Role of prostaglandin e2 in the development of periostosis

BackgroundPeriostosis is an important imaging hallmark of primary hypertrophic osteoarthropahty (PHO) which refers to the abnormal and pathological woven bone formation on the surface of the cortical bone of the long bones, is considered one of the major diagnostic criteria of PHO patients. Homozygous and compound heterozygous mutations in 15-hydroxyprostaglandin dehydrogenase (HPGD) or in the prostaglandin transporter encoding gene (SLCO2A1) which results in the disturbance of prostaglandin E2 (PGE2) catabolism and consequently leads to persistent elevation of circulation of PGE2 levels. The increased levels of PGE2 involved in the pathogenesis of PHO. Numerous studies have revealed that PGE2 played a critical role in stimulating the activity of osteoblast and osteoclast. PGE2 has also been reported to affect bone structure in vivo. The typical periostosis image features in the cortex of long bones combined with high level of PGE2 in PHO patients suggested bone microstructure and bone strength of the long bones would be compromised in PHO patients. However, up to date, detailed information on bone quality changes in long bones of PHO patients have not be reported. Recently, PGE2 has been shown to bind its EP4 receptor (PGE2 receptor 4) in sensory nerve to stimulate bone formation and augment bone mass upon systemic administration. Sensory neuropeptide calcitonin gene-related peptide (CGRP), which are richer in periosteum, played key role in the regulation of periosteum formation. Whether CGRP had an essential role on periosteum cells osteogenesis by PGE2/EP4 axis remains unclear. Therefore, this study aimed to 1) evaluate bone microstructure and bone strength in PHO patients by using high resolution-peripheral quantitative computed tomography (HR-pQCT). 2) In vitro explore the role of CGRP in promoting osteogenic differentiation of PGE2 in periosteum-derived stem cells.MethodsClinical studyThe cohort comprised 20 male PHO patients of the average age of 27.0 years and 20 age and sex-matched healthy controls. Areal bone mineral density (aBMD) was assessed by using dual-energy X-ray absorptiometry (DXA) Bone geometry, volumetric bone mineral density (vBMD), microstructure parameters at distal tibia were evaluated by using HR-pQCT. Bone strength was evaluated by the finite element analysis (FEA) based on HR-pQCT screening at distal tibia. PGE2, serum phosphatase (ALP), beta-C-telopeptide of type I collagen (β-CTX), soluble receptor activator of nuclear factor-kappaB Ligand (sRANKL), osteoprotegerin (OPG), and CGRP were also investigated.Experiment studyIn vivo, PGE2 at 3mg/kg per day or vehicle was systematically given by daily subcutaneous injections at back and locally given by daily along the periosteum injection at bilateral lower extremities for 30 days to 1-month-old intact male rats and 3-month-old male rats. At time of the end point (30th day), in vivo X-ray analysis were performed in these rats to evaluate periostosis. Subsequently, the tibia and femur of each sacrificed rat was collected to perform micro-CT analysis and histopathological analysis. Periosteum derived stem cells (PDSCs) and bone mesenchymal stem cells (BMSCs) were isolated from the PGE2 treated rats and vehicle-treated rats to perform osteogenic differentiation and mineralization analysis by gene expression, western blot and alizarin red staining. In vitro, PDSCs and BMSCs were isolated from the vehicle-treated rats. And then the cells were treated with 50nM and 100nM concentration of PGE2 decided by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) analysis. Osteogenic differentiation and mineralization were analyzed by gene expression, western blot, and alizarin red staining.ResultsClinical studyBy comparing healthy controls, PHO patients had larger bone cross-sectional areas (total area: +75.8%, p<0.001; cortical area: +25.8%, p<0.001; trabecular area: +87.3%, p<0.001), lower vBMD (total vBMD: -35.5%, p<0.001; trabecular vBMD: -31.0%, p<0.001; cortical vBMD: -22.6%, p<0.001) and compromising bone microstructures with more porous cortices (cortical porosity: +121.2%, p<0.001), thinned trabeculae (trabecular thickness: -11.0%, p<0.001), reduced trabecular