Matrix Metalloprotease Activity Research Articles

In Vivo Study on Doxycycline Protective Mechanisms during Myocardial Ischemia Injury in Rats.

The fact that during myocardial ischemia/reperfusion (I/R) injury, myosin light chain 1 (MLC1) and troponin I (TnI) are degraded by matrix metalloproteases activity has already been well established in both in vitro and ex vivo studies. However, I/R injury is a complex issue based on several overlapping mechanisms. Increased activity of myosin light chain kinase and nitric oxide synthase due to oxidative stress leads to post-translational modifications of MLC1, thus leading to the increased degradation of these proteins. Wistar rats were subjected to left anterior descending coronary artery occlusion. To measure the pharmacological effect of doxycycline, transthoracic echocardiography as well as biochemical tests, concentrations of TnI, LDH, MLC1, MMP-2 and MMP-9 were performed. Gelatinize activity and cytotoxicity level were also assessed; Results: I.p., administration of doxycycline before LAD occlusion surgery increased TnI and LDH content in the heart and decreased cytotoxicity. A reduction of MMP-2 and MMP-9 concentration and MMP-2 activity after administration of Doxy was also observed, as well as improvement in echocardiographic parameters just 7 days after surgery. Inhibition of MMPs by doxycycline, in vivo, may serve as a protective agent in future therapy.

Inhibition of the histone methyltransferase EZH2 induces vascular stiffness.

Vascular stiffness increases with aging, obesity and hypertension and predicts cardiovascular risk. The levels of histone H3-lysine-27 methylation (H3K27me) and the histone methyltransferase EZH2 both decrease in aging vessels, driving vascular stiffness. The impact of EZH2 inhibitors on vascular stiffness is unknown. We tested the hypothesis that the EZH2 inhibitor GSK126, currently in development for cancer treatment, increases vascular stiffness and explored underlying molecular mechanisms. Young (3 month) and middle-aged (12 month) male mice were treated with GSK126 for 1-2 months and primary human aortic smooth muscle cells (HASMCs) from young male and female donors were treated with GSK126 for 24-48 h. Stiffness was measured in vivo by pulse wave velocity and in vitro by atomic force microscopy (AFM) and vascular structure was quantified histologically. Extracellular matrix proteins were studied by qRT-PCR, immunoblotting, zymography and chromatin immunoprecipitation. GSK126 treatment decreased H3K27 methylation (H3K27me) and increased acetylation (H3K27ac) in mouse vessels and in HASMCs. In GSK126-treated mice, aortic stiffness increased without changes in vascular fibrosis. EZH2 inhibition enhanced elastin fiber degradation and matrix metalloprotease-2 (MMP2) expression. In HASMCs, GSK126 treatment increased synthetic phenotype markers and intrinsic HASMCs stiffness by AFM with altered cytoskeletal structure and increased nuclear actin staining. GSK126 also increased MMP2 protein expression, activity and enrichment of H3K27ac at the MMP2 promoter in HASMCs. GSK126 causes vascular stiffening, inducing MMP2 activity, elastin degradation, and modulation of SMC phenotype and cytoskeletal stiffness. These findings suggest that EZH2 inhibitors used to treat cancer could negatively impact the vasculature by enhancing stiffness and merits examination in human trials.

Antisense oligonucleotide targeting hepatic Serum Amyloid A limits the progression of angiotensin II-induced abdominal aortic aneurysm formation

Background and aimsObesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. MethodsMale C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. ResultsPlasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. ConclusionsWe demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

The activity, distribution, and colocalization of cathepsin K and matrix metalloproteases in intact and eroded dentin.

