Osteocyte apoptosis regulates osteoclast precursor adhesion via osteocytic IL-6 secretion and endothelial ICAM-1 expression

Osteocyte apoptosis precedes osteoclast resorption, and may act as a critical signal to trigger bone remodeling. While osteoclast precursors are known to travel via the circulation, the specific mechanisms by which they accumulate at remodeling sites are unclear. We hypothesized that osteocyte apoptosis mediates osteoclast precursor adhesion to vascular endothelium by regulating osteocytic secretion of IL-6 and soluble IL-6 receptor (sIL-6R) to promote endothelial ICAM-1 expression. We found that conditioned media from TNF-α-induced apoptotic MLO-Y4 osteocytes promoted RAW264.7 osteoclast precursor adhesion onto D4T endothelial cells (P < 0.05). Blocking osteocyte apoptosis with a pan-caspase inhibitor (ZVAD-FMK) reduced osteoclast precursor adhesion to baseline levels (P < 0.001). Endothelial cells treated with apoptotic osteocyte conditioned media had elevated surface expression of ICAM-1 (P < 0.05), and blocking ICAM-1 abolished apoptosis-induced osteoclast precursor adhesion. Apoptotic osteocyte conditioned media contained more IL-6 (P < 0.05) and sIL-6R (P < 0.05) than non-apoptotic osteocyte conditioned media. When added exogenously, both IL-6 and sIL-6R were required for endothelial activation, and blocking IL-6 reduced apoptosis-induced osteoclast precursor adhesion to baseline levels (P < 0.05). Therefore, we conclude that osteocyte apoptosis can promote osteoclast precursor adhesion to endothelial cells via ICAM-1; this is likely through increased osteocytic IL-6 and sIL-6R secretion, both of which are indispensible to endothelial activation.     

Differential research of inflammatory and related mediators in BPH,histological prostatitis and PCa

Prostate cancer (PCa) is one of the most common male malignancies in the world. It was aimed to investigate differential expression of inflammatory and related factors in benign prostatic hyperplasia (BPH), prostate cancer (PCa), histological prostatitis (HP) and explore the role of Inducible nitric oxide synthase (iNOS), (VEGF) Vascular endothelial growth factor, androgen receptor (AR) and IL‐2, IL‐8 and TNF‐α in the occurrence and development of prostate cancer. RT‐PCR was used to detect the mRNA expression level of iNOS, VEGF, AR and IL‐2, IL‐8 and TNF‐α in BPH, PCa and BPH+HP. Western blotting and immunohistochemical staining were used to detect the protein levels of various proteins in three diseases. The results showed the mRNA and protein levels of iNOS, VEGF and IL‐2, IL‐8 and TNF‐α were significantly increased in PCa and BPH+HP groups compared with BPH group (p < .05), while the AR was significantly lower than those in PCa and BPH+HP groups (p < .05). There was no significant difference in the mRNA and protein levels of iNOS, VEGF, AR and IL‐2, IL‐8 and TNF‐α between PCa and BPH+HP groups (p > .05). iNOS, VEGF, AR and IL‐2, IL‐8 and TNF‐α are involved in the malignant transformation of prostate tissue and play an important role in the development and progression of Prostate cancer (PCa).     

Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF‐κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan‐caspase apoptosis inhibitor, QVD (quinolyl‐valyl‐O‐methylaspartyl‐[‐2,6‐difluorophenoxy]‐methylketone). Immunohistochemistry was used to identify apoptotic and RANKL‐producing osteocytes in femoral diaphysis and distal trabecular bone, and µCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3‐ and 4‐fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3‐fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5‐fold versus Ctrl, p < 0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU‐triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, µCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse. © 2016 American Society for Bone and Mineral Research.     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

Augmentation of surgical angiogenesis in vascularized bone allotransplants with host‐derived a/v bundle implantation,fibroblast growth factor‐2, and vascular endothelial growth factor administration

