Potential Molecular Mechanism of Retrograde Aortic Arch Stenosis in the Hybrid Approach to Hypoplastic Left Heart Syndrome

Abstract

Background

The hybrid palliation for hypoplastic left heart syndrome (HLHS) has emerged as an alternative approach to the Norwood procedure. The development of patent ductus arteriosus (PDA) in-stent stenosis can cause retrograde aortic arch stenosis (RAAS), leading to significant morbidity. This study aimed to identify potential mechanisms of PDA in-stent stenosis contributing to RAAS.

METHODS

Tissues from stented PDA were collected from 17 patients undergoing comprehensive stage 2 repair between 2009 and 2014. Patients requiring RAAS intervention based on cardiology–surgery consensus were defined as RAAS (+) (n=10), whereas patients without any RAAS intervention were defined as RAAS (−) (n=7). Tissues were examined by qPCR analysis for vascular smooth muscle cell (VSMC) differentiation and proliferation markers.

RESULTS

Patient characteristics were: HLHS with aortic atresia: 6; HLHS with aortic stenosis: 3; unbalanced AVC: 3; DILV/TGA: 3; DORV: 2. VSMC differentiation markers (β–actin, SM22, and calponin) and signaling pathways for VSMC modulation (TGFβ1, Notch, and PDGF-BB) were significantly higher in the RAAS (+) than in RAAS (−). The proliferation marker Ki67 was increased in RAAS (+). Cell cycle markers were comparable in both groups.

CONCLUSION

Increased VSMC differentiation and proliferation markers suggest a mechanism for inward neointima formation of the PDA in RAAS. The apparent lack of change in cell cycle markers is contrary to coronary artery in-stent stenosis, suggesting further targets should be examined. Combined primary in vitro PDA cell culture and proteomics can be strong tools to elucidate targets to reduce PDA in-stent stenosis for RAAS in the future.

INTRODUCTION

Single ventricle anomalies (SVAs) comprise the largest groups of congenital heart diseases (CHDs) and are the leading causes of death associated with congenital anomalies in the newborn period [1]. Hypoplastic left heart syndrome (HLHS) is currently one of the most challenging SVAs to treat due to the inadequate development of left heart structures to support the systemic circulation [2]. Surgical methods have improved since the first successful report of the Norwood procedure to manage HLHS in 1983, but this procedure still carries the highest surgical risk among all congenital heart surgeries with a high early mortality of 17-50% [3, 4]. The hybrid procedure for HLHS was first developed in 1993 in response to poor outcomes following the Norwood procedure [5]. Stage I of the hybrid procedure combines surgical placement of bilateral branch pulmonary artery bands, placement of a stent in the patent ductus arteriosus (PDA), and catheter-based atrial septostomy.

One serious complication of the hybrid procedure is the development of retrograde aortic arch obstruction. The obstruction of blood flow through retrograde arch can cause multiple hemodynamic sequelae, including decreased retrograde perfusion of the head and neck vessels, as well as the coronary arteries. This increases morbidity and mortality immediately postoperatively or prior to subsequent surgeries. Previous reports suggested several potential mechanisms of retrograde aortic arch stenosis such as degree of branch pulmonary arterial banding [6], size of ascending aorta [7], aortic root size, and angiographic angle between the aortic isthmus and PDA [8]. We have identified an additional mechanism to account for restriction of retrograde flow into the aortic arch – effectively causing RAAS. At the time of stent deployment in the PDA, part of the strategy to prevent hemodynamically-important constriction of unsupported native ductal tissue involves positioning the stent(s) in such a way that part of the ductal stent actually protrudes into, and often across the lumen of the adjacent aorta. As such, retrograde blood flow into the aortic arch is dependent upon flow through the interstices of the most distal (intra-aortic) portion of the stent. Thus, in instances of in-stent intimal hyperplasia or tissue ingrowth, the interstices within the stent can be narrowed (or some may be occluded), which results in reduced retrograde blood flow into the aortic arch, effectively causing RAAS. Understanding the biology and mechanics of in-stent stenosis is an important step to prevent reduction of retrograde aortic arch flow.

