Dimethyl Sulfoxide as a Novel Therapy in a Murine Model of Acute Lung Injury.

INTRODUCTION: The endothelial glycocalyx (EGX) on the luminal surface of endothelial cells contributes to the permeability barrier of the pulmonary vasculature. Dimethyl sulfoxide (DMSO) has a disordering effect on plasma membranes, which prevents the formation of ordered membrane domains important in the shedding of the EGX. We hypothesized that DMSO would protect against protein leak by preserving the EGX in a murine model of acute respiratory distress syndrome (ARDS). METHODS: C57BL/6 mice were given ARDS via intra-tracheally administered lipopolysaccharide (LPS). DMSO (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed post-injury day 4 after bronchoalveolar lavage (BAL). BAL cell counts and protein content was quantified. Lung sections were stained with FITC-labelled wheat germ agglutinin (FITC-WGA) to quantify the EGX. Cultured endothelial cells (HUVECs) were exposed to LPS. EGX was measured using FITC-WGA, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. RESULTS: DMSO treatment resulted in greater EGX staining intensity in the lung when compared to sham (9,641 vs. 36,659 A.U. p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 x 106 vs. 2.49 x 106 cells, p = 0.04). The treated group had less BAL macrophages (189.2 vs. 76.9 cells, p = 0.02) and lymphocytes (527.7 vs. 200.0 cells, p = 0.02). Interleukin-6 levels were lower in DMSO treated. Animals that received DMSO had less protein leak in BAL (1.48 vs. 1.08 ug/ul, p = 0.02). DMSO prevented LPS-induced EGX loss in HUVECs, and reduced the interaction between Matrix Metalloproteinase (MMP) 16 and syndecan-1. CONCLUSIONS: Systemically administered DMSO protects the EGX in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. DMSO also results in decreased inflammatory response. DMSO reduced the interaction between MMP16 and Syndecan-1 and prevented LPS-induced glycocalyx damage in cultured endothelial cells. DMSO may be a novel therapeutic for ARDS. LEVEL OF EVIDENCE: Not applicable (animal studies).

Dimethyl malonate protects the lung in a murine model of acute respiratory distress syndrome.

BACKGROUND: Succinate is a proinflammatory citric acid cycle metabolite that accumulates in tissues during pathophysiological states. Oxidation of succinate after ischemia-reperfusion leads to reversal of the electron transport chain and generation of reactive oxygen species. Dimethyl malonate (DMM) is a competitive inhibitor of succinate dehydrogenase, which has been shown to reduce succinate accumulation. We hypothesized that DMM would protect against inflammation in a murine model of ARDS. METHODS: C57BL/6 mice were given ARDS via 67.7 µg of intratracheally administered lipopolysaccharide. Dimethyl malonate (50 mg/kg) was administered via tail vein injection 30 minutes after injury, then daily for 3 days. The animals were sacrificed on day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts were performed to examine cellular influx. Supernatant protein was quantified via Bradford protein assay. Animals receiving DMM (n = 8) were compared with those receiving sham injection (n = 8). Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify the endothelial glycocalyx (EGX). RESULTS: Total cell counts in BAL was less for animals receiving DMM (6.93 × 10 6 vs. 2.46 × 10 6 , p = 0.04). The DMM group had less BAL macrophages (168.6 vs. 85.1, p = 0.04) and lymphocytes (527.7 vs. 248.3; p = 0.04). Dimethyl malonate-treated animals had less protein leak in BAL than sham treated (1.48 vs. 1.15 µg/µl, p = 0.03). Treatment with DMM resulted in greater staining intensity of the EGX in the lung when compared with sham (12,016 vs. 15,186 arbitrary units, p = 0.03). Untreated animals had a greater degree of weight loss than treated animals (3.7% vs. 1.1%, p = 0.04). Dimethyl malonate prevented the upregulation of monocyte chemoattractant protein-1 (1.66 vs. 0.92 RE, p = 0.02) and ICAM-1 (1.40 vs. 1.01 RE, p = 0.05). CONCLUSION: Dimethyl malonate reduces lung inflammation and capillary leak in ARDS. This may be mediated by protection of the EGX and inhibition of monocyte chemoattractant protein-1 and ICAM-1. Dimethyl malonate may be a novel therapeutic for ARDS.

Succinate metabolism and membrane reorganization drives the endotheliopathy and coagulopathy of traumatic hemorrhage.

Acute hemorrhage commonly leads to coagulopathy and organ dysfunction or failure. Recent evidence suggests that damage to the endothelial glycocalyx contributes to these adverse outcomes. The physiological events mediating acute glycocalyx shedding are undefined, however. Here, we show that succinate accumulation within endothelial cells drives glycocalyx degradation through a membrane reorganization-mediated mechanism. We investigated this mechanism in a cultured endothelial cell hypoxia-reoxygenation model, in a rat model of hemorrhage, and in trauma patient plasma samples. We found that succinate metabolism by succinate dehydrogenase mediates glycocalyx damage through lipid oxidation and phospholipase A2-mediated membrane reorganization, promoting the interaction of matrix metalloproteinase 24 (MMP24) and MMP25 with glycocalyx constituents. In a rat hemorrhage model, inhibiting succinate metabolism or membrane reorganization prevented glycocalyx damage and coagulopathy. In patients with trauma, succinate levels were associated with glycocalyx damage and the development of coagulopathy, and the interaction of MMP24 and syndecan-1 was elevated compared to healthy controls.

