Objective Constrictive extracellular matrix (ECM) remodeling contributes significantly to restenosis after

Objective Constrictive extracellular matrix (ECM) remodeling contributes significantly to restenosis after arterial reconstruction but its molecular regulation is usually poorly defined. +/+ (WT) controls 1 month after carotid ligation. Results HA increased SMC attachment to collagen-coated plates but blocking RHAMM reduced adhesion (p=0.025). rKO SMC also exhibited Tandutinib reduced adhesion (% adherent: 36.1±2.2 vs. 76.3±1.9 p< 0.05). SMC contraction of collagen gels was enhanced by HA and further increased by RHAMM blockade (p< 0.01) or knockout (gel diameter mm: rKO 6.7 vs. WT 9.8 p=0.015). RHAMM Rabbit polyclonal to ACD. promoted constrictive remodeling as carotid artery size was significantly larger in rKO mice 1 month after ligation. Neointimal thickening however was not affected in rKO (p=NS vs WT) but lumen size was significantly larger (lumen area μm2: 52.4±1.4 × 103 vs. 10.4±1.8 × 103 p=0.01) because artery size constricted less (EEL area μm2: rKO 92.4 vs. WT 51.3 × 103 p=0.015). Adventitial thickening and collagen deposition were also more extensive in ligated rKO carotids (adventitial thickness μm: 218±12.2 vs. 109±7.9 p=0.01). Conclusion HA activation of RHAMM significantly impacts SMC-ECM adhesive interactions and contributes to constrictive artery wall remodeling in mice. Strategies to block RHAMM at sites of vessel injury may show useful in the prevention of clinical restenosis. and artery wall remodeling in mice. Our findings suggest a critical role for RHAMM in mediating adhesive interactions between SMC and ECM and constrictive artery wall remodeling assays were run with multiple samples per condition and repeated at least three times to establish consistency. Data were averaged within and between experiments to determine a mean of means±SD for each endpoint. morphometric data were averaged from 5 cross sections/artery and mean of means±SD decided for each group. Differences between groups were compared using 2-tailed Student’s t test with significance assigned at p=0.05. RESULTS Arterial SMC culture and genotype SMC cultured from rat and mouse aortas exhibited common hill-and-valley morphology and expressed α-actin and sm-MHC. Growth rates and morphology were comparable for rKO and WT SMC and loss of RHAMM expression confirmed in knockout lines (Physique 2). Physique 2 RHAMM mRNA and protein expression in aortic SMCs cultured from WT and rKO mice measured with reverse transcriptase polymerase chain reaction (RT-PCR) and western blot. RHAMM promotes SMC adhesion to collagen and HA We established the timing of SMC adhesion to plastic pre-coated with collagen and found ~50% Tandutinib attached within one hour. HA significantly increased adhesion to collagen (% adherent: collagen+HA 83.2 vs. collagen 66.4 p=0.01 N=4) while blocking RHAMM with antibody significantly reduced adhesion (% adherent: 48.8±1.9 vs. 80.6±2.4 p= 0.025 N=4). This effect was mirrored by loss of RHAMM expression which significantly inhibited adhesion to collagen with/without Tandutinib HA for rKO SMC (p< 0.05 Determine 3). Physique 3 Chart shows number of cells adherent to plastic coated with either type-I collagen alone or collagen with HA. Adherent cell number expressed as percent of total Tandutinib SMCs loaded per well. HA increased adhesion by 35.5±1.9% compared to collagen alone ... RHAMM stimulates SMC migration HA signaling through RHAMM can promote SMC migration.26 27 We confirmed that blocking RHAMM inhibited migration as scratch wound coverage at 48 hours was significantly reduced by blocking antibody (% wound area covered: 8.2±2.3 vs. 92.2±2.2 p=0.008 N=4). The impact of RHAMM deletion on SMC migration has not previously been described. WT SMCs migrated into and nearly completely resurfaced scrape wounds within 48 hours whereas migration was significantly less for rKO (% wound covered: 36.3±3.5 vs. 82.6±2.7 p=0.01 Physique 4). Physique 4 Scrape Wound Migration Assay. A. Photomicrographs show representative scrape wounds acutely and then 48 hours later for SMC treated with control IgG (above) or anti-RHAMM blocking antibody (below). RHAMM blockade inhibited migration into scrape wounds ... RHAMM modulates HA-induced collagen gel remodeling We previously reported HA enhances contraction of collagen gels by primate SMCs16. We confirmed a similar effect of HA on rat SMC (Physique 5A) and to address the contribution of RHAMM SMC were pre-treated with blocking antibody or control IgG. Surprising to us gel contraction increased after RHAMM blockade (gel diameter mm: 6.8±0.1 vs..