Dual Inhibition of the Wnt Inhibitors DKK1 and Sclerostin Promotes Fracture Healing and Increases the Density and Strength of Uninjured Bone: An Experimental Study in Nonhuman Primates.

BACKGROUND: Fracture repair involves the reactivation of developmental signaling cascades, including Wnt signaling that stimulates bone formation and bone regeneration. Rodent data indicate that dual inhibition of the Wnt signaling antagonists sclerostin and Dickkopf-1 (DKK1) increases callus bone volume and strength while increasing bone mass systemically. METHODS: We evaluated the effects of 16 weeks of subcutaneously administered carrier solution (vehicle, VEH), anti-sclerostin antibody (Scl-Ab), anti-DKK1 antibody (DKK1-Ab), or Scl-Ab plus DKK1-Ab combination therapy (COMBO) on ulnar osteotomy healing in nonhuman primates (cynomolgus monkeys; 20 to 22 per group). RESULTS: Scl-Ab and COMBO therapy increased systemic markers of bone formation versus VEH, with COMBO leading to synergistic increases versus Scl-Ab or DKK1-Ab monotherapies. The COMBO and Scl-Ab groups showed reduced serum markers of bone resorption versus VEH. The COMBO and DKK1-Ab groups exhibited greater callus bone mineral density (BMD), torsional stiffness, and torsional rigidity versus VEH. Lumbar vertebrae from the Scl-Ab and COMBO groups showed greater BMD and bone formation rate versus VEH, and the femoral mid-diaphysis of the Scl-Ab and COMBO groups showed greater periosteal and endocortical bone formation rates versus VEH. CONCLUSIONS: DKK1-Ab increased BMD and strength at the ulnar osteotomy site, Scl-Ab increased bone formation and BMD at uninjured skeletal sites, and Scl-Ab plus DKK1-Ab combination therapy induced all of these effects, in some cases to a greater degree versus 1 or both monotherapies. These results in nonhuman primates suggest that DKK1 preferentially regulates bone healing while sclerostin preferentially regulates systemic bone mass. CLINICAL RELEVANCE: Combination therapy with antibodies against sclerostin and DKK1 may offer a promising therapeutic strategy for both fracture treatment and fracture prevention.

Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease.

Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 (PNPLA3) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While Pnpla3-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type PNPLA3 in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous PNPLA3 rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA). Limiting our search to SNP-spanning triggers, a series of chemically modified siRNA were tested in vitro for activity and selectivity toward PNPLA3 rs738409 mRNA. Conjugation of the siRNA to a triantennary N-acetylgalactosamine (GalNAc) ligand enabled in vivo screening using adeno-associated virus to overexpress human PNPLA3I148M versus human PNPLA3I148I in mouse livers. Structure-activity relationship optimization yielded potent and minor allele-specific compounds that achieved high levels of mRNA and protein knockdown of human PNPLA3I148M but not PNPLA3I148I. Testing of the minor allele-specific siRNA in PNPLA3I148M-expressing mice fed a NASH-inducing diet prevented PNPLA3I148M-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Notch1 signalling regulates endothelial proliferation and apoptosis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterised by excessive pulmonary vascular remodelling involving deregulated proliferation of cells in intima, media as well as adventitia. Pulmonary arterial endothelial cell (PAEC) hyperproliferation and survival underlies the endothelial pathobiology of the disease.The indispensable involvement of Notch1 in the arterial endothelial phenotype and angiogenesis provides intriguing prospects for its involvement in the pathogenesis of PAH.We observed an increased expression of Notch1 in lungs of idiopathic PAH (IPAH) patients and hypoxia/SU5416 (SUHx) rats compared with healthy subjects. In vitro loss- and gain-of-function studies demonstrated that Notch1 increased proliferation of human PAECs (hPAECs) via downregulation of p21 and inhibited apoptosis via Bcl-2 and Survivin. Inhibition of Notch signalling using the γ-secretase inhibitor dibenzazepine dose-dependently decreased proliferation and migration of hPAECs. Notably, Notch1 expression and transcriptional activity were increased under hypoxia in hPAECs and knockdown of Notch1 inhibited hypoxia-induced proliferation of the cells. Furthermore, in vivo treatment with a γ-secretase inhibitor (AMG2008827) significantly reduced the right ventricular systolic pressure and right heart hypertrophy in SUHx rats.Here, we conclude that Notch1 plays a critical role in PAH and Notch inhibitors may be a promising therapeutic option for PAH.

TGF-ß prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/ß-catenin pathways.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-ß induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/ß-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-ß to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-ß pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/ß-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-ß may not be achieved when extracellular phosphate levels are high. Moreover, TGF-ß prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/ß-catenin pathway.

Discovery of a calcimimetic with differential effects on parathyroid hormone and calcitonin secretion.

Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.