Further elucidation of the mechanism of action of trabodenoson using validated 3D‐human trabecular meshwork constructs
DS Albers1, R Noecker2, A Unser3, F Ahmed3, KY Torrejon3, R Baumgartner1, and WK McVicar1
1Inotek Pharmaceuticals Corporation, Lexington, MA; 2Ophthalmic Consultants of Connecticut; 3Glauconix, Inc., Albany, NY
INTRODUCTION
Glaucoma is characterized by elevated intraocular pressure (IOP)
resulting in the loss of retinal ganglion cells. Adenosine A1 receptor
stimulation has been shown to lower IOP by increasing total outflow via
the conventional trabecular meshwork (TM) pathway through a matrix
metalloproteinase (MMP)‐mediated mechanism.1 Trabodenoson is an
adenosine mimetic that selectively binds the A1 receptor, increases
outflow through the conventional TM pathway2, and lowers IOP in
glaucoma patients.3 The purpose of the current study was to elucidate
further trabodenoson’s mechanism of action in a validated 3D‐human
trabecular meshwork (3D‐HTMTM) ex‐vivo healthy tissue model.
METHODS
The 3D‐HTMTM constructs were cultured in 10% FBS‐IMEM, using primary
cells from 4 individual donor eyes and standard validated protocols, over
a three week period. Upon confluence, the constructs were serum‐
starved (1% FBS‐IMEM) for 6 days prior to initiation of treatment:
vehicle, 1 µM and 10 µM trabodenoson. Supernatant and tissue lysates
were collected on days 2 and 8 post‐initiation of treatment and analyzed
as follows: Total MMP‐2 and TIMP‐2 levels in supernatant by
immunoassay; MMP‐2 activity in supernatant via zymography; active
MMP‐14, collagen IV, and fibronectin levels in tissue lysates via western
blot analysis.
Figure 1: Collagen IV and fibronectin levels are reduced 48 hrs post‐trabodenoson or exogenous MMP‐2 treatments
DAPI
Collagen IV Fibronectin
Figure 4: MMP‐2 activation via MMP‐14 (MT‐1)
Merged
Control
10 µM
INO‐8875
Robert Visse, and Hideaki Nagase Circ Res. 2003;92:827‐839
Figure 5: Increased MMP‐14 levels and decreased fibronectin and collagen IV levels by trabodenoson at days 2 and 8
1 µg/mL
MMP‐2
3 µg/mL
MMP‐2
Figure 2: No measurable increases in MMP‐2 or TIMP‐2 release by trabodenoson 2 or 8 days after treatment initiation
RESULTS
1.
2.
3.
4.
5.
Confocal photomicrographs of 3D‐HTM constructs following 48 hrs
of treatment with trabodenoson (10 µM) or exogenous MMP‐2 (1 or 3 µg/ml). The reduction in staining of both fibronectin and collagen IV by trabodenoson treatment appears more effective than exogenous MMP‐2 in reducing ECM protein (Fig. 1)
Western blot analysis indicates levels of MMP‐2 and TIMP‐2 released are unaffected on days 2 and 8 post‐initiation of 1 or 10 µM trabodenoson treatment (Fig. 2).
Zymographic analysis of supernatant demonstrates significant increases in MMP‐2 activity on days 2 and 8 following initiation of 1 and 10 µM trabodenoson treatment (Fig. 3).
Significant increases in the presence of MMP‐14 were detected in tissue lysates from constructs treated with 1 µM (day 2 and 8) and 10 µM (day 8) trabodenoson as compared to constructs treated with placebo (Fig. 5).
Both fibronectin and collagen IV levels were decreased by trabodenoson treatment as compared to vehicle treatment as measured by western blot analysis (Fig. 5). Both of these ECM proteins are substrates for MMP‐2 as well as MMP‐14.
1Myers et al (2016) JOPT 32, 555‐562; 2Cunha (2005) Pur
Data are presented as mean±SD; *p<0.05 vs control
Figure 3: Zymographic analysis of supernatants reveal increases in MMP‐2 activity by trabodenoson at days 2 and 8
*p<0.05 vs control
Active MMP-2
Day 2
Vehicle
1 µM
Day 8
10 µM
Donor 1
Donor 2
Donor 3
Donor 4
Signaling 1, 111‐134; 3Kalesnykas et al (2012) IOVS 53, 3847‐3857. Vehicle
1 µM
10 µM
CONCLUSIONS
• Trabodenoson treatment reduces ECM protein levels similarly, if not better than, exogenous MMP‐2
• Trabodenoson treatment does not appear to increase MMP‐2 or TIMP‐2 release from TM cells
• Instead, trabodenoson appears to increase MMP‐2 activity
• This trabodenoson‐mediated increase in MMP‐2 activity appears to come from an increase in MMP‐14 presence/activity causing further MMP‐2 activation.