EXOSOMES AND MICROVESICLES FROM ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROTECTS THE ENDOTHELIAL GLYCOCALYX FROM LPS INJURY.

ABSTRACT: Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 µg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.

Glycocalyx degradation and the endotheliopathy of viral infection.

The endothelial glycocalyx (EGX) contributes to the permeability barrier of vessels and regulates the coagulation cascade. EGX damage, which occurs in numerous disease states, including sepsis and trauma, results in endotheliopathy. While influenza and other viral infections are known to cause endothelial dysfunction, their effect on the EGX has not been described. We hypothesized that the H1N1 influenza virus would cause EGX degradation. Human umbilical vein endothelial cells (HUVECs) were exposed to varying multiplicities of infection (MOI) of the H1N1 strain of influenza virus for 24 hours. A dose-dependent effect was examined by using an MOI of 5 (n = 541), 15 (n = 714), 30 (n = 596), and 60 (n = 653) and compared to a control (n = 607). Cells were fixed and stained with FITC-labelled wheat germ agglutinin to quantify EGX. There was no difference in EGX intensity after exposure to H1N1 at an MOI of 5 compared to control (6.20 vs. 6.56 Arbitrary Units (AU), p = 0.50). EGX intensity was decreased at an MOI of 15 compared to control (5.36 vs. 6.56 AU, p<0.001). The degree of EGX degradation was worse at higher doses of the H1N1 virus; however, the decrease in EGX intensity was maximized at an MOI of 30. Injury at MOI of 60 was not worse than MOI of 30. (4.17 vs. 4.47 AU, p = 0.13). The H1N1 virus induces endothelial dysfunction by causing EGX degradation in a dose-dependent fashion. Further studies are needed to characterize the role of this EGX damage in causing clinically significant lung injury during acute viral infection.

Dimethyl malonate slows succinate accumulation and preserves cardiac function in a swine model of hemorrhagic shock.

BACKGROUND: Succinate (SI) is a citric acid cycle metabolite that accumulates in tissues during hemorrhagic shock (HS) due to electron transport chain uncoupling. Dimethyl malonate (DMM) is a competitive inhibitor of SI dehydrogenase, which has been shown to reduce SI accumulation and protect against reperfusion injury. Whether DMM can be therapeutic after severe HS is unknown. We hypothesized that DMM would prevent SI buildup during resuscitation (RES) in a swine model of HS, leading to better physiological recovery after RES. METHODS: The carotid arteries of Yorkshire pigs were cannulated with a 5-Fr catheter. After placement of a Swan-Ganz catheter and femoral arterial line, the carotid catheters were opened and the animals were exsanguinated to a mean arterial pressure (MAP) of 45 mm. After 30 minutes in the shock state, the animals were resuscitated to a MAP of 60 mm using lactated ringers. A MAP above 60 mm was maintained throughout RES. One group received 10 mg/kg of DMM (n = 6), while the control received sham injections (n = 6). The primary end-point was SI levels. Secondary end-points included cardiac function and lactate. RESULTS: Succinate levels increased from baseline to the 20-minute RES point in control, while the DMM cohort remained unchanged. The DMM group required less intravenous fluid to maintain a MAP above 60 (450.0 vs. 229.0 mL; p = 0.01). The DMM group had higher pulmonary capillary wedge pressure at the 20-minute and 40-minute RES points. The DMM group had better recovery of cardiac output and index during RES, while the control had no improvement. While lactate levels were similar, DMM may lead to increased ionized calcium levels. DISCUSSION: Dimethyl malonate slows SI accumulation during HS and helps preserve cardiac filling pressures and function during RES. In addition, DMM may protect against depletion of ionized calcium. Dimethyl malonate may have therapeutic potential during HS.

Dynamic modulation of Leishmania cytochrome c oxidase subunit IV (LmCOX4) expression in response to mammalian temperature.

The Leishmania LACK antigen is a ribosome-associated protein that facilitates expression of mitochondrial cytochrome c oxidase subunit IV (LmCOX4) to support parasite mitochondrial fitness and virulence within the vertebrate host. To further examine the relationship between LACK, its putative ribosome binding motif and LmCOX4, we compared the kinetics of LmCOX4 expression following temperature elevation in wildtype LACK (LACK WT) and LACK-putative ribosome-binding mutant (LACKDDE) L. major. We found that, after initial exposure to mammalian temperature, LmCOX4 levels became undetectable in LACKDDE L. major and also, surprisingly, in wild type (WT) control strains. Upon sustained exposure to mammalian temperature, LmCOX4 expression returned in WT control strains only. The initial loss of LmCOX4 in WT L. major was substantially reversed by treatment with the proteasome inhibitor MG132. Our findings indicate that initial loss of LmCOX4 under mammalian conditions is dependent upon proteasome degradation and LmCOX4 re-expression is dependent upon LACK possessing a WT putative ribosome binding motif.