To date, there is very little known about the mechanism of in-stent stenosis of the PDA. We have previously shown that this occurs as the result of neointima formation that is associated with local inflammation, vascular smooth muscle cell (VSMC) deposition, and ECM deposition [9]. In the current study, we hypothesized that PDA instent stenosis occurs as a result of the exacerbated innate proliferative nature of the PDA, contributing to neointimal formation stemming from VSMC proliferation. To test this hypothesis, we analyzed stented PDAs harvested from patients undergoing stage II palliation of HLHS with the presence or absence of retrograde aortic arch stenosis (RAAS) for genetic markers of cellular differentiation and proliferation.

Material and Methods

Patient Population

After hybrid stage I surgery, patients were followed regularly with echocardiography to measure PDA and retrograde aortic arch flow. When abnormal echocardiographic findings or clinical symptoms of cardiac failure or ischemia presented, patients underwent cardiac intervention including balloon dilatation and/or stent placement for the treatment of RAAS based on the consensus in cardiology– surgery conference [7]. Patients requiring RAAS intervention were defined as Group RAAS (+) (n=10), whereas patients without any observable RAAS formed Group RAAS (−) (n =7). RAAS (+) patients showed significantly high echocardiogram peak velocity across the PDA stent (in general more than 3 m/s) and signs of worsening clinical status including electrocardiogram changes and poor weight gain. Detail data of pressure gradient was described previously[10].

The comprehensive stage II operation was performed at an average age of 6 months. Tissues from stented PDA were collected from 17 patients with HLHS or HLHS variant undergoing timely or premature comprehensive stage II repair from 2009 to 2014. The study was approved by the institutional review board at Nationwide Children’s Hospital.

Surgical Technique

Our hybrid strategy and results were recently described in detail [11]. Briefly, an initial hybrid stage 1 palliation consists of the bilateral pulmonary artery band using a 3.0- or a 3.5-mm Gore-Tex tube graft (W.L. Gore & Assoc, Flagstaff, AZ) depending on the patient’s body weight and the stent to cover the PDA completely [7, 10]. The comprehensive stage 2 surgery consists of removal of the PDA stent and PA bands, reconstruction of the aortic arch and diminutive ascending aorta with main pulmonary artery, atrial septectomy, and a bidirectional cavopulmonary anastomosis. Procedures were performed on cardiopulmonary bypass without circulatory arrest [11]. One patient in RAAS (−) group underwent biventricular repair after stage 1 palliation because of the optimal growth of left ventricle.

RNA Isolation and Quantitation

Stented PDAs were collected during comprehensive stage 2 surgery, snap frozen in liquid nitrogen and stored at −80°C. Froz en tissues were pulverized in liquid nitrogen, stents removed, and tissues solubilized by mechanical lysis in Trizol at 4°C. Following centrifugation to remove unlysed material, samples were chloroform extracted and aqueous fractions containing RNA purified using RNeasy columns (Qiagen, Inc., Valencia, CA) and the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 (Thermo Fisher) and equivalent amounts reverse transcribed using the Maxima First Strand Synthesis Kit (Thermo Fisher). Quantitative PCR reactions were performed in duplicate using Thermo Maxima Probe/ROX qPCR Master Mix and Roche Universal Probe/Primer sets. Relative expression was determined using ΔΔC_t analysis with the ribosomal transcript Rpl13a (NM_012423.2) serving as the housekeeping gene (ref below). Genes analyzed for expression were beta-actin (NM_001101.3), alpha-SMA (NM_001141945), transgelin (NM_001001522), smooth muscle calponin (NM_001299), TGFβ1 (NM_000660), Notch 1 (NM_017617.3), Notch 2 (NM_024408), Notch 3 (NM_000435), PDGF-B (NM_002608), Ki67 (NM_002417), PCNA (NM_002592.2), Cyclin D1 (NM_053056.2), p27^Kip1 (NM_004064) and p21^Waf (NM_001220778.1). Primer sequences are listed in Table 4 [12].