We have previously shown experimental transplantation of living allogeneic bone to be feasible without long‐term immunosuppression by development of a recipient‐derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L‐lactide‐co‐glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient‐derived arteriovenous (a/v) bundle. FK‐506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGF+VEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1015–1021, 2010     

TNF‐α–induced NF‐κB signaling reverses age‐related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RI^null), or TNF receptor 2‐null (TNF‐RII^null) mice. VEGF induction was inhibited when we used TNF‐RI^null‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229–235, 2009     

Role and regulation of VEGF and its receptors 1 and 2 in the aseptic loosening of total hip implants

It was hypothesized that vascular endothelial growth factor (VEGF) in fibroblasts participates in aseptic loosening of total hip replacement (THR) implants. Therefore, osteoarthritic (OA) samples (n = 11) were compared with synovial membrane‐like interface tissues from revision THR (n = 10). VEGF‐A and its receptors were stained using streptavidin‐immunoperoxidase method. Their regulation by hypoxia and cytokines were studied in cultured fibroblasts using quantitative real‐time polymerase chain reaction (qRT‐PCR). VEGFR1⁺ lining cells (p < 0.01), stromal fibroblast‐like cells (p = 0.001) and stromal macrophage‐like cells (p < 0.05) were more numerous in rTHR than in OA. As to VEGFR2⁺, only stromal fibroblast‐like cells in rTHR outnumbered those found in OA (p < 0.05). VEGFRs in synovial fibroblasts were not affected by hypoxia, but VEGF increased 2.4‐fold (p < 0.05). Interleukin‐4 up‐regulated VEGFR1 expression 23‐fold. This is the first study to describe a difference between rTHR and OA in VEGF receptors, particularly VEGFR1. Hypoxia increased VEGF, but the VEGFR1 increase in the lining and stroma is probably IL‐4 driven, in accordance with the M2‐type macrophage dominance in interface tissues. VEGF/VEGFR system is also affected by hypoxia and may play a role in angiogenesis and bone pathology in aseptic loosening of total hip implants. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1830–1836, 2012     

Increased IL‐1β expression and myofibroblast recruitment in subacromial bursa is associated with rotator cuff lesions with shoulder stiffness

We evaluated whether proinflammatory cytokine expression and myofibroblast recruitment in subacromial bursa was linked to rotator cuff lesions with shoulder stiffness. We analyzed expressions of IL‐1β, IL‐6, and TNF‐α in subacromial bursa and joint fluid collected from 14 patients with cuff tears with stiffness as a study group (Group I) and 14 patients with rotator cuff tears without shoulder stiffness as a control group (Group II) using real‐time RT‐PCR, immunohistochemistry, and ELISA. Myofibroblast apoptosis in subacromial bursa was analyzed using terminal deoxynucleotidyl transferase ‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) and α‐smooth muscle actin immunofluorescence staining. Shoulder function was evaluated using the Constant score. Group I had higher mRNA expression (p < 0.001) and immunoreactivities (p < 0.001) of IL‐1β. They also had higher levels of IL‐1β, IL‐6, and TNF‐α in joint fluid. Increased IL‐1β mRNA expression in the subacromial bursa and IL‐1β levels in joint fluid were correlated with a preoperative deficit in shoulder motion (p < 0.001) and preoperative Constant scores (p < 0.001). Immunofluorescence observations showed that Group I subjects had more myofibroblasts (p < 0.001) than Group II. In Group II, a significant correlation was found between apoptotic myofibroblasts and total myofibroblasts (p = 0.002), but not in Group I (p = 0.510). Increased expression of IL‐1β and myofibroblast recruitment in the subacromial bursa in rotator cuff lesions are linked to shoulder stiffness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1090–1097, 2008     

Innate immune molecule surfactant protein D attenuates sepsis‐induced acute kidney injury through modulating apoptosis and NFκB‐mediated inflammation

