Prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles.

RATIONALE: Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. OBJECTIVE: Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. METHODS AND RESULTS: Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. CONCLUSIONS: Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression.

Rat aortic smooth muscle cells cultured on hydroxyapatite differentiate into osteoblast-like cells via BMP-2-SMAD-5 pathway.

Vascular calcification is an important pathological condition associated with increased risk of cardiovascular mortality. Hydroxyapatite (HA) found in such deposits is the same polymorph of calcium (Ca) found in bone, indicating calcification may involve mechanisms akin to bone formation. Vascular smooth muscle cells (Vsmcs) have been shown to undergo phenotypic change to osteoblast-like cells. However, the mechanisms underlying this phenotypic change are unclear, and whether the stimulus to become osteogenic is a result of loss of mineralization inhibitors or early mineral deposits is not known. Our aim in this study is to identify mechanisms and signal transduction pathways that cause differentiation of Vsmcs into osteoblast-like cells in the presence of HA. We first characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22α at both mRNA and protein levels. Vsmcs grown on HA exhibited progressive change in cellular morphology at 3-, 7-, and 14-day time points. Culturing of Vsmcs on HA disc resulted in decrease in media Ca levels and increased expression of Ca-sensing receptor (CaSR) on Vsmcs resulting in upregulation of intracellular CaSR signaling leading to increased BMP-2 secretion. BMP-2 pathway mediated differentiation of Vsmcs to osteoblast-like cells shown by expression of osteogenic markers like runt-related transcription factor 2, osteocalcin, and alkaline phosphatase at mRNA and protein levels. Blocking CaSR by NPS-2143 reduced BMP-2 secretion and blocking the BMP-2 pathway by LDN-193189, a BMP inhibitor, modulated expression of osteogenic markers confirming their role in osteogenesis of Vsmcs.

RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein-protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by (3) H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.

Novel cannabidiol sunscreen protects keratinocytes and melanocytes against ultraviolet B radiation.

Cannabidiol (CBD), a natural occurring phytocannabinoid, is used extensively in consumer products ranging from foods to shampoos, topical oils and lotions. Several studies demonstrated the anti-inflammatory and antioxidative properties of cannabidiol. Nevertheless, the role of cannabidiol use in sunscreens is largely unknown as no studies on its effect on keratinocytes or melanocytes exist. As such, we aimed to explore the effect of CBD on keratinocyte and melanocyte viability following ultraviolet B (UVB) irradiation. CBD exhibited a dose-dependent protective effect on both keratinocytes and melanocyte viability. Further, since CBD does not demonstrate absorption in the UVB spectra, we speculate that the protective effect is due to reduction in reactive oxygen species. To our knowledge, this is the first study demonstrating the protective effect of CBD on keratinocytes and melanocytes irradiated with UVB.

Identification of gamma-synuclein as a new PCBP1-interacting protein.

OBJECTIVES: PolyC binding protein 1 (PCBP1) is a transcriptional regulator of human mu-opioid receptor (hMOR) gene in the CNS and is also related to cancer/diseases. It possesses multi-roles that can be mediated by protein-protein interactions. To understand the mechanism controlling PCBP1 functions, PCBP1-interacting protein was investigated. METHODS: Using PCBP1 as the bait, a human brain cDNA library was screened via two-hybrid system. DNA sequence of candidate protein was confirmed using NCBI/SNP databases. Candidate protein in various cell lines was examined by RT-PCR. Glutathione-S-transferase (GST) pull-down and co-immunoprecipitation were used to validate the physical interaction. Its effects on hMOR gene regulation were examined. RESULTS: One clone was identified as gamma-synuclein110E, an SNP of gamma-synuclein110V. The interaction between PCBP1 and gamma-synuclein110E was confirmed by further validation and GST pull-down assay. Confocal analysis showed gamma-synuclein110E mainly expressing in the cytosol of human neuronal NMB cells. This interaction was confirmed by co-immunoprecipitation with NMB lysates, containing both proteins endogenously. Ectopic expression of gamma-synuclein110E or 110V did not alter hMOR mRNA level or promoter activity, suggesting no involvement of gamma-synuclein in modulating hMOR expression. Co-immunoprecipitation using gamma-synuclein110E or 110V overexpressed NMB cells with anti-PCBP1 antibody revealed a stronger intensity of co-immunoprecipitated gamma-synuclein band using gamma-synuclein110E-overexpressed cells as compared to that using gamma-synuclein110V-overexpressed cells. Synuclein110E was also identified in H292 (lung), HT29 (colon) and T47D (breast) cells, and this physical interaction was confirmed. CONCLUSION: We report a newly identified PCBP1-interacting protein, gamma-synuclein110E, and provide some insight into its complex role as well as discuss potential roles of this interaction.