Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX-725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and non-aromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists.

P2Y2 promotes fibroblasts activation and skeletal muscle fibrosis through AKT, ERK, and PKC

Background Skeletal muscle atrophy and fibrosis are pathological conditions that contribute to morbidity in numerous conditions including aging, cachexia, and denervation. Muscle atrophy is characterized as reduction of muscle fiber size and loss of muscle mass while muscle fibrosis is due to fibroblasts activation and excessive production of extracellular matrix. Purinergic receptor P2Y2 has been implicated in fibrosis. This study aims to elucidate the roles of P2Y2 in sleketal muscle atrophy and fibrosis. Methods Primary muscle fibroblasts were isolated from wild type and P2Y2 knockout (KO) mice and their proliferating and migrating abilities were assessed by CCK-8 and Transwell migration assays respectively. Fibroblasts were activated with TGF-β1 and assessed by western blot of myofibroblast markers including α-SMA, CTGF, and collagen I. Muscle atrophy and fibrosis were induced by transection of distal sciatic nerve and assessed using Masson staining. Results P2Y2 KO fibroblasts proliferated and migrated significantly slower than WT fibroblasts with or without TGF-β1.The proliferation and ECM production were enhanced by P2Y2 agonist PSB-1114 and inhibited by antagonist AR-C118925. TGF-β1 induced fibrotic activation was abolished by P2Y2 ablation and inhibited by AKT, ERK, and PKC inhibitors. Ablation of P2Y2 reduced denervation induced muscle atrophy and fibrosis. Conclusions P2Y2 is a promoter of skeletal muscle atrophy and activation of fibroblasts after muscle injury, which signaling through AKT, ERK and PKC. P2Y2 could be a potential intervention target after muscle injury.

The Combination of Electroacupuncture and Massage Therapy Alleviates Myofibroblast Transdifferentiation and Extracellular Matrix Production in Blunt Trauma-Induced Skeletal Muscle Fibrosis

Complementary therapies, such as acupuncture and massage, had been previously reported to have therapeutic effects on skeletal muscle contusions. However, the recovery mechanisms on skeletal muscles after blunt trauma via the combination of electroacupuncture (EA) and massage therapy remain unclear. In the present study, a rat model of the skeletal muscle fibrosis following blunt trauma to rat skeletal muscle was established, and the potential molecular mechanisms of EA + massage therapy on the skeletal muscle fibrosis were investigated. The results suggested that EA + massage therapy could significantly decrease inflammatory cells infiltration and collagenous fiber content and ameliorate the disarrangement of sarcomeres within myofibrils compared to the model group. Further analysis revealed that EA + massage therapy could reduce the degree of fibrosis and increase the degree of myofibroblast apoptosis by downregulating the mRNA and protein expression of transforming growth factor- (TGF-) β1 and connective tissue growth factor (CTGF). Furthermore, the fibrosis of injured skeletal muscle was inhibited after treatment through the normalization of balance between matrix metalloproteinase- (MMP-) 1 and tissue inhibitor of matrix metalloproteinase (TIMP). These findings suggested that the combination of electroacupuncture and massage therapy could alleviate the fibrotic process by regulating TGF β1-CTGF-induced myofibroblast transdifferentiation and MMP-1/TIMP-1 balance for extracellular matrix production.

Role of Matricellular CCN Proteins in Skeletal Muscle: Focus on CCN2/CTGF and Its Regulation by Vasoactive Peptides

The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins’ role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides.

Influence of Platelet Rich Plasma on the Skeletal Muscle Fibrosis after Limb Lengthening in Mice

Category: Basic Sciences/Biologics Introduction/Purpose: Skeletal muscle fibrosis induced by the increase of collagen occurs after limb lengthening which is also called distraction osteogenesis. Although there are studies about influence of platelet rich plasma (PRP) on tissues healing process, its effectiveness is still controversial. The aim of this study was to examine whether PRP decreased the skeletal muscle fibrosis induced by limb lengthening. Methods: Tibial osteotomy was done to 8-week-old wild type mice. Tibia was lengthened at a rate of 0.42 mm/day during 2 weeks, launching 1 week after tibial osteotomy. Just after lengthening completed (3 weeks after tibial osteotomy), PRP was injected into the gastrocnemius muscle (PRP group). As a sham group, phosphate buffered saline (PBS) was injected into the gastrocnemius muscle (non-PRP group). The gastrocnemius (GC) muscles were taken and analyzed at 4, 6, 8 and 10 weeks after tibial osteotomy. Results: The fibrotic area of the GC muscles in the both groups increased at 4 weeks after tibial osteotomy in histological analysis (Figure). Then, it gradually decreased at 6, 8, and 10 weeks after tibial osteotomy. There were no significant differences between the both groups at 6, 8, and 10 weeks after tibial osteotomy. Hydroxyproline, which was a major constituent of collagen, increased in the non-PRP and PRP groups by limb lengthening as well. However, significant changes were not found between the both groups at all any points. Conclusion: At first, we anticipated that PRP should reduce the skeletal muscle fibrosis after limb lengthening significantly. But our results implied that PRP did not decrease the skeletal muscle fibrosis induced by limb lengthening.