Denosumab compared with zoledronic acid on PFS in multiple myeloma: exploratory results of an international phase 3 study.

An exploratory end point from a recent trial in patients with newly diagnosed multiple myeloma showed that median progression-free survival (PFS) was increased by 10.7 months with denosumab vs zoledronic acid. We performed additional analyses to identify factors that may have contributed to the favorable PFS with denosumab. Ad hoc analyses were performed for patients intending to undergo autologous stem cell transplantation (ASCT; ASCT intent), not intending to undergo ASCT (ASCT no intent), and intent-to-treat according to age (<70 or ≥70 years) and baseline renal function (≤60 mL/min or >60 mL/min creatinine clearance [CrCl]). Of 1718 patients, 930 (54.1%) were in the ASCT-intent subgroup, and 788 (45.9%) were in the ASCT-no-intent subgroup. In the ASCT-intent subgroup, frontline triplet (median PFS, not estimable vs 35.7 months; hazard ratio [HR] [95% confidence interval (CI)], 0.65 [0.47-0.90]; descriptive P = .009) or bortezomib-only (median PFS, not estimable vs not estimable; HR [95% CI], 0.61 [0.39-0.95]; descriptive P = .029) induction regimens demonstrated the strongest PFS benefit favoring denosumab vs zoledronic acid. In the ASCT-no-intent subgroup, no benefit with denosumab vs zoledronic acid was observed. PFS favored denosumab vs zoledronic acid in patients with CrCl >60 mL/min and in patients <70 years old, but no difference was observed in patients with CrCl ≤60 mL/min or patients ≥70 years old. The PFS difference observed with denosumab is one of the notable benefits reported in newly diagnosed multiple myeloma and was most pronounced in patients intending to undergo ASCT and those who received proteasome inhibitor (PI)-based triplet regimens. This study was registered at www.clinicaltrials.gov as #NCT01345019.

Thrombin as important factor for cutaneous wound healing: comparison of fibrin biomatrices in vitro and in a rat excisional wound healing model.

Fibrin biomatrices have been used for many years for hemostasis and sealing and are a well-established surgical tool. The objective of the present study was to compare two commercially available fibrin biomatrices regarding the effect of their thrombin concentration on keratinocytes and wound healing in vitro and in vivo. Keratinocytes showed significant differences in adhesion, viability, and morphology in the presence of the fibrin matrices in vitro. A high thrombin concentration (800-1,200 IU/mL) caused deteriorated cell compatibility. By using a thrombin inhibitor, those differences could be reversed. In a rat excisional wound healing model, we observed more rapid wound closure and less wound severity in wounds treated with a fibrin matrix containing a lower concentration of thrombin (4 IU/mL). Furthermore, fewer new functional vessels and a lower level of vascular endothelial growth factor were measured in wounds after 7 days treated with the matrix with higher thrombin concentration. These in vivo results may be partially explained by the in vitro biocompatibility data. Additionally, results show that low thrombin biomatrices were degraded faster than the high thrombin material. Hence, we conclude that the composition of fibrin biomatrices influences keratinocytes and therefore has an impact on wound healing.

High thrombin concentrations in fibrin sealants induce apoptosis in human keratinocytes.

Over the last century many studies have been performed to assess the impact of fibrin sealant (FS) components on cells. Because of the noncovalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to further evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in three different, commercially available FS. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 to 820 IU mL(-1), but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU mL(-1)) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation (as already published by others), it also does induce NHEK apoptosis when present at high concentrations.

Self-organization phenomena in embryonic stem cell-derived embryoid bodies: axis formation and breaking of symmetry during cardiomyogenesis.

Aggregation of embryonic stem cells gives rise to embryoid bodies (EBs) which undergo developmental processes reminiscent of early eutherian embryonic development. Development of the three germ layers suggests that gastrulation takes place. In vivo, gastrulation is a highly ordered process but in EBs only few data support the hypothesis that self-organization of differentiating cells leads to morphology, reminiscent of the early gastrula. Here we demonstrate that a timely implantation-like process is a prerequisite for the breaking of the radial symmetry of suspended EBs. Attached to a surface, EBs develop a bilateral symmetry and presumptive mesodermal cells emerge between the center of the EBs and a horseshoe-shaped ridge of cells. The development of an epithelial sheet of cells on one side of the EBs allows us to define an 'anterior' and a 'posterior' end of the EBs. In the mesodermal area, first cardiomyocytes (CMCs) develop mainly next to this epithelial sheet of cells. Development of twice as many CMCs at the 'left' side of the EBs breaks the bilateral symmetry and suggests that cardiomyogenesis reflects a local or temporal asymmetry in EBs. The asymmetric appearance of CMCs but not the development of mesoderm can be disturbed by ectopic expression of the muscle-specific protein Desmin. Later, the bilateral morphology becomes blurred by an apparently chaotic differentiation of many cell types. The absence of comparable structures in aggregates of cardiovascular progenitor cells isolated from the heart demonstrates that the self-organization of cells during a gastrulation-like process is a unique feature of embryonic stem cells.

Peptide Bbeta(15-42) preserves endothelial barrier function in shock.

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bbeta15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bbeta15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bbeta15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bbeta15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bbeta15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bbeta15-42 is confirmed in Fyn(-/-) mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bbeta15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.

The fibrin-derived peptide Bbeta15-42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury.

OBJECTIVE: The fibrin-derived peptide Bbeta15-42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of Bbeta15-42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of Bbeta15-42 in several species, including humans. DESIGN: Animal study and phase I trial. SETTING: University hospital and contract research laboratories. SUBJECTS: Pigs/healthy volunteers. INTERVENTIONS: Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, Bbeta15-42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of Bbeta15-42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study. MEASUREMENTS AND MAIN RESULTS: Bbeta15-42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. Bbeta15-42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide Bbeta15-42 were comparable to those of animals, and no serious adverse effects were observed. CONCLUSIONS: Bbeta15-42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide Bbeta15-42.

Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro.

Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.