Table 4.

Primers for qPCR Analysis

Target	Accession number	Forward primer sequence	Reverse primer sequence
beta-actin	NM_001101.3	CCAACCGCGAGAAGATGA	CCAGAGGCGTACAGGGATAG
alpha- SMA	NM_001141945	TGATCACCATCGGAAATGAA	TGATGCTGTTGTAGGTGGTTTC
transgelin	NM_001001522	GGCCAAGGCTCTACTGTCTG	CCCTTGTTGGCCATGTCT
calponin	NM_001299	TGTCAGCCGAGGTTAAGAACA	CCTCGATCCACTCTCTCAGC
TGFb1	NM_000660	GCAGCACGTGGAGCTGTA	CAGCCGGTTGCTGAGGTA
Notch 1	NM_017617.3	CGGGGCTAACAAAGATATGC	CACCTTGGCGGTCTCGTA
Notch 2	NM_024408	GCAGGAGGTGGATGTGTTAGA	CTCGGAGAGAAGCCAACATC
Notch 3	NM_000435	GCCAAGCGGCTAAAGGTA	CACTGACGGCAATCCACA
PDGF-B	NM_002608	CCTGGCATGCAAGTGTGA	CGAATGGTCACCCGAGTTT
Ki67	NM_002417	AGTAACGCGGAGTGTCAAGAG	TCACTGTCCCTATGACTTCTGG
PCNA	NM_002592.2	TGGCGCTAGTATTTGAAGCA	CAGAAGGCATCTTTACTACACAGC
Cyclin D1	NM_053056.2	GCTGTGCATCTACACCGACA	TTGAGCTTGTTCACCAGGAG
p27Kip1	NM_004064	GCACATAAACTTTGGGGAAGG	TCAAAGCAAGCTCTTCATACCC
p21Waf	NM_001220778.1	TCACTGTCTTGTACCCTTGTGC	GGCGTTTGGAGTGGTAGAAA

Statistical Analysis

All data are represented as mean ± SEM and were analyzed using GraphPad Prism 6.0 software using a Mann-Whitney unpaired t test for comparing between groups. The criterion used for statistical significance was a two-tailed p value less than or equal to 0.05.

Results

Patient Characteristics

The characteristics of the two experimental groups are presented in Table 1. Patients undergoing stage I surgery were of the same age. Patients who presented with RAAS were generally younger and smaller than those who did not. Anatomically, all patients in the RAAS (−) group were diagnosed as HLHS variant or single ventricle physiology, while most patients in the RAAS (+) group were diagnosed as true HLHS (Table 2). High-risk anatomic features such as low body weight, prematurity, restrictive atrial septal defect (ASD), tricuspid valve regurgitation, low cardiac function, and associated cardiac or non-cardiac anomalies were distributed equally in both groups (Table 3).

Table 1.

The Characteristics of the Patients

Retrograde Aortic Arch Stenosis due to in stent stenosis	(−) (N=7)	(+) (N=10)	p
Age at stage I (days)	5.8±5.2	5.1±2.3	0.72
Age at stage II (months)	6.8±3.3	4.4±0.8	0.04
Weight at stage II (kg)	7.3±1.2	5.7±0.7	0.02

Table 2.

Diagnosis of the patients

Retrograde Aortic Arch Stenosis due to in stent stenosis		(−) (N=7)	(+) (N=10)
HLHS
	AA/MA	0	1
	AA/MS	0	5
	AS/MA	0	0
	AS/MS	0	3
Unbalanced AVC		2	1
DILV, TGA		3	0
DORV		2	0

Table 3.