The objective of this study is to investigate the mechanism whereby innate immune molecule surfactant protein D (SP‐D) attenuates sepsis‐induced acute kidney injury (AKI) through modulating apoptosis and nuclear factor kappa‐B (NFκB)‐mediated inflammation. In the present study, a mouse sepsis model was established by cecal ligation and puncture in SP‐D knockout (KO) mice and wild‐type (WT) mice. A sham‐operated group was included as the control. The experimental materials were extracted 6 and 24 hours postoperatively. The plasma levels of tumour necrosis factor alpha (TNF‐α) and MCP‐1 were determined by enzyme‐linked immunosorbent assay (ELISA). Apoptosis was measured by double staining with Annexin V/propidium iodide and flow cytometry. The levels of NFκB in renal tissues were measured by ELISA and Western blotting assay. Apoptosis was detected by TUNEL assays. There were no significant differences in plasma TNF‐α levels between the WT sham group and the KO sham group at 6 and 24 hours postoperatively (P < .05), but the levels of TNF‐α in the WT sepsis and KO sepsis groups were significantly higher than those in controls (P < .05). The levels of TNF‐α in the KO sepsis group were significantly higher than those of the WT sepsis group (P < .05). TNF‐α levels in the WT sepsis group and the KO sepsis group at 24 hours postoperatively were significantly higher than those at 6 hours postoperatively (P < .05). The levels of MCP‐1 in the WT sepsis group and the KO sepsis group at 6 and 24 hours postoperatively were significantly higher than those in the control group (P < .05), and MCP‐1 levels in the KO sepsis group were significantly higher than those in the WT sepsis group (P < .05). MCP‐1 levels in the WT sepsis group and the KO sepsis group at 24 hours postoperatively were significantly higher than those at 6 hours postoperatively (P < .05). The expression of SP‐D in WT kidneys was significantly lower at 6 and 24 hours postoperatively (P < .05). The number of TUNEL‐positive cells in the kidneys from septic SP‐D KO mice was significantly higher (P < .05). The levels of NFκB in septic mice were significantly increased at 6 and 24 hours after induction of sepsis compared with the sham‐operated group compared with those of septic SP‐D KO mice and WT mice (P < .05). Innate immune molecule SP‐D significantly decreased plasma levels of inflammatory cytokines in mice and attenuated sepsis‐induced AKI by inhibiting NFκB activity and apoptosis.     

Co–Cr–Mo alloy particles induce tumor necrosis factor alpha production in MLO-Y4 osteocytes: A role for osteocytes in particle-induced inflammation

Wear debris-induced osteolysis is purportedly the limiting problem affecting the long term results of joint arthroplasty. Pathogenic effects of wear debris in peri-implant cells such as macrophages, osteoblasts and osteoclasts have been well studied. In contrast, the effects of wear debris on osteocytes, which make up over 90% of all bone cells, remain unknown. We hypothesized that metal implant debris can induce the pro-inflammatory response in osteocytes. This study demonstrated the effects of cobalt–chromium–molybdenum alloy (Co–Cr–Mo) particles on a well-characterized MLO-Y4 osteocyte cell line. Co–Cr–Mo alloy particle treatment significantly (p < 0.05) up-regulated tumor necrosis factor alpha (TNFα) gene expression after 3 and 6 h and TNFα protein production after 24 h, but down-regulated interleukin-6 (IL-6) gene expression after 6 h. Co–Cr–Mo alloy particle treatment also induced osteocyte apoptosis after 24 h. This apoptotic effect was partially (40%) dependent on TNFα. Therefore, our results suggest that osteocytes play a role in particle-induced inflammation and bone resorption following total joint arthroplasty by inducing pro-inflammatory cytokines and inducing osteocyte apoptosis.     

