Characterization of Laminins in Healthy Human Aortic Valves and a Modified Decellularized Rat Scaffold

Aortic valve stenosis is one of the most common cardiovascular diseases in western countries and can only be treated by replacement with a prosthetic valve. Tissue engineering is an emerging and promising treatment option, but in-depth knowledge about the microstructure of native heart valves is lacking, making the development of tissue-engineered heart valves challenging. Specifically, the basement membrane (BM) of heart valves remains incompletely characterized, and decellularization protocols that preserve BM components are necessary to advance the field. This study aims to characterize laminin isoforms expressed in healthy human aortic valves and establish a small animal decellularized aortic valve scaffold for future studies of the BM in tissue engineering. Laminin isoforms were assessed by immunohistochemistry with antibodies specific for individual α, β, and γ chains. The results indicated that LN-411, LN-421, LN-511, and LN-521 are expressed in human aortic valves (n = 3), forming a continuous monolayer in the endothelial BM, whereas sparsely found in the interstitium. Similar results were seen in rat aortic valves (n = 3). Retention of laminin and other BM components, concomitantly with effective removal of cells and residual DNA, was achieved through 3 h exposure to 1% sodium dodecyl sulfate and 30 min exposure to 1% Triton X-100, followed by nuclease processing in rat aortic valves (n = 3). Our results provide crucial data on the microenvironment of valvular cells relevant for research in both tissue engineering and heart valve biology. We also describe a decellularized rat aortic valve scaffold useful for mechanistic studies on the role of the BM in heart valve regeneration.


Introduction
Aortic valve stenosis is one of the most prevalent cardiovascular disorders in the western world. 1 To date, no medical therapies are available, making treatment options limited to surgical or endovascular replacement with a biological or mechanical prosthetic valve. However, biological valves have limited durability due to gradual structural deterioration eventually leading to graft failure, and mechanical valves necessitate lifelong treatment with potent anticoagulants. 2 The ideal heart valve replacement is considered to be an offthe-shelf valve that does not exert an immune response and has the capacity to grow and be repopulated by living cells. Therefore, tissue-engineered heart valves, both decellularized and in vitro-produced scaffolds, have been developed and extensively studied in animal models, [3][4][5][6][7][8][9] and decellularized aortic valve allografts have even reached clinical trials. 10,11 Although initial studies have been favorable, tissue-engineered heart valves suffer from structural deterioration similar to conventional allografts and may even be associated with a higher reoperation rate. 12 What causes degeneration of tissue-engineered valves is unknown. 12,13 Despite the widely recognized importance of the extracellular matrix (ECM), and the significant interest in heart valve tissue engineering, the important basement membrane (BM) of heart valves remains poorly characterized. The BM plays key roles in cell adhesion, migration, function, and differentiation. 14 Laminins, consisting of an a, b, and c chain, are the first BM components to be expressed in mammalian tissues. 15 Their importance in cell function and tissue formation is illustrated by the fact that several laminin chain isoforms are vital for normal embryonic development. 14 However, the laminin isoforms and their expression pattern in heart valves have not been characterized, and the role of laminins in tissue engineering remains largely unknown.
Decellularized scaffolds provide an opportunity to investigate the effects of individual ECM components in a regenerative context relevant for tissue engineering. They have previously been used to study how heparan sulfate proteoglycans affect the differentiation of endodermderived stem cells on a decellularized lung scaffold. 16 However, it is well known that different tissues require different methods for decellularization [17][18][19][20] as the process inevitably has detrimental effects on the ECM. 21 Therefore, optimization of a decellularization protocol for the specific tissue in question to achieve effective cell removal while limiting detergent exposure is critical.
The aim of the present study was two-fold. First, to characterize the laminin isoforms present in human and rat aortic valves, and their distribution patterns. Second, to develop a decellularized rat aortic valve scaffold that preserves laminin and other BM components for future studies on how the BM influences cell behavior in heart valve regeneration.

Human tissues
Whole human hearts were obtained from Legacy Donor Services Foundation, Florida. Legacy Donor Services Foundation receives donor referrals from hospitals and medical examiners serving the state of Florida. Written informed consent was obtained from the donor through registration as a tissue and organ donor with Donate Life America, Donate Life Florida, or registering to be a donor when renewing or obtaining a driver's license. Alternatively, written informed consent was obtained from the donor's next-of-kin by a Donor Management Coordinator from Legacy Donor Services Foundation. Hearts intended for biomedical research were offered to Nova Southeastern University by the Legacy Donor Services Foundation. Inclusion criteria were donor age 18-60 years and no history of valvular heart disease. Institutional Review Board approval was obtained from the Regional Ethics Review Committee in Stockholm, Sweden (Protocol Number: 2017/2268-31). Hearts (n = 3) were processed within 48 h from the time of death. The aortic valve was inspected for gross morphologic abnormalities, the cusps were excised at their origin and snap frozen in optimal cutting temperature (OCT) compound.

