Chitosan Covalently Functionalized with Peptides Mapped on Vitronectin and BMP-2 for Bone Tissue Engineering

Worldwide, over 20 million patients suffer from bone disorders annually. Bone scaffolds are designed to integrate into host tissue without causing adverse reactions. Recently, chitosan, an easily available natural polymer, has been considered a suitable scaffold for bone tissue growth as it is a biocompatible, biodegradable, and non-toxic material with antimicrobial activity and osteoinductive capacity. In this work, chitosan was covalently and selectively biofunctionalized with two suitably designed bioactive synthetic peptides: a Vitronectin sequence (HVP) and a BMP-2 peptide (GBMP1a). Nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR) investigations highlighted the presence of the peptides grafted to chitosan (named Chit-HVP and Chit-GBMP1a). Chit-HVP and Chit-GBMP1a porous scaffolds promoted human osteoblasts adhesion, proliferation, calcium deposition, and gene expression of three crucial osteoblast proteins. In particular, Chit-HVP highly promoted adhesion and proliferation of osteoblasts, while Chit-GBMP1a guided cell differentiation towards osteoblastic phenotype.

Ameliorative effect of allicin on vascular calcification via inhibiting endoplasmic reticulum stress

Objectives Vascular calcification (VC) is an independent predictor for cardiovascular events and mortality. However, there are currently no effective methods to reverse or prevent it. The present study aimed to determine the ameliorative effect of allicin on VC. Methods VC model of rats was induced by high-dose vitamin D3, which was valued by Alizarin Red staining, calcium contents, and alkaline phosphatase in the aorta. Systolic blood pressure, pulse pressure, and pulse wave velocity were measured to determine aortic stiffness. Protein levels were detected by Western blot. Results Allicin treatment rescued aortic VC and stiffness. The increased protein levels of RUNX2 and BMP2, two markers of osteoblastic phenotype of vascular smooth muscle cells, in the calcified aorta were attenuated by allicin, whereas the decreased levels of calponin and SM22α induced by calcification were improved. Allicin treatment significantly attenuated the increased protein levels of GRP78, GRP94, and CHOP, which are key markers of endoplasmic reticulum stress, in the calcified aorta. The activation of PERK/eIF2α/ATF4 cascades was also prevented by allicin. Conclusions Allicin could ameliorate aortic VC and stiffness. The ameliorative effect of allicin on VC might be mediated by inhibiting PERK/eIF2α/ATF4 cascades. Our results might provide a new proof for VC treatment.

P4485The number of lipoprotein(a) kringle IV-type 2 repeats is associated with the osteogenic profile of aortic valvular interstitial cells induced by plasma from patients with calcific aortic stenosis

Background Considerable progresses have been made in the invasive treatment of calcific aortic stenosis (AS), but there is still no pharmacological treatment available because the exact mechanism leading to the initiation of valvular calcification remains unknown. An increasing number of evidences, including large-scale genetic studies, have linked Lipoprotein(a) (Lp(a)) to AS but its pathogenic role in the osteoblastic transition of valvular interstitial cells (VIC) has remained undeciphered. Objective We sought to study the mechanistic link between the transition of VICs towards an osteoblastic phenotype leading to intraleaflet calcium deposition and the type of Lp(a) isoform, defined by the number of kringle IV-type 2 (KIV 2) repeats, in the plasma of patients with AS compared with healthy controls. Methods VICs isolated from healthy aortic valves were cultured in the presence of plasma samples deriving from 100 patients with severe AS included in the prospective cohort GENERAC and 50 matched control patients exempt from any aortic valve disease. We evaluated the number of Lp(a) KIV 2 repeats of each plasma preparation by liquid chromatography-mass spectrometry. The phenotypic changes of VICs towards an osteoblastic phenotype were assessed by immunofluorescence microscopy (osteocalcin expression) and Alizarin red staining (calcium deposition). Results Incubation of VICs with the plasma of AS patients triggered their transformation towards an osteoblastic phenotype, evidenced by the production of osteocalcin, and calcium deposition. There was no association between the plasma levels of Lp(a) and the extent of calcium deposition in the study population. However, a negative and significant correlation was found between calcium deposition and the number of KIV-2 repeats in the Lp(a) of the different plasma preparations (r=−0.20, p=0.038). A direct, causal role of Lp(a) isoforms containing a low number of KIV-2 repeats (5 to 6) in the transition of VICs towards an osteoblastic phenotype was supported by experiments performed with preparations of these isoforms, isolated from the plasma of blood donors. Conclusion A low number of KIV–2 repeats in plasma Lp(a) triggers the acquisition of an osteoblastic phenotype by VICs. The isoform, rather than the concentration of Lp(a) may play a pathogenic role in AS. Determining the number of KIV-2 repeats in the Lp(a) of patients may allow to identify subgroups of patients with an increased risk of developing AS. Acknowledgement/Funding ANR-16-RHUS-0003_STOP-AS. PHRC National 2005 and 2010, and PHRC regional 2007.

Mechanisms of Osteoblastic Bone Metastasis in Prostate Cancer: Role of Prostatic Acid Phosphatase

Prostate cancer (PCa) preferentially metastasizes to bone, leading to complications including severe pain, fractures, spinal cord compression, bone marrow suppression, and a mortality of ∼70%. In spite of recent advances in chemo-, hormonal, and radiation therapies, bone-metastatic, castrate-resistant PCa is incurable. PCa is somewhat unique among the solid tumors in its tendency to produce osteoblastic lesions composed of hypermineralized bone with multiple layers of poorly organized type I collagen fibrils that have reduced mechanical strength. Many of the signaling pathways that control normal bone homeostasis are at play in pathologic PCa bone metastases, including the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin system. A number of PCa-derived soluble factors have been shown to induce the dysfunctional osteoblastic phenotype. However, therapies directed at these osteoblastic-stimulating proteins have yielded disappointing clinical results to date. One of the soluble factors expressed by PCa cells, particularly in bone metastases, is prostatic acid phosphatase (PAP). Human PAP is a prostate epithelium-specific secretory protein that was the first tumor marker ever described. Biologically, PAP exhibits both phosphatase activity and ecto-5′-nucleotidase activity, generating extracellular phosphate and adenosine as the final products. Accumulating evidence indicates that PAP plays a causal role in the osteoblastic phenotype and aberrant bone mineralization seen in bone-metastatic, castrate-resistant PCa. Targeting PAP may represent a therapeutic approach to improve morbidity and mortality from PCa osteoblastic bone metastases.

Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A

Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A. Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940–overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment.