Examining lysyl oxidase-like modulation of collagen architecture in 3D spheroid models of idiopathic pulmonary fibrosis via second-harmonic generation microscopy

Abstract

Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored.

Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition).

Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids.

Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group.

Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

Keywords: collagen; crosslinking; polarization; second-harmonic generation; stiffness.

Fig. 1

Enhancement or inhibition of pyridinoline crosslinking in a 3D in vitro model of fibrosis. Total mature trivalent (PYD + DPD) collagen crosslinks determined by ELISA. $n=6$ samples from three IPF donors. ** indicates $p<0.01$ and *** indicates $p<0.0001$.

Fig. 2

Representative SHG images of in vitro IPF samples for 42- (top row) and 60-day (bottom row) cultures. The collagen morphology for the control [(a) and (e)], crosslinking inhibitor [(b) and (f)], crosslinking promoter [(c) and (g)], and inhibitor + promoter [(d) and (h)] treatment groups are shown. $\text{Scalebar}=30\mu \mathrm{m}$.

Fig. 3

Representative collagen fiber/fiber bundle map. Average collagen fiber straightness, length, and width for control (black), LOXL inhibitor (red), crosslinking promoter (blue), and inhibitor in combination with promoter (magenta) in vitro samples quantified by CT-FIRE software. Standard error bars are shown. * indicates $p<0.05$, ** indicates $p<0.01$ and **** indicates $p<0.00001$. $\text{Scale bar}=30\mu \mathrm{m}$.

Fig. 4

Forward attenuation as a function of depth for control (black), LOXL inhibitor (red), crosslinking promoter (blue), and inhibitor in combination with promoter (magenta) in vitro samples. Standard error bars are shown.

Fig. 5

Linear polarization analysis of Ctrl (black), LOXL inhibitor (red), crosslinking promoter (blue) and inhibitor in combination with promoter (magenta) in vitro samples, where the reconstructed pixel-based response and the extracted pitch angles are in (a) and (b), respectively. Standard error bars are shown. ** indicates $p<0.01$ and **** indicates $p<0.00001$.

Fig. 6

Pixel-based SHG signal anisotropy responses for Ctrl (black), LOXL inhibitor (red), crosslinking promoter (blue), and inhibitor in combination with promoter (magenta) in vitro samples. Reconstructed anisotropies at (a) all excitation angles and (b) individual 0-deg angle. Standard error bars are shown. * indicates $p<0.01$ and ** indicates $p<0.001$.

Fig. 7

Normalized SHG-CD data of optically cleared Ctrl (black), LOXL inhibitor (red), crosslinking promoter (blue), and inhibitor in combination with promoter (magenta) in vitro samples. * indicates $p<0.01$ and ** indicates $p<0.001$.