Animals
Rat aortic roots were procured from female Sprague-Dawley rats 8-12 weeks of age (Charles River). All animals were cared for in accordance with the rules and regulations of Karolinska University Hospital and Karolinska Institutet. Institutional Review Board approvals for animal experiments were obtained from the Regional Committee on the Ethics of Animal Experiments, Linköping, Sweden (Protocol Numbers: ID 6-17 and S190-12).
Rat aortic valve procurement Rats were anesthetized through isoflurane inhalation and then immediately euthanized by inhalation of CO 2 or decapitation. The thoracic cavity was accessed by dividing the ribs and sternoclavicular joints and excising the sternum. All intrathoracic organs were removed en bloc. The aortic valve was dissected from the heart under a microscope as a U-shaped aortic conduit as described by others. 22,23 Briefly, a myocardial cuff of *1-2 mm and part of the anterior mitral valve leaflet were preserved, whereas the brachiocephalic artery, left common carotid artery, and left subclavian artery were ligated with 6-0 sutures (Ethicon) at their origin. The descending aorta was divided at the level of the aortic valve. For normal controls, the ascending aorta was divided at the origin of the brachiocephalic artery and the aortic root was then embedded in OCT and snap frozen.

Decellularization
A perfusion decellularization method based on earlier protocols 5,17 was used. In brief, the aortic conduit was cannulated with a 1.1 mm intravenous catheter (B. Braun Medical), which was connected to a tubing pump (Ismatec) through a custom-built stainless steel bottle cap to enable recirculation of reagents in a 250-mL bottle ( Supplementary Fig. S1A). Aortic conduits were perfused with 1% sodium dodecyl sulfate (SDS; Merck Sigma-Aldrich) in deionized water for 3 h at a flow rate of 2 mL/min, followed by 15 min with deionized water, 30 min with 1% Triton X-100 (Merck Sigma-Aldrich) in deionized water and 12-24 h with phosphate-buffered saline (PBS), changed twice. The aorta was divided at the origin of the brachiocephalic artery to facilitate enzymatic treatment. Removal of residual nucleic acids was achieved by processing aortic roots with 90 U benzonase endonuclease (E1014; Merck Sigma-Aldrich) on a see-saw rocker (Cole-Parmer) overnight at room temperature (RT). The remaining aortic roots were processed on the see-saw rocker overnight in PBS and then snap frozen in OCT or used for DNA quantification.
DNA quantification DNA content per decellularized aortic valve (n = 3) was compared with normal controls (n = 6). To provide an estimation of residual cellular contents other than nucleic acids, aortic valves treated with SDS for 3 h, but not processed with benzonase endonuclease, were included (n = 3). Aortic roots treated with SDS for 12 h (n = 3) were also included to quantify difference in residual cellular contents and nucleic acids between the original protocol 17 and our modified protocol. The ascending aorta was opened longitudinally along one of the commissures and the aortic valve leaflets were excised in their entirety under a microscope. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Purified DNA was eluted twice in 25 lL buffer and quantified with NanoDrop (Implen).

Statistics
One-way analysis of variance with Bonferroni corrections for multiple comparisons was used to identify differences between groups. p < 0.05 was considered statistically significant.

Results
Healthy human aortic valves express laminin a4, a5, b1, b2, and c1 chains Aortic valves were from male donors 50-60 years of age. Upon gross morphologic assessment, one of the hearts was hypertrophic and had significant atherosclerosis in the main coronary arteries (Table 1). All three aortic valves were tricuspid and macroscopically normal. A single aortic valve cusp was used from each donor, that is, one right coronary cusp, one left coronary cusp, and one noncoronary cusp. H&E staining and double staining for elastin and collagen I confirmed the absence of plaques and an overall normal histology with an intact three-layered structure (Fig. 1). The noncoronary cusp had a markedly vascu-larized interstitium, the left coronary-and right noncoronary cusp had limited or no vascularization.
The endothelial BM of the human aortic valve cusps stained positive for laminin a4, a5, b1, b2, and c1 chains (Fig. 2). All chains were uniformly present in all three cusps, forming a continuous monolayer. The same chains were also expressed in the BM of the interstitial vessels observed in two of the cusps. The interstitium itself was mostly negative for the laminin chains, except in minor regions. Laminin a1, a2, a3, b3, and c2 could not be detected in any of the valve cusps. This suggests that the human aortic valve endothelial BM consists of LN-411, LN-421, LN-511, and LN-521.
To assess the relevance and utility of the rat model, three rat aortic roots were stained for laminin a4, a5, and c1. Rat aortic valve cusps also stained positive for

