Name: Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition
Uploaded: 2023-07-21T14:00:00
Description: Watch this Scientific Journal Video about A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition at JoVE.com

This research was funded by Additional Ventures - Single Ventricle Research Fund (SVRF) and Single Ventricle Expansion Fund (to I.F.) and a Marietta Blau scholarship of the OeAD-GmbH from funds provided by the Austrian Federal Ministry of Education, Science and Research BMBWFC (to G.G.).

All the animal procedures were conducted in accordance with the National Research Council. 2011. Guide for the Care and Use of Laboratory Animals: Eighth Edition. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Boston Children&#39;s Hospital.
Prior to surgery, all the surgical instruments are steam-autoclaved, and modified Krebs-Henseleit buffer, with a final concentration of 22 mmol/L KCl, is prepared as a cardioplegic solution (Ta...

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

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M., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+atrioventricular+valvuloseptal+morphogenesis.">Molecular regulation of atrioventricular valvuloseptal morphogenesis.</a> Circulation Research. 77 (1), 1-6 (1995).</li><li>Illigens, B. 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=Vascular+endothelial+growth+factor+prevents+endothelial-to-mesenchymal+transition+in+hypertrophy.">Vascular endothelial growth factor prevents endothelial-to-mesenchymal transition in hypertrophy.</a> Annals of Thoracic Surgery. 104 (3), 932-939 (2017).</li><li>Zeisberg, E. M., Potenta, S. 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This animal model of heterotopic transplantation of a neonatal donor rat heart into the recipient&#39;s abdomen creates the possibility to study EndMT-derived fibrosis through detailed histological tissue evaluation, identify regulatory signaling pathways, and test treatment options. Since EndMT is the underlying mechanism for fibrotic diseases of the heart, this model has great value in the field of pediatric cardiac surgery and beyond. In this model, many factors can negatively influence the outcome of the procedure. T...

