Immunogenicity of intensively decellularized equine carotid arteries is conferred by the extracellular matrix protein collagen type VI.

The limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. Here, we demonstrate the residual immunogenicity of an extensively decellularized equine carotid artery (dEAC(intens)) and identify the involved immunogenic components. EAC were submitted to an elaborated intensified decellularization protocol with SDS/sodium desoxycholate for 72 h using increased processing volumes (dEAC(intens)), and compared to dEAC(ord) prepared by an ordinary protocol (40 h, normal volumes). Matrix integrity was checked via correlative volumetric visualization which revealed only minor structural changes in the arterial wall. In dEAC(intens), a substantial depletion of cellular components was obvious for smooth muscle actin (100%), MHC I complexes (97.8%), alphaGal epitops (98.4% and 91.3%) and for DNA (final concentration of 0.34 ± 0.16 ng/mg tissue). However, dEAC(intens) still evoked antibody formation in mice after immunization with dEAC(intens) extracts, although to a lower extent than dEAC(ord). Mouse plasma antibodies recognized a 140 kDa band which was revealed to contain collagen VI alpha1 and alpha2 chains via mass spectrometry of both 2D electrophoretically separated and immunoprecipitated proteins. Thus, even the complete removal of cellular proteins did not yield non-immunogenic dEAC as the extracellular matrix still conferred immunogenicity by collagen VI. However, as lower antibody levels were achieved by the intensified decellularization protocol, this seems to be a promising basis for further development

Correlative volumetric visualization of the carotid wall for native EAC, dEAC_ord and dEAC_intens by Scanning Laser Optical Tomography (SLOT).

<p>SLOT (A–C) was performed in transmission mode displaying autofluorescence at 532 nm on tissue pieces of 1.5 cm in length from the indicated tissues and by Multi Photon Microscopy (D–F) with maximum intensity projections of axial cross sections of the indicated tissues representing the autofluorescence at 800 nm excitation wavelength. TA: tunica adventitia; TM: tunica media; TI: tunica intima.</p

Quantification of specific antibodies in mouse plasma after immunization with dEAC_ord (plasma_ord) and dEAC_intens (plasma_intens).

<p>On the western blots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105964#pone-0105964-g004" target="_blank">Fig. 4</a>, the 140 and the 240 kDa bands of the dEAC_ord extracts (n = 7) and of the dEAC_intens extracts (n = 9) probed with the respective plasma were quantified densitometrically. Shown are th means ± SEM. *: p<0.05 and **: p<0.01 between plasma_ord and plasma_intens by Student’s t-test (plasma_intens) or Mann-Whitney U test (plasma_ord).</p

Detection of specific antibodies in mouse plasma after immunization with dEAC_ord (plasma_ord) and dEAC_intens (plasma_intens).

<p>NMRI mice were injected with aqueous extracts of dEAC_ord or dEAC_intens over 17 days and plasma was probed as a primary antibody on 30 µg of extracts of dEAC_ord and dEAC_intens seperated by SDS-PAGE and transferred to PVDF membrane. Bound antibodies were visualized with anti-mouse peroxidase-labeled secondary antibodies and the enhanced chemiluminescence system. A typical blot of three independent similar experiments is shown.</p

Immunogenic Proteins identified by mass spectrometry.

<p>From gels containing 2D electrophoresis, separated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105964#pone-0105964-g005" target="_blank">Fig. 5</a>) or immunoprecipitated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105964#pone-0105964-g006" target="_blank">Fig. 6</a>) proteins spots or bands were excised, trypsin-digested and submitted to mass spectrometry.</p>a<p>Processed mass spectra were searched for in the “equus caballus” database downloaded from UniProtKB.</p>b<p>Mass spectra were processed with ProteinLynx global server software V2.1 (Waters).</p>c<p>Sample number as indicated on the respective figure.</p>d<p>Mean error of all peptides measured.</p><p>Immunogenic Proteins identified by mass spectrometry.</p

Immunoprecipitation of immunogenic proteins.

<p>1 mg of dEAC_ord and dEAC_intens extracts were incubated with plasma from mice immunized with dEAC_ord (plasma_ord) and dEAC_intens (plasma_intens) overnight, immune complexes were precipitated with protein A agarose, separated by SDS-PAGE and coomassie-stained. Bands form the gel were excised as indicated by the brackets and submitted as samples S2–S9 to mass spectrometry.</p