Patsy R. Carter

Effects of the endothelin-converting enzyme inhibitor SM-19712 in a mouse model of dextran sodium sulfate-induced colitis.

Background: Ingestion by mice of dextran sodium sulfate (DSS) induces colonic vasoconstriction and inflammation, with some of the effects potentially mediated by the vasoconstrictor endothelin‐1 (ET‐1). Methods: In this study, mice given 5% 40 kD DSS for 5–6 days had elevated colonic immunostaining for ET‐1 and platelet endothelial cell adhesion molecule‐1 (PECAM‐1). Increased ET‐1 can induce microvascular constriction; however, the increase in PECAM‐1 is consistent with angiogenesis that could decrease flow resistance. Results: Our measurements of intestinal blood flow, via infused microspheres, suggests that these 2 factors may offset each other, with only a nonsignificant tendency for a DSS‐induced decrease in flow. Daily administration of the endothelin converting enzyme inhibitor SM‐19712 (15 mg/kg) attenuated DSS‐induced increases in colonic immunostaining of ET‐1 and PECAM‐1. Conclusions: SM‐19712 attenuated histologic signs of tissue injury and inflammation induced by DSS, and decreased the extent of loose stools and fecal blood. However, the inhibitor did not significantly decrease DSS‐induced colon shortening or tissue levels of myeloperoxidase (an indicator of neutrophil infiltration).

Relationship between inflammation and tissue hypoxia in a mouse model of chronic colitis

Background: Hypoxia has been reported to be associated with the colonic inflammation observed in a chemically induced mouse model of self‐limiting colitis, suggesting that low tissue oxygen tension may play a role in the pathophysiology of inflammatory tissue injury. However, no studies have been reported evaluating whether tissue hypoxia is associated with chronic gut inflammation. Therefore, the objective of the present study was to determine whether hypoxia is produced within the colon during the development of chronic gut inflammation. Methods: Adoptive transfer of CD4+ T cells obtained from interleukin‐10‐deficient (IL‐10−/−) mice into lymphopenic recombinase‐activating gene‐1‐deficient (RAG−/−) mice induces chronic colonic inflammation, with the inflammation ranging from mild to severe as determined by blinded histological analyses. Colonic blood flow, hematocrit, and vascular density were determined using standard protocols, whereas tissue hypoxia was determined using the oxygen‐dependent probe pimonidazole. Results: Adoptive transfer of IL‐10−/− CD4+ T cells into RAG−/− recipients induced chronic colonic inflammation that ranged from mild to severe at 8 weeks following T‐cell transfer. The colitis was characterized by bowel wall thickening, goblet cell dropout, and inflammatory infiltrate. Surprisingly, we found that animals exhibiting mild colonic inflammation had increased hypoxia and decreased systemic hematocrit, whereas mice with severe colitis exhibited levels of hypoxia and hematocrit similar to healthy controls. In addition, we observed that the extent of hypoxia correlated inversely with hematocrit and vascular density. Conclusions: Changes in hematocrit, vascular density, and inflammatory state appear to influence the extent of tissue oxygenation in the T‐cell‐mediated model of chronic gut inflammation. (Inflamm Bowel Dis 2011;)

Antecedent Ethanol Ingestion Prevents Postischemic P-Selectin Expression in Murine Small Intestine

Objective: Ethanol ingestion 24 h prior to ischemia and reperfusion (I/R) prevents postischemic leukocyte rolling and adhesion in postcapillary venules of the small bowel. Since I/R‐induced leukocyte rolling is critically dependent on the expression of P‐selectin by endothelial cells lining postcapillary venules, the authors hypothesized that antecedent ethanol consumption would attenuate postischemic expression of this adhesive ligand.

Association between blood flow and inflammatory state in a T-cell transfer model of inflammatory bowel disease in mice

Background:Adoptive transfer of naive T‐lymphocyte subsets into lymphopenic mice initiates chronic gut inflammation that mimics several aspects of inflammatory bowel disease (IBD). Patients with IBD can have profound alterations in intestinal blood flow, but whether the same is true in the T‐cell transfer model has yet to be determined. Methods:In the current study, chronic intestinal inflammation was induced in recombinase‐activating gene‐1‐deficient (RAG−/−) mice by adoptive transfer of CD4+ T‐lymphocytes obtained from interleukin‐10 deficient (IL‐10−/−) mice. Results:Four weeks later, widespread colonic inflammation was observed in the reconstituted recipients, in contrast to 2 control sets of mice injected with a different subset of lymphocytes or with vehicle alone. We observed that the resulting pathology induced in the reconstituted RAG−/− mice was divided distinctly into 2 subsets: 1 with blood flow near normal with very high inflammation scores, and the other with severely attenuated blood flow but with much lower signs of inflammation. Colonic and ileal blood flow rates in the latter subset of CD4+ mice averaged only ≈30% compared to the mice with higher inflammation scores. The lower blood flow rates were associated with greatly reduced red blood cell concentrations in the tissue, suggesting a possible loss of vascular density. Conclusions:In this model of chronic intestinal inflammation, mild inflammation was associated with significant decreases in blood flow. Inflamm Bowel Dis 2009

Polynitroxylated starch/TPL attenuates cachexia and increased epithelial permeability associated with TNBS colitis

Free radicals play an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, the reduction or elimination of adverse oxidant effects can provide novel therapy for IBD. Here, the antioxidant capacity and protective effects of a new class of chemically modified hetastarch (polynitroxyl starch, or PNS) plus 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol or TPL) (PNS/TPL) were assessed in a model of colitis. The superoxide scavenging capacity of PNS/TPL—that is, the inhibition of the reduction of cytochrome c in the presence of xanthine/xanthine oxidase (X/XO)—was evaluated in vitro. The effects of PNS/TPL on X/XO–induced neutrophil endothelial adhesion in vitro were investigated. Also, this study tested the protection produced by PNS/TPL in a mouse model of trinitrobenzene sulfonic acid (TNBS)–induced colitis. PNS/TPL was given intravenously immediately before (<30 min) and intraperitoneally at 24 and 72 hr after TNBS induction. The body weight and survival rate of the mice were checked daily. Colonic mucosal damage was assessed on the 7th day by measuring intestinal permeability to Evans blue (EB) in vivo. The ability of PNS to reoxidize bioreduced TPL was documented by whole-body electron paramagnetic resonance (EPR) detection. We found that PNS or TPL exhibits superoxide dismutase (SOD)–like activity, with approximately 2% of SOD activity occurring on a molar basis. The endothelial–neutrophil adherence induced by X/XO was significantly inhibited by PNS/TPL but not by TPL alone. PNS/TPL protected against cachexia and mortality, both usually induced by TNBS. Epithelial permeability was increased significantly in TNBS mice but was ameliorated by the administration of PNS/TPL. In conclusion, PNS/TPL may be beneficial in the treatment or prevention of IBD through its antioxidant effects, which inhibit oxidant-mediated leukocyte adhesion and injury to endothelial cells.

Altered microvascular hemodynamics during the induction and perpetuation of chronic gut inflammation

Adoptive transfer of naïve CD4+ T cells into lymphopenic mice induces chronic small and large bowel inflammation similar to Crohns disease. Although much is now known regarding the immunopathology in this model of inflammatory bowel disease, virtually nothing is known about the microvascular hemodynamic changes during the induction and perpetuation of chronic gut inflammation. In this study, CD4+CD45RBhigh T cells obtained from healthy C57BL/6 donor mice were transferred into lymphopenic recombinase-activating gene-1-deficient (RAG knockout) mice, which induced small and large bowel inflammation. At various time points following reconstitution (3 days-9 wk), intravital microscopy was used to examine the microvessels in the submucosa of the ileum and proximal colon following infusion of fluorescently labeled platelets and injection of rhodamine 6G (to label leukocytes). Hemodynamic measurements and the extent of blood cell adhesion to the venular wall were compared with measurements in unreconstituted RAG knockout controls. In <1 wk following reconstitution, velocity and wall shear rate of the arterioles decreased by >50% compared with controls, with this decrease also observed at 4-5 and 7-9 wk postreconstitution. At 7-9 wk, arteriolar diameters were found to be approximately 15% larger than in controls, but, despite this dilation, flow rates in the individual vessels were decreased by approximately 30%. Venular platelet and leukocyte adherence were not significantly elevated above controls; however, an association was found between platelet adherence and venular shear rate. In summary, significant decreases in arteriolar velocity and shear rates are observed in this model of chronic gut inflammation.

Glutamate transport asymmetry and metabolism in the functioning kidney

Renal glutamate extraction in vivo shows a preference for the uptake ofd-glutamate on the antiluminal and l-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either d- orl-glutamate alone or in combination with the system [Formula: see text]specific transport inhibitor,d-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion withd-glutamate alone resulted in a removal rate that equaled or exceeded thel-glutamate removal rate, with uptake predominantly across the antiluminal surface;l-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake ofd-glutamate at the antiluminal and l-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolard-aspartate concentration blocked most of the antiluminald-glutamate uptake and a significant portion of the luminall-glutamate uptake, consistent with system [Formula: see text] activity at both sites. d-Glutamate uptake was associated with 5-oxo-d-proline production, whereas l-glutamate uptake supported both glutamine and 5-oxo-l-proline formation;d-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system[Formula: see text] activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity towardl- andd-glutamate suggests that functional asymmetry may reflect two different[Formula: see text] transporter subtypes.Renal glutamate extraction in vivo shows a preference for the uptake of D-glutamate on the antiluminal and L-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either D- or L-glutamate alone or in combination with the system X-AG specific transport inhibitor, D-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion with D-glutamate alone resulted in a removal rate that equaled or exceeded the L-glutamate removal rate, with uptake predominantly across the antiluminal surface; L-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake of D-glutamate at the antiluminal and L-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolar D-aspartate concentration blocked most of the antiluminal D-glutamate uptake and a significant portion of the luminal L-glutamate uptake, consistent with system X-AG activity at both sites. D-Glutamate uptake was associated with 5-oxo-D-proline production, whereas L-glutamate uptake supported both glutamine and 5-oxo-L-proline formation; D-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system X-AG activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity toward L- and D-glutamate suggests that functional asymmetry may reflect two different X-AG transporter subtypes.