This study aimed to assess the activity, distribution, and colocalization of cathepsin K (catK) and matrix metalloproteases (MMPs) in both intact and eroded dentin in vitro. Eroded dentin was obtained by consecutive treatment with 5% citric acid (pH = 2.3) for 7days, while intact dentin remained untreated. Pulverized dentin powder (1.0g) was extracted from both intact and eroded dentin using 5mL of 50mM Tris-HCl buffer (0.2g/1mL, pH = 7.4) for 60h to measure the activity of catK and MMPs spectrofluorometrically. In addition, three 200-μm-thick dentin slices were prepared from intact and eroded dentin for double-labeling immunofluorescence to evaluate the distribution and colocalization of catK and MMPs (MMP-2 and MMP-9). The distribution and colocalization of enzymes were analyzed using inverted confocal laser scanning microscopy (CLSM), with colocalization rates quantified using Leica Application Suite Advanced Fluorescent (LAS AF) software. One-way analysis of variance (ANOVA) was used to analyze the fluorescence data related to enzyme activity (α = 0.05). The activity of catK and MMPs was significantly increased in eroded dentin compared with intact dentin. After erosive attacks, catK, MMP-2, and MMP-9 were prominently localized in the eroded regions. The colocalization rates of catK with MMP-2 and MMP-9 were 13- and 26-fold higher in eroded dentin, respectively, than in intact dentin. Erosive attacks amplified the activity of catK and MMPs in dentin while also altering their distribution patterns. Colocalization between catK and MMPs increased following erosive attacks. CatK, MMP-2, and MMP-9 likely play synergistic roles in the pathophysiology of dentin erosion.

Gelatin In Situ Zymography to Study Gelatinase Activity in Colon Cancer Cells Treated with Platelet Microparticles (PMPs).

The platelet-derived microparticles (PMPs) have been connected with tumor progression and metastatic dissemination. PMPs infiltrate solid tumors and transfer platelet-derived cargo to cancer cells. The functional roles of PMPs in cancer progression are still poorly understood. PMPs, incorporated by colorectal cancer (CRC) cells, were shown to upregulate the expression and activity of matrix metalloproteases (MMPs). To investigate the impact of PMPs on the invasive potential of CRC, we established the protocol of dequenched gelatin(DG), fluorescein conjugate assay. The procedure confirms the activity of two gelatinases, namely, MMP2 and MMP9, that digest denatured collagen (gelatin). This "step-by-step" protocol, with notes and comments implemented to human CRC lines with different phenotypes and migratory potentials, should be sufficient to obtain representative and elegant results.

Regnase-1 overexpression as a therapeutic approach of Marfan syndrome

Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells invitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.

Multimodal imaging of the role of hyperglycemia following experimental subarachnoid hemorrhage

Hyperglycemia has been linked to worsening outcomes after subarachnoid hemorrhage (SAH). Nevertheless, the mechanisms involved in the pathogenesis of SAH have been scarcely evaluated so far. The role of hyperglycemia was assessed in an experimental model of SAH by T2 weighted, dynamic contrast-enhanced magnetic resonance imaging (T2W and DCE-MRI), [18F]BR-351 PET imaging and immunohistochemistry. Measures included the volume of bleeding, the extent of cerebral infarction and brain edema, blood brain barrier disruption (BBBd), neutrophil infiltration and matrix metalloprotease (MMP) activation. The neurofunctional outcome, neurodegeneration and myelinization were also investigated. The induction of hyperglycemia increased mortality, the size of the ischemic lesion, brain edema, neurodegeneration and worsened neurological outcome during the first 3 days after SAH in rats. In addition, these results show for the first time the exacerbating effect of hyperglycemia on in vivo MMP activation, Intercellular Adhesion Molecule 1 (ICAM-1) expression and neutrophil infiltration together with increased BBBd, bleeding volume and fibrinogen accumulation at days 1 and 3 after SAH. Notably, these data provide valuable insight into the detrimental effect of hyperglycemia on early BBB damage mediated by neutrophil infiltration and MMP activation that could explain the worse prognosis in SAH.

