Mesenchymal stem cells modulate IL-17 and IL-9 production induced by Th17-inducing cytokine conditions in autoimmune arthritis: an explorative analysis.

BACKGROUND: The importance of proinflammatory T-cells and their cytokine production in patients with autoimmune arthritis has been widely described. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have come into focus as a potential therapeutic concept. The aim of this study was to investigate the influence of MSCs on the phenotype, cytokine profile, and functionality of naive and non-naive CD4+ T-cells from healthy donors (HD) and patients with autoimmune arthritis under Th17-cytokine polarizing conditions in an explorative way using a transwell system prohibiting any cell-cell-contact. METHODS: Magnetically isolated naive and non-naive CD4+ T-cells were stimulated under Th17-polarizing proinflammatory cytokine conditions in presence and absence of bone marrow derived mesenchymal stromal cells (MSCs). After an incubation period of 6 days, the proportions of the T-cell subpopulations TEMRA (CD45RA+CD27-), memory (CD45RA-CD27+), effector (CD45RA-CD27-) and naive cells (CD45RA+CD27+) were determined. Quantitative immunofluorescence intensity was used as a measure for IL-9, IL-17 and IFN-γ production in each subpopulation. RESULTS: In isolated naive CD4+ T-cells from HD and patients, MSCs suppressed the differentiation of naive towards an effector phenotype while memory and naive cells showed higher percentages in culture with MSCs. In patients, MSCs significantly decreased the proportion of IL-9 and IL-17 producing effector T-cells. MSCs also reduced IFN-γ production in the naive and memory phenotype from HD. CONCLUSION: The results of the study indicate significant immunomodulatory properties of MSCs, as under Th17-polarizing conditions MSCs are still able to control T-cell differentiation and proinflammatory cytokine production in both HD and patients with autoimmune arthritis.

Mesenchymal stem cells modulate IL-17 and IL-9 production induced by Th17-inducing cytokine conditions in autoimmune arthritis: an explorative analysis

Abstract Background The importance of proinflammatory T-cells and their cytokine production in patients with autoimmune arthritis has been widely described. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have come into focus as a potential therapeutic concept. The aim of this study was to investigate the influence of MSCs on the phenotype, cytokine profile, and functionality of naive and non-naive CD4+ T-cells from healthy donors (HD) and patients with autoimmune arthritis under Th17-cytokine polarizing conditions in an explorative way using a transwell system prohibiting any cell-cell-contact. Methods Magnetically isolated naive and non-naive CD4+ T-cells were stimulated under Th17-polarizing proinflammatory cytokine conditions in presence and absence of bone marrow derived mesenchymal stromal cells (MSCs). After an incubation period of 6 days, the proportions of the T-cell subpopulations TEMRA (CD45RA+CD27−), memory (CD45RA−CD27+), effector (CD45RA−CD27−) and naive cells (CD45RA+CD27+) were determined. Quantitative immunofluorescence intensity was used as a measure for IL-9, IL-17 and IFN-γ production in each subpopulation. Results In isolated naive CD4+ T-cells from HD and patients, MSCs suppressed the differentiation of naive towards an effector phenotype while memory and naive cells showed higher percentages in culture with MSCs. In patients, MSCs significantly decreased the proportion of IL-9 and IL-17 producing effector T-cells. MSCs also reduced IFN-γ production in the naive and memory phenotype from HD. Conclusions The results of the study indicate significant immunomodulatory properties of MSCs, as under Th17-polarizing conditions MSCs are still able to control T-cell differentiation and proinflammatory cytokine production in both HD and patients with autoimmune arthritis.

CYR61 improves muscle force recreation in a rabbit trauma model.

