. Effects of hyperoxia on local and remote microcirculatory inflammatory response after splanchnic ischemia and reperfusion. Am J Physiol Heart Circ Physiol 285: H643-H652, 2003. First published April 24, 2003 10.1152/ajpheart.00900.2002-Splanchnic ischemia-reperfusion (I/R) causes tissue hypoxia that triggers local and systemic microcirculatory inflammatory responses. We evaluated the effects of hyperoxia in I/R induced by 40-min superior mesenteric artery (SMA) occlusion and 120-min reperfusion in four groups of rats: 1) control (anesthesia only), 2) sham operated (all surgical procedures without vascular occlusion; air ventilation), 3) SMA I/R and air, 4) SMA I/R and 100% oxygen ventilation started 10 min before reperfusion. Leukocyte rolling and adhesion in mesenteric microvessels, pulmonary microvascular blood flow velocity (BFV), and macromolecular (FITC-albumin) flux into lungs were monitored by intravital videomicroscopy. We also determined pulmonary leukocyte infiltration. SMA I/R caused marked decreases in mean arterial blood pressure (MABP) and blood flow to the splanchnic and hindquarters vascular beds and pulmonary BFV and shear rates, followed by extensive increase in leukocyte rolling and adhesion and plugging of Ͼ50% of the mesenteric microvasculature. SMA I/R also caused marked increase in pulmonary sequestration of leukocytes and macromolecular leak with concomitant decrease in circulating leukocytes. Inhalation of 100% oxygen maintained MABP at significantly higher values (P Ͻ 0.001) but did not change regional blood flows. Oxygen therapy attenuated the increase in mesenteric leukocyte rolling and adherence (P Ͻ 0.0001) and maintained microvascular patency at values not significantly different from sham-operated animals. Hyperoxia also attenuated the decrease in pulmonary capillary BFV and shear rates, reduced leukocyte infiltration in the lungs (P Ͻ 0.001), and prevented the increase in pulmonary macromolecular leak (P Ͻ 0.001), maintaining it at values not different from sham-operated animals. The data suggest that beneficial effects of normobaric hyperoxia in splanchnic I/R are mediated by attenuation of both local and remote inflammatory microvascular responses. reperfusion injury; acute lung injury; reoxygenation injury; intravital videomicroscopy; multiorgan failure; systemic inflammatory response syndrome HYPOPERFUSION AND ISCHEMIA of the bowel is a common, important pathological process that affects adults and infants. In both groups the morbidity and mortality related to these processes are high. In adults, occlusive and nonocclusive conditions may reduce splanchnic blood flow (8,35