Risk Factors of the Patients

	Retrograde Aortic Arch Stenosis due to in stent stenosis
Risk Factor	− (N=7)	+ (N=10)
Low Birth Weight (<2.5 kg)	1	0
Premature (<35 week)	1	0
Cardiac Anomaly	0	1
Non Cardiac Anomaly	0	0
Restrictive ASD	0	0

Expression Analysis

Vascular smooth muscle cell differentiation markers such as beta-actin, transgelin (SM22) and calponin were significantly higher in the RAAS (+) than in RAAS (−) group (beta-actin: p=0.048, transgelin: p=0.010, calponin: p=0.045) (Figure 1). Signaling pathways known to be involved in smooth muscle cell modulation – TGFβ1, Notch receptors and PDGF-B – were significantly higher in the RAAS (+) group compared with the RAAS (−) group (TGFβ1: p=0.030, Notch1: p=0.011, Notch2: p=0.045, Notch3: p<0.01, PDGF-B: p<0.01) (Figure 1). The cell proliferation marker Ki67 and cell cycle marker p27^Kip1 were increased, and decreased, respectively, in RAAS (+) PDA tissue, although they did not achieve statistical significance (Ki67: p=0.06; p27^Kip1: p=0.13). Other cell cycle markers PCNA, cyclin D1 and cyclin-dependent kinase inhibitors p21^Waf) were not significantly different between the RAAS (+) and RAAS (−) groups (PCNA: p=0.43, cyclin D1: p=0.52, , p21^Waf: p=0.43) (Figure 2).

Figure 1.

Cells from RAAS(+) PDA tissue express increased differentiation and VSMC-modulation markers. Cell differentiation markers (beta actin, SM22, and calponin) and signaling pathways for VSMC modulation (TGFβ1, Notch, and PDGF-B) were significantly higher in the RAAS (+) group compared with the RAAS (−) group. ^*p<0.05, **p<0.01 vs. RAAS(−). n=7-10 per group. Error bars are standard error of the mean.

Figure 2.

Cells from RAAS(+) PDA tissue exhibit signs of proliferation. Cell proliferation marker Ki67 and cell cycle marker p27^Kip1 were increased, and decreased, respectively, although they did not achieve statistical significance. n=7-10 per group. Error bars are standard error of the mean.

Comment

In this study, we demonstrated that retrograde aortic arch stenosis of the stented PDA in patients who underwent the hybrid palliation of HLHS was associated with increased expression of VSMC differentiation and proliferation markers.

In its primal form, the PDA serves to bypass the pulmonary circulation in utero. After birth, the PDA normally closes in two phases: a “functional” vasoconstriction phase followed by permanent “anatomical” closure, the latter of which involves a rapid deposition of extracellular matrix into which VSMC can proliferate and migrate luminally. This process permanently occludes blood flow through the vessel. The hybrid approach to the palliation of HLHS harnesses this anatomical feature to maintain retrograde aortic arch perfusion to the head and neck by using a stent to maintain its patency after birth. A subset of these patients manifests clinical RAAS, which we postulate may be due in part to the innate plasticity of PDA VSMCs. As there is no clear definition of RAAS, we used a practical definition of the requirement for intervention based on the consensus in both cardiologist and cardiac surgeon from echocardiographic or clinical symptoms. A recent study showed that approximately 10% of patients with HLHS have RAAS at birth and 25% of patients with stage I procedure developed RAAS at a mean age of 74 days. Patients who developed RAAS demonstrate a significantly lower survival rate [7], therefore it is important to understand the molecular and physiological mechanisms of RAAS so that it can be better managed and/or prevented.

