Severe degeneration of a sub-coronary pulmonary autograft in a young adult

Background. The pulmonary autograft is currently the best valve substitute in terms of longevity and performance. However, there is no agreement about the optimal method of insertion (sub-coronary position or freestanding root). Objectives. We sought to examine the clinical status, detailed imaging and morphometric changes in an explanted pulmonary autograft 22 years after sub-coronary implantation. Methods. A 30-year-old female underwent pulmonary autograft replacement of a severely stenotic valve at the age of 7 years, after presenting to us with signs of moderate to severe heart failure. She underwent clinical examination, detailed imaging including echocardiographic and CT examination with computerised image analysis. The explanted valve was examined by morphometry. Results. Clinical examination showed signs of heart failure (NYHA III). Trans-thoracic and trans-oesophageal 2D echo showed severe malfunction of both the aortic and pulmonary valves associated with dilatation and hypertrophy of both the right and left ventricles. Surgical correction was performed by replacing both the pulmonary and aortic valves with Medtronic 27mm Freestyle valves. The pulmonary autograft showed degeneration of the trilamellar layering of the leaflets, loss and disorganisation of GAGs, increased collagen with fibrotic overgrowth, and markers of fibrosis, inflammation, and calcification. Post-operative imaging showed good correction of the haemodynamic lesions. Conclusion. The pulmonary autograft implanted into the sub-coronary position presented with adverse remodelling, which was detrimental to the functionality and longevity of the valve. Authorship. NL, AM, MN all contributed equally to this paper.


INTRODUCTION
Pulmonary autograft replacement of the aortic valve (Ross operation) is currently the most durable form of aortic valve replacement [1][2][3][4][5][6] . This is closely linked to the capacity of the valve substitute to remain viable and adaptive to local and systemic conditions, both in the short and long term 1,7,8 . The original operation was configured in the subcoronary position by Donald Ross 9 and was modified to a freestanding root replacement (previously described for homografts) 10 .
However, varying degrees of degenerative changes, resulting in malfunction of the pulmonary autograft, have been reported. The exact incidence and, importantly, the factors contributing to the degenerative changes, have not been adequately defined. We describe the evolution of long-term severe degeneration of a pulmonary autograft, inserted in the sub-coronary position and discuss the possible role of the method of insertion on this serious complication. In addition, we compare these changes to those seen following the root replacement technique 7 .

Sample preparation
Paraffin-embedded sections of valve leaflets were examined with histochemical and immunohistochemical staining as previously described 7 .

Clinical course and imaging
A 30-year-old female had a sub-coronary root replacement at the age of 7. This patient presented with progressive dyspnea on exertion from the age of 16 (NYHA III). On examination she was found to have mild central cyanosis, congested neck veins, pan-systolic murmur over the pericardium, and marked hepatosplenomegaly.
The ICU course necessitated prolonged mechanical ventilation due to a lung collapse, which gradually improved. Additionally, the patient suffered from atrial fibrillation and atrial flutter attacks, which were successfully cardioverted back to a normal sinus rhythm. The sub-coronary root replacement was explanted 23 years later ( Figure 3) after a redo sternotomy, and both pulmonary and aortic valves were replaced by 27 mm Freestyle valves.
At follow up, the patient was asymptomatic with normal sinus rhythm, normal LV function and dimensions and no significant gradients across either valve ( Figure 4).

EVG
The amount and distribution of elastin was increased ( Figure 5). This was mostly in the radial direction with many separate parallel layers of elastin at both ventricular ( Figure 5E) and fibrosal sides ( Figure 5G) of the leaflets however in thickened regions, fibres of elastin were discernible in anteroposterior direction ( Figure 5F) and disorganised ( Figure 5G). The elastin in the normal pulmonary leaflet was restricted predominantly to the ventricularis ( Figure 5I). Collagen was increased in all layers of the Ross leaflets ( Figure 5C) compared to the fibrosal pattern in the normal pulmonary leaflet ( Figure 5J).