connectivity (Tb.BV/TV: -27.3%, p<0.001), and relatively more significant resorption of rod-like trabeculae (+301.6%, p<0.001) at distal tibia. Changes in apparent young`s modulus (-35.8%, p<0.001) was significantly lower in PHO patients. PGE2, β-CTX, sRANKL/OPG ratio and CGRP were significantly higher in PHO patients versus healthy controls. PGE2 correlated negatively to vBMD, bone microstructure and estimated bone strength, and positively to bone geometry at distal tibia. aBMD of femoral neck and total hip were found closely to vBMD, bone microstructure and bone strength.Experiment studyIn vivo, significant differences showed between 1-month-old rats and 3-month-old rats in cortical bone changes under PGE2 treatment. PGE2 administration induced a significant increase in cortical bone area, bone volume and cortical bone strength in 1-month-old rapid growing male rats. While these aberrant cortical changes were not found in 3-month-old rats. Histological and molecular analysis of periosteum isolated from the 1-month-old PGE2-treated rats revealed that high levels of PGE2 combined with EP4 receptor enhanced CGRP receptor expression in promoting osteogenesis and mineralization in the peripheral cortex of the femora. Furthermore, vitro studies revealed that PGE2 treatment upregulated the expression of CGRP throughout the osteogenesis differentiation in PDSCs and activated cell osteogenesis differentiation at quite the earlier stage, and therefore completed the differentiation of the whole cells in advance, shorten the whole stage of osteogenesis.ConclusionThis clinical study was the first one illustrating the microstructure and bone strength features in long bones, characterized by PGE2, β-CTX, sRANKL/OPG ratio and CGRP related alterations of long bone quality in PHO patients. The significant high level of CGRP in PHO patients suggested the association of CGRP with PHO in periostosis progression of PHO. Experimentally, both in vivo and in vitro studies confirmed the essential role of CGRP in PGE2/EP4 axis in promoting periosteal formation and mineralization.SignificanceThe clinical findings provided new insight into structural changes in PHO patients that were not captured by DXA assessment along, which the new findings were essential for the further study in skeletal disease development in the PHO patients. As the study confirmed the essential role of CGRP in PGE2/EP4 induced osteogenesis differentiation in the periosteum, suggesting CGRP might be a potential treatment target for PGE2 induced proliferative periostosis, which would provide new insight on identification of potential therapies or develop new drugs for PHO treatment.研究背景骨膜肥厚是原發性肥厚性骨關節炎（PHO）的重要影像學標誌，由包圍長骨皮質骨下病理性的編織骨組成，被認為是PHO患者的主要影像學診斷標準之一。 15-羥基前列腺素脫氫酶（HPGD）或前列腺素轉運蛋白編碼基因（SLCO2A1）純合突變或復合雜合突變導致前列腺素E2（PGE2）代謝障礙，從而導致迴圈中PGE2水準持續升高。目前研究表明，持續高水準的PGE2與PHO的發病機理密切相關。大量研究表明，PGE2在刺激成骨細胞和破骨細胞的活性中起著關鍵作用。動物體內研究也證實了PGE2治療可影響其骨骼結構。PHO患者骨皮質典型的骨膜肥厚結構和其體內持續高水準的PGE2，提示PHO患者的骨結構和骨質量改變。然而，到目前為止，尚未有關於PHO患者長骨骨微結構和骨強度的相關報導。近期研究表明，成骨分泌的PGE2在骨重建位點上調感覺神經纖維啟動PGE2相關EP4受體來促進骨形成。感覺神經肽降鈣素基因相關肽（CGRP）在骨膜中廣泛存在，我們研究小組前期研究表明CGRP在骨膜形成中起關鍵作用。但是，CGRP是否通過PGE2/EP4軸對骨膜成骨起重要作用目前尚不清楚。因此，本研究1）通過高高分辨率外周定量計算機斷層掃描（HR-pQCT）評估PHO患者的骨微結構和骨強度。 2）體外体内研究CGRP在促進骨膜來源幹細胞中PGE2的成骨分化中的作用。研究方法臨床研究本臨床研究於2015年至2018年收集了20名平均年齡為27.0歲的男性PHO患者和20名年齡和性別匹配的健康對照者。使用雙能X線骨密度儀（DXA）評估研究對象的骨骼礦物質密度（aBMD），使用HR-pQCT評估脛骨遠端的骨幾何形狀，體積骨礦物質密度（vBMD）和骨骼微結構。最後通過基於脛骨遠端的HR-pQCT圖像的有限元分析（FEA）進行骨強度評估。此外，該臨床研究還測定患者及其健康對照者的PGE2，血鹼性磷酸酶（ALP），I型膠原蛋白β-C-端肽（β-CTX），核因數-κB配體的可溶性受體啟動劑（sRANKL），骨保護素（OPG）和感覺神經肽降鈣素基因相關肽（CGRP）。实验研究動物研究中，給與1個月大的雄性大鼠和3個月大的雄性大鼠分為兩組，每組實驗組和對照組，實驗組予大鼠背部皮下系統性以及順著股骨方向貼骨膜局部每日注射3mg / kg PGE2，對照組予同等方式注射生理鹽水，連續30天。注射期間每週觀察及給大鼠稱重兩次，预防PGE2不良反應（腹瀉，嘔吐，體重減輕等）。在終點時間（第30天），實驗組和對照組大鼠均進行體內X線檢查以評估骨膜增生情況。隨後，收集每隻處死大鼠的脛骨和股骨，以進行micro-CT分析和組織病理學分析。從PGE2處理的大鼠和經媒介物處理的大鼠的股骨中分離骨膜來源幹細胞（PDSC）和骨髓間充質幹細胞（BMSC），通過基因表達，蛋白質印跡和茜素紅染色進行成骨分化和礦化分析。體外研究中，通過不同濃度的PGE2誘導正常大鼠股骨來源的骨膜幹細胞和間充質幹細胞，通過基因表達，蛋白質印跡和茜素紅染色分析PGE2體外誘導下細胞的成骨分化和礦化。结果臨床研究臨床研究中，与健康對照组相比，PHO患者的骨横截面积较大(总横截面积：+75.8%, p<0.001；皮质骨横截面积：+25.8%, p<0.001；小梁骨横截面积：+87.3%, p<0.001）。vBMD較低（总体积骨密度：-35.5%, p<0.001；皮质骨体积骨密度： -22.6%, p<0.001；小梁骨体积骨密度：-31.0%, p<0.001），以及骨微结构损害较为严重表现在：皮質孔隙增多（+121.2%, p<0.001），小梁骨变薄（-11.0%, p<0.001），骨小梁連接度降低（-27.3%, p<0.001）以及桿狀小梁骨样改变。骨强度分析显示，PHO患者的表觀楊氏模量较正常对照组明顯降低（-35.8%, p