Background/Purpose: We reported previously that inflammation in TNF-Tg mice, a model of rheumatoid arthritis (RA), inhibits lymphatic vessel maturation, resulting in decreased lymphatic smooth muscle cell (LSMC) coverage and lymph leakage, thereby reduced draining function. Here, we investigatedthe mechanisms responsible for LSMCdysfunction in TNF-Tg mice and if existing drugs could be used asnew lymphatic-based therapy for RA.Wetestedthe hypotheses:1) chronic jointinflammation stimulateslymphatic endothelial cells(LECs) to produce iNOS, leading to reduced draining functionvia inhibition of LSMC contraction; and 2)Ferulic acid (FLA) andPanaxNotoginsengSaponins(PNS), herbal drugs that have been used to treat patients with vascular diseases, reduce LEC iNOSand restoreLSMC/lymphatic vessel function toattenuate RA.

Methods: TNF-Tg mice and WT littermates were used. 1)iNOS expression was examined by qPCR in synovial LECs that were purified with anti-Podoplanin Ab-conjugated beads, and by immunostaining in the collecting lymphatic vessels. (2) The effect of LECs on LSMCs that were isolated from rat mesenteric lymphatic vessels were assessed by pre-treating LECs with TNF on glass coverslips, transferring LECs to LSMC cultures, and measuring expression of functional muscle genes in LSMCs by qPCR and Western blot. 3)TNF-Tg mice were treated with the herbal drugs,FLA orPNS, or vehicle for 3 months. Lymphatic draining function, including clearance and pulse,was measured by indocyanine green near-infrared lymphatic imaging, and tissue damage was determined by histology. 4) The effects of FLA and PNS on LEC inhibition of LSMCs were tested in co-cultures, as in (3).

Results: 1) Synovial LECs from TNF-Tg mice expressed higher levels of nos2thanWT LECs (8.4+2.7 vs. 1+0.4) and these were greater than those observed fornos1and tnf(nos1: 2.4+SD,tnf: 3.8+0.7). Low levels of TNF (0.1ng/mL) stimulated the expression of nos2,but not nos1and tnf, in LECs (nos2: 40+11, nos1: 6.5+2.5, tnf:2.4+0.9). Immunostainingrevealedthat LECs in the collecting lymphatic vessels from TNF-Tg mouse leg stained strongly positively for iNOS.2) TNF pre-treated LECssignificantly decreased expression of multiple functional muscle genes (h1-calponin, sMYH11, SMα2 and SMα22)in LMSCs at mRNA and protein levels, which was blocked by a NO inhibitor. 3) FLA and PNS significantly improved lymph clearance of ankle tissues, restored lymphatic pulses, and markedly reduced joint inflammation, bone erosion and cartilage loss inTNF-Tg mice in comparison with vehicle-treated TNF-Tg mice. 4) FLA and PNS blocked the inhibitory effects on expression of LSMC functional genes in LECs withpre-treated TNF.

Conclusion: In the setting ofchronic inflammation in TNF-Tg mice, LECs express high levels of iNOS and produce NO, which negatively affects the structure and contractile function of LSMCs, leading to impaired lymphatic drainagefrom collecting lymphatic vessels, and exacerbates inflammation and tissue damage in afferent joints.Herbal drugscommonly used to treat patients with vascular diseases have potential as anti-RA agents based on theirinhibition of NO production and preservation of lymphatic function.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-lymphatic-muscle-contraction-by-lymphatic-endothelial-inos-impairs-lymph-drainage-from-arthritic-joints-in-tnf-tg-mice-and-is-prevented-by-herbal-drugs/