Indeed, our previous histological analysis of stented human PDA has shown significant neointimal formation containing VSMCs and was associated with local inflammation and extracellular matrix (ECM) deposition similar to what is observed in coronary in-stent restenosis [9]. Our current PDA data are in keeping with many previous studies showing that VSMC differentiation and proliferation plays a critical role in neointimal hyperplasia in coronary in-stent stenosis through inflammation and extracellular matrix deposition [13, 14]. However, it is interesting that PDA tissue composition differs from coronary artery tissue in that the PDA is programmed to close spontaneously through a complex process involving both vasoconstriction (“functional” closure) and neointima cushion formation (“anatomical” closure) [15]. Because of this fundamental difference, lessons learned from coronary restenosis may not be applicable to the PDA. Therefore, in this study, we performed a molecular approach to help better understand and mitigate in-stent PDA restenosis.

Our observed significant increases in beta-actin, alpha-SMA, transgelin and calponin expression, in addition to increased Ki67 and decreased p27^Kip1 expression, in stenotic PDA tissue suggest that their resident VSMCs are activated to a hybrid differentiated and proliferative phenotype in response to increased stimulatory growth factors and cytokines such as TGFβ1, Notch and PDGF-B [16-18] in a manner identical with observations from coronary in-stent stenosis [19]. However, the lack of significant expression differences in cell cycle markers such as cyclin D1and p21^Waf in stenotic PDA tissue is contrary to what has been observed in the coronary in-stent stenosis.

Under normal conditions, VSMCs are in a non-proliferative resting phase (G0) of the cell cycle. After vessel injury induced by stenting, growth factors are released and stimulate cells to (G1) phase that activate cell proliferation [20-23], a process controlled by cyclins and their respective cyclin-dependent kinases (CDKs) [21]. CDK inhibitors such as p27^Kip1 and p21^Waf are also critical negative regulators of cell cycle progression [24] and halt cellular proliferation at the G1-S phase cell cycle transition. p27^Kip1 binds to and inhibits G1 cyclin/CDK complexes and mitigates cell cycle progression [25]. p21^Waf accumulates in late G1, and provides a counterbalance to increased cyclin/CDK activities. Overexpression of p27^Kip1 results in cell cycle arrest in the late G1 phase [26], and gene transfer of p27^Kip1 into balloon-injured vessels significantly reduced VSMC proliferation and neointimal formation [25, 27]. Conversely, reduction in p27^Kip1 levels increased neointimal formation and inflammatory cell accumulation after mechanical vascular injury. Our current data showing reduced p27^Kip1, combined with increased Ki67, are in keeping with these previous findings and suggest a heightened proliferative response in RAAS (+) tissues. Indeed Ki67 is expressed in all phases of the cell cycle except the quiescent G0 phase. The lack of concomitant increased PCNA in our study may suggest that PDA cells from RAAS (+) patients were primarily at the G2-M phase of the cell cycle, a point at which PCNA expression can be low in the face of increased Ki67. Finally, Rapamycin and Paclitxel that are commonly used in drug eluting stents to prevent in-stent stenosis significantly reduce the activity of cyclin/CDK complexes by increasing p27^Kip1 [28-31].

Our lack of observable differences in cyclin D1, p21^Waf and PCNA expression in stenotic PDAs relative to non-stenotic PDAs suggests that stenting the PDA may elicit a different molecular response than stenting a coronary artery. If true, different targets for intervention may need to be identified in the PDA to prevent in-stent stenosis. Alternatively, the lack of expression differences in these markers may be due to the timing of PDA excision. It may be that active cellular proliferation occurs earlier in the restenosis process and that the cells residing in the PDA tissue at excision are relatively less proliferative, although they still retain features of proliferation. Future studies will be required to explore this possibility.

Additional limitations to this study warrant discussion. The primary limitation is the small number of samples. The population of each group is also biased: the number of patients presenting with a small aortic arch was significantly higher in RAAS (+) group. With the limited sample number we did not excluded HLHS variant patients in this study, which may have biased the comparison between non-HLHS vs. HLHS patients. Future in vitro cell studies, increasing the number of PDA samples, and extending our studies to full proteomic analysis of expression changes will provide stronger supportive data for clinical application to investigate therapeutic targets for prevention of RAAS.

