Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells

Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.

Factors affecting the quantity and quality of platelet-rich plasma and platelet-derived growth factor-BB:an observational study

Objective:Platelet-rich plasma(PRP)releases growth factors upon activation,which in turn accelerates healing and regeneration of the target tissue.However,PRP composition may vary according to the patient’s demographics,and wider applications of PRP warrant product standardization.The current study aimed to examine variables influencing the platelet-derived growth factor BB(PDGF-BB)concentration in PRP.Methods:This observational study was conducted in the Department of Pathology and Dentistry at Swami Rama Himalayan University,a tertiary care hospital in northern India from December 2016 to November 2017.PRP was prepared from 40mL of whole blood from 35 individuals(22 women,13 men).Platelet counts,platelet indices(platelet distribution width,mean platelet volume)and PDGF-BB levels were measured,and platelet yield,platelet dose,and growth factor dose in PRP were also calculated.All parameters were analyzed using Pearson’s correlation coefficient.The association between PDGF-BB and PRP platelet count was evaluated using logistic regression.This study was approved by the Ethics Committee of Swami Rama Himalayan University(SRHU/HIMS/ETHICS/2016/103)on September 7,2016.Results:The mean platelet count,PDGF-BB concentration,platelet yield,platelet dose,and growth factor dose in PRP were 1317×10^(9)/L,30±9.89ng/mL,71.62±28.34%,6.5±3.5×10^(9),and 159.62±52.39ng/mL,respectively.Linear regression analysis indicated that PRP platelet counts were a good predictor for PGDF-BB(P<0.05;adjusted R^(2)=0.96.PRP platelet count was significantly positively correlated with PDGF-BB concentration(r=0.74,P<0.001),platelet yield(r=0.80,P<0.001),platelet dose(r=1,P<0.001),and growth factor dose(r=0.74,P<0.001).Conclusions:PRP has wide clinical applications associated with its healing and regenerative properties,and both the quality and quantity of PRP thus need to be standardized as per the requirements.Evaluating variables affecting PRP will thus aid pathologists and clinical practitioners.