Igor Erjavec

The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing

PurposeThe purpose of this study was to revise the clinical use of commercial BMP2 (Infuse) and BMP7 (Osigraft) based bone devices and explore the mechanism of action and efficacy of low BMP6 doses in a novel whole blood biocompatible device OSTEOGROW.MethodsComplications from the clinical use of BMP2 and BMP7 have been systemically reviewed in light of their role in bone remodeling. BMP6 function has been assessed in Bmp6-/- mice by μCT and skeletal histology, and has also been examined in mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and osteoclasts. Safety and efficacy of OSTEOGROW have been assessed in rats and rabbits.ResultsClinical use issues of BMP2 and BMP7 have been ascribed to the limited understanding of their role in bone remodeling at the time of device development for clinical trials. BMP2 and BMP7 in bone devices significantly promote bone resorption leading to osteolysis at the endosteal surfaces, while in parallel stimulating exuberant bone formation in surrounding tissues. Unbound BMP2 and BMP7 in bone devices precipitate on the bovine collagen and cause inflammation and swelling. OSTEOGROW required small amounts of BMP6, applied in a biocompatible blood coagulum carrier, for stimulating differentiation of MSCs and accelerated healing of critical size bone defects in animals, without bone resorption and inflammation. BMP6 decreased the number of osteoclasts derived from HSC, while BMP2 and BMP7 increased their number.ConclusionsCurrent issues and challenges with commercial bone devices may be resolved by using novel BMP6 biocompatible device OSTEOGROW, which will be clinically tested in metaphyseal bone fractures, compartments where BMP2 and BMP7 have not been effective.

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E(1) osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair.

Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography-mass spectrometry, we identified BMP6, GDF15, and the BMP1-3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1-3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1-3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1-3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFβ1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGFβ1, β-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin β1 subunit. The profibrotic effect of BMP1-3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1-3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD.

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

Aim To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Methods Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Results Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. Conclusion MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema.

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.

Urodilatin reverses the detrimental influence of bradykinin in acute ischemic stroke

Occlusion of cerebral arteries leads to ischemic stroke accompanied by subsequent brain edema. Bradykinin (BK) is involved in the formation of cerebral edema, and natriuretic peptides (NPs) potentially have beneficial effects on brain edema formation via a still unknown mechanism. The aim of this study was clarifying the mechanisms of action of NPs on BK signaling, and their interactive effects after ischemic brain injury. We used a mouse model for stroke, the middle cerebral artery (MCA) occlusion. Brain lesion and edema were measured by microcomputerized tomography volumetric measurements. To determine the effects of NPs on the BK signaling pathway in the MCAs we measured changes in vessel diameter and membrane potentials in endothelial cells. To determine the effects of NPs on BK signaling pathway in isolated astrocytes and neurons, membrane potentials and intercellular Ca2+ concentrations were measured. Urodilatin inhibited and when applied together with BK, reduced the formation of the ischemic lesion via activation of G-Protein-Signaling Protein Type 4 at the cellular (atrocities, neurons) and blood vessel (endothelial cells and isolated MCA) level as well as in in vivo experiments. The results of this study show the existence of a natural antagonist of BK in the brain, and the possible use of NPs in the treatment of stroke.

Soluble type III TGFβ receptor in diagnosis and follow-up of patients with breast cancer.

Abstract Type III transforming growth factor (TGFβ) receptor (TGFβrIII) modulates TGFβ superfamily signaling. Its tumor tissue expression is downregulated in human breast cancer. We determined (indirect ELISA) plasma levels of the soluble receptor (sTGFβrIII) in 47 women with breast cancer (AJCC stages 0–IIB) (cases) pre-surgery and over two months after the surgery, and in 36 healthy women (controls). Plasma sTBFβrIII was lower in cases than in the controls (age-adjusted difference −29.7 ng/mL, p < 0.001), and discriminated between disease and health (sensitivity and specificity 100% at 16.6 ng/mL). With adjustment for age, AJCC stage, lymph node involvement, HER2 and hormone receptor status, higher pre-surgery sTBFβrIII was associated with better progression-free survival (HR = 0.68, 95%CI 0.49–0.89, p = 0.004). An increasing trend in plasma sTBFβrIII was observed over 2 months after the surgery (0.6% increase/day, p < 0.001), consistently across the patient subsets. Data suggest a high potential of plasma sTBFβrIII as a novel diagnostic and prognostic biomarker in breast cancer.