Extracellular matrix metalloproteinases inducer gene polymorphism and reduced serum matrix metalloprotease-2 activity in preeclampsia patients.

Preeclampsia increases the risk of pregnancy-related complications, nevertheless a successful spiral vessel remodeling, and trophoblast invasion reduces disorders of pregnancy. Matrix metalloproteinase-2 (MMP-2) clears the path for trophoblast invasion, and activation of MMP-2 largely depends on extracellular matrix metalloproteinases inducer (EMMPRIN) protein. This study aimed to investigate EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia patients. Archival whole blood and serum samples of 74 preeclampsia and 66 normotensive pregnant women age-matched were used in this case-control study. Genomic DNA was extracted from the whole blood samples and EMMPRIN gene amplified with specific primers following fragments sequence mutation analysis. Serum MMP-2 activity was determined using enzyme-linked immunosorbent assay (ELISA) and socio-demographic data of participants retrieved from the database. Age of preeclampsia patients (32.78 ± 6.39) years and body mass index (BMI) (33.09 ± 7.27) kg/m2 compared with the normotensive counterparts (32.33 ± 5.56) years and (32.33 ± 5.56) kg/m2,respectively, were not statistically significant (P > 0.05). Serum matrix metalloprotease-2 (MMP-2) activity was significantly reduced in preeclampsia group (16.34 ± 7.07) compared with the normotensives (25.63 ± 4.56) (P < 0.001), and rs424243T/G variant (55.6%) was overrepresented among the cases compared with the normotensives (16.7%). The single-nucleotide polymorphism T/G was found to be associated with preeclampsia (odds ratio [OR] = 7.63; 95% confidence interval [CI] = 3.95-14.75; P < 0.0001). Decreased activity of MMP-2 and rs424243T/G SNP of EMMPRIN gene was reported in preeclampsia. These preliminary data warrant a further investigation into the relationship between EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia.

How the mechanobiology orchestrates the iterative and reciprocal ECM-cell cross-talk that drives microtissue growth.

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo-grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5β1 to α2β1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Natural Killer Cell-Derived Interferon-γ Regulates Macrophage-Mediated Immunopathology During Viral Infection.

Regulation of immune responses during viral infection is critical to preventing the development immunopathology that impairs host survival. Natural killer (NK) cells are well known for their antiviral functions that promote viral clearance; however, their roles in limiting immune-mediated pathology are still unclear. Using a mouse model for genital herpes simplex virus type 2 infection, we find that NK cell-derived interferon-γ directly counteracts interleukin-6-mediated matrix metalloproteases (MMPs) activity in macrophages to limit MMP-mediated tissue damage. Our findings uncover a key immunoregulatory function of NK cells during host-pathogen interactions that highlight the potential of NK cell therapy for treatment of severe viral infections.

Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions.

Suprabasin (SBSN) is a secreted protein that is isolated as a novel gene expressed in differentiated keratinocytes in mice and humans. It induces various cellular processes such as proliferation, invasion, metastasis, migration, angiogenesis, apoptosis, therapy and immune resistance. The role of SBSN was investigated in oral squamous cell carcinoma (OSCC) under hypoxic conditions using the SAS, HSC‑3, and HSC‑4 cell lines. Hypoxia induced SBSN mRNA and protein expression in OSCC cells and normal human epidermal keratinocytes (NHEKs), and this was most prominent in SAS cells. The function of SBSN in SAS cells was analyzed using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT); 5‑bromo‑2'‑deoxyuridine (BrdU); cell cycle, caspase 3/7, invasion, migration, and tube formation assays; and gelatin zymography. Overexpression of SBSN decreased MTT activity, but the results of BrdU and cell cycle assays indicated upregulation of cell proliferation. Western blot analysis for cyclin‑related proteins indicated involvement of cyclin pathways. However, SBSN did not strongly suppress apoptosis and autophagy, as revealed by caspase 3/7 assay and western blotting for p62 and LC3. Additionally, SBSN increased cell invasion more under hypoxia than under normoxia, and this resulted from increased cell migration, not from matrix metalloprotease activity or epithelial‑mesenchymal transition. Furthermore, SBSN induced angiogenesis more strongly under hypoxia than under normoxia. Analysis using reverse transcription‑quantitative PCR showed that vascular endothelial growth factor (VEGF) mRNA was not altered by the knockdown or overexpression of SBSN VEGF, suggesting that VEGF is not located downstream of SBSN. These results demonstrated the importance of SBSN in the maintenance of survival and proliferation, invasion and angiogenesis of OSCC cells under hypoxia.

Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting.

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in ~45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.

Reversal of Neuralgia Effect of Beta Carotene in Streptozotocin-Associated Diabetic Neuropathic Pain in Female Zebrafish via Matrix Metalloprotease-13 Inhibition.

Beta carotene is a natural anti-oxidant agent, and it inhibits the matrix metalloprotease (MMP) activity. Diabetic neuropathic pain (DNP) is produced by cellular oxidative stress. The role of the beta carotene effect in diabetic neuropathic pain is not explored yet. The present study is designed for the evaluation of the palm oil mill effluent-derived beta carotene (PBC) effect in DNP in zebrafish. The DNP was induced by the intraperitoneal administration of streptozotocin (STZ). Blood glucose levels of above 15 mM were considered to be diabetic conditions. The zebrafish were exposed to test compound PBC (25, 50, and 100 µM), pregabalin (PG: 10 μM), and an MMP-13 inhibitor (CL-82198; 10 μM) for 10 consecutive days from day 11. The neuralgic behavioral parameters, i.e., temperature test, acetic acid test, and fin clip test were assessed on day 0 and the 7th, 14th, and 21st days. On the 22nd day, the blood glucose and MMP-13 levels and brain thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and MMP-13 activity levels were estimated. The treatment of PBC ameliorated the DNP-associated behavioral and biochemical changes. The results are similar to those of PG and CL-82198 treatments. Hence, the PBC possesses a potentially ameliorative effect against DNP due to its potential anti-oxidant, anti-lipid peroxidation, and MMP-13 inhibitory actions.

Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes.

Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)-CB1R agonist, rimonabant-CB1R antagonist, John W. Huffman (JWH133)-CB2R agonist and CB2R antagonist-6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation.

Cerebral microvascular matrix metalloproteinase-3 (MMP3) contributes to vascular injury after stroke in female diabetic rats

Diabetes exacerbates hemorrhagic transformation (HT) after stroke and worsens clinical outcomes. Female patients with diabetes are at a greater risk of stroke and worsened recovery. We have shown that activation of matrix metalloprotease 3 (MMP3) in hyperglycemic settings mediates HT in male rats. In light of our recent findings that diabetic female rats develop greater HT, the current study was designed to test the hypotheses that: 1) cerebral microvascular MMP3 activation contributes to poor functional outcomes and increased hemorrhagic transformations (HT) after ischemic stroke, and 2) MMP3 inhibition can improve functional outcomes in female diabetic rats. Female control and diabetic Wistar rats were subjected to 60 min of middle cerebral artery occlusion (MCAO). One cohort of diabetic animals received a single dose of MMP3 inhibitor (UK356618; 15 mg/kg; iv) or vehicle after reperfusion. Neurobehavioral outcomes, brain infarct size, edema, HT, and MMPs were measured in brain tissue. Diabetic rats had significant neurological deficits on Day 3 after stroke. MMP3 expression and enzyme activity were significantly increased in both micro and macro vessels of diabetic animals. MMP3 inhibition improved functional outcomes and reduced brain edema and HT scores. In conclusion, cerebral endothelial MMP3 activation to vascular injury in female diabetic rats. Our findings identify MMP3 as a potential therapeutic target in diabetic stroke.

