Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Abstract

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTR^S52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α₂-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.

Fig. 1. Human TTR protein in the circulation of TTR^S52P transgenic mice.

a Human TTR concentrations in the sera of transgenic mice were measured by electroimmunoassay calibrated with isolated pure human wild-type TTR; non-transgenic mice gave no signal in this assay. Results (individual values, mean and SD) are presented for male and female transgenic mice of three independent lines that were also homozygous for a mouse ttr null allele (n = 26, 29 and 14 for male mice of lines O5, N1 and N4, respectively, and n = 24, 23 and 19 for female mice of lines O5, N1 and N4, respectively). The reported ranges of TTR concentrations in healthy adult women and men are indicated in pink and blue, respectively. b N-glycosylation of human TTR in serum of human TTR^S52P transgenic mice demonstrated by western blot probed with anti-human TTR antiserum. Transgenic mouse serum was treated with PNGaseF (lanes 7 & 8) under denaturing conditions for 1 or 24 h, as indicated; lanes 10 and 11 contained serum samples treated identically except for the omission of the PNGaseF enzyme, and lanes 5 and 12 contained untreated serum. The asterisk indicates the N-glycosylated human TTR^S52P protein. Other lanes contained recombinant human TTR (R), human serum (Hu), wild-type mouse serum (WT), mouse ttr knockout serum (KO) or molecular weight protein markers (m). The signal in lane 9 is a result of spillover from an adjacent lane. This experiment was not repeated. Source data are provided as a Source Data file.

Fig. 2. Amyloid in seeded mice of lines O5 and N4.

Amyloid demonstrated by the pathognomonic red-green birefringence of Congo red stained sections viewed by polarising microscopy under intense illumination; background white birefringence was generated by myofibrils. The full extent of the amyloid is readily appreciated in the Congo red fluorescence images. a Minor but unequivocal amyloid deposits in the heart and tongue of a male line O5 mouse 7 months after seeding, representative of the maximum amyloid loads in mice of this line (n = 53 amyloidotic mice). The electron micrograph shows the unbranching ~10 nm diameter morphology characteristic of genuine amyloid fibrils. b Copious amyloid deposits in heart and tongue of a line N4 transgenic male mouse 6 months after seeding. c Low magnification Congo red fluorescence images showing the typical extent of amyloid deposits in heart and tongue of line N4 transgenic mice seeded 6 months prior to sample collection. b, c are representative of typical amyloid loads in mice of this line analysed 6-7 months after seeding (n = 10). Scale bars: a, 100 µm (light micrographs) and 500 nm (EM); b, 100 µm; c, 1 mm.

Fig. 3. Immunohistochemical demonstration of ATTR amyloid in the transgenic model.

To determine the amyloid type, heart (a) and tongue (b) were stained with anti-human TTR, anti-mouse apoAII or anti-mouse SAA antisera, as indicated, followed by alcoholic alkaline Congo red. Bright field images show the immunohistochemical signal (brown), and faint red Congo red stained amyloid in a representative amyloidotic line N4 mouse (n = 15). The amyloid deposits exhibited the characteristic red-green birefringence when viewed by polarising microscopy. The coincidence of TTR signal and amyloid, together with the absence of apoAII and SAA signal, demonstrates that the amyloid is of ATTR type. In places, the intense anti-TTR staining obscured the Congo red staining, the extent of which is clearly visible in the closely adjacent sections stained for apoAII and SAA. Scale bar, 100 µm (a) and 200 µm (b). hTTR human TTR, mApoAII mouse apolipoprotein A2, mSAA mouse serum amyloid A protein.

Fig. 4. Presence of cleaved human TTR in transgenic human ATTR amyloid.

Homogenates of heart, tongue and liver from amyloidotic line O5 and line N4 mice (a and b, respectively) were fractionated into soluble and insoluble fractions, and analysed by western blotting. Lanes labelled H contain unfractionated homogenates, lanes labelled S1 contained the soluble fraction (first supernatant), and lanes labelled P contained the insoluble fraction (pellet). The line O5 mouse (a) was homozygous wild-type for mouse ttr and the line N4 mouse (b) was homozygous for the mouse ttr knockout allele. The same proportion of the entire sample was loaded in each lane; two exposures are shown in a to reveal all relevant bands. Data are representative of n = 5 (a) and n = 2 (b) biological replicates. Lanes labelled R contained recombinant human TTR^S52P that had undergone limited tryptic cleavage; the positions of full-length TTR protomer, the 49–127 cleavage product and mouse TTR are indicated. Glycosylated human TTR protomer is indicated by asterisks. Source data are provided as a Source Data file.

