Substance P Induces Proliferation And Differentiation Of Tendon Stem Cells And Effects On The Development Of Tendinopathy

BackgroundTendinopathy refers to a heterogeneous group of musculoskeletal diseases caused by overuse, with an incidence of 2%~65%, accounting for 30% ~ 50% in all the sports-related diseases. Tendinopathy is one of the main diseases impairing humans’ kinetism and quality of life. However, there is no golden standard in the treatment of tendinopathy. The clinical effect of teratment for tendinopathy is poor, which has a high recurrence rate. The fundamental reason is that the pathogenesis of tendinopathy has not been very clear, resulting in lack of specificity in treatment.Now increasing interests were focused on neurogenic inflammation caused by substance P(SP). SP, as a critical proinflammatory mediator, was thought to involve in pain transmission and aggravate neurogenic inflammation. Many studies have f ound that expression of SP and its receptor(NK-1R) increased in tendinopathy. SP-positive nerve fibers have been found to be correlated with histopathological changes and clinical symptom in tendinopathy. SP can promote tendon tissue hyperplasia, collagen remodeling, hypercellularity, angiogenesis and accelerate paratendinitis. Moreover, SP can promote the bone marrow stromal cells to differentiate into osteocytes.Tendon stem cells(TSCs), identified as the unique precursor cells of tenocytes, possess several universal characters of general stem cells including clonogenicity, self-renewal and multilineage differentiation potential. Differentiation of TSCs is the key factor to decide the quality of repair, which is affected by a variety of mechanical and biochemical factors. Moderate mechanical loading(4%) induces TSCs to differentiate into tenocytes so as to be beneficial to the repair of tendon injury. However, excessive mechanical loading(8%) promotes TSCs differentiate into non-tenocyte lineages such as adipocytes, chondrocytes, and osteocytes, which may contribute to the development of tendinopathy. Furthermore, studies have confirmed that mechanical loading lead TSCs to produce inflammatory mediators,and some types of inflammatory mediators may also affect the TSCs proliferation and differentiation, which provides a new way of thinking to explore the pathogenesis of tendinopathy. Reviewing the literature, the effects of SP on the proliferation and differentiation of TSCs have yet not been reported.ObjectivesWe aimed to investigate SP expression and histopathological characteristics in rotator cuff tendinopathy; and to explore the effects of excessive mechanical loading on TSCs’ differentiation and SP expression; also to evaluate the effects of SP on TSCs proliferation, differentiation in vitro and the formation of tendinopathy in vivo.Materials and Methods1. SP expression and histopathological characteristics of tendinopathyFrom May 2013 to April 2014, samples were obtained from tendinopathy group and control group arthroscopically. In the tendinopathy group, samples were obtained from supraspinatus tendons from 15 patients diagnosed as rotator cuff tendinopathy. And in the control group, samples were obtained from subscapularis tendons from 8 patients undergoing arthroscopic surgery for shoulder stabilisation. First, we detected SP expression by immunofluorescence staining and semi-quantitative statistical analysis. Then we evaluated histopathological characteristics by H-E staining, Masson staining, and histopathological scores.2. Effects of excessive mechanical loading on differentiation and SP expression of TSCs.First, TSCs were identified by their morphology and clonogenicity,stem cell markers(Nanog, Nucleostemin, and Oct-4), cell surface makers(CD31,CD34,CD44, and CD90), and multilineage differentiation potential. Second, we performed the 8% stretch at 1hz on the TSCs, then detected expression of SP and NK-1R by RT-PCR, Western blot, and ELISA. Finally, we performed the 8% stretch at 1hz on the TSCs with or without NK-1R antagonist, then detected gene expression of multilineage differentiation by RT-PCR.3. Effects of SP on TSCs’ proliferation and apoptosis and abnormal differentiation.3.1 Effects of SP on TSCs’ proliferation and apoptosis.First, we investigated the TSCs’ proliferation by CCK-8 and Ki67 staining respond to different concentrations of SP and the antagonist of NK-1R.(Control, 10-6M, 10-7M,10-8M, 10-9M, 10-10M)Second, we defined the viability of TSCs by crystal violet staining respond to SP and NK-1R antagonist. Third, we examined apoptosis of TSCs by flow cytometry cultured with SP or NK-1R antagonist.(Control, 5×10-6M NK-1R antagonit+10-6M SP、10-6M SP)3.2 Effects of SP on TSCs’ abnormal differentiation.First, we investigated the stemness of TSCs respond to(10-6M)SP by RT-PCR and immunofluorescence staining. Second, we examed the effects of(10-6M)SP on TSCs’ differentiation by multilineage induced differentiation test.Third, we detected gene expression of TSCs’ differentiation cultured with SP and/or NK-1R antagonist by RT-PCR. Finally, we detected protein expression of TSCs’ differentiation cultured with SP and/or NK-1R antagonist by immunofluorescence staining.