Thrombospondin-2 regulates extracellular matrix production, LOX levels, and cross-linking via downregulation of miR-29.

Collagen fibrillogenesis and crosslinking have long been implicated in extracellular matrix (ECM)-dependent processes such as fibrosis and scarring. However, the extent to which matricellular proteins influence ECM protein production and fibrillar collagen crosslinking has yet to be determined. Here we show that thrombospondin 2 (TSP2), an anti-angiogenic matricellular protein, is an important modulator of ECM homeostasis. Specifically, through a fractionated quantitative proteomics approach, we show that loss of TSP2 leads to a unique ECM phenotype characterized by a significant decrease in fibrillar collagen, matricellular, and structural ECM protein production in the skin of TSP2 KO mice. Additionally, TSP2 KO skin displays decreased lysyl oxidase (LOX), which manifests as an increase in fibrillar collagen solubility and decreased levels of LOX-mediated fibrillar collagen crosslinking. We show that these changes are indirectly mediated by miR-29, a major regulator of ECM proteins and LOX, as miR-29 expression is increased in the TSP2 KO. Altogether, these findings indicate that TSP2 contributes to ECM production and assembly by regulating miR-29 and LOX.

A hidden structural vulnerability in the thrombospondin-2 deficient aorta increases the propensity to intramural delamination.

Mice lacking thrombospondin-2 (TSP2) represent an animal model of impaired collagen fibrillogenesis. Collagen constitutes ~1/3 of the wall of the normal murine descending thoracic aorta (DTA) and is thought to confer mechanical strength at high pressures. Microstructural analysis of the DTA from TSP2-null mice revealed irregular and disorganized collagen fibrils in the adventitia and at the interface between the media and adventitia. Yet, biaxial mechanical tests performed under physiologic loading conditions showed that most mechanical metrics, including stress and stiffness, were not different between mutant and control DTAs at 20- and 40-weeks of age, thus suggesting that the absence of TSP2 is well compensated under normal conditions. A detailed bilayered analysis of the wall mechanics predicted, however, that the adventitia of TSP2-null DTAs fails to engage at high pressures, which could render the media vulnerable to mechanical damage. Failure tests confirmed that the pressure at which the DTA ruptures is significantly lower in 20-week-old TSP2-null mice compared to age-matched controls (640±37 vs. 1120±45mmHg). Moreover, half of the 20-week-old and all 40-week-old mutant DTAs failed by delamination, not rupture. This delamination occurred at the interface between the media and the adventitia, with separation planes often observed at ~45 degrees with respect to the circumferential/axial directions. Combined with the observed microstructural anomalies, our theoretical-experimental biomechanical results suggest that TSP2-null DTAs are more susceptible to material failure when exposed to high pressures and this vulnerability may result from a reduced resistance to shear loading at the medial/adventitial border.