Bioactivity of a polyhydroxy gorgostane steroid from Xenia umbellata

Abstract A C-30 steroid, 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol was isolated from the soft coral Xenia umbellata Lamarck (Xeniidae). The chemical structure was elucidated by examining the NMR spectral data and comparison with the previously published data. Compound 1 inhibited the growth of ovarian cancer (SKOV-3), breast cancers (MCF-7 and MDA-MB-231) and hepatocellular carcinoma (HepG2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Notably against HepG2, compound 1 showed significant effect with an IC50 value of 19.70 ± 1.98 µg/mL. It significantly increased the population in the SubG1 phase for 2.01- and 2.05-folds, respectively, compared to untreated cells. Additionally, it showed potent inhibitory activities of superoxide dismutase (384.6 vs 8594.2 U/g protein in dimethyl sulfoxide-treated cells), catalase (0.3 vs 0.07 U/g protein), decreased the level of reduced glutathione (1.7 vs 0.6 mg/g protein) and the activity of matrix metalloproteases (MMP-2 and MMP-9 [0.5-fold of change in MMP activity]) in HepG2 cells. The results indicated the potent antiproliferative activity of the gorgostane derivative (1) against HepG2 cells. This study provides a scientific basis of the antiproliferative effects of steroidal compound with gorgostane nucleus against hepatocellular carcinoma cells.

Dentine biomodification by sulphonamides pre-treatment: bond strength, proteolytic inhibition, and antimicrobial activity

We evaluated the effects of dentine biomodification after pre-treatment with two sulphonamide carbonic anhydrase inhibitors (CAIs) of the N-[4-sulphamoylphenethylcarbamoyl]benzenesulphonamide type, investigating matrix metalloproteases activity, resin–dentine micro tensile bond strength, dentine surface wettability, and antimicrobial activities. Ninety-five sound-extracted human molars were selected for the study. Inhibitory effects were evaluated by gelatinase and collagenase activity tests and collagen degradation FT-IR spectroscopic analysis. Pre-treatment with the two CAIs kept the micro tensile values after 12 months of storage (32.23 ± 5.95) and cariogenic challenge (34.13 ± 2.71) similar to the initial, pre-treatment values (33.56 ± 4.34). A decreased Streptococcus mutans biofilm formation on dentine surfaces and antibacterial activity against planktonic bacteria were observed after CAI treatment. Dentine pre-treatment with sulphonamide CAIs maintained adhesion strength stability, allowed better dentine wettability, maintained matrix collagen, and showed anti-S. mutans activity.

Role of NADPH Oxidases in Blood-Brain Barrier Disruption and Ischemic Stroke.

NADPH oxidases (Nox) are one of the main sources of reactive oxygen species (ROS) in the central nervous system (CNS). While these enzymes have been shown to be involved in physiological regulation of cerebral vascular tone, excessive ROS produced by Nox1-5 play a critical role in blood–brain barrier (BBB) dysfunction in numerous neuropathologies. Nox-derived ROS have been implicated in mediating matrix metalloprotease (MMP) activation, downregulation of junctional complexes between adjacent brain endothelial cells and brain endothelial cell apoptosis, leading to brain microvascular endothelial barrier dysfunction and consequently, increases in BBB permeability. In this review, we will highlight recent findings on the role played by these enzymes in BBB disruption induced by ischemic stroke.

HDL metabolism and functions impacting on cell cholesterol homeostasis are specifically altered in patients with abdominal aortic aneurysm.