(Control, 5×10-6M NK-1R antagonit+10-6M SP、10-6M SP)4. Effects of exogenous SP on the development of rat tendinopathy.36 SD rats were randomely divided into three groups, including control group(saline), low-dose SP group(0.5 × 10-6M) and high-dose SP group(5 × 10-6M). Each group included 12 rats. 18 rats were sacrificed after 14 days and 28 days, respectively. Then, Achilles tendons were havested. First, we evaluated histopathological features by H-E staining, Masson staining, and histological scores. Second, we detected expression of SP on TSCs’ differentiation by immunofluorescence staining.Results1. Immunofluorescence staining demonstrated that SP and NK-1R expressed both in tendinopathy and control group, and the fracion of SP positive area of tendinopathy group was greater than the fracion of SP positive area of control group(P﹤0.05). The mean histopathologic scores of tendinopathy group were greater than the mean histopathologic scores of control group(P﹤0.05). The histopathologic appearance of tendinopathy group included destruction of collagen structure, disordered arrangement of collagen fibers, hyperplasia, hypercellularity, angiogenesis, pimelosis and hyalinization.2. SP expression of TSCs was significantly up-regulated by excessive mechanical loading both at gene level and at protein level(P < 0.05). Gene expression of TSCs’ abnormal differentiation including Runx2, PPARγ, Sox9 and Collagen type II, were significantly up-regulated by excessive mechanical loading(P<0.05). However,the gene expression of PPARγ, Sox9 and Collagen type II were significantly decreased by NK-1R antagonist(P<0.05).3. CCK-8 test demonstrated that the effect of SP promoted TSCs proliferation was dose and time dependent. Crystal violet staining demonstrated that SP enhanced TSCs’ viability. Flow cytometry detected that SP inhibited TSCs apoptosis. However, these effects of SP on TSCs proliferation and apoptosis were effectively blocked by NK-1R antagonist(P<0.05).SP decreased TSCs’ stemness. RT-PCR demonstrated that gene expression of Nanog and Nuclestemin in SP group was significantly lower than that in control group(P<0.05). Immunofluorescence staining demonstrated that the ratio of positive cells respond to Nanog and Nuclestemin were reduced by SP(P<0.05).SP promoted TSCs’ adipogenic and chondrogenic differentiation. RT-PCR demonstrated that gene expression of PPARγ, ColⅡand Sox9 in SP group was significantly higher than that in control group(P<0.05), and gene expression of TNC and Tnmd was down-regulated(P<0.05). Gene expression of PPARγ, ColⅡand Sox9 in SP + NK-1R antagonist group was lower than that in SP group(P<0.05). And the difference was not significant between SP + NK-1R antagonist group and control group(P>0.05). These results were further confirmed by immunofluorescence staining.4. The mean histopathologic scores of high-dose SP group were greater than the mean histopathologic scores of low-dose SP group and control group(P﹤0.05). The difference of mean histopathologic scores between low-dose SP group and control group was not significant(P>0.05). Immunofluorescence staining demonstrated that PPARγ and ColⅡexpression of high-dose SP group were significantly higher than that of low-dose SP group and control group(P﹤0.05). PPARγ、ColⅡand Runx2 expression between low-dose SP group and control group was not significant different(P>0.05).Conclusion1. Confirmed that SP was high expressed in rotator cuff tendinopathy and verified that the histopathological characteristics of rotator cuff tendinopathy was consistant with that of general tendinopathy, including destruction of collagen structure, disordered arrangement of collagen fibers, hyperplasia, hypercellularity, angiogenesis, pimelosis and hyalinization.2. We had successfully isolated and identified TSCs, which also expressed SP and NK-1R;excessive mechanical loading enhanced SP expression of TSCs in vitro. Excessive mechanical loading induced differentiation of TSCs into adipocytes and chondrocytes. However, these effects can be effectively inhibited by NK-1R antagonist.3. SP enhanced TSCs’ proliferation and viability; and inhibited TSCs apoptosis through NK-1R, which may contribute to tendon tissue hyperplasia, hypercellularity, and develop tendinopathy. SP promoted differentiation of TSCs into adipocytes and chondrocytes through NK-1R, which may contribute to the development of tendinopathy such as fatty degeneration and chondrification.4. In vivo, SP lead to tendon tissue hyperplasia, hypercellularity, angiogenesis and disorganized collagen in rat tendons; and promoted fatty degeneration and chondrification in a manner of dose-dependent. The effect of low-dose SP on the formation of tendinopathy is not obvious. However, high-dose SP promoted the formation of tendinopathy. We concluded that SP-induced TSCs proliferation and abnormal differentiation may play a key role on the development of tendinopathy.