width:145px;">Miltenyi</td>
			<td style="width:123px;">130-094-183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">PBS</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:123px;">BP2438-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Scissors</td>
			<td style="width:145px;">any</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:222px;">Syringes</td>
			<td style="width:145px;">BD</td>
			<td style="width:123px;">309597</td>
			<td></td>
		</tr>
	</tbody>
</table>

harvest of endothelial cells from the balloon tips of swan-ganz catheters after right heart catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol results in the purification of cells, from Swan-Ganz catheter tips after right heart catheterization, that bind to a CD146 selection column and have forward/side scatter profiles that are indistinguishable from cultured primary human pulmonary artery endothelial cells (Figure 1). Given that the catheter balloon is in physical contact with only the introducer sheath (a cell-free material), the pulmonary artery vessel wall, and the blood, data is provided herein to demonstrate that these cells are not derived from the blood (Figure 2), and therefore it is assumed that they are indeed derived from the pulmonary artery vessel wall. The reader should note that the anti-CD146 magnetic beads that are used to purify the cells remain adherent to the cell surface after the procedure, and thus it is suboptimal to attempt to analyze the cell harvests for CD146 as a marker of endothelial cells. CD31 or other endothelial markers would presumably be available for labeling and could be considered as alternatives.
Key elements of this protocol have been identified that may affect results. First, it is important to rinse the catheter tip at the bedside in order to remove blood. Figure 3 shows Side scatter (SSC)/Forward scatter (FSC) profiles of flow-through from the anti-CD146 column for one sample that was properly rinsed and one that was not. The column appears to exclude blood-derived cells from the harvests (Figure 2), but the rinsing step will ensure minimal possible contamination from circulating endothelial cells (which are normally present at &#60;50 cells/mL of blood4). We have observed that eluate from the CD146 column is completely clear, even in the case of very bloody catheter tip samples. This suggests that the column is able to handle significant amounts of blood without clogging or retaining non-CD146+ bloodborne cells.
Second, it is important to inflate the balloon tip at least once during the cell detachment solution/cell scraper step. Given that the putative PAECs on the balloon tip remain adherent despite the saline rinse step, it seems likely they may be partially trapped by folds in the vinyl balloon as it is deflated in preparation for removal from the patient. Presumably, the inflation step frees the PAECs, allowing them to be gently scraped away. It is not immediately clear one might test this hypothesis, but lower yields of cells have been observed when the balloon is not inflated (data not shown).
Finally, it is important to note that yields of PAECs derived from this method can vary considerably. Presumably, patient-, disease state- and physician-specific factors contribute to this variability, as the protocol does not contain steps that would be expected to result in major loss of source material. Table 1 shows representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58353/58353fig1.jpg" /> 
Figure 1: SSC/FSC profiles of a typical catheter balloon tip harvest from a pulmonary hypertension patient (A) and a similar number of cells from a commercially-acquired primary human PAEC culture (B). Both samples were purified with the anti-CD146 column, fixed, and permeablized as described in the protocol, prior to FACS analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58353/58353fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58353/58353fig2.jpg" /> 
Figure 2: SSC/FSC profiles of a typical catheter balloon tip harvest from a pulmonary hypertension patient (A) and serial 1:10 dilution of Ficoll gradient-purified human buffy coat (B, C, D). (A) was purified with the anti-CD146 column whereas (B-D) were not. All cell samples were fixed and permeablized as described in the protocol, prior to FACS analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58353/58353fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58353/58353fig3.jpg" /> 
Figure 3: SSC/FSC profiles of a catheter balloon tip harvest from a pulmonary hypertension patient in which blood was visible on the catheter tip prior to processing. Prior to step 3.1 of the protocol, the sample was split and lysis buffer was added to one aliquot and withheld from the other. (A) and (C) show the SSC/FSC profiles of the lysed sample, both the cells retained by the column (A) and the flow-through from the column. (B) and (D) show the SSC/FSC profiles of the unlysed sample, both the cells retained by the column (B) and the flow-through from the column (D). We conclude that lysis removes some but not all of the leukocytes but that leukocytes are not retained by the column and are thus excluded from the final harvest, regardless of lysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58353/58353fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Date</td>
			<td>Cell Yield</td>
			<td>Diagnosis</td>
		</tr>
		<tr>
			<td>433</td>
			<td>4/17/2018</td>
			<td>7364</td>
			<td>PH-Interstitial lung disease</td>
		</tr>
		<tr>
			<td>279</td>
			<td>4/17/2018</td>
			<td>7130</td>
			<td>PH-Scleroderma</td>
		</tr>
		<tr>
			<td>432</td>
			<td>4/13/2018</td>
			<td>3976</td>
			<td>PH-Multifactorial</td>
		</tr>
		<tr>
			<td>431</td>
			<td>4/13/2018</td>
			<td>3634</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>390</td>
			<td>4/13/2018</td>
			<td>5666</td>
			<td>PAH</td>
		</tr>
		<tr>
			<td>423</td>
			<td>3/23/2018</td>
			<td>9024</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>272</td>
			<td>3/23/2018</td>
			<td>7098</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>408</td>
			<td>2/27/2018</td>
			<td>4784</td>
			<td>HFrEF</td>
		</tr>
		<tr>
			<td>403</td>
			<td>2/14/2018</td>
			<td>10376</td>
			<td>PH-ASD+mitral disease</td>
		</tr>
		<tr>
			<td>396</td>
			<td>1/26/2018</td>
			<td>4890</td>
			<td>HFpEF-IPCPH</td>
		</tr>
		<tr>
			<td>397</td>
			<td>1/26/2018</td>
			<td>7672</td>
			<td>Normal hemodynamics</td>
		</tr>
		<tr>
			<td>318</td>
			<td>1/4/2018</td>
			<td>7002</td>
			<td>HFrEF-LVAD present</td>
		</tr>
	</tbody>
</table>
Table 1: CD146+ yields from a variety of PAEC harvests. Representative CD146+ yields from a variety of PAEC harvests utilizing catheter tips obtained from a variety of patients with different disease states of pulmonary hypertension and a variety of physician operators.

