Renal cortex microperfusion evaluated by laser speckle contrast imaging in an ex vivo perfused kidney model-A proof-of-concept study.

BACKGROUND: Validated quantitative biomarkers for assessment of renal graft function during normothermic machine perfusion (NMP) conditions are lacking. The aim of this project was to quantify cortex microperfusion during ex vivo kidney perfusion using laser speckle contrast imaging (LSCI), and to evaluate the sensitivity of LSCI when measuring different levels of renal perfusion. Furthermore, we aimed to introduce LSCI measurements during NMP in differentially damaged kidneys. METHODS: Eleven porcine kidneys were nephrectomized and perfused ex vivo. Cortex microperfusion was simultaneously monitored using LSCI. First, a flow experiment examined the relationship between changes in delivered renal flow and corresponding changes in LSCI-derived cortex microperfusion. Second, renal cortical perfusion was reduced stepwise by introducing a microembolization model. Finally, LSCI was applied for measuring renal cortex microperfusion in kidneys exposed to minimal damage or 2 h warm ischemia (WI). RESULTS: Cortex microperfusion was calculated from the LSCI-obtained data. The flow experiment resulted in relatively minor changes in cortex microperfusion compared to the pump-induced changes in total renal flow. Based on stepwise injections of microspheres, we observed different levels of cortex microperfusion that correlated with administrated microsphere dosages (r2 = 0.95-0.99). We found no difference in LSCI measured cortex microperfusion between the kidneys exposed to minimal damage (renal cortex blood flow index, rcBFI = 2090-2600) and 2 h WI (rcBFI = 2189-2540). CONCLUSIONS: Based on this preliminary study, we demonstrated the feasibility of LSCI in quantifying cortex microperfusion during ex vivo perfusion. Furthermore, based on LSCI-measurements, cortical microperfusion was similar in kidneys exposed to minimal and 2 h WI.

Dosing Limitation for Intra-Renal Arterial Infusion of Mesenchymal Stromal Cells.

The immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) make MSC therapy a promising therapeutic strategy in kidney disease. A targeted MSC administration via the renal artery offers an efficient delivery method with limited spillover to other organs. Although local administration alleviates safety issues with MSCs in systemic circulation, it introduces new safety concerns in the kidneys. In a porcine model, we employed intra-renal arterial infusion of ten million allogenic adipose tissue-derived MSCs. In order to trigger any potential adverse events, a higher dose (hundred million MSCs) was also included. The kidney function was studied by magnetic resonance imaging after the MSC infusion and again at two weeks post-treatment. The kidneys were assessed by single kidney glomerular filtration rate (skGFR) measurements, histology and inflammation, and fibrosis-related gene expression. None of the measured parameters were affected immediately after the administration of ten million MSCs, but the administration of one hundred million MSCs induced severe adverse events. Renal perfusion was reduced immediately after MSC administration which coincided with the presence of microthrombi in the glomeruli and signs of an instant blood-mediated inflammatory reaction. At two weeks post-treatment, the kidneys that were treated with one hundred million MSCs showed reduced skGFR, signs of tissue inflammation, and glomerular and tubular damage. In conclusions, the intra-renal administration of ten million MSCs is well-tolerated by the porcine kidney. However, higher concentrations (one hundred million MSCs) caused severe kidney damage, implying that very high doses of intra-renally administered MSCs should be undertaken with caution.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Dynamics of circulating dendritic cells and cytokines after kidney transplantation-No effect of remote ischaemic conditioning.

Inflammation resulting from ischaemia/reperfusion injury can cause kidney graft dysfunction, increase the risk of delayed graft function and possibly reduce long-term graft survival. Remote ischaemic conditioning may protect against ischaemia/reperfusion injury and mitigate the immunological response to the graft. We investigated the immunological effects of remote ischaemic conditioning on kidney transplantation from deceased donors in the randomized CONTEXT study. Three circulating dendritic cell (DC) subtypes identified in peripheral blood from kidney transplant recipients [myeloid DCs, plasmacytoid DCs and immunoglobulin-like transcript (ILT)3+ DCs] were measured at baseline, days 1, 3 and 5 and 1 and 3 months after transplantation. We also quantified 21 cytokines at baseline, days 1 and 5 and 3 months after transplantation. Neither DC counts nor cytokine levels differed between patients receiving remote ischaemic conditioning and controls; however, several parameters exhibited dynamic and parallel alterations in the two groups over time, reflecting the immunological response to the kidney transplantation and immunosuppression.