ABSR
The central glycosaminoglycan (GAG) layer was disrupted in all leaflets with a random distribution of GAGs and abundant collagen ( Figures 5C, 6C and 7C). GAGs were increased in 2 leaflets ( Figures 6C and 7C) and in one of these leaflets, these were expressed abnormally at the edges of the leaflet (Figures 6C, 6E) despite having a normal thickness. Collagen was distributed throughout leaflet A ( Figure 5C), present in the central and thickened region of leaflet B (Figures 6C, 6E) and disorganised throughout leaflet C ( Figure 7C).

Immunostaining
Cellularity was reduced markedly in the central regions of all the thickened leaflets and absent in some regions (Figures 7B, 7F). However, the fibrotic overgrowth regions on both sides of the leaflets contained numerous cells ( Figure 7E). The only region of normal thickness on leaflet B showed a normal distribution of valve interstitial cells, however these cells showed an abnormal phenotype with a similar pattern of expression of calcification markers, Runx2 and osteocalcin (Figure 8).
On higher magnification, the native valve interstitial cells, with larger, elongated nuclei, could be discerned from the smaller, infiltrating polymorphonuclear cells ( Figure 7E) and these latter cells were abundant in all leaflets. Many myofibroblasts (staining for α-smooth muscle actin) and leucocytes (CD45) were present in the cellular regions ( Figure 8) with a small population of smooth muscle cells (Figure 8, SMM). No T cells were identified, however macrophages (CD68), and monocytes (CD14) were identified, with macrophages being predominantly of the M1 phenotype (CCR7). Activation of valve  interstitial cells was evident by expression of α-SMA and Runx2 (Figure 8) as well as in endothelial cells (Figures 6G, 6H), even in macroscopically-normal tissue.

Calcification
Despite being markedly thicker and stiffer than normal aortic valves (Figure 3), there was minimal alizarin red staining in the leaflets (Figures 5D, 5H and 7D). The fibrosal sides of leaflets B and C showed some darker staining for alizarin red ( Figure 7D), while leaflet A showed a spotted pattern on both sides of the leaflet ( Figure 5H). Markers for calcification, Runx2 and osteocalcin were, however abundant in all leaflets in valve interstitial cells (Figure 8) and also valve endothelial cells ( Figure 6G).

Discussion and Conclusion
This manuscript documents the long-term adverse effects of the use of the sub-coronary position for the Ross operation, which can potentially introduce distortion both in the aortic root and components of the valve culminating in late failure. In contrast, the use of the freestanding aortic root, which was introduced for homografts very early on 10 , guarantees an optimal relationship between the component parts of the valve. Interestingly, long term follow-up of homografts showed that the use of a freestanding root was associated with better survival 11 . There is a continuing concern about the use of the freestanding root for pulmonary autografts because of the reports of dilatation of the root, resulting in malfunction of the valve [12][13][14][15][16] . This has stimulated the use of the sub-coronary implantation as the autograft is supported by the native valve preventing dilatation 17 . However as is illustrated from the current patient, accurate positioning of the pulmonary autograft in the sub-coronary position requires both skill and judgement, particularly when there is a mismatch between the size and shape of the root as compared to the autograft (Figure 9). Regarding the dilatation following the free root replacement, this is quite variable and occurs in only a small fraction of patients ( Figure 9). To safeguard against this complication the technique of loose jacket is being used routinely in our centre ( Figure 10).
To date, there has been no randomised trials comparing the two techniques, however a systematic review performed by Berdajs and Von Segesser showed better results with the root replacement, both in terms of longevity and incidence of reoperation 18 . It is hoped that the data presented in this manuscript can be of some help in decision making in the choice of technique in the Ross operation.

Lessons Learnt
1. The Ross operation continues to be the best aortic valve substitute, particularly in children. 2. Sub-coronary implantation can result in both distortion of the root and degeneration of the component parts. 3. The use of root replacement guarantees appropriate relationship between the component parts of the aortic root 10 and valve, and has been shown to have a survival advantage following homograft valve replacement 11 . 4. Late dilatation of the aortic root when inserted as freestanding can be prevented (at least in theory) with the use of an autologous loose jacket (19).