Watch this Scientific Journal Video about Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization at JoVE.com

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

8.2K Views.  Allegheny Health Network. This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body. 

Video: Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ventricular (RV) function has strong diagnostic and prognostic power in various cardiovascular disorders. Due to its complex morphology and function, assessment of the RV by conventional two-dimensional echocardiography is limited: the everyday clinical practice usually relies on simple linear dimensions and functional measures. Three-dimensional (3D) echocardiography overcame these limitations by providing volumetric quantification of the RV free of geometrical assumptions. Here, we offer a step-by-step guide to obtain and analyze 3D echocardiographic data of the RV using the leading commercially available software. We will quantify 3D RV volumes and ejection fraction. Several technical aspects may help to improve the quality of RV acquisition and analysis as well, which we present in a practical manner. We review the current opportunities and the limiting factors of this method and also highlight the potential applications of 3D RV assessment in current clinical practice.

Here, we provide a step-by-step acquisition and analysis protocol for the 3D volumetric assessment of the right ventricle, mainly focusing on the practical aspects that maximize the feasibility of this technique.

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ...

The Intra-Aortic Balloon Pump

Cardiogenic shock remains one of the most challenging clinical syndromes in modern medicine. Mechanical support is being increasingly used in the management of cardiogenic shock. Intra-aortic balloon pump (IABP) is one of the earliest and most widely used types of mechanical circulatory support. The device acts by external counterpulsation and uses systolic unloading and diastolic augmentation of aortic pressure to improve hemodynamics. Although IABP provides less hemodynamic support when compared with newer mechanical circulatory support devices, it can still be the mechanical support device of choice in appropriate situations because of its relative simplicity of insertion and removal, need for smaller size vascular access and better safety profile. In this review, we discuss the equipment, procedural and technical aspects, hemodynamic effects, indications, evidence, current status and recent advances in the use of IABP in cardiogenic shock.

We describe the steps for the percutaneous implantation of the intra-aortic balloon pump (IABP), a mechanical circulatory support device. It acts by counterpulsation, inflating at the onset of diastole, augmenting diastolic aortic pressure and improving coronary blood flow and systemic perfusion, and deflating before systole, reducing left ventricular afterload.

Cardiogenic shock remains one of the most challenging clinical syndromes in modern medicine. Mechanical support is being increasingly used in the ...

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) using acetylcholine (ACh) spasm provocation testing. CAS can further be divided into different subtypes (e.g., focal, diffuse epicardial, or microvascular spasm), each with different pathophysiological mechanisms that may require tailored drug treatment. The evidence behind the role of nitrates in the setting of each CAS subtype is lacking, and the effectivity can vary on a per-patient basis. In order to assess on a per-patient level whether nitroglycerine (NTG) can prevent inducible spasm, the vasospastic ACh dose can be readministered after NTG administration as part of the spasm provocation test. The preventive effect of NTG is assessed by evaluating improvements in the severity of induced symptoms, ischemic ECG changes, and by reassessing the site and mode of spasm on angiography. This technique can therefore be used to assess the nitrate responsiveness on a per-patient level and unmask co-existing microvascular spasm in patients with epicardial spasm that is prevented with NTG. The NTG rechallenge, therefore, allows to further guide targeted therapy for CAS and provide new insights into the pathophysiological mechanism behind vasospastic disorders.

This protocol presents the acetylcholine rechallenge after nitroglycerine as an add-on procedure to spasm provocation testing. The purpose of this technique is to unmask co-existing microvascular spasm&#160;in patients with epicardial spasm&#160;and to assess the protective efficacy of nitroglycerine on a per-patient level to guide medical therapy.

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) ...