Two-day-treatment of Activin-A leads to transient change in SV-HFO osteoblast gene expression and reduction in matrix mineralization.

Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin-A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression profile. A single 2-day treatment of Activin-A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A-treated osteoblasts responded with transient change in gene expression, in a 2-wave-fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1-2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin-A. In summary, 2-day treatment with Activin-A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes.

Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics.

BACKGROUND: Remote ischaemic conditioning (RIC) is currently being explored as a non-invasive method to attenuate ischaemia/reperfusion injuries in organs. A randomised clinical study (CONTEXT) evaluated the effects of RIC compared to non-RIC controls in human kidney transplants. METHODS: RIC was induced prior to kidney reperfusion by episodes of obstruction to arterial flow in the leg opposite the transplant using a tourniquet (4 × 5 min). Although RIC did not lead to clinical improvement of transplant outcomes, we explored whether RIC induced molecular changes through precision analysis of CONTEXT recipient plasma and kidney tissue samples by high-resolution tandem mass spectrometry (MS/MS). RESULTS: We observed an accumulation of muscle derived proteins and altered amino acid metabolism in kidney tissue proteomes, likely provoked by RIC, which was not reflected in plasma. In addition, MS/MS analysis demonstrated transient upregulation of several acute phase response proteins (SAA1, SAA2, CRP) in plasma, 1 and 5 days post-transplant in RIC and non-RIC conditions with a variable effect on the magnitude of acute inflammation. CONCLUSIONS: Together, our results indicate sub-clinical systemic and organ-localised effects of RIC.

A follistatin-based molecule increases muscle and bone mass without affecting the red blood cell count in mice.

Inhibitors of the activin receptor signaling pathway (IASPs) have become candidate therapeutics for sarcopenia and bone remodeling disorders because of their ability to increase muscle and bone mass. However, IASPs utilizing activin type IIA and IIB receptors are also potent stimulators of erythropoiesis, a feature that may restrict their usage to anemic patients because of increased risk of venous thromboembolism. Based on the endogenous TGF-ß superfamily antagonist follistatin (FST), a molecule in the IASP class, FSTΔHBS-mFc, was generated and tested in both ovariectomized and naive BALB/c and C57BL/6 mice. In ovariectomized mice, FSTΔHBS-mFc therapy dose-dependently increased cancellous bone mass up to 42% and improved bone microstructural indices. For the highest dosage of FSTΔHBS-mFc (30 mg/kg, 2 times/wk), the increase in cancellous bone mass was similar to that observed with parathyroid hormone therapy (1-34, 80 µg/kg, 5 times/wk). Musculus quadriceps femoris mass dose-dependently increased up to 21% in ovariectomized mice. In both ovariectomized and naive mice, FSTΔHBS-mFc therapy did not influence red blood cell count or hematocrit or hemoglobin levels. If the results are reproduced, a human FSTΔHBS-mFc version could be applicable in patients with musculoskeletal conditions irrespective of hematocrit status.-Lodberg, A., van der Eerden, B. C. J., Boers-Sijmons, B., Thomsen, J. S., Brüel, A., van Leeuwen, J. P. T. M., Eijken, M. A follistatin-based molecule increases muscle and bone mass without affecting the red blood cell count in mice.

Early Immunological Effects of Ischemia-Reperfusion Injury: No Modulation by Ischemic Preconditioning in a Randomised Crossover Trial in Healthy Humans.

Ischemic preconditioning (IPC) has been protective against ischemia-reperfusion injury (IRI), but the underlying mechanism is poorly understood. We examined whether IPC modulates the early inflammatory response after IRI. Nineteen healthy males participated in a randomised crossover trial with and without IPC before IRI. IPC and IRI were performed by cuff inflation on the forearm. IPC consisted of four cycles of five minutes followed by five minutes of reperfusion. IRI consisted of twenty minutes followed by 15 min of reperfusion. Blood was collected at baseline, 0 min, 85 min and 24 h after IRI. Circulating monocytes, T-cells subsets and dendritic cells together with intracellular activation markers were quantified by flow cytometry. Luminex measured a panel of inflammation-related cytokines in plasma. IRI resulted in dynamic regulations of the measured immune cells and their intracellular activation markers, however IPC did not significantly alter these patterns. Neither IRI nor the IPC protocol significantly affected the levels of inflammatory-related cytokines. In healthy volunteers, it was not possible to detect an effect of the investigated IPC-protocol on early IRI-induced inflammatory responses. This study indicates that protective effects of IPC on IRI is not explained by direct modulation of early inflammatory events.

Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli.

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

Using the Connectivity Map to discover compounds influencing human osteoblast differentiation.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. Identification of factors influencing osteoblast differentiation and bone formation is very important. Previously, we identified parbendazole to be a novel compound that stimulates osteogenic differentiation of human mesenchymal stromal cells (hMSCs), using gene expression profiling and bioinformatic analyzes, including the Connectivity Map (CMap), as an in-silico approach. The aim for this paper is to identify additional compounds affecting osteoblast differentiation using the CMap. Gene expression profiling was performed on hMSCs differentiated to osteoblasts using Illumina microarrays. Our osteoblast gene signature, the top regulated genes 6 hr after induction by dexamethasone, was uploaded into CMap (www.broadinstitute.org/cmap/). Through this approach we identified compounds with gene signatures positively correlating (withaferin-A, calcium folinate, amylocaine) or negatively correlating (salbutamol, metaraminol, diprophylline) to our osteoblast gene signature. All positively correlating compounds stimulated osteogenic differentiation, as indicated by increased mineralization compared to control treated cells. One of three negatively correlating compounds, salbutamol, inhibited dexamethasone-induced osteoblastic differentiation, while the other two had no effect. Based on gene expression data of withaferin-A and salbutamol, we identified HMOX1 and STC1 as being strongly differentially expressed . shRNA knockdown of HMOX1 or STC1 in hMSCs inhibited osteoblast differentiation. These results confirm that the CMap is a powerful approach to identify positively compounds that stimulate osteogenesis of hMSCs, and through this approach we can identify genes that play an important role in osteoblast differentiation and could be targets for novel bone anabolic therapies.

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.

Activin A suppresses osteoblast mineralization capacity by altering extracellular matrix (ECM) composition and impairing matrix vesicle (MV) production.

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-ß (TGF-ß) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

Calcifying vascular smooth muscle cells and osteoblasts: independent cell types exhibiting extracellular matrix and biomineralization-related mimicries.

BACKGROUND: Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are largely unknown hampering the development of therapies to tackle this life threatening medical condition. RESULTS: In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype. CONCLUSIONS: The analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa.

Protein kinase C-induced activin A switches adrenocortical steroidogenesis to aldosterone by suppressing CYP17A1 expression.

Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin ßA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin ßA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Does the route matter? A preclinical review of mesenchymal stromal cell delivery to the kidney.

Mesenchymal stromal/stem cell (MSC) therapy has been thoroughly tested in preclinical animal models and holds great promise for the treatment of kidney diseases. It is becoming increasingly evident that the efficacy of MSC therapy is dependent on several factors including dosage, the tissue source of MSCs, the route of delivery and timing of administration. In a time where MSC therapy is moving from preclinical research to clinically therapeutic use, the importance of choice of delivery method, modality, and administration route increases. In this review, we provide an overview of the different MSC delivery routes used in preclinical kidney disease models, highlight the recent advances in the field, and summarize studies comparing delivery routes of MSCs to the kidney.

Reconstitution and post-thaw storage of cryopreserved human mesenchymal stromal cells: Pitfalls and optimizations for clinically compatible formulants.

Introduction: The regenerative and immunomodulatory properties of multipotent mesenchymal stromal cells (MSCs) make them an intriguing asset for therapeutic applications. An off-the-shelf approach, using pre-expanded cryopreserved allogenic MSCs, bypasses many practical difficulties of cellular therapy. Reconstitution of a MSC product away from cytotoxic cryoprotectants towards a preferred administration solution might be favorable for several indications. Variations in MSC handling accompanied by a non-standardized use of reconstitution solutions complicate a general clinical standardization of MSC cellular therapies. In this study, we aimed to identify a simple and clinically compatible approach for thawing, reconstitution, and post-thaw storage of cryopreserved MSCs. Methods: Human adipose tissue-derived MSCs were expanded in human platelet lysate (hPL) supplemented culture medium and cryopreserved using a dimethyl sulfoxide (DMSO)-based cryoprotectant. Isotonic solutions (saline, Ringer's acetate and phosphate buffered saline (PBS)) with or without 2% human serum albumin (HSA) were used as thawing, reconstitution, and storage solutions. MSCs were reconstituted to 5 × 106 MSCs/mL for evaluating MSC stability. Total MSC numbers and viability were determined using 7-aminoactinomycin D (7-AAD) and flow cytometry. Results: For thawing cryopreserved MSCs the presence of protein was proven to be essential. Up to 50% of MSCs were lost when protein-free thawing solutions were used. Reconstitution and post-thaw storage of MSCs in culture medium and widely used PBS demonstrated poor MSC stability (>40% cell loss) and viability (<80%) after 1 h of storage at room temperature. Reconstitution in simple isotonic saline appeared to be a good alternative for post-thaw storage, ensuring >90% viability with no observed cell loss for at least 4 h. Reconstitution of MSCs to low concentrations was identified as critical. Diluting MSCs to <105/mL in protein-free vehicles resulted in instant cell loss (>40% cell loss) and lower viability (<80%). Addition of clinical grade HSA could prevent cell loss during thawing and dilution. Conclusion: This study identified a clinically compatible method for MSC thawing and reconstitution that ensures high MSC yield, viability, and stability. The strength of the method lies within the simplicity of implementation which offers an accessible way to streamline MSC therapies across different laboratories and clinical trials, improving standardization in this field.

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/- H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice.

Bone is the major store for Ca(2+) in the body and plays an important role in Ca(2+) homeostasis. During bone formation and resorption Ca(2+) must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca(2+) transport machinery in these bone cells. In this study, we examined the epithelial Ca(2+) channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca(2+) transport genes, calbindin-D(9k) and/or -D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+) ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6(D541A/D541A) mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca(2+) transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice.

Opposing actions of rosiglitazone and resveratrol on mineralization in human vascular smooth muscle cells.

Arteriosclerotic vascular disease is a major cardiac health problem in westernized countries and the primary cause of mortality in diabetic patients. Recent data have raised serious safety concerns with the antidiabetic rosiglitazone, a thiazolidinedione with peroxisome proliferator-activated receptor γ (PPAR-γ) agonistic activity, in regard to cardiovascular risks. A common feature of atherosclerosis is vascular mineralization. The latter is formed by vascular smooth muscle cells (VSMC) through complex processes that are similar to mineralization in bone. The aim of the current study was to investigate the effect of rosiglitazone on mineralization in cultured human VSMCs. We found that rosiglitazone stimulated mineralization by, at least in part, induction of caspase-dependent apoptosis. Furthermore, rosiglitazone-induced oxidative stress was correlated with stimulated osteoblast-like differentiation of VSMCs. Treatment of rosiglitazone-supplemented VSMC cultures with the caloric restriction mimetic and antioxidant resveratrol diminished rosiglitazone-induced oxidative stress, osteoblast-like differentiation and mineralization. In conclusion, this study reveals novel insights into the relationship of rosiglitazone and cardiovascular events by providing a model that links rosiglitazone-induced osteoblast-like differentiation, oxidative stress and apoptosis with mineralization in VSMCs. In addition, we position resveratrol in this model acting to reduce rosiglitazone-induced oxidative stress, osteoblast-like VSMC differentiation and mineralization.

Unraveling the human bone microenvironment beyond the classical extracellular matrix proteins: a human bone protein library.

A characteristic feature of bone, differentiating it from other connective tissues, is the mineralized extracellular matrix (ECM). Mineral accounts for the majority of the bone tissue volume, being the remainder organic material mostly derived from collagen. This, and the fact that only a limited number of noncollagenous ECM proteins are described, provides a limited view of the bone tissue composition and bone metabolism, the more so considering the increasing understanding of ECM significance for cellular form and function. For this reason, we set out to analyze and extensively characterize the human bone proteome using large-scale mass spectrometry-based methods. Bone samples of four individuals were analyzed identifying 3038 unique proteins. A total of 1213 of these were present in at least 3 out of 4 bone samples. For quantification purposes, we were limited to noncollagenous proteins (NCPs) and we could quantify 1051 NCPs. Most classical bone matrix proteins mentioned in literature were detected but were not among the highly abundant ones. Gene ontology analyses identified high-abundance groups of proteins with a functional link to mineralization and mineral metabolism such as transporters, pyrophosphatase activity, and Ca(2+)-dependent phospholipid binding proteins. ECM proteins were as well overrepresented together with nucleosome and antioxidant activity proteins, which have not been extensively characterized as being important for bone. In conclusion, our data clearly demonstrates that human bone tissue is a reservoir of a wide variety of proteins. In addition to the classical osteoblast-derived ECM, we have identified many proteins from different sources and of unknown function in bone. Thus, this study represents an informative library of bone proteins forming a source for novel bone formation modulators as well as biomarkers for bone diseases such as osteoporosis.