Aptamer-functionalized hybrid nanoparticle for the treatment of breast cancer.

PURPOSE: Resistance to chemotherapeutic agents such as doxorubicin is a major reason for cancer treatment failure. At present the treatment option for metastatic breast cancer is very poor. Therefore, development of an effective therapeutic strategy to circumvent MDR of metastatic breast cancer is highly anticipated. The MDR of metastatic breast cancer cells was accompanied with the overexpression of P-gp transporter. Even though the overexpression of P-gp could be minimized by silencing with siRNA, the question is how they can be selectively targeted to the cancer cells. We propose that aptamer surface labeling of the nanoparticles could enhance the selectively delivery of p-gp siRNA into the metastatic breast cancer cells. Our hypothesis is that conjugating nanoparticles with a cancer cell specific aptamer should allow selective delivery of therapeutic drugs to tumor cells leading to enhanced cellular toxicity and antitumor effect as compared to unconjugated nanoparticles. The primary objective of this study is to develop a targeted nanocarrier delivery system for siRNA into breast cancer cells. DESIGN METHODS: For targeted delivery, Aptamer A6 has been used which can bind to Her-2 receptors on breast cancer cells. For aptamer binding to particle surface, maleimide-terminated PEG-DSPE (Mal-PEG) was incorporated into the nanoparticles. Initially, three blank hybrid nanoparticles (i.e. F21, F31, and F40) out of nine different formulations prepared by high pressure homogenization (HPH) using different amount of DOTAP, cholesterol, PLGA or PLGA-PEG and Mal-PEG were chosen. Then protamine sulfate-condensed GAPDH siRNA (TRITC conjugated; red) or P-gp siRNA was encapsulated into those nanoparticles. Finally, the particles were incubated with aptamer A6 (FITC conjugated; green) for surface labeling. RESULTS: Aptamer labeled-nanoparticles having PLGA are smaller in size than those having PLGA-PEG. Surface charge was reduced when the particles were labeled with aptamer. Cell transfection was increased significantly in Her-2 (+) SKBR-3 and 4T1-R cells but not in Her-2 poorly expressed MDA MB-231 and MCF-7 cells. The knockdown of P-gp was increased significantly when the particles were labeled with aptamer. No significant cellular toxicity was observed for any of these formulations. CONCLUSION: This preliminary study concludes that aptamer-functionalized hybrid nanoparticles could be used to deliver P-gp targeted siRNA into the breast cancer cells to overcome chemoresistance.

Preparation, structure and spasmolytic activities of some derivatives of harmine series of alkaloids.

Keeping in view the interesting chemistry and pharmacological importance of harmine series of bases -- the beta-carboline alkaloids, a number of new derivatives of tetrahydroharmine and harmalol have been prepared and characterized through spectral studies. Some of these derivatives showed spasmolytic activity. It was observed that all the N-acyl tetrahydroharmine derivatives are stable, not labile and no ring opening occurs in these compounds, as reported recently.

Structure and spasmolytic activity of eucalyptanoic acid from Eucalyptus camaldulensis var. obtusa and synthesis of its active derivative from oleanolic acid.

A new triterpenoid acid named eucalyptanoic acid (1) has been isolated from the fresh uncrushed leaves of Eucalyptus camaldulensis var. obtusa along with two known constituents, beta-sitosterol (2) and betulinic acid (3). The structure of 1 has been established as 3beta-hydroxyolean-9(11),12-dien-28-oic acid through spectral studies including 1D and 2D NMR. 1 and its acetyl (1a) and acetylmethyl (1b) derivatives were tested for spasmolytic activity. 1b was found to be the most active spasmolytic, mediated through blockade of calcium influx at 1 mg/mL. In the present study 1b was also prepared starting from oleanolic acid (4). Acetylation of 4 gave 4a, which on methylation afforded 4b. Reaction of 4b with N-bromosuccinimide (NBS) furnished 1b. Hence 4 may be regarded as the biogenetic precursor of 1. Compounds 4 and 4a were found inactive at 1 mg/mL, while 4b was moderately active in showing spasmolytic activity.

Triterpenoids from the leaves of Psidium guajava.

Two triterpenoids, 20beta-acetoxy-2alpha,3beta-dihydroxyurs-12-en-28-oic acid (guavanoic acid, 3), and 2alpha,3beta-dihydroxy-24-p-z-coumaroyloxyurs-12-en-28-oic acid (guavacoumaric acid, 7), along with six known compounds 2alpha-hydroxyursolic acid (1), jacoumaric acid (2), isoneriucoumaric acid (4), asiatic acid (5), ilelatifol D (6) and beta-sitosterol-3-O-beta-D-glucopyranoside (8), have been isolated from the leaves of Psidium guajava. Their structures were determined through spectroscopic methods. Compound 5 showed dose-dependent (10-500 microg/ml) spasmolytic activity in spontaneously contracting isolated rabbit jejunum preparations.