BackgroundThe etiopathogenesis of abdominal aortic aneurysm (AAA) is still unclarified, but vascular inflammation and matrix metalloproteases activation have a recognized role in AAA development and progression. Circulating lipoproteins are involved in tissue inflammation and repair, particularly through the regulation of intracellular cholesterol, whose excess is associated to cell damage and proinflammatory activation. We analyzed lipoprotein metabolism and function in AAA and in control vasculopathic patients, to highlight possible non-atherosclerosis-related, specific abnormalities.MethodsWe measured fluorometrically serum esterified/total cholesterol ratio, as an index of lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in patients referred to vascular surgery either for AAA (n=30) or stenotic aortic/peripheral atherosclerosis (n=21) having similar burden of cardiovascular risk factors and disease. We measured high-density lipoprotein (HDL)-cholesterol efflux capacity (CEC), through the ATP-binding cassette G1 (ABCG1) and A1 (ABCA1) pathways and serum cell cholesterol loading capacity (CLC), by radioisotopic and fluorimetric methods, respectively.ResultsWe found higher LCAT (+23%; p < 0.0001) and CETP (+49%; p < 0.0001) activity in AAA sera. HDL ABCG1-CEC was lower (−16%; p < 0.001) and ABCA1-CEC was higher (+31.7%; p < 0.0001) in AAA. Stratification suggests that smoking may partly contribute to these modifications. CEC and CETP activity correlated with CLC only in AAA.ConclusionsWe demonstrated that compared to patients with stenotic atherosclerosis, patients with AAA had altered HDL metabolism and functions involved in their anti-inflammatory and tissue repair activity, particularly through the ABCG1-related intracellular signaling. Clarifying the relevance of this mechanism for AAA evolution might help in developing new diagnostic parameters and therapeutic targets for the early management of this condition.

Intra-articular injection of flavopiridol-loaded microparticles for treatment of post-traumatic osteoarthritis

Rapid joint clearance of small molecule drugs is the major limitation of current clinical approaches to osteoarthritis and its subtypes, including post-traumatic osteoarthritis (PTOA). Particulate systems such as nano/microtechnology could provide a potential avenue for improved joint retention of small molecule drugs. One drug of interest for PTOA treatment is flavopiridol, which inhibits cyclin-dependent kinase 9 (CDK9). Herein, polylactide-co-glycolide microparticles encapsulating flavopiridol were formulated, characterized, and evaluated as a strategy to mitigate PTOA-associated inflammation through the inhibition of CDK9. Characterization of the microparticles, including the drug loading, hydrodynamic diameter, stability, and release profile was performed. The mean hydrodynamic diameter of flavopiridol particles was ∼15 µm, indicating good syringeability and low potential for phagocytosis. The microparticles showed no cytotoxicity in-vitro, and drug activity was maintained after encapsulation, even after prolonged exposure to high temperatures (60 °C). Flavopiridol-loaded microparticles or blank (unloaded) microparticles were administered by intraarticular injection in a rat knee injury model of PTOA. We observed significant joint retention of flavopiridol microparticles compared to the soluble flavopiridol, confirming the sustained release behavior of the particles. Matrix metalloprotease (MMP) activity, an indicator of joint inflammation, was significantly reduced by flavopiridol microparticles 3 days post-injury. Histopathological analysis showed that flavopiridol microparticles reduced PTOA severity 28 days post-injury. Taken altogether, this work demonstrates a promising biomaterial platform for sustained small molecule drug delivery to the joint space as a therapeutic measure for post-traumatic osteoarthritis. Statement of significancePost-traumatic osteoarthritis (PTOA) begins with the deterioration of subchondral bone and cartilage after acute injuries. In spite of the prevalence of PTOA and its associated financial and psychological burdens, therapeutic measures remain elusive. A number of small molecule drugs are now under investigation to replace FDA-approved palliative measures, including cyclin-dependent kinase 9 (CDK9) inhibitors which work by targeting early inflammatory programming after injury. However, the short half-life of these drugs is a major hurdle to their success. Here, we show that biomaterial encapsulation of Flavopiridol (CDK9 inhibitor) in poly (lactic-co-glycolic acid) microparticles is a promising route for direct delivery and improved drug retention time in the knee joint. Moreover, administration of the flavopiridol microparticles reduced the severity of PTOA.