Molecular angiogenic events of a two‐herb wound healing formula involving MAPK and Akt signaling pathways in human vascular endothelial cells

The emergence of electric cell‐substrate impedance sensing (ECIS) technology has provided new insight in advanced cell behavioral study by its nanometer sensitivity, precise electrical wounds generation, and high reproducibility that can be monitored in real time in a noninvasive way. However, little is known regarding pro‐angiogenic agents in wound healing studies using endothelial cells evaluated with ECIS technology. Our previous studies showed a prominent wound healing effect of a two‐herb formula (NF3) comprising of Astragali Radix and Rehmanniae Radix in a rat chronic wound model through actions including angiogenesis. Here we further investigated the angiogenic effect and its underlying molecular mechanism through proliferation, motility, and tubule formation of human vascular endothelial cells (HECV) using ECIS technology. It was first shown that HECV treated with NF3 had a higher resistance than that of control using ECIS cell attachment and cell migration model (p < 0.01). We further validated in a scratch assay that NF3 treatment significantly stimulated HECV cell migration (p < 0.01–0.05). Also, NF3‐treated HECV were observed to develop into a significantly more branched tubular structure when compared with control (p < 0.05–0.01). Meanwhile, Western blot analysis of NF3‐treated HECV revealed the activated expression of p‐Akt, and mitogen‐activated protein (MAP) kinases for p‐ERK, p‐p38, and p‐JNK. We propose that the effect of NF3 in the promotion of endothelial cell migration and tubule formation could be mediated through pathways involving p‐Akt and activated MAP kinases. Hence, we demonstrated the complexity of the angiogenic effect activated by NF3 molecularly and functionally. NF3 treatment could offer therapeutic value to chronic wound healing for its pro‐angiogenic efficacy.     

The effects of local vanadium treatment on angiogenesis and chondrogenesis during fracture healing

This study quantified the effects of local intramedullary delivery of an organic vanadium salt, which may act as an insulin‐mimetic on fracture healing. Using a BB Wistar rat femoral fracture model, local vanadyl acetylacetonate (VAC) was delivered to the fracture site and histomorphometry, mechanical testing, and immunohistochemistry were performed. Callus percent cartilage was 200% higher at day 7 (p < 0.05) and 88% higher at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Callus percent mineralized tissue was 37% higher at day 14 (p < 0.05) and 31% higher at day 21 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Maximum torque to failure was 104% and 154% higher at 4 weeks post‐fracture (p < 0.05) for the healing femurs from the VAC‐treated (1.5 and 3.0 mg/kg) animals. Animals treated with other VAC doses demonstrated increased mechanical parameters at 4 weeks (p < 0.05). Immunohistochemistry detected 62% more proliferating cells at days 7 (p < 0.05) and 94% more at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg VAC. Results showed 100% more vascular endothelial growth factor‐C (VEGF‐C) positive cells and 80% more blood vessels at day 7 (p < 0.05) within the callus subperiosteal region of VAC‐treated animals (1.5 mg/kg) compared to controls. The results suggest that local VAC treatment affects chondrogenesis and angiogenesis within the first 7–10 days post‐fracture, which leads to enhanced mineralized tissue formation and accelerated fracture repair as early as 3–4 weeks post‐fracture. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1971–1978, 2012     

参黄合剂对大鼠瘘管创面血管及内皮细胞的影响

目的:观察参黄合剂对大鼠瘘管创面组织中血管生成及内皮细胞凋亡的影响。方法:选用180只雄性SD大鼠用于瘘管术后模型,造模成功后,按随机数字表法分为空白对照组、空白模型组、模型对照组、生理盐水组、高锰酸钾组、参黄合剂组等6组,每组30只。分别于术后第l、7、14 d从各组取出10只大鼠处死取标本,观察各组大鼠创面肉芽形态变化及血管内皮细胞凋亡情况。结果:术后1 d、7 d和14 d空白对照组镜下毛细血管及凋亡细胞无明显变化:其余各组术后1 d可见凋亡细胞、毛细血管及大量渗出液及炎性细胞,术后7 d、14 d可见大量由内皮细胞增生形成的实性细胞索及扩张的毛细血管,在毛细血管周围有许多新生的成纤维细胞,凋亡细胞、渗出液及炎性细胞逐渐减少。在术后第7 d时参黄合剂组VEGF mRNA(13.64±3.19)开始增加,凋亡指数(1.92±0.15)开始下降,均达到高速增长期;第14 d参黄合剂组VEGF mRNA(18.48±3.18)继续上升、凋亡指数(1.48±0.16)继续下降,速度放缓。治疗7 d时,参黄合剂组大鼠创面肉芽组织中VEGF mRNA及凋亡指数与空白对照组VEGF mRNA(7.37±1.32)、凋亡指数(2.42±0.16),生理盐水组VEGF mRNA(7.76±1.05)、凋亡指数(2.53±0.17)及高锰酸钾组VEGF mRNA(10.53±1.24)、凋亡指数(2.16±0.17)比较,差异具有统计学意义(P0.05)。治疗14 d时,参黄合剂组创面VEGF mRNA及凋亡指数与空白对照组VEGF mRNA(9.46±1.25)、凋亡指数(2.15±0.14),生理盐水组VEGF mRNA(9.47±1.73)、凋亡指数(2.22±0.15)及高锰酸钾组VEGF mRNA(14.65±1.45)、凋亡指数(1.76±0.11)比较,差异具有统计学意义(P0.05)。结论:参黄合剂促进创面肉芽组织中血管生成,抑制了血管内皮细胞的凋亡,调节了VEGF mRNA的表达,加速创面修复。     

Neovascular potential of adipose‐derived stromal cells (ASCs) from diabetic patients

We explored the vascular biology of adipose‐derived stromal cells (ASCs) from diabetic patients and applied these cells to a murine ischemic flap model to assess the comparative angiogenic potentials between normal and diabetic human ASCs. ASCs were obtained from diabetic patients (n = 5) and controls (n = 5). Secretion and expression of angiogenic cytokines were measured under normoxic and hypoxic condition in vitro. Conditioned media harvested from ASC cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation and tubulization. The control and diabetic ASCs were injected into the murine ischemic flaps, and the surviving area was measured. Diabetic adipose‐derived stromal cells showed a lower level of vascular endothelial growth factor expression and cell proliferation rates than the control cells (p < 0.05). However, vascular endothelial growth factor, hepatocyte growth factor secretion, tubulogenesis, and cell proliferation in diabetic conditioned media were increased in response to hypoxic stimuli (p < 0.05), and it was similar to those of control cells. In an animal study, diabetic and normal ASCs significantly increased flap survival (p < 0.05); however, the functional difference was not found between the two groups. Diabetic ASCs were impaired in their ability to produce vascular endothelial growth factors and to induce cellular proliferation under hypoxic conditions. However, diabetic ASCs showed similar flap salvaging effect compared with controls. These findings may be important in the context of future study of autologous cell‐based therapy in diabetic patients.     

Superior effect of combination vs. single steroid therapy in keloid disease: A comparative in vitro analysis of glucocorticoids

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids—dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol)—on primary keloid fibroblasts (KFs) (n = 27) and normal skin (n = 19) fibroblasts at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p < 0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid‐associated fibrotic markers were significantly (p < 0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial growth factor expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p < 0.04) apoptosis, whereas Dex inhibited the UV‐induced apoptosis and up‐regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p < 0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well‐known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD.     

Alteration of testicular regulatory and functional molecules following long‐time exposure to 900 MHz RFW emitted from BTS