FIG. 2.
Laminin chain expression in human aortic valves. The endothelial basement membrane covering the valve cusps were positive for laminin a4, a5, b1, b2, and c1, as well as the endothelial lining of interstitial vessels and minor patches in the interstitium. Images of a1, a2, a3, b3, and c2 show absence of immunoreactivity. A representative image of ‡3 tissue sections from each of the three donor valve cusps is shown. All images shown at 20 · magnification. Scale bar represents 100 lm.

FIG. 3.
Expression of laminin a4, a5, and c1 in rat aortic valves. The endothelial basement membrane stained positive for all three laminin chains. Minimal immunoreactivity was seen in the interstitium. A representative image of ‡4 tissue sections from three animals per group is shown. All images captured with 20 · magnification. Scale bar represents 100 lm. a4, a5, and c1, indicating the presence of the same laminin isoforms as in human valves (Fig. 3). The distribution pattern was uniform for all three laminin chains, which formed a convergent monolayer, continuous with the endocardium and aortic endothelium.

Decellularization with SDS and Triton X-100 preserves BM components
To establish a small animal model for studies of celllaminin interactions, we sought to assess the effectiveness and effects on the BM of a decellularization protocol for rat aortic valves adapted from earlier studies. 5,17 Aortic roots were exposed to 1% SDS for 1, 3, 6, or 12 h (n = 1 for each time point). After 3 h, the myocardial rim remained incompletely decellularized macroscopically, whereas the entire specimen was completely translucent after 12 h (Supplementary Fig. S1B-D). No clear difference could be observed in terms of DAPI-positive intact nuclei or residual DNA in the valve cusps at the different time points, (Supplementary Fig. S3) and intact nuclei could still be seen in the aortic annulus even after 12 h. To ensure adequate SDS treatment while avoiding prolonged exposure, 3 h was chosen as an intermediate incubation time for further assessment. As residual DNA has been associated with an adverse immune response, 25  with benzonase endonuclease was performed, 16,18 resulting in an almost complete removal of nucleic acids in the valve (Fig. 4). Occasionally, isolated intact cell nuclei could be observed in the valve cusps (Supplementary Fig. S3).
To confirm the integrity of the BM, tissue sections of decellularized rat aortic valves (n = 3) and normal controls (n = 3) were stained for laminin c1, common to all laminin isoforms found in the aortic valve, as well as perlecan, heparan sulfate, and collagen IV (Fig. 4). All of the analyzed BM components, as well as fibronectin, could be visualized after decellularization, with a largely intact distribution pattern.
Limited detergent exposure combined with benzonase endonuclease results in minimal residual DNA Due to their extremely small mass, we were unable to obtain reliable weight measurements from the valve cusps. However, by using fine dissection tools, the three valve cusps could be excised from the aortic root in a highly reproducible fashion, enabling assessment of DNA content per valve, that is, all three cusps excised from the same specimen. Normal control aortic valves (n = 6) had a mean DNA content of 1.04 lg (Fig. 5; Supplementary Table S2). This was reduced to 413.8 ng in aortic valves after 3 h of SDS perfusion (39.8% of normal controls, p < 0.001). The DNA content was similarly reduced to 414.4 ng after 12 h of SDS perfusion (39.8% of normal controls, p < 0.001). Additional processing with benzonase endonuclease resulted in a mean aortic valve DNA content of 63.4 ng (6.1% of normal controls), a significant decrease compared with 3 h SDS exposure without enzymatic treatment ( p = 0.0137).