The high incidence of RAAS in aortic atresia patients may be attributed to accelerated flow in small diameter vessels, which promotes smooth muscle cell proliferation and differentiation in PDA tissue. Patients with RAAS underwent stage II surgery earlier in order to prevent the risk of ischemic complications in the brain and heart as a result of impaired retrograde arch blood flow. However, every attempt was made to extend the time of stage II surgery as long as possible given that it is a more complex procedure than a simple Glenn surgery following a Norwood operation. Accordingly, RAAS (+) and RAAS (−) tissues could not be collected at specific equivalent time points, which could impact the expression of VSMC phenotype markers measured in our study. Indeed, we can only get the tissue at the time of scheduled surgery, which may not allow us to observe the progression of in-stent PDA stenosis. In addition, it is difficult to determine if this in-stent stenosis originates from the aortic side, the stented PDA side, or both sides in this study because intimal hyperplasia appears to be either well underway or mostly complete.

In conclusion, increased VSMC differentiation and the expression of proliferation signaling markers suggest that VSMCs residing within RAAS (+) PDA tissue exhibit a hybrid phenotype that may be a mechanism for inward neointimal formation. Furthermore, these markers could be therapeutic targets to prevent RAAS. Although the histology of PDA instent stenosis is similar to coronary artery [9], the molecular mechanisms appear to be distinct, which suggests new targets could be required to inhibit in-stent PDA stenosis. Combined primary in vitro PDA cell culture and proteomics can be a strong tool to elucidate targets to reduce RAAS in the future.

Discussion

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Paper presented by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

Discussion by Sitaram M. Emani, M.D., Boston, Massachusetts, sitaram.emani@cardio.chboston.org, sitaram.emani@childrens.harvard.edu

DR. S. EMANI (Boston, Massachusetts): I assume that in these patients, this is an acquired phenomenon. So there is obviously some patients who had retrograde arch obstruction from the very get-go. That is, they apply the stent and they develop it. That is a mechanical phenomenon.

And in all these patients, I assume, you did not have retrograde arch obstruction to begin with, but then developed it over time?

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Response by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

DR. HIBINO: Yes, exactly.

DR. EMANI: And you distinguished those two groups?

DR. HIBINO: Yes.

we followed patients after stage I surgery with an ultrasound, and then if significant stenosis was found, we decided to take the patient to the cath lab. The patients who required intervention for retrograde aortic arch stenosis were assigned as stenosis group. The PDA stent tissue was collected at the time of comprehensive surgery. So, this is a retrospective analysis.

DR. EMANI: What is your management of a patient with retrograde stenosis at this point? Would you stent within it? How do you decide how to manage these patients?

DR. HIBINO: So, as I said, cardiology follows the patient very closely after stage 1 almost every month by using ultrasound and clinical findings.

If they find significant stenosis in retrograde aortic arch or PDA, we decide to take the patient to the cath lab. Then if there is a significant stenosis in the cath, we place the stent there.

Because we want to wait to do surgery as late as possible until 6 months due to the complexity of stage II procedure, we extend the surgery by placing the stent.

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Paper presented by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

Discussion by Christopher Caldarone, MD, Toronto, Ontario, Canada. chris.caldarone@sickkids.ca, Christopher.caldarone@sickkids.ca

DR. CALDARONE: We have a different strategy.

If a quarter of the patients are developing the problem of retrograde arch hypoplasia, I think it is reasonable to undertake a prophylactic strategy.

So when we do a hybrid with a limited prograde flow across the aortic valve, we use a reverse BT shunt, although we have not been doing as many hybrids recently.

Using that strategy, we have not had to take a patient to the cath lab for retrograde arch obstruction, except in two patients. One of whom we did not do the reverse BT shunt first, and another who had some other issues as well.