BACKGROUND: Critically elevated compartment pressures after complicated tibial fractures may result in fibrosis and therefore scarring of muscles with impaired function. Several studies have shown a relationship between angiogenesis and more effective muscle regeneration. Cysteine-rich angiogenic inducer 61 (CYR61) is associated with angiogenesis but it is not clear whether it would restore muscle force, reduce scarring or improve angiogenesis after acute musculoskeletal trauma. OBJECTIVE: We researched whether local application of CYR61 (1) restores muscle force, (2) reduces scar tissue formation, and (3) improves angiogenesis. METHODS: We generated acute soft tissue trauma with temporary ischemia and increased compartment pressure in 22 rabbits and shortened the limbs to simulate surgical fracture debridement. In the test group, a CYR61-coated collagen matrix was applied locally around the osteotomy site. After 10 days of limb shortening, gradual distraction of 0.5 mm per 12 hours was performed to restore the original length. Muscle force was measured before trauma and on every fifth day after trauma. Forty days after trauma we euthanized the animals and histologically determined the percentage of connective and muscle tissue. Immunohistology was performed to analyze angiogenesis. RESULTS: Recovery of preinjury muscle strength was significantly greater in the CYR61 group (2.8 N; 88%) as compared to the control (1.8 N; 53%) with a moderate reduction of connective tissue (9.9% vs. 8.5%). Immunohistochemical staining showed that blood vessel formation increased significantly (trauma vs. control 38.75 ± 27.45 mm2 vs. 24.16 ± 19.81 mm2). CONCLUSIONS: Local application of CYR61 may improve restoration of muscle force and accelerate muscle force recovery by improving angiogenesis and moderately reducing connective tissue.

Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to the very early phase of regeneration.

Multipotent adult stem cells/precursor cells, especially of the mesenchymal and endothelial lineage, may have great potential for bone tissue engineering. Although their potential is highly recognized, not much is known about the underlying molecular mechanisms that initiate the regeneration process, connect osteogenesis, and angiogenesis and, finally, orchestrate renewal of bone tissue. Our study addressed these questions by generating two in vitro cell culture models to examine the changes in the global gene expression patterns of endothelial precursor cells and mesenchymal stem cells after 24 hours of either humoral (conditioned medium) or direct cell-cell interaction (co-culture). Endothelial precursor cells were isolated from human buffy coat and mesenchymal stem cells from the bone marrow of the femoral head. The comparison of the treated and control cells by microarray analyses revealed in total more than 1500 regulated genes, which were analyzed for their affiliation to angiogenesis and osteogenesis. Expression array analyses at the RNA and protein level revealed data with respect to regulated genes, pathways and targets that may represent a valid basis for further dissection of the systems biology of regeneration processes. It may also be helpful for the reconstitution of the natural composition of a regenerative microenvironment when targeting tissue regeneration both in vitro and in situ.

Dissection of mechanoresponse elements in promoter sites of the mechanoresponsive CYR61 gene.

Mechanotransduction is important for mesenchymal regeneration and differentiation. Exaggerated high or very low impact yields pathological outcome resulting in fracture or tissue atrophy. Pathological strain in animal models was described but tools to dissect the respective stimuli and downstream pathways are limited. We expand the analytical tools to describe DNA strain response elements in a reporter gene approach. Deletion constructs of the human cysteine-rich protein 61 (CYR61) promoter were cloned into luciferase vectors and stably transfected into human telomerase-immortalised mesenchymal stem cells (hMSC-TERT). Cells were mechanically stimulated with variable frequencies, amplitudes and durations. Promoter activity was determined as well as CYR61 mRNA and protein expression. In silico promoter analysis identified putative transcription factor binding sites, one of which was a cAMP response element, verified by electrophoretic mobility shift assay. We demonstrate for the first time that the activity of promoter regions is inhibited in low, but stimulated in high frequency stimulations. We conclude that by varying conditions of mechanical strain it is possible to characterize stimulatory versus inhibitory strain on cellular levels. Our work may be helpful in future studies to dissect the molecular pathways of physiological versus pathological strain and may have implications for clinical exercise based treatment strategies.

Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263-275, 2017. © 2016 Wiley Periodicals, Inc.

Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells -Implications for myeloma bone disease.

Physical interaction of skeletal precursors with multiple myeloma cells has been shown to suppress their osteogenic potential while favoring their tumor-promoting features. Although several transcriptome analyses of myeloma patient-derived mesenchymal stem cells have displayed differences compared to their healthy counterparts, these analyses insufficiently reflect the signatures mediated by tumor cell contact, vary due to different methodologies, and lack results in lineage-committed precursors. To determine tumor cell contact-mediated changes on skeletal precursors, we performed transcriptome analyses of mesenchymal stem cells and osteogenic precursor cells cultured in contact with the myeloma cell line INA-6. Comparative analyses confirmed dysregulation of genes which code for known disease-relevant factors and additionally revealed upregulation of genes that are associated with plasma cell homing, adhesion, osteoclastogenesis, and angiogenesis. Osteoclast-derived coupling factors, a dysregulated adipogenic potential, and an imbalance in favor of anti-anabolic factors may play a role in the hampered osteoblast differentiation potential of mesenchymal stem cells. Angiopoietin-Like 4 (ANGPTL4) was selected from a list of differentially expressed genes as a myeloma cell contact-dependent target in skeletal precursor cells which warranted further functional analyses. Adhesion assays with full-length ANGPTL4-coated plates revealed a potential role of this protein in INA-6 cell attachment. This study expands knowledge of the myeloma cell contact-induced signature in the stromal compartment of myelomatous bones and thus offers potential targets that may allow detection and treatment of myeloma bone disease at an early stage.

The KISS1 Receptor as an In Vivo Microenvironment Imaging Biomarker of Multiple Myeloma Bone Disease.

Multiple myeloma is one of the most common hematological diseases and is characterized by an aberrant proliferation of plasma cells within the bone marrow. As a result of crosstalk between cancer cells and the bone microenvironment, bone homeostasis is disrupted leading to osteolytic lesions and poor prognosis. Current diagnostic strategies for myeloma typically rely on detection of excess monoclonal immunoglobulins or light chains in the urine or serum. However, these strategies fail to localize the sites of malignancies. In this study we sought to identify novel biomarkers of myeloma bone disease which could target the malignant cells and/or the surrounding cells of the tumor microenvironment. From these studies, the KISS1 receptor (KISS1R), a G-protein-coupled receptor known to play a role in the regulation of endocrine functions, was identified as a target gene that was upregulated on mesenchymal stem cells (MSCs) and osteoprogenitor cells (OPCs) when co-cultured with myeloma cells. To determine the potential of this receptor as a biomarker, in vitro and in vivo studies were performed with the KISS1R ligand, kisspeptin, conjugated with a fluorescent dye. In vitro microscopy showed binding of fluorescently-labeled kisspeptin to both myeloma cells as well as MSCs under direct co-culture conditions. Next, conjugated kisspeptin was injected into immune-competent mice containing myeloma bone lesions. Tumor-burdened limbs showed increased peak fluorescence compared to contralateral controls. These data suggest the utility of the KISS1R as a novel biomarker for multiple myeloma, capable of targeting both tumor cells and host cells of the tumor microenvironment.

Fibroblast growth factors 1 and 2 inhibit adipogenesis of human bone marrow stromal cells in 3D collagen gels.

Multipotent human bone marrow stromal cells (hBMSCs) are the common progenitors of osteoblasts and adipocytes. A shift in hBMSC differentiation in favor of adipogenesis may contribute to the bone loss and marrow fat accumulation observed in aging and osteoporosis. Hence, the identification of factors modulating marrow adipogenesis is of great therapeutic interest. Fibroblast growth factors 1 (FGF1) and 2 (FGF2) play important roles in several cellular processes including differentiation. Their role in adipogenesis is, however, still unclear given the contradictory reports found in the literature. In this work, we investigated the effect of FGF signaling on hBMSC adipogenesis in a 3D collagen gel system to mimic the natural microenvironment. We successfully established adipogenic differentiation of hBMSC embedded in type I collagen gels. We found that exogenous FGF1 and FGF2 exerted an inhibitory effect on lipid droplet accumulation and gene expression of adipogenic markers, which was abolished by pharmacological blocking of FGF receptor (FGFR) signaling. FGF treatment also affected the expression of the matrix metalloproteinase 13 (MMP13) and the tissue inhibitor of metalloproteinases 1 (TIMP1), altering the MMP/TIMP balance, which modulates collagen processing and turnover. FGF1- and FGF2-mediated inhibition of differentiation was, however, not restricted to adipogenesis since FGF1 and FGF2 treatment also resulted in the inhibition of the osteogenic differentiation in collagen gels. We conclude that FGFR signaling inhibits the in vitro adipogenic commitment of hBMSCs, downregulating core differentiation markers and altering ECM composition.

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion.

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.

WISP 1 is an important survival factor in human mesenchymal stromal cells.

WNT-induced secreted protein 1 (WISP1/CCN4), a member of the CCN protein family, acts as a downstream factor of the canonical WNT signaling pathway. Its expression is known to affect proliferation and differentiation of human mesenchymal stromal cells (hMSCs), which are fundamental for the development and maintenance of the musculoskeletal system. Whereas a dysregulated, excessive expression of WISP1 often reflects its oncogenic potential via the inhibition of apoptosis, our study emphasizes the importance of WISP1 signaling for the survival of primary human cells. We have established the efficient and specific down-regulation of endogenous WISP1 transcripts by gene silencing in hMSCs and observed cell death as a consequence of WISP1 deficiency. This was confirmed by Annexin V staining for apoptotic cells. DNA microarray analyses of WISP1 down-regulated versus control samples revealed several clusters of differentially expressed genes important for apoptosis induction such as TNF-related apoptosis-inducing ligand 1 (TRAIL) and the corresponding apoptosis-inducing receptors TRAIL-R1 and -R2. An increased expression of TRAIL and its receptors TRAIL-R1 and -R2 in WISP1-deficient hMSCs was confirmed by immunocytofluorescence. Accordingly, WISP1 deficiency is likely to cause TRAIL-induced apoptosis. This is an important novel finding, which suggests that WISP1 is indispensable for the protection of healthy hMSCs against TRAIL-induced apoptosis.

Mesenchymal stem cell contact promotes CCN1 splicing and transcription in myeloma cells.

CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.

Effects of phytoestrogens and other plant-derived compounds on mesenchymal stem cells, bone maintenance and regeneration.

Phytoestrogens and other plant-derived compounds and extracts have been developed for the treatment of menopause-related complaints and disorders, e.g. hot flushes and osteoporosis. Since estrogens have been discussed to enhance the risk for hormone-sensitive cancers, research activities try to find alternatives. Phytoestrogens like genistein and resveratrol as well as other plant-derived compounds are capable of substituting for estrogens to some extent. Their effects on mesenchymal stem cells and the tissues derived therefrom have been investigated in vitro and in preclinical settings. Besides their well-known estrogenic, i.e. mainly antiresorptive effects on bone via estrogen receptor (ER) signalling, they also directly or indirectly affect osteogenic and adipogenic pathways. As a novel mechanism, phytoestrogens and plant-derived saponins and flavonoids like kaempferol and xanthohumol have been described to reciprocally affect the osteogenic versus the adipogenic differentiation pathway. Both, ER-mediated and other pathways mediate a shift towards osteogenesis by inhibiting PPARγ and C/EBPα, the key adipogenic transcription factors (TFs), while stimulating the key osteogenic TFs Runx2 and Sp7. Besides ER signalling, the broad spectrum of molecular mechanisms supporting osteogenesis comprises the modulation of PPARγ, Wnt/ß-catenin, and Sirt1 signalling, which inversely influence the transcription or transactivation of osteogenic versus adipogenic TFs. Preventing the age- and hormone deficiency-related shift towards adipogenesis without provoking adverse estrogenic effects represents a very promising strategy for treating bone loss and other metabolic diseases beyond bone. Research on plant-derived compounds will have to be pursued in vitro as well as in preclinical studies and controlled clinical trials in humans are urgently needed. This article is part of a Special Issue entitled 'Phytoestrogens'.

Zoledronic acid and atorvastatin inhibit αvß3-mediated adhesion of breast cancer cells.

Bone metastases represent common long term complications of patients with breast cancer. Zoledronic acid, an amino-bisphosphonate and mevalonate pathway inhibitor, is an established agent for the treatment of bone metastases. Direct antitumor effects of zoledronic acid have been proposed in breast cancer. Statins are another group of mevalonate pathway inhibitors that have been repeatedly discussed for potential anti-tumor activity. In this study, we tested the hypothesis, whether these agents regulate adhesion of breast cancer cells to extracellular matrix components. Treatment of breast cancer cells with zoledronic acid and atorvastatin, significantly impaired MDA-MB-231 breast cancer cell adhesion on the αvß3 ligands gelatin and vitronectin, but had no effect on collagen type 1 (α2ß1-ligand) and fibronectin (α5ß1-ligand). Anti-adhesive effects of zoledronic acid were fully reversed by geranylgeranyl pyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Furthermore, effects of zoledronic acid and atorvastatin were mimicked by a specific inhibitor of geranylgeranylation GGTI-298. Functional (using integrin array) and quantitative (using FACS) integrin analyses on MDA-231 cells following zoledronic acid exposure revealed decreased levels of αv and αvß3 expression. In addition to its effects on integrin mediated adhesion of breast cancer cells, the presence of zoledronic acid caused pronounced morphological changes in MDA-231 cells as seen by F-actin and vinculin rearrangement. Furthermore, phosphorylation of the focal adhesion kinase was inhibited by zoledronic acid. In both cases, changes were fully reversed by GGPP. These results emphasize the role of mevalonate pathway mediated impairment of geranylgeranylation in the anti-adhesive effects of zoledronic acid in breast cancer cells.

IFN-γ and TNF-α synergize to inhibit CTGF expression in human lung endothelial cells.

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-ß, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma.

Differential regulation of blood vessel formation between standard and delayed bone healing.

Blood vessel formation is a prerequisite for bone healing. In this study, we tested the hypothesis that a delay in bone healing is associated with an altered regulation of blood vessel formation. A tibial osteotomy was performed in two groups of sheep and stabilized with either a rigid external fixator leading to standard healing or with a highly rotationally unstable one leading to delayed healing. At days 4, 7, 9, 11, 14, 21, and 42 after surgery, total RNA was extracted from the callus. Gene expressions of vWF, an endothelial cell marker, and of several molecules related to blood vessel formation were studied by qPCR. Furthermore, histology was performed on fracture hematoma and callus sections. Histologically, the first blood vessels were detected at day 7 in both groups. mRNA expression levels of vWF, Ang1, Ang2, VEGF, CYR61, FGF2, MMP2, and TIMP1 were distinctly lower in the delayed compared to the standard healing group at several time points. Based on differential expression patterns, days 7 and 21 postoperatively were revealed to be essential time points for vascularization of the ovine fracture callus. This work demonstrates for the first time a differential regulation of blood vessel formation between standard and mechanically induced delayed healing in a sheep osteotomy model.

In vitro investigation of orthopedic titanium-coated and brushite-coated surfaces using human osteoblasts in the presence of gentamycin.

Anti-infective coatings have been developed to protect the surfaces of cementless implants from bacterial colonization that is known to be a prerequisite for device-related infection. The aim of this study is to investigate the effect of brushite-coated arthroplasty surfaces on human osteoblasts and to evaluate the impact of concomitant exposure to gentamycin. We cultured human osteoblasts (hFOB 1.19) on brushite-coated and uncoated titanium alloy in the presence of gentamycin and analyzed cell function and vitality. Our results show that brushite-coated titanium alloy surfaces supported the function of osteoblasts and the expression of extracellular matrix even in the presence of highly dosed gentamycin. Brushite-coated titanium alloy surfaces supported osteogenic function, indicating that this coating could enhance implant osteointegration in vivo. Concomitant exposure to gentamycin slightly decreased osteoblastic activity in vitro, suggesting that there might also be negative effects in vivo. However, in vivo studies are necessary to validate these in vitro findings.

Cysteine-rich protein 61 and connective tissue growth factor induce deadhesion and anoikis of retinal pericytes.

Loss of retinal pericytes is one of the distinctive features of diabetic retinopathy (DR), which is characterized by retinal capillary obliteration. The matricellular proteins, cysteine-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF), are aberrantly expressed in the retinal vasculature from the early stages of DR, but their effects on retinal pericytes are unknown. We show herein that rat retinal pericytes (RRPs) exposed to advanced glycosylation-end products, an important injurious stimulus of diabetes, express increased levels of both Cyr61 and CTGF, and concomitantly undergo anoikis, a form of apoptosis by loss of cell-matrix interactions. Adenovirus-mediated expression of Cyr61 and/or CTGF conferred an anoikis-prone phenotype to rat retinal pericytes, including decreased phosphotyrosine protein levels at focal adhesion points and formation of cortical actin rings. When used as substrates for pericyte attachment and compared with other matrix proteins (e.g. type IV collagen), recombinant Cyr61 and CTGF proteins exhibited antiadhesive and apoptogenic activities. Phosphatase inhibitors reversed these effects, suggesting that Cyr61 and CTGF promote dephosphorylation events. Furthermore, Cyr61- and CTGF-induced apoptosis was mediated through the intrinsic pathway and involved the expression of genes that have been functionally grouped as p53 target genes. Expression of the matrix metalloproteinase-2 gene, a known target of p53, was increased in pericytes overexpressing either Cyr61 or CTGF. Inhibition of matrix metalloproteinase-2 had, at least in part, a protective effect against Cyr61- and CTGF-induced apoptosis. Taken together, these findings support the involvement of Cyr61 and CTGF in pericyte detachment and anoikis, implicating these proteins in the pathogenesis of DR.