Discussion
The present study describes, for the first time, the specific laminin chains present in healthy human aortic valves. We show that the main laminin isoforms expressed in the endothelial BM of heart valves are LN-411, LN-421, LN-511, and LN-521, and that the isoforms are conserved across mammalian species. The same laminin isoforms were also expressed in small interstitial vessels that may exist in valve cusps. 26,27 Interestingly, small traces of laminin that appeared to be located in the interstitium rather than in the endothelial BM were observed. These findings are consistent with previous reports on laminin isoforms expressed in vascular endothelial BMs. [28][29][30][31][32][33] However, cardiac valvular cells and their ECM are unique in structure and function, [34][35][36] and our study provides critical data on the BM structure and microenvironment of valvular cells necessary for recreating the native BM in tissue-engineered heart valves.
Laminins are key components of vascular BMs and have important, isoform-specific functions. 37 Laminin a4 is ubiquitous in the BM of blood vessels and most endothelial BMs also express a5. [29][30][31] However, their distribution pattern and relative expression levels vary considerably between different vessel types, which affects the properties of the BM. For example, the expression patterns of a4 and a5 are known to determine the tightness of endothelial junctions and thus the permeability of the endothelium in the context of lymphocyte extravasation, with a4 isoforms being permissive and a5 isoforms being restrictive. [38][39][40][41] Furthermore, evidence suggests that a4 has a role in angiogenesis as well as the regulation of endothelial cell survival, 42,43 indicating a role in endothelialization of tissue-engineered valves and vascular grafts. Indeed, laminin coating, although of an undefined isoform, has been shown to promote endothelialization while decreasing intimal hyperplasia in aortic grafts in vivo. 6 On the other hand, the a5-containing LN-511 has been shown to be important in the shear stress response of resistance arteries 44 and may be of similar importance in heart valves during the cardiac cycle. The b-chain is also significant in endothelial BMs as an isoform switch from LN-421 to LN-411 may lead to decreased adhesion and increased endothelial-tomesenchymal transition. 33 Furthermore, laminins may have additional regulatory effects on valvular interstitial cells. 45,46 In conclusion, laminins play key roles in the cardiovascular system, and the presence or lack of specific laminin isoforms in tissue-engineered heart valves may thus have a profound impact on their viability. However, the role of laminins in tissue engineering needs to be defined to assess their true relevance in this context, and further studies on small animal scaffolds are an appropriate next step. While decellularized rat aortic valves have been described by others, 5,23,[47][48][49] we present a protocol with reduced detergent exposure, which results in both effective cell removal and preservation of laminin and other key BM components. Scaffolds produced through limited detergent exposure, and thus with a better preserved BM, 20,21,50 will be a useful tool in future mechanistic studies.
The number of donated heart valves in our study was limited due to the sparse availability of healthy human heart valves. We did not obtain clinical data, such as cause of death, comorbidities, and clinical risk factors, which could have added insights into whether valves were at risk of calcific degeneration. However, all valves had a normal gross morphology and histology. Furthermore, the laminin expression in all human aortic valves was fully consistent between specimens, and data are supported by current literature on laminins in the cardiovascular system. [28][29][30][31][32][33]37 To the best of our knowledge, this is the first report to describe the laminin chains expressed in aortic valves and their localization. Given the importance of laminins for a number of cellular functions, this provides crucial data for future attempts to recreate the aortic valve through tissue engineering. The presence or absence of laminins, and other ECM components such as perlecan, 51-54 heparan sulfate, 16 and fibronectin 4,55 is bound to affect the viability of tissueengineered heart valve grafts, but other factors that influence graft viability also need to be considered. Aside from a lack of appropriate ECM components, structural deterioration of tissue-engineered heart valve grafts may be due to (i) immunogenicity, 56 (ii) thrombogenicity, 57 or (iii) incomplete or inappropriate valvular endothelial or interstitial cell repopulation. 3,4,8 The relative importance of laminins and other ECM components, as well as other factors that may affect the long-term viability of a tissue-engineered heart valve, will need to be determined in future studies.

Conclusion
We have shown that the aortic valve BM contains LN-411, LN-421, LN-511, and LN-521. We also describe a modified decellularized rat scaffold with a largely preserved BM for future mechanistic studies. Further studies are required to quantify the laminin isoforms and determine their role in heart valve tissue engineering. Additional studies are also necessary to assess the effect of detergents used during decellularization on the functional properties of ECM components in heart valve regeneration.

Funding Information
This study was supported by grants from the Swedish Research Council, Young Investigator (2013-03590), the Magnus Bergvall Foundation (2017-02416), the Wallenius Foundation and a private donation from Mr. Fredrik Lundberg. None of the funding sources had any role in study design, data collection or analysis, decision to publish, or preparation of the article.  Table S1  Supplementary Table S2