So we try to prevent the problem and inherit the problems associated with that strategy, versus waiting for retrograde arch malperfusion to develop and then using a detect-and-treat strategy.

Can I ask you, where are you going to go with this data that you have, this evidence of TGF-beta signaling and more evidence of myeloproliferation? Are you going to look at the downstream markers of TGF-beta signaling to see whether you are signaling through canonical or non-canonical signaling pathways? Because that would indicate potentially different medical therapies and maybe can prevent the problem all together.

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Response by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

DR. HIBINO: Based on these results, we found that TGF-beta was an important marker to contribute smooth muscle cell proliferation. We are looking for the difference of downstream between groups; and at the same time, we are working on proteomics analysis.

We particularly are interested in the integlin pathway, which shows the significant difference in preliminary proteomics analysis. If we find the specific target, we can test the effect of the drug for these proteins.

In addition, we have a system to take cells from the PDA tissue for primary culture.

So once we find the specific target, we can test the drug using this cell line to examine response.

DR. CALDARONE: That is terrific work, because you have a high incidence of a very significant lesion. You have the tissue and the capability of developing a medical therapy to prevent the problem. I really think a prevention strategy is going to be much better than a detect-and-treat strategy.

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Paper presented by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

Discussion by John E. Mayer, M.D., Boston, Massachusetts. john.mayer@cardio.chboston.org

DR. J. MAYER (Boston, Massachusetts): If I understand correctly, you are actually sampling tissue from the duct, but the obstruction is actually retrograde in the arch; is that correct?

060. POTENTIAL MOLECULAR MECHANISM OF RETROGRADE AORTIC ARCH STENOSIS IN THE HYBRID APPROACH TO HYPOPLASTIC LEFT HEART SYNDROME. Response by Narutoshi Hibino, MD, PhD, Baltimore, Maryland. nhibino1@jhmi.edu

DR. HIBINO: In case there was a significant obstruction in the retrograde aortic arch, usually there was a very thick intima in the stent. We took out stent with this thick intima for analysis.

DR. MAYER: But is the hypothesis then that it is displacement of ductal tissue retrograde into the arch?

I am trying to figure out if there is a phenomenon in the arch that is occurring, but the tissue you are sampling is in the duct, or within the stent that is in the duct.

Is what is going on in the arch assumed to reflect what happens with the stent and what happens in the arch? Or is it all of the retrograde stenosis due to the reaction to the stent?

DR. HIBINO: We usually place the stent beyond the retrograde arch. So after the placement of the stent, the blood flow into retrograde arch go through the mesh of the stent. If there is an intimal hyperplasia in the stent, the perfusion to the retrograde arch should be reduced.

DR. MAYER: So the mechanism for the retrograde stenosis actually is related to the reaction to the stent?

DR. HIBINO: Yes, exactly.

DR. MAYER: Not something actually happening in the arch itself?

DR. HIBINO: No, we hypothesize that PDA tissue reaction with stent could cause intimal hyperplasia and retrograde aortic arch stenosis.

DR. MAYER: Okay.

DR. HIBINO: But actually, as I showed in this presentation, most of the patients who had retrograde aortic arch stenosis had a significantly small ascending aorta or aortic atresia. So that would be another factor. But now we are focusing to just the reaction with the stent.

DR. EMANI: Thank you.

Abbreviations

ASD

atrial septal defect

AVC

atrioventricular canal

CDKs

cyclin dependent kinases

CHD

congenital heart disease

DILV

double inlet left ventricle

DORV

double outlet right ventricle

ECM

extracellular matrix

HLHS

hypoplastic left heart syndrome

PA

pulmonary artery

PDA

patent ductus arteriosus

PDGF

platelet derived growth factor

RAAS

retrograde aortic arch stenosis

SVA

single ventricle anomaly

TGA

transposition of the great arteries

TGF-β

transforming growth factor beta

VSMC

vascular smooth muscle cell