Osteogrow: A Novel Bone Graft Substitute for Orthopedic Reconstruction

Complications associated with the clinical use of BMP2 and BMP7 result from the limited understanding of their molecular mechanisms in bone remodeling. Recently, a novel BMP6-based approach has been developed with superior healing results and reduced side effects in preclinical studies. BMP6-containing osteogenic medicinal product called Osteogrow, which is aimed to induce and accelerate bone formation, is currently being tested in clinical studies. It comprises of a biologically compatible autologous carrier made from the patient’s peripheral blood and of rhBMP6 as an active ingredient. Such formulation circumvents the use of animal-derived materials, significantly limits inflammatory processes common in commercial bone devices, and renders the carrier flexible and injectable ensuring the ease of use. The ongoing clinical trial results will provide a more detailed insight into the safety, tolerability, pharmacokinetics, and bone healing effects in humans and hopefully provide novel and valuable therapeutic options in the field of bone regeneration.

Systemic inhibition of BMP1-3 decreases progression of CCl4-induced liver fibrosis in rats

Abstract Liver fibrosis is a progressive pathological process resulting in an accumulation of excess extracellular matrix proteins. We discovered that bone morphogenetic protein 1-3 (BMP1-3), an isoform of the metalloproteinase Bmp1 gene, circulates in the plasma of healthy volunteers and its neutralization decreases the progression of chronic kidney disease in 5/6 nephrectomized rats. Here, we investigated the potential role of BMP1-3 in a chronic liver disease. Rats with carbon tetrachloride (CCl4)-induced liver fibrosis were treated with monoclonal anti-BMP1-3 antibodies. Treatment with anti-BMP1-3 antibodies dose-dependently lowered the amount of collagen type I, downregulated the expression of Tgfb1, Itgb6, Col1a1, and Acta2 and upregulated the expression of Ctgf, Itgb1, and Dcn. Mehanistically, BMP1-3 inhibition decreased the plasma levels of transforming growth factor beta 1(TGFβ1) by prevention of its activation and lowered the prodecorin production further suppressing the TGFβ1 profibrotic effect. Our results suggest that BMP1-3 inhibitors have significant potential for decreasing the progression of fibrosis in liver cirrhosis.

The effect of natriuretic peptides and bradykinin on development of brain oedema after ischemic stroke

Background Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain oedema formation, contributes to further deterioration of the already impaired brain function. Bradykinin (BK), which levels increase during ischemic stroke, promotes blood– brain barrier permeability and raises intracranial capillary blood pressure, leading to brain oedema formation. Furthermore, BK induces glutamate release from neurons and astrocytes via activation of BK receptor type 2. suggesting involvement of BK in glutamate neurotoxicity. It has been recently shown that humans without functional natriuretic peptides (NPs) suffer from massive stokes [1,2]. NPs can reduce brain oedema and have a neuroprotective role in acute ischemic stroke as well as during recovery after stroke. Although mechanisms are still not clear, it appears that NPs enhance angiogenesis, neurogenesis and oligodenrogenesis [3,4]. One of the possible beneficiary effects of NPs during the stroke could be an inhibition of BK pathological function. Materials and methods Aim of our study is to determine beneficial effects of the NPs in stroke development in murine model (middle cerebral artery occlusion – MCAO). The symptoms of the stroke are determined by behavioural studies. The sizes of the lesion and brain oedema are established by μCT. Furthermore, we determined the effects of NPs on the BK signalling pathway in primary culture of neurons and astrocytes using whole cell patch clamp experiments to measure membrane potential and measurements of intracellular Ca 2+ concentration.