Determining the Mechanism of Propofol-induced Neurotoxicity

Background and Hypothesis: Propofol is an IV anesthetic agent with many clinical applications, including general anesthesia, sedation, delirium, and palliative care. Despite its versatility and effectiveness, Propofol has been shown to have detrimental effects on the vascular interface between the brain and circulation. The Blood-Brain Barrier (BBB) acts as a selective interface that serves to protect the brain from its cerebrovascular network. BBB dysfunction can have lethal complications such as cerebral edema and stroke. Matrix Metalloproteinases (MMP’s), specifically MMP-2 and MMP-9 have been linked to BBB breakdown. Using human induced pluripotent stem cell (IPSC) derived brain microvascular endothelial cells (BMECs), we evaluated the impact of inhibiting MMP-2 on restoring BBB integrity following exposure to Propofol with the intent to reveal the mechanism by which Propofol disrupts the BBB. We hypothesized that inhibiting MMP-2 would lead to phenotype recovery after exposure to Propofol. Methods: IPSC-differentiated BMECs were treated with MMP inhibitors at varying time points and concentrations relative to exposure to Propofol for 3 hours. Trans-endothelial electrical resistance (TEER) and sodium fluorescein permeability was used to assess BBB structural integrity. A MTT assay was conducted to assess cell viability. Results: Inhibiting MMP-2 did not result in in recovery of BBB integrity following exposure to Propofol as no significant differences were observed in TEER and sodium fluorescein permeability between the Propofol control and Propofol + MMP-2 inhibitor groups. Conclusions & Future Directions: Propofol-induced disruption of BBB integrity does not appear to be through MMP-2 activity. High concentrations of MMP-inhibitor compounds result in increased disruption of BBB tightness and permeability. It is plausible to suspect that Propofol may act through other MMP’s to facilitate BBB break-down, thus future studies should investigate the effects of the other selective MMP inhibitors in their ability to achieve phenotype recovery following exposure to Propofol.

Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Expression pattern and prognostic impact of glycoprotein non-metastatic B (GPNMB) in triple-negative breast cancer

Glycoprotein non-metastatic B (GPNMB) is a transmembrane protein overexpressed in numerous cancers including triple-negative breast cancers (TNBC). It has been linked to promote cancer aggressiveness and implicated as a novel target for GPNMB-expressing cancers. In current study, we aimed to explore the clinical significance of GPNMB in TNBC. Among 759 specimens, immunohistochemistry (IHC) exhibited GPNMB expressions were variable in different subtypes and significantly higher in TNBC. Kaplan–Meier analysis revealed GPNMB overexpression in TNBC was associated with worse prognosis especially distant metastasis (P = 0.020, HR = 2.515, CI 1.154–5.480). Multivariate analysis showed GPNMB expression was an independent prognostic factor in terms of recurrence and distant metastasis (P = 0.008, HR = 3.22, CI 1.36–7.61; P = 0.017, HR = 3.08, CI 1.22–7.74). In silico analysis showed high mRNA expression of GPNMB was associated with distant metastasis and GPNMB was overexpressed in TNBC. Furthermore, GPNMB positively correlated with epithelial–mesenchymal transition (EMT) regulators, mesenchymal marker vimentin, MMP and integrins. The protein levels of Twist and MMP2 were upregulated by GPNMB overexpression in TNBC cells. GPNMB-enhanced cell invasion was attenuated by broad spectrum MMP inhibitor (GM 6001) and the selective inhibitor of MMP-2 (ARP100). In summary, GPNMB expression is prevalent in TNBC and may be implicated as a prognostic biomarker in patients with TNBC.

Cionin, a vertebrate cholecystokinin/gastrin homolog, induces ovulation in the ascidian Ciona intestinalis type A

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.

Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

Basal-like breast cancer is characterized by an aggressive clinical outcome and presence of metastasis, for which effective therapies are unavailable. We have previously shown that chondroitin 4-O-sulfotransferase-1 (C4ST-1) controls the invasive properties of the basal-like breast cancer cell line BT-549 by inducing matrix metalloproteinase (MMP) expression through the N-cadherin/β-catenin pathway. Here we report that C4ST-1 controls the proliferation of BT-549 cells via the MMP-dependent cleavage of syndecan-1. Syndecan-1 is a membrane-bound proteoglycan associated with an aggressive phenotype and poor prognosis in breast cancer. In addition, the cleavage of syndecan-1 at a specific juxtamembrane cleavage site is implicated in the pathophysiological response in breast cancer. Knockout of C4ST-1 remarkably suppressed both the cleavage of syndecan-1 and proliferation of BT-549 cells. Kinases (AKT1, ERK1/2, PI3K, and STAT3) comprising cancer proliferative pathways are phosphorylated in C4ST-1 knockout cells at a level similar to that in parental BT-549 cells, whereas levels of phosphorylated S6 kinase and SUMOylated AKT (hyperactivated AKT observed in breast cancer) decreased in C4ST-1 knockout cells. An MMP inhibitor, GM6001, suppressed the small ubiquitin-like modifier (SUMO) modification of AKT, suggesting that cleavage of syndecan-1 by MMPs is involved in the SUMO modification of AKT. Forced expression of the cytoplasmic domain of syndecan-1, which is generated by MMP-dependent cleavage, increased the SUMO modification of AKT and global protein SUMOylation. Furthermore, syndecan-1 C-terminal domain-expressing BT-549 cells were more proliferative and sensitive to a potent SUMOylation inhibitor, tannic acid, compared with BT-549 cells transfected with an empty expression vector. These findings assign new functions to the C-terminal fragment of syndecan-1 generated by MMP-dependent proteolysis, thereby broadening our understanding of their physiological importance and implying that the therapeutic inhibition of syndecan-1 cleavage could affect the progression of basal-like breast cancer.