The aim of this investigation was to evaluate changes in testosterone and some of the functional and regulatory molecules of testis such as P450scc, steroidogenic acute regulatory protein (StAR), tumour necrosis factor‐α (TNF‐α), interleukin‐1α (IL‐1α), interleukin‐1β (IL‐1β) and nerve growth factor (NGF) following exposure to 900 MHz radio frequency (RF). Thirty adult male Sprague Dawley rats (190 ± 20 g BW) were randomly classified in three equal groups, control (sham, without any exposure), short‐time exposure (2 hr) (STE) and long‐time exposure (4 hr) (LTE). The exposure was performed for 30 consecutive days. The testosterone level in both exposed groups was significantly less than control (p < .05). Level of TNF‐α in both exposed groups was significantly greater than control (p < .05). IL‐1α and NGF levels in LTE were significantly higher than the STE and control groups (p < .05). Level of IL‐1β in LTE was significantly higher than control (p < .05). Expression of both P450scc and StAR mRNA was significantly down‐regulated in both exposed groups compared to control (p < .05). Our results showed that RFW can affect testis and reproductive function through changes in factors, which are important during steroidogenesis, and also through changes in inflammatory factors, which regulate Leydig cell functions.     

The protective role of melatonin and curcumin in the testis of young and aged rats

We aimed to investigate the effect of melatonin and curcumin treatment on oxidative stress, apoptosis, and histology of testicular tissue in our study. Four groups were formed using young (4 months old, n = 6) and aged (20–22 months old, n = 18) male Wistar albino rats: (a) Young control (1% ethanol:phosphate‐buffered saline [PBS], subcutaneously [s.c.]); (b) Aged control (CTL; n = 6, 1% ethanol:PBS, s.c.); (c) Aged Melatonin (MLT; n = 6, 10 mg/kg, s.c.); (d) Aged Curcumin (CUR; n = 6, 30 mg/kg, i.p.). At the end of 21 days, the rats were sacrificed, and testicular tissues were removed. Malondialdehyde (MDA) in the testicular tissue was determined with thiobarbituric acid reactive substances formation, and glutathione (GSH) was determined with modified Ellman method; testosterone level was determined with chemiluminescence method and histologic changes were determined with Haematoxylin‐Eosin and Johnsen's scoring; Apoptotic cell counts were made with TUNEL staining of seminiferous tubule in testis. With ageing, MDA level increased in testicular tissue, but GSH and blood testosterone levels decreased. Melatonin treatment for aged rats significantly decreased Paired total testicular/body weight ratio compared to aged control group (p < 0.05). Curcumin treatment for aged rats significantly increased GSH level compared to the aged control group (p < 0.05). Besides, melatonin and curcumin treatment significantly decreased the number of apoptotic cells and significantly increased Johnsen's score (p < 0.05).     

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading

Osteocytes’ mechano‐regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well‐established MLO‐Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross‐talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin‐expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano‐sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ‐B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate‐resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts’ ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large‐sized TRAP‐positive osteoclasts when osteoclast precursors were treated with CM from flow‐stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1681–1689, 2019     

Selective intra-graft apoptosis and down-regulation of lymphocyte bcl-2, iNOs and CD95L expression in kidney–pancreas transplanted patients after anti-Thymoglobulin induction

Intra-graft infiltrating cells apoptosis was evaluated in 20 consecutive kidney–pancreas transplanted (KP) patients without kidney rejection. Two fine-needle aspirated biopsy (FNAB) and two peripheral blood lymphocytes (PBL) samples were obtained 14 days after transplantation. Immunosuppression was based on anti-Thymoglobulins (ATG) induction for 7 days and cyclosporine/mofetil mycophenolate as maintenance therapy. Ten matched healthy subjects were chosen as controls for PBL. Lymphocyte phenotypes and activation markers, apoptotic rate and lymphocyte expression of pro/anti-apoptotic molecules were analysed by flow cytometry analysis (FACS). Lymphocyte phenotypes and activation markers: higher levels of CD8 and CD4DR were evident in the graft (p<0.05) than in PBL, CD3CD25 in PBL were higher in transplanted patients than in controls. Apoptotic rate and lymphocyte expression of pro- and anti-apoptotic molecules: a higher expression of annexin V, together with reduced lymphocytes CD95L, iNOs and Bcl-2 expression (PBL=97.7±1.1% vs FNAB=81.9±15.1%; p<0.05) were evident in the graft than in PBL. In KP patients intra-graft apoptosis and reduced anti-apoptotic molecules were evident after ATG induction.