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1.
J Invest Dermatol ; 141(1): 36-47, 2021 01.
Article in English | MEDLINE | ID: mdl-32504614

ABSTRACT

Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD because it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion proteins, and basement membrane proteins. Minimally expressed in normal skin, GzmB is elevated in AD and is positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- mice with wild-type mice, showing significant reductions in inflammation, epidermal thickness, and lesion formation in GzmB-/- mice. Topical administration of a small-molecule GzmB inhibitor reduced disease severity compared with vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and FLG cleavage. GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.


Subject(s)
Cadherins/metabolism , Dermatitis, Atopic/metabolism , Granzymes/metabolism , Oxazolone/adverse effects , S100 Proteins/metabolism , Animals , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/pathology , Epidermis/metabolism , Extracellular Matrix/metabolism , Filaggrin Proteins , Humans
2.
Sci Rep ; 9(1): 10027, 2019 07 11.
Article in English | MEDLINE | ID: mdl-31296909

ABSTRACT

Wildfire smoke induces acute pulmonary distress and is of particular concern to risk groups such as the sick and elderly. Wood smoke (WS) contains many of the same toxic compounds as those found in cigarette smoke (CS) including polycyclic aromatic hydrocarbons, carbon monoxide, and free radicals. CS is a well-established risk factor for respiratory diseases such as asthma and COPD. Limited studies investigating the biological effects of WS on the airway epithelium have been performed. Using a cell culture-based model, we assessed the effects of a WS-infused solution on alveolar epithelial barrier function, cell migration, and survival. The average geometric mean of particles in the WS was 178 nm. GC/MS analysis of the WS solution identified phenolic and cellulosic compounds. WS exposure resulted in a significant reduction in barrier function, which peaked after 24 hours of continuous exposure. The junctional protein E-cadherin showed a prominent reduction in response to increasing concentrations of WS. Furthermore, WS significantly repressed cell migration following injury to the cell monolayer. There was no difference in cell viability following WS exposure. Mechanistically, WS exposure induced activation of the p44/42, but not p38, MAPK signaling pathway, and inhibition of p44/42 phosphorylation prevented the disruption of barrier function and loss of E-cadherin staining. Thus, WS may contribute to the breakdown of alveolar structure and function through a p44/42 MAPK-dependent pathway and may lead to the development and/or exacerbation of respiratory pathologies with chronic exposure.


Subject(s)
Alveolar Epithelial Cells/pathology , Epithelium/physiopathology , MAP Kinase Signaling System/drug effects , Smoke/adverse effects , Tight Junctions/pathology , A549 Cells , Alveolar Epithelial Cells/drug effects , Cadherins/metabolism , Cell Line , Cell Movement/drug effects , Cell Survival/drug effects , Humans , Lung Diseases/chemically induced , Tight Junctions/drug effects , Wildfires
3.
Sci Rep ; 7(1): 15857, 2017 Nov 20.
Article in English | MEDLINE | ID: mdl-29158532

ABSTRACT

Decorin (DCN) is a small-leucine rich proteoglycan that mediates collagen fibrillogenesis, organization, and tensile strength. Adventitial DCN is reduced in abdominal aortic aneurysm (AAA) resulting in vessel wall instability thereby predisposing the vessel to rupture. Recombinant DCN fusion protein CAR-DCN was engineered with an extended C-terminus comprised of CAR homing peptide that recognizes inflamed blood vessels and penetrates deep into the vessel wall. In the present study, the role of systemically-administered CAR-DCN in AAA progression and rupture was assessed in a murine model. Apolipoprotein E knockout (ApoE-KO) mice were infused with angiotensin II (AngII) for 28 days to induce AAA formation. CAR-DCN or vehicle was administrated systemically until day 15. Mortality due to AAA rupture was significantly reduced in CAR-DCN-treated mice compared to controls. Although the prevalence of AAA was similar between vehicle and CAR-DCN groups, the severity of AAA in the CAR-DCN group was significantly reduced. Histological analysis revealed that CAR-DCN treatment significantly increased DCN and collagen levels within the aortic wall as compared to vehicle controls. Taken together, these results suggest that CAR-DCN treatment attenuates the formation and rupture of Ang II-induced AAA in mice by reinforcing the aortic wall.


Subject(s)
Aortic Aneurysm, Abdominal/genetics , Apolipoproteins E/genetics , Decorin/genetics , Recombinant Proteins/genetics , Angiotensin II/genetics , Animals , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/physiopathology , Disease Models, Animal , Humans , Mice , Mice, Knockout , Recombinant Proteins/administration & dosage
4.
Oncotarget ; 7(17): 24284-302, 2016 Apr 26.
Article in English | MEDLINE | ID: mdl-27015368

ABSTRACT

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.


Subject(s)
Biomarkers, Tumor/genetics , Cell Differentiation , Hypoxia/genetics , Neuroendocrine Cells/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Retinoblastoma Protein/metabolism , Apoptosis , Cell Movement , Cell Proliferation , Gene Expression Profiling , Gene Regulatory Networks , Humans , Male , Neoplasm Invasiveness , Neuroendocrine Cells/metabolism , Prostatic Neoplasms/metabolism , Retinoblastoma Protein/genetics , Tumor Cells, Cultured
5.
Inorg Chem ; 55(1): 177-90, 2016 Jan 04.
Article in English | MEDLINE | ID: mdl-26652771

ABSTRACT

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.


Subject(s)
Dimethyl Sulfoxide/analogs & derivatives , Ferrous Compounds/chemistry , Neoplasm Metastasis/prevention & control , Organometallic Compounds/chemistry , Pyridines/chemistry , Crystallography, X-Ray , Dimethyl Sulfoxide/chemistry , Dimethyl Sulfoxide/pharmacology , Electron Spin Resonance Spectroscopy , Metallocenes , Molecular Structure , Organometallic Compounds/pharmacology , Ruthenium Compounds
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