Cionin, A Vertebrate Cholecystokinin/Gastrin Homolog, Induces Ovulation in the Ascidian Ciona Intestinalis Type A.

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44–MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Loss of mutually protective effects between osteoclasts and chondrocytes in damaged joints drives osteoclast-mediated cartilage degradation via matrix metalloproteinases

Osteoclasts are large multinucleated cells that resorb bone to regulate bone remodelling during skeletal maintenance and development. It is overlooked that osteoclasts also digest cartilage during this process, as well as in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcomas such as giant cell tumour of bone. This study explores the poorly understood mechanisms behind the interaction between osteoclasts and cartilage. Morphologically, osteoclasts differentiated on acellular human cartilage formed multinucleated cells expressing characteristic osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR) that visibly damaged the cartilage surface by SEM, but without the formation of resorption pits. Osteoclasts caused increased glycosaminoglycan (GAG) release from acellular and cellular human cartilage that was dependent on direct contact with the substrate. Direct co-culture with chondrocytes during osteoclast differentiation increased the number of large osteoclasts formed. When osteoclasts were cultured on dentine, direct co-culture with chondrocytes inhibited osteoclast formation and reduced basal degradation of cartilage. This suggests a mutually protective effect on their ‘native’ tissue between bone-resident osteoclasts and chondrocytes, that is reversed when the joint structure breaks down and osteoclasts are in contact with non-native substrates. Mechanistically, osteoclast-mediated cartilage degradation was inhibited by the pan-MMP inhibitor GM6001 and by TIMP1, indicative of a role for soluble MMPs. RNA sequencing and RT-qPCR analysis identified MMP8 as overexpressed in osteoclasts differentiated on cartilage versus dentine, while MMP9 was the most highly expressed MMP on both substrates. Inhibition of either MMP8 or MMP9 by siRNA in mature osteoclasts reduced GAG release, confirming their involvement in cartilage degradation. Immunohistochemical expression of MMP8 and MMP9 was evident in osteoclasts in osteosarcoma tissue sections. Understanding and controlling the activity of osteoclasts might represent a new therapeutic approach for pathologies characterized by cartilage degeneration and presents an attractive target for further research.

Attenuation of pulmonary injury by an inhaled MMP inhibitor in the endotoxin lung injury model

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema and poor gas exchange resulting from severe inflammatory lung injury. Neutrophilic infiltration and increased pulmonary vascular permeability are hallmarks of early ARDS and precipitate a self-perpetuating cascade of inflammatory signaling. The biochemical processes initiating these events remain unclear. Typically associated with extracellular matrix degradation, recent data suggest matrix metalloproteinases (MMPs) are regulators of pulmonary inflammation. To demonstrate that inhalation of a broad MMP inhibitor attenuates LPS induced pulmonary inflammation. Nebulized CGS27023AM (CGS) was administered to LPS-injured mice. Pulmonary CGS levels were examined by mass spectroscopy. Inflammatory scoring of hematoxylin-eosin sections, examination of vascular integrity via lung wet/dry and bronchoalveolar lvage/serum FITC-albumin ratios were performed. Cleaved caspase-3 levels were also assessed. Differential cell counts and pulse-chase labeling were utilized to determine the effects of CGS on neutrophil migration. The effects of CGS on human neutrophil migration and viability were examined using Boyden chambers and MTT assays. Nebulization successfully delivered CGS to the lungs. Treatment decreased pulmonary inflammatory scores, edema, and apoptosis in LPS treated animals. Neutrophil chemotaxis was reduced by CGS treatment, with inhalation causing significant reductions in both the total number and newly produced bromodeoxyuridine-positive cells infiltrating the lung. Mechanistic studies on cells isolated from humans demonstrate that CGS-treated neutrophils exhibit decreased chemotaxis. The protective effect observed following treatment with a nonspecific MMP inhibitor indicates that one or more MMPs mediate the development of pulmonary edema and neutrophil infiltration in response to LPS injury. In accordance with this, inhaled MMP inhibitors warrant further study as a potential new therapeutic avenue for treatment of acute lung injury.