Endocardial fibroelastosis (EFE), defined by the accumulation of collagen and elastic fibers in the subendocardial tissue, presents as a pearly or opaque thickened endocardium; EFE undergoes most active growth during the fetal period and early infancy1. In an autopsy study, 70% of cases with hypoplastic left heart syndrome (HLHS) were associated with the presence of EFE2.
Cells expressing markers for fibroblasts are the main cell population in EFE, but these cells also concomitantly express endocardial endothelial markers, which is an indication of the origin of these EFE cells. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts through endothelial-to-mesenchymal transition (EndMT)3. EndMT can be detected using immunohistochemical double-staining for endothelial markers such as cluster of differentiation (CD) 31 or vascular endothelial (VE)-cadherin (CD144) and fibroblast markers (e.g., alpha-smooth muscle actin, &#945;-SMA). Furthermore, we also previously established the regulatory role of the TGF-&#223; pathway in this process with activation of the transcription factors SLUG, SNAIL, and TWIST3.
EndMT is a physiological process that occurs during embryonic cardiac development and leads to the formation of the septa and valves from endocardial cushions4, but it also causes organ fibrosis in heart failure, kidney fibrosis, or cancer and plays a key role in vascular atherosclerosis5,6,7,8. EndMT in cardiac fibrosis is mainly regulated through the TGF-&#946; pathway, as we and others have reported3,9. Various stimuli have been described to induce EndMT: inflammation10, hypoxia11, mechanical alterations12, and flow disturbances, including alterations of the intracavitary blood flow13, and EndMT may also be a consequence of a genetic disease14.
This animal model was developed using the key components of cardiac EFE development, which are immaturity and alterations of the intracavitary blood flow, specifically flow stagnation. Immaturity was fulfilled by using neonatal rat hearts as donors, since neonatal rats are known to be developmentally immature immediately after birth. Heterotopic heart transplantation offered the provision of intracavitary flow restriction15.
From a clinical point of view, this animal model allows for better investigating the impact of EndMT on the growing left ventricle (LV). The growth restriction imposed on the fetal and neonatal heart through EndMT-induced EFE formation16 precludes patients with left ventricular outflow tract obstructions (LVOTO) such as congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair17. This animal model facilitates the study of the cellular mechanisms and regulation of tissue formation through EndMT and allows for the testing of pharmacological treatment options3,18.
Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, EFE formation can be macroscopically observed within 3 days after transplantation. EFE tissue shows the same histopathological characteristics as human EFE tissue from patients with LVOTO.
Hence, this animal model, though developed for pediatric use in the spectrum of HLHS, can be applied when studying various diseases based on the molecular mechanism of EndMT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced Ventilator System For Rodents, SAR-1000</td>
			<td>CWE, Inc.</td>
			<td>12-03100</td>
			<td>small animal ventilator</td>
		</tr>
		<tr>
			<td>aSMA</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Axio observer Z1&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>inverted microscope</td>
		</tr>
		<tr>
			<td>Betadine&#160;Solution</td>
			<td>Avrio Health L.P.</td>
			<td>367618150092</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Invitrogen</td>
			<td>MA1-80069</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DemeLON Nylon black 10-0</td>
			<td>DemeTECH</td>
			<td>NL76100065F0P</td>
			<td>10-0 Nylon suture</td>
		</tr>
		<tr>
			<td>ETFE IV Catheter, 18G x 2</td>
			<td>TERUMO SURFLO</td>
			<td>SR-OX1851CA</td>
			<td>intubation cannula</td>
		</tr>
		<tr>
			<td>Micro Clip 8mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-6471</td>
			<td>microvascular clamps</td>
		</tr>
		<tr>
			<td>Nylon black monofilament 11-0</td>
			<td>SURGICAL SPECIALTIES CORP</td>
			<td>AA0130</td>
			<td>11-0 Nylon</td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Embedding medium for frozen tissue specimen</td>
		</tr>
		<tr>
			<td>p-SMAD2/3</td>
			<td>Invitrogen</td>
			<td>PA5-110155</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Rodent, Tilting WorkStand</td>
			<td>Hallowell EMC.</td>
			<td>000A3467</td>
			<td>oblique shelf for intubation</td>
		</tr>
		<tr>
			<td>Silk Sutures, Non-absorbable, 7-0</td>
			<td>Braintree Scientific</td>
			<td>NC9201231</td>
			<td>Silk suture</td>
		</tr>
		<tr>
			<td>Slug/Snail</td>
			<td>Abcam</td>
			<td>ab180714</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 5-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>5-0 Vicryl</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 6-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J492G</td>
			<td>6-0 Vicryl</td>
		</tr>
		<tr>
			<td>VE-Cadherin</td>
			<td>Abcam</td>
			<td>ab231227</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Zeiss OPMI 6-SFR</td>
			<td>Zeiss</td>
			<td></td>
			<td>Surgical microscope</td>
		</tr>
		<tr>
			<td>Zen, Blue Edition, 3.6</td>
			<td>Zen&#160;</td>
			<td></td>
			<td>inverted microscope software</td>
		</tr>
	</tbody>
</table>

a neonatal heterotopic rat heart transplantation model for the study of endothelial-to-mesenchymal transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition  

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibro elastosis hinders left ventricular growth and prevents biventricular surgical repair for patients with congenital critical aortic stenosis and hypoplastic left heart syndrome. While surgical resection is currently the only available treatment option, EFE frequently recurs. Therefore, we aimed to investigate the mechanisms behind EFE and develop new therapeutic strategies using an animal model.The main developments in the field of EndMT research are restricted to cell culture models which have inherent limitations, and research has been hampered due to a lack of in vivo models. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts, a process called endothelial to mesenchymal transition. This animal model allows for a more generalized examination of EndMT induced fibrosis in the heart, whereas other models are designed to induce EndMT through genetic modifications, hypertension, or dietary restrictions.As EFE often recurs after primary resection, sometimes with an even more infiltrative growth pattern, we are especially interested in the molecular interaction with the underlying myocardium.

Graft viability and beating 
In this work, the graft viability was visually assessed after all the clamps had been removed, and an approximate reperfusion time of 10-15 min was allowed with an open abdomen for observation of the graft. The same scoring system to objectively verify graft viability was used for visual assessment at the end of surgery and for the echocardiography on POD 1, POD 7, and POD 14.
0 = no organ function; 1 = (rest) organ function, only minimal cont...

Watch this Scientific Journal Video about A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition at JoVE.com

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...