Mechanism of aortic medial matrix remodeling is distinct in patients with bicuspid aortic valve.

Aortic specimens were collected during ascending aortic replacement, aortic valve replacement and heart transplants from non-aneurysmal (NA) donors and recipients. Matrix architecture of the aortic media was assessed qualitatively using multi-photon microscopy followed by quantification of collagen and elastin fiber orientation. α-elastin was determined and matrix maturity was assessed by quantifying immature and mature collagen and lysyl oxidase (Lox) expression and activity in aortic specimens. Matrix metalloproteinase (MMP)-2/9 activity was quantified in aortic smooth muscle cells.

Methods Patient selection and specimen acquisition

Ascending thoracic aortic specimens were collected during elective surgery for ascending aortic replacement and/or aortic valve replacement from patients with BAV or tricuspid aortic valve (TAV) with IRB approval and informed patient consent. Specimens were also collected from heart transplant donors and recipients with TAV and absence of aortic disease with approval from the Center for Organ Recovery and Education or IRB approval and informed patient consent, respectively. Patient demographics of sex, age, maximal orthogonal aortic diameter, degree of aortic insufficiency and stenosis and history of hypertension and cigarette smoking were documented ( Table 1 ). TAA patients enrolled in our tissue bank with BAV (median age= 53, n=142) presenting for elective surgery at the University of Pittsburgh Medical Center for ascending aortic replacement do so at least 10 years earlier than patients with TAV (median age=63, n=99). Specimens chosen for each experiment were diameter-matched (median diameters within 2 mm) for TAV-TAA and BAV-TAA patients and ages span the distribution given above. Specimens of both sexes were included in the study. Upon excision, the specimens were placed in ice-cold saline and transported to the laboratory. A portion of tissue was processed for isolation of primary aortic SMC cultures as previously described. 4 , 7 . Multi-photon microscopy of aortic matrix architecture A 1 cm 2 portion of the specimen was dissected, noting proximal/distal orientation. The adventitia was left intact to avoid potential deformation in the native matrix architecture. The specimen was kept in PBS on ice and imaged immediately (within 2 hr of harvest) using multi-photon microscopy (MPM) or fixed in 2% paraformaldehyde for 1 hr, followed by storage in PBS at 4°C until MPM was performed. Specimens were imaged using an Olympus FV1000 MPE (Tokyo, Japan) equipped with a Spectra-Physics DeepSee Mai Tai Ti-Sapphire laser (Newport, Mountain View, CA) using an excitation wavelength of 830 nm and 1.12 NA 25X MPE water immersion objective. Epi-detectors were utilized to collect the second harmonic generation at 400±50 nm for collagen fibers and autofluorescence of elastin at 525±25 nm. Images were captured using a dwell time of 4 μs/pixel and a Kalman filter of 2. Images of the media were captured as 2 μm slices starting just beyond the intimal surface sequentially moving towards the adventitia. An attempt to capture images spanning the full thickness of the medial layer was made with each specimen; however, the number of images collected varied with each sample due to thickness and geometry of the specimen and ranged from 14–108 μm.

Determination of collagen and elastin fiber alignment Following

MPM analysis, projected image stacks were assembled for each channel (collagen and elastin separately) using ImageJ (National Institutes of Health). To quantify collagen and elastin fiber orientation on superimposed 2D image stacks, the gradient of image intensity at each pixel taken above a threshold, was determined for an edge detection algorithm using a custom MATLAB program previously described. 8 Briefly, these values were entered into an accumulator bin defined over a subregion and for each angle the summed gradient-weighted contribution of each pixel was calculated. Histograms of dominant orientation, defined as the angle associated with the maximum accumulator bin value were plotted, and orientation indices (OI) were determined based on variance of fiber angle (Δθ) of 50% of the total number of fibers as one-half the area under the curve of fiber angle distribution histogram ( Figure 2D ). For images with a high degree of fiber alignment, it was necessary to rotate the image clockwise 90° to generate half-area calculations from fiber-count angle histograms. The normalized orientation index (NOI) was then calculated from Equation 1 ( Eq. 1 ) as described previously 9 where OI=Δθ. (Eq. 1) NOI = 90 - OI / 90 × 100 so that NOI € [ 0 , 100 ] RNA isolation and quantitative real-time PCR A portion of each specimen was placed in RNA later (Life Technologies, Carlsbad, CA) and stored at −20°C until use. Total RNA was isolated using the RNeasy Plus kit (Qiagen, Valencia, CA). Gene expression of Lysyl oxidase ( Lox ) was quantified by real-time PCR as previously described 7 using the One-Step Taqman ® PCR kit and custom-designed Taqman ® gene expression assay for human Lox (Assay ID# Hs00184700_m1) (Life Technologies). Histology Specimens (~0.5 × 1cm 2 ) were fixed in 10% buffered formalin and paraffin embedded. Four micron sections were stained using Masson’s Trichrome and Verheoff’s van Giemsa (Research Histology Services, University of Pittsburgh Thomas E. Starzl Transplantation Institute) to ascertain collagen and elastin composition respectively. Slides were stained using picrosirius red stain kit (Polysciences Inc., Warrington, PA) according to the manufacturer’s instructions to assess collagen fiber thickness. Slides were visualized using a Nikon TE-2000-E inverted microscope under polarized light and captured using a Nikon DS-Fi1 5MP color camera and NIS Elements Software 3.2 (Nikon Corporation, Melville, NY). Mean area fractions of red/orange and green fibers were quantified.

Determination of soluble and insoluble collagen

Snap-frozen aortic specimens were pulverized under liquid nitrogen and weighed. Pepsin-soluble collagen was extracted using 5mg/mL pepsin in 0.5M glacial acetic acid (20 μL/g tissue) overnight at 4°C. Samples were centrifuged at 2,100 × g for 6 min at RT. Undenatured soluble “immature” collagen was detected from the supernatant fraction using the Sircol ™ assay according to the manufacturer’s instructions and a standard curve of Type I collagen (Accurate Chemical and Scientific Corporation, Westbury, NY) and normalized to tissue mass. Insoluble collagen content was estimated from the pellet fraction following pepsin digestion using a colorimetric hydroxyproline (HYP) assay. 10 Pelleted samples with a minimal volume (