Glutamate transport asymmetry in renal glutamine metabolism

d-Glutamate (Glu) was previously shown to block l-Glu uptake and accelerate glutaminase flux in cultured kidney cells [Welbourne, T. C., and D. Chevalier. Am. J. Physiol. 272 ( Endocrinol. Metab. 35): E367-E370, 1997]. To test whether d-Glu would be taken up by the intact functioning kidney and effect the same response in vivo, male Sprague-Dawley rats were infused withd-Glu (2.6 μmol/min), and renal uptake of d- andl-Glu was determined from chemical and radiolabeled arteriovenous Glu concentration differences times renal plasma flow. The amount removed was then compared with that amount filtered to obtain the antiluminal contribution. In the controls, l-Glu uptake measured as net removal was 33% of the arteriall-Glu load and not different from that filtered, 27%; however, the unidirectional uptake was actually 58% of the arterial load, indicating that antiluminal uptake contributes at least half to the overall Glu consumption. Surprisingly, the kidneys showed a more avid removal ofd-Glu, removing 73% of the arterial load, indicating uptake predominantly across the antiluminal cell surface. Furthermore, uptake ofd-Glu was associated with a 55% reduction in l-Glu uptake, with the residual amount taken up equivalent to that filtered;d-Glu did not increase the excretion of the l-isomer. However, elevating plasma l-Glu concentration reduced uptake of thed-isomer, suggesting a shared antiluminal transporter. Thus there is an apparent asymmetrical distribution of the d-Glu transporter. Under these conditions, kidney cortexl-Glu content decreased 44%, whereas net glutamine (Gln) uptake increased sevenfold (170 ± 89 to 1,311 ± 219 nmol/min, P < 0.01) and unidirectional uptake nearly threefold (393 ± 121 to 1,168 ± 161 nmol/min, P < 0.05); this large Gln consumption was paralleled by an increase in ammonium production so that the ratio of production to consumption approaches 2, consistent with accelerated Gln deamidation and subsequent Glu deamination. These results point to a functional asymmetry (antiluminal vs. luminal) for Glu transporter activity, which potentially plays an important role in modulating Gln metabolism and renal function.

Relationship among circulating leukocytes, platelets, and microvascular responses during induction of chronic colitis

The mechanisms by which microvascular alterations contribute to the pathogenesis of the inflammatory bowel diseases (IBDs; Crohns disease, ulcerative colitis) have not been clearly delineated. The purpose of the current study was to characterize the inflammatory events, microvascular alterations, and blood cell changes that occur in a mouse model of IBD. In this model, CD4(+) T-lymphocytes obtained from interleukin-10-deficient mice were injected intraperitoneally into lymphopenic, recombinase-activating gene-1 deficient (RAG(-/-)) mice. Two groups of control mice were also included: RAG(-/-) mice and C57BL/6 mice that were injected with phosphate-buffered saline but did not receive the T-cells. Four weeks later, the RAG(-/-) mice that had received the T-cell transfer showed significant signs of colonic inflammation, but without significant decreases in either body weight or mean arterial blood pressure. T-cell transfer increased the volume % of circulating platelets, while decreasing the number of circulating red blood cells. Additionally, the T-cell transfer tended to increase the circulating numbers of both lymphocytes and neutrophils when compared to unmanipulated RAG(-/-) mice. First-order colonic arterioles and venules tended to dilate in the colitic mice; however, the dilation was considerably more substantial with higher numbers of circulating leukocytes. The possibility that circulating inflammatory cells initiate the microvascular alterations in colitis warrants further investigation.

Decreased retinal blood flow in experimental colitis; improvement by eye drop administration of losartan.

Patients with inflammatory bowel disease suffer not only from gut inflammation, but also from extraintestinal manifestations of the disease, including ocular pathology. The mechanisms causing ocular inflammation in these patients are unknown. The purpose of the current study was to investigate the possible vascular changes occurring in the retina using a mouse model of acute colitis, that is, ingestion of dextran sodium sulfate (DSS). Intravital microscopy of anesthetized mice revealed that DSS caused a significant 30-40% decrease in retinal red blood cell velocities, and a 45% decrease in total retinal blood flow, but no changes in intraocular pressure. To determine whether the decreases in retinal perfusion could be inhibited by an angiotensin II receptor antagonist, losartan was administered by eye drops in a subset of the mice prior to the intravital microscopy measurements. Topical losartan was able to largely attenuate the altered hemodynamics induced by DSS. We conclude that angiotensin II might be a possible target for reducing the vascular changes occurring distantly in the eye during colitis.