This study investigated mechanisms of the hemodynamic effects of oxygen in hemorrhagic shock induced by bleeding 30% of the total blood volume in anesthetized rats. An ultrasonic flowmeter was used to monitor regional blood flow. Changes in tissue perfusion were assessed by the laser-Doppler technique. The inhalation of 100% oxygen induced a significant increase in mean arterial blood pressure (MABP) and vascular resistance in the hindquarters, with a concomitant decrease in blood flow in the distal aorta and biceps femoris muscle. In contrast, oxygen did not change vascular resistance in the superior mesenteric artery (SMA) and renal beds and induced a significant increase in blood flow to the renal artery, SMA, and small bowel in hemorrhaged rats. L-Arginine (100 mg/kg iv) but not D-arginine or the vehicle (0.9% NaCl) completely abolished the effects of oxygen on blood pressure and reversed its effects on blood flow and resistance in the hindquarters and biceps femoris muscle. Administration of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (50 mg/kg iv) significantly increased MABP and the resistance in the three vascular beds. Pretreatment of hemorrhaged rats with a superoxide dismutase mimic, the NO-stable radical 2,2,6,6-tetramethylpiperidine-N-oxyl (5 mg/kg iv), resulted in significantly diminished effects of oxygen on hindquarter hemodynamics. These results demonstrate a differential effect of oxygen, which increases vascular resistance in the hindquarters without a significant effect in the splanchnic and renal beds, thus favoring an increase in splanchnic and renal perfusion. It is suggested that inactivation of NO by reactive oxygen species may underlie the effects of oxygen on hindquarter vascular tone during shock.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) mediates tumor cell-macrophage interactions, and has been shown to induce both matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). However, the epitope responsible for MMP induction is controversial, and the epitope responsible for VEGF induction is yet unknown. We generated a novel anti-EMMPRIN antibody directed against a specific epitope that successfully inhibited the production of both MMP-9 and VEGF in tumor cellmacrophage in vitro co-culture systems, exhibiting a U-shaped dose response. Furthermore, this antibody efficiently inhibited in vivo tumor progression in both the RENCA renal cell carcinoma and CT26 colon carcinoma subcutaneous tumor models, and reduced tumor size and number of metastatic foci in the 4T1 orthotopic model. This was achieved by inhibiting angiogenesis as assessed by immunohistochemical staining for the endothelial marker CD31, by inhibiting tumor cell proliferation as assessed by the staining for Ki-67, and by enhancing tumor cell apoptosis as assessed in the TUNEL assay. Moreover, administration of the antibody recruited more macrophages into the tumor, and skewed the tumor microenvironment for macrophages from TGFb-dominated anti-inflammatory microenvironment, to a less immunosuppressive one. The antibody improved the ability of stimulated macrophages to perform antibody-dependent cell cytotoxicity (ADCC) and kill tumor cells. Thus, our new antibody maps the epitope capable of inducing both MMPs and VEGF, and places EMMPRIN as a good target for cancer therapy.

The behavior of white and red blood cells, platelets and circulating injected particles is one of the main focuses in physiological studies. Most methods used to analyze the circulatory patterns of cells are time consuming. We describe a system named CellTrack, designed for a fully automated tracking of circulating cells and micro-particles and retrieval of their behavioral characteristics.The task of automated blood cell tracking in vessels from in-vivo video is particularly challenging because of the blood cells' non-rigid shapes, the instability inherent in in-vivo videos, the abundance of moving objects and their frequent superposition.To tackle this, the CellTrack system operates on two levels: first, a global processing module extracts vessel borders and center lines based on color and temporal patterns. This enables the computation of the approximate direction of the blood flow in each vessel. Second, a local processing module extracts the locations and velocities of circulating cells. This is performed by artificial neural network classifiers that are designed to detect specific types of blood cells and micro-particles. The motion correspondence problem is then resolved by a novel algorithm that incorporates both the local and the global information. The system has been tested on a series of in-vivo color video recordings of rat mesentery. Our results show that the synergy between the global and local information enables CellTrack to overcome many of the difficulties inherent in tracking methods that rely solely on local information. A comparison was made between manual measurements and the automatically extracted measurements of leukocytes and fluorescent microspheres circulatory velocities. This comparison revealed an accuracy of 97%. CellTrack also enabled a much larger volume of sampling in a fraction of time compared to the manual measurements. All these results suggest that our method can in fact constitute a reliable replacement for manual extraction of blood flow characteristics from in-vivo videos.

Hyperoxia reduces small bowel injury, accelerates enterocyte turnover, and improves intestinal rehabilitation after IR.

Previously, we have identified a new epitope in EMMPRIN, a multifunctional protein that mediates tumor cell-macrophage interactions and induces both MMP-9 and VEGF. Here, we synthesized this epitope as an octa-branched multiple antigenic peptide (MAP) to vaccinate mice implanted with subcutaneous syngeneic colon (CT26), prostate (TRAMP-C2) or renal (RENCA) cell line carcinomas. Vaccination inhibited, and sometimes regressed, tumor growth in a dose-dependent manner, reaching 94%, 71% and 72% inhibition, respectively, at a 50 μg dose ( < 0.01). Mice with regressed tumors demonstrated immune memory, preventing tumor recurrence upon re-implantation ( < 0.001). When tumor cells were administered through the tail vein to generate lung metastases, vaccination reduced the number of metastatic foci (by 15- and 23-folds, < 0.001), and increased the median survival time by 25% and 53% in RENCA and CT26 metastases, respectively ( < 0.01) relative to scrambled-MAP controls. No significant adverse responses were observed in all experiments. We show that the tumor microenvironment was immune modulated, as vaccination induced production of EMMPRIN-specific antibodies, increased CD8 T cells infiltration and cytotoxicity, alleviated immune suppression by decreasing TGFβ concentrations, reduced angiogenesis and cell proliferation, and enhanced apoptosis. Thus, our successful active peptide vaccination strategy differs from previous, unsuccessful attempts, both in the selected target (the EMMPRIN epitope) and in the use of a modified, MAP configuration, and demonstrates that this may be an efficient approach for the treatment and prevention of some types of cancer.

Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME) that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS), miR-146a-5p inhibits nitric oxide (NO) production in a mouse renal carcinoma cell line (RENCA), thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p’s activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001). Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001), leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05), and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001) and angiogenesis (twofolds, p < 0.01), and increased apoptosis (twofolds, p < 0.01). This combination therapy increased nitrites and reduced TGFβ concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage therapy approach to induce the immune system to successfully attack the tumor, and should be further explored as a new therapy for the treatment of cancer.

lar endothelial injury is the major mechanism for multiorgan dysfunction in sepsis. Following this process, the permeability of the alveolar capillaries is augmented with subsequent increase in water content and acute respiratory distress syndrome (ARDS). Nevertheless, the role of alveolar epithelium is less known. Therefore, we examined alveolar fluid clearance (AFC) using isolated perfused rat lung model in septic rats without ARDS. Sepsis was induced by ligating and puncturing the cecum with a 21-gauge needle. AFC was examined 24 and 48 h later. The expression of Na-K-ATPase proteins was examined in type II alveolar epithelial cells (ATII) and basolateral membrane (BLM). The rate of AFC in control rats was 0.51 Ϯ 0.02 ml/h (means Ϯ SE) and decreased to 0.3 Ϯ 0.02 and 0.33 Ϯ 0.03 ml/h in 24 and 48 h after sepsis induction, respectively (P Ͻ 0.0001). Amiloride, significantly decreased AFC in sepsis; conversely, isoproterenol reversed the inhibitory effect of sepsis. The alveolar-capillary barrier in septic rats was intact; therefore the finding of increased extravascular lung water in early sepsis could be attributed to accumulation of protein-poor fluid. The expression of epithelial sodium channel and Na-K-ATPase proteins in whole ATII cells was not different in both cecal ligation and puncture and control groups; however, the abundance of Na-K-ATPase proteins was significantly decreased in BLMs of ATII cells in sepsis. Early decrease in AFC in remote sepsis is probably related to endocytosis of the Na-K-ATPase proteins from the cell plasma membrane into intracellular pools, with resultant inhibition of active sodium transport in ATII cells. acute respiratory distress syndrome; alveolar fluid clearance; active sodium transport; endocytosis SEPSIS AND RELATED SYSTEMIC inflammatory reaction is a lifethreatening disease that affects ϳ750,000 patients a year in the United States; the frequency is increasing, given an aging population with increasing numbers of patients with comorbid conditions (29). The acute respiratory distress syndrome (ARDS) is a common, devastating clinical syndrome of acute lung injury (ALI) that affects both medical and surgical patients. Sepsis that is not caused by pneumonia is associated with the highest risk of progression to ALI or ARDS (12, 42).Widespread vascular endothelial injury is thought to be the major mechanism for multiorgan dysfunction and ARDS in sepsis, thereby augmenting the permeability of alveolar capillaries with subsequent influx of protein-rich edema fluid into the air spaces (17).The resolution of both cardiogenic pulmonary edema and ARDS depends on the clearance of fluid from the alveolar space, a process that requires an intact, functional alveolar epithelium (22, 24). The primary driving force for alveolar fluid clearance (AFC) is the active transport of sodium from the alveolar space to the interstitium by alveolar epithelial type II cells (ATII cells) (2,21,23,34,36). Little is known about ATII function in sepsis. Therefore, we aimed to examine the effects of r...