Page 88 - Annual report 2021-22

Annual Report 2021-22 |

control blood samples from 60 healthy individual as healthy control (HC) was completed. To
understand the etiology behind the pathogenesis of OA plasma, proteome profile of OA (KL grade 4)
cases (total knee replacement-TKR and uni-compartmental knee replacement-UKR) were studied. The
study was designed to identify novel OA-associated proteins. 52 differentially expressed proteins
(DEPs) were identified by 2-DE and iTRAQ, more novel proteins were identified by SWATHanalysis.
DEPs were analyzed by DAVID online tool. Haptoglobin (Hp) was identified as a significant DEP, 71
validated by western-blot and ELISA. Results revealed 1.5 times increased levels of Hp in OA cases
compared to HC. Further, glycosylated Hp tetramer protein was found to be down regulated and
autoantibody against Hp-beta subunit was up regulated in TKR/UKR plasma samples compared to HC.
Glycosylated active Hp tetramer was found to have higher binding affinity with the free hemoglobin.
The data suggests that poor clearance of free haemoglobin-haemoglobin may be associated with OA
pathogenesis.

Leucine-rich alpha-2-glycoprotein (LRG1) was found highly differentially expressed by iTRAQ and
SWATH analysis. LRG1 was validated in OA (N=36) and HC(N=36). Four groups of pooled plasma
samples from TKR/UKR (n=12) and HC (n=12) were used. Western-Blot analysis revealed 2.1-fold
higher expression of LRG1 in TKR/UKR patient compared to HC with significant p value (p <0.0001).
LRG1 was further validated by ELISA using (N=80) and was found 1.66-fold upregulation with
significant p value (p<0.0001) in TKR/UKR compared to HC.

Knockdown of LRG1 in primary FLS: The knockdown of LRG1 led to reduced IL-6 as well as COMP level
in Primary FLS cells. This signifies that LRG1 is upregulated in OA synovium and plasma, might be
involved in OA synovium fibrosis which might be responsible for the joint stiffness.
Down regulation of active Hp tetramer accompanied by increased levels of free hemoglobin indicated
the poor clearance of free hemoglobin in patients with severe OA and may be responsible for disease
severity. Similarly, LRG1 was found to be another most significant up regulated protein in TKR/UKR
plasma samples as well as in SF samples compared to HC. LRG-1 was found to be involved in elevation
of fibrosis markers in primary cells and its knockdown enhanced the reduced expression of pro-
inflammatory cytokines and COMP level significantly.