Fig. 5. Amyloid-associated protease activity.

Protease activity in heart and tongue was visualised by in situ zymography using DQ-gelatin substrate overlaid on unfixed cryosections. a protease activity was readily detected in hearts and tongues of amyloidotic mice, but not of control mice. b the amyloid-associated protease activity was insensitive to inhibition by EDTA, but was completely inhibited by a cocktail of serine protease inhibitors (aprotinin, PMSF and Pefabloc SC). The protease activity was associated with amyloid deposits, but the activity was not as extensively distributed as the amyloid (Fig. 2c). Scale bars: 250 µm (a); 1 mm (b). Data are representative of a minimum of two technical replicates of two biological replicates.

Fig. 6. Association of plasmin(ogen) with amyloid deposits.

a, b Western blots probed with an affinity-purified anti-plasminogen (PLG) antiserum, comparing amyloidotic and control heart (a) and tongue (b) from three different mice. The amounts of plasmin heavy and light chains in amyloidotic hearts and tongues was greater than in the control tissues. GAPDH was used as a sample processing control for the tissue extracts. c, d Adjacent tissue sections of heart (c) or tongue (d) of amyloidotic and control non-amyloidotic mice were probed with an affinity-purified anti-plasminogen antiserum, or stained with Congo red. c In amyloidotic heart, the pattern of immunoreactivity closely parallels the distribution of amyloid deposits. In control heart, the signal is much lower and almost exclusively within the vasculature. d In control tongue, moderate immunoreactivity is evident in connective tissue, consistent with the presence of plasminogen in the extracellular space. In amyloid-containing tongue, additional strong immunoreactivity was evident in the same distribution as the amyloid seen here, for example, surrounding muscle fibres (circled), in the adventitia of an arteriole (arrowhead), and within a small nerve (asterisk). Images in c and d are representative of findings in three different mice. Scale bar: 100 µm. Source data are provided as a Source Data file.

Fig. 7. Effect of α₂-antiplasmin deficiency on amyloid deposition.

Amyloid deposition was enhanced by α₂-antiplasmin deficiency (P = 0.0068, Mann–Whitney test, two-tailed). Estimates of lingual amyloid content (individual values, mean and SD) are shown for male line N4 human TTR^S52P mice that were wild-type (control) or homozygous for an α₂-antiplasmin knockout allele (n = 10 each group). The tissues were collected 2 months after amyloid deposition was seeded by injection of human TTR^S52P amyloidotic spleen homogenate. Similar results were obtained when comparing α₂-antiplasmin deficient (homozygous knockout) with control heterozygous α₂-antiplasmin knockout male mice (P = 0.0047, Mann–Whitney test, two-tailed, n = 7 controls, 6 knockouts). Source data are provided as a Source Data file.

Fig. 8. Relationship between plasminogen activators and amyloid.

a Adjacent sections of human TTR^S52P amyloid-containing heart and tongue stained with Congo red to show amyloid or probed with anti-tPA or anti-uPA affinity-purified antisera (representative of results seen with six mice). The patterns of anti-tPA immunoreactivity were indistinguishable from those of normal control tissues (Supplementary Fig. 9). The amyloid deposits in heart and tongue were strongly immunoreactive when probed for uPA. Additional strong immunoreactivity in tongue was observed in mast cells, indistinguishable from that seen in normal control tissue (Supplementary Fig. 9). b, c Western blot showing greater amounts of uPA in amyloidotic hearts (b) and tongues (c) than in controls (n = 3 amyloidotic and 3 control mice). Amyloidotic tissues contained more pro-uPA, the precursor of active uPA, as well as the heavy chain of active uPA; the antibody used for this analysis does not react with uPA light chain. GAPDH was used as a sample processing control for the tissue extracts. Scale bar: 50 µm (heart); 100 µm (